CN107216363A - The vitamin B12 derivative of FITC marks and its synthetic method and application - Google Patents
The vitamin B12 derivative of FITC marks and its synthetic method and application Download PDFInfo
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- CN107216363A CN107216363A CN201710089234.1A CN201710089234A CN107216363A CN 107216363 A CN107216363 A CN 107216363A CN 201710089234 A CN201710089234 A CN 201710089234A CN 107216363 A CN107216363 A CN 107216363A
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- 150000001867 cobalamins Chemical class 0.000 title claims abstract description 9
- 238000010189 synthetic method Methods 0.000 title description 3
- 229930003779 Vitamin B12 Natural products 0.000 claims abstract description 53
- 239000011715 vitamin B12 Substances 0.000 claims abstract description 53
- 235000019163 vitamin B12 Nutrition 0.000 claims abstract description 53
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 claims abstract description 52
- 238000002360 preparation method Methods 0.000 claims abstract description 19
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 18
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 17
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 238000013459 approach Methods 0.000 claims abstract description 8
- 230000008878 coupling Effects 0.000 claims abstract description 6
- 238000010168 coupling process Methods 0.000 claims abstract description 6
- 238000005859 coupling reaction Methods 0.000 claims abstract description 6
- 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 claims abstract description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 44
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- 229910052757 nitrogen Inorganic materials 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 10
- 238000005576 amination reaction Methods 0.000 claims description 9
- 238000001291 vacuum drying Methods 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 239000012190 activator Substances 0.000 claims description 5
- 239000003054 catalyst Substances 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 4
- 150000003462 sulfoxides Chemical class 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 229940124597 therapeutic agent Drugs 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims description 2
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- 239000011261 inert gas Substances 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000003384 imaging method Methods 0.000 abstract description 13
- 238000001215 fluorescent labelling Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 239000007850 fluorescent dye Substances 0.000 abstract description 4
- 238000012632 fluorescent imaging Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 231100000053 low toxicity Toxicity 0.000 abstract description 2
- 238000011017 operating method Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 230000008685 targeting Effects 0.000 abstract description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 9
- 208000005718 Stomach Neoplasms Diseases 0.000 description 7
- 206010017758 gastric cancer Diseases 0.000 description 7
- 201000011549 stomach cancer Diseases 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 229930003231 vitamin Natural products 0.000 description 4
- 235000013343 vitamin Nutrition 0.000 description 4
- 239000011782 vitamin Substances 0.000 description 4
- 229940088594 vitamin Drugs 0.000 description 4
- 108091005461 Nucleic proteins Proteins 0.000 description 3
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 2
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- 210000004185 liver Anatomy 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 229910001414 potassium ion Inorganic materials 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000006386 Myelin Proteins Human genes 0.000 description 1
- 108010083674 Myelin Proteins Proteins 0.000 description 1
- 208000031845 Pernicious anaemia Diseases 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- FEZWOUWWJOYMLT-DSRCUDDDSA-M cobalt;[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2,7,1 Chemical compound [Co].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O FEZWOUWWJOYMLT-DSRCUDDDSA-M 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- -1 hydrogen ions Chemical class 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000000752 ionisation method Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 229960005321 mecobalamin Drugs 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 235000007672 methylcobalamin Nutrition 0.000 description 1
- 239000011585 methylcobalamin Substances 0.000 description 1
- JEWJRMKHSMTXPP-BYFNXCQMSA-M methylcobalamin Chemical compound C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O JEWJRMKHSMTXPP-BYFNXCQMSA-M 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000005012 myelin Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
-
- 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
-
- 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
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0052—Small organic molecules
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- 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
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
-
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- 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
- C09K2211/187—Metal complexes of the iron group metals, i.e. Fe, Co or Ni
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/061—Sources
- G01N2201/06113—Coherent sources; lasers
Abstract
The present invention relates to the vitamin B12 derivative of a kind of vitamin B12 derivative of new fluorescence labeling, i.e. fluorescein isothiocynate (FITC) mark, referred to as VB12 FITC.VB12 FITC of the present invention preparation method includes：On the low reaction activity OH groups of VB12 structures, the double amino of coupling, and then reacted with FITC isosulfocyanate radical, synthesize VB12 FITC derivatives.The preparation condition of the vitamin B12 of FITC marks provided by the present invention is gentle (can carry out at normal temperatures), and raw material low toxicity, operating method is easy, and combined coefficient is high, and reaction product property is stable, with good fluorescent imaging effect.The vitamin B12 of FITC marks provided by the present invention has tumor uptake targeting, and the research in terms of VB12 Absorption And Metabolisms approach, tumor imaging treatment has important value.
Description
Technical field
The present invention relates to a kind of vitamin B12 derivative, more particularly to a kind of vitamin of new fluorescence labeling
B12 derivatives, referred to as VB12-FITC, and the derivative synthetic method and application.
Background technology
Vitamin B12, also known as Mecobalamin, are a kind of water soluble vitamins needed by human, it is impossible to which itself is closed in vivo
Into being only capable of by food intake, and be stored in liver.Lack the situation for continuing supplemented with exogenous vitamin B12 in human body
Under, the vitamin B12 total amount stored in liver was consumed totally in 3-6 months.Human body cell be metabolized methylate,
Safeguard the metabolism of neural myelin and function, promote the development of red blood cell with it is ripe, participate in the synthesizing of picodna (DNA), fat
And multiple important steps such as the synthesis of the metabolism of carbohydrate, nucleic acid and protein, it be unable to do without the participation of vitamin B12.
With the shortage of vitamin B12, the complication such as pernicious anaemia, irreversibility DPN are ultimately resulted in.
Vitamin is as the coenzyme for participating in carbon-based group metabolism, and the synthesis with nucleic acid and protein is closely related.Cell
In propagation and growth course, the synthesis of nucleic acid and protein is dramatically speeded up, thus in fast breeding tissue --- tumor focus
In, significantly raised for vitamin B12 receptor number, show to dramatically increase vitamin intake, thus tumour is to VB12
There is high intake phenomenon.This characteristic make vitamin B12 in itself have certain tumor-targeting, tumour Absorption And Metabolism approach,
Research in terms of tumor imaging treatment, vitamin B12 definite meaning.
Vitamin B12 normal absorption metabolism, tumor metabolic feature increasingly paid close attention to, its Absorption And Metabolism approach and should
Novel targeted administration, is to pay close attention to study hotspot in recent years under approach, so the imaging of vitamin B12, visualization seem particularly heavy
Will.Current VB12 imaging label, mainly marked by radionuclide Co+, 111In, 10B, 99mTc or positive electron Cu+ etc.
Approach realizes, such method needs that special picture reproducer, cost be high, operation difficulty is big, in addition radionuclide, positive electron
To the active infringement of human body.Fluorescent imaging is comparatively safe, with low cost at present, without ultra-large type instrument, behaviour
Make easy developing method, if can greatly reduce the difficulty of operation by fluorescence labelling in vitamin B12 Absorption Study
And cost.
However, in vitamin B12 structure can reactive group it is few, it is difficult to directly with the direct reaction bonded of fluorescer, it is necessary to first
Increase by one " bridge " in this structure, can be combined simultaneously with VB12, fluorescer, and then complete the fluorescence mark of vitamin B12
Note.But vitamin B12 structural instability itself, is shown in the possibility that light, heat are easily decomposed, and then have degraded under the conditions of vigorous reaction,
Therefore vitamin B12 fluorescence labeling has great difficulty.The present invention is by being coupled diamino compounds, by vitamin B12 structure
- OH change into the higher amino of reactivity, reacted again with FITC isosulfocyanate radical at room temperature, so as to realize in temperature
FITC fluorescence labelings VB12 is prepared with the conditions of.
The content of the invention
First purpose of the present invention is to provide a kind of vitamin B12 derivative of FITC marks, constructed fluorescence
The VB12 of mark activates its low reaction activity group, so as to solve mesh on the premise of VB12 original structures are maintained under normal temperature
There is the problems such as cost is high, operation difficulty is big in preceding nucleic, the vitamin B12 imaging of positive electron mark.
The vitamin B12 derivative (VB12-FITC) of FITC marks of the present invention, with following molecular structure
Formula：
Second object of the present invention is to provide the preparation method of the vitamin B12 derivative of described FITC marks.
VB12-FITC of the present invention preparation method includes：On the low reaction activity-OH groups of VB12 structures,
The double amino of coupling, and then reacted with FITC isosulfocyanate radical, synthesize VB12-FITC derivatives.
In a preferred embodiment, VB12-FITC of the present invention preparation method comprises the following steps：
(A) amination of vitamin B12：Under inert gas gas shielded, vitamin B12 is dissolved in through except water process
Dimethyl sulfoxide (DMSO) (DMSO) in, according to 1:1-1:2 ratios are added at activator N, N'- carbonyl dimidazoles (CDI), 20-25 DEG C
Lucifuge is activated 6-12 hours, according to 1:2-1:5 ratios add diamino compounds, continue the reaction 3-12 at 20-25 DEG C small
When；Precipitate and separate reaction product, amidized vitamin B12 is dried to obtain at 20-25 DEG C；
(B) FITC and amination vitamin B12 coupling：Obtained amination vitamin B12 will be reacted in step A molten
Xie Yujing removes the dimethyl sulfoxide (DMSO) (DMSO) of water process, according to 1:1-1:2 ratios add catalyst of triethylamine, according to 1:1.5 than
Example adds lucifuge at fluorescein isothiocynate (FITC), 20-25 DEG C and reacted 4-12 hours, precipitates and separates reaction product, 20-
The vitamin B12 of FITC marks is dried to obtain at 25 DEG C.
According to the further feature of VB12-FITC of the present invention preparation method, two selected in the step A
Amino-compound is 1,8- diaminourea -3,6- dioxaoctanes.
According to the further feature of VB12-FITC of the present invention preparation method, in the step A and B, heavy
Acetone is added during shallow lake and is used as precipitating reagent.
According to the further feature of VB12-FITC of the present invention preparation method, in the step A and B, point
During from reaction product, 10min is centrifuged under the conditions of 10000r/min using centrifuge, unreacted compound is removed.
It is described in the step A and B according to the further feature of VB12-FITC of the present invention preparation method
Drying is vacuum drying.
3rd purpose of the invention is the application of the VB12-FITC described in offer, that is, described VB12-FITC
Application in the imaging therapeutic agent in preparing diagnosis and monitoring VB12 Absorption And Metabolisms approach and tumour.
Examined based on the imaging therapeutic agent prepared by VB12-FITC of the present invention in assessment VB12-FITC derivatives
Had a good application prospect in disconnected and monitoring VB12 Absorption And Metabolisms new way and tumour.
The present invention has the advantages that：
(1) preparation condition of the vitamin B12 of FITC marks provided by the present invention is gentle (can carry out at normal temperatures),
Raw material low toxicity, operating method is easy, and combined coefficient is high, and reaction product property is stable, with good fluorescent imaging effect；
(2) vitamin B12 of FITC marks provided by the present invention has tumor uptake targeting, and generation is absorbed in VB12
Thank to the research in terms of approach, tumor imaging are treated to have a good application prospect.
Brief description of the drawings
Fig. 1 is FITC marks vitamin B12 of the present invention1HMR mass spectrograms.
Fig. 2 is the HPLC figures of FITC marks vitamin B12 of the present invention.
Fig. 3 is the flow cytometer showed result figure that FITC of the present invention marks vitamin B12.
Fig. 4 is the Laser Scanning Confocal Microscope result figure that FITC of the present invention marks vitamin B12.
Fig. 5 is the imaging in vivo figure that FITC of the present invention marks vitamin B12.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not
It is limited to this.
Embodiment 1：VB12-FITC of the present invention preparation
(A) 200mg vitamin B12 is dissolved in 4ml through the dimethyl sulfoxide (DMSO) except water process under nitrogen protection
(DMSO), add lucifuge at 200mg activator N, N'- carbonyl dimidazoles (CDI), 20 DEG C to activate 12 hours, add 400mg
21,8- amino -3,6- dioxaoctane, continue to react 6 hours at 20 DEG C.With 500ml acetone precipitation reaction products, and
Vacuum drying at 10min separation reaction products, 20 DEG C is centrifuged under the conditions of 10000r/min and obtains amidized vitamin B12.
(B) the amination vitamin B12 for obtaining step A, weighs 150ng and is dissolved in 4ml through the dimethyl except water process
Sulfoxide (DMSO), adds 0.1ml catalyst of triethylamine, according to 1：1 ratio adds lucifuge at FITC, 20 DEG C and reacted 6 hours,
Precipitate and separate reaction product, vacuum drying obtains the vitamin B12 of FITC marks at 20 DEG C.
Embodiment 2：VB12-FITC of the present invention preparation
(A) 200mg vitamin B12 is dissolved in 4ml through the dimethyl sulfoxide (DMSO) except water process under nitrogen protection
(DMSO), add lucifuge at 300mg activator N, N'- carbonyl dimidazoles (CDI), 22 DEG C to activate 12 hours, add 600mg
1,8- diaminourea -3,6- dioxaoctanes, continue at 22 DEG C react 6 hours.With 500ml acetone precipitation reaction products, and
Vacuum drying at 10min separation reaction products, 22 DEG C is centrifuged under the conditions of 10000r/min and obtains amidized vitamin B12.
(B) the amination vitamin B12 for obtaining step A, weighs 150ng and is dissolved in 4ml through the dimethyl except water process
Sulfoxide (DMSO), adds 0.1ml catalyst of triethylamine, according to 1：It is small that 1.25 ratios add lucifuge reaction 6 at FITC, 22 DEG C
When, precipitate and separate reaction product, vacuum drying obtains the vitamin B12 of FITC marks at 22 DEG C.
Embodiment 3：VB12-FITC of the present invention preparation
(A) 200mg vitamin B12 is dissolved in 4ml through the dimethyl sulfoxide (DMSO) except water process under nitrogen protection
(DMSO), add lucifuge at 400mg activator N, N'- carbonyl dimidazoles (CDI), 25 DEG C to activate 12 hours, add 800mg
21,8- amino -3,6- dioxaoctane, continue to react 6 hours at 25 DEG C.With 500ml acetone precipitation reaction products, and
Vacuum drying at 10min separation reaction products, 20 DEG C is centrifuged under the conditions of 10000r/min and obtains amidized vitamin B12.
(B) the amination vitamin B12 for obtaining step A, weighs 150ng and is dissolved in 4ml through the dimethyl except water process
Sulfoxide (DMSO), adds 0.1ml catalyst of triethylamine, according to 1：It is small that 1.5 ratios add lucifuge reaction 6 at FITC, 25 DEG C
When, precipitate and separate reaction product, vacuum drying obtains the vitamin B12 of FITC marks at 20 DEG C.
Embodiment 4：VB12-FITC of the present invention analysis
(1) VB12-FITC synthesis checking：Using high performance liquid chromatograph (HPLC), mass spectrometer, detect respectively
The respective wave spectrum of VB12, FITC, molecular weight, verify VB2-FITC coupling.
(2) VB12-FITC tumour absorbs flow cytometer showed：Stomach cancer cell (AGS) is cultivated to 70-80% density, is added
VB12-FITC, is incubated after 4h altogether, after PBS is resuspended twice, using flow cytomery cell to fluorescence labeling vitamin B12
Absorptivity.
(3) VB12-FITC tumour absorbs imaging：Culture stomach cancer cell (AGS) is to 70-80%'s in the burnt ware of copolymerization
Density, adds VB12-FITC, and cell is fixed in different time sections, contaminates nucleus with DAPI, and observed with laser co-focusing
VB12-FITC absorption site and uptake.
(4) VB12-FITC in body tumor imaging：It is stable in stomach cancer cell (AGS) using slow-virus transfection technology
Expression feux rouges carrier segments are transferred to, and then expand culture cell.Take 1 × 107Cell, is injected into nude mice by subcutaneous, builds stomach cancer naked
Mouse model.Before and after tail vein injection VB12-FITC, VB12 tumor-targeting is observed with living imaging instrument respectively.
Mass spectrometry results are as shown in Figure 1：The vitamin B12 of FITC marks, takes divalent charge after ionization,
The fragment ion (1007.41,1020.92,1034.42 abundance highest) for different mass-to-charge ratioes occur is because the dimension that FITC is marked
Raw element B12 molecular structures are more complicated, the cation (Na of varying number on ionization process band+、K+Or H+), such as 1034 from
Two potassium ions, three potassium ions and 3 hydrogen ions on sub- fragment band：1034 × 2-1918 (FITC-VB12)=39 (K+)
×2+23(Na+)×3+3×1(H+)。
High-efficient liquid phase chromatogram technique analysis are as shown in Figure 2：Fluorescence detector FLD is used, using FITC excitation wavelength 490nm,
Launch wavelength is 520nm, in terms of the elution curve of efficient liquid phase, and after FITC modifications VB12, molecular polarity changes, and compares
FITC, faster flows out pillar, FITC-VB12 delivery time is 15.1min, and FITC delivery time is 16.8min.From
FITC-VB12 elution curve sees that the residual for the FITC not reacted is considerably less, illustrates that labeling effciency is higher.
Flow cytometer showed result is as shown in Figure 3：AGS has 90.9% positive rate in FITC passages, and this prompting AGS stomach cancer is thin
Born of the same parents have height intake ability to VB12-FITC.
Laser Scanning Confocal Microscope result is as shown in Figure 4：Increase over time, the stomach cancer cell of FITC mark vitamin B12s
Uptake gradually increases.
Living imaging result is as shown in Figure 5：In gastric cancer in nude mice model, FITC marks vitamin B12 high in tumor locus
Degree aggregation.
Claims (8)
1. a kind of vitamin B12 derivative (VB12-FITC) of fluorescein isothiocynate (FITC) mark, it is characterised in that institute
Stating derivative has following molecular structural formula：
2. VB12-FITC as claimed in claim 1 preparation method, it is characterised in that including：In the low reaction of VB12 structures
On reactive-OH groups, the double amino of coupling, and then reacted with FITC isosulfocyanate radical, synthesize VB12-FITC derivatives.
3. VB12-FITC according to claim 2 preparation method, it is characterised in that comprise the following steps：
(A) amination of vitamin B12：Under inert gas gas shielded, vitamin B12 is dissolved in through the diformazan except water process
In base sulfoxide (DMSO), according to 1:1-1:2 ratios add lucifuge at activator N, N'- carbonyl dimidazoles (CDI), 20-25 DEG C and lived
Change 6-12 hours, according to 1:2-1:5 ratios add diamino compounds, continue to react 3-12 hours at 20-25 DEG C；Precipitation is simultaneously
Separate and be dried to obtain amidized vitamin B12 at reaction product, 20-25 DEG C；
(B)) FITC and amination vitamin B12 coupling：Obtained amination vitamin B12 will be reacted in step A to be dissolved in
Through the dimethyl sulfoxide (DMSO) (DMSO) except water process, according to 1:1-1:2 ratios add catalyst of triethylamine, according to 1:1.5 ratios add
Enter lucifuge at fluorescein isothiocynate (FITC), 20-25 DEG C to react 4-12 hours, precipitate and separate reaction product, at 20-25 DEG C
It is dried to obtain the vitamin B12 of FITC marks.
4. VB12-FITC according to claim 3 preparation method, it is characterised in that：The diamino selected in the step A
Based compound is 1,8- diaminourea -3,6- dioxaoctanes.
5. VB12-FITC according to claim 3 preparation method, it is characterised in that：In the step A and B, in precipitation
When add acetone be used as precipitating reagent.
6. VB12-FITC according to claim 3 preparation method, it is characterised in that：In the step A and B, in separation
During reaction product, 10min is centrifuged under the conditions of 10000r/min using centrifuge, unreacted compound is removed.
7. VB12-FITC according to claim 3 preparation method, it is characterised in that：It is described dry in the step A and B
Dry is vacuum drying.
8. VB12-FITC as claimed in claim 1 is in diagnosis and monitoring VB12 Absorption And Metabolisms approach and tumour is prepared
Image the application in therapeutic agent.
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| CN108299555A (en) * | 2018-02-09 | 2018-07-20 | 武汉伊艾博科技有限公司 | A kind of preparation method of VB12 comlete antigens |
| CN111467322A (en) * | 2020-04-08 | 2020-07-31 | 南方医科大学南方医院 | Synthesis method and application of VB12 targeted sildenafil nano-drug |
| CN111620918A (en) * | 2020-06-19 | 2020-09-04 | 辽宁中医药大学 | 8-beta-D-glucopyranose-4', 7-dihydroxyisoflavone FAM derivative and synthetic method thereof |
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| WO2007027796A2 (en) * | 2005-08-30 | 2007-03-08 | Board Of Regents, The University Of Texas System | Proximity ligation assays with peptide conjugate 'burrs' and aptamers for the sensitive detection of spores and cancer cells |
| US20130316926A1 (en) * | 2012-05-25 | 2013-11-28 | Health Diagnostic Laboratory, Inc. | Method for the monitoring of smoking cessation compliance and recovery, therapeutic intervention, and risk management |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007027796A2 (en) * | 2005-08-30 | 2007-03-08 | Board Of Regents, The University Of Texas System | Proximity ligation assays with peptide conjugate 'burrs' and aptamers for the sensitive detection of spores and cancer cells |
| US20130316926A1 (en) * | 2012-05-25 | 2013-11-28 | Health Diagnostic Laboratory, Inc. | Method for the monitoring of smoking cessation compliance and recovery, therapeutic intervention, and risk management |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108299555A (en) * | 2018-02-09 | 2018-07-20 | 武汉伊艾博科技有限公司 | A kind of preparation method of VB12 comlete antigens |
| CN111467322A (en) * | 2020-04-08 | 2020-07-31 | 南方医科大学南方医院 | Synthesis method and application of VB12 targeted sildenafil nano-drug |
| CN111620918A (en) * | 2020-06-19 | 2020-09-04 | 辽宁中医药大学 | 8-beta-D-glucopyranose-4', 7-dihydroxyisoflavone FAM derivative and synthetic method thereof |
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