CN103540312A - Rhodamine fluorescent probe with pseudo nucleic acid base as recognition site and preparation thereof and application to nucleotide image - Google Patents
Rhodamine fluorescent probe with pseudo nucleic acid base as recognition site and preparation thereof and application to nucleotide image Download PDFInfo
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- CN103540312A CN103540312A CN201310470715.9A CN201310470715A CN103540312A CN 103540312 A CN103540312 A CN 103540312A CN 201310470715 A CN201310470715 A CN 201310470715A CN 103540312 A CN103540312 A CN 103540312A
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- rhodamine
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- 239000002773 nucleotide Substances 0.000 title claims abstract description 35
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 35
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 18
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 12
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 12
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 35
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 6
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 238000001953 recrystallisation Methods 0.000 claims description 6
- 238000001291 vacuum drying Methods 0.000 claims description 6
- VHNQIURBCCNWDN-UHFFFAOYSA-N pyridine-2,6-diamine Chemical compound NC1=CC=CC(N)=N1 VHNQIURBCCNWDN-UHFFFAOYSA-N 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- TWCNIAOHBHOUEM-UHFFFAOYSA-N 3-methyl-1,8-naphthyridin-2-amine Chemical class C1=CN=C2N=C(N)C(C)=CC2=C1 TWCNIAOHBHOUEM-UHFFFAOYSA-N 0.000 claims description 3
- PJCCSZUMZMCWSX-UHFFFAOYSA-N 4,4-Dimethoxy-2-butanone Chemical compound COC(OC)CC(C)=O PJCCSZUMZMCWSX-UHFFFAOYSA-N 0.000 claims description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 229910052786 argon Inorganic materials 0.000 claims description 3
- 239000012300 argon atmosphere Substances 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000007789 gas Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 3
- 238000006386 neutralization reaction Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- -1 rhodamine acyl chlorides Chemical class 0.000 claims description 3
- 238000002390 rotary evaporation Methods 0.000 claims description 3
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 229960001866 silicon dioxide Drugs 0.000 claims description 3
- 235000012239 silicon dioxide Nutrition 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000010792 warming Methods 0.000 claims description 3
- 150000003951 lactams Chemical group 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 238000007363 ring formation reaction Methods 0.000 claims description 2
- 238000006467 substitution reaction Methods 0.000 claims description 2
- ZIFGWWCUONMOLI-UHFFFAOYSA-N 7-methyl-1,8-naphthyridin-2-amine Chemical compound C1=CC(N)=NC2=NC(C)=CC=C21 ZIFGWWCUONMOLI-UHFFFAOYSA-N 0.000 claims 2
- 239000000523 sample Substances 0.000 abstract description 52
- ZWIADYZPOWUWEW-XVFCMESISA-N CDP Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O1 ZWIADYZPOWUWEW-XVFCMESISA-N 0.000 abstract description 29
- 238000003384 imaging method Methods 0.000 abstract description 13
- 230000004044 response Effects 0.000 abstract description 10
- 238000002474 experimental method Methods 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 7
- 239000001257 hydrogen Substances 0.000 abstract description 5
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 5
- 230000000295 complement effect Effects 0.000 abstract description 4
- 150000002500 ions Chemical class 0.000 abstract description 4
- 230000009881 electrostatic interaction Effects 0.000 abstract description 3
- CRADWWWVIYEAFR-UHFFFAOYSA-N 1,8-naphthyridin-2-amine Chemical compound C1=CC=NC2=NC(N)=CC=C21 CRADWWWVIYEAFR-UHFFFAOYSA-N 0.000 abstract description 2
- ACNUVXZPCIABEX-UHFFFAOYSA-N 3',6'-diaminospiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(N)C=C1OC1=CC(N)=CC=C21 ACNUVXZPCIABEX-UHFFFAOYSA-N 0.000 abstract description 2
- 238000007142 ring opening reaction Methods 0.000 abstract 2
- 230000001171 adenosinetriphosphoric effect Effects 0.000 abstract 1
- 230000007541 cellular toxicity Effects 0.000 abstract 1
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- 239000012847 fine chemical Substances 0.000 abstract 1
- 238000012632 fluorescent imaging Methods 0.000 abstract 1
- 150000002466 imines Chemical class 0.000 abstract 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 abstract 1
- 229940048102 triphosphoric acid Drugs 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 16
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 10
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 10
- 238000010586 diagram Methods 0.000 description 10
- 239000012530 fluid Substances 0.000 description 9
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 7
- PCDQPRRSZKQHHS-XVFCMESISA-N CTP Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-XVFCMESISA-N 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- 230000009514 concussion Effects 0.000 description 5
- 230000005311 nuclear magnetism Effects 0.000 description 5
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- 230000000536 complexating effect Effects 0.000 description 3
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- 238000009825 accumulation Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
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- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000003068 molecular probe Substances 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
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- 238000005406 washing Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000218636 Thuja Species 0.000 description 1
- CLWRFNUKIFTVHQ-UHFFFAOYSA-N [N].C1=CC=NC=C1 Chemical group [N].C1=CC=NC=C1 CLWRFNUKIFTVHQ-UHFFFAOYSA-N 0.000 description 1
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- 231100000135 cytotoxicity Toxicity 0.000 description 1
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- 229910052805 deuterium Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 238000005649 metathesis reaction Methods 0.000 description 1
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- 239000008055 phosphate buffer solution Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a fluorescent probe, which belongs to the technical field of fine chemical engineering, and discloses a rhodamine fluorescent probe with a pseudo nucleic acid base as a recognition site and preparation thereof and application to a nucleotide image. The ring opening of rhodamine lactone is induced between amino naphthyridine and nucleic acid base through a base analogue complementary hydrogen bond effect, and the selective response of specific nucleotide is realized by using the weakness of electrostatic interaction between imine positive ions generated by a ring opening product and phosphoric acid link negative ions with proper length. The probe shows good selectivity to cytidine diphosphate (CDP) and adenosine triphosphoric acid (ATP). Hela cell copolymerization focal imaging experiments show that the probe can produce a sensitive imaging effect on the nucleotide in the cell, the cell state is good after and before imaging, and the probe has small cell toxicity. Since the probe has good bioavailability and a high-sensitivity imaging effect, the probe is widely applied to medical fluorescent imaging.
Description
Technical field
The present invention relates to a kind of fluorescent probe, specifically, relate to rhodamine fluorescent probe and preparation and the application on Nucleotide image that a kind of nucleic acid analogue base is recognition site.
Background technology
Nucleotide is as the important biological micromolecule of a class, synthetic at human DNA, cell signal transmission, and various material cross-films transport, and transmission ofenergy plays an important role in many vital processes such as genetic expression.And abnormal in many physiological processs, or be all accompanied by the unconventionality expression of certain Nucleotide during body generation pathology.Therefore realize to intracellular nucleic thuja acid in real time, original position, not damaged high resolution image provide effective way directly perceived for people study the many physiological functions of cell.It is to realize one of the most effective approach of the sensitive image of high score that design and development has the molecular probe of specificity response to Nucleotide.Fluoroscopic image technology is because it is convenient to operation, noninvasive, and there is highly sensitive, can realize high-throughput scanning and can obtain real-time original position information welcomed by the people more and more.Therefore design and develop the molecular probe that Nucleotide is had to a fluorescence response and seem particularly important.The Nucleotide fluorescent probe molecule of developing is at present mainly based on three kinds of strategies: (1) constructs multi-arm load positive charge and large conjugated radicle regimental commander chain acceptor, utilizes accumulation and electrostatic interaction between itself and Nucleotide to realize the detection to Nucleotide; (2) utilize the phosphoric acid chain in Nucleotide to realize the response identification to Nucleotide to the coordination of the metal ion in metal complexes (being generally zine ion title complex).This strategy is in fact the identification to phosphoric acid chain realizing, rather than the identification of Nucleotide truly; (3) utilize the metathesis of the fluorophore in the large ring inclusion compound of nucleotide pair to realize the detection to Nucleotide, the accumulation in fact or between simple conjugated system.The excitation wavelength of the above-mentioned fluorescent probe that Nucleotide is detected and emission wavelength are mostly in shortwave district, and during fluoroscopic image, background signal is strong, and shortwave excitation-emission is larger to cell photo-damage, are difficult to investigate accurately the normal physiological function of cell.Meanwhile, the probe that the complementary pairing effect between real analog D NA base realizes the detection of Nucleotide will provide more real biological information for people.Therefore the fluorescent probe that builds excitation-emission on base analog binding mode long wave is expected to address the above problem, and will in clinical medicine image, show huge application prospect.
Summary of the invention
In order to overcome the deficiencies in the prior art, the object of the invention is to provide rhodamine fluorescent probe and preparation and the application on Nucleotide image that a kind of nucleic acid analogue base is recognition site.It is nucleic acid base to be had to the 2-amino-1 of recognition function, the switching mode rhodamine fluorescent signal group that the grafting of 8-naphthyridines excites and launches to long wave, prepare the low damage fluorescent probe of hyperfluorescenceZeng Yongminggaoyingguang Nucleotide to specific respone, this probe not only has special response to Nucleotide, and is applied to the highly sensitive fluorescence imaging of active somatic cell.
In order to realize foregoing invention object, solve existing problem in prior art, the technical scheme that the present invention takes is: the rhodamine fluorescent probe that a kind of nucleic acid analogue base is recognition site, by 7-methyl-2-amino-1,2 bit aminos of 8-naphthyridines and the generation substitution reaction of rhodamine acyl chlorides and ring-closure reaction and be connected on rhodamine five membered lactams rings, have following A molecular structural formula.
Base is a preparation method for the rhodamine fluorescent probe of recognition site, comprises the following steps:
(a) by 2, 6-diamino-pyridine is dissolved in the first reactor that fills appropriate strong phosphoric acid, in argon atmosphere, be heated to 90-95 degree, take 4, 4-dimethoxy-2-butanone is placed in constant voltage separating funnel and dropwise adds 2, in the phosphoric acid solution of 6-diamino-pyridine, be warming up to the rear stirring reaction of 110～115 degree 5～10 hours, be cooled to room temperature, with the phosphoric acid in 10～15% ammonia neutralization reaction solution to pH=8～9, with chloroform extraction 5～6 times, extraction liquid is washed 2～3 times with saturated nacl aqueous solution, with anhydrous magnesium sulfate drying, rotary evaporation chloroform obtains dark red solid matter, with toluene recrystallization, obtain pale yellow powder shape intermediate material 2-amino-methyl-1, 8-naphthyridines AMND, described 2, 6-diamino-pyridine and 4, the mol ratio of 4-dimethoxy-2-butanone is 1:1,
(b) taking rhodamine solid is dissolved in and fills appropriate anhydrous 1, in the second reactor of 2-ethylene dichloride, logical argon gas is also used magnetic stirrer, in the second reactor, dropwise add phosphorus oxychloride again, then be heated to back flow reaction 4～5 hours, vacuum-drying is dissolved in the 3rd reactor that fills appropriate dry acetonitrile after removing reaction solution, take appropriate AMND add in dry acetonitrile, dissolve and add appropriate triethylamine to mix after dropwise join in the 3rd reactor, reflux 5～6 hours, be chilled to room temperature, vacuum-drying, the silicon-dioxide chromatography column purification that the ethyl acetate that the volume ratio of take is 1:1～2 and methylene dichloride are moving phase makes faint yellow solid, with the mixing solutions recrystallization that volume ratio is 1:20～25 normal hexane and methylene dichloride, make the pulverous target product RBS of white solid again, the mol ratio of described rhodamine and phosphorus oxychloride is 1:10～20, and the mol ratio of rhodamine and ANDM is 1:1～1.5, and the mol ratio of rhodamine and triethylamine is 1:10～30.
Base is a rhodamine fluorescent probe for recognition site, the application on Nucleotide image.
Beneficial effect of the present invention is: the rhodamine fluorescent probe that a kind of nucleic acid analogue base is recognition site is by realizing the fluorescence response to Nucleotide by the open loop of the complementary hydrogen bond action induction of base analog rhodamine lactone between amino naphthyridines and nucleotide base, utilizes electrostatic interaction power between cationic imide that open-loop products generates and the phosphoric acid chain negatively charged ion of appropriate length to realize the selective response of specific nucleotide.This probe shows good selectivity to cytidine diphosphate(CDP) (CDP) and adenosine triphosphate (ATP).The experiment of Hela cell co-focusing imaging shows that this probe can produce sensitive imaging effect to intracellular Nucleotide, and before and after imaging, cell state is good, illustrates that this probe cytotoxicity is little.Because this probe has good biocompatibility and high-sensitive imaging effect, so applied widely in medical science fluorescence imaging.
Accompanying drawing explanation
Fig. 1 is the selective response figure of Nucleotide.
Fig. 2 is that probe is to the complementary hydrogen bond action figure of CDP.
Fig. 3 is that fluorescence intensity and absorbancy are with the variation diagram of CDP concentration.
Fig. 4 is that fluorescence intensity and absorbancy are with the variation diagram of ATP concentration.
Fig. 5 is that fluorescence intensity and absorbancy are with the variation diagram of ADP concentration.
Fig. 6 is that fluorescence intensity and absorbancy are with the variation diagram of CTP concentration.
Fig. 7 is the nuclear-magnetism titration experiments figure of probe to CDP.
Fig. 8 is the mass spectrum titration experiments figure of probe to CDP.
Fig. 9 is that probe compares lab diagram to the complexing of CDP.
Figure 10 is RB0053 cell image lab diagram.
Embodiment
Below by embodiment, the invention will be further described.
The preparation of embodiment 1(probe RBS)
By 3.0g(27.5mM) 2, 6-diamino-pyridine is dissolved in the first reactor that fills 35mL strong phosphoric acid, in argon atmosphere, be heated to 90 ℃, take 3.70 grams of (27.5mM) 4, 4-dimethoxy-2-butanone is placed in constant voltage separating funnel and dropwise adds 2, in the phosphoric acid solution of 6-diamino-pyridine, after being warming up to 115 ℃, stirring reaction is 5 hours, be cooled to room temperature, with the phosphoric acid in 15% ammonia neutralization reaction solution to pH=8, with chloroform extraction 5 times, chloroformic solution after extraction is used anhydrous magnesium sulfate drying after washing 3 times with saturated nacl aqueous solution, rotary evaporation chloroform obtains dark red solid matter, with toluene recrystallization, make pale yellow powder shape intermediate material 2-amino-methyl-1, 8 naphthyridines (AMND) 2.30g (52%), 1H NMR (CDCl
3, ppm): 7.82 (d, 1H, J=4.0Hz), 7.80 (d, 1H, J=4.0Hz), 7.07 (d, 1H, J=8.0Hz), 6.71 (d, 1H, J=8.4Hz), 5.08 (s, 2H), 2.69 (s, 3H).Taking 1.0g(2.1mM) rhodamine solid is dissolved in and fills 15mL anhydrous 1, in the second reactor of 2-ethylene dichloride, logical argon gas is also used magnetic stirrer, in the second reactor, dropwise add 1.9mL(21mM again) phosphorus oxychloride, then be heated to back flow reaction 4 hours, vacuum-drying is dissolved in the 3rd reactor that fills the dry acetonitrile of 5mL after removing reaction solution; Take 0.5g(3.15mM) AMND add in the dry acetonitrile of 15mL, dissolve and add 0.5mL(42mM) triethylamine dropwise joins after mixing in the 3rd reactor, reflux 5 hours, be chilled to room temperature, vacuum-drying, the silicon-dioxide chromatography column purification that the ethyl acetate that the volume ratio of take is 1:1 and methylene dichloride are moving phase makes faint yellow solid, then the methylene dichloride that is 25:1 by volume ratio and normal hexane recrystallization make the Powdered target product RBS0.275 of white solid gram (productive rate 22%).1H?NMR(d
6-DMSO,ppm):1.03(12H,m),2.57(3H,s),3.24–3.34(8H,m),6.15(1H,d,J=2.6Hz),6.17(1H,d,2.6,J=8.8Hz),6.40(4H,m),7.05(1H,d,J=8.8Hz),7.32(1H,d,J=7.7Hz),7.59(1H,t,8.8Hz),7.64(1H,t,J=7.7Hz),7.97(1H,d,J=7.2Hz),8.12(1H,d,J=8.0Hz),8.25(1H,d,J=8.8Hz),8.47(1H,d,J=8.8Hz);
13C?NMR168.67,162.27,154.63,154.20,153.52,152.57,148.50,137.32,135.75,133.73,129.90,128.12,128.05,124.30,123.41,121.35,118.25,116.04,107.98,107.40,98.01,66.85,44.26,25.60,12.61;TOF-ESI-MS:Calcd?for[M+2H]
2+:292.6552m/z.Found:m/z292.7144;Calcd?for[M+H]
+:m/z584.3026.Found:m/z584.4264。
Embodiment 2(probe is measured the selective response of Nucleotide)
Take probe 30mg, be mixed with the standard reserving solution of the acetonitrile of 10mM, Nucleotide ADP, ATP, AMP, CDP, CTP, CMP, GDP, GTP, GMP, UDP, UTP, UMP is made into respectively 10mM storing solution.Getting probe storing solution 2 μ L, to join 2mL concentration be that 50mM, pH are in 6.04 TRIS-HCl buffered soln, and concussion shakes up measures its fluorescence intensity (F
0), then add the Nucleotide of 30 μ L, measure under the same conditions fluorescence intensity (F), calculate the relative changing value of its fluorescence intensity, result is as shown in Figure 1.Probe has response in various degree to all Nucleotide, wherein CDP, ATP, ADP and CTP cause that the relative variation of fluorescence intensity is more obvious, and CDP can with probe in naphthyridines form the three strong deuterium bonds of class Nucleotide base complementrity, act on the strongest, therefore change in fluorescence is the most obvious, as shown in Figure 2, in figure, (1), (2), (3) three dotted lines represent three hydrogen bonds to its binding mode, and "+" represents that pyridine nitrogen atom is protonated and be with a positive charge.
Embodiment 3 (fluorescence probe intensity and absorbancy are with the variation of CDP concentration)
Take probe 10mg, be mixed with the standard reserving solution (1) of the water of 10mM, then configure the storing solution (2) of the CDP of 10mM.Measuring storing solution (1) 2 μ L, to join 2mL concentration be that 50mM, pH are that in 6.04 TRIS-HCl buffered soln, concussion shakes up, and adds the storing solution (2) of calculated amount, is mixed with standard testing solution.Measure respectively its fluorescence intensity and absorbancy, test result as shown in Figure 3.Along with the increase of CDP concentration, fluorescence intensity and the absorbance of probe increase gradually.Wherein, figure a is change in fluorescence figure; Figure b is absorbancy variation diagram, and in figure, arrow direction represents the direction that image intensity increases.
Embodiment 4 (fluorescence probe intensity and absorbancy are with the variation of ATP concentration)
Take probe 10mg, be mixed with the standard reserving solution (1) of the water of 10mM, then configure the storing solution (2) of the ATP of 10mM.Measuring storing solution (1) 2 μ L, to join 2mL concentration be that 50mM, pH are that in 6.04 TRIS-HCl buffered soln, concussion shakes up, and adds the storing solution (2) of calculated amount, is mixed with standard testing solution.Measure respectively its fluorescence intensity and absorbancy, test result as shown in Figure 4.Along with the increase of ATP concentration, fluorescence intensity and the absorbance of probe increase gradually.Wherein, figure a is change in fluorescence figure; Figure b is absorbancy variation diagram, and in figure, arrow direction represents the direction that image intensity increases.
Embodiment 5 (fluorescence probe intensity and absorbancy are with the variation of ADP concentration)
Take probe 10mg, be mixed with the standard reserving solution (1) of the water of 10mM, then configure the storing solution (2) of the ADP of 10mM.Measuring storing solution (1) 2 μ L, to join 2mL concentration be that 50mM, pH are that in 6.04 TRIS-HCl buffered soln, concussion shakes up, and adds the storing solution (2) of calculated amount, is mixed with standard testing solution.Measure respectively its fluorescence intensity and absorbancy, test result as shown in Figure 5.Along with the increase of ADP concentration, fluorescence intensity and the absorbance of probe increase gradually.Wherein, figure a is change in fluorescence figure; Figure b is absorbancy variation diagram, and in figure, arrow direction represents the direction that image intensity increases.
Embodiment 6 (fluorescence probe intensity and absorbancy are with the variation of CTP concentration)
Take probe 10mg, be mixed with the standard reserving solution (1) of the water of 10mM, then configure the storing solution (2) of the CTP of 10mM.Measuring storing solution (1) 2 μ L, to join 2mL concentration be that 50mM, pH are that in 6.04 TRIS-HCl buffered soln, concussion shakes up, and adds the storing solution (2) of calculated amount, is mixed with standard testing solution.Measure respectively its fluorescence intensity and absorbancy, test result as shown in Figure 6.Along with the increase of CTP concentration, fluorescence intensity and the absorbance of probe increase gradually.Wherein, figure a is change in fluorescence figure; Figure b is absorbancy variation diagram, and in figure, arrow direction represents the direction that image intensity increases.
The nuclear-magnetism titration experiments of embodiment 7(probe to CDP)
Take probe 0.0015g and be dissolved in the deuterated DMSO/D that 0.5mL volume ratio is 4:1
2in O, obtain 5mM test fluid (1), get GMP and be dissolved in the deuterated DMSO/D that 0.5mL volume ratio is 4:1
2o, in 10mM test fluid (2).Measure test fluid (1)
1hNMR spectrum and
31pNMR, then adds the test fluid (2) of equivalent, measures
1hNMR spectrum
31pNMR, as shown in Figure 7.In figure, a is CDP nuclear-magnetism curve, and b is the nuclear-magnetism curve of CDP and probe mixed solution, and c is probe nuclear-magnetism curve.Experimental result signify hydrogen nuclear magnetic resonance spectrum and phosphorus nuclear magnetic resonance spectrum have all produced obvious displacement, illustrate between probe and CDP and interact.
The mass spectrum titration experiments of embodiment 8(probe to CDP)
Take probe 0.0015g and be dissolved in 0.5mL acetonitrile, obtain 5mM test fluid (1), get CDP and be dissolved in 0.5mL water, obtain 5mM test fluid (2).To test (1) equal-volume and join in test fluid (2), direct injection negative mode acquired signal, can obtain total mass number and be 1007.0087 mass spectra peak is [probe+CDP-Na]
-fignal center, as shown in Figure 8.Illustrate between probe and CDP and can form 1:1 complex compound.Wherein (a) is Theoretical Calculation simulation curve; (b) be experimental calculation simulation curve.
The complexing ratio experiment of embodiment 9(probe to CDP)
Take and get probe 0.0015g and be dissolved in 0.5mL acetonitrile, obtain 10mM test solution (1), get CDP and be dissolved in 0.5mL water, obtain 10mM test fluid (2).The total concn of stationary probe and CDP is 10mM, continuously change the concentration ratio of the two and be mixed with concentration ratio and be respectively 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8 test soln, and measure its absorbance, with concentration ratio to absorbancy mapping as shown in Figure 9, at concentration ratio, be that 0.5 o'clock test fluid absorbance reaches maximum, illustrate that the complexing ratio of probe and CDP is 1.
The experiment of embodiment 10(probe cell imaging)
With the HeLa cultivating 24 hours, carry out cell imaging experiment.First the HeLa cell of cultivating in cell culture incubator 24 hours is hatched 30 minutes with 10 μ M probes in the phosphate buffer solution of PH=6.04,40 times of eyepiece imagings on Olympus FV1000 fluorescence co-focusing imager, then use physiological saline drip washing cell three times, with CDP and the ATP of pH=6.04, hatch 30 minutes again, be placed in again 40 times of eyepiece imagings on fluorescence co-focusing imager, front and back twice imaging contrast, can see obvious image reinforced effects.As shown in figure 10.(a) be the cell striograph of probe; (b) be the cell striograph of RBS+CDP; (c) be the cell striograph of probe+ATP; (d) be the cell striograph of probe+ADP; (e) be the cell striograph of probe+CTP.
Claims (3)
1. the rhodamine fluorescent probe that nucleic acid analogue base is recognition site, it is characterized in that: by 7-methyl-2-amino-1,2 bit aminos of 8-naphthyridines and the generation substitution reaction of rhodamine acyl chlorides and ring-closure reaction and be connected on rhodamine five membered lactams rings, have following A molecular structural formula.
2. a preparation method for the rhodamine fluorescent probe that nucleic acid analogue base is recognition site, is characterized in that comprising the following steps:
(a) by 2, 6-diamino-pyridine is dissolved in the first reactor that fills appropriate strong phosphoric acid, in argon atmosphere, be heated to 90-95 degree, take 4, 4-dimethoxy-2-butanone is placed in constant voltage separating funnel and dropwise adds 2, in the phosphoric acid solution of 6-diamino-pyridine, be warming up to the rear stirring reaction of 110～115 degree 5～10 hours, be cooled to room temperature, with the phosphoric acid in 10～15% ammonia neutralization reaction solution to pH=8～9, with chloroform extraction 5～6 times, extraction liquid is washed 2～3 times with saturated nacl aqueous solution, with anhydrous magnesium sulfate drying, rotary evaporation chloroform obtains dark red solid matter, with toluene recrystallization, make pale yellow powder shape intermediate material 2-amino-methyl-1, 8-naphthyridines AMND, described 2, 6-diamino-pyridine and 4, the mol ratio of 4-dimethoxy-2-butanone is 1:1,
(b) taking rhodamine solid is dissolved in and fills appropriate anhydrous 1, in the second reactor of 2-ethylene dichloride, logical argon gas is also used magnetic stirrer, in the second reactor, dropwise add phosphorus oxychloride again, then be heated to back flow reaction 4～5 hours, vacuum-drying is dissolved in the 3rd reactor that fills appropriate dry acetonitrile after removing reaction solution, take appropriate AMND add in dry acetonitrile, dissolve and add appropriate triethylamine to mix after dropwise join in the 3rd reactor, reflux 5～6 hours, be chilled to room temperature, vacuum-drying, the silicon-dioxide chromatography column purification that the ethyl acetate that the volume ratio of take is 1:1～2 and methylene dichloride are moving phase makes faint yellow solid, with the mixing solutions recrystallization that volume ratio is 1:20～25 normal hexane and methylene dichloride, make the pulverous target product RBS of white solid again, the mol ratio of described rhodamine and phosphorus oxychloride is 1:10～20, and the mol ratio of rhodamine and ANDM is 1:1～1.5, and the mol ratio of rhodamine and triethylamine is 1:10～30.
3. the rhodamine fluorescent probe that nucleic acid analogue base is recognition site, is characterized in that the application on Nucleotide image.
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| CN104447774A (en) * | 2014-11-12 | 2015-03-25 | 南京理工大学 | Rhodamine B-based fluorescence sensor and preparation |
| CN105567216A (en) * | 2015-10-27 | 2016-05-11 | 西北大学 | Lysosome targeted fluorescent probe and preparation method and application thereof |
| CN105820597A (en) * | 2016-04-24 | 2016-08-03 | 河南师范大学 | Fluorescent dye containing high-energy phosphate bond |
| CN115636853A (en) * | 2022-09-14 | 2023-01-24 | 南昌大学 | Nucleotide anion recognition ferrocene receptor based on fluorescent indicator displacement method and preparation method and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357045A (en) * | 2014-11-05 | 2015-02-18 | 北京化工大学 | Synthesis method of spiropyrane small-molecule fluorescent probe with extreme acid/extreme alkaline switch response and application of spiropyrane small-molecule fluorescent probe |
| CN104447774A (en) * | 2014-11-12 | 2015-03-25 | 南京理工大学 | Rhodamine B-based fluorescence sensor and preparation |
| CN104447774B (en) * | 2014-11-12 | 2016-07-06 | 南京理工大学 | Rhodamine B-based fluorescence sensor and preparation |
| CN105567216A (en) * | 2015-10-27 | 2016-05-11 | 西北大学 | Lysosome targeted fluorescent probe and preparation method and application thereof |
| CN105820597A (en) * | 2016-04-24 | 2016-08-03 | 河南师范大学 | Fluorescent dye containing high-energy phosphate bond |
| CN105820597B (en) * | 2016-04-24 | 2017-09-29 | 河南师范大学 | Fluorescent dye containing energy-rich phosphate bond |
| CN115636853A (en) * | 2022-09-14 | 2023-01-24 | 南昌大学 | Nucleotide anion recognition ferrocene receptor based on fluorescent indicator displacement method and preparation method and application thereof |
| CN115636853B (en) * | 2022-09-14 | 2024-06-04 | 南昌大学 | Nucleotide anion recognition ferrocene receptor based on fluorescent indicator substitution method and preparation method and application thereof |
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