CN105601675B - The synthesis of a kind of phosphorescent iridium complex and its fluorescent marker for imago of blood fluke - Google Patents
The synthesis of a kind of phosphorescent iridium complex and its fluorescent marker for imago of blood fluke Download PDFInfo
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- CN105601675B CN105601675B CN201610060560.5A CN201610060560A CN105601675B CN 105601675 B CN105601675 B CN 105601675B CN 201610060560 A CN201610060560 A CN 201610060560A CN 105601675 B CN105601675 B CN 105601675B
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- 241001442514 Schistosomatidae Species 0.000 title claims abstract description 38
- 229910052741 iridium Inorganic materials 0.000 title claims abstract description 32
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 title claims abstract description 22
- 239000003550 marker Substances 0.000 title claims abstract description 20
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 19
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 19
- 241000754688 Cercaria Species 0.000 claims abstract description 23
- 238000000799 fluorescence microscopy Methods 0.000 claims abstract description 18
- 239000003446 ligand Substances 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 9
- 208000015181 infectious disease Diseases 0.000 claims abstract description 8
- 239000007853 buffer solution Substances 0.000 claims abstract description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 210000001015 abdomen Anatomy 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 238000003384 imaging method Methods 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 6
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- 230000005284 excitation Effects 0.000 claims description 4
- DKAGJZJALZXOOV-UHFFFAOYSA-N hydrate;hydrochloride Chemical compound O.Cl DKAGJZJALZXOOV-UHFFFAOYSA-N 0.000 claims description 4
- 239000012046 mixed solvent Substances 0.000 claims description 4
- 241000699670 Mus sp. Species 0.000 claims description 3
- 241000565675 Oncomelania Species 0.000 claims description 3
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 3
- 238000005286 illumination Methods 0.000 claims description 3
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- 239000003960 organic solvent Substances 0.000 claims description 2
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 9
- 239000007850 fluorescent dye Substances 0.000 abstract description 8
- -1 nitrogenous aromatic compounds Chemical class 0.000 abstract description 8
- 238000001215 fluorescent labelling Methods 0.000 abstract description 5
- 239000000523 sample Substances 0.000 abstract description 5
- 238000013461 design Methods 0.000 abstract description 4
- 229910001873 dinitrogen Inorganic materials 0.000 abstract description 2
- 230000000886 photobiology Effects 0.000 abstract description 2
- 150000001491 aromatic compounds Chemical class 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 26
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 8
- 229940079593 drug Drugs 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000004440 column chromatography Methods 0.000 description 6
- 201000004409 schistosomiasis Diseases 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 5
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 4
- 241000242677 Schistosoma japonicum Species 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000012044 organic layer Substances 0.000 description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- 244000309464 bull Species 0.000 description 3
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- 230000010148 water-pollination Effects 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 150000005642 2-chloroquinolines Chemical class 0.000 description 2
- 0 CC(**C*C*)[P+]*C*CCNC Chemical compound CC(**C*C*)[P+]*C*CCNC 0.000 description 2
- 201000004624 Dermatitis Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000237858 Gastropoda Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- 208000030852 Parasitic disease Diseases 0.000 description 2
- YNPNZTXNASCQKK-UHFFFAOYSA-N Phenanthrene Natural products C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 2
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- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
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- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 2
- 239000013110 organic ligand Substances 0.000 description 2
- 230000003071 parasitic effect Effects 0.000 description 2
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 description 2
- 239000000047 product Substances 0.000 description 2
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- 238000010992 reflux Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- LCPVQAHEFVXVKT-UHFFFAOYSA-N 2-(2,4-difluorophenoxy)pyridin-3-amine Chemical compound NC1=CC=CN=C1OC1=CC=C(F)C=C1F LCPVQAHEFVXVKT-UHFFFAOYSA-N 0.000 description 1
- FSVJFNAIGNNGKK-UHFFFAOYSA-N 2-[cyclohexyl(oxo)methyl]-3,6,7,11b-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4-one Chemical class C1C(C2=CC=CC=C2CC2)N2C(=O)CN1C(=O)C1CCCCC1 FSVJFNAIGNNGKK-UHFFFAOYSA-N 0.000 description 1
- YYROPELSRYBVMQ-UHFFFAOYSA-N 4-toluenesulfonyl chloride Chemical class CC1=CC=C(S(Cl)(=O)=O)C=C1 YYROPELSRYBVMQ-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
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- 208000002720 Malnutrition Diseases 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241000935974 Paralichthys dentatus Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 208000034189 Sclerosis Diseases 0.000 description 1
- 208000035472 Zoonoses Diseases 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
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- 239000012670 alkaline solution Substances 0.000 description 1
- 230000000347 anti-schistosomal effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
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- 230000006470 autoimmune attack Effects 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
- 239000002027 dichloromethane extract Substances 0.000 description 1
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- 238000010438 heat treatment Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 150000002503 iridium Chemical class 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
- 235000000824 malnutrition Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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- 210000003205 muscle Anatomy 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Substances [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- JRMUNVKIHCOMHV-UHFFFAOYSA-M tetrabutylammonium bromide Chemical compound [Br-].CCCC[N+](CCCC)(CCCC)CCCC JRMUNVKIHCOMHV-UHFFFAOYSA-M 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 206010048282 zoonosis Diseases 0.000 description 1
Classifications
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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
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0033—Iridium compounds
- C07F15/004—Iridium compounds without a metal-carbon linkage
-
- 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"
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- 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
- C09K2211/18—Metal complexes
- C09K2211/185—Metal complexes of the platinum group, i.e. Os, Ir, Pt, Ru, Rh or Pd
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to the Photobiology marker fields of preventing and treating verminosis, are related to the synthesis of a kind of phosphorescent iridium complex, and for the fluorescent marker of imago of blood fluke.Realize the fluorescent marker of design, synthesis and such complex of iridium to imago of blood fluke of phosphorescent complexes.It is main ligand that the structure of phosphorescent iridium complex, which is by two nitrogenous aromatic compounds, and an aromatic compound containing dinitrogen is the phosphorescent iridium complex for assisting ligands synthesis.Imago of blood fluke fluorescence labeling method includes the following steps：(1) blood fluke cercaria obtains；(2) miracidium infection mouse；(3) imago of blood fluke obtains；(4) at room temperature, imago of blood fluke fluorescent marker is carried out with the phosphorescent complexes PBS buffer solution of 5 μm of ol/L；(5) phosphorescent signal is acquired by laser confocal fluorescence microscope and carries out imago of blood fluke fluorescence imaging.By design and synthesis, such phosphorescent iridium complex is used for imago of blood fluke fluorescent marker as fluorescence probe, successfully solves the problem of fluorescent marker effect difference and anti-light bleachability difference during imago of blood fluke fluorescence imaging.
Description
Technical field
The invention belongs to the Photobiology marker fields of preventing and treating verminosis, more particularly it relates to a kind of phosphorescence
The synthesis of complex of iridium and its be used for imago of blood fluke fluorescent marker, the present invention provides a kind of novel imago of blood fluke fluorescence
Labeling method, such phosphorescent iridium complex have the advantageous effect of imago of blood fluke fluorescent marker.
Background technology
Snail fever is a kind of ancient parasitic disease, has huge harm to the production and living of the mankind, is to be only second to malaria
The second largest tropical parasitic parasitosis of disease.Schistosomiasis endemic has 6.52 hundred million populations by prestige at present in 74 countries and regions, estimation
The side of body, has 1.93 hundred million the infecteds, there is symptom case about 1.2 hundred million, wherein 20,000,000 be several cases, China is the main of snail fever
Endemic Area.Snail fever is a kind of parasitic zoonoses typically propagated through water source, and blood fluke cercaria is infection people and animals
Unique phases develop for the virgin worm stage, entered back by 20 to 60 days time after blood fluke cercaria enters human body
The worm stage can cause acute and chronic snail fever and related complication in the whole process, cause greatly to injure to human body, from skin
Skin allergy dermatitis, malnutrition, apocleisis, jaundice or even hepatic sclerosis, late period severe patient can be with causing deaths.Researcher is logical
It is studied after constantly, has developed a series of antischistosomal drugs for having good killing effect to fluke adult, wherein pyrrole quinoline
Ketone is as main representative, and because with safety, adverse reaction is few, and instructions of taking is simple and easy to do, good effect, and degradation is fast in human body
The features such as and at home or even the world is widely applied.But due to largely making for a long time to the extensive of praziquantel class drug
With blood fluke generates drug resistance to it；Such drug kills the definite mechanism of action of adult and is still not clear simultaneously, so needing
Such drug is carried out to continue in-depth study.It is explored according to traditional research method (detection predominantly to dead polypide)
The possibility mechanism of action of these drugs cannot directly reflect that the mode of action and site of the drug on live body, intermediate link can shadows
Ring the accuracy and confidence level of result, thus need to seek some new research methods make up presently, there are deficiency.Currently,
Fluorescence imaging is sensitive, non-intrusion type, a low-cost Visual retrieval approach, and resolution ratio, can up to hundred nanometers
Realize living imaging from cell to animal, fluorescence imaging means have that sensitive monitoring, imaging is rapid, can observe polymolecular simultaneously
The advantages that event, therefore be with a wide range of applications in terms of preventing blood fluke drug mechanism study.
Imago of blood fluke body wall is made of body quilt, basement membrane and body by lower layer, i.e., the plasomidum and muscle layer that body is formed by cell
It is interleaved together, and is connected with body by cytoplasm tubule, body is made to be covered by the body of the seedless acellular separation in surface layer by lower layer
Lid, therefore imago of blood fluke does not have cuticula.Imago of blood fluke parasitizes in blood vessel, and body surface directly connects with host blood
It touches, in order to escape the autoimmune attack of host, body has been capped a kind of specific carbohydrate identical with host's red blood cell ab antigens,
So stronger hydrophily is presented in adult body surface.Currently, using traditional organic molecule fluorescent dye as fluorescence probe pair
Organism is studied, and haves the shortcomings that following：Short wavelength's transmitting has the interference of archebiosis fluorescence, photostability not enough to cause not
It can observe for a long time.Meanwhile most of organic molecule fluorescent dye hydrophily is poor, to the coloring of imago of blood fluke
It is undesirable, therefore it is a challenge to develop to fluorescent dye of the imago of blood fluke with good fluorescence label effect.
Phosphorescent iridium complex compares other fluorescent materials, has a greater degree of MLCT features, has luminous efficiency height, light
The advantages that stability is good, luminescent color is adjustable.Present invention combination phosphorescent iridium complex excellent luminance performance, inventor match by iridium
The application that object is used for imago of blood fluke fluorescent marker as phosphorescence probe is closed, selects suitable organic ligand and to organic ligand
Hydrophilic modification is carried out, fluorescent marker effect difference and anti-light bleaching during imago of blood fluke fluorescence imaging can be successfully solved
The problem of poor performance.
Invention content
The present invention provides a kind of phosphorescent iridium complexes, by being chemically modified upper hydrophily base to this kind of complex
Group makes it have the performance that phosphorescence markers are carried out to Schistosoma japonicum adult, and improves such phosphorescent iridium complex and shine
Quantum efficiency can be used as Schistosoma japonicum adult phosphorescence markers reagent.
The structure of the complex of iridium of the present invention be by using two nitrogenous aromatic compounds as bidentate ligand, be aided with one and contain
The ligands of dinitrogen aromatic compound synthesize, and more specific structure is as follows：
Wherein n=0,1,2,3,4
The compounds of this invention can be prepared according to following synthetic route：
It is known compound to prepare the raw material of the compounds of this invention and agents useful for same, can be obtained on the market, or can
It is prepared with methods known in the art.In synthetic route second step can be prepared by methods known in the art (referring to Nonoyama,
K.Bull.Chem.Soc.Jpn.1974,47,467-468.)
LigandIt is heated to reflux in acetonitrile alkaline solution with PEG-Ts and can be obtained the bidentate ligand containing hydrophilic groupWherein PEG-Ts structural formulas are as follows：
LigandStructural formula is as follows：
By iridous chloride hydrate and containing the bidentate ligand of hydrophilic groupIn the mixed solution of cellosolvo and water
Middle heating is reacted 12 to 48 hours, can synthesize to obtain corresponding iridium dichloro bridge complex；Then, the iridium dichloro bridge complex obtained exists
Organic solvent, water or in the mixed solvent and corresponding assistant ligandReaction 4 to 12 hours synthesizes final product.
For any one of the above phosphorescent iridium complex as imago of blood fluke fluorescence labeling probe, fluorescence labeling method includes such as
Lower step：
(1) blood fluke cercaria obtains：Fresh cercaria obtains from the positive oncomelania of infection miracidium, take positive spiral shell in
In 100mL conical flasks, addition clear water to bottleneck, 2 hours or so the effusion cercarias of illumination under incandescent lamp.
(2) mouse infection：Six week old female mice four limbs are fixed on self-control shelf, abdomen unhairing takes warm water to moisten
Mouse part skin, and the coverslip containing 50 or so cercarias is affixed on the plucked skin of abdomen of mouse, it is kept for 20 minutes
Make cercaria penetrate mouse skin to be infected.
(3) imago of blood fluke obtains：After metainfective Mouse feeder 6 weeks, put to death using cervical dislocation, in strict accordance with nothing
Bacterium operation requires, and splits skin of abdomen, peritonaeum successively, perfusion is taken to collect adult in liver portal-mesentcric vein.It will collect
To adult rinse 3 times in physiological saline after be assigned in culture dish, RPMI-1640 (Roswell Park are added
Memorial Institute) after cell culture fluid culture 2 hours, the good polypide of microscopy screening activity is used for fluorescence imaging.
(4) imago of blood fluke fluorescence imaging：Phosphorescent complexes dimethyl sulfoxide (DMSO) (DMSO) dissolving is accurately configured to 1mmol
Mother liquor, 5 μM of dilution is then diluted to PBS buffer solution again.The dilution for pipetting 200 μ L is added in culture dish, is used
Tweezers picking adult is added in phosphorescent complexes dilution, after incubation at room temperature 5 minutes to 6 hours, adult is transferred to glimmering
Imaging apparatus ware, confocal fluorescent microscopic observes the phosphorescence markers situation of cercaria under 405nm excitations, acquires 520-620nm
Phosphorescent signal carry out fluorescence imaging.
Phosphorescent iridium complex is used for the research of imago of blood fluke fluorescent marker by inventor by design and synthesis, can be with
Successfully solve adult fluorescence imaging during fluorescent marker effect difference and anti-light bleachability difference deficiency.
Description of the drawings
The absorption spectrum and phosphorescence emission spectra of Fig. 1 complexs Ir-1.
The absorption spectrum and phosphorescence emission spectra of Fig. 2 complexs Ir-2.
The laser co-focusing fluorescence imaging figure of Fig. 3 complexs Ir-2 label Schistosoma japonicum adults.
The laser co-focusing fluorescence imaging figure of Fig. 4 complexs Ir-2 label Schistosoma japonicum adult head devices.
Specific implementation mode
Set forth below is the specific embodiments of the compounds of this invention, they are of the invention with example in detail, but to this hair
It is bright to be not limited in any way.Raw material used in the present embodiment is known compound, can be obtained by commercial sources, or can be pressed
Pertinent literature design method synthesizes.
In the following embodiments, involved physical and chemical parameter is by following Instrument measurings：1H H NMR spectroscopies are in Bruker
It is measured using 400MHz on Mercuryplus, is internal standard with TMS；Mass spectrometric data MALDI-TOF-MS is in 5800 types of AB SCIEX
It is obtained on mass spectrograph；Ultraviolet-visible absorption spectroscopy is completed on Shimadzu UV-2700 uv-visible absorption spectra instrument；Phosphorescence
Emission spectrum in PerkinElmer LS-55 Fluorescence Spectrometer by measuring；Cercaria Phosphorescence imaging swashs in OLYMPUS FV1000 types
It is acquired in light confocal fluorescent microscopic, laser provides the excitation light source that wavelength is 405nm, according to the phosphorus of complex of iridium
Light emitting property collects the transmitting phosphorescent signal within the scope of 520nm-620nm.
Embodiment 1
[Ir(HO-pq)2(phen)][PF6] (Ir-1) synthesis
(1) synthesis of HO-pq：By the 4- phenolic group phenyl boric acids of 1mmol, the 2- chloroquinolines of 1mmol, four (3- benzene of 7% equivalent
Base phosphine) potassium carbonate of palladium (0) and 1mmol is added in three-neck flask, 30mL THF and H is then added2The mixed solvent of O.
Under the protection of nitrogen, reaction system is reacted for 24 hours at 70 DEG C.It is cooled to room temperature, reaction solution is extracted with dichloromethane, water phase dichloro
Methane extracts 3 times (10mL × 3), merges organic layer, is dried with anhydrous sodium sulfate, column chromatography for separation obtains.
(2) synthesis of HO-pq iridium dichloro bridge complex：Bibliography (Nonoyama,
K.Bull.Chem.Soc.Jpn.1974,47,467-468. it) synthesizes.The iridous chloride hydrate of 0.5mmol and matching for 1mmol
Body HO-pq is dissolved in (v/v=3 in the cellosolvo of 25mL and the mixed liquor of water：1), for 24 hours, room temperature cools down for 110 DEG C of reactions.
Collected by suction solid is washed for several times with distilled water and absolute ethyl alcohol respectively, obtains HO-pq iridium dichloro bridge complexs.
(3)[Ir(HO-pq)2(bpy)][PF6] (Ir-1) synthesis：By the HO-pq iridium dichloro bridge complexs of 0.2mmol,
1, the 10- Phens of 0.4mmol are added in round-bottomed flask, and 30mL dichloromethane and methanol (v/v=2 is then added：1).
Under the protection of nitrogen, reaction system reacts 10h at 50 DEG C.Add the KPF of 10 times of equivalents6, the reaction was continued 4h.Room temperature cools down,
Collected by suction filtrate is depressurized, column chromatography for separation obtains product.The Absorption and fluorescence spectrum of complex Ir-1 is as shown in Figure 1.
Nuclear-magnetism characterize data：1H NMR(400MHz,d6- DMSO) δ 9.49 (s, 2H), 8.71 (dd, J=8.2,1.2Hz, 2H), 8.52
(t, J=12.8Hz, 2H), 8.34 (q, J=9.0Hz, 4H), 8.16 (d, J=8.7Hz, 2H), 8.09-7.97 (m, 4H),
7.84-7.56 (m, 2H), 7.14 (t, J=7.5Hz, 2H), 7.01 (d, J=8.9Hz, 2H), 6.78-6.69 (m, 2H), 6.63
(dd, J=8.6,2.3Hz, 2H), 5.98 (d, J=2.2Hz, 2H).
Embodiment 2
[Ir(PEG-pq)2(phen)][PF6] (Ir-2) synthesis
(1) synthesis of HO-pq：By the 4- phenol phenyl boric acids of 1mmol, the 2- chloroquinolines of 1mmol, four (3- phenyl of 7% equivalent
Phosphine) potassium carbonate of palladium (0) and 1mmol is added in three-neck flask, 30mL THF and H is then added2The mixed solvent of O.In nitrogen
Under the protection of gas, reaction system is reacted for 24 hours at 70 DEG C.It is cooled to room temperature, reaction solution is extracted with dichloromethane, water phase dichloromethane
Alkane extracts 3 times (10mL × 3), merges organic layer, is dried with anhydrous sodium sulfate, column chromatography for separation.
The synthesis of the single-ended p-methyl benzenesulfonic acid ester of (2) three diglycol ethylenes：Three necks are added in tri- diglycol ethylenes of 1mmol to burn
In bottle, the pyridine of 1mmoL is added, is dissolved with the dichloromethane of 20mL, is stirred in 0 DEG C of ice bath.It is molten to be slowly dropped into dichloromethane
The 1mmoL p-methyl benzene sulfonic chlorides of solution.Reaction uses salt acid elution after 3 hours, takes organic layer to add and is washed to aqueous solution and is in neutrality.Nothing
Aqueous sodium persulfate is dried, and column chromatography for separation obtains.
(3) synthesis of ligand PEG-pq：By the single-ended p-methyl benzenesulfonic acid ester of tri- diglycol ethylenes of 1mmol, 1mmol carbonic acid
The tetrabutylammonium bromide of potassium, 10% equivalent is added in three-necked bottle, is dissolved with the acetonitrile of 20mL.It is stirred at reflux in 80 DEG C of oil baths.
It is slowly dropped into the 1mmol HO-pq of acetonitrile dissolving.After reacting 10h, it is cooled to room temperature.Reaction solution is extracted with dichloromethane, water phase
3 times (10mL × 3) are extracted with dichloromethane, merges organic layer, is dried with anhydrous sodium sulfate, column chromatography for separation obtains.
(4) PEG-pq iridium dichloro bridge complex synthesis (referring to document Nonoyama,
K.Bull.Chem.Soc.Jpn.1974,47,467-468.)：The iridous chloride hydrate of 0.5mmol and the ligand of 1mmol
PEG-pq is dissolved in (v/v=3 in the cellosolvo of 25mL and the mixed liquor of water：1) it, reacts 24 hours for 110 DEG C, terminates cooling
Room temperature.Collected by suction solid is washed for several times with distilled water and absolute ethyl alcohol respectively, obtains PEG-pq iridium dichloro bridge complexs.
(5)[Ir(PEG-pq)2(bpy)][PF6] (Ir-2) synthesis：The PEG-pq iridium dichloro bridges of 0.2mmol are coordinated
1, the 10- Phens of object, 0.4mmol are added in round-bottomed flask, and 30mL dichloromethane and methanol (v/v=2 is then added：1).
Under the protection of nitrogen, reaction system reacts 10h at 50 DEG C.Add the KPF of 10 times of equivalents6, the reaction was continued 4h.Room temperature is cold
But, collected by suction filtrate is depressurized, column chromatography for separation obtains product.The Absorption and fluorescence spectrum of complex Ir-2 such as Fig. 2 institutes
Show.Complex Ir-2 characterize datas：1H NMR(400MHz,CDCl3) δ 8.73 (dd, J=8.2,1.2Hz, 2H), 8.51 (dd, J
=5.1,1.2Hz, 2H), 8.45 (d, J=9.0Hz, 2H), 8.38 (d, J=8.9Hz, 2H), 8.30 (d, J=8.9Hz, 2H),
8.08 (s, 2H), 8.02 (dt, J=13.9,7.0Hz, 2H), 7.75 (d, J=8.0Hz, 2H), 7.19 (t, J=7.7Hz, 2H),
7.03 (d, J=8.9Hz, 2H), 6.86 (dd, J=8.8,2.4Hz, 2H), 6.80-6.72 (m, 2H), 5.96 (d, J=2.5Hz,
2H), 4.50 (t, J=5.5Hz, 2H), 3.95-3.78 (m, 2H), 3.76-3.62 (m, 2H), 3.68-3.61 (m, 8H), 3.57
(s,8H),3.55–3.50(m,4H)；MALDI-TOF MS:m/z 1077.6443([M-PF6]+)。
Embodiment 3
Phosphorescent complexes Ir-2 is imaged the phosphorescence markers of imago of blood fluke
(1) reagent and material
Reagent：Dimethyl sulfoxide (DMSO) (Tianjin Kermel Chemical Reagent Co., Ltd.), PBS buffer solution autogamy.
Cercaria：Snails escape to obtain, and Snails are provided by Jiangsu Prov. Bilharziasis Prevention and Control Inst..
Mouse：Kunming mice is provided by Gannan Medical College of Jiangxi Province.
(2) blood fluke cercaria obtains
Fresh cercaria obtains from the positive oncomelania of infection miracidium, takes positive spiral shell in 100mL conical flasks, and clear water is added
To bottleneck, 2 hours or so the effusion cercarias of illumination under incandescent lamp.
(3) mouse infection
Six week old female mice four limbs are fixed on self-control shelf, abdomen unhairing takes warm water to moisten mouse part skin,
And the coverslip containing 50 or so cercarias is affixed on the plucked skin of abdomen of mouse, it keeps making cercaria penetrate mouse in 20 minutes
Skin is infected.
(4) adult is obtained
After metainfective Mouse feeder 6 weeks, is put to death using cervical dislocation, required in strict accordance with sterile working, cutd open successively
Skin of abdomen, peritonaeum are opened, perfusion is taken to collect adult in liver portal-mesentcric vein.By the adult being collected into physiology salt
It is assigned in culture dish after being rinsed 3 times in water, it is thin that RPMI-1640 (Roswell Park Memorial Institute) is added
After born of the same parents' culture solution culture 2 hours, the good polypide of microscopy screening activity is used for fluorescence imaging.
(5) adult fluorescent marker imaging method
Phosphorescent complexes Ir-2 is chosen as fluorescence labeling probe, phosphorescent complexes Ir-2 is molten with dimethyl sulfoxide (DMSO) (DMSO)
Solution is accurately configured to the mother liquor of 1mmol, and 5 μM of dilution is then diluted to PBS buffer solution.Pipette the addition of 200 μ L dilutions
Into culture dish, it is added in phosphorescent complexes dilution with tweezers picking adult, after being incubated 5 minutes to 6 hours at room temperature,
Adult is transferred to fluorescence imaging special utensil, cercaria is observed using laser confocal fluorescence microscope under 405nm excitations
Phosphorescence markers situation, and the phosphorescent signal for acquiring 520-620nm carries out fluorescence imaging.
(6) fluorescent marker result
Specific label result can be obtained referring to Fig. 3 and Fig. 4 from fluorescence imaging figure：Phosphorescent complexes Ir-2 can be to blood
Fluke adult carries out fluorescent marker imaging.
Claims (4)
1. a kind of phosphorescent iridium complex, it is characterised in that：Phosphorescent compound with formula：
Wherein n=3.
2. a kind of method preparing phosphorescent iridium complex as described in claim 1, it is characterised in that：The phosphorescent iridium complex
It can be synthesized by following route：
Wherein PEG-Ts structural formulas are as follows：
Bidentate ligandStructural formula is as follows：
By iridous chloride hydrate and containing the bidentate ligand of hydrophilic groupAdd in the mixed solution of cellosolvo and water
Thermal response 12 to 48 hours, synthesis obtain iridium dichloro bridge complex；Then, the iridium dichloro bridge complex obtained organic solvent,
Water or in the mixed solvent and corresponding assistant ligandReaction 4 to 12 hours synthesizes final product.
3. a kind of application to phosphorescent iridium complex as described in claim 1, it is characterised in that：The phosphorescent iridium complex is used
In the imago of blood fluke fluorescent marker for the purpose of non-diagnostic treatment.
4. application according to claim 3, which is characterized in that imago of blood fluke fluorescent marker imaging method includes following step
Suddenly：
Blood fluke cercaria obtains：Fresh cercaria obtains from the positive oncomelania of infection miracidium, takes positive spiral shell in 100mL conical flasks
In, addition clear water to bottleneck, 2 hours or so the effusion cercarias of illumination under incandescent lamp；
Mouse infection：Six week old female mice four limbs are fixed on self-control shelf, abdomen unhairing takes warm water to moisten mouse web portion
Skin, and the coverslip containing 45-55 cercaria is affixed on the plucked skin of abdomen of mouse, it keeps making cercaria penetrate in 20 minutes
Mouse skin is infected；
Imago of blood fluke obtains：After metainfective Mouse feeder 6 weeks, put to death using cervical dislocation, in strict accordance with sterile working
It is required that splitting skin of abdomen, peritonaeum successively, perfusion is taken to collect adult in liver portal-mesentcric vein；To be collected at
Worm is assigned to after rinsing 3 times in physiological saline in culture dish, and RPMI-1640 is added
(RoswellParkMemorialInstitute-1640) after cell culture fluid culture 2 hours, the good worm of microscopy screening activity
Body is used for fluorescence imaging；
Imago of blood fluke fluorescence imaging：Phosphorescent complexes dimethyl sulfoxide (DMSO) (DMSO) dissolving is accurately configured to the mother liquor of 1mmol,
Then 5 μM of dilution is diluted to PBS buffer solution again；The dilution for pipetting 200 μ L is added in culture dish, with tweezers picking
Adult is added in phosphorescent complexes dilution, and after incubation at room temperature 5 minutes to 6 hours, adult is transferred to fluoroscope imager
Ware, confocal fluorescent microscopic observes the phosphorescence markers situation of cercaria under 405nm excitations, and acquires the phosphorescence of 520-620nm
Signal carries out fluorescence imaging.
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