WO2015167216A1 - Photocleavable mass tag material and use thereof - Google Patents

Photocleavable mass tag material and use thereof Download PDF

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WO2015167216A1
WO2015167216A1 PCT/KR2015/004242 KR2015004242W WO2015167216A1 WO 2015167216 A1 WO2015167216 A1 WO 2015167216A1 KR 2015004242 W KR2015004242 W KR 2015004242W WO 2015167216 A1 WO2015167216 A1 WO 2015167216A1
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antigen
maldi
mmol
antibody
dichloromethane
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French (fr)
Korean (ko)
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문봉진
오한빈
강나나
전애란
박계신
박형순
방주용
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다이아텍코리아 주식회사
서강대학교 산학협력단
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Publication of WO2015167216A1 publication Critical patent/WO2015167216A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/02—Iron compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/622—Ion mobility spectrometry
    • G01N27/623—Ion mobility spectrometry combined with mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/26—Mass spectrometers or separator tubes

Definitions

  • the present invention relates to a photodegradable mass labeling substance and its use, and more particularly, to a photodegradable mass labeling substance capable of easily photolysing to release a cation labeled with a specific mass and a matrix-free condition using the substance.
  • So-called "soft ionization” mass spectroscopy methods including matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI), allow for complete ionization, detection and mass measurement of macromolecules whose mass exceeds 300 kDa [ See Fenn et al., Science 246: 64-71 (1989); Karas and Hillenkamp, Anal. Chem. 60: 2299-3001 (1988).
  • MALDI-MS requires incorporation into the matrix of the macromolecules to be analyzed and has been performed on polypeptides and nucleic acids mixed in a solid (ie crystalline) matrix.
  • a laser is used to exfoliate the biopolymer / matrix mixture that crystallizes on the probe tip, which affects the desorption and ionization of the biopolymer.
  • MALDI-MS uses organic acids or glycerol such as hydrated water (i.e. ice) benzoic acid as a matrix, and the complexity of the matrix impedes the analysis of trace polypeptides, and when using hydrated water as a matrix, MALDI-MS is used as a matrix.
  • the protein Before performing, the protein must first be lyophilized or air dried. See Berkenkamp et al. (1996) Proc. Natl. Acad. Sci. USA 93: 7003-7007.
  • MALDI-TOF matrix-assisted laser desorption / ionization-time-of-flight
  • the current MALDI-TOF method is not enough to obtain information on the amount as well as the presence or absence of a biomarker. Therefore, it is absolutely necessary to develop a MALDI-TOF high-sensitivity quantitative method to overcome this problem, and furthermore, to develop a mass spectrometry method capable of amplifying signals such as quantitative analysis of trace biomolecules even if the matrix-assisted method is not used.
  • the present invention has been made in view of the above necessity, and an object of the present invention is to provide a photodegradable mass labeling material which can be measured in matrix-free conditions, unlike conventional MALDI-TOF MS.
  • the present invention to achieve the above object
  • R1 and R2 are H, alkyl, alkenyl, and Carney day, alkoxy, or aryl group
  • R3 is R 3 is a reactor that can react with the amine group of a protein or non-protein molecule
  • the reactor is N-hydroxysuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, benzotriazol-1-yloxyl ester, pentahalobenzyl ester and 4- It is preferably selected from the group consisting of nitrophenyl esters, but is not limited thereto.
  • the material is preferably a compound of any one of the following formulas (2) to (4);
  • the yellow circle portion is the mass changer portion
  • the yellow circle portion is the mass changer portion
  • R1 and R2 are H, alkyl, alkenyl, alkaniyl, alkoxy, or an aryl group
  • R ' is N.
  • n is 0 Is an integer of 20, but is not limited thereto.
  • the material is preferably a compound of Formula 5, but is not limited thereto;
  • n is preferably 0 to 20, but is not limited thereto.
  • the present invention also provides a substance comprising an antibody or an amine group conjugated with the labeling substance of the present invention.
  • the present invention is to treat the antigen-labeled sample on a maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface capable of binding the capture antibody to generate an antigen antibody complex between the antigen and the capture antibody. And treating the detection antibody labeled with the photodegradable mass labeling substance of the present invention to the complex to bind to another portion of the antigen to which the capture antibody does not bind only when the detection antibody is present in the antigen to be detected. It provides a method for performing laser desorption ionization time-of-flight mass spectrometry (LDI-TOF MS) without matrix.
  • LBI-TOF MS laser desorption ionization time-of-flight mass spectrometry
  • the present invention is to treat a sample containing a different type of antigen in a maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface to which the capture antibody can be bound to selectively select between the antigen and the capture antibody.
  • An antigen antibody complex is generated, and the complex is treated with a detection antibody labeled with the photodegradable mass label of the present invention, so that only when the antigen to be detected is present in the antigen, the capture antibody does not bind to another part of the antigen to which the capture antibody does not bind.
  • the plate is subjected to laser desorption ionization time-of flight mass spectrometry (LDI-TOF MS) without a matrix to provide a method for quantitative detection of multiple antigens.
  • LTI-TOF MS laser desorption ionization time-of flight mass spectrometry
  • the present invention is to treat the sample containing the antigen in a maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface capable of binding the capture antibody to generate an antigen antibody complex between the antigen and the capture antibody And treating the detection antibody labeled with the photodegradable mass labeling substance of the present invention to the complex to bind to another portion of the antigen to which the capture antibody does not bind only when the detection antibody is present in the antigen to be detected. It provides a method for quantitative analysis of antigen by performing laser desorption ionization time-of-flight mass spectrometry (LDI-TOF MS) without matrix.
  • LBI-TOF MS laser desorption ionization time-of-flight mass spectrometry
  • An object of the present invention is photolysis capable of releasing a specific mass labeled cation by easily photolysing under a matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer (MALDI-TOF MS) under irradiation with a 355 nm laser.
  • MALDI-TOF MS matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer
  • a MALDI plate treated with a capture antibody (or a primary antibody) and a detection antibody (or a secondary antibody) labeled with a photodegradable mass labeling agent are prepared, respectively.
  • the biomarkers are sprayed onto the prepared MALDI plate, only the antigen to be detected is selectively bound to the capture antibody immobilized by the antigen-antibody reaction, and when the labeled antibody is processed again, the detection antibody is like a sandwich. Bind to the antigen.
  • the labeled detection antibody is immobilized on the MALDI plate only when there is a biomarker (antigen) to be detected in the sample.
  • the mass-labeling material is photolyzed to generate cations, thereby analyzing the presence of antigen.
  • the biggest advantage of this method is that there is no signal contamination due to the non-specific antigen-antibody binding which is always present in the sandwich type detection method using antigen-antibody binding. Even if unwanted biomaterials are captured, their mass values do not appear at all because the matrix is not used. In this case, the measured signal intensity is proportional to the amount of labeling material, so that quantitative analysis of captured biomarkers is possible, and even if the labeling materials having different structures (mass values) are labeled with different antibodies, they can be used at once. Can be used to detect multiple markers because it releases cations of different mass values independently without affecting.
  • the new photodegradable mass labeling substance should basically have a structure capable of producing stable cations by light.
  • a reactor capable of binding the antibody and a mass changer capable of varying the mass for detecting multiple markers should be provided at the same time.
  • they must be materials that can be synthesized in a relatively simple and high yield protocol.
  • Ferrocene a lead compound used in the present invention, has been studied for a long time that the ⁇ -carbon cation of this ligand has a very stable metallocene structure. Based on this fact, the inventors devised ferrocene derivatives as shown in FIG. 3.
  • This derivative can be decomposed into stable carbon cations and sulfur anions while the CS bond at the ligand ⁇ position is unevenly decomposed by a 355 nm laser.
  • the mass labeling material of the present invention can be used for the multiplexing of multiple markers (FIG. 7).
  • the present inventors synthesized ferrocene derivatives having three different mass change groups as shown in FIG. 8 and compared their matrix-less LDI-TOF MS results to find the most suitable structure for the purpose.
  • Suzuki cross-coupling reaction of an intermediate made through a boronation reaction between a 9-BBN (9-borabicyclo [3.3.1] nonane) and a hydrocarbon compound having a terminal double (C C) as shown in FIG. 9.
  • the three compounds have two carbon chains of mass changer. Irrespective of the structure difference of the mass changer, matrix-less LDI-TOF MS was measured after mixing 6a-c in the same number of moles to verify whether the matrix-less LDI-TOF MS detection intensity was the same.
  • Conjugation of a novel mass labeling substance to a detection antibody requires a reactor capable of pairing with an amine group of a protein in high yield.
  • the most widely used of the N - hydroxy mugwort god imide ester (N -hydroxysuccinimide ester, NHS ester) It is known that this reactor reacts very effectively with the amine groups of a protein.
  • N - hydroxy mugwort god imide N -hydroxysuccinimide, NHS
  • N, N'- dicyclohexyl carbodiimide N, N '-dicyclohexylcarbodiimide, DCC
  • ferrocene derivatives 6-carboxylic acid and thereby the reaction of the terminal NHS ester is introduced at the site in high yield.
  • novel photodegradable mass labeling materials 17a, 17b, and 17c as shown in FIG. 14 were synthesized, and the compound was actually conjugated to a detection antibody and applied to detection of a biomarker.
  • the photodegradable mass labeling substances were dissolved in tetrahydrofuran (1.0 ⁇ 10 ⁇ 6 ⁇ 1.0 ⁇ 10 ⁇ 15 ) at various concentrations, then 1 ⁇ l was taken at each spot on the plate and exposed to air at room temperature to dry the solvent.
  • the dried samples on the plates were analyzed with a MALDI-TOF mass spectrometer (matrix assisted laser desorption time-of-flight mass spectrometer, Autoflex Speed series, BrukerDaltonics, Leipzig, Germany). All spectra were measured in cationic reflecton mode using lasers of 335 nm wavelength of varying intensity in the range of 10% to 70%.
  • the mass range was set at 0-800 Da, after which data analysis was performed using the flexAnalysis program.
  • the photodegradable mass labeling material of the present invention can be used for detection and quantitative analysis of biomarkers, and can be used for simultaneous detection of multiple markers.
  • FIG. 1 is a schematic view of the principle of operation of the photodegradable mass labeling material to be developed in the present invention
  • FIG. 3 is a diagram showing a photolysis reaction of a ferrocene mass labeling material
  • FIG. 7 is a schematic diagram of a principle of simultaneous detection of multiple markers using photodegradable mass labeling substances
  • 11 to 13 are matrix-less LDI-TOF MS detection limit measurement spectra of ferrocene derivatives 6a-c (laser power: 70%): detection signal intensity comparison spectra according to the absolute moles of molecules placed on a plate;
  • 15 is a diagram showing the results of LDI experiments in a matrix free state of the BSA conjugated tags
  • 16 is a verification diagram of tag signal ratio change according to protein mixing ratio
  • Figure 17 is a tag signal detection in the antibody system conjugated with leptin conjugated with a tag.
  • 18 is a diagram showing a method for synthesizing derivatives of photodegradable labeling substances of the present invention.
  • 19 is a diagram showing a method for synthesizing derivatives of photodegradable labeling substances of the present invention.
  • Ferrocene carboxyaldehyde (0.100 g, 0.467 mmol) was dissolved in ethanol (8 mL), and sodium borohydride (0.090 g, 2.4 mmol) was slowly added slowly at 0 ° C in small portions. The reaction mixture was stirred at room temperature for 3 hours. Water (3 mL) and dichloromethane (10 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (15 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure.
  • Acetyl ferrocene ( 1b , 0.100 g, 0.438 mmol) was dissolved in ethanol (5 mL), and sodium borohydride (0.087 g, 2.2 mmol) was slowly added in small portions at 0 ° C.
  • the reaction mixture was stirred at room temperature for 3 hours.
  • Water (3 mL) and dichloromethane (10 mL) were added sequentially to terminate the reaction.
  • the organic layer was separated and the remaining water layer was extracted three times with dichloromethane (15 mL x 3).
  • the combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure.
  • Acetyl ferrocene ( 1b , 0.080 g, 0.28 mmol) was dissolved in ethanol (5 mL), and sodium borohydride (0.052 g, 1.4 mmol) was slowly added in small portions at 0 ° C.
  • the reaction mixture was stirred at room temperature for 3 hours.
  • the reaction was terminated by adding water (2 mL) and dichloromethane (8 mL) in this order.
  • the organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • the combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure.
  • Acetyl ferrocene ( 1b , 0.100 g, 0.438 mmol) was dissolved in anhydrous tetrahydrofuran (4 mL) and cooled to -78 ° C, and then methyllithium solution (1.67 M diethyl ether solution, 1.1 mL, 1.8 mmol) was added to a nitrogen atmosphere. Slowly put in. After stirring at ⁇ 78 ° C. for 11 hours, water (2 mL) and dichloromethane (8 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Benzoylferrocene ( 1c , 0.100 g, 0.345 mmol) was dissolved in anhydrous tetrahydrofuran (3.5 mL) and cooled to -78 ° C, and then methyllithium solution (1.67 M diethyl ether solution, 0.46 mL, 0.77 mmol) was added to a nitrogen atmosphere. Slowly put in. After stirring at ⁇ 78 ° C. for 2.5 hours, water (2 mL) and dichloromethane (8 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Benzoylferrocene ( 1c , 0.100 g, 0.345 mmol) was dissolved in anhydrous tetrahydrofuran (3.5 mL), cooled to -78 ° C, and the phenyllithium solution (1.35 M dinormalbutylether solution, 0.51 mL, 0.69 mmol) was added to a nitrogen atmosphere. Slowly put in. After stirring at -78 ° C for 20 minutes, water (2 mL) and dichloromethane (8 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocenemethanol ( 2a , 0.020 g, 0.093 mmol) is dissolved in dichloromethane (0.4 mL), and thiophenol (0.031 g, 0.28 mmol) is added thereto. To this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.034 mL, 0.18 mmol). The reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocenyl (phenyl) methanol ( 2c , 0.030 g, 0.10 mmol) is dissolved in dichloromethane (1.0 mL), and then thiophenol (0.012 g, 0.11 mmol) is added thereto.
  • dichloromethane 1.0 mL
  • thiophenol 0.012 g, 0.11 mmol
  • the reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocenyldiphenylmethanol ( 2f, 0.042 g, 0.11 mmol) is dissolved in dichloromethane (0.8 mL) and thiophenol (0.029 g, 0.26 mmol) is added.
  • thiophenol 0.029 g, 0.26 mmol
  • the reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocene (0.500 g, 2.69 mmol) was dissolved in anhydrous dichloromethane (5 mL), and then paraanisoyl chloride ( p- anisoyl chloride, 0.504 g, 2.96 mmol) was added to the solution.
  • Aluminum chloride (0.394 g, 2.96 mmol) was slowly added in small portions at 0 ° C. The reaction mixture was stirred at room temperature for 11 hours, and then ice water (5 mL) and dichloromethane (10 mL) were added sequentially. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (20 mL x 3).
  • Ferrocenyl (4-methoylphenyl) methanone ( 4 , 0.152 g, 0.475 mmol) was dissolved in anhydrous dichloromethane (1.5 mL), cooled to -78 ° C, and boron tribromide (0.054 mL, 0.57 mmol) was added slowly under nitrogen atmosphere. . The temperature of the reaction mixture was slowly raised to room temperature and stirred for 8 hours. The reaction mixture was poured into iced water (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocenyl (4-hydroxylphenyl) methanone ( 5 , 0.094 g, 0.24 mmol) was dissolved in ethanol (1 mL), and sodium borohydride (0.027 g, 0.71 mmol) was slowly added slowly at 0 ° C. The reaction mixture was stirred at room temperature for 3 hours. Water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure.
  • Ferrocenyl (4-methoylphenyl) methanone ( 4 , 0.060 g, 0.19 mmol) was dissolved in a mixture of ethanol (0.5 mL) / tetrahydrofuran (0.5 mL), followed by a small amount of sodium borohydride (0.035 g, 0.94 mmol) at 0 ° C. Slowly put in. The reaction mixture was stirred at room temperature for 3 hours. Water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3).
  • Ferrocenyl (4-methoxyphenyl) methanol ( 7b , 0.059 g, 0.18 mmol) is dissolved in dichloromethane (0.8 mL) and thiophenol (0.022 g, 0.20 mmol) is added.
  • thiophenol 0.022 g, 0.20 mmol
  • the reaction mixture is stirred at room temperature for 3 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3).
  • Ferrocenyl (4-iodophenyl) methanone 9 , 0.100 g, 0.240 mmol
  • tetrakis (triphenylphosphine) palladium 0.028 g, 0.024 mmol
  • copper iodide I
  • 0.009 g, 0.05 mmol It was dissolved in furan (1 mL).
  • Triethylamine (0.27 mL, 1.9 mmol) was added to this solution in a nitrogen atmosphere. After removing the oxygen inside by blowing argon gas into the solution for 20 minutes, 1-decane (1-decyne, 0.037 g, 0.26 mmol) was put in an argon atmosphere, and stirred at room temperature for 12 hours.
  • Ferrocenyl (4-hexylphenyl) methanone ( 14a , 0.040 g, 0.11 mmol) was dissolved in anhydrous tetrahydrofuran (1 mL) and cooled to -78 ° C, followed by phenyllithium solution (1.35 M dinormalbutylether solution, 0.09 mL, 0.1 mmol) was slowly added in a nitrogen atmosphere. After stirring at ⁇ 78 ° C. for 1 minute, water (1 mL) and dichloromethane (3 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (5 mL x 3).
  • Ferrocenyl (4-octylphenyl) methanone ( 14b , 0.121 g, 0.301 mmol) was dissolved in anhydrous tetrahydrofuran (2 mL) and cooled to -78 ° C, followed by phenyllithium solution (1.35 M dinormalbutylether solution, 0.22 mL, 0.30 mmol) was slowly added in a nitrogen atmosphere. After stirring at ⁇ 78 ° C. for 1 minute, water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocenyl (4-hexylphenyl) (phenyl) methanol 15a , 0.048 g, 0.11 mmol was dissolved in dichloromethane (1 mL), and 3-mercaptopropionic acid (3-mercaptopropionic acid, 0.012 g, 0.12 mmol) was added thereto.
  • an aqueous solution of fluoroboric acid 48 wt%, 0.025 mg, 0.14 mmol.
  • the reaction mixture is stirred at room temperature for 3 hours, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL).
  • Ferrocenyl (4-octylphenyl) (phenyl) methanol 15b , 0.138 g, 0.287 mmol was dissolved in dichloromethane (1 mL) and 3-mercaptopropionic acid (3-mercaptopropionic acid, 0.037 g, 0.35 mmol) was added thereto.
  • an aqueous solution of fluoroboric acid 48 wt%, 0.074 mg, 0.40 mmol.
  • the reaction mixture is stirred at room temperature for 3 hours, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL).
  • N, N'-dicyclohexylcarbodiimide N, N'-dicyclohexylcarbodiimide 0.014 g, 0.14 mmol
  • anhydrous dichloromethane 0.3 mL
  • the temperature of the final reaction mixture was slowly raised to room temperature and stirred for 8 hours.
  • the reaction mixture was cooled to 0 ° C.
  • the solid that had settled by precipitation was washed with cold dichloromethane under reduced pressure.
  • N, N'-dicyclohexylcarbodiimide N, N'-dicyclohexylcarbodiimide 0.028 g, 0.14 mmol
  • anhydrous dichloromethane 0.4 mL
  • the temperature of the final reaction mixture was slowly raised to room temperature and stirred for 8 hours.
  • the reaction mixture was cooled to 0 ° C.
  • the solid that had settled by precipitation was washed with cold dichloromethane under reduced pressure.
  • N, N'-dicyclohexylcarbodiimide N, N'-dicyclohexylcarbodiimide 0.028 g, 0.14 mmol
  • anhydrous dichloromethane 0.4 mL
  • the temperature of the final reaction mixture was slowly raised to room temperature and stirred for 8 hours.
  • the reaction mixture was cooled to 0 ° C., and the solids which settled by precipitation were washed with cold dichloromethane under reduced pressure.
  • NHS-Tag was prepared by dissolving 50 times the amount of BSA mole number in 100% DMSO (50nmoles).
  • Conjugation reaction was carried out in 50mM MES, pH 6, 10% Acetonitrile, the final volume of the reaction was 80uL, 1hr reaction at room temperature. After conjugation, Bio-Spin (Bio Rad) P-30 was buffered with water, and the remaining free NHS-Tag was removed. Dry in vacuo and concentrate to 50 uL. (MA) LDI-MS without matrix was confirmed that the tagging on the BSA.
  • Table 1 Tag1 Tag2 Tag3 Tag4 Remarks BSA 1 nmole 7 uL 7 uL 7 uL 7 uL 10ug / uL stock in MES MES 62.2 uL 61.8 uL 61.6 uL 61.5uL 50 mM, pH 6 ACN 8uL 8uL 8uL 8uL 10% ACN Tag 50nmoles 2.8uL 3.2 uL 3.4 uL 3.5 uL 10ug / uL stock in DMSO Total Vol. 80 uL 80 uL 80 uL 80 uL 80 uL 80 uL
  • the AB Scix 4800 instrument and ASTA micro focus 384 well plate were used to determine if the tag was conjugated.
  • Tag4 1 1 10: 1 1:10 Tag2 Tag4 Tag2 Tag4 Tag2 Tag4 m / z 435.29 491.37 435.27 491.35 435.25 491.32 Height 2149.64 683.92 2508.49 252.18 411.24 2454.64 Area 112.5 39.16 147.33 13.14 22.88 130.52 Ratio 2.87 11.21 5.7
  • Antibody-antigen system condition experiment to detect antigens with conjugated tag

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Abstract

The present invention relates to a photocleavable mass tag material and a use thereof. The photocleavable mass tag material of the present invention can be used for the detection and quantitative analysis of a biomarker, and can be used for the simultaneous detection of multiple markers.

Description

광분해성 질량 표지 물질 및 그 용도Photodegradable mass labeling substance and its use
본 발명은 광분해성 질량 표지 물질 및 그 용도에 관한 것으로 더욱 상세하게는 쉽게 광분해되어 특정 질량으로 표지된 양이온을 방출할 수 있는 광분해성 질량 표지 물질 및 그 물질을 이용하여 매트릭스가 없는 조건(matrix-free)에서 레이저 탈착 이온화-비행시간(laser desorption/ionization-time-of-flight, LDI-TOF) 질량 분석을 수행하는 방법에 관한 것이다. The present invention relates to a photodegradable mass labeling substance and its use, and more particularly, to a photodegradable mass labeling substance capable of easily photolysing to release a cation labeled with a specific mass and a matrix-free condition using the substance. free desorption / ionization-time-of-flight (LDI-TOF) mass spectrometry.
매트릭스-보조된 레이저 탈착/이온화(MALDI) 및 전기분무 이온화(ESI)를 포함하여 소위 "연성 이온화" 질량 분광 방법은 질량이 300kDa를 초과하는 거대 분자의 완전한 이온화, 검출 및 질량 측정을 가능케 한다[참조: Fenn et al., Science 246: 64-71(1989); Karas and Hillenkamp, Anal. Chem. 60: 2299-3001(1988)].So-called "soft ionization" mass spectroscopy methods, including matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI), allow for complete ionization, detection and mass measurement of macromolecules whose mass exceeds 300 kDa [ See Fenn et al., Science 246: 64-71 (1989); Karas and Hillenkamp, Anal. Chem. 60: 2299-3001 (1988).
MALDI-MS는 분석되는 거대분자의 매트릭스내 도입을 필요로 하며, 고체(즉, 결정질) 매트릭스에서 혼합된 폴리펩타이드 및 핵산에 대해 수행되어 왔다. 이들 방법에 있어서, 레이저는 프로브 팁 위에 결정화되는 생체고분자/매트릭스 혼합물을 박리하기 위해 사용되는데, 이는 생체고분자의 탈착 및 이온화에 영향을 미친다. MALDI-MS requires incorporation into the matrix of the macromolecules to be analyzed and has been performed on polypeptides and nucleic acids mixed in a solid (ie crystalline) matrix. In these methods, a laser is used to exfoliate the biopolymer / matrix mixture that crystallizes on the probe tip, which affects the desorption and ionization of the biopolymer.
또한, MALDI-MS는 매트릭스로서 수화 물(즉, 아이스) 벤조산 등의 유기산 또는 글리세롤을 사용하며, 매트릭스의 복잡성이 미량 폴리펩타이드 분석에 장애가 되며, 수화 물을 매트릭스로서 사용하는 경우, MALDI-MS를 수행하기 전에 먼저 단백질을 동결 건조시키나 공기 건조시켜야 한다[참조: Berkenkamp et al. (1996) Proc. Natl. Acad. Sci. USA 93:7003-7007]. In addition, MALDI-MS uses organic acids or glycerol such as hydrated water (i.e. ice) benzoic acid as a matrix, and the complexity of the matrix impedes the analysis of trace polypeptides, and when using hydrated water as a matrix, MALDI-MS is used as a matrix. Before performing, the protein must first be lyophilized or air dried. See Berkenkamp et al. (1996) Proc. Natl. Acad. Sci. USA 93: 7003-7007.
또한 질량 분석은 시료의 이온화와 이온의 검출 방식에 따라 여러 종류가 있는데, 이러한 질량 분석법 중에서도 매트릭스 보조 레이저 탈착 이온화-비행시간(matrix-assisted laser desorption/ionization-time-of-flight, MALDI-TOF) 방법이 생체 시료의 질량 분석에 널리 쓰여 왔다. MALDI-TOF 방식의 질량 분석법은 많은 수의 시료를 빠르게 측정하여야 하는 초고속 진단을 위해 가장 적합한 질량 분석법으로 알려져 있다. 하지만 MALDI-TOF 방법으로 표적 분자 질량을 직접 측정하는 것은 민감도가 떨어져 적은 양의 생체 표지를 측정하기 힘들다는 한계가 있다. 또한 생체 표지의 존재 유무만이 아니라 그 양에 대한 정보까지 얻기 위해서는 현재 쓰이는 MALDI-TOF방법만으로는 부족한 실정이다. 그러므로 이를 극복하기 위한 MALDI-TOF 고감도 정량법의 개발, 나아가서는 매트릭스 보조 방식이 아니더라도 미량 생분자를 정량적으로 분석할 수 있는 등 신호 증폭이 가능한 질량 분석방법의 개발이 절대적으로 필요하다.There are several types of mass spectrometry, depending on the ionization of the sample and the detection of ions. Among these mass spectrometry, matrix-assisted laser desorption / ionization-time-of-flight (MALDI-TOF) The method has been widely used for mass spectrometry of biological samples. MALDI-TOF mass spectrometry is known to be the most suitable mass spectrometry for ultra-fast diagnostics where a large number of samples must be measured quickly. However, direct measurement of the target molecular mass by the MALDI-TOF method has a limitation that it is difficult to measure a small amount of biomarker due to its low sensitivity. In addition, the current MALDI-TOF method is not enough to obtain information on the amount as well as the presence or absence of a biomarker. Therefore, it is absolutely necessary to develop a MALDI-TOF high-sensitivity quantitative method to overcome this problem, and furthermore, to develop a mass spectrometry method capable of amplifying signals such as quantitative analysis of trace biomolecules even if the matrix-assisted method is not used.
[선행 특허 문헌][Previous Patent Document]
대한민국 특허공개번호 특2001-0043409Korean Patent Publication No. 2001-0043409
본 발명은 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 기존의 MALDI-TOF MS 와는 다르게 매트릭스가 없는 조건(matrix-free conditions)에서 측정할 수 있는 광분해성 질량 표지 물질을 제공하는 것이다.The present invention has been made in view of the above necessity, and an object of the present invention is to provide a photodegradable mass labeling material which can be measured in matrix-free conditions, unlike conventional MALDI-TOF MS.
상기의 목적을 달성하기 위하여 본 발명은 The present invention to achieve the above object
하기 화학식 1의 광분해성 질량표지 물질을 제공한다;It provides a photodegradable mass labeling material of the formula (1);
[화학식 1][Formula 1]
Figure PCTKR2015004242-appb-I000001
Figure PCTKR2015004242-appb-I000001
상기 화학식 1에서 R1 및 R2는 H, 알킬, 알케닐, 알카니일, 알콕시, 또는 아릴기이고, R3는 R3는 단백질 분자 또는 단백질 이외의 아민기와 반응할 수 있는 반응기이고,In Formula 1 R1 and R2 are H, alkyl, alkenyl, and Carney day, alkoxy, or aryl group, R3 is R 3 is a reactor that can react with the amine group of a protein or non-protein molecule,
본 발명의 일 구현예에 있어서, 상기 반응기는 N-하이드록시숙신이미딜 에스터, N-하이드록시설포숙신이미딜 에스터, 벤조트리아졸-1-일옥실 에스터, 펜타할로벤질 에스터 및 4-니트로페닐 에스터로 이루어진 그룹으로부터 선택되는 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the invention, the reactor is N-hydroxysuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, benzotriazol-1-yloxyl ester, pentahalobenzyl ester and 4- It is preferably selected from the group consisting of nitrophenyl esters, but is not limited thereto.
본 발명의 일 구현예에 있어서, 상기 물질은 하기 화학식 2 내지 화학식 4의 중 하나의 화합물인 것이 바람직하고;In one embodiment of the present invention, the material is preferably a compound of any one of the following formulas (2) to (4);
[화학식 2][Formula 2]
Figure PCTKR2015004242-appb-I000002
Figure PCTKR2015004242-appb-I000002
[화학식 3][Formula 3]
Figure PCTKR2015004242-appb-I000003
Figure PCTKR2015004242-appb-I000003
[화학식 4][Formula 4]
Figure PCTKR2015004242-appb-I000004
Figure PCTKR2015004242-appb-I000004
상기 화학식에서 노란 원 부분은 질량변화기 부위이고, 노란 원 부분은 질량변화기 부위이고, 상기 화학식에서 R1 및 R2는 H, 알킬, 알케닐, 알카니일, 알콕시, 또는 아릴기이고, R'는 N-하이드록시숙신이미딜 에스터, N-하이드록시설포숙신이미딜 에스터, 벤조트리아졸-1-일옥실 에스터, 펜타할로벤질 에스터 및 4-니트로페닐 에스터로 이루어진 그룹으로부터 선택되고, n은 0에서 20의 정수인 것이 바람직하나 이에 한정되지 아니한다.In the formula, the yellow circle portion is the mass changer portion, the yellow circle portion is the mass changer portion, and in the formula, R1 and R2 are H, alkyl, alkenyl, alkaniyl, alkoxy, or an aryl group, and R 'is N. -Hydroxysuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, benzotriazol-1-yloxyl ester, pentahalobenzyl ester and 4-nitrophenyl ester, n is 0 Is an integer of 20, but is not limited thereto.
본 발명의 다른 구현예에 있어서, 상기 물질은 하기 화학식 5의 화합물인 것이 바람직하나 이에 한정되지 아니한다;In another embodiment of the present invention, the material is preferably a compound of Formula 5, but is not limited thereto;
[화학식 5][Formula 5]
Figure PCTKR2015004242-appb-I000005
Figure PCTKR2015004242-appb-I000005
상기 화학식에서 n은 0 내지 20인 것이 바람직하나 이에 한정되지 아니한다. In the formula, n is preferably 0 to 20, but is not limited thereto.
또 본 발명은 상기 본 발명의 표지 물질과 컨쥬게이션된 항체 혹은 아민기를 포함한 물질을 제공한다.The present invention also provides a substance comprising an antibody or an amine group conjugated with the labeling substance of the present invention.
또 본 발명은 포획 항체가 처리된 말디(MALDI) 플레이트 또는 포획 항체를 결합시킬 수 있는 표면을 포함하는 말디(MALDI) 플레이트에 항원이 표함된 시료를 처리하여 항원과 포획항체간의 항원 항체 복합체를 생성하고, 상기 복합체에 상기 본 발명의 광분해성 질량표지 물질로 표지된 검출항체를 처리하여 검출항체가 검출하고자 하는 항원이 존재하는 경우에만 포획항체가 결합하지 않은 항원의 다른 부분에 결합되고, 상기 플레이트를 매트릭스 없이 레이저 이탈 이온화 비행시간형 질량분석법(laser desorption ionization time-of-flight mass spectrometry/LDI-TOF MS)을 수행하는 방법을 제공한다.In another aspect, the present invention is to treat the antigen-labeled sample on a maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface capable of binding the capture antibody to generate an antigen antibody complex between the antigen and the capture antibody. And treating the detection antibody labeled with the photodegradable mass labeling substance of the present invention to the complex to bind to another portion of the antigen to which the capture antibody does not bind only when the detection antibody is present in the antigen to be detected. It provides a method for performing laser desorption ionization time-of-flight mass spectrometry (LDI-TOF MS) without matrix.
또 본 발명은 포획 항체가 처리된 말디(MALDI) 플레이트 또는 포획 항체를 결합시킬 수 있는 표면을 포함하는 말디(MALDI) 플레이트에 다른 종류의 항원이 포함된 시료를 처리하여 항원과 포획항체간의 선택적인 항원 항체 복합체를 생성하고, 상기 복합체에 상기 본 발명의 광분해성 질량표지물질로 표지된 검출항체를 처리하여 검출항체가 검출하고자 하는 항원이 존재하는 경우에만 포획항체가 결합하지 않은 항원의 다른 부분에 결합되고, 상기 플레이트를 매트릭스 없이 레이저 이탈 이온화 비행시간형 질량분석법(laser desorption ionization time-of flight mass spectrometry/LDI-TOF MS)을 수행하여 다중 항원을 정량적으로 검출하는 방법을 제공한다.In another aspect, the present invention is to treat a sample containing a different type of antigen in a maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface to which the capture antibody can be bound to selectively select between the antigen and the capture antibody. An antigen antibody complex is generated, and the complex is treated with a detection antibody labeled with the photodegradable mass label of the present invention, so that only when the antigen to be detected is present in the antigen, the capture antibody does not bind to another part of the antigen to which the capture antibody does not bind. When combined, the plate is subjected to laser desorption ionization time-of flight mass spectrometry (LDI-TOF MS) without a matrix to provide a method for quantitative detection of multiple antigens.
또 본 발명은 포획 항체가 처리된 말디(MALDI) 플레이트 또는 포획 항체를 결합시킬 수 있는 표면을 포함하는 말디(MALDI) 플레이트에 항원이 포함된 시료를 처리하여 항원과 포획항체간의 항원 항체 복합체를 생성하고, 상기 복합체에 상기 본 발명의 광분해성 질량표지 물질로 표지된 검출항체를 처리하여 검출항체가 검출하고자 하는 항원이 존재하는 경우에만 포획항체가 결합하지 않은 항원의 다른 부분에 결합되고, 상기 플레이트를 매트릭스 없이 레이저 이탈 이온화 비행시간형 질량분석법(laser desorption ionization time-of-flight mass spectrometry/LDI-TOF MS)을 수행하여 항원을 정량적으로 분석하는 방법을 제공한다. In another aspect, the present invention is to treat the sample containing the antigen in a maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface capable of binding the capture antibody to generate an antigen antibody complex between the antigen and the capture antibody And treating the detection antibody labeled with the photodegradable mass labeling substance of the present invention to the complex to bind to another portion of the antigen to which the capture antibody does not bind only when the detection antibody is present in the antigen to be detected. It provides a method for quantitative analysis of antigen by performing laser desorption ionization time-of-flight mass spectrometry (LDI-TOF MS) without matrix.
이하 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 발명의 목적은 355 nm 의 레이저가 조사되는 MALDI-TOF MS (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer) 조건에서 쉽게 광분해됨으로써 특정 질량으로 표지된 양이온을 방출할 수 있는 광분해성 질량 표지물질(photo-cleavable mass tag)을 개발하는 것이다. An object of the present invention is photolysis capable of releasing a specific mass labeled cation by easily photolysing under a matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer (MALDI-TOF MS) under irradiation with a 355 nm laser. The development of photo-cleavable mass tags.
이러한 기술의 핵심은 기존의 MALDI-TOF MS 와는 다르게 매트릭스가 없는 조건(matrix-less)에서 측정한다는 것인데, 조사되는 레이저에 의해서만 질량 표지물질로부터 양이온이 생성되기 때문에 오직 이 질량 표지물질로 표지된 항체만을 선택적으로 검출할 수 있다는 장점을 가진다.The key to this technology is that, unlike conventional MALDI-TOF MS, it measures in matrix-less conditions. Antibodies labeled with this mass labeler only because cations are generated from the mass labeler by the irradiated laser. It has the advantage of being able to selectively detect only bays.
본 발명의 작동 원리는 도 1 에 간단히 기술되어 있다. 간단하게 설명하면 포획항체(또는 1차 항체)가 처리된 MALDI 플레이트와 광분해성 질량표지물질로 표지한 검출항체(또는 2차 항체)를 각각 준비한다. 상기 준비된 MALDI 플레이트 위에 바이오 마커들을 뿌려주게 되면 항원-항체 반응에 의하여 고정된 포획항체에 검출하고자 하는 항원만이 선택적으로 결합되며, 여기에 다시 표지된 검출항체를 처리하게 되면 마치 샌드위치처럼 검출항체가 항원 위에 결합하게 된다. 결과적으로 시료 내에 검출하고자 하는 바이오 마커(항원)가 존재할 경우에만 표지된 검출항체가 MALDI 플레이트 위에 고정되는 것이다. 이 플레이트를 이용한 matrix-less LDI-TOF MS 조건에서 질량표지 물질이 광분해되어 양이온을 생성시키므로 이를 통해 항원의 존재 유무를 분석할 수 있다. The operating principle of the invention is briefly described in FIG. Briefly, a MALDI plate treated with a capture antibody (or a primary antibody) and a detection antibody (or a secondary antibody) labeled with a photodegradable mass labeling agent are prepared, respectively. When the biomarkers are sprayed onto the prepared MALDI plate, only the antigen to be detected is selectively bound to the capture antibody immobilized by the antigen-antibody reaction, and when the labeled antibody is processed again, the detection antibody is like a sandwich. Bind to the antigen. As a result, the labeled detection antibody is immobilized on the MALDI plate only when there is a biomarker (antigen) to be detected in the sample. In this matrix-less LDI-TOF MS conditions, the mass-labeling material is photolyzed to generate cations, thereby analyzing the presence of antigen.
이 방법의 가장 큰 장점은 이와 같이 항원-항체 결합을 이용한 샌드위치 타입의 검출법에서 늘 존재하게 되는 비 특이적인 항원-항체 결합에 따른 신호 오염이 전혀 없다는 것이다. 비록 원하지 않는 바이오물질이 포획되었더라도 매트릭스를 사용하지 않는 조건이므로 이들의 질량값은 전혀 나타나지 않기 때문이다. 이 때 측정되는 신호의 세기는 표지물질의 양에 비례하므로 포획된 바이오 마커의 정량적 분석이 가능하며, 서로 다른 구조(질량값)를 갖는 표지물질을 각각 다른 항체에 표지시킨 후 한꺼번에 사용하여도 각각은 영향을 끼치지 않고 독립적으로 서로 다른 질량값의 양이온을 방출하므로 다중 마커 검출에도 이용될 수 있다.The biggest advantage of this method is that there is no signal contamination due to the non-specific antigen-antibody binding which is always present in the sandwich type detection method using antigen-antibody binding. Even if unwanted biomaterials are captured, their mass values do not appear at all because the matrix is not used. In this case, the measured signal intensity is proportional to the amount of labeling material, so that quantitative analysis of captured biomarkers is possible, and even if the labeling materials having different structures (mass values) are labeled with different antibodies, they can be used at once. Can be used to detect multiple markers because it releases cations of different mass values independently without affecting.
이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
신규 광분해성 질량표지 물질의 탐색Exploration of New Photodegradable Mass Labeling Materials
선행연구 결과를 종합해보면 신규 광분해성 질량 표지 물질은 기본적으로 빛에 의해서 안정한 양이온을 생성시킬 수 있는 구조이어야 한다. 또한 항체와 결합시킬 수 있는 반응기와 다중 마커 검출을 위하여 질량을 변화시킬 수 있는 질량 변화기를 동시에 갖추어야 한다. 이와 더불어 상업적인 진단 키트 개발을 위해서는 비교적 간단하고 높은 수율을 갖는 프로토콜로 합성될 수 있는 물질이어야 한다. Taken together, the new photodegradable mass labeling substance should basically have a structure capable of producing stable cations by light. In addition, a reactor capable of binding the antibody and a mass changer capable of varying the mass for detecting multiple markers should be provided at the same time. In addition, for the development of commercial diagnostic kits, they must be materials that can be synthesized in a relatively simple and high yield protocol.
본 발명에서 사용된 리드 화합물인 페로센은 매우 안정한 메탈로센 구조로써 이 리간드의 α-탄소 양이온이 매우 안정하다는 것은 오래전부터 연구되어 왔다. 본 발명자들은 이러한 사실에 근거하여 도 3 과 같은 페로센 유도체를 고안하였다.Ferrocene, a lead compound used in the present invention, has been studied for a long time that the α -carbon cation of this ligand has a very stable metallocene structure. Based on this fact, the inventors devised ferrocene derivatives as shown in FIG. 3.
이 유도체는 355 nm의 레이저에 의해서 리간드 α 위치의 C-S 결합이 불균일 분해되면서 안정한 탄소 양이온과 황 음이온으로 분해될 수 있다.This derivative can be decomposed into stable carbon cations and sulfur anions while the CS bond at the ligand α position is unevenly decomposed by a 355 nm laser.
시중에서 비교적 싼 값에 구입할 수 있는 페로센 또는 페로세닐 알데히드(1a)로부터 다음과 같은 간단한 유기합성을 통해 총 6개의 페로센 유도체(3a-f)를 높은 수율로 합성할 수 있었다(도 4). 그 중에서도 α-탄소의 수소가 두 개의 페닐기로 치환된 유도체(3f)가 가장 큰 세기를 나타냄을 알 수 있었다. From the ferrocene or ferrocenyl aldehyde (1a) which can be purchased at a relatively low price on the market, a total of six ferrocene derivatives (3a-f) were synthesized in high yield through the following simple organic synthesis (FIG. 4). Among them, the derivative (3f) in which hydrogen of α -carbon was substituted with two phenyl groups showed the greatest intensity.
다중 마커 동시 검출(multiplexing)을 위한 질량 변화기의 도입Introduction of mass changers for multiplexing multiple markers
거의 유사한 구조이지만 특정 부분의 작용기 차이로 인하여 다른 분자량을 갖는 질량표지 물질을 각기 다른 검출 항체에 결합시키게 되면, 각 항체로부터 광분해 되는 양이온은 각각 서로 다른 검출값을 나타내게 된다. 이 때 이들의 질량 변화기가 질량표지 물질의 광분해 효율에 크게 영향을 미치지 않는다면 검출 세기는 질량 변화기의 구조에 관계없이 오직 검출 항체의 양에만 정량적으로 비례할 것이다. 그러므로 본 발명의 질량표지 물질은 다중 마커의 동시 검출(multiplexing)에 이용할 수 있다(도 7). 본 발명자들은 도 8에서와 같이 세 가지의 각기 다른 질량 변화기를 갖는 페로센 유도체를 합성하였고 그들의 matrix-less LDI-TOF MS 결과를 비교하여 목적에 가장 적합한 구조를 가려내었다. When a mass-labeling substance having a similar molecular weight but having different molecular weights due to functional groups of a specific moiety is bound to different detection antibodies, the cations photolyzed from the respective antibodies show different detection values. At this time, if their mass changers do not significantly affect the photolysis efficiency of the mass labeling substance, the detection intensity will be quantitatively proportional to the amount of the detection antibody regardless of the structure of the mass changer. Therefore, the mass labeling material of the present invention can be used for the multiplexing of multiple markers (FIG. 7). The present inventors synthesized ferrocene derivatives having three different mass change groups as shown in FIG. 8 and compared their matrix-less LDI-TOF MS results to find the most suitable structure for the purpose.
도 9 와 같이 9-BBN (9-borabicyclo[3.3.1]nonane) 과 말단 이중(C=C)이 있는 탄화수소 화합물 사이의 붕수소화 반응을 통해 만들어진 중간체를 스즈키 짝지음 반응 (Suzuki cross-coupling reaction)을 통해 페로센 유도체 11에 도입할 수 있다. 우리는 이것을 다중 마커 동시 검출에 실제로 적용해보기 위하여 세가지 종류의 페로센 유도체 6a, 6b, 6c를 합성하였다. 세 화합물은 질량 변화기의 탄소 사슬이 2 개씩 차이가 나는 구조이다. 이러한 질량 변화기의 구조 차이에 관계없이 matrix-less LDI-TOF MS 검출 세기가 동일한 세기로 나오는지 검증하기 위해서 6a-c를 각각 같은 몰수가 되도록 혼합한 후 matrix-less LDI-TOF MS를 측정하였다. 그 결과 다른 노이즈는 거의 발견되지 않았고 오직 6a-c로부터 생성되는 탄소 양이온 값만이 검출되었으며, 특히 검출 세기가 모두 비슷한 수준으로 측정되는 것이 확인되었다(도 10). 이 결과를 통해 우리는 질량 변화기로써 이와 같은 포화 탄화 수소 사슬의 작용기를 사용하기로 결정하였다.Suzuki cross-coupling reaction of an intermediate made through a boronation reaction between a 9-BBN (9-borabicyclo [3.3.1] nonane) and a hydrocarbon compound having a terminal double (C = C) as shown in FIG. 9. Can be introduced into ferrocene derivative 11). We synthesized three types of ferrocene derivatives 6a, 6b and 6c to apply this to the simultaneous detection of multiple markers. The three compounds have two carbon chains of mass changer. Irrespective of the structure difference of the mass changer, matrix-less LDI-TOF MS was measured after mixing 6a-c in the same number of moles to verify whether the matrix-less LDI-TOF MS detection intensity was the same. As a result, almost no other noise was found and only carbon cation values generated from 6a-c were detected, and in particular, it was confirmed that the detection intensities were all measured at similar levels (FIG. 10). From these results, we decided to use these functional groups of saturated hydrocarbon chains as mass changers.
신규 질량표지 물질의 검출 한계 측정 실험Experiment to detect limit of detection of new mass labeling substance
본 발명을 통해서 바이오 마커의 다중 검출에 실제로 적용해 보기로 한 구조인 페로센 유도체 6의 matrix-less LDI-TOF MS 검출 한계를 알아보는 실험을 진행하였다. 테트라하이드로퓨란 (tetrahydrofuran, THF) 용매 1 μL에 유도체 6a-c 각각을 10-8에서 10-15 mol 까지 1/10 씩 양을 줄여가면서 녹인 용액을 차례로 플레이트 위에 올린 다음 50, 60, 70 % 의 레이저 세기를 이용하여 matrix-less LDI-TOF MS를 측정하였다. 그 결과 유도체의 종류와 레이저의 세기에 따라서 미세하게 차이를 보이기는 하지만 대체적으로 10-10에서 10-11 mol 정도 수준까지의 질량표지 물질을 충분히 확인할 수 있었다. 또한 각 몰수의 로그값에 따른 matrix-less LDI-TOF MS 검출 세기를 도시화한 결과 선형의 그래프가 얻어졌는데, 이를 통하여 우리가 기대했던 바와 같이 바이오 마커의 정량적 분석에 이용될 수 있음을 확인할 수 있었다. Through the present invention, an experiment was conducted to find the limit of detection of matrix-less LDI-TOF MS of ferrocene derivative 6, which is a structure that is actually applied to multiple detection of biomarkers. Dissolve each of the derivatives 6a-c in 1 μL of tetrahydrofuran (THF) solvent in increments of 10 -8 to 10 -15 mol in increments of 1/10, and then add 50, 60, 70% of the solution. Matrix-less LDI-TOF MS was measured using laser intensity. As a result, although there was a slight difference depending on the type of the derivative and the laser intensity, it was possible to sufficiently identify the mass labeling material from about 10 -10 to about 10 -11 mol. In addition, as a result of plotting the matrix-less LDI-TOF MS detection intensity according to the logarithm of each mole number, a linear graph was obtained, which can be used for the quantitative analysis of biomarkers as expected. .
검출 항체와의 컨쥬게이션을 위한 반응기의 도입Introduction of Reactor for Conjugation with Detection Antibody
신규 질량표지 물질을 검출 항체에 컨쥬게이션 시키기 위해서는 단백질의 아민기와 높은 수율로 짝지음 반응을 할 수 있는 반응기가 필요하다. 그 중 가장 널리 쓰이는 것이 N-하이드록시쑥신이미드에스터(N-hydroxysuccinimide ester, NHS ester)이다. 이 반응기는 단백질의 아민기와 매우 효과적으로 짝지음 반응을 하는 것이 알려져 있다. N-하이드록시쑥신이미드(N-hydroxysuccinimide, NHS)를 N,N’-다이사이클로헥실카르보디이미드(N,N’-dicyclohexylcarbodiimide, DCC) 존재하에서 페로센 유도체 6 말단의 카르복실산과 반응시키게 되면 이 위치에 NHS ester 가 높은 수율로 도입된다. 이를 통해 도 14와 같은 신규 광분해성 질량표지 물질 17a, 17b, 17c를 합성하였으며, 이 화합물을 실제로 검출 항체에 컨쥬게이션하여 바이오 마커의 검출에 적용해 보았다. Conjugation of a novel mass labeling substance to a detection antibody requires a reactor capable of pairing with an amine group of a protein in high yield. The most widely used of the N - hydroxy mugwort god imide ester (N -hydroxysuccinimide ester, NHS ester) . It is known that this reactor reacts very effectively with the amine groups of a protein. N - hydroxy mugwort god imide (N -hydroxysuccinimide, NHS) of N, N'- dicyclohexyl carbodiimide (N, N '-dicyclohexylcarbodiimide, DCC ) in the presence When ferrocene derivatives 6-carboxylic acid and thereby the reaction of the terminal NHS ester is introduced at the site in high yield. Through this, novel photodegradable mass labeling materials 17a, 17b, and 17c as shown in FIG. 14 were synthesized, and the compound was actually conjugated to a detection antibody and applied to detection of a biomarker.
광 분해성 질량 표지 물질의 Matrix-less LDI-TOF MS 실험 방법Matrix-less LDI-TOF MS Experimental Method for Photodegradable Mass Labeling Materials
광 분해성 질량 표지 물질들을 다양한 농도로 테트라하이드로퓨란(1.0x10-6-1.0x10-15)에 녹인 후, 1 μl 를 취해 플레이트 위의 각 점에 떨어뜨리고 실온의 공기 중에 노출 시킴으로써 용매를 건조시켰다. 플레이트 위의 건조된 시료를 MALDI-TOF mass spectrometer (매트릭스 보조 레이저 탈착 비행시간형 질량분석기, Autoflex Speed series, BrukerDaltonics, Leipzig, Germany) 로 분석하였다. 모든 스펙트럼은 10% 에서 70% 범위 내의 다양한 세기의 335 nm 파장의 레이저를 이용하여 양이온 리플렉트론 모드에서 측정되었다. 질량 범위는 0-800 Da 로 설정되었고, 이후 데이터 분석은 flexAnalysis 프로그램을 사용하였다.The photodegradable mass labeling substances were dissolved in tetrahydrofuran (1.0 × 10 −6 −1.0 × 10 −15 ) at various concentrations, then 1 μl was taken at each spot on the plate and exposed to air at room temperature to dry the solvent. The dried samples on the plates were analyzed with a MALDI-TOF mass spectrometer (matrix assisted laser desorption time-of-flight mass spectrometer, Autoflex Speed series, BrukerDaltonics, Leipzig, Germany). All spectra were measured in cationic reflecton mode using lasers of 335 nm wavelength of varying intensity in the range of 10% to 70%. The mass range was set at 0-800 Da, after which data analysis was performed using the flexAnalysis program.
본 발명을 통하여 알 수 있는 바와 같이 본 발명의 광분해성 질량 표지물질은 바이오마커의 검출 및 정량적인 분석에 이용될 수 있으며, 다중마커의 동시 검출에 이용할 수 있다. As can be seen through the present invention, the photodegradable mass labeling material of the present invention can be used for detection and quantitative analysis of biomarkers, and can be used for simultaneous detection of multiple markers.
도 1은 본 과제에서 개발하려고 하는 광분해성 질량표지 물질의 작동 원리 모식도;1 is a schematic view of the principle of operation of the photodegradable mass labeling material to be developed in the present invention;
도 2는 신규 광분해성 질량 태그의 구조를 나타낸 그림;2 shows the structure of a novel photodegradable mass tag;
도 3은 페로센 질량표지 물질의 광분해 반응을 나타낸 그림;3 is a diagram showing a photolysis reaction of a ferrocene mass labeling material;
도 4는 다양한 α-탄소 위치의 치환기를 갖는 페로센 유도체의 합성을 나타낸 그림;4 shows the synthesis of ferrocene derivatives having substituents at various α -carbon positions;
도 5는 다양한 α-탄소 위치 치환기를 갖는 페로센 유도체(3a-f)의 matrix-less LDI-TOF MS 스펙트럼; 5 is a matrix-less LDI-TOF MS spectrum of ferrocene derivatives (3a-f) having various α -carbon position substituents;
도 6은 3f 분자의 matrix-less LDI-TOF MS 스펙트럼; 6 is a matrix-less LDI-TOF MS spectrum of 3f molecules;
도 7은 광분해성 질량표지 물질을 이용한 다중 마커의 동시 검출 작동 원리 모식도;7 is a schematic diagram of a principle of simultaneous detection of multiple markers using photodegradable mass labeling substances;
도 8은 다양한 질량 변화기가 도입된 광분해성 질량표지 물질의 예;8 is an example of a photodegradable mass label material having various mass changers introduced therein;
도 9는 페로센 유도체의 합성 과정을 나타낸 그림; 9 is a diagram showing the synthesis process of ferrocene derivatives;
도 10은 질량 변화기가 도입된 페로센 유도체 6a-c 혼합물의 matrix-less LDI-TOF MS 스펙트럼; 10 is a matrix-less LDI-TOF MS spectrum of a ferrocene derivative 6a-c mixture in which a mass changer is introduced;
도 11 내지 13은 페로센 유도체 6a-c의 matrix-less LDI-TOF MS 검출 한계 측정 실험 스펙트럼 (laser power: 70%): 플레이트 위에 올린 분자의 절대 몰수에 따른 검출 신호 세기 비교 스펙트럼; 11 to 13 are matrix-less LDI-TOF MS detection limit measurement spectra of ferrocene derivatives 6a-c (laser power: 70%): detection signal intensity comparison spectra according to the absolute moles of molecules placed on a plate;
도 14는 바이오 마커와의 컨쥬게이션을 위한 반응기의 도입 과정을 나타낸 그림; 14 shows the introduction of a reactor for conjugation with a biomarker;
도 15는 tag들이 컨쥬게이션된 BSA를 matrix free 상태로 LDI 실험한 결과를 나타낸 그림; 15 is a diagram showing the results of LDI experiments in a matrix free state of the BSA conjugated tags;
도 16은 단백질 혼합 비율에 따른 tag 신호 비율 변화 검증 그림;16 is a verification diagram of tag signal ratio change according to protein mixing ratio;
도 17은 tag으로 conjugation된 leptin과 결합한 항체 시스템에서 tag 신호 검출 그림이다.Figure 17 is a tag signal detection in the antibody system conjugated with leptin conjugated with a tag.
도 18은 본 발명의 광분해성 표지물질의 유도체 합성 방법을 나타낸 그림.18 is a diagram showing a method for synthesizing derivatives of photodegradable labeling substances of the present invention.
도 19는 본 발명의 광분해성 표지물질의 유도체 합성 방법을 나타낸 그림.19 is a diagram showing a method for synthesizing derivatives of photodegradable labeling substances of the present invention.
이하 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재된 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the invention and the scope of the present invention is not to be construed as limited by the following examples.
실시예 1:광 분해성 질량 표지 물질의 합성 방법Example 1 Synthesis of Photodegradable Mass Labeling Material
본 발명의 광분해성 질량 표지물질의 합성 방법 및 반응기 도입 등에 관하여서는 도 4, 도 9 및 도 16 내지 도 17에서 설명하였다.Synthesis of the photodegradable mass labeling substance of the present invention, introduction of a reactor, and the like have been described with reference to FIGS. 4, 9 and 16 to 17.
해당 도면에 기재된 각 화합물의 합성 방법에 대해서 상술하면,When the synthesis method of each compound described in the said figure is explained in full detail,
Acetylferrocene (1b) 합성Acetylferrocene (1b) Synthesis
페로센 (0.500 g, 2.69 mmol) 을 아세틱 안하이드라이드 (1.7 mL) 에 녹인 후, 0 ℃ 에서 인산 수용액 (85%, 0.53 g, 0.30 mL, 4.8 mmol) 을 넣었다. 이 반응 혼합물을 15 분 동안 환류 하에 가열한 후, 20 g 의 얼음이 들어있는 비커에 부었다. 모든 얼음이 녹은 후 기포가 더 이상 발생하지 않을 때까지 포화 탄산수소나트륨 수용액을 넣었다. 반응 혼합물을 0 ℃ 로 냉각시킨 후 생성된 고체를 물로 씻으면서 걸러준 후 감압 하에서 건조시켜 화합물 1b를 붉은색 고체를 얻었다. (0.598 g, 97%): 1H NMR (400 MHz, CDCl3) δ 4.78 (s, 2H), 4.51 (s, 2H), 4.21 (s, 5H), 2.41 (s, 3H).Ferrocene (0.500 g, 2.69 mmol) was dissolved in acetic anhydride (1.7 mL), and an aqueous phosphoric acid solution (85%, 0.53 g, 0.30 mL, 4.8 mmol) was added at 0 ° C. The reaction mixture was heated at reflux for 15 minutes and then poured into a beaker containing 20 g of ice. After all the ice melted, saturated aqueous sodium hydrogen carbonate was added until no more bubbles were generated. After the reaction mixture was cooled to 0 ° C., the resulting solid was filtered off with washing with water and dried under reduced pressure to obtain Compound 1b , a red solid. (0.598 g, 97%): 1 H NMR (400 MHz, CDCl 3 ) δ 4.78 (s, 2H), 4.51 (s, 2H), 4.21 (s, 5H), 2.41 (s, 3H).
Benzoylferrocene (1c) 합성Benzoylferrocene (1c) synthesis
페로센 (0.300 g, 1.61 mmol) 을 무수 디클로로메탄 (3 mL) 에 녹인 후, 이 용액에 벤조일 클로라이드 (0.249 g, 1.77 mmol) 를 넣었다. 0 ℃ 로 냉각 시킨 후 알루미늄 클로라이드 (0.323 g, 2.42 mmol) 를 소량씩 천천히 넣었다. 이 반응 혼합물을 상온에서 3 시간 동안 교반한 후 얼음물 (5 mL) 과 디클로로메탄 (10 mL) 을 차례대로 넣었다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (20 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산마그네슘을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=6:1) 로 정제하여 화합물 1c를 붉은색 고체로 얻었다. (0.361 g, 77%): 1H NMR (400 MHz, CDCl3) δ 7.90 (d, J = 7.2 Hz, 2H), 7.55 (t, J = 7.4 Hz, 1H), 7.47 (d, J = 7.4 Hz, 2H), 4.91 (t, J = 2.0 Hz, 2H), 4.59 (t, J = 1.8 Hz, 2H), 4.21 (s, 5H).Ferrocene (0.300 g, 1.61 mmol) was dissolved in anhydrous dichloromethane (3 mL), and benzoyl chloride (0.249 g, 1.77 mmol) was added to the solution. After cooling to 0 ° C., aluminum chloride (0.323 g, 2.42 mmol) was slowly added in small portions. The reaction mixture was stirred at room temperature for 3 hours, and then ice water (5 mL) and dichloromethane (10 mL) were added sequentially. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (20 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous magnesium sulfate was added and filtered under reduced pressure. The solvent of the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 6: 1) to give compound 1c as a red solid. (0.361 g, 77%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.90 (d, J = 7.2 Hz, 2H), 7.55 (t, J = 7.4 Hz, 1H), 7.47 (d, J = 7.4 Hz, 2H), 4.91 (t, J = 2.0 Hz, 2H), 4.59 (t, J = 1.8 Hz, 2H), 4.21 (s, 5H).
Ferrocenemethanol (2a) 합성Ferrocenemethanol (2a) synthesis
페로센 카복실알데히드 (0.100 g, 0.467 mmol) 을 에탄올 (8 mL) 에 녹인 후, 0 ℃ 에서 수소화붕소나트륨 (0.090 g, 2.4 mmol) 을 소량씩 천천히 넣었다. 이 반응 혼합물을 상온에서 3 시간 동안 교반 하였다. 물 (3 mL) 과 디클로로메탄 (10 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (15 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트:메탄올=15:5:1) 로 정제하여 화합물 2a를 노란색 고체로 얻었다. (0.090 g, 89%): 1H NMR (400 MHz, CDCl3) δ 4.31 (d, J = 6.0 Hz, 2H), 4.22 (s, 2H), 4.16 (s, 5H) 1.48 (t, J = 5.8 Hz, 1H).Ferrocene carboxyaldehyde (0.100 g, 0.467 mmol) was dissolved in ethanol (8 mL), and sodium borohydride (0.090 g, 2.4 mmol) was slowly added slowly at 0 ° C in small portions. The reaction mixture was stirred at room temperature for 3 hours. Water (3 mL) and dichloromethane (10 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (15 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate: methanol = 15: 5: 1) to give compound 2a as a yellow solid. (0.090 g, 89%): 1 H NMR (400 MHz, CDCl 3 ) δ 4.31 (d, J = 6.0 Hz, 2H), 4.22 (s, 2H), 4.16 (s, 5H) 1.48 (t, J = 5.8 Hz, 1 H).
1-(Ferrocenyl)ethan-1-ol (2b) 합성1- (Ferrocenyl) ethan-1-ol (2b) synthesis
아세틸페로센 (1b, 0.100 g, 0.438 mmol) 을 에탄올 (5 mL) 에 녹인 후, 0 ℃ 에서 수소화붕소나트륨 (0.087 g, 2.2 mmol) 을 소량씩 천천히 넣었다. 이 반응 혼합물을 상온에서 3 시간 동안 교반 하였다. 물 (3 mL) 과 디클로로메탄 (10 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (15 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 헥산으로 두 번 재결정하여 화합물 2b를 노란색 고체로 얻었다. (0.048 g, 48%): 1H NMR (400 MHz, CDCl3) δ 4.55 (qd, J = 6.4, 5.4 Hz, 1H), 4.17-4.22 (br m, 9H) 1.84 (d, J = 4.4 Hz, 1H), 1.44 (d, J = 6.0 Hz, 3H).Acetyl ferrocene ( 1b , 0.100 g, 0.438 mmol) was dissolved in ethanol (5 mL), and sodium borohydride (0.087 g, 2.2 mmol) was slowly added in small portions at 0 ° C. The reaction mixture was stirred at room temperature for 3 hours. Water (3 mL) and dichloromethane (10 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (15 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was all removed under reduced pressure and then recrystallized twice with hexane to give compound 2b as a yellow solid. (0.048 g, 48%): 1 H NMR (400 MHz, CDCl 3 ) δ 4.55 (qd, J = 6.4, 5.4 Hz, 1H), 4.17-4.22 (br m, 9H) 1.84 (d, J = 4.4 Hz , 1H), 1.44 (d, J = 6.0 Hz, 3H).
Ferrocenyl(phenyl)methanol (2c)합성Ferrocenyl (phenyl) methanol (2c) Synthesis
아세틸페로센 (1b, 0.080 g, 0.28 mmol) 을 에탄올 (5 mL) 에 녹인 후, 0 ℃ 에서 수소화붕소나트륨 (0.052 g, 1.4 mmol) 을 소량씩 천천히 넣었다. 이 반응 혼합물을 상온에서 3 시간 동안 교반 하였다. 물 (2 mL) 과와 디클로로메탄 (8 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 헥산으로 두 번 재결정하여 화합물 2c를 노란색 고체로 얻었다. (0.056 g, 70%): 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 6.8 Hz, 2H), 7.32 (t, J = 6.2 Hz, 2H), 7.24 (t, J = 7.6 Hz, 1H), 5.47 (d, J = 2.8 Hz, 1H), 4.17-4.23 (m, 9H), 2.44 (d, J = 3.2 Hz, 1H).Acetyl ferrocene ( 1b , 0.080 g, 0.28 mmol) was dissolved in ethanol (5 mL), and sodium borohydride (0.052 g, 1.4 mmol) was slowly added in small portions at 0 ° C. The reaction mixture was stirred at room temperature for 3 hours. The reaction was terminated by adding water (2 mL) and dichloromethane (8 mL) in this order. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was all removed under reduced pressure and then recrystallized twice with hexane to give compound 2c as a yellow solid. (0.056 g, 70%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.39 (d, J = 6.8 Hz, 2H), 7.32 (t, J = 6.2 Hz, 2H), 7.24 (t, J = 7.6 Hz, 1H), 5.47 (d, J = 2.8 Hz, 1H), 4.17-4.23 (m, 9H), 2.44 (d, J = 3.2 Hz, 1H).
2-(Ferrocenyl)propan-2-ol (2d) 합성2- (Ferrocenyl) propan-2-ol (2d) Synthesis
아세틸페로센 (1b, 0.100 g, 0.438 mmol) 을 무수 테트라하이드로퓨란 (4 mL) 에 녹이고 -78 ℃ 로 냉각시킨 후, 메틸리튬 용액 (1.67 M 디에틸에테르 용액, 1.1 mL, 1.8 mmol) 을 질소 분위기에서 천천히 넣었다. -78 ℃ 에서 11 시간 동안 교반한 후, 물 (2 mL) 과 디클로로메탄 (8 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=3:1)로 정제하여 화합물 2d를 노란색 고체로 얻었다. (0.060 g, 56%): 1H NMR (400 MHz, CDCl3) δ 4.17-4.21 (m, 7H), 4.16 (s, 2H), 2.06 (s, 1H), 1.50 (s, 6H).Acetyl ferrocene ( 1b , 0.100 g, 0.438 mmol) was dissolved in anhydrous tetrahydrofuran (4 mL) and cooled to -78 ° C, and then methyllithium solution (1.67 M diethyl ether solution, 1.1 mL, 1.8 mmol) was added to a nitrogen atmosphere. Slowly put in. After stirring at −78 ° C. for 11 hours, water (2 mL) and dichloromethane (8 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent of the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 3: 1) to give compound 2d as a yellow solid. (0.060 g, 56%): 1 H NMR (400 MHz, CDCl 3 ) δ 4.17-4.21 (m, 7H), 4.16 (s, 2H), 2.06 (s, 1H), 1.50 (s, 6H).
1-(Ferrocenyl)-1-phenylethan-1-ol (2e) 합성Synthesis of 1- (Ferrocenyl) -1-phenylethan-1-ol (2e)
벤조일페로센 (1c, 0.100 g, 0.345 mmol) 을 무수 테트라하이드로퓨란 (3.5 mL) 에 녹이고 -78 ℃ 로 냉각시킨 후, 메틸리튬 용액 (1.67 M 디에틸에테르 용액, 0.46 mL, 0.77 mmol) 을 질소 분위기에서 천천히 넣었다. -78 ℃ 에서 2.5 시간 동안 교반한 후, 물 (2 mL) 과 디클로로메탄 (8 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=6:1) 로 정제하여 화합물 2e를 노란색 고체로 얻었다. (0.085 g, 80%): 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 7.2 Hz, 2H), 7.26 (t, J = 7.6 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 4.31 (s, 1H), 4.25 (s, 6H), 4.16 (s, 1H), 4.08 (s, 1H), 2.73 (s, 1H), 1.83 (s, 3H).Benzoylferrocene ( 1c , 0.100 g, 0.345 mmol) was dissolved in anhydrous tetrahydrofuran (3.5 mL) and cooled to -78 ° C, and then methyllithium solution (1.67 M diethyl ether solution, 0.46 mL, 0.77 mmol) was added to a nitrogen atmosphere. Slowly put in. After stirring at −78 ° C. for 2.5 hours, water (2 mL) and dichloromethane (8 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 6: 1) to give compound 2e as a yellow solid. (0.085 g, 80%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.38 (d, J = 7.2 Hz, 2H), 7.26 (t, J = 7.6 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 4.31 (s, 1H), 4.25 (s, 6H), 4.16 (s, 1H), 4.08 (s, 1H), 2.73 (s, 1H), 1.83 (s, 3H).
Ferrocenyldiphenylmethanol (2f) 합성Ferrocenyldiphenylmethanol (2f) Synthesis
벤조일페로센 (1c, 0.100 g, 0.345 mmol) 을 무수 테트라하이드로퓨란 (3.5 mL) 에 녹이고 -78 ℃로 냉각시킨 후 페닐리튬 용액 (1.35 M 디노르말부틸에테르 용액, 0.51 mL, 0.69 mmol) 을 질소 분위기에서 천천히 넣었다. -78 ℃ 에서 20 분 동안 교반한 후, 물 (2 mL) 과 디클로로메탄 (8 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=50:1) 로 정제하여 화합물 2f를 노란색 고체로 얻었다. (0.061 g, 48%): 1H NMR (400 MHz, CDCl3) δ 7.22-3.58 (m, 10H), 4.27 (s, 2H), 4.17 (s, 5H), 4.04 (s, 2H), 3.45 (s, 1H).Benzoylferrocene ( 1c , 0.100 g, 0.345 mmol) was dissolved in anhydrous tetrahydrofuran (3.5 mL), cooled to -78 ° C, and the phenyllithium solution (1.35 M dinormalbutylether solution, 0.51 mL, 0.69 mmol) was added to a nitrogen atmosphere. Slowly put in. After stirring at -78 ° C for 20 minutes, water (2 mL) and dichloromethane (8 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 50: 1) to give compound 2f as a yellow solid. (0.061 g, 48%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.22-3.58 (m, 10H), 4.27 (s, 2H), 4.17 (s, 5H), 4.04 (s, 2H), 3.45 (s, 1 H).
(Ferrocenylmethyl)(phenyl)sulfane (3a) 합성(Ferrocenylmethyl) (phenyl) sulfane (3a) synthesis
Ferrocenemethanol (2a, 0.020 g, 0.093 mmol) 을 디클로로메탄 (0.4 mL)에 녹인 후 싸이오페놀 (0.031 g, 0.28 mmol) 을 넣는다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.034 mL, 0.18 mmol) 을 넣는다. 반응 혼합물을 상온에서 5 분 동안 교반한 후, 포화 탄산수소나트륨 수용액 (5 mL) 에 붓고, 디클로로메탄 (10 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3)으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=30:1) 로 정제하여 화합물 3a를 노란색 고체로 얻었다. (0.028 g, 98%): 1H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 7.6 Hz, 2H), 7.29 (d, J = 7.29 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 4.15 (s, 7H), 4.10 (s, 2H), 3.92 (s, 2H).Ferrocenemethanol ( 2a , 0.020 g, 0.093 mmol) is dissolved in dichloromethane (0.4 mL), and thiophenol (0.031 g, 0.28 mmol) is added thereto. To this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.034 mL, 0.18 mmol). The reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 30: 1) to give compound 3a as a yellow solid. (0.028 g, 98%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.34 (d, J = 7.6 Hz, 2H), 7.29 (d, J = 7.29 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 4.15 (s, 7H), 4.10 (s, 2H), 3.92 (s, 2H).
(1-Ferrocenylethyl)(phenyl)sulfane (3b) 합성Synthesis of (1-Ferrocenylethyl) (phenyl) sulfane (3b)
1-(Ferrocenyl)ethan-1-ol (2b, 0.030 g, 0.13 mmol) 을 디클로로메탄 (0.6 mL)에 녹인 후 싸이오페놀 (0.016 g, 0.14 mmol) 을 넣는다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.057 mL, 0.20 mmol) 을 넣는다. 반응 혼합물을 상온에서 5 분 동안 교반한 후, 포화 탄산수소나트륨 수용액 (5 mL) 에 붓고, 디클로로메탄 (10 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3)으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피(헥산:에틸아세테이트=20:1)로 정제하여 화합물 3b를 노란색 고체로 얻었다. (0.035 g, 83%): 1H NMR (400 MHz, CDCl3) δ 7.35 (br m, 5H), 4.10-4.16 (m, 9H), 4.03 (s, 2H), 1.64 (d, J = 6.4 Hz, 3H).1- (Ferrocenyl) ethan-1-ol ( 2b , 0.030 g, 0.13 mmol) is dissolved in dichloromethane (0.6 mL), and thiophenol (0.016 g, 0.14 mmol) is added thereto. Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.057 mL, 0.20 mmol). The reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 20: 1) to obtain compound 3b as a yellow solid. (0.035 g, 83%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.35 (br m, 5H), 4.10-4.16 (m, 9H), 4.03 (s, 2H), 1.64 (d, J = 6.4 Hz, 3H).
(Ferrocenyl(phenyl)methyl)(phenyl)sulfane (3c) 합성(Ferrocenyl (phenyl) methyl) (phenyl) sulfane (3c) synthesis
Ferrocenyl(phenyl)methanol (2c, 0.030 g, 0.10 mmol) 을 디클로로메탄 (1.0 mL)에 녹인 후 싸이오페놀 (0.012 g, 0.11 mmol) 을 넣는다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.028 g, 0.15 mmol) 을 넣는다. 반응 혼합물을 상온에서 5 분 동안 교반한 후, 포화 탄산수소나트륨 수용액 (5 mL) 에 붓고, 디클로로메탄 (10 mL) 을넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피(헥산:에틸아세테이트=20:1)로 정제하여 화합물 3c를 노란색 고체로 얻었다. (0.012 g, 40%): 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 7.2 Hz, 2H), 7.15-7.30 (m, 8H), 4.13-4.15 (m, 4H), 4.09 (s, 5H).Ferrocenyl (phenyl) methanol ( 2c , 0.030 g, 0.10 mmol) is dissolved in dichloromethane (1.0 mL), and then thiophenol (0.012 g, 0.11 mmol) is added thereto. Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.028 g, 0.15 mmol). The reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 20: 1) to obtain compound 3c as a yellow solid. (0.012 g, 40%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.39 (d, J = 7.2 Hz, 2H), 7.15-7.30 (m, 8H), 4.13-4.15 (m, 4H), 4.09 (s, 5H).
(2-Ferrocenylpropan-2-yl)(phenyl)sulfane (3d) 합성Synthesis of (2-Ferrocenylpropan-2-yl) (phenyl) sulfane (3d)
2-(Ferrocenyl)propan-2-ol (2d, 0.030 g, 0.12 mmol) 을 디클로로메탄 (1.0 mL)에 녹인 후 싸이오페놀 (0.012 g, 0.11 mmol) 을 넣는다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.015 mg, 0.18 mmol) 을 넣는다. 반응 혼합물을 상온에서 5 분 동안 교반한 후, 포화 탄산수소나트륨 수용액 (5 mL) 에 붓고, 디클로로메탄 (10 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=50:1) 로 정제하여 화합물 3d를 노란색 고체로 얻었다. (0.030 g, 73%): 1H NMR (400 MHz, CDCl3) δ 7.29-7.32 (m, 1H), 7.19-7.24 (m, 4H), 4.13 (s, 5H) 4.10 (s, 2H), 3.94 (s, 2H), 1.64 (s, 6H).2- (Ferrocenyl) propan-2-ol Dissolve ( 2d, 0.030 g, 0.12 mmol) in dichloromethane (1.0 mL) and add thiophenol (0.012 g, 0.11 mmol). Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.015 mg, 0.18 mmol). The reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 50: 1) to give compound 3d as a yellow solid. (0.030 g, 73%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.29-7.32 (m, 1H), 7.19-7.24 (m, 4H), 4.13 (s, 5H) 4.10 (s, 2H), 3.94 (s, 2 H), 1.64 (s, 6 H).
(1-Ferrocenyl-1-phenylethyl)(phenyl)sulfane (3e) 합성Synthesis of (1-Ferrocenyl-1-phenylethyl) (phenyl) sulfane (3e)
1-(Ferrocenyl)-1-phenylethan-1-ol (2e, 0.040 g, 0.13 mmol) 을 디클로로메탄 (0.6 mL)에 녹인 후 싸이오페놀 (0.016 g, 0.14 mmol) 을 넣는다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.036 mg, 0.20 mmol) 을 넣는다. 반응 혼합물을 상온에서 5 분 동안 교반한 후, 포화 탄산수소나트륨 수용액 (5 mL) 에 붓고, 디클로로메탄 (10 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=50:1) 로 정제하여 화합물 3e를 노란색 고체로 얻었다. (0.045 g, 86%): 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 8.0 Hz, 2H), 7.17-7.26 (m, 4H), 7.12 (t, J = 7.6 Hz, 2H), 7.06 (d, J = 7.6 Hz, 2H), 4.24 (s, 1H), 4.16 (s, 8H), 2.03 (s, 3H).1- (Ferrocenyl) -1-phenylethan-1-ol Dissolve ( 2e, 0.040 g, 0.13 mmol) in dichloromethane (0.6 mL) and add thiophenol (0.016 g, 0.14 mmol). Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.036 mg, 0.20 mmol). The reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 50: 1) to give compound 3e as a yellow solid. (0.045 g, 86%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.53 (d, J = 8.0 Hz, 2H), 7.17-7.26 (m, 4H), 7.12 (t, J = 7.6 Hz, 2H ), 7.06 (d, J = 7.6 Hz, 2H), 4.24 (s, 1H), 4.16 (s, 8H), 2.03 (s, 3H).
(Ferrocenyldiphenylmethyl)(phenyl)sulfane (3f) 합성(Ferrocenyldiphenylmethyl) (phenyl) sulfane (3f) synthesis
Ferrocenyldiphenylmethanol (2f, 0.042 g, 0.11 mmol) 을 디클로로메탄 (0.8 mL)에 녹인 후 싸이오페놀 (0.029 g, 0.26 mmol) 을 넣는다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.031 mg, 0.17 mmol) 을 넣는다. 반응 혼합물을 상온에서 5 분 동안 교반한 후, 포화 탄산수소나트륨 수용액 (5 mL) 에 붓고, 디클로로메탄 (10 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=20:1) 로 정제하여 화합물 3f를 노란색 고체로 얻었다. (0.034 g, 65%): 1H NMR (400 MHz, CDCl3) δ 7.33-7.35 (m, 4H), 7.16-7.17 (m, 7H), 7.00-7.02 (m, 4H), 4.22 (s, 2H), 4.19 (s, 5H), 4.06 (s, 2H).Ferrocenyldiphenylmethanol ( 2f, 0.042 g, 0.11 mmol) is dissolved in dichloromethane (0.8 mL) and thiophenol (0.029 g, 0.26 mmol) is added. Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.031 mg, 0.17 mmol). The reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 20: 1) to give compound 3f as a yellow solid. (0.034 g, 65%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.33-7.35 (m, 4H), 7.16-7.17 (m, 7H), 7.00-7.02 (m, 4H), 4.22 (s, 2H), 4.19 (s, 5H), 4.06 (s, 2H).
Ferrocenyl(4-methoylphenyl)methanone (4) 합성Ferrocenyl (4-methoylphenyl) methanone (4) Synthesis
페로센 (0.500 g, 2.69 mmol) 을 무수 디클로로메탄 (5 mL) 에 녹인 후, 이 용액에 파라아니소일 클로라이드 (p-anisoyl chloride,0.504 g, 2.96 mmol) 를 넣었다. 0 ℃에서 알루미늄 클로라이드 (0.394 g, 2.96 mmol) 를 소량씩 천천히 넣었다. 이 반응 혼합물을 상온에서 11 시간 동안 교반한 후 얼음물 (5 mL) 과 디클로로메탄 (10 mL) 을 차례대로 넣었다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (20 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산마그네슘을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=3:1) 로 정제하여 화합물 4를 붉은색 고체로 얻었다. (0.318 g, 37%): 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 6.8 Hz, 2H), 6.96 (d, J = 6.4 Hz, 2H), 4.90 (s, 2H), 4.56 (s, 2H), 4.20 (s, 5H), 3.89 (s, 3H).Ferrocene (0.500 g, 2.69 mmol) was dissolved in anhydrous dichloromethane (5 mL), and then paraanisoyl chloride ( p- anisoyl chloride, 0.504 g, 2.96 mmol) was added to the solution. Aluminum chloride (0.394 g, 2.96 mmol) was slowly added in small portions at 0 ° C. The reaction mixture was stirred at room temperature for 11 hours, and then ice water (5 mL) and dichloromethane (10 mL) were added sequentially. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (20 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous magnesium sulfate was added and filtered under reduced pressure. The solvent of the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 3: 1) to give compound 4 as a red solid. (0.318 g, 37%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.95 (d, J = 6.8 Hz, 2H), 6.96 (d, J = 6.4 Hz, 2H), 4.90 (s, 2H), 4.56 (s, 2H), 4.20 (s, 5H), 3.89 (s, 3H).
Ferrocenyl(4-hydroxylphenyl)methanone (5) 합성Ferrocenyl (4-hydroxylphenyl) methanone (5) Synthesis
Ferrocenyl(4-methoylphenyl)methanone (4, 0.152 g, 0.475 mmol) 을 무수 디클로로메탄 (1.5 mL) 에 녹이고 -78 ℃ 로 냉각 시킨 후, 보론 트라이브로마이드 (0.054 mL, 0.57 mmol) 을 질소 분위기에서 천천히 넣었다. 반응 혼합물의 온도를 상온으로 서서히 올린 후 8 시간 동안 교반 하였다. 반응 혼합물을 얼음물 (3 mL) 에 붓고, 디클로로메탄 (5 mL) 을 넣어 희석시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산마그네슘을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=1:1) 로 정제하여 화합물 5를 갈색 고체로 얻었다. (0.105 g, 72%): 1H NMR (400 MHz, DMSO-d 6) δ 10.22 (br s, 1H), 7.84 (s, 2H), 6.87 (s, 2H), 4.81 (s, 2H), 4.63 (s, 2H), 4.23 (s, 5H).Ferrocenyl (4-methoylphenyl) methanone ( 4 , 0.152 g, 0.475 mmol) was dissolved in anhydrous dichloromethane (1.5 mL), cooled to -78 ° C, and boron tribromide (0.054 mL, 0.57 mmol) was added slowly under nitrogen atmosphere. . The temperature of the reaction mixture was slowly raised to room temperature and stirred for 8 hours. The reaction mixture was poured into iced water (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous magnesium sulfate was added and filtered under reduced pressure. The solvent of the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 1: 1) to give compound 5 as a brown solid. (0.105 g, 72%): 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.22 (br s, 1H), 7.84 (s, 2H), 6.87 (s, 2H), 4.81 (s, 2H), 4.63 (s, 2 H), 4.23 (s, 5 H).
(4-(Benzyloxy)phenyl)(ferrocenyl)methanone (6) 합성(4- (Benzyloxy) phenyl) (ferrocenyl) methanone (6) Synthesis
Ferrocenyl(4-hydroxylphenyl)methanone (5, 0.097 g, 0.32 mmol) 을 에탄올 (1 mL) 에 녹이고, 벤질 클로라이드 (0.044 g, 0.35 mmol) 와 포타슘 카보네이트 (0.110 g, 0.793 mmol) 를 넣었다. 이 반응 혼합물을 환류하에 2.5 시간 동안 가열하였다. 가열이 끝난 혼합물을 상온까지 식히고 모든 고체가 녹을 때까지 물 (3 mL) 을 넣어준 후, 디클로로메탄 (5 mL) 을 넣어서 희석시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산마그네슘을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=6:1) 로 정제하여 화합물 6을 붉은색 고체로 얻었다. (0.100 g, 80%): 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 8.4 Hz, 2H), 7.36-7.47 (m, 5H), 7.04 (d, J = 8.8 Hz, 2H), 5.16 (s, 2H), 4.09 (s, 2H), 4.56 (s, 2H), 4.20 (s, 5H).Ferrocenyl (4-hydroxylphenyl) methanone ( 5 , 0.097 g, 0.32 mmol) was dissolved in ethanol (1 mL), and benzyl chloride (0.044 g, 0.35 mmol) and potassium carbonate (0.110 g, 0.793 mmol) were added thereto. The reaction mixture was heated at reflux for 2.5 h. The heated mixture was cooled to room temperature, water (3 mL) was added until all the solids dissolved, and then diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous magnesium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 6: 1) to give compound 6 as a red solid. (0.100 g, 80%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.95 (d, J = 8.4 Hz, 2H), 7.36-7.47 (m, 5H), 7.04 (d, J = 8.8 Hz, 2H ), 5.16 (s, 2H), 4.09 (s, 2H), 4.56 (s, 2H), 4.20 (s, 5H).
(4-(Benzyloxy)phenyl)(ferrocenyl)methanol (7a) 합성Synthesis of (4- (Benzyloxy) phenyl) (ferrocenyl) methanol (7a)
Ferrocenyl(4-hydroxylphenyl)methanone (5, 0.094 g, 0.24 mmol) 을 에탄올 (1 mL) 에 녹인 후, 0 ℃ 에서 수소화붕소나트륨 (0.027 g, 0.71 mmol) 을 소량씩 천천히 넣었다. 이 반응 혼합물을 상온에서 3 시간 동안 교반 하였다. 물 (2 mL) 과 디클로로메탄 (5 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (7 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=3:1) 로 정제하여 화합물 7a를 노란색 고체로 얻었다. (0.084 g, 89%): 1H NMR (400 MHz, CDCl3) 7.29-4.43 (m, 7H), 6.93 (d, J = 8.4Hz, 2H), 5.44 (s, 1H), 5.05 (s, 2H), 4.22 (s, 6H), 4.18 (s, 3H), 2.38 (s, 1H).Ferrocenyl (4-hydroxylphenyl) methanone ( 5 , 0.094 g, 0.24 mmol) was dissolved in ethanol (1 mL), and sodium borohydride (0.027 g, 0.71 mmol) was slowly added slowly at 0 ° C. The reaction mixture was stirred at room temperature for 3 hours. Water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 3: 1) to give compound 7a as a yellow solid. (0.084 g, 89%): 1 H NMR (400 MHz, CDCl 3 ) 7.29-4.43 (m, 7H), 6.93 (d, J = 8.4 Hz, 2H), 5.44 (s, 1H), 5.05 (s, 2H), 4.22 (s, 6H), 4.18 (s, 3H), 2.38 (s, 1H).
Ferrocenyl(4-methoxyphenyl)methanol (7b) 합성Ferrocenyl (4-methoxyphenyl) methanol (7b) Synthesis
Ferrocenyl(4-methoylphenyl)methanone (4, 0.060 g, 0.19 mmol) 을 에탄올 (0.5 mL)/테트라하이드로퓨란 (0.5 mL) 혼합액에 녹인 후, 0 ℃ 에서 수소화붕소나트륨 (0.035 g, 0.94 mmol) 을 소량씩 천천히 넣었다. 이 반응 혼합물을 상온에서 3시간 동안 교반 하였다. 물 (2 mL) 과 디클로로메탄 (5 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (7 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=3:1) 로 정제하여 화합물 7b를 주홍색 액체로 얻었다. (0.060 g, 99%): 1H NMR (400 MHz, CDCl3) δ 7.30 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 5.45 (s, 1H), 4.22 (s, 6H), 4.17 (s, 3H), 3.79 (s, 3H), 2.38 (s, 1H).Ferrocenyl (4-methoylphenyl) methanone ( 4 , 0.060 g, 0.19 mmol) was dissolved in a mixture of ethanol (0.5 mL) / tetrahydrofuran (0.5 mL), followed by a small amount of sodium borohydride (0.035 g, 0.94 mmol) at 0 ° C. Slowly put in. The reaction mixture was stirred at room temperature for 3 hours. Water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 3: 1) to give compound 7b as a scarlet liquid. (0.060 g, 99%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.30 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 5.45 (s, 1H), 4.22 (s, 6H), 4.17 (s, 3H), 3.79 (s, 3H), 2.38 (s, 1H).
((4-(Benzyloxy)phenyl)(ferrocenyl)methyl)(phenyl)sulfane (8a) 합성((4- (Benzyloxy) phenyl) (ferrocenyl) methyl) (phenyl) sulfane (8a) Synthesis
(4-(Benzyloxy)phenyl)(ferrocenyl)methanol (7a, 0.020 g, 0.050 mmol) 을 디클로로메탄 (0.8 mL) 에 녹인 후 싸이오페놀 (0.006 g, 0.06 mmol) 을 넣는다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.014 mg, 0.075 mmol) 을 넣는다. 반응 혼합물을 상온에서 3 분 동안 교반한 후, 포화 탄산수소나트륨 수용액 (3 mL) 에 붓고, 디클로로메탄 (5 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (7 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=15:1) 로 정제하여 화합물 8a를 노란색 고체로 얻었다. (0.018 g, 73%): 1H NMR (400 MHz, CDCl3) δ 7.37-7.44 (m, 4H), 7.29-7.35 (m, 3H), 7.22-7.26 (m, 2H), 7.16-7.17 (m, 3H), 6.89 (d, J = 8.4 Hz, 2H), 5.11 (s, 1H), 5.04 (s, 2H), 4.13 (s, 3H), 4.10 (s, 6H).(4- (Benzyloxy) phenyl) (ferrocenyl) methanol Dissolve ( 7a , 0.020 g, 0.050 mmol) in dichloromethane (0.8 mL) and add thiophenol (0.006 g, 0.06 mmol). Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.014 mg, 0.075 mmol). The reaction mixture is stirred at room temperature for 3 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 15: 1) to give compound 8a as a yellow solid. (0.018 g, 73%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.37-7.44 (m, 4H), 7.29-7.35 (m, 3H), 7.22-7.26 (m, 2H), 7.16-7.17 ( m, 3H), 6.89 (d, J = 8.4 Hz, 2H), 5.11 (s, 1H), 5.04 (s, 2H), 4.13 (s, 3H), 4.10 (s, 6H).
((Ferrocenyl)(4-methoxyphenyl)methyl)(phenyl)sulfane (8b) 합성((Ferrocenyl) (4-methoxyphenyl) methyl) (phenyl) sulfane (8b) synthesis
Ferrocenyl(4-methoxyphenyl)methanol (7b, 0.059 g, 0.18 mmol) 을 디클로로메탄 (0.8 mL) 에 녹인 후 싸이오페놀 (0.022 g, 0.20 mmol) 을 넣는다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.050 mg, 0.28 mmol) 을 넣는다. 반응 혼합물을 상온에서 3 분 동안 교반한 후, 포화 탄산수소나트륨 수용액 (3 mL) 에 붓고, 디클로로메탄 (5 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (7 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=15:1) 로 정제하여 화합물 8b를 노란색 고체로 얻었다. (0.068 g, 90%): 1H NMR (400 MHz, CDCl3)δ 7.30 (d, J = 8.4 Hz, 2H), 7.20-7.22 (m, 2H), 7.16-7.20 (m, 3H), 6.82 (d, J = 8.8 Hz, 2H), 5.11 (s, 1H), 4.08-4.13 (m, 9H), 3.79 (s, 3H).Ferrocenyl (4-methoxyphenyl) methanol ( 7b , 0.059 g, 0.18 mmol) is dissolved in dichloromethane (0.8 mL) and thiophenol (0.022 g, 0.20 mmol) is added. Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.050 mg, 0.28 mmol). The reaction mixture is stirred at room temperature for 3 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent of the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 15: 1) to give compound 8b as a yellow solid. (0.068 g, 90%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.30 (d, J = 8.4 Hz, 2H), 7.20-7.22 (m, 2H), 7.16-7.20 (m, 3H), 6.82 (d, J = 8.8 Hz, 2H), 5.11 (s, 1H), 4.08-4.13 (m, 9H), 3.79 (s, 3H).
Ferrocenyl(4-iodophenyl)methanone (9) 합성Ferrocenyl (4-iodophenyl) methanone (9) Synthesis
페로센 (1.00 g, 5.38 mmol) 을 무수 디클로로메탄 (10 mL) 에 녹인 후, 이 용액에 4-아이오도벤조일 클로라이드 (4-iodobenzoyl chloride, 1.58 g, 5.91 mmol) 를 넣었다. 0 ℃ 로 냉각 시킨 후, 알루미늄 클로라이드 (1.01 g, 8.06 mmol) 를 소량씩 천천히 넣었다. 이 반응 혼합물을 상온에서 14.5 시간 동안 교반한 후 얼음물 (10 mL) 과 디클로로메탄 (15 mL) 을 차례대로 넣었다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (30 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산마그네슘을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=6:1) 로 정제하여 화합물 9를 붉은색 고체로 얻었다. (1.38 g, 62%): 1H NMR (400 MHz, CDCl3) 7.83 (d, J = 7.4 Hz, 2H), 7.63 (d, J = 7.4 Hz, 2H), 4.87 (s, 2H), 4.61 (s, 2H), 4.20 (s, 5H).Ferrocene (1.00 g, 5.38 mmol) was dissolved in anhydrous dichloromethane (10 mL), and then 4-iodobenzoyl chloride (4-58), 5.91 mmol) was added to the solution. After cooling to 0 ° C., aluminum chloride (1.01 g, 8.06 mmol) was slowly added in small portions. The reaction mixture was stirred at room temperature for 14.5 hours, and then ice water (10 mL) and dichloromethane (15 mL) were added sequentially. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (30 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous magnesium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 6: 1) to give compound 9 as a red solid. (1.38 g, 62%): 1 H NMR (400 MHz, CDCl 3 ) 7.83 (d, J = 7.4 Hz, 2H), 7.63 (d, J = 7.4 Hz, 2H), 4.87 (s, 2H), 4.61 (s, 2H), 4.20 (s, 5H).
(4-(Dec-1-yn-1-yl)phenyl)(ferrocenyl)methanone (10) 합성(4- (Dec-1-yn-1-yl) phenyl) (ferrocenyl) methanone (10) Synthesis
Ferrocenyl(4-iodophenyl)methanone (9, 0.100 g, 0.240 mmol), 테트라키스(트리페닐포스핀)팔라듐 (0.028 g, 0.024 mmol), 요오드화 구리(Ⅰ) (0.009 g, 0.05 mmol) 를 무수 테트라하이드로퓨란 (1 mL) 에 녹였다. 이 용액에 트리에틸아민 (0.27 mL, 1.9 mmol) 을 질소분위기에서 넣었다. 20 분 동안 용액에 아르곤 가스를 불어줌으로써 내부의 산소를 제거한 후 1-데카인 (1-decyne, 0.037 g, 0.26 mmol) 을 아르곤 분위기에서 넣고, 상온에서 12 시간 동안 교반 하였다. 포화 염화암모늄 수용액 (1 mL) 과 디클로로메탄 (5 mL) 을 넣어서 반응을 종료시킨 후, 유기 층을 물 층으로부터 분리하였다. 남은 물 층을 디클로로메탄 (8 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산마그네슘을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=9:1 )로 정제하여 화합물 10을 붉은색 액체로 얻었다. (0.100 g, 98%): 1H NMR (400 MHz, CDCl3) δ 7.83 (d, J = 7.6 Hz, 2H), 7.48 (d, J = 7.6 Hz, 2H), 5.30 (d, J = 1.6 Hz, 2H), 4.59 (d, J = 1.6 Hz, 2H), 4.19 (s, 5H), 2.44 (t, J = 7.2 Hz, 2H), 1.59-1.67 (m, 2H), 1.47 (br m, 2H), 1.30-1.32 (br m, 8H), 0.89 (t, J = 6.2 Hz, 3H).Ferrocenyl (4-iodophenyl) methanone ( 9 , 0.100 g, 0.240 mmol), tetrakis (triphenylphosphine) palladium (0.028 g, 0.024 mmol), copper iodide (I) (0.009 g, 0.05 mmol) It was dissolved in furan (1 mL). Triethylamine (0.27 mL, 1.9 mmol) was added to this solution in a nitrogen atmosphere. After removing the oxygen inside by blowing argon gas into the solution for 20 minutes, 1-decane (1-decyne, 0.037 g, 0.26 mmol) was put in an argon atmosphere, and stirred at room temperature for 12 hours. After the reaction was terminated by adding saturated aqueous ammonium chloride solution (1 mL) and dichloromethane (5 mL), the organic layer was separated from the water layer. The remaining water layer was extracted three times with dichloromethane (8 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous magnesium sulfate was added and filtered under reduced pressure. The solvent of the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 9: 1) to give compound 10 as a red liquid. (0.100 g, 98%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.83 (d, J = 7.6 Hz, 2H), 7.48 (d, J = 7.6 Hz, 2H), 5.30 (d, J = 1.6 Hz, 2H), 4.59 (d, J = 1.6 Hz, 2H), 4.19 (s, 5H), 2.44 (t, J = 7.2 Hz, 2H), 1.59-1.67 (m, 2H), 1.47 (br m, 2H), 1.30-1.32 (br m, 8H), 0.89 (t, J = 6.2 Hz, 3H).
(4-(Dec-1-yn-1-yl)phenyl)(ferrocenyl)(phenyl)methanol (11) 합성(4- (Dec-1-yn-1-yl) phenyl) (ferrocenyl) (phenyl) methanol (11) Synthesis
(4-(Dec-1-yn-1-yl)phenyl)(ferrocenyl)methanone (10, 0.078 g, 0.18 mmol) 을 무수 테트라하이드로퓨란 (0.7 mL) 에 녹이고 -78 ℃ 로 냉각시킨 후 페닐리튬 용액 (1.35 M 디노르말부틸에테르 용액, 0.16 mL, 0.22 mmol) 을 질소 분위기에서 천천히 넣었다. -78 ℃ 에서 5 분 동안 교반한 후, 물 (1 mL) 과 디클로로메탄 (5 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (8 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=9:1) 로 정제하여 화합물 11을 노란색 액체로 얻었다. (0.080 g, 87%): 1H NMR (400 MHz, CDCl3)δ 7.31 (d, J = 8.0 Hz, 2H), 7.21-7.26 (m, 8H), 4.27 (s, 2H), 4.17 (s, 5H), 4.04 (s, 1H), 3.99 (s, 1H), 3.44 (s, 1H), 2.38 (t, J = 6.8 Hz, 2H), 1.56-1.60 (m, 2H), 1.43-1.45 (br m, 2H), 1.29 (br m, 8H), 0.88 (t, J = 6.4 Hz, 3H).(4- (Dec-1-yn-1-yl) phenyl) (ferrocenyl) methanone ( 10 , 0.078 g, 0.18 mmol) was dissolved in anhydrous tetrahydrofuran (0.7 mL) and cooled to -78 ° C, followed by phenyllithium solution (1.35 M dinormalbutylether solution, 0.16 mL, 0.22 mmol) was slowly added in a nitrogen atmosphere. After stirring at -78 ° C for 5 minutes, water (1 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (8 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 9: 1) to obtain compound 11 as a yellow liquid. (0.080 g, 87%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.31 (d, J = 8.0 Hz, 2H), 7.21-7.26 (m, 8H), 4.27 (s, 2H), 4.17 (s , 5H), 4.04 (s, 1H), 3.99 (s, 1H), 3.44 (s, 1H), 2.38 (t, J = 6.8 Hz, 2H), 1.56-1.60 (m, 2H), 1.43-1.45 ( br m, 2H), 1.29 (brm, 8H), 0.88 (t, J = 6.4 Hz, 3H).
3-(((4-(Dec-1-yn-1-yl)phenyl)(ferrocenyl)(phenyl)methyl)thio)propanoic acid (12) 합성 Synthesis of 3-(((4- (Dec-1-yn-1-yl) phenyl) (ferrocenyl) (phenyl) methyl) thio) propanoic acid (12)
(4-(Dec-1-yn-1-yl)phenyl)(ferrocenyl)(phenyl)methanol (11, 0.075 g, 0.15 mmol) 을 디클로로메탄 (0.8 mL) 에 녹인 후 3-머캅토프로피오닉산 (3-mercaptopropionic acid, 0.017 g, 0.16 mmol) 을 넣는다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.035 mg, 0.19 mmol) 을 넣는다. 반응 혼합물을 상온에서 3 시간 동안 교반한 후, 포화 탄산수소나트륨 수용액 (3 mL) 에 붓고, 디클로로메탄 (5 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (7 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=1:1) 로 정제하여 화합물 12를 노란색 액체로 얻었다. (0.042 g, 48%): 1H NMR (400 MHz, CDCl3) δ 7.23-7.41 (m, 9H), 4.20 (s, 2H), 4.05-4.10 (m, 7H), 2.50 (t, J = 7.4 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H), 2.26 (t, J = 7.4 Hz, 2H), 1.56-1.62 (m, 2H), 1.44 (br m, 2H), 1.26-1.30 (br m, 8H), 0.87 (t, J = 6.8 Hz, 3H).(4- (Dec-1-yn-1-yl) phenyl) (ferrocenyl) (phenyl) methanol ( 11 , 0.075 g, 0.15 mmol) is dissolved in dichloromethane (0.8 mL), and 3-mercaptopropionic acid (3-mercaptopropionic acid, 0.017 g, 0.16 mmol) is added thereto. Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.035 mg, 0.19 mmol). The reaction mixture is stirred at room temperature for 3 hours, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 1: 1) to give compound 12 as a yellow liquid. (0.042 g, 48%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.23-7.41 (m, 9H), 4.20 (s, 2H), 4.05-4.10 (m, 7H), 2.50 (t, J = 7.4 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H), 2.26 (t, J = 7.4 Hz, 2H), 1.56-1.62 (m, 2H), 1.44 (br m, 2H), 1.26-1.30 (br m, 8H), 0.87 (t, J = 6.8 Hz, 3H).
Methyl 3-(((4-(dec-1-yn-1-yl)phenyl)(ferrocenyl)(phenyl)methyl)thio) propanoate (13) 합성 Methyl 3-(((4- (dec-1-yn-1-yl) phenyl) (ferrocenyl) (phenyl) methyl) thio) propanoate (13) Synthesis
3-(((4-(Dec-1-yn-1-yl)phenyl)(ferrocenyl)(phenyl)methyl)thio)propionic acid (12, 0.035 g, 0.059 mmol) 를 무수 벤젠 (0.5 mL) 과 메탄올 (0.1 mL) 혼합액에 녹인 후, (트리메틸실릴)디아조메탄 용액 ((trimethylsilyl)diazomethane, 2.0 M 헥산 용액, 0.060 mL, 0.12 mmol) 을 질소 분위기에서 넣었다. 이 반응 혼합물을 5 분 동안 상온에서 교반한 후, 감압하에 모든 용매를 제거하였다. 컬럼크로마토그래피 (헥산:에틸아세테이트=15:1) 로 정제하여 화합물 13을 노란색 액체로 얻었다. (0.036 g, 100%): 1H NMR (400 MHz, CDCl3) δ 7.26-7.40 (m, 9H), 4.20 (s, 2H), 4.06-4.08 (m, 7H), 3.66 (s, 3H), 2.49 (t, J = 7.4 Hz, 2H), 2.40 (t, J = 7.0 Hz, 2H), 2.26 (t, J = 7.2 Hz, 2H), 1.58-1.62 (m, 2H), 1.44 (br m, 2H), 1.30 (br m, 8H), 0.87 (t, J = 6.4 Hz, 3H).3-(((4- (Dec-1-yn-1-yl) phenyl) (ferrocenyl) (phenyl) methyl) thio) propionic acid ( 12 , 0.035 g, 0.059 mmol) was dissolved in anhydrous benzene (0.5 mL) and methanol. After dissolving in (0.1 mL) mixed solution, (trimethylsilyl) diazomethane solution ((trimethylsilyl) diazomethane, 2.0 M hexane solution, 0.060 mL, 0.12 mmol) was added in a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 5 minutes and then all solvents were removed under reduced pressure. Purification by column chromatography (hexane: ethyl acetate = 15: 1) gave compound 13 as a yellow liquid. (0.036 g, 100%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.26-7.40 (m, 9H), 4.20 (s, 2H), 4.06-4.08 (m, 7H), 3.66 (s, 3H) , 2.49 (t, J = 7.4 Hz, 2H), 2.40 (t, J = 7.0 Hz, 2H), 2.26 (t, J = 7.2 Hz, 2H), 1.58-1.62 (m, 2H), 1.44 (br m , 2H), 1.30 (br m, 8H), 0.87 (t, J = 6.4 Hz, 3H).
Ferrocenyl(4-hexylphenyl)methanone (14a) 합성Ferrocenyl (4-hexylphenyl) methanone (14a) Synthesis
9-보라바이사이클로[3.3.1]노네인 (0.052 g, 0.43 mmol) 을 무수 테트라하이드로퓨란 (0.9 mL) 에 녹이고 0 ℃ 로 냉각 시킨 후, 1-헥센 (1-hexene, 0.030 g, 0.36 mmol) 을 질소분위기에서 천천히 넣었다. 이 반응 혼합물을 상온에서 40 분 동안 교반 하였다. 이와 별개로 ferrocenyl(4-iodophenyl)methanone (9, 0.099 g, 0.24 mmol), 테트라키스(트리페닐포스핀)팔라듐 (0.014 g, 0.012 mmol), 포타슘 카보네이트 (0.099 g, 0.71 mmol) 를 질소 분위기에서 무수 디메틸포름아미드 (1.5 mL) 에 녹이고, 이 용액에 먼저 반응시켜 놓은 반응 혼합물을 질소 분위기에서 넣었다. 최종 반응 혼합물을 환류하에 70 ℃ 에서 12 시간 동안 가열하였다. 물 (1 mL) 과 디클로로메탄 (5 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=9:1) 로 정제하여 화합물 14a를 붉은색 액체로 얻었다. (0.044 g, 49%): 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 4.91 (t, J = 3.6 Hz, 2H), 4.57 (t, J = 3.2 Hz, 2H), 4.21 (s, 5H), 2.68 (t, J = 7.8 Hz, 2H), 1.62-1.70 (m, 2H), 1.30-1.38 (br m, 6H), 0.90 (t, J = 7.0 Hz, 3H).9-Vorabicyclo [3.3.1] nonane (0.052 g, 0.43 mmol) was dissolved in anhydrous tetrahydrofuran (0.9 mL) and cooled to 0 ° C., followed by 1-hexene (1-hexene, 0.030 g, 0.36 mmol). ) Was slowly added in a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 40 minutes. Separately ferrocenyl (4-iodophenyl) methanone ( 9 , 0.099 g, 0.24 mmol), tetrakis (triphenylphosphine) palladium (0.014 g, 0.012 mmol), potassium carbonate (0.099 g, 0.71 mmol) in a nitrogen atmosphere The reaction mixture, which was dissolved in anhydrous dimethylformamide (1.5 mL) and reacted first with this solution, was placed in a nitrogen atmosphere. The final reaction mixture was heated at reflux at 70 ° C. for 12 h. Water (1 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent of the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 9: 1) to give compound 14a as a red liquid. (0.044 g, 49%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.84 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 4.91 (t, J = 3.6 Hz, 2H), 4.57 (t, J = 3.2 Hz, 2H), 4.21 (s, 5H), 2.68 (t, J = 7.8 Hz, 2H), 1.62-1.70 (m, 2H), 1.30-1.38 (br m, 6H), 0.90 (t, J = 7.0 Hz, 3H).
Ferrocenyl(4-octylphenyl)methanone (14b) 합성Ferrocenyl (4-octylphenyl) methanone (14b) synthesis
9-보라바이사이클로[3.3.1]노네인 (0.110 g, 0.901 mmol) 을 무수 테트라하이드로퓨란 (0.4 mL) 에 녹이고 0 ℃ 로 냉각 시킨 후, 1-옥텐 (1-octene, 0.081 g, 0.72 mmol) 을 질소분위기에서 천천히 넣었다. 이 반응 혼합물을 상온에서 40 분 동안 교반 하였다. 이와 별개로 ferrocenyl(4-iodophenyl)methanone (9, 0.150 g, 0.361 mmol), 테트라키스(트리페닐포스핀)팔라듐 (0.015 g, 0.361 mmol), 포타슘 카보네이트 (0.199 g, 1.44 mmol) 를 질소 분위기에서 무수 디메틸포름아미드 (1.0 mL) 에 녹이고, 이 용액에 먼저 반응시켜 놓은 반응 혼합물을 질소 분위기에서 넣었다. 최종 반응 혼합물을 환류하에 70 ℃ 에서 12 시간 동안 가열하였다. 물 (2 mL) 과 디클로로메탄 (5 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=9:1) 로 정제하여 화합물 14b를 붉은색 액체로 얻었다. (0.123 g, 85%): 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 7.6 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 4.91 (t, J = 2.0 Hz, 2H), 4.57 (t, J = 1.6 Hz, 2H), 4.21 (s, 5H), 2.68 (t, J = 7.8 Hz, 2H), 1.62-1.68 (m, 2H), 1.28-1.33 (br m, 10H), 0.89 (t, J = 6.6 Hz, 3H).9-Vorabicyclo [3.3.1] nonane (0.110 g, 0.901 mmol) was dissolved in anhydrous tetrahydrofuran (0.4 mL) and cooled to 0 ° C., followed by 1-octene (1-octene, 0.081 g, 0.72 mmol). ) Was slowly added in a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 40 minutes. Separately, ferrocenyl (4-iodophenyl) methanone ( 9 , 0.150 g, 0.361 mmol), tetrakis (triphenylphosphine) palladium (0.015 g, 0.361 mmol), potassium carbonate (0.199 g, 1.44 mmol) in a nitrogen atmosphere The reaction mixture which was dissolved in anhydrous dimethylformamide (1.0 mL) and reacted first with this solution was put in a nitrogen atmosphere. The final reaction mixture was heated at reflux at 70 ° C. for 12 h. Water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 9: 1) to give compound 14b as a red liquid. (0.123 g, 85%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.84 (d, J = 7.6 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 4.91 (t, J = 2.0 Hz, 2H), 4.57 (t, J = 1.6 Hz, 2H), 4.21 (s, 5H), 2.68 (t, J = 7.8 Hz, 2H), 1.62-1.68 (m, 2H), 1.28-1.33 (br m, 10H), 0.89 (t, J = 6.6 Hz, 3H).
(4-Decylphenyl)(ferrocenyl)methanone (14c) 합성(4-Decylphenyl) (ferrocenyl) methanone (14c) Synthesis
9-보라바이사이클로[3.3.1]노네인 (0.110 g, 0.901 mmol) 을 무수 테트라하이드로퓨란 (0.4 mL) 에 녹이고 0 ℃ 로 냉각 시킨 후, 1-데센 (1-decene, 0.101 g, 0.721 mmol) 을 질소분위기에서 천천히 넣었다. 이 반응 혼합물을 상온에서 40 분 동안 교반 하였다. 이와 별개로 ferrocenyl(4-iodophenyl)methanone (9, 0.150 g, 0.361 mmol), 테트라키스(트리페닐포스핀)팔라듐 (0.021 g, 0.018 mmol), 포타슘 카보네이트 (0.249 g, 1.80 mmol) 를 질소 분위기에서 무수 디메틸포름아미드 (1 mL) 에 녹이고, 이 용액에 먼저 반응시켜 놓은 반응 혼합물을 질소 분위기에서 넣었다. 최종 반응 혼합물을 환류하에 70 ℃ 에서 12 시간 동안 가열하였다. 물 (2 mL) 과 디클로로메탄 (5 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=9:1) 로 정제하여 화합물 14c를 붉은색 액체로 얻었다. (0.125 g, 80%): 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 4.91 (t, J = 2.0 Hz, 2H), 4.57 (t, J = 1.8 Hz, 2H), 4.21 (s, 5H), 2.68 (t, J = 7.8 Hz, 2H), 1.64-1.68 (m, 2H), 1.27-1.33 (br m, 14H), 0.88 (t, J = 6.8 Hz, 3H).9-Vorabicyclo [3.3.1] nonane (0.110 g, 0.901 mmol) was dissolved in anhydrous tetrahydrofuran (0.4 mL) and cooled to 0 ° C., followed by 1-decene (1-decene, 0.101 g, 0.721 mmol). ) Was slowly added in a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 40 minutes. Separately ferrocenyl (4-iodophenyl) methanone ( 9 , 0.150 g, 0.361 mmol), tetrakis (triphenylphosphine) palladium (0.021 g, 0.018 mmol), potassium carbonate (0.249 g, 1.80 mmol) in a nitrogen atmosphere The reaction mixture dissolved in anhydrous dimethylformamide (1 mL) and reacted first with this solution was put in a nitrogen atmosphere. The final reaction mixture was heated at reflux at 70 ° C. for 12 h. Water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 9: 1) to give compound 14c as a red liquid. (0.125 g, 80%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.84 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 4.91 (t, J = 2.0 Hz, 2H), 4.57 (t, J = 1.8 Hz, 2H), 4.21 (s, 5H), 2.68 (t, J = 7.8 Hz, 2H), 1.64-1.68 (m, 2H), 1.27-1.33 (br m, 14H), 0.88 (t, J = 6.8 Hz, 3H).
Ferrocenyl(4-hexylphenyl)(phenyl)methanol (15a) 합성Ferrocenyl (4-hexylphenyl) (phenyl) methanol (15a) Synthesis
Ferrocenyl(4-hexylphenyl)methanone (14a, 0.040 g, 0.11 mmol) 을 무수 테트라하이드로퓨란 (1 mL) 에 녹이고 -78 ℃ 로 냉각시킨 후 페닐리튬 용액 (1.35 M 디노르말부틸에테르 용액, 0.09 mL, 0.1 mmol) 을 질소 분위기에서 천천히 넣었다. -78 ℃ 에서 1 분 동안 교반한 후, 물 (1 mL) 과 디클로로메탄 (3 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (5 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=9:1) 로 정제하여 화합물 15a를 노란색 액체로 얻었다. (0.064 g, 100%): 1H NMR (400 MHz, CDCl3) δ 7.26-7.32 (m, 4H), 7.21-7.24 (m, 1H), 7.15-7.18 (m, 2H), 7.07 (d, J = 8.4 Hz, 2H), 4.26 (s, 2H), 4.16 (s, 5H), 4.06 (s, 1H), 4.01 (s, 1H), 3.40 (s, 1H), 2.56 (t, J = 7.8 Hz, 2H), 1.55-1.62 (m, 2H), 1.24-1.33 (br m, 6H), 0.87 (t, J = 6.6 Hz, 3H).Ferrocenyl (4-hexylphenyl) methanone ( 14a , 0.040 g, 0.11 mmol) was dissolved in anhydrous tetrahydrofuran (1 mL) and cooled to -78 ° C, followed by phenyllithium solution (1.35 M dinormalbutylether solution, 0.09 mL, 0.1 mmol) was slowly added in a nitrogen atmosphere. After stirring at −78 ° C. for 1 minute, water (1 mL) and dichloromethane (3 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (5 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 9: 1) to give compound 15a as a yellow liquid. (0.064 g, 100%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.26-7.32 (m, 4H), 7.21-7.24 (m, 1H), 7.15-7.18 (m, 2H), 7.07 (d, J = 8.4 Hz, 2H), 4.26 (s, 2H), 4.16 (s, 5H), 4.06 (s, 1H), 4.01 (s, 1H), 3.40 (s, 1H), 2.56 (t, J = 7.8 Hz, 2H), 1.55-1.62 (m, 2H), 1.24-1.33 (br m, 6H), 0.87 (t, J = 6.6 Hz, 3H).
Ferrocenyl(4-octylphenyl)(phenyl)methanol (15b) 합성Ferrocenyl (4-octylphenyl) (phenyl) methanol (15b) Synthesis
Ferrocenyl(4-octylphenyl)methanone (14b, 0.121 g, 0.301 mmol) 을 무수 테트라하이드로퓨란 (2 mL) 에 녹이고 -78 ℃ 로 냉각시킨 후 페닐리튬 용액 (1.35 M 디노르말부틸에테르 용액, 0.22 mL, 0.30 mmol) 을 질소 분위기에서 천천히 넣었다. -78 ℃ 에서 1 분 동안 교반한 후, 물 (2 mL) 과 디클로로메탄 (5 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=9:1) 로 정제하여 화합물 15b를 노란색 액체로 얻었다. (0.138 g, 95%): 1H NMR (400 MHz, CDCl3) δ 7.26-7.31 (m, 4H), 7.20-7.23 (m, 1H), 7.17 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 4.25 (s, 2H), 4.16 (s, 5H), 4.06 (s, 1H), 4.01 (s, 1H), 3.40 (s, 1H), 2.56 (t, J = 7.8 Hz, 2H), 1.55-1.60 (m, 2H), 1.25-1.28 (br m, 10H), 0.87 (t, J = 6.6 Hz, 3H).Ferrocenyl (4-octylphenyl) methanone ( 14b , 0.121 g, 0.301 mmol) was dissolved in anhydrous tetrahydrofuran (2 mL) and cooled to -78 ° C, followed by phenyllithium solution (1.35 M dinormalbutylether solution, 0.22 mL, 0.30 mmol) was slowly added in a nitrogen atmosphere. After stirring at −78 ° C. for 1 minute, water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 9: 1) to obtain compound 15b as a yellow liquid. (0.138 g, 95%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.26-7.31 (m, 4H), 7.20-7.23 (m, 1H), 7.17 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 4.25 (s, 2H), 4.16 (s, 5H), 4.06 (s, 1H), 4.01 (s, 1H), 3.40 (s, 1H), 2.56 (t, J = 7.8 Hz, 2H), 1.55-1.60 (m, 2H), 1.25-1.28 (br m, 10H), 0.87 (t, J = 6.6 Hz, 3H).
(4-Decylphenyl)(ferrocenyl)(phenyl)methanol (15c) 합성(4-Decylphenyl) (ferrocenyl) (phenyl) methanol (15c) Synthesis
(4-Decylphenyl)(ferrocenyl)methanone (14c, 0.125 g, 0.290 mmol) 을 무수 테트라하이드로퓨란 (2 mL) 에 녹이고 -78 ℃ 로 냉각시킨 후 페닐리튬 용액 (1.35 M 디노르말부틸에테르 용액, 0.22 mL, 0.30 mmol) 을 질소 분위기에서 천천히 넣었다. -78 ℃ 에서 1 분 동안 교반한 후, 물 (2 mL) 과 디클로로메탄 (5 mL) 을 차례대로 넣어 반응을 종료시켰다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=15:1) 로 정제하여 화합물 15c를 노란색 액체로 얻었다. (0.147 g, 100%): 1H NMR (400 MHz, CDCl3) δ 7.27-7.32 (m, 4H), 7.20-7.23 (m, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 4.25 (t, J = 1.0 Hz, 2H), 4.16 (s, 5H), 4.05 (s, 1H), 4.01 (s, 1H), 3.40 (s, 1H), 2.58 (t, J = 7.8 Hz, 2H), 1.55-1.58 (m, 2H), 1.25-1.28 (br m, 14H), 0.88 (t, J = 6.8 Hz, 3H).(4-Decylphenyl) (ferrocenyl) methanone ( 14c , 0.125 g, 0.290 mmol) was dissolved in anhydrous tetrahydrofuran (2 mL) and cooled to -78 ° C, followed by phenyllithium solution (1.35 M dinormalbutylether solution, 0.22 mL , 0.30 mmol) was slowly added under a nitrogen atmosphere. After stirring at −78 ° C. for 1 minute, water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 15: 1) to obtain compound 15c as a yellow liquid. (0.147 g, 100%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.27-7.32 (m, 4H), 7.20-7.23 (m, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 4.25 (t, J = 1.0 Hz, 2H), 4.16 (s, 5H), 4.05 (s, 1H), 4.01 (s, 1H), 3.40 (s, 1H) , 2.58 (t, J = 7.8 Hz, 2H), 1.55-1.58 (m, 2H), 1.25-1.28 (br m, 14H), 0.88 (t, J = 6.8 Hz, 3H).
3-(((Ferrocenyl)(4-hexylphenyl)(phenyl)methyl)thio)propanoic acid (16a) 합성 Synthesis of 3-(((Ferrocenyl) (4-hexylphenyl) (phenyl) methyl) thio) propanoic acid (16a)
Ferrocenyl(4-hexylphenyl)(phenyl)methanol (15a, 0.048 g, 0.11 mmol) 을 디클로로메탄 (1 mL) 에 녹인 후 3-머캅토프로피오닉산 (3-mercaptopropionic acid, 0.012 g, 0.12 mmol) 을 넣었다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.025 mg, 0.14 mmol) 을 넣는다. 반응 혼합물을 상온에서 3 시간 동안 교반한 후, 포화 탄산수소나트륨 수용액 (3 mL) 에 붓고, 디클로로메탄 (5 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (7 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=3:1+MeOH 10%) 로 정제하여 화합물 16a를 노란색 액체로 얻었다. (0.051 g, 88%): 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 4.8, 2H), 7.23-7.31 (m, 5H), 7.06 (d, J = 8.4 Hz, 2H), 4.19 (t, J = 2.0 Hz, 2H), 4.06-4.12 (m, 7H), 2.59 (t, J = 8.0 Hz, 2H), 2.52 (br t, J = 7.4 Hz, 2H), 2.21 (br t, J = 7.4 Hz, 2H), 1.55-1.62 (m, 2H), 1.29-1.31 (br m, 6H), 0.88 (t, J = 6.8 Hz, 3H).Ferrocenyl (4-hexylphenyl) (phenyl) methanol ( 15a , 0.048 g, 0.11 mmol) was dissolved in dichloromethane (1 mL), and 3-mercaptopropionic acid (3-mercaptopropionic acid, 0.012 g, 0.12 mmol) was added thereto. Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.025 mg, 0.14 mmol). The reaction mixture is stirred at room temperature for 3 hours, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 3: 1 + MeOH 10%) to give compound 16a as a yellow liquid. (0.051 g, 88%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.39 (d, J = 4.8, 2H), 7.23-7.31 (m, 5H), 7.06 (d, J = 8.4 Hz, 2H) , 4.19 (t, J = 2.0 Hz, 2H), 4.06-4.12 (m, 7H), 2.59 (t, J = 8.0 Hz, 2H), 2.52 (br t, J = 7.4 Hz, 2H), 2.21 (br t, J = 7.4 Hz, 2H), 1.55-1.62 (m, 2H), 1.29-1.31 (br m, 6H), 0.88 (t, J = 6.8 Hz, 3H).
3-(((Ferrocenyl)(4-octylphenyl)(phenyl)methyl)thio)propanoic acid (16b) 합성Synthesis of 3-(((Ferrocenyl) (4-octylphenyl) (phenyl) methyl) thio) propanoic acid (16b)
Ferrocenyl(4-octylphenyl)(phenyl)methanol (15b, 0.138 g, 0.287 mmol) 을 디클로로메탄 (1 mL) 에 녹인 후 3-머캅토프로피오닉산 (3-mercaptopropionic acid, 0.037g, 0.35 mmol) 을 넣었다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.074 mg, 0.40 mmol) 을 넣는다. 반응 혼합물을 상온에서 3 시간 동안 교반한 후, 포화 탄산수소나트륨 수용액 (3 mL) 에 붓고, 디클로로메탄 (5 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=3:1+MeOH 10%) 로 정제하여 화합물 16b를 노란색 액체로 얻었다. (0.100 g, 61%): 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.6, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.21-7.29 (m, 3H), 7.08 (d, J = 8.0 Hz, 2H), 4.19 (s, 2H), 4.10 (s, 2H), 4.06 (s, 5H), 2.58 (t, J = 7.6 Hz, 2H), 2.49 (br t, J = 7.8 Hz, 2H), 2.20 (br t, J = 7.8 Hz, 2H), 1.58-1.62 (m, 2H), 1.24-1.30 (br m, 10H), 0.88 (t, J = 6.8 Hz, 3H).Ferrocenyl (4-octylphenyl) (phenyl) methanol ( 15b , 0.138 g, 0.287 mmol) was dissolved in dichloromethane (1 mL) and 3-mercaptopropionic acid (3-mercaptopropionic acid, 0.037 g, 0.35 mmol) was added thereto. Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.074 mg, 0.40 mmol). The reaction mixture is stirred at room temperature for 3 hours, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 3: 1 + MeOH 10%) to give compound 16b as a yellow liquid. (0.100 g, 61%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.43 (d, J = 7.6, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.21-7.29 (m, 3H) , 7.08 (d, J = 8.0 Hz, 2H), 4.19 (s, 2H), 4.10 (s, 2H), 4.06 (s, 5H), 2.58 (t, J = 7.6 Hz, 2H), 2.49 (br t , J = 7.8 Hz, 2H), 2.20 (br t, J = 7.8 Hz, 2H), 1.58-1.62 (m, 2H), 1.24-1.30 (br m, 10H), 0.88 (t, J = 6.8 Hz, 3H).
3-(((4-Decylphenyl)(ferrocenyl)(phenyl)methyl)thio)propanoic acid (16c)3-(((4-Decylphenyl) (ferrocenyl) (phenyl) methyl) thio) propanoic acid (16c) 합성synthesis
(4-Decylphenyl)(ferrocenyl)(phenyl)methanol (15c, 0.147 g, 0.290 mmol) 을 디클로로메탄 (3 mL) 에 녹인 후 3-머캅토프로피오닉산 (3-mercaptopropionic acid, 0.046g, 0.44 mmol) 을 넣었다. 이 용액에 플루오로붕산 수용액 (48 wt%, 0.095 mg, 0.52 mmol) 을 넣는다. 반응 혼합물을 상온에서 3 시간 동안 교반한 후, 포화 탄산수소나트륨 수용액 (3 mL) 에 붓고, 디클로로메탄 (5 mL) 을 넣어 희석시킨다. 유기 층은 분리하고 남은 물 층을 디클로로메탄 (10 mL x 3) 으로 세 번 추출 하였다. 모아진 유기 층은 포화 염화나트륨 수용액으로 씻고, 무수 황산나트륨을 넣고 감압 여과 하였다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=3:1+MeOH 10%) 로 정제하여 화합물 16c를 노란색 액체로 얻었다. (0.060 g, 35%): 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.6, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.20-7.33 (m, 3H), 7.08 (d, J = 8.0 Hz, 2H), 4.18 (s, 2H), 4.09 (s, 2H), 4.06 (s, 5H), 2.58 (t, J = 7.86 Hz, 2H), 2.50 (br t, J = 7.2 Hz, 2H), 2.20 (br t, J = 7.0 Hz, 2H), 1.59-1.62 (m, 2H), 1.26-1.31 (br m, 14H), 0.88 (t, J = 6.6 Hz, 3H).(4-Decylphenyl) (ferrocenyl) (phenyl) methanol ( 15c , 0.147 g, 0.290 mmol) was dissolved in dichloromethane (3 mL) and then 3-mercaptopropionic acid (0.046 g, 0.44 mmol) Put it. Into this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.095 mg, 0.52 mmol). The reaction mixture is stirred at room temperature for 3 hours, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure. The solvent in the filtrate was completely removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 3: 1 + MeOH 10%) to give compound 16c as a yellow liquid. (0.060 g, 35%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.43 (d, J = 7.6, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.20-7.33 (m, 3H) , 7.08 (d, J = 8.0 Hz, 2H), 4.18 (s, 2H), 4.09 (s, 2H), 4.06 (s, 5H), 2.58 (t, J = 7.86 Hz, 2H), 2.50 (br t , J = 7.2 Hz, 2H), 2.20 (br t, J = 7.0 Hz, 2H), 1.59-1.62 (m, 2H), 1.26-1.31 (br m, 14H), 0.88 (t, J = 6.6 Hz, 3H).
Methyl 3-(((ferrocenyl)(4-hexylphenyl)(phenyl)methyl)thio)propanoate (17a)합성 Methyl 3-(((ferrocenyl) (4-hexylphenyl) (phenyl) methyl) thio) propanoate (17a) Synthesis
3-(((Ferrocenyl)(4-hexylphenyl)(phenyl)methyl)thio)propanoic acid (16a, 0.035 g, 0.059 mmol), N-하이드록시 숙신이미드 (N-hydroxysuccinimide, 0.008 g, 0.07 mmol), N,N-디메틸피리디늄 p-톨루엔설포네이트 (N,N-dimethylpyridinium p-toluenesulfonate (DPTS) 0.001 g, 0.003 mmol) 를 무수 디클로로메탄 (0.3 mL) 에 질소 분위기에서 녹인 후 0 ℃ 로 냉각시켰다. 무수 디클로로메탄 (0.3 mL) 에 녹인 N,N'-디사이클로헥실카르보디이미드 (N,N'-dicyclohexylcarbodiimide 0.014 g, 0.14 mmol) 용액을 질소 분위기에서 위 반응 혼합물에 천천히 넣어 주었다. 최종 반응 혼합물의 온도를 상온으로 서서히 올리고 8 시간 동안 교반 하였다. 반응 혼합물을 0 ℃ 로 냉각시킨 후 침전으로 가라앉은 고체를 감압 여과하면서 차가운 디클로로메탄으로 씻어 주었다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=3:1) 로 정제하여 화합물 17a를 끈적한 노란색 액체로 얻었다. (0.029 g, 77%): 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 7.6, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.24-7.31 (m, 3H), 7.10 (d, J = 8.0 Hz, 2H), 4.19 (s, 2H), 4.13 (s, 1H), 4.10 (s, 1H), 4.08 (s, 5H), 2.81 (br s, 4H), 2.60 (m, 4H), 2.36 (t, J = 7.4 Hz, 2H), 1.59-1.63 (m, 2H), 1.26-1.31 (br m, 6H), 0.88 (t, J = 6.2 Hz, 3H).3 - (((Ferrocenyl) ( 4-hexylphenyl) (phenyl) methyl) thio) propanoic acid (16a, 0.035 g, 0.059 mmol), N - hydroxy-succinimide (N -hydroxysuccinimide, 0.008 g, 0.07 mmol), N , N -dimethylpyridinium p -toluenesulfonate ( N , N- dimethylpyridinium p- toluenesulfonate (DPTS) 0.001 g, 0.003 mmol) was dissolved in anhydrous dichloromethane (0.3 mL) in a nitrogen atmosphere and cooled to 0 ° C. A solution of N, N'-dicyclohexylcarbodiimide (N, N'-dicyclohexylcarbodiimide 0.014 g, 0.14 mmol) dissolved in anhydrous dichloromethane (0.3 mL) was slowly added to the above reaction mixture in a nitrogen atmosphere. The temperature of the final reaction mixture was slowly raised to room temperature and stirred for 8 hours. After the reaction mixture was cooled to 0 ° C., the solid that had settled by precipitation was washed with cold dichloromethane under reduced pressure. The solvent of the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 3: 1) to give compound 17a as a sticky yellow liquid. (0.029 g, 77%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.47 (d, J = 7.6, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.24-7.31 (m, 3H) , 7.10 (d, J = 8.0 Hz, 2H), 4.19 (s, 2H), 4.13 (s, 1H), 4.10 (s, 1H), 4.08 (s, 5H), 2.81 (br s, 4H), 2.60 (m, 4H), 2.36 (t, J = 7.4 Hz, 2H), 1.59-1.63 (m, 2H), 1.26-1.31 (br m, 6H), 0.88 (t, J = 6.2 Hz, 3H).
Methyl 3-(((ferrocenyl)(4-octylphenyl)(phenyl)methyl)thio)propanoate (17b)합성 Methyl 3-(((ferrocenyl) (4-octylphenyl) (phenyl) methyl) thio) propanoate (17b) Synthesis
3-(((Ferrocenyl)(4-octylphenyl)(phenyl)methyl)thio)propanoic acid (16b, 0.065 g, 0.11 mmol), N-하이드록시 숙신이미드 (N-hydroxysuccinimide, 0.016 g, 0.14 mmol), N,N-디메틸피리디늄 p-톨루엔설포네이트 (N,N-dimethylpyridinium p-toluenesulfonate (DPTS), 0.002 g, 0.006 mmol) 를 무수 디클로로메탄 (0.4 mL) 에 질소 분위기에서 녹인 후 0 ℃ 로 냉각시켰다. 무수 디클로로메탄 (0.4 mL) 에 녹인 N,N'-디사이클로헥실카르보디이미드 (N,N'-dicyclohexylcarbodiimide 0.028 g, 0.14 mmol) 용액을 질소 분위기에서 위 반응 혼합물에 천천히 넣어 주었다. 최종 반응 혼합물의 온도를 상온으로 서서히 올리고 8 시간 동안 교반 하였다. 반응 혼합물을 0 ℃ 로 냉각시킨 후 침전으로 가라앉은 고체를 감압 여과하면서 차가운 디클로로메탄으로 씻어 주었다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=3:1) 로 정제하여 화합물 17b를 끈적한 노란색 액체로 얻었다. (0.062 g, 82%): 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 8.0, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.24-7.31 (m, 3H), 7.10 (d, J = 8.0 Hz, 2H), 4.19 (s, 2H), 4.13 (s, 1H), 4.11 (s, 1H), 4.08 (s, 5H), 2.81 (br s, 4H), 2.60 (m, 4H), 2.36 (t, J = 7.4 Hz, 2H), 1.59-1.63 (m, 2H), 1.26-1.31 (br m, 10H), 0.88 (t, J = 6.4 Hz, 3H).3 - (((Ferrocenyl) ( 4-octylphenyl) (phenyl) methyl) thio) propanoic acid (16b, 0.065 g, 0.11 mmol), N - hydroxy-succinimide (N -hydroxysuccinimide, 0.016 g, 0.14 mmol), N , N -dimethylpyridinium p -toluenesulfonate ( N , N- dimethylpyridinium p- toluenesulfonate (DPTS), 0.002 g, 0.006 mmol) was dissolved in anhydrous dichloromethane (0.4 mL) in a nitrogen atmosphere and cooled to 0 ° C. . A solution of N, N'-dicyclohexylcarbodiimide (N, N'-dicyclohexylcarbodiimide 0.028 g, 0.14 mmol) dissolved in anhydrous dichloromethane (0.4 mL) was slowly added to the above reaction mixture in a nitrogen atmosphere. The temperature of the final reaction mixture was slowly raised to room temperature and stirred for 8 hours. After the reaction mixture was cooled to 0 ° C., the solid that had settled by precipitation was washed with cold dichloromethane under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 3: 1) to give compound 17b as a sticky yellow liquid. (0.062 g, 82%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.47 (d, J = 8.0, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.24-7.31 (m, 3H) , 7.10 (d, J = 8.0 Hz, 2H), 4.19 (s, 2H), 4.13 (s, 1H), 4.11 (s, 1H), 4.08 (s, 5H), 2.81 (br s, 4H), 2.60 (m, 4H), 2.36 (t, J = 7.4 Hz, 2H), 1.59-1.63 (m, 2H), 1.26-1.31 (br m, 10H), 0.88 (t, J = 6.4 Hz, 3H).
Methyl 3-(((ferrocenyl)(9H-fluoren-2-yl)(phenyl)methyl)thio)propanoate (18c) 합성 Synthesis of methyl 3-(((ferrocenyl) (9H-fluoren-2-yl) (phenyl) methyl) thio) propanoate (18c)
3-(((4-Decylphenyl)(ferrocenyl)(phenyl)methyl)thio)propanoic acid (16c, 0.065 g, 0.11 mmol), N-하이드록시 숙신이미드 (N-hydroxysuccinimide, 0.015 g, 0.13 mmol), N,N-디메틸피리디늄 p-톨루엔설포네이트 (N,N-dimethylpyridinium p-toluenesulfonate (DPTS), 0.002 g, 0.006 mmol) 를 무수 디클로로메탄 (0.4 mL) 에 질소 분위기에서 녹인 후 0 ℃ 로 냉각시켰다. 무수 디클로로메탄 (0.4 mL) 에 녹인 N,N'-디사이클로헥실카르보디이미드 (N,N'-dicyclohexylcarbodiimide 0.028 g, 0.14 mmol) 용액을 질소 분위기에서 위 반응 혼합물에 천천히 넣어 주었다. 최종 반응 혼합물의 온도를 상온으로 서서히 올리고 8 시간 동안 교반 하였다. 반응 혼합물을 0 ℃로 냉각시킨 후 침전으로 가라앉은 고체를 감압 여과하면서 차가운 디클로로메탄으로 씻어 주었다. 여액의 용매를 감압하에 모두 제거한 후 컬럼크로마토그래피 (헥산:에틸아세테이트=3:1) 로 정제하여 화합물 17c를 끈적한 노란색 액체로 얻었다. (0.064 g, 85%): 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 7.2 2H), 7.35 (d, J = 8.8 Hz, 2H), 7.24-7.31 (m, 3H), 7.10 (d, J = 8.0 Hz, 2H), 4.19 (s, 2H), 4.13 (s, 1H), 4.11 (s, 1H), 4.08 (s, 5H), 2.81 (br s, 4H), 2.60 (m, 4H), 2.37 (t, J = 7.4 Hz, 2H), 1.61-1.63 (m, 2H), 1.26-1.31 (br m, 14H), 0.88 (t, J = 6.8 Hz, 3H).3 - (((4-Decylphenyl ) (ferrocenyl) (phenyl) methyl) thio) propanoic acid (16c, 0.065 g, 0.11 mmol), N - hydroxy-succinimide (N -hydroxysuccinimide, 0.015 g, 0.13 mmol), N , N -dimethylpyridinium p -toluenesulfonate ( N , N- dimethylpyridinium p- toluenesulfonate (DPTS), 0.002 g, 0.006 mmol) was dissolved in anhydrous dichloromethane (0.4 mL) in a nitrogen atmosphere and cooled to 0 ° C. . A solution of N, N'-dicyclohexylcarbodiimide (N, N'-dicyclohexylcarbodiimide 0.028 g, 0.14 mmol) dissolved in anhydrous dichloromethane (0.4 mL) was slowly added to the above reaction mixture in a nitrogen atmosphere. The temperature of the final reaction mixture was slowly raised to room temperature and stirred for 8 hours. The reaction mixture was cooled to 0 ° C., and the solids which settled by precipitation were washed with cold dichloromethane under reduced pressure. The solvent in the filtrate was removed under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate = 3: 1) to give compound 17c as a sticky yellow liquid. (0.064 g, 85%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.47 (d, J = 7.2 2H), 7.35 (d, J = 8.8 Hz, 2H), 7.24-7.31 (m, 3H), 7.10 (d, J = 8.0 Hz, 2H), 4.19 (s, 2H), 4.13 (s, 1H), 4.11 (s, 1H), 4.08 (s, 5H), 2.81 (br s, 4H), 2.60 ( m, 4H), 2.37 (t, J = 7.4 Hz, 2H), 1.61-1.63 (m, 2H), 1.26-1.31 (br m, 14H), 0.88 (t, J = 6.8 Hz, 3H).
실시예 2:신규 질량표지 물질을 이용한 바이오 마커의 검출 방법 검증Example 2 Verification of Detection Methods for Biomarkers Using New Mass Labeling Substances
tag 컨쥬게이션 검증을 위한 조건 실험 Conditional experiments for tag conjugation validation
BSA에 NHS-Tag들을 conjugation 하는 방법 개발 Development of conjugation of NHS-tags in BSA
BSA는 1nmole에 해당하는 단백량을 준비하였고(BSA mw = 66KDa 1nmole = 70ug), NHS-Tag는 BSA mole 수의 50배에 해당하는 양을 100% DMSO에 녹여 준비하였다.(50nmoles) BSA prepared a protein amount corresponding to 1nmole (BSA mw = 66KDa 1nmole = 70ug), NHS-Tag was prepared by dissolving 50 times the amount of BSA mole number in 100% DMSO (50nmoles).
Conjugation 반응은 50mM MES, pH6, 10% Acetonitrile에서 진행하였고, 반응의 최종 부피는 80uL로 하였으며, 상온에서 1hr 반응시켰다. Conjugation 후, Bio-Spin(Bio Rad) P-30을 물로 버퍼 교체한 다음, 남은 Free한 NHS-Tag를 제거하였다. 진공으로 건조하여 50uL까지 농축하였다다. Matrix 없이 (MA)LDI-MS를 수행하여 BSA에 Tagging 된 것을 확인하였다.Conjugation reaction was carried out in 50mM MES, pH 6, 10% Acetonitrile, the final volume of the reaction was 80uL, 1hr reaction at room temperature. After conjugation, Bio-Spin (Bio Rad) P-30 was buffered with water, and the remaining free NHS-Tag was removed. Dry in vacuo and concentrate to 50 uL. (MA) LDI-MS without matrix was confirmed that the tagging on the BSA.
표 1
Tag1 Tag2 Tag3 Tag4 비고
BSA 1nmole 7uL 7uL 7uL 7uL 10ug/uL stock in MES
MES 62.2uL 61.8uL 61.6uL 61.5uL 50mM, pH6
ACN 8uL 8uL 8uL 8uL 10% ACN
Tag 50nmoles 2.8uL 3.2uL 3.4uL 3.5uL 10ug/uL stock in DMSO
Total Vol. 80uL 80uL 80uL 80uL
Table 1
Tag1 Tag2 Tag3 Tag4 Remarks
BSA 1 nmole 7 uL 7 uL 7 uL 7 uL 10ug / uL stock in MES
MES 62.2 uL 61.8 uL 61.6 uL 61.5uL 50 mM, pH 6
ACN 8uL 8uL 8uL 8uL 10% ACN
Tag 50nmoles 2.8uL 3.2 uL 3.4 uL 3.5 uL 10ug / uL stock in DMSO
Total Vol. 80 uL 80 uL 80 uL 80 uL
상기 표에서 tag1: 3f, tag2: 6a, tag3: 6b, tag4: 6c를 의미함.In the table, tag1: 3f, tag2: 6a, tag3: 6b, and tag4: 6c.
LDI-MS 검출- matrix 사용하지 않고 AB Scix 4800 기기와 ASTA micro focus 384 well plate를 사용하여 tag 이 컨쥬게이션이 되어 있는 가를 확인하였다. Without the LDI-MS detection matrix, the AB Scix 4800 instrument and ASTA micro focus 384 well plate were used to determine if the tag was conjugated.
Tag과 컨쥬게이션 단백질의 농도에 따른 검출 신호 변화 분석 Analysis of Detection Signal Change According to Concentration of Tag and Conjugated Protein
BSA에 Tag2와 Tag4를 conjugation한 후, 아래 테이블의 비율대로 섞어서 1uL 를 MALDI Palte에 올려 (MA)LDI-MS 측정하였다.After conjugation of Tag2 and Tag4 to BSA, 1uL was added to MALDI Palte by mixing according to the ratio of the table below and measured (MA) LDI-MS.
표 2
Tag2 : Tag4 1:1 10:1 1:10
Tag2 Tag4 Tag2 Tag4 Tag2 Tag4
m/z 435.29 491.37 435.27 491.35 435.25 491.32
Height 2149.64 683.92 2508.49 252.18 411.24 2454.64
Area 112.5 39.16 147.33 13.14 22.88 130.52
Ratio 2.87 11.21 5.7
TABLE 2
Tag2: Tag4 1: 1 10: 1 1:10
Tag2 Tag4 Tag2 Tag4 Tag2 Tag4
m / z 435.29 491.37 435.27 491.35 435.25 491.32
Height 2149.64 683.92 2508.49 252.18 411.24 2454.64
Area 112.5 39.16 147.33 13.14 22.88 130.52
Ratio 2.87 11.21 5.7
tag이 커쥬게이션 된 항원을 검출하는 항체-항원 시스템 조건 실험Antibody-antigen system condition experiment to detect antigens with conjugated tag
NC 막(membrane) 위에서 항체와 NHS-Tagging 된 항원 사이에서 항원항체 반응이 일어나고, Tagging된 항원이 (MA)LDI-MS에서 확인되는지 알아보고자 실험하였다.To examine whether antigen-antibody reactions occur between the antibody and NHS-tagged antigen on the NC membrane, and whether the tagged antigen is identified in (MA) LDI-MS.
항원검출 방법Antigen Detection Method
Leptin(항원)에 NHS-Tag2를 위와 같은 방법으로 Tagging하고, Vacuum으로 Dry 하여 25uL까지 농축하였다. 2% BSA, 0.2% Tween-20 으로 2배 희석하였다. Leptin 항체는 10mM PB, pH 7.4로 버퍼 교체 후 NC membrane(5mm x 5mm)위에 loading 하여 37℃에서 건조하고, Tag2가 Tagging된 Leptin을 NC membrane 위에 올렸다. 상온에서 30-60min 반응시키고, 남은 항원 용액을 뽑아내고, memebrane 위에서 50uL의 물로 3번 막 상에서 세척을 한 후, 37℃에서 건조시켰다. AB-Sciex 4800을 이용하여 matrix free 상태에서 LDI-MS 검출 실험을 수행하였다.Tagging NHS-Tag2 on leptin (antigen) in the same manner as above, dried by vacuum and concentrated to 25uL. Diluted 2-fold with 2% BSA, 0.2% Tween-20. Leptin antibody was loaded on NC membrane (5mm x 5mm) after replacing buffer with 10mM PB, pH 7.4, dried at 37 ℃, and tagptylated Leptin was placed on NC membrane. After reacting for 30-60 min at room temperature, the remaining antigen solution was extracted, washed three times with 50 uL of water on a memebrane, and then dried at 37 ° C. The LDI-MS detection experiment was performed in matrix free state using AB-Sciex 4800.

Claims (9)

  1. 하기 화학식 1의 광분해성 질량표지 물질;A photodegradable mass labeling material of Formula 1;
    [화학식 1][Formula 1]
    Figure PCTKR2015004242-appb-I000006
    Figure PCTKR2015004242-appb-I000006
    상기 화학식 1에서 R1 및 R2는 H, 알킬, 알케닐, 알카니일, 알콕시, 또는 아릴기이고, R3는 단백질 분자 또는 단백질 이외의 아민기와 반응할 수 있는 반응기임.In Formula 1, R 1 and R 2 are H, alkyl, alkenyl, alkynyl, alkoxy, or aryl groups, and R 3 is a reactor capable of reacting with protein molecules or amine groups other than proteins.
  2. 제1항에 있어서, 상기 반응기는 N-하이드록시숙신이미딜 에스터, N-하이드록시설포숙신이미딜 에스터, 벤조트리아졸-1-일옥실 에스터, 펜타할로벤질 에스터 및 4-니트로페닐 에스터로 이루어진 그룹으로부터 선택되는 것을 특징으로 하는 물질.The reactor of claim 1 wherein the reactor is N-hydroxysuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, benzotriazol-1-yloxyl ester, pentahalobenzyl ester, and 4-nitrophenyl ester Substances, characterized in that selected from the group consisting of.
  3. 제1항에 있어서, 상기 물질은 하기 화학식 2 내지 화학식 4의 중 하나의 화합물인 것을 특징으로 하는 물질;According to claim 1, wherein the material is a material, characterized in that the compound of any one of formulas (2) to (4);
    [화학식 2][Formula 2]
    Figure PCTKR2015004242-appb-I000007
    Figure PCTKR2015004242-appb-I000007
    [화학식 3][Formula 3]
    Figure PCTKR2015004242-appb-I000008
    Figure PCTKR2015004242-appb-I000008
    [화학식 4][Formula 4]
    Figure PCTKR2015004242-appb-I000009
    Figure PCTKR2015004242-appb-I000009
    상기 화학식에서 노란 원 부분은 질량변화기 부위이고, 상기 화학식에서 R1 및 R2는 H, 알킬, 알케닐, 알카니일, 알콕시, 또는 아릴기이고, R'는 N-하이드록시숙신이미딜 에스터, N-하이드록시설포숙신이미딜 에스터, 벤조트리아졸-1-일옥실 에스터, 펜타할로벤질 에스터 및 4-니트로페닐 에스터로 이루어진 그룹으로부터 선택되고, n은 0에서 20의 정수. The yellow circle in the formula is the mass changer moiety, R 1 and R 2 in the formula are H, alkyl, alkenyl, alkynyl, alkoxy, or aryl groups, and R 'is N-hydroxysuccinimidyl ester , N-hydroxysulfosuccinimidyl ester, benzotriazol-1-yloxyl ester, pentahalobenzyl ester and 4-nitrophenyl ester, n is an integer from 0 to 20.
  4. 제3항에 있어서, 상기 물질은 하기 화학식 5의 화합물인 것을 특징으로 하는 물질;The material of claim 3, wherein the material is a compound of Formula 5;
    [화학식 5][Formula 5]
    Figure PCTKR2015004242-appb-I000010
    Figure PCTKR2015004242-appb-I000010
    상기 화학식에서 n은 0 내지 20임.N in the formula is 0 to 20.
  5. 제1항 내지 제4항 중 어느 한 항의 물질과 컨쥬게이션된 항체 혹은 아민기를 포함한 물질.A substance comprising an antibody or amine group conjugated with the substance of any one of claims 1 to 4.
  6. 포획 항체가 처리된 말디(MALDI) 플레이트 또는 포획 항체를 결합시킬 수 있는 표면을 포함하는 말디(MALDI) 플레이트에 항원이 표함된 시료를 처리하여 항원과 포획항체간의 항원 항체 복합체를 생성하고, 상기 복합체에 제5항의 광분해성 질량표지 물질로 표지된 검출항체를 처리하여 검출항체가 검출하고자 하는 항원이 존재하는 경우에만 포획항체가 결합하지 않은 항원의 다른 부분에 결합되고, 상기 플레이트를 매트릭스 없이 레이저 이탈 이온화 비행시간형 질량분석법(laser desorption ionization time-of-flight mass spectrometry/LDI-TOF MS)을 수행하는 방법. A sample containing an antigen is treated on a Maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface capable of binding a capture antibody to generate an antigen-antibody complex between the antigen and the capture antibody, and the complex The detection antibody labeled with the photodegradable mass labeling substance of claim 5 is bound to another part of the antigen to which the detection antibody does not bind only when the antigen to be detected is present, and the plate is laser-deleted without a matrix. A method for performing laser desorption ionization time-of-flight mass spectrometry / LDI-TOF MS.
  7. 포획 항체가 처리된 말디(MALDI) 플레이트 또는 포획 항체를 결합시킬 수 있는 표면을 포함하는 말디(MALDI) 플레이트에 다른 종류의 항원이 포함된 시료를 처리하여 항원과 포획항체간의 선택적인 항원 항체 복합체를 생성하고, 상기 복합체에 상기 제5항의 광분해성 질량표지물질로 표지된 검출항체를 처리하여 검출항체가 검출하고자 하는 항원이 존재하는 경우에만 포획항체가 결합하지 않은 항원의 다른 부분에 결합되고, 상기 플레이트를 매트릭스 없이 레이저 이탈 이온화 비행시간형 질량분석법(laser desorption ionization time-of flight mass spectrometry/LDI-TOF MS)을 수행하여 다중 항원을 정량적으로 검출하는 방법.Samples containing different types of antigens are treated on a Maldi (MALDI) plate treated with a capture antibody or on a Maldi (MALDI) plate including a surface to which the capture antibody can be bound to form a selective antigen antibody complex between the antigen and the capture antibody. And the detection antibody labeled with the photodegradable mass labeling substance of claim 5 in the complex to bind to another part of the antigen to which the capture antibody does not bind only when the antigen to be detected is present. A method for quantitatively detecting multiple antigens by performing plate desorption ionization time-of flight mass spectrometry (LDI-TOF MS) without a matrix.
  8. 포획 항체가 처리된 말디(MALDI) 플레이트 또는 포획 항체를 결합시킬 수 있는 표면을 포함하는 말디(MALDI) 플레이트에 항원이 포함된 시료를 처리하여 항원과 포획항체간의 항원 항체 복합체를 생성하고, 상기 복합체에 제5항의 광분해성 질량표지 물질로 표지된 검출항체를 처리하여 검출항체가 검출하고자 하는 항원이 존재하는 경우에만 포획항체가 결합하지 않은 항원의 다른 부분에 결합되고, 상기 플레이트를 매트릭스 없이 레이저 이탈 이온화 비행시간형 질량분석법(laser desorption ionization time-of-flight mass spectrometry/LDI-TOF MS)을 수행하여 항원을 정량적으로 분석하는 방법.A sample containing an antigen is treated on a Maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface capable of binding a capture antibody to generate an antigen antibody complex between the antigen and the capture antibody, and the complex The detection antibody labeled with the photodegradable mass labeling substance of claim 5 is bound to another part of the antigen to which the detection antibody does not bind only when the antigen to be detected is present, and the plate is laser-deleted without a matrix. A method for quantitative analysis of antigen by performing laser desorption ionization time-of-flight mass spectrometry (LDI-TOF MS).
  9. 제6항 내지 제8항 어느 한 항에 있어서, 상기 포획 항체를 결합시킬 수 있는 표면은 나이트로셀루로스(NC) 막, PVDF 막, 또는 글래스 슬라이드인 것을 특징으로 하는 방법. The method of claim 6, wherein the surface capable of binding the capture antibody is a nitrocellulose (NC) membrane, a PVDF membrane, or a glass slide.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005320270A (en) * 2004-05-07 2005-11-17 Tum-Gene Inc New electrochemical response compound, method for producing the compound and method for detecting bond between target substance and probe molecule using the same
JP2012041317A (en) * 2010-08-23 2012-03-01 Panasonic Corp Ferrocene modified antibody

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100534190B1 (en) * 1996-10-07 2005-12-08 신젠타 파티서페이션즈 아게 Chiral ferrocenyls

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005320270A (en) * 2004-05-07 2005-11-17 Tum-Gene Inc New electrochemical response compound, method for producing the compound and method for detecting bond between target substance and probe molecule using the same
JP2012041317A (en) * 2010-08-23 2012-03-01 Panasonic Corp Ferrocene modified antibody

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FREIESLEBEN, D ET AL.: "Metal Complexes of biologically important ligands. CLXVI[1] Metal complexes with ferrocenylmethylcycteinate and 1, 1'-ferrocenylbis (methylcycteinate) as ligands.", Z. ANORG. ALLEG. CHEM., vol. 633, 2007, pages 1100 - 1106 *
SCUTARU, D. ET AL.: "Monosubstituted derivatives of ferrocene. Ferrocene-containing penicillins and cephalosporins.", JOURNAL OF ORGANOMETALLIC CHEMISTRY, vol. 401, no. 1-2, 1991, pages 81 - 85, XP055235084, ISSN: 0022-328x *

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