CN115490674A - 选择性检测同型半胱氨酸的荧光探针的合成及应用 - Google Patents
选择性检测同型半胱氨酸的荧光探针的合成及应用 Download PDFInfo
- Publication number
- CN115490674A CN115490674A CN202211195448.4A CN202211195448A CN115490674A CN 115490674 A CN115490674 A CN 115490674A CN 202211195448 A CN202211195448 A CN 202211195448A CN 115490674 A CN115490674 A CN 115490674A
- Authority
- CN
- China
- Prior art keywords
- hcy
- fluorescent probe
- substituted
- serum
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 title claims abstract description 71
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 36
- 230000015572 biosynthetic process Effects 0.000 title claims description 20
- 238000003786 synthesis reaction Methods 0.000 title claims description 20
- 239000000523 sample Substances 0.000 claims abstract description 30
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 210000002966 serum Anatomy 0.000 claims abstract description 20
- 238000000799 fluorescence microscopy Methods 0.000 claims abstract description 8
- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical compound C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 claims abstract description 8
- 125000000547 substituted alkyl group Chemical group 0.000 claims abstract description 8
- 239000000126 substance Substances 0.000 claims abstract description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 22
- -1 nitro, hydroxyl Chemical group 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 10
- DEQMCKPKPIHOJO-UHFFFAOYSA-N CCCCSC(C(C(O1)=C2)=CC([N+]([O-])=O)=C2N(CC)CC)=C(C=O)C1=O Chemical compound CCCCSC(C(C(O1)=C2)=CC([N+]([O-])=O)=C2N(CC)CC)=C(C=O)C1=O DEQMCKPKPIHOJO-UHFFFAOYSA-N 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- JPXXIBZATGUHGO-UHFFFAOYSA-N CCN(CC)C(C=C(C(C(Cl)=C1C=O)=C2)OC1=O)=C2[N+]([O-])=O Chemical compound CCN(CC)C(C=C(C(C(Cl)=C1C=O)=C2)OC1=O)=C2[N+]([O-])=O JPXXIBZATGUHGO-UHFFFAOYSA-N 0.000 claims description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000012472 biological sample Substances 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 5
- WQAQPCDUOCURKW-UHFFFAOYSA-N butanethiol Chemical compound CCCCS WQAQPCDUOCURKW-UHFFFAOYSA-N 0.000 claims description 4
- 239000012065 filter cake Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000011002 quantification Methods 0.000 claims description 4
- 125000001424 substituent group Chemical group 0.000 claims description 4
- 238000002835 absorbance Methods 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- CYXXVTLWYIGJJW-UHFFFAOYSA-N 4-chloro-7-(diethylamino)-2-oxochromene-3-carbaldehyde Chemical compound ClC1=C(C=O)C(=O)OC2=CC(N(CC)CC)=CC=C21 CYXXVTLWYIGJJW-UHFFFAOYSA-N 0.000 claims description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 2
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims description 2
- 239000007795 chemical reaction product Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000005457 ice water Substances 0.000 claims description 2
- 229910017604 nitric acid Inorganic materials 0.000 claims description 2
- 239000012265 solid product Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims 2
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims 2
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 claims 2
- 125000003545 alkoxy group Chemical group 0.000 claims 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims 2
- 229910052794 bromium Inorganic materials 0.000 claims 2
- 125000004432 carbon atom Chemical group C* 0.000 claims 2
- 229910052801 chlorine Inorganic materials 0.000 claims 2
- 239000000460 chlorine Substances 0.000 claims 2
- 125000004093 cyano group Chemical group *C#N 0.000 claims 2
- 229910052731 fluorine Inorganic materials 0.000 claims 2
- 239000011737 fluorine Substances 0.000 claims 2
- 125000001188 haloalkyl group Chemical group 0.000 claims 2
- 229910052740 iodine Inorganic materials 0.000 claims 2
- 239000011630 iodine Substances 0.000 claims 2
- 239000007858 starting material Substances 0.000 claims 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims 1
- 239000012046 mixed solvent Substances 0.000 claims 1
- 238000002156 mixing Methods 0.000 claims 1
- 239000012085 test solution Substances 0.000 claims 1
- 238000000825 ultraviolet detection Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract description 2
- 239000013543 active substance Substances 0.000 abstract 1
- 238000000338 in vitro Methods 0.000 abstract 1
- 230000036632 reaction speed Effects 0.000 abstract 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 10
- 238000012360 testing method Methods 0.000 description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 239000008346 aqueous phase Substances 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 229960003180 glutathione Drugs 0.000 description 5
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 4
- 229960004308 acetylcysteine Drugs 0.000 description 4
- 239000012074 organic phase Substances 0.000 description 4
- HYHCSLBZRBJJCH-UHFFFAOYSA-M sodium hydrosulfide Chemical compound [Na+].[SH-] HYHCSLBZRBJJCH-UHFFFAOYSA-M 0.000 description 4
- 108010024636 Glutathione Proteins 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 229960001570 ademetionine Drugs 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- HYHCSLBZRBJJCH-UHFFFAOYSA-N sodium polysulfide Chemical compound [Na+].S HYHCSLBZRBJJCH-UHFFFAOYSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 208000018262 Peripheral vascular disease Diseases 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0039—Coumarin dyes
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开了一类选择性检测同型半胱氨酸（Hcy）的荧光探针，探针的化学结构通式如下：
Description
技术领域
本发明属于分析化学技术领域，具体涉及一类选择性检测Hcy荧光探针的合成，以及该探针在制备直接定量血清中总Hcy、在环境中定量检测Hcy、在生物样品中选择性荧光成像Hcy的器件的应用。
背景技术
同型半胱氨酸是一种含硫分子的氨基酸，属于人体非必需氨基酸，是人体摄入蛋白质中的蛋氨酸在体内代谢为谷胱甘肽和S-腺苷蛋氨酸（SAMe）的一种中间产物。正常情况下血浆中Hcy处于较低水平，但是当血液中Hcy升高（≥ 10 μmol/L）时，将对人体的健康产生重大影响。近年来研究发现血清Hcy水平对心肌梗塞的预测准确性几乎是胆固醇的40倍；高浓度的Hcy是冠状动脉疾病、脑血管疾病、外周血管疾病独立的危险因子。目前定量血清中Hcy的方法主要包括色谱法、酶联免疫、化学发光和循环酶法等，但这些方法存在耗时长、成本高、需专用仪器等不足。因此，开发高效的检测方法/技术用于直接快速定量血清总Hcy水平，具有十分重要的临床意义与广阔的应用前景。
近年来，荧光探针因选择性好、灵敏度高、操作简便且能够监测和可视化活细胞中的生物分子而受到广泛的关注。截至目前，已报道了一些荧光探针用于检测Hcy。但已报道的Hcy荧光探针存在灵敏度低、选择性差等问题，难以用于实际样品的直接快速定量。
发明内容
鉴于上述情况，克服一些现有技术的不足，本发明的目的在于提供一类选择性检测同型半胱氨酸的荧光探针的合成，以及该类探针在制备直接定量血清中总Hcy试剂的应用方法，制备在环境中定量检测Hcy，在生物样品中选择性荧光成像Hcy的器件的应用。
本发明的目的还在于提供一种制备方法简单的选择性检测Hcy的荧光探针的合成方法。
本发明解决问题采取的具体技术方案为，选择性检测同型半胱氨酸的荧光探针的合成及应用，其探针的化学结构通式如下：
选择性检测同型半胱氨酸的荧光探针的合成，其特征在于，所述荧光探针的制备方法包括以下步骤：
将原料1与含有R取代基的原料2加入适量有机溶剂中，40–95°C搅拌反应5–48小时，反应完毕后纯化得权利要求1所示的荧光探针，合成路线如下所示：
所述的原料1的制备方法包括以下步骤：
步骤1. 合成4-氯-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛
步骤2. 合成4-(丁硫基)-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛
a. 将适量4-氯-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛加入无水二氯甲烷中，再加入适量的正丁硫醇和三乙胺，室温搅拌反应5–24小时；
b. 待反应进行完全后，在旋转蒸发仪上旋干溶剂，柱层析纯化得黄色固体产物4-(丁硫基)-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛。
本发明的荧光探针检测Hcy的使用方法：无特殊说明，通常在室温下将探针分子溶解在有机相和水相体积比为4:6的环境下用于检测Hcy，有机相为二甲基亚砜（DMSO），水相为pH = 7.4的磷酸盐缓冲溶液（PBS）和分析物的水溶液。
具体特征如下：荧光探针用二甲基亚砜(DMSO)溶解，探针检测Hcy的检测环境为有机相和水相体积比为4:6的缓冲溶液。具体测试方式为：取20 μL的1 mM的探针溶液，780 μL的分析纯DMSO，所需量PBS缓冲水溶液和所需量的1mM的Hcy水溶液于2 mL的样品管中，所有测试均保持有机相和水相的体积比为4:6 (每一个测试样品总体积为2 mL)。例如当要求测试Hcy浓度为10 μM时探针与Hcy反应后的光谱变化情况，配制样品情况为：取20 μL的1 mM的探针溶液，780 μL的分析纯DMSO，1180 μL的PBS缓冲溶液和20 μL的1 mM的Hcy水溶液于2mL的样品管中，室温下震荡摇匀后即可测量其光谱变化。该探针检测Hcy具有极高的选择性，对其他常见氨基酸、金属离子、活性氧和活性氮物种均无明显响应，对Hcy的检测灵敏度高。该类探针能够实现长波长吸收、红色/近红外荧光选择性检测Hcy（图1，图2）。因此，本发明公开的荧光探针能够实现对Hcy的高灵敏选择性检测。
附图说明
图1为本发明实施例1合成的荧光探针检测半胱氨酸（Cys）、Hcy、谷胱甘肽（GSH）、N-乙酰半胱氨酸（NAC）、NaHSO3、硫氢化钠（NaHS）和多硫化钠（Na2Sn）的紫外-可见吸收光谱图。
图2为本发明实施例1合成的荧光探针检测半胱氨酸（Cys）、Hcy、谷胱甘肽（GSH）、N-乙酰半胱氨酸（NAC）、NaHSO3、硫氢化钠（NaHS）和多硫化钠（Na2Sn）的荧光发射光谱图。
具体实施方式
本发明所述的荧光探针的合成路线如下所示：
实施例1. 合成探针2-(2-(4-(丁硫基)-7-(二乙基氨基)-6-硝基-2-氧代-2H-色烯-3-基)-1-氰基乙烯基)-1-甲基吡啶-1-碘化鎓，合成方法如下所示：
将100.0 mg（264.2 μmol）4-(丁硫基)-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛和103.1 mg（396.4 μmol）2-(氰基甲基)-1-甲基吡啶-1-碘化鎓加入10 mL无水乙醇中，80°C搅拌反应24小时；待反应进行完全后，过滤，滤饼用无水乙醇重结晶得橙黄色固体110.0 mg，产率67.1%，该探针检测Hcy的最大吸收和发射峰分别为543 nm和627 nm。
实施例2. 合成3-溴-4-(2-(4-(丁硫基)-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-基)-1-氰基乙烯基)-1- (4-羟基丁基)吡啶-1-碘化鎓，合成方法如下所示：
将100.0 mg（264.2 μmol）4-(丁硫基)-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛和209.8 mg（528.5 μmol）3-溴-4-(氰基甲基)-1-(4-羟基丁基)吡啶-1-碘化鎓加入10 mL无水乙醇中，70°C搅拌反应48小时；待反应进行完全后，过滤，滤饼用无水乙醇重结晶得红黑色固体98.0 mg，产率48.9%，该探针能够近红外荧光检测Hcy。
实施例3. 合成3-(2-(4-(丁硫基)-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-基)-1-氰基乙烯基)-1-(3-羧丙基) )吡啶-1-碘化鎓，合成方法如下所示：
将100.0 mg（264.2 μmol）4-(丁硫基)-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛和219.4 mg（660.6 μmol）1-(3-羧丙基)-3-(氰基甲基)吡啶-1-碘化鎓加入10 mL无水乙醇中，90°C搅拌反应48小时；待反应进行完全后，柱层析纯化得橙红色固体25.0 mg，产率13.7%。
实施例4. 本发明的荧光探针检测Hcy的应用
本发明所述的荧光探针检测Hcy的光谱性质实验：将探针溶解在二甲基亚砜（DMSO）中配制成浓度为1 mM的探针溶液，配制浓度为1 mM的Hcy的水溶液。检测Hcy的具体测试方式为：取20 μL的探针溶液（1 mM），780 μL的分析纯DMSO，所需量的1 mM的Hcy水溶液和所需量的PBS缓冲水溶液于2 mL的样品管中，所有测试均保持有机相和水相的最终体积比为4:6（每一个测试样品总体积为2 mL)，室温下震荡摇匀后即可测定特定时间的反应速率或单位时间内的反应产物的吸光度或荧光强度。
实施例5. 本发明的荧光探针在直接定量血清中总Hcy的应用
直接定量血清中总Hcy的操作方法如下：取0.2 mL的血清，加入三(2-羧乙基)膦（40 μL, 10 mM）还原处理30分钟。取20 μL的探针溶液（10 mM，以实施例1所示合成的探针为例），780 μL的分析纯DMSO，960 μL的PBS缓冲液，然后加入上述血清溶液，室温下反应2分钟后测量其吸收光谱，根据标准曲线计算血清中总Hcy的浓度，5个血清样本的实验结果如下表：
| 序号 | Hcy浓度（μM） |
| 1 | 7.6 |
| 2 | 12.8 |
| 3 | 20.3 |
| 4 | 27.7 |
| 5 | 36.9 |
实施例4. 荧光成像细胞中内源性的Hcy的应用
将SH-SY5Y细胞传代至共聚焦皿细胞培养基中，在标准生长条件下培养24 小时后，加入适量探针（5 μM，以实施例2所示合成的探针为例）继续在标准生长条件下培养30分钟，然后在共聚焦荧光显微镜下照相，用近红外荧光通道进行荧光成像，可观察到明显的红色荧光，本发明的荧光探针能够实现细胞中内源性Hcy的荧光成像分析，在生物化学、分析检测和环境科学等领域极具应用价值。
本发明的选择性检测同型半胱氨酸的荧光探针的合成及应用，报道了一类选择性检测Hcy的荧光探针及其合成与应用方法，可实现直接快速定量Hcy，生物样品中Hcy的选择性荧光成像分析，可用于制备直接定量血清中总Hcy、在环境中定量检测Hcy、在生物样品中选择性荧光成像Hcy的器件。尽管本发明的内容已经通过上述优选实施例作了详细的介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，具有本文所述技术特征的选择性检测同型半胱氨酸的荧光探针的合成及应用，均落入本专利的保护范围。
Claims (8)
1.选择性检测同型半胱氨酸的荧光探针，其特征在于，所述的荧光探针的化学结构通式如下所示：
2.如权利要求1所述的选择性检测同型半胱氨酸的荧光探针，其特征在于，所述的R中的烷基为：1至10个碳原子的烷基；
所述的R中的取代烷基为：羟基、羧基或磺酸基取代的1至10个碳原子的烷基；
所述的R中的取代1-烷基吡啶鎓盐基是吡啶鎓盐基被一个或多个选自烷基、烷氧基、硝基、羟基、氨基、羧基、磺酸基、氰基、卤代烷基、氟、氯、溴或碘的取代基取代；
所述的R中的取代1-（取代烷基）吡啶鎓盐基是吡啶鎓盐基被一个或多个选自烷基、烷氧基、硝基、羟基、氨基、羧基、磺酸基、氰基、卤代烷基、氟、氯、溴或碘的取代基取代。
3.如权利要求1所述的荧光探针的合成，其特征在于，所述荧光探针的制备方法如下：
将原料1与含有R取代基的原料2加入适量有机溶剂中，40–95°C搅拌反应5–48小时，反应完毕后纯化得权利要求1所示的荧光探针，合成路线如下所示：
4.如权利要求3所述的荧光探针的合成，其特征在于，所述的原料1的制备方法包括以下步骤：
步骤1. 合成4-氯-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛
步骤2. 合成4-(丁硫基)-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛
a. 将适量4-氯-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛加入无水二氯甲烷中，再加入适量的正丁硫醇和三乙胺，室温搅拌反应5–24小时；
b. 待反应进行完全后，在旋转蒸发仪上旋干溶剂，柱层析纯化得黄色固体产物4-(丁硫基)-7-(二乙氨基)-6-硝基-2-氧代-2H-色烯-3-甲醛。
5.如权利要求3所述的荧光探针的合成，其特征在于，所述的原料1和原料2的摩尔比为1：(1~3)；所述的有机溶剂是乙醇、甲醇或它们的混合溶剂。
6.如权利要求1所述的荧光探针的应用，其特征在于，所述的荧光分子探针在制备直接定量血清中总Hcy试剂的应用。
7.如权利要求6所述的荧光探针在直接定量血清中总Hcy中的应用，其特征在于，直接定量包含的方法包括如下步骤：
i. 血清中氧化Hcy的还原：将血清与适量的三(2-羧乙基)膦混匀，20–37°C温育10–30分钟；
ii. 紫外检测：取适量的步骤i中血清溶液加入二甲基亚砜和PBS缓冲液体积比为4:6的探针溶液中，得到测试液后即可通过测定特定时间的反应速率或单位时间内的反应产物的吸光度；
iii. 结果计算：使用作图软件，根据实验制作的标准曲线，结合步骤ii中检测到的反应速率或吸光度值计算出血清中总Hcy的浓度。
8.如权利要求1所述的荧光探针的应用，其特征在于，所述的荧光分子探针能够制备在环境中定量检测Hcy，在生物样品中荧光成像Hcy的器件的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211195448.4A CN115490674A (zh) | 2022-09-29 | 2022-09-29 | 选择性检测同型半胱氨酸的荧光探针的合成及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211195448.4A CN115490674A (zh) | 2022-09-29 | 2022-09-29 | 选择性检测同型半胱氨酸的荧光探针的合成及应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115490674A true CN115490674A (zh) | 2022-12-20 |
Family
ID=84472502
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211195448.4A Pending CN115490674A (zh) | 2022-09-29 | 2022-09-29 | 选择性检测同型半胱氨酸的荧光探针的合成及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115490674A (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115490664A (zh) * | 2022-09-29 | 2022-12-20 | 湖南师范大学 | 一类选择性检测同型半胱氨酸的荧光探针的合成及应用 |
| CN117229285A (zh) * | 2023-09-26 | 2023-12-15 | 湖南师范大学 | 一种同时区分检测同型半胱氨酸和淀粉样蛋白低聚物的荧光探针的合成与应用 |
| CN117229265A (zh) * | 2023-09-26 | 2023-12-15 | 湖南师范大学 | 一种同时区分检测同型半胱氨酸和硫化氢的荧光探针的合成与应用 |
-
2022
- 2022-09-29 CN CN202211195448.4A patent/CN115490674A/zh active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115490664A (zh) * | 2022-09-29 | 2022-12-20 | 湖南师范大学 | 一类选择性检测同型半胱氨酸的荧光探针的合成及应用 |
| CN115490664B (zh) * | 2022-09-29 | 2024-02-13 | 湖南师范大学 | 一类选择性检测同型半胱氨酸的荧光探针的合成及应用 |
| CN117229285A (zh) * | 2023-09-26 | 2023-12-15 | 湖南师范大学 | 一种同时区分检测同型半胱氨酸和淀粉样蛋白低聚物的荧光探针的合成与应用 |
| CN117229265A (zh) * | 2023-09-26 | 2023-12-15 | 湖南师范大学 | 一种同时区分检测同型半胱氨酸和硫化氢的荧光探针的合成与应用 |
| CN117229285B (zh) * | 2023-09-26 | 2024-07-16 | 湖南师范大学 | 一种同时区分检测同型半胱氨酸和淀粉样蛋白低聚物的荧光探针的合成与应用 |
| CN117229265B (zh) * | 2023-09-26 | 2024-07-16 | 湖南师范大学 | 一种同时区分检测同型半胱氨酸和硫化氢的荧光探针的合成与应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115490674A (zh) | 选择性检测同型半胱氨酸的荧光探针的合成及应用 | |
| CN113185506B (zh) | 多信号荧光探针的合成及其在检测Cys、GSH和Hcy中的应用 | |
| CN111349070B (zh) | 一种用于检测生物细胞粘度的近红外荧光分子探针及其制备方法和应用 | |
| Peng et al. | A novel fluorescent probe for selective detection of hydrogen sulfide in living cells | |
| CN108844931B (zh) | Lzq荧光探针在同时检测so2衍生物和hsa中的应用 | |
| CN114181204A (zh) | 一种检测粘度的近红外荧光探针及其制备和应用 | |
| CN110878085B (zh) | 一种快速高选择性次溴酸荧光探针、制备方法与应用 | |
| CN111116573B (zh) | 一种双通道下同时检测dna和rna的近红外荧光探针及其制备方法和应用 | |
| CN111285836A (zh) | 一种近红外荧光探针的制备和应用 | |
| CN110615786B (zh) | 用于检测粘度的近红外荧光化合物及其制备与应用 | |
| CN102633694B (zh) | 一种检测巯基化合物的荧光探针及其制备方法与使用方法 | |
| CN114230494A (zh) | 大斯托克斯位移近红外荧光探针的合成及其在检测硫化氢中的应用 | |
| CN110964044B (zh) | 一种基于双香豆素衍生物的过氧化亚硝酸盐荧光探针、制备方法与应用 | |
| CN114736199B (zh) | 一种基于亚甲基蓝的近红外荧光探针及其合成方法和应用 | |
| CN113637048A (zh) | 一种γ-谷氨酰转肽酶的双光子荧光探针及其制备方法和应用 | |
| CN114835658B (zh) | 一种用于检测硫化氢的荧光探针及其制备方法和应用 | |
| CN114394986B (zh) | 一种比率型过氧亚硝酰荧光探针、制备方法与应用 | |
| CN114249743B (zh) | 多信号荧光探针的合成及其同时区分Hcy、Cys、SO2和GSH的应用 | |
| CN113121552A (zh) | 一种选择性检测半胱氨酸荧光探针的合成与应用 | |
| CN115490664B (zh) | 一类选择性检测同型半胱氨酸的荧光探针的合成及应用 | |
| CN112500382A (zh) | 一种可逆检测二氧化硫/甲醛的比率荧光探针、制备方法与应用 | |
| CN113512034B (zh) | 多信号荧光探针的合成及其同时区分Cys、SO2、GSH和Hcy的应用 | |
| CN108840818B (zh) | 一种用于检测硫化氢的比色型咔唑类荧光探针的合成与应用 | |
| CN117229265B (zh) | 一种同时区分检测同型半胱氨酸和硫化氢的荧光探针的合成与应用 | |
| CN112457249A (zh) | 一种新型细胞膜特异性甲醛荧光探针、制备方法与应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |