CN107033252A - A kind of glycan molecule containing uronic acid of fluorescence labeling and its preparation method and application - Google Patents
A kind of glycan molecule containing uronic acid of fluorescence labeling and its preparation method and application Download PDFInfo
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- CN107033252A CN107033252A CN201710150098.2A CN201710150098A CN107033252A CN 107033252 A CN107033252 A CN 107033252A CN 201710150098 A CN201710150098 A CN 201710150098A CN 107033252 A CN107033252 A CN 107033252A
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- glycan molecule
- fluorescence labeling
- uronic acid
- fluorescence
- containing uronic
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 87
- 238000001215 fluorescent labelling Methods 0.000 title claims abstract description 57
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 56
- 239000002253 acid Substances 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000000502 dialysis Methods 0.000 claims abstract description 12
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 11
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims description 22
- 238000001514 detection method Methods 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 229920001542 oligosaccharide Polymers 0.000 claims description 8
- 150000002482 oligosaccharides Chemical class 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000002372 labelling Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 229920001282 polysaccharide Polymers 0.000 claims description 5
- 239000005017 polysaccharide Substances 0.000 claims description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- 229920005654 Sephadex Polymers 0.000 claims description 4
- 239000012507 Sephadex™ Substances 0.000 claims description 4
- 238000011033 desalting Methods 0.000 claims description 4
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000000975 dye Substances 0.000 claims description 3
- 238000001727 in vivo Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims description 3
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 claims description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 210000003722 extracellular fluid Anatomy 0.000 claims description 2
- 238000005227 gel permeation chromatography Methods 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000007689 inspection Methods 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 11
- 210000000056 organ Anatomy 0.000 abstract description 9
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 9
- 238000003384 imaging method Methods 0.000 abstract description 8
- 230000004060 metabolic process Effects 0.000 abstract description 8
- 229920001223 polyethylene glycol Polymers 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 6
- 150000007513 acids Chemical class 0.000 abstract description 5
- 150000005846 sugar alcohols Chemical class 0.000 abstract description 5
- 238000002523 gelfiltration Methods 0.000 abstract 1
- 229920000855 Fucoidan Polymers 0.000 description 7
- 239000003814 drug Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 235000014633 carbohydrates Nutrition 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 description 4
- -1 mannose ester Chemical class 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 3
- 238000006482 condensation reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 238000011580 nude mouse model Methods 0.000 description 3
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 2
- 229920001287 Chondroitin sulfate Polymers 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 229940059329 chondroitin sulfate Drugs 0.000 description 2
- 229940109262 curcumin Drugs 0.000 description 2
- 235000012754 curcumin Nutrition 0.000 description 2
- 239000004148 curcumin Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N 2,3,4,5-tetrahydroxypentanal Chemical compound OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 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
- KKAJSJJFBSOMGS-UHFFFAOYSA-N 3,6-diamino-10-methylacridinium chloride Chemical compound [Cl-].C1=C(N)C=C2[N+](C)=C(C=C(N)C=C3)C3=CC2=C1 KKAJSJJFBSOMGS-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- 229920000288 Keratan sulfate Polymers 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010029240 Neuritis Diseases 0.000 description 1
- IGLKELDWPZFFKF-UHFFFAOYSA-N OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.P.P Chemical compound OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.P.P IGLKELDWPZFFKF-UHFFFAOYSA-N 0.000 description 1
- 241000199919 Phaeophyceae Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 241000234314 Zingiber Species 0.000 description 1
- 235000006886 Zingiber officinale Nutrition 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229920002892 amber Polymers 0.000 description 1
- 238000001949 anaesthesia Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 229940040387 citrus pectin Drugs 0.000 description 1
- 239000009194 citrus pectin Substances 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000008397 ginger Nutrition 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000000055 hyoplipidemic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- KXCLCNHUUKTANI-RBIYJLQWSA-N keratan Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@H](COS(O)(=O)=O)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H]([C@@H](COS(O)(=O)=O)O[C@@H](O)[C@@H]3O)O)[C@H](NC(C)=O)[C@H]2O)COS(O)(=O)=O)O[C@H](COS(O)(=O)=O)[C@@H]1O KXCLCNHUUKTANI-RBIYJLQWSA-N 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 210000005084 renal tissue Anatomy 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
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
-
- 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
- A61K49/0032—Methine dyes, e.g. cyanine dyes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Materials Engineering (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention relates to glycan molecule containing uronic acid of a kind of fluorescence labeling and its preparation method and application.Carboxyl in the present invention in the glycan molecule containing uronic acid is hydroxysuccinimide-activated through N, it is condensed again with the fluorescence molecule containing amino, fluorescence labeling glycan molecule is purified using dialysis or gel filtration method, the glycan molecule of acids containing alditol of the fluorescence labeling of purifying is obtained.The method of the present invention is used to study tissue and organ distribution and metabolism behavior of the glycan molecule in living animal.The fluorescence labeling method that the present invention is provided is simple to operate, reproducible, with low cost, it is adaptable to all glycan molecule fluorescence labelings containing uronic acid, and for the metabolism and tissue and organ distribution using living animal imaging device research glycan molecule in animal body.
Description
Technical field
The invention belongs to biomedicine technical field, and in particular to a kind of glycan molecule containing uronic acid of fluorescence labeling and its
Preparation method and application.
Background technology
The sugar subcategory containing uronic acid is more in animals and plants and microorganism, glycosaminoglycan such as in animal body (heparin,
Heparan, chondroitin sulfate A (CSA) BCDE, keratan sulfate etc.), it is the pectin in terrestrial plant, the algin in marine algae, brown
Algae carbohydrate gum and its oligomer and derivative etc., these acidic polysaccharoses have various biological activity.Such as heparin and LMWHs
With anti-freezing and antithrombotic acitivity, chondroitin sulfate has anti-neuritis and hypolipidemic activity, and citrus pectin, which has, suppresses tumour
The function of transfer, ocean Carbohydrate drugs PSS, mannose ester by development of raw materials of algin etc. have anti-cerebral thrombus, brain
The disease such as ischemic and hypertension, hyperlipidemia, coronary heart diseases and angina pectoris.Extra large elder brother's kidney by development of raw materials of fucoidan
Happiness capsule has anti-chronic renal failure disease etc., containing abundant carboxyl in these sugared drug molecules.But carbohydrate chemical combination
Thing especially polysaccharide is due to complicated, without specific ultra-violet absorption spectrum so that trace detection is difficult, and then limits it
Internal pharmacokinetic and the bottleneck for turning into sugared drug development.Traditional Carbohydrate drugs dynamic metabolism research method has together
The plain labelling method in position, Immunological Method, red, orange, green, blue, yellow (ROGBY) etc., because process is cumbersome, sensitivity is low, as a result poor repeatability and limited
System.
Fluorescence labeling and living imaging detection technique are due to sensitivity height, and testing result is directly perceived, is widely used in life section
Learn in research and drug development.Research shows that Cy7 can carry out fluorescence labeling to curcumin, and using bioluminescence imaging technology to ginger
Flavine is detected in real time in rat kidney tissue situation, shows that the technology can be studied curcumin in Mice Body intracellular metabolite
(Sun Yong etc., Nanjing University of Traditional Chinese Medicine's journal, 2011,27 (6):539-541).But, for saccharide compound be particularly containing
Whether the glycan molecule of uronic acid can carry out fluorescence labeling, and study its pharmacokinetic characteristics in vivo using similar techniques
And tissue and organ distribution there is no patent or document report.
The content of the invention
For metabolism and the insensitive problem of Histological and organic distribution research method in the glycan molecule body of acids containing alditol, the present invention
Glycan molecule containing uronic acid there is provided a kind of fluorescence labeling and its preparation method and application, the present invention can overcome the disadvantages that prior art is deposited
Weak point.Fluorescence labeling and purification process provided by the present invention are simple, efficiency high, and fluorescence signal is strong, detect sensitive
Degree is high, and as a result reappearance and visuality are good, has significantly especially for the glycan molecule pharmacokinetic of unknown structure
Advantage.
For achieving the above object, the present invention is achieved using following technical proposals：
The invention provides a kind of glycan molecule containing uronic acid of fluorescence labeling, the glycan molecule containing uronic acid of the fluorescence labeling
Obtained by following labeling process：
Wherein, the R is the polysaccharide containing carboxyl, oligosaccharide, oligosaccharides or sugar derivatives molecule, and the fluorescence molecule is with ammonia
At least one of cyanine dye Cy3, Cy5 and Cy7 of base.
Present invention also offers the preparation method of the glycan molecule containing uronic acid of described fluorescence labeling, it includes following step
Suddenly：(1) activated carboxylic：Glycan molecule containing uronic acid is dissolved in 2- morpholino ethane sulfonic acid buffer solutions, 1- ethyls -3- is added
(3- dimethyl aminopropyls)-carbodiimides and n-hydroxysuccinimide, are well mixed and form reaction system, stirring reaction,
Reaction purify after terminating with bag filter dialysis or gel chromatographic columnses, and freezing is done after dialyzate or glycan molecule elution fraction are concentrated under reduced pressure
The dry glycan molecule that must be activated；
(2) fluorescence labeling：The glycan molecule that step (1) is activated is dissolved in phosphate buffer, is added and is contained-NH2Fluorescence
Molecule, forms overall reaction solution, and concentrate is transferred in dialysis desalting in bag filter, dialysis by 2~24h of stirring reaction, concentration
Liquid is concentrated under reduced pressure, and the glycan molecule containing uronic acid of fluorescence labeling is obtained after drying.
Further：1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides and N- hydroxyl ambers in the step (1)
Molar concentration of the amber acid imide in reaction system is 2~20 times of the molar concentration that carboxyl accounts for reaction system in glycan molecule,
Reaction time is 2~24h.
Further：The fluorescence molecule added in the step (2) is carboxyl in the final molar concentration of overall reaction solution
Account for 0.1~10 times of the molar concentration of reaction system.
Further：The glycan molecule containing uronic acid of step (2) described fluorescence labeling is subjected to following purification step：Will be glimmering
The glycan molecule containing uronic acid of signal is purified with gel chromatographic columnses, is detected using Composition distribution and fluorescence detector combination,
Collect Composition distribution and fluorescence detector coincidence signal peak；
Or the glycan molecule of fluorescence labeling obtained by step (2) is dialysed through bag filter, detection extracellular fluid dialysis electrical conductivity no longer changes, sugar
Molecular components be freeze-dried after being concentrated under reduced pressure fluorescence labeling glycan molecule.
Further：The gel chromatography column packing is Sephadex G-10, Sephadex G-15, Sephadex G-
25 or Bio Gel P2, P4 or P6.
Further：The molecular cut off of the step bag filter is 1kDa~10kDa.
Prepared present invention also offers the glycan molecule containing uronic acid of described fluorescence labeling for detecting that glycan molecule exists
Application in the detection agent being metabolized in vivo.
Prepared present invention also offers the glycan molecule containing uronic acid of described fluorescence labeling for detecting glycan molecule in tissue
Application in the detection agent being distributed in organ.
Further：The detection time of the glycan molecule containing uronic acid of the fluorescence labeling is 0.01~24h.
Compared with prior art, advantages of the present invention and good effect are：The glycan molecule of acids containing alditol that the present invention is provided
Fluorescent labelling techniques it is simple to operate, it is adaptable to any polysaccharide containing uronic acid, oligosaccharide, oligosaccharides and its derivative molecular,
With universal applicability, and marked product purity is high, and molecule prototype is kept after mark.Fluorescence labeling glycan molecule prepared by the present invention
Detect that tissue in animal body and organ distribution confirm have fluorescence signal strong through living animal imaging technique, detection sensitivity
Height, the advantages of visual strong, provides new method, with wide application for the sugared medicine live body metabolism research of acids containing alditol
Prospect.
After the embodiment that the present invention is read in conjunction with the figure, further advantage of the invention and feature will become more clear
It is clear.
Brief description of the drawings
Fig. 1 is fluorescence labeling glycan molecule of the present invention different time Histological and organic distribution figure in Mice Body.
Fig. 2 is fluorescence labeling glycan molecule of the present invention in Different Organs distribution detection figure.
Embodiment
Technical scheme is described in further detail with reference to the accompanying drawings and detailed description.
The present invention uses 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides (EDC) and n-hydroxysuccinimide
(NHS) carboxyl of glycan molecule is activated, then be condensed with the fluorescence molecule containing amino, obtain fluorescence labeling glycan molecule, tool
Body labeling process is as follows：
Wherein, the R be the polysaccharide containing uronic acid, oligosaccharide, oligosaccharides or sugar derivatives molecule, the fluorescence molecule be with
At least one of cyanine dye Cy3, Cy5 and Cy7 of amino.
Embodiment 1：The fluorescence labeling of the glycan molecule of acids containing alditol
1st, the 2- morpholino ethane sulfonic acids (MES) that 50mg fucoidans are dissolved in into 1mL 0.1mol/L pH=5.0 are buffered
In liquid, 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides (EDC) and n-hydroxysuccinimide (NHS) are added,
Well mixed to form reaction system, the molar concentration for making EDC and NHS is carboxyl mole in fucoidan molecule respectively
5 times of concentration, stirring reaction 12h, reaction terminates the bag filter dialysis desalting with 7kDa, is freeze-dried after dialyzed solution is concentrated
Obtain the glycan molecule of NHS activation.
2nd, take the fucoidan activated in step 1 through NHS appropriate, add 1mL 0.1mol/L pH=7.2 phosphorus
Phthalate buffer dissolve, add the fluorescence molecule Cy7 containing amino, stirring reaction 12h, using in glycan molecule through overactivation
The amino of carboxyl and fluorometric reagent occurs condensation reaction and fluorescence labeling is carried out to glycan molecule, and concentrate is transferred to 7kDa by concentration
Dialysis desalting in bag filter, dialyzed solution is concentrated under reduced pressure, and the fucoidan of fluorescence labeling is obtained after drying.
3rd, fucoidan described in step 1, and the fucoidan of fluorescence labeling described in step 2 is taken to carry out
Molecular weight is analyzed, and chromatographic column is that OHpak SB-802.5HQ connect with OHpak SB-804HQ gel chromatographic columnses, with 0.1mol/L
Aqueous sodium persulfate solution is mobile phase, and on-line checking is combined using Composition distribution-multi-angle laser scatterometer, calculates fucosan sulphur
Weight average molecular weight and number-average molecular weight in acid esters labeling process.
The fluorescence molecule Cy7 containing amino described in the present embodiment can use instead fluorescence molecule Cy3, Cy5 with amino or
Other fluorometric reagents available for living animal image checking；The molecular cut off of the purifying bag filter can use instead 1kDa~
Any one of 10kDa；The gel chromatographic columnses of the molecular weight determination can use OHpak SB-802HQ, OHpak SB- instead
One or both of 806HQ, TSK-GEL G3000PW/G3000PWXL or SPL aquagel-OH mixed connect.
The present invention carboxylic group of alditol acids glycan molecule is activated by NHS, further with fluorometric reagent molecule
Condensation reaction occurs for amino, so that fluorescence molecule introducing glycan molecule realized to the fluorescence labeling of glycan molecule, due to reaction product
It is water-soluble strong, it can be purified by dialysis or gel filtration chromatography, reaction efficiency is high, simple to operate.
Embodiment 2：Dynamic metabolism and tissue and device of the glycan molecule containing uronic acid of fluorescence labeling in detection glycan molecule
Application in official's distribution
1st, be the 5mg/mL aqueous solution with normal saline by the glycan molecule of fluorescence labeling, physiological saline as blank control group,
According to 0.1mL/10g body weight dose nude mice tail vein injections.
2nd, respectively at 0,15min, 30min, 45min, 1h, 1.5h, 2h, 2.5h and 3h anaesthesia experiment after step 1 injection
Tissue distribution patterns of each experimental group by reagent in Mice Body are observed by nude mice using small animal living body imaging system,
The fluorescence signal of mice belly and back different time is photographed to record respectively.
3rd, each group is tested post mortem at nude mice by living imaging observation after terminating, and brain, heart, lungs, liver are taken respectively
Taken pictures with kidney using living imaging system observation, record Different Organs fluorescence signal.
In the present invention fluorescence labeling glycan molecule different time in experimental mouse body distribution situation as shown in figure 1, can be with from Fig. 1
After finding out that the glycan molecule intravenous injection of mark enters in Mice Body, fluorescence signal can be detected in 15min~3h, and with
The extension of time, signal is gradually assembled to kidney, and the feature of renal excretion is presented.Mark glycan molecule is found simultaneously within 3h, sugar
Molecule does not depart from fluorescence molecule, shows that the fluorescence labeling method is adapted to the Tracing detection of drug molecule in metabolic process.
Present invention mark glycan molecule is as shown in Figure 2 in the fluorescence signal distribution testing result of each organ.It can be further illustrated from Fig. 2 results
Renal tract is focused primarily upon after mark glycan molecule 3h.As needed, the metabolism of mark glycan molecule and Histological and organic distribution feelings
Condition can be detected continuously 24 hours.
To sum up, the method for the invention is activated by the carboxyl to glycan molecule, then is sent out with the amino in fluorescence molecule
Raw condensation reaction, and purified by dialysis and gel filtration chromatography, the rapid fluorescence mark to the glycan molecule containing uronic acid is realized,
The Pharmacokinetic Characteristics and tissue and organ point of mark glycan molecule in animal body are realized using living animal imaging technique
Cloth is analyzed.
The fluorescence labeling method that the present invention is set up is applied to any glycan molecule containing uronic acid, strong with universality, spirit
Sensitivity is high, and the features such as visual result is reliable has a extensive future.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to foregoing reality
Apply example the present invention is described in detail, for the person of ordinary skill of the art, can still implement to foregoing
Described technical scheme is modified in example, or carries out equivalent substitution to which part technical characteristic；And these modification or
Replace, the essence of appropriate technical solution is departed from the spirit and scope of claimed technical solution of the invention.
Claims (10)
1. a kind of glycan molecule containing uronic acid of fluorescence labeling, it is characterised in that the glycan molecule containing uronic acid of the fluorescence labeling
Obtained by following labeling process：
Wherein, the R is the polysaccharide containing carboxyl, oligosaccharide, oligosaccharides or sugar derivatives molecule, and the fluorescence molecule is with ammonia
At least one of cyanine dye Cy3, Cy5 and Cy7 of base.
2. the preparation method of the glycan molecule containing uronic acid of the fluorescence labeling described in claim 1, it is characterised in that it include with
Lower step：
(1) activated carboxylic：Glycan molecule containing uronic acid is dissolved in 2- morpholino ethane sulfonic acid buffer solutions, add 1- ethyls-
3- (3- dimethyl aminopropyls)-carbodiimides and n-hydroxysuccinimide, well mixed to form reaction system, stirring is anti-
Should, reaction is purified after terminating with bag filter dialysis or gel chromatographic columnses, and dialyzate or glycan molecule elution fraction are cold after being concentrated under reduced pressure
Freeze the dry glycan molecule that must be activated；
(2) fluorescence labeling：The glycan molecule that step (1) is activated is dissolved in phosphate buffer, is added and is contained-NH2Fluorescence point
Son, forms overall reaction solution, and concentrate is transferred to dialysis desalting in bag filter, dialyzed solution by 2~24h of stirring reaction, concentration
It is concentrated under reduced pressure, the glycan molecule containing uronic acid of fluorescence labeling is obtained after drying.
3. the preparation method of the glycan molecule containing uronic acid of the fluorescence labeling according to claims 2, it is characterised in that：Institute
1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides and n-hydroxysuccinimide are stated in step (1) in reaction system
Molar concentration be 2~20 times of the molar concentration that carboxyl accounts for reaction system in glycan molecule, the reaction time is 2~24h.
4. the preparation method of the glycan molecule containing uronic acid of the fluorescence labeling according to claims 2, it is characterised in that：Institute
State the molar concentration that the fluorescence molecule added in step (2) accounts for reaction system in the final molar concentration of overall reaction solution for carboxyl
0.1~10 times.
5. the preparation method of the glycan molecule containing uronic acid of the fluorescence labeling according to claims 2, it is characterised in that：Will
The glycan molecule containing uronic acid of step (2) described fluorescence labeling carries out following purification step：By fluorescence labeling containing uronic acid
Glycan molecule is purified with gel chromatographic columnses, is detected using Composition distribution and fluorescence detector combination, is collected Composition distribution and glimmering
Photodetector coincidence signal peak；
Or the glycan molecule of fluorescence labeling obtained by step (2) is dialysed through bag filter, detection extracellular fluid dialysis electrical conductivity no longer changes, sugar
Molecular components be freeze-dried after being concentrated under reduced pressure fluorescence labeling glycan molecule.
6. the preparation method of the glycan molecule containing uronic acid of the fluorescence labeling according to claims 5, it is characterised in that：Institute
State gel chromatography column packing for Sephadex G-10, Sephadex G-15, Sephadex G-25 or Bio Gel P2, P4 or
P6。
7. the preparation method of the glycan molecule containing uronic acid of the fluorescence labeling according to claims 5, it is characterised in that：Institute
The molecular cut off for stating step bag filter is 1kDa~10kDa.
8. the glycan molecule containing uronic acid of the fluorescence labeling described in claims 1 is used to detect glycan molecule generation in vivo preparing
Application in the detection agent thanked.
9. the glycan molecule containing uronic acid of the fluorescence labeling described in claims 1 is being prepared for detecting glycan molecule in organizer
Application in the detection agent being distributed in official.
10. the glycan molecule containing uronic acid of the fluorescence labeling according to claims 9 is being prepared for detecting that glycan molecule exists
Application in the detection agent being distributed in histoorgan, it is characterised in that：The inspection of the glycan molecule containing uronic acid of the fluorescence labeling
The survey time is 0.01~24h.
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| CN107727762A (en) * | 2017-09-28 | 2018-02-23 | 山东大学 | A kind of method of nitrous acid degradation analysis LMWHs disaccharides structure |
| CN109265573A (en) * | 2018-08-20 | 2019-01-25 | 华中科技大学 | A kind of dendrobium polysaccharide fluorescent marker and its synthetic method near infrared imaging |
| CN113588943A (en) * | 2020-11-04 | 2021-11-02 | 北京北方生物技术研究所有限公司 | Natural polymeric polysaccharide used for preparing sample treatment fluid and application thereof in immunochromatography detection kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107727762A (en) * | 2017-09-28 | 2018-02-23 | 山东大学 | A kind of method of nitrous acid degradation analysis LMWHs disaccharides structure |
| CN109265573A (en) * | 2018-08-20 | 2019-01-25 | 华中科技大学 | A kind of dendrobium polysaccharide fluorescent marker and its synthetic method near infrared imaging |
| CN113588943A (en) * | 2020-11-04 | 2021-11-02 | 北京北方生物技术研究所有限公司 | Natural polymeric polysaccharide used for preparing sample treatment fluid and application thereof in immunochromatography detection kit |
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Application publication date: 20170811 |