CN117736171A - Preparation method of AF488TSA - Google Patents
Preparation method of AF488TSA Download PDFInfo
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- CN117736171A CN117736171A CN202410155739.3A CN202410155739A CN117736171A CN 117736171 A CN117736171 A CN 117736171A CN 202410155739 A CN202410155739 A CN 202410155739A CN 117736171 A CN117736171 A CN 117736171A
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- Prior art keywords
- af488tsa
- solvent
- base
- preparation
- diphenylphosphine
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- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 24
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical compound OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 11
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 10
- 125000001834 xanthenyl group Chemical group C1=CC=CC=2OC3=CC=CC=C3C(C12)* 0.000 claims abstract description 9
- 150000001408 amides Chemical class 0.000 claims abstract description 8
- 239000000376 reactant Substances 0.000 claims abstract description 7
- 238000006277 sulfonation reaction Methods 0.000 claims abstract description 7
- 230000018044 dehydration Effects 0.000 claims abstract description 6
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 42
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 39
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 38
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 27
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- 239000002585 base Substances 0.000 claims description 27
- -1 bicyclic amidine Chemical class 0.000 claims description 27
- 239000002904 solvent Substances 0.000 claims description 27
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 19
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- DZGWFCGJZKJUFP-UHFFFAOYSA-N Tyramine Natural products NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 claims description 15
- 238000002156 mixing Methods 0.000 claims description 15
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 14
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 claims description 12
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 12
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-diisopropylethylamine Substances CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 10
- 125000003277 amino group Chemical group 0.000 claims description 10
- 229910052763 palladium Inorganic materials 0.000 claims description 10
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 9
- 239000003054 catalyst Substances 0.000 claims description 9
- MZRVEZGGRBJDDB-UHFFFAOYSA-N N-Butyllithium Chemical compound [Li]CCCC MZRVEZGGRBJDDB-UHFFFAOYSA-N 0.000 claims description 8
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 8
- 239000003446 ligand Substances 0.000 claims description 8
- WGOPGODQLGJZGL-UHFFFAOYSA-N lithium;butane Chemical compound [Li+].CC[CH-]C WGOPGODQLGJZGL-UHFFFAOYSA-N 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 229960003732 tyramine Drugs 0.000 claims description 8
- 230000008878 coupling Effects 0.000 claims description 7
- 238000010168 coupling process Methods 0.000 claims description 7
- 238000005859 coupling reaction Methods 0.000 claims description 7
- 229910000073 phosphorus hydride Inorganic materials 0.000 claims description 7
- XJHCXCQVJFPJIK-UHFFFAOYSA-M caesium fluoride Chemical compound [F-].[Cs+] XJHCXCQVJFPJIK-UHFFFAOYSA-M 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- GPAYUJZHTULNBE-UHFFFAOYSA-N diphenylphosphine Chemical compound C=1C=CC=CC=1PC1=CC=CC=C1 GPAYUJZHTULNBE-UHFFFAOYSA-N 0.000 claims description 6
- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 claims description 6
- 239000011261 inert gas Substances 0.000 claims description 6
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 claims description 6
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 6
- LPNYRYFBWFDTMA-UHFFFAOYSA-N potassium tert-butoxide Chemical compound [K+].CC(C)(C)[O-] LPNYRYFBWFDTMA-UHFFFAOYSA-N 0.000 claims description 6
- MFRIHAYPQRLWNB-UHFFFAOYSA-N sodium tert-butoxide Chemical compound [Na+].CC(C)(C)[O-] MFRIHAYPQRLWNB-UHFFFAOYSA-N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 claims description 4
- 229910000024 caesium carbonate Inorganic materials 0.000 claims description 4
- LNAMMBFJMYMQTO-FNEBRGMMSA-N chloroform;(1e,4e)-1,5-diphenylpenta-1,4-dien-3-one;palladium Chemical compound [Pd].[Pd].ClC(Cl)Cl.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1 LNAMMBFJMYMQTO-FNEBRGMMSA-N 0.000 claims description 4
- 238000009833 condensation Methods 0.000 claims description 4
- 230000005494 condensation Effects 0.000 claims description 4
- SXZIXHOMFPUIRK-UHFFFAOYSA-N diphenylmethanimine Chemical compound C=1C=CC=CC=1C(=N)C1=CC=CC=C1 SXZIXHOMFPUIRK-UHFFFAOYSA-N 0.000 claims description 4
- UBJFKNSINUCEAL-UHFFFAOYSA-N lithium;2-methylpropane Chemical compound [Li+].C[C-](C)C UBJFKNSINUCEAL-UHFFFAOYSA-N 0.000 claims description 4
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 4
- PWNHMYPRQAUDMJ-UHFFFAOYSA-N (2,3-dimethylphenyl)-phenylphosphane Chemical compound CC1=CC=CC(PC=2C=CC=CC=2)=C1C PWNHMYPRQAUDMJ-UHFFFAOYSA-N 0.000 claims description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 125000003963 dichloro group Chemical group Cl* 0.000 claims description 3
- LVKCSZQWLOVUGB-UHFFFAOYSA-M magnesium;propane;bromide Chemical compound [Mg+2].[Br-].C[CH-]C LVKCSZQWLOVUGB-UHFFFAOYSA-M 0.000 claims description 3
- 230000003472 neutralizing effect Effects 0.000 claims description 3
- PIBWKRNGBLPSSY-UHFFFAOYSA-L palladium(II) chloride Chemical compound Cl[Pd]Cl PIBWKRNGBLPSSY-UHFFFAOYSA-L 0.000 claims description 3
- YJVFFLUZDVXJQI-UHFFFAOYSA-L palladium(ii) acetate Chemical compound [Pd+2].CC([O-])=O.CC([O-])=O YJVFFLUZDVXJQI-UHFFFAOYSA-L 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 3
- 235000011009 potassium phosphates Nutrition 0.000 claims description 3
- PNGLEYLFMHGIQO-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical compound O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 PNGLEYLFMHGIQO-UHFFFAOYSA-M 0.000 claims description 3
- 239000008096 xylene Substances 0.000 claims description 3
- 239000003513 alkali Substances 0.000 claims description 2
- 150000008064 anhydrides Chemical class 0.000 claims description 2
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 claims 1
- DZGWFCGJZKJUFP-UHFFFAOYSA-O tyraminium Chemical compound [NH3+]CCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-O 0.000 claims 1
- 239000007850 fluorescent dye Substances 0.000 abstract description 11
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 abstract description 7
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 abstract description 7
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 4
- RWCCWEUUXYIKHB-UHFFFAOYSA-N benzophenone Chemical compound C=1C=CC=CC=1C(=O)C1=CC=CC=C1 RWCCWEUUXYIKHB-UHFFFAOYSA-N 0.000 abstract description 2
- 239000012965 benzophenone Substances 0.000 abstract 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 8
- 239000000243 solution Substances 0.000 description 6
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000012065 filter cake Substances 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- WJKHJLXJJJATHN-UHFFFAOYSA-N triflic anhydride Chemical compound FC(F)(F)S(=O)(=O)OS(=O)(=O)C(F)(F)F WJKHJLXJJJATHN-UHFFFAOYSA-N 0.000 description 4
- 230000003321 amplification Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XPDWGBQVDMORPB-UHFFFAOYSA-N Fluoroform Chemical compound FC(F)F XPDWGBQVDMORPB-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 2
- 229940125782 compound 2 Drugs 0.000 description 2
- 229940126214 compound 3 Drugs 0.000 description 2
- 229940125898 compound 5 Drugs 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- HIFJUMGIHIZEPX-UHFFFAOYSA-N sulfuric acid;sulfur trioxide Chemical compound O=S(=O)=O.OS(O)(=O)=O HIFJUMGIHIZEPX-UHFFFAOYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000012317 TBTU Substances 0.000 description 1
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229960000956 coumarin Drugs 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000004066 metabolic change Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000012038 nucleophile Substances 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 125000001889 triflyl group Chemical group FC(F)(F)S(*)(=O)=O 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
Landscapes
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)
Abstract
The invention discloses a preparation method of AF488TSA, which relates to the technical field of fluorescent probe preparation, wherein 2, 4-tetrahydroxy benzophenone is used as a reactant, a xanthene ring is constructed by dehydration and ether formation, activated hydroxyl is introduced into amino and coupled and connected with phthalic acid, amide is condensed, and the AF488TSA is obtained after sulfonation; wherein the structural formula of AF488TSA is. The preparation method provided by the invention avoids the use of expensive rhodamine, fluorescein or derivatives thereof, and can realize gram-grade preparation of AF488TSA from commercially available low-cost raw materials 2, 4-tetrahydroxybenzophenone, and can amplify synthesis.
Description
Technical Field
The invention relates to the technical field of fluorescent probe preparation, in particular to a preparation method of AF488TSA.
Background
Fluorescent probes (Fluorescent Probes) are an important tool in biochemical research and are a technique that can be used to detect and track certain substances in living cells. Fluorescent probes are used in a wide variety of applications, and can be used to detect and analyze a variety of substances in living cells, including proteins, sugars, amino acids, nucleic acids, lipids, and the like. It can also be used to detect intracellular activities such as apoptosis, proliferation, differentiation, migration, etc., and can further use fluorescent probes to study metabolic changes in living cells and the response of cells to external stimuli.
In 1999, molecular Probes reported a series of fluorochromes prepared from coumarin and rhodamine-modified derivatives (Journal of Histochemistry & Cytochemistry, 1999, 47 (9): 1179-1188), specifically as shown by Alexa350-Alexa 594:
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the fluorescent dyes have the characteristics of good sensitivity, specificity, light stability and the like, can be combined with antibodies, generate strong fluorescent signals in cells and tissues, and are used for detecting and positioning biomolecules such as proteins, nucleic acids and the like.
In 2011, jonathan B et al reported a series of rhodamine derivatives (org. Lett. 2011, 13, 24, 6354-6357) that were directly obtained by coupling fluorescein diacylate with nucleophiles such as amines, amides, etc., as shown in the following formula:
。
most of the synthesis methods of the fluorescent probes or the fluorescent dyes with the same skeleton fluorescent groups are based on expensive fluorescein, rhodamine or derivatives thereof, so that the natural products are modified, and the cost is high; in addition, fuming sulfuric acid and a complex post-treatment means are required in the sulfonation process, and the synthesis is difficult to amplify.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a method for preparing AF488TSA, which aims to solve the problems of high cost and expensive fluorescent yellow, rhodamine or derivatives thereof as reactants in the preparation of the existing fluorescent probes.
The technical scheme of the invention is as follows:
the invention provides a preparation method of AF488TSA, wherein 2, 4-tetrahydroxy diphenyl ketone is used as a reactant, a xanthene ring is constructed by dehydration and ether formation, an activated hydroxyl group is introduced into an amino group and coupled with phthalic acid, amide condensation and sulfonation are carried out, and AF488TSA is obtained;
wherein the structural formula of AF488TSA is；
The step of dehydrating into ether to construct a xanthene ring specifically comprises the following steps:
mixing 2, 4-tetrahydroxybenzophenone and water, and reacting at 100-200 ℃ for 3-24 h to obtain；
The steps of introducing an amine group into the activated hydroxyl group and coupling the attached phthalic acid specifically comprise:
under the condition of-20 to 0 ℃ and inert gas protection, the catalyst is preparedAfter being mixed with the first solvent, the trifluoromethane is added dropwise under the condition of stirringThe sulfonic anhydride is then added with the first alkali dropwise, and the +.>；
Will beMixing with a second solvent, adding a palladium catalyst, a phosphine ligand, a second base and benzophenone imine under the protection of inert gas, and reacting at 60-120 ℃ to obtain；
Mixing 4-bromo-1, 3-phthalic acid with a third solvent, adding a third base at-78 to-30 ℃ with stirring, and then addingAfter the reaction, trifluoroacetic acid and water are added, and after the reaction again, the +.>；
The amide condensation step specifically comprises:
will beMixing tyramine, fourth base and fourth solvent, adding condensing agent at 0deg.C, and reacting to obtain +.>；
The sulfonation step specifically comprises the following steps:
at a temperature of 0 DEG CAdding concentrated sulfuric acid, reacting, adding water and triethylamine under stirring at-20 ℃, and neutralizing the pH of the system to 6-8 to obtain the AF488TSA.
Optionally, the first solvent comprises at least one of dichloromethane, acetonitrile, 1, 4-dioxane, tetrahydrofuran, toluene, benzene, acetone;
the first base comprises at least one of pyridine, triethylamine, N-diisopropylethylamine and bicyclic amidine.
Optionally, the second solvent comprises at least one of toluene, dioxane, benzene, tetrahydrofuran, N-dimethylformamide;
the second base comprises at least one of cesium carbonate, potassium tert-butoxide, potassium phosphate, sodium tert-butoxide, sodium methoxide, triethylamine, cesium fluoride and potassium carbonate.
Optionally, the palladium catalyst comprises at least one of tris (dibenzylideneacetone) dipalladium-chloroform complex, tetrakis (triphenylphosphine) palladium, dichloro [1,1' -bis (diphenylphosphine) ferrocene ] palladium, palladium acetate, palladium dichloride;
the phosphine ligand comprises at least one of 4, 5-bis-diphenylphosphine-9, 9-dimethyl xanthene, triphenylphosphine, dimethyl diphenylphosphine, 1' -bis (diphenylphosphine) ferrocene and 1,1' -binaphthyl-2, 2' -bis-diphenylphosphine.
Optionally, the third solvent comprises at least one of tetrahydrofuran, 1, 4-dioxane, dichloromethane, toluene, xylene;
the third base comprises at least one of sec-butyllithium, n-butyllithium, tert-butyllithium and isopropyl magnesium bromide.
Optionally, the fourth base comprises at least one of N, N-diisopropylethylamine, triethylamine, dicycloamidine, potassium tert-butoxide, sodium methoxide, potassium carbonate;
the fourth solvent comprises at least one of N, N-dimethylformamide, toluene, benzene and tetrahydrofuran;
the condensing agent comprises at least one of 1H-benzotriazole-1-yloxy tripyrrolidinyl hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1, 3-tetramethylurea hexafluorophosphate, O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea tetrafluoroborate, N, N, N ', N' -tetramethyl-O- (N-succinimidyl) urea tetrafluoroborate, hexafluorophosphoric acid (7-azabenzotriazole-1-oxy) tripyrrolidinyl phosphate.
The beneficial effects are that: the invention starts from commercially available 2, 4-tetrahydroxybenzophenone, and the target product AF488TSA is obtained after the steps of constructing a xanthene ring by dehydration and ether formation, introducing an amino group into an activated hydroxyl group, coupling with phthalic acid, condensing the amino group and sulfonating the amino group. The preparation method provided by the invention avoids the use of expensive rhodamine, fluorescein or derivatives thereof, and can realize gram-grade preparation of AF488TSA from commercially available low-cost raw materials 2, 4-tetrahydroxybenzophenone, and can amplify synthesis.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of AF488TSA prepared in example 1 of the present invention.
Detailed Description
The invention provides a preparation method of AF488TSA, which is used for making the purposes, technical schemes and effects of the invention clearer and more definite, and is further described in detail below. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
AF488TSA is a fluorescent dye based on AF488, and Tyramine Signal Amplification (TSA) is a particularly universal and powerful enzyme amplification technology with higher detection sensitivity. The principle of TSA is that horseradish peroxidase (HRP) catalyzes hydroxyl free radicals generated by hydrogen peroxide, phenol groups of tyrosine residues of tyramine and protein can be converted into free radical intermediates, the free radical intermediates are mutually and covalently combined, and tyramine is mutually crosslinked or connected with protein, so that signal amplification can be caused after the tyramine is coupled with signal substances such as fluorescein and the like to trigger phenol polymerization reaction. Therefore, the embodiment of the invention provides a preparation method of AF488TSA, wherein 2, 4-tetrahydroxybenzophenone is used as a reactant, a xanthene ring is constructed by dehydration and ether formation, activated hydroxyl is introduced into amino and coupled and connected with phthalic acid, amide is condensed, and the AF488TSA is obtained after sulfonation;
wherein the structural formula of AF488TSA is。
The invention starts from commercially available 2, 4-tetrahydroxybenzophenone, and the target product AF488TSA is obtained after the steps of constructing a xanthene ring by dehydration and ether formation, introducing an amino group into an activated hydroxyl group, coupling with phthalic acid, condensing the amino group and sulfonating the amino group. The preparation method provided by the invention avoids the use of expensive rhodamine and fluorescein or derivatives thereof, and can realize gram-grade preparation of AF488TSA from commercially available low-cost raw materials 2, 4-tetrahydroxybenzophenone, and can amplify synthesis.
In some embodiments, the step of dehydrating to ether to build a xanthene ring specifically comprises:
mixing 2, 4-tetrahydroxybenzophenone and water, and reacting at 100-200deg.C (such as 100deg.C, 120deg.C, 140deg.C, 150deg.C, 160deg.C, 180deg.C or 200deg.C) for 3-24 hr (such as 3 hr, 5 hr, 10 hr, 15 hr, 20 hr or 24 hr)。
In some embodiments, the step of activating the hydroxyl group to introduce an amine group and coupling the attached phthalic acid specifically comprises:
s11 is prepared by reacting at-20-0deg.C (e.g., -20deg.C, -15deg.C, -10deg.C, -5deg.C or 0deg.C) with inert gas (such as nitrogen gas)Mixing with the first solvent, dropwise adding trifluoromethanesulfonic anhydride under stirring, then dropwise adding the first base, and reacting to obtain +.>(Tf has the same meaning as is well known to those skilled in the art and is trifluoromethanesulfonyl)A base);
s12, willMixing with a second solvent, adding palladium catalyst, phosphine ligand, second base and benzophenone imine under the protection of inert gas (such as nitrogen, etc.), and reacting at 60-120deg.C (such as 60deg.C, 70deg.C, 80deg.C, 90deg.C, 100deg.C, 110deg.C or 120deg.C, etc.), to obtain ∈>(Ph is phenyl, as is well known to those skilled in the art);
s13, mixing 4-bromo-1, 3-phthalic acid and a third solvent, adding a third base (specifically, adding the third base in a solution form, for example, mixing the third base with tetrahydrofuran, and then adding the mixture under stirring at-78 to-30 ℃ (for example, -78 ℃, -70 ℃, -60 ℃, -50 ℃, -40 ℃ or-30 ℃), etc.), and then adding the mixture(specifically, it can be added in the form of a solution, for example, it is mixed with tetrahydrofuran and then added), then trifluoroacetic acid and water are added, and after the reaction again, the +.>。
In step S11, in some embodiments, the first solvent includes at least one of dichloromethane, acetonitrile, 1, 4-dioxane, tetrahydrofuran, toluene, benzene, and acetone, but is not limited thereto.
In some embodiments, the first base includes at least one of pyridine, triethylamine, N-diisopropylethylamine, and bicyclic amidine (DBU), but is not limited thereto.
In step S12, in some embodiments, the second solvent includes at least one of toluene, dioxane, benzene, tetrahydrofuran, N-dimethylformamide, but is not limited thereto.
In some embodiments, the palladium catalyst includes at least one of tris (dibenzylideneacetone) dipalladium-chloroform complex, tetrakis (triphenylphosphine) palladium, dichloro [1,1' -bis (diphenylphosphine) ferrocene ] palladium, palladium acetate, palladium dichloride, but is not limited thereto.
In some embodiments, the phosphine ligand includes at least one of 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene, triphenylphosphine, dimethyldiphenylphosphine, 1' -bis (diphenylphosphine) ferrocene, 1' -binaphthyl-2, 2' -bis-diphenylphosphine, but is not limited thereto. The phosphine ligand is used as a ligand matched with a palladium catalyst.
In some embodiments, the second base includes at least one of cesium carbonate, potassium t-butoxide, potassium phosphate, sodium t-butoxide, sodium methoxide, triethylamine, cesium fluoride, potassium carbonate, but is not limited thereto. The second base may also be a non-nucleophilic, highly sterically hindered organic base.
In step S13, in some embodiments, the third solvent includes at least one of tetrahydrofuran, 1, 4-dioxane, dichloromethane, toluene, and xylene, but is not limited thereto.
In some embodiments, the third base includes at least one of sec-butyllithium, n-butyllithium, tert-butyllithium, and isopropylmagnesium bromide, but is not limited thereto. When the third base is sec-butyllithium, n-butyllithium or tert-butyllithium, the terephthalic acid is attached by lithium halide exchange coupling.
In some embodiments, the step of amide condensation specifically comprises:
will beMixing tyramine, fourth base and fourth solvent, adding condensing agent at 0deg.C, and reacting to obtain +.>。
In some embodiments, the fourth base includes at least one of N, N-diisopropylethylamine, triethylamine, bicyclic amidine, potassium tert-butoxide, sodium methoxide, potassium carbonate, but is not limited thereto.
In some embodiments, the fourth solvent includes at least one of N, N-Dimethylformamide (DMF), toluene, benzene, tetrahydrofuran, but is not limited thereto.
In some embodiments, the condensing agent comprises 1H-benzotriazol-1-yloxy tripyrrolidinyl hexafluorophosphate, 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurone Hexafluorophosphate (HATU), benzotriazol-N, N, N ', N' -tetramethylurone Hexafluorophosphate (HBTU), 6-chlorobenzotriazol-1, 3-tetramethylurone Hexafluorophosphate (HCTU), O- (benzotriazol-1-yl) -N, at least one of N, N '-tetramethyl urea tetrafluoroborate (TBTU), N' -tetramethyl-O- (N-succinimidyl) urea tetrafluoroborate (TSTU), hexafluorophosphoric acid (7-azabenzotriazole-1-oxy) tripyrrolidine phosphate (PyAOP), but not limited thereto.
In some embodiments, the step of sulfonating specifically comprises:
at a temperature of 0 DEG CAdding concentrated sulfuric acid, reacting, adding water and triethylamine under stirring at-20 ℃, and neutralizing the pH of the system to 6-8 to obtain the AF488TSA.
The following is a detailed description of specific examples.
Example 1
The synthetic route for AF488TSA is as follows:
。
according to the above synthetic route, the preparation method of AF488TSA comprises the following steps:
step 1, adding the compound 1 (6.34 g,25.75 mmol) and deionized water (76.00 mL) into a polytetrafluoroethylene high-pressure reaction tank, sealing a pipe, reacting at a high temperature of 200 ℃ for 6 hours, filtering the product, washing a filter cake with deionized water, and drying at 60 ℃ to obtain a crude product compound 2, LRMS (ESI) m/z, calcd for C 13 H 8 O 4 [M+H] + 229.04; found,29.13 (LRMS means low resolution mass spectrometry, ESI means electrospray ion)The source, calculated, found, experimental, and the like) was used directly in the next step without purification.
Step 2, mixing Compound 2 (2.9 g,12.40 mmol) with dry dichloromethane (62.00 mL) under nitrogen protection at 0deg.C, dropwise adding trifluoromethanesulfonic anhydride (6.26 mL,37.20 mmol) under stirring, stirring for ten minutes, slowly dropwise adding pyridine (9.98 mL,124.00 mmol), stirring for one hour, slowly cooling to room temperature, monitoring the reaction by LC-MS (liquid chromatography-mass spectrometry), quenching with water at 0deg.C, recovering room temperature, separating liquid extraction, washing the organic phase with 1M HCl solution, washing with saturated saline, drying with anhydrous sodium sulfate, spin-drying solvent, purifying by silica gel column chromatography to obtain 5.00g pure compound 3, LRMS (ESI) M/z, calcd for C 15 H 6 F 6 O 8 S 2 [M+H] + ，492.94；found，492.99。
Step 3, compound 3 (1.00 g,2.0 mmol) and dried toluene (30 mL) were added to a three-necked flask, and tris (dibenzylideneacetone) dipalladium-chloroform complex (0.3110 g,0.0300 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (0.2310 g,0.4380 mmol), cesium carbonate (2.0 g,6.10 mmol) and benzophenone imine (1.00 mL,6.00 mmol) were sequentially added under nitrogen protection, the reaction was monitored at 100℃overnight under nitrogen protection, after the completion of the reaction, filtration was performed, the filtrate was diluted with dichloromethane, washed with water, dried over anhydrous sodium sulfate, the spin-dried solvent was purified by silica gel column chromatography to give 1.24g of pure Compound 4, LRMS (ESI) m/z, calcd for C 39 H 26 N 2 O 2 [M+H] + ，555.20；found，555.32。
Step 4, 4-bromo-1, 3-phthalic acid (6.68 g,27.26 mmol) and dried tetrahydrofuran (500 mL) were added to a three-necked flask, and a solution of sec-butyllithium in tetrahydrofuran (59.85 mL, sec-butyllithium 77.81mmol, sec-butyllithium concentration 1.3M) was slowly added dropwise with stirring at-78 ℃; compound 4 (2.16 g,3.89mmol,2.16g of Compound 4 can be obtained by repeating step 3 a plurality of times) was previously charged in another flaskObtained, or obtained by amplifying each reactant in step 3) and tetrahydrofuran (100 mL) in equal proportions to obtain a tetrahydrofuran solution of compound 4, which is maintained in an anhydrous and anaerobic state throughout; stirring for 1h after sec-butyllithium addition, slowly adding tetrahydrofuran solution of compound 4, allowing reaction at room temperature, LC-MS monitoring, quenching with deionized water at-30deg.C, spin drying solvent, adding trifluoroacetic acid (20 mL) and deionized water (5 mL), reacting overnight, adding cold diethyl ether, filtering, washing filter cake with small amount of acetonitrile and dichloromethane, washing filter cake with diethyl ether, oven drying filter cake at 30deg.C to obtain 1.38g of compound 5, LRMS (ESI) m/z: calcd for C 21 H 15 N 2 O 5 [M] + ，375.10；found，375.31。
Step 5, adding Compound 5 (0.5 g,1.33 mmol), tyramine (0.19 g,1.40 mmol), N-diisopropylethylamine (0.93 mL,5.32 mmol), DMF (1 mL) to a flask, stirring well, adding 1H-benzotriazol-1-yloxytripyrrolidine hexafluorophosphate (0.69 g,1.33 mmol) at 0deg.C, returning to room temperature for reaction, LC-MS monitoring the reaction, after the reaction, silica gel column chromatography gives 0.41g of Compound 6, LRMS (ESI) m/z calcd for C 29 H 24 N 3 O 5 [M] + ，494.17；found，494.25。
Step 6, compound 6 (1.10 g,2.23mmol,1.10g of compound 6 can be obtained by repeating step 5 a plurality of times, or by amplifying each reactant in step 5 in equal proportion) is added into a flask, concentrated sulfuric acid (the mass content of sulfuric acid is 98%,1.5 mL) is added at the temperature of 0 ℃ and stirred for 1h, the reaction is slowly restored to room temperature, the reaction is carried out overnight, LC-MS monitoring is carried out after sampling and dilution, 20mL of water and 3-8 mL of triethylamine (the amount of triethylamine is 5mL in the embodiment) are added at the temperature of-20 ℃ and under the stirring condition after the reaction is finished, the mixture is neutralized to pH of 6-8, a C-18 silica gel column is separated and purified, an eluent is acetonitrile aqueous solution (containing 0.1% formic acid and the volume content) with the volume percentage concentration of acetonitrile of 50% -90%, and AF488TSA (0.70 g, the yield is 80%) is obtained after the purification. The nuclear magnetic resonance hydrogen spectrum is shown in figure 1. Its high scoreMass spectrum data is identified as HRMS (ESI) m/z, calculated for C 29 H 23 N 3 O 11 S 2 [M+H] + ，654.08；found，654.11。
In summary, the invention provides a preparation method of AF488TSA, which reduces the use of fuming sulfuric acid, avoids the use of expensive rhodamine, fluorescein or derivatives thereof, realizes gram-grade preparation of AF488TSA from commercially available and low-cost raw materials 2, 4-tetrahydroxybenzophenone, and can amplify synthesis.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (6)
1. A preparation method of AF488TSA is characterized in that 2, 4-tetrahydroxybenzophenone is used as a reactant, a xanthene ring is constructed by dehydration and ether formation, activated hydroxyl is introduced into amino and coupled connection phthalic acid, amide is condensed, and the AF488TSA is obtained after sulfonation;
wherein the structural formula of AF488TSA is；
The step of dehydrating into ether to construct a xanthene ring specifically comprises the following steps:
mixing 2, 4-tetrahydroxybenzophenone and water, and reacting at 100-200 ℃ for 3-24 h to obtain；
The steps of introducing an amine group into the activated hydroxyl group and coupling the attached phthalic acid specifically comprise:
under the condition of-20 to 0 ℃ and inert gas protection, the catalyst is preparedAfter being mixed with the first solvent, the trifluoromethanesulfonic acid is added dropwise under the condition of stirringAcid anhydride, then first alkali is added dropwise, and after reaction, the +.>；
Will beMixing with a second solvent, adding a palladium catalyst, a phosphine ligand, a second base and benzophenone imine under the protection of inert gas, and reacting at 60-120 ℃ to obtain +.>；
Mixing 4-bromo-1, 3-phthalic acid with a third solvent, adding a third base at-78 to-30 ℃ with stirring, and then addingAfter the reaction, trifluoroacetic acid and water are added, and after the reaction again, the product is obtained；
The amide condensation step specifically comprises:
will beMixing tyramine, fourth base and fourth solvent, adding condensing agent at 0deg.C, and reacting to obtain +.>；
The sulfonation step specifically comprises the following steps:
at a temperature of 0 DEG CAdding concentrated sulfuric acid, reacting, adding water and triethylamine under stirring at-20 ℃, and neutralizing the pH of the system to 6-8 to obtain the AF488TSA.
2. The method according to claim 1, wherein the first solvent comprises at least one of dichloromethane, acetonitrile, 1, 4-dioxane, tetrahydrofuran, toluene, benzene, and acetone;
the first base comprises at least one of pyridine, triethylamine, N-diisopropylethylamine and bicyclic amidine.
3. The method according to claim 1, wherein the second solvent comprises at least one of toluene, dioxane, benzene, tetrahydrofuran, N-dimethylformamide;
the second base comprises at least one of cesium carbonate, potassium tert-butoxide, potassium phosphate, sodium tert-butoxide, sodium methoxide, triethylamine, cesium fluoride and potassium carbonate.
4. The method of preparation of claim 1, wherein the palladium catalyst comprises at least one of tris (dibenzylideneacetone) dipalladium-chloroform complex, tetrakis (triphenylphosphine) palladium, dichloro [1,1' -bis (diphenylphosphine) ferrocene ] palladium, palladium acetate, palladium dichloride;
the phosphine ligand comprises at least one of 4, 5-bis-diphenylphosphine-9, 9-dimethyl xanthene, triphenylphosphine, dimethyl diphenylphosphine, 1' -bis (diphenylphosphine) ferrocene and 1,1' -binaphthyl-2, 2' -bis-diphenylphosphine.
5. The method according to claim 1, wherein the third solvent comprises at least one of tetrahydrofuran, 1, 4-dioxane, dichloromethane, toluene, and xylene;
the third base comprises at least one of sec-butyllithium, n-butyllithium, tert-butyllithium and isopropyl magnesium bromide.
6. The method according to claim 1, wherein the fourth base comprises at least one of N, N-diisopropylethylamine, triethylamine, dicycloamidine, potassium tert-butoxide, sodium methoxide, potassium carbonate;
the fourth solvent comprises at least one of N, N-dimethylformamide, toluene, benzene and tetrahydrofuran;
the condensing agent comprises at least one of 1H-benzotriazole-1-yloxy tripyrrolidinyl hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1, 3-tetramethylurea hexafluorophosphate, O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea tetrafluoroborate, N, N, N ', N' -tetramethyl-O- (N-succinimidyl) urea tetrafluoroborate, hexafluorophosphoric acid (7-azabenzotriazole-1-oxy) tripyrrolidinyl phosphate.
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