CN111606892B - Fluorescein couplet and synthesis method and application thereof - Google Patents
Fluorescein couplet and synthesis method and application thereof Download PDFInfo
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- CN111606892B CN111606892B CN202010324004.0A CN202010324004A CN111606892B CN 111606892 B CN111606892 B CN 111606892B CN 202010324004 A CN202010324004 A CN 202010324004A CN 111606892 B CN111606892 B CN 111606892B
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- 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 title claims abstract description 88
- 238000001308 synthesis method Methods 0.000 title abstract description 7
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 71
- 230000001580 bacterial effect Effects 0.000 claims abstract description 24
- 241000894006 Bacteria Species 0.000 claims abstract description 13
- 150000001875 compounds Chemical class 0.000 claims abstract description 13
- 239000000523 sample Substances 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 28
- KWGRBVOPPLSCSI-WCBMZHEXSA-N pseudoephedrine Chemical class CN[C@@H](C)[C@@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WCBMZHEXSA-N 0.000 claims description 12
- 230000002194 synthesizing effect Effects 0.000 claims description 7
- 238000006467 substitution reaction Methods 0.000 claims description 6
- 238000007259 addition reaction Methods 0.000 claims description 5
- 108010090343 pseudopaline Proteins 0.000 claims description 4
- 238000010189 synthetic method Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 11
- 230000015572 biosynthetic process Effects 0.000 abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 8
- 238000011161 development Methods 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract description 2
- 125000000542 sulfonic acid group Chemical group 0.000 abstract description 2
- 239000007864 aqueous solution Substances 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 53
- 239000002609 medium Substances 0.000 description 24
- 239000000872 buffer Substances 0.000 description 20
- 230000000694 effects Effects 0.000 description 18
- 229910000368 zinc sulfate Inorganic materials 0.000 description 18
- 239000011686 zinc sulphate Substances 0.000 description 18
- 238000011534 incubation Methods 0.000 description 17
- 239000007788 liquid Substances 0.000 description 17
- 230000005284 excitation Effects 0.000 description 15
- 239000007787 solid Substances 0.000 description 13
- 239000000725 suspension Substances 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical group CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- 238000012258 culturing Methods 0.000 description 8
- 238000000799 fluorescence microscopy Methods 0.000 description 8
- 238000002372 labelling Methods 0.000 description 8
- 241000588626 Acinetobacter baumannii Species 0.000 description 7
- 241000588747 Klebsiella pneumoniae Species 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 241000588697 Enterobacter cloacae Species 0.000 description 6
- 241000194031 Enterococcus faecium Species 0.000 description 6
- 241000191967 Staphylococcus aureus Species 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000003384 imaging method Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000006862 quantum yield reaction Methods 0.000 description 5
- 210000002784 stomach Anatomy 0.000 description 5
- 206010059866 Drug resistance Diseases 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 229940125898 compound 5 Drugs 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- WEVYAHXRMPXWCK-FIBGUPNXSA-N acetonitrile-d3 Chemical compound [2H]C([2H])([2H])C#N WEVYAHXRMPXWCK-FIBGUPNXSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 229940125782 compound 2 Drugs 0.000 description 3
- 239000006059 cover glass Substances 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- 239000012737 fresh medium Substances 0.000 description 3
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- 239000002207 metabolite Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 238000005842 biochemical reaction Methods 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- KWGRBVOPPLSCSI-UHFFFAOYSA-N d-ephedrine Natural products CNC(C)C(O)C1=CC=CC=C1 KWGRBVOPPLSCSI-UHFFFAOYSA-N 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- -1 fluorescein compound Chemical class 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 description 2
- 125000002924 primary amino group Chemical class [H]N([H])* 0.000 description 2
- 229960003908 pseudoephedrine Drugs 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical group [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 206010014665 endocarditis Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
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- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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Abstract
The invention belongs to the technical field of bacteria identification materials, and particularly relates to a fluorescein couplet and a synthesis method and application thereof. The structure of the fluorescein couplet is shown in a general formula I, wherein R represents fluorescein containing electronegative phenolic hydroxyl or sulfonic acid groups; m is an integer of 1 to 10. The obtained fluorescein couplet has the advantages of selection specificity on pseudomonas aeruginosa, high stability and easy reaction in aqueous solution, and can be used as a fluorescein probe for identifying the pseudomonas aeruginosa. The synthesis method adopts a modular synthesis route, can be applied to the chemical synthesis of compounds with similar structures, and opens up a wide development space for novel bacterial diagnostic reagents.
Description
Technical Field
The invention belongs to the technical field of bacteria identification materials, and particularly relates to a fluorescein couplet and a synthesis method and application thereof; further relates to a pseudoplaine derivative coupled fluorescein couplet with a specific stereo configuration, a synthetic method and application thereof.
Background
Compared with gram-positive bacteria, gram-negative bacteria have the outer membrane protection, are easier to generate drug resistance, and are more difficult to treat. The reason for the generation of drug resistance is mainly that the bacteria causing infection cannot be identified rapidly, so that the clinical treatment often selects broad-spectrum antibiotics, and further the generation of cross-drug resistance is caused.
The pseudomonas aeruginosa is one of three pathogenic bacteria with the most serious drug resistance, and the currently common clinical identification methods of the pseudomonas aeruginosa mainly comprise biochemical reaction based on bacterial isolation culture, immunological detection, molecular biological detection based on nucleic acid hybridization and PCR and the like. Identification methods based on biochemical reactions of bacterial isolation and culture require long treatment times and are also not suitable for the diagnosis of deep-seated infections, such as infections associated with biological materials or endocarditis. Other methods or procedures for identification are complicated, or have insignificant specificity, and the effect is not very desirable.
The prior document CN110627727A discloses a metabolite of pseudomonas aeruginosa and a derivative thereof, and a preparation method and application thereof, and particularly discloses that the metabolite of pseudomonas aeruginosa and the derivative thereof form an amide bond by coupling primary amine and antibiotic, and the obtained ferricin antibiotic has higher antibacterial activity on gram-negative bacteria. Based on this, we attempted to exploit this property of metabolites of pseudomonas aeruginosa and derivatives thereof to achieve the development of bacterial detection reagents.
Disclosure of Invention
In order to solve the above problems, the present invention provides a pseudocaline derivative-conjugated fluorescein conjugate having a specific steric configuration; the fluorescent probe has selection specificity on pseudomonas aeruginosa and can be used as a fluorescent probe for identifying the pseudomonas aeruginosa.
The structure of the fluorescein couplet is shown in a general formula I:
wherein (S) represents that the spatial configuration of the chiral carbon at the corresponding position is S;
r represents fluorescein containing a negatively charged phenolic hydroxyl group or a sulfonic acid group;
m is an integer of 1 to 10.
The fluorescein probe used for the thallus identification needs to meet a plurality of conditions, such as higher fluorescence intensity and stability, easy water solubility, higher selection specificity to thallus, simpler and easier synthetic process and the like; the technical personnel of the invention find that not all sites with higher reactivity in the pseudoephedrine derivatives are suitable for being coupled with fluorescein when researching the structures of all pseudoephedrine derivatives obtained by the existing artificial synthesis; meanwhile, the existing fluorescein has more types, and although part of the fluorescein has higher fluorescence intensity, the requirement of stable coupling with pseudocaline derivatives and obvious selection specificity on pseudomonas aeruginosa is difficult to meet at the same time.
The technical personnel of the invention screen out pseudoephedrine derivatives with the structure shown in the general formula I after carrying out a large number of tests, and the primary amine site of the pseudoephedrine derivatives is coupled with fluorescein containing negative phenolic hydroxyl or sulfonic group, so that the obtained fluorescein coupler not only has the advantages of high fluorescence intensity and high stability, but also has selection specificity to pseudomonas aeruginosa and good water solubility, and can be used as a fluorescein probe for identifying the pseudomonas aeruginosa.
As one embodiment of the present invention, the fluorescein couplet is selected from one of the following structural formulas, m is an integer of 1 to 10; n is an integer of 1 to 10:
the research shows that all the four fluorescein couplets can specifically recognize pseudomonas aeruginosa; of these, I-1 and I-2 are preferable, and I-1 is more preferable. Further research shows that the I-1 fluorescein couplet has higher fluorescence quantum yield, longer service life and better stability compared with other structural formulas.
The invention also provides a synthetic method of the fluorescein couplet, which comprises the following steps: carrying out addition or substitution reaction on pseudoephedrine derivatives and fluorescein to obtain a fluorescein couplet;
the pseudoplaline derivative has the following structure:
wherein m is an integer of 1 to 10.
The invention provides a synthetic route of pseudoephedrine derivatives and fluorescein couplets with specific three-dimensional configurations, and the modularized synthetic route can be applied to chemical synthesis of compounds with similar structures, thereby opening up a wide development space for novel bacterial diagnostic reagents.
In one embodiment of the present invention, the method for synthesizing a fluorescein couplet comprises: when the fluorescein is a compound 2, obtaining a fluorescein couplet I-1 through an addition reaction between the fluorescein and the pseudoephedrine derivative under an alkaline condition;
the specific synthetic route is as follows:
specifically, the synthesis method comprises the following steps: dissolving pseudoplaline derivative compound 1 in water, adding an alkaline reagent and a fluorescein compound 2 at room temperature to perform addition reaction, and purifying to obtain the fluorescein couplet I-1.
Wherein the alkaline reagent is selected from triethylamine, potassium carbonate, sodium bicarbonate or diisopropylethylamine and the like.
Wherein the time of the addition reaction is 11-13h, and preferably 12 h.
In another embodiment of the present invention, the method for synthesizing a fluorescein couplet comprises: when the fluorescein is a compound 4, obtaining a fluorescein couplet I-2 through substitution reaction between the fluorescein and the pseudoephedrine derivative under alkaline conditions;
the specific synthetic route is as follows:
wherein n is an integer of 1 to 10.
Further, the synthesis method comprises the following steps: dissolving pseudoplaline derivative compound 1 and an alkaline reagent in water, adding acetonitrile and a fluorescein compound 4 to carry out substitution reaction, and purifying to obtain the fluorescein couplet I-2.
Wherein the alkaline reagent is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, triethylamine or diisopropylethylamine and the like.
Wherein the time of the substitution reaction is 1-3h, preferably 2 h.
In the present invention, the above-mentioned fluorescein couplet I-3 and fluorescein couplet I-4 can be synthesized according to the method for synthesizing fluorescein couplet I-2 (i.e., compound 5), except that the corresponding compound is synthesized according to step (2) of synthesizing I-2 (i.e., compound 5) by replacing fluorescein compound 4 with the corresponding fluorescein structural formula.
The method provides a synthetic route of pseudocaline coupled fluorescein with a specific three-dimensional configuration, and the modularized synthetic route can be applied to the chemical synthesis of compounds with similar structures and related derivatives, so that a wide development space is opened up for novel bacterial diagnostic reagents.
The invention also provides application of the fluorescein couplet in identification of bacteria, wherein the bacteria are pseudomonas aeruginosa.
According to some embodiments of the invention, the fluorescein conjugate is conjugated to Zn2+Forming a complex to realize the identification of pseudomonas aeruginosa; preferably, the fluorescein conjugate is conjugated with the Zn2+The molar ratio of (a) to (b) is 1:1 to 1:5, more preferably 1: 1.
According to some embodiments of the invention, the fluorescein conjugate enables identification of pseudomonas aeruginosa by an exogenous transport system for pseudocaline molecules. Preferably, the concentration of the Pseudopaline molecule is 10-100. mu.M, preferably 10-50. mu.M.
According to some embodiments of the invention, the fluorescein conjugate is used at a concentration of no more than 50 μ M. At this concentration, the fluorescein couplet had no effect on cell survival, and the safety was high.
The invention has the following beneficial effects: the invention provides a pseudoplaline derivative coupled fluorescein couplet with a specific stereo configuration; the fluorescent probe can be used for diagnosing bacterial infection as a fluorescein probe, can obtain an identification result more quickly and accurately, and has important significance for developing a novel pseudocaline detection technology.
Drawings
FIG. 1 shows the labeling effect of P-FL (I-1) and P-Cy7(I-2) on P.aeruginosa.
FIG. 2 is a confocal laser scanning microscope image of P-FL/Zn, P-FL and FL treated P-M.aeruginosa. Wherein, the left graph: 485 plus or minus 20nm excitation/516 plus or minus 20nm emission fluorescence imaging; right panel: and (4) bright field imaging. The scale unit in the figure is 50 μm.
FIG. 3 is a P-FL (I-1) marker Pseudomonas aeruginosa dependent Pseudomonas aeruginosa transport system. (A) P-FL/Zn pairs in CDM, CDM +5 μ M ZnSO4LB or LB + 5. mu.M ZnSO4Marking effect of cultured pseudomonas aeruginosa. (B) The marking effect of P-FL/Zn on the pseudomonas aeruginosa intracellular after the exogenous addition of 10 mu M, 50 mu M or 100 mu M pseudoephedrine.
FIG. 4 is fluorescence imaging of P-FL/Zn treated Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, enterococcus faecium, Raw264.7 cells, and Hela cells. The method comprises the following steps: 485 plus or minus 20nm excitation/516 plus or minus 20nm emission fluorescence imaging; the following: and (4) bright field imaging. The scale units in the figure are 20 μm except for Acinetobacter baumannii and Klebsiella pneumoniae which are 25 μm.
FIG. 5 shows fluorescence images of P-FL/Zn treated Pseudomonas aeruginosa mixed with Staphylococcus aureus (20 μm in scale), Enterobacter cloacae (12 μm in scale), Acinetobacter baumannii (10 μm in scale), Klebsiella pneumoniae (25 μm in scale), and enterococcus faecium (20 μm in scale). The method comprises the following steps: fluorescence and bright field overlay images; the method comprises the following steps: 485 plus or minus 20nm excitation/516 plus or minus 20nm emission fluorescence imaging; the following: and (4) bright field imaging.
FIG. 6 is fluorescence imaging of P-FL/Zn treated Pseudomonas aeruginosa infected Raw264.7 cells and Hela cells. Left: fluorescence and bright field overlay images; the method comprises the following steps: 485 plus or minus 20nm excitation/516 plus or minus 20nm emission fluorescence imaging; and (3) right: and (4) bright field imaging. The scale unit in the figure is 20 μm.
FIG. 7 is fluorescence imaging of P-FL/Zn treated mice after infection of stomach tissue sections with P.aeruginosa. Left: fluorescence and bright field overlay images; the method comprises the following steps: 485 plus or minus 20nm excitation/516 plus or minus 20nm emission fluorescence imaging; and (3) right: and (4) bright field imaging. The scale unit in the figure is 15 μm.
FIG. 8 shows the cytotoxicity test of P-FL/Zn on Raw264.7 cells and Hela cells.
FIG. 9 shows nuclear magnetic hydrogen spectrum of fluorescein couplet I-1.
FIG. 10 shows nuclear magnetic carbon spectra of a fluorescein couplet I-1.
FIG. 11 shows nuclear magnetic hydrogen spectra of fluorescein couplet I-2.
FIG. 12 shows nuclear magnetic carbon spectra of a fluorescein couplet I-2.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
EXAMPLE 1 Synthesis of fluorescein couplet I-1
Synthesis of fluorescein couplet I-1:
compound 2(4.8mg, 12.3. mu. mol,1eq) and 1(5.6mg, 12.3. mu. mol,1eq) were dissolved in water (1.5mL)), and triethylamine (8.5. mu.L, 61.5. mu. mol,5eq) was added at room temperature, followed by stirring for 10 hours to obtain yellow solid 3(2.3mg, 2.71. mu. mol, 22%) by high performance liquid purification.
1H NMR(400MHz,D2O) δ 8.27(s,1H),7.97(s,1H),7.78(d, J ═ 8.4Hz,1H),7.62(d, J ═ 9.8Hz,2H),7.41(d, J ═ 8.1Hz,1H),7.35(d, J ═ 2.0Hz,2H),7.17(dd, J ═ 9.2,2.0Hz,2H),4.48(t, J ═ 5.6Hz,2H),4.36(d, J ═ 5.7Hz,1H),4.12(t, J ═ 6.4Hz,1H),4.08(t, J ═ 6.4Hz,1H), 3.64-3.56 (m,2H), 3.50-3.38 (m,4H), 2.72-2.57 (m), 2.57 (m-2H), 2.48 (m-3.29H), 2H) (fig. 93-3.48 (m,2H) (fig. 29.3.7 (m, 2H);13C NMR(239MHz,D2O/CD3CN) δ 180.8,176.2,171.3,170.8,170.2,168.7,166.0,156.7,141.0,140.7,131.8,129.8,129.6,128.5,124.9,117.3,114.4,102.9,100.0,62.8,60.3,60.0,59.0,50.3,44.0,29.9,27.1,26.6,25.5,25.2,25.1 (fig. 10); IR (neat) vmax 3209,2292,2851,1608,1393,1115cm–1；HRMS(ESI):[M+H]+Calculated values: c39H40N7O13846.2410, found value 846.2440; [ alpha ] to]22 D+18.0(c 0.05,H2O/ACN)；m.p.＞330℃。
EXAMPLE 2 Synthesis of fluorescein couplet I-2
Synthesis of fluorescein couplet I-2:
compound 1(3.5mg, 6.4. mu. mol,1eq) and sodium bicarbonate (2.7mg, 32. mu. mol,5eq) were dissolved in water (1mL), acetonitrile (1mL) and compound 4(6.0mg, 7.7. mu. mol,1.2eq) were added at room temperature, stirred for 2 hours, and the gel column was purified to give green solid 5(3.8mg, 3.0. mu. mol, 47%).
1H NMR(600MHz,D2O/30％CD3CN) δ 8.66(s,1H), 8.21-8.13 (m,2H), 8.11-8.04 (m,5H), 7.88-7.77 (s,1H),7.55(d, J ═ 30.5Hz,2H), 6.89-6.73 (m,2H),6.53(dd, J ═ 37.9,12.1Hz,2H),4.60(br.s,2H),4.33(br.s,2H),4.27(br.s,2H),4.18(br.s,1H), 3.96-3.82 (m,2H),3.54(br.s,4H),3.33(br.s,2H),2.72(br.s,2H),2.41(br.s,2H),2.08 (br.08 (br.s,2H), 2.33 (br.s,2H),2.72(br.s,2H),2.41(br.s,2H),2.08 (br.3.s, 2H) (1H), 3.3.3.3.3.3.3.3.3.3.3.3H) (br.s, 3.3.3.3.3H), 3.3.s, 3.3.3.3.;13C NMR(151MHz,D2O/30％CD3CN) δ 175.9,172.8,172.7,172.3,172.2,156.7,152.6,151.8,144.6,144.0,142.0,141.6,141.5,140.2,139.9,139.8,126.9,126.8,126.7,126.6,124.7,120.0,120.0,110.9,104.6,104.4,63.0,62.8,50.1,49.8,49.3,49.2,49.1,49.0,44.0,39.5,39.0,38.6,35.7,27.1,26.9,26.8,26.8,26.6,26.5,25.9,25.7,25.3,23.9,11.8 (fig. 12); HRMS (ESI) [ M-H ]]2-Calculated values: c53H68N8O15S2560.2128, found 560.2130.
The fluorescein couplet I-3 and I-4 can be synthesized according to the method of the compound I-2 (namely the compound 5), except that the synthesis block 4 is replaced by a corresponding synthesis block, and the target compound is synthesized according to the step of synthesizing the I-2 (namely the compound 5).
Effect verification
1. Determination of fluorescence quantum yield and lifetime of fluorescein couplet I-1 and I-2
mu.L of 10mM ZnSO was added to 10. mu.L of 1mM P-FL (I-1), FL (5-carboxyfluorescein), P-Cy7(I-2) or Cy74After incubation at 37 ℃ for 15 minutes, 990. mu.L of PBS was added and diluted to 10. mu.M. The fluorescence quantum yield of the compound was measured by Nanolog FL3-2iHR, and the fluorescence lifetime of the compound was measured by Deltaflex.
As a result, as shown in Table 1, the fluorescence quantum yield and lifetime of fluorescein conjugated to pseudoephedrine were not affected.
TABLE 1 fluorescence quantum yield and lifetime of fluorescein conjugates I-1, I-2
2. Activity test of fluorescein couplet I-1 and I-2
The labeling effect of the pseudoplaine fluorescein couplet P-FL (I-1) and P-Cy7(I-2) on Pseudomonas aeruginosa. The specific method comprises the following steps: pseudomonas aeruginosa was inoculated on LB solid medium plate and cultured overnight at 37 ℃. Single colonies were picked from the plate, inoculated into LB liquid medium, and cultured overnight at 37 ℃ with shaking at 200 rpm. 1mL of overnight culture was centrifuged, and the cells were washed 2 times with PBS buffer and then resuspended in 1mL of PBS buffer. Inoculating 100 μ L of the resuspended bacterial solution into 10mL CDM medium[6]In (c), the cells were cultured at 37 ℃ for 12 hours with shaking at 200 rpm. P-FL (I-1) and P-Cy7(I-2) with equimolar amounts of ZnSO4Incubate at 37 ℃ for 15 minutes, using FL (5-carboxyfluoroscein) or Cy7 as control. 1mL of cultured Pseudomonas aeruginosa bacterial liquid was centrifuged, the supernatant was discarded, and the resulting solution was resuspended in 1mL of PBS, and then 10. mu.M P-FL (I-1) or 30. mu.M P-Cy7(I-2) was added to the resulting suspension, followed by incubation at 37 ℃ for 15 minutes. After incubation, the cells were collected by centrifugation, washed 3 times with PBS, and then resuspended in PBS. The labeling effect of P-FL (I-1) on Pseudomonas aeruginosa was detected at 485nm/535nm (excitation/emission) and the labeling effect of P-Cy7(I-2) on Pseudomonas aeruginosa was detected at 749nm/788nm (excitation/emission) using a TECAN F200 PRO microplate reader (FIG. 1).
The results show that: after the pseudomonas aeruginosa is incubated with P-FL (I-1) or P-Cy7(I-2), the fluorescence intensity of the thallus is obviously enhanced, which indicates that the two compounds can mark the pseudomonas aeruginosa.
3. Pseudoplaline fluorescein couplet P-FL (I-1) requires Zn2+Can form a complex to mark pseudomonas aeruginosa
The specific method comprises the following steps: pseudomonas aeruginosa was inoculated on LB solid medium plate and cultured overnight at 37 ℃. Single colonies were picked from the plate, inoculated into LB liquid medium, and cultured overnight at 37 ℃ with shaking at 200 rpm. 1mL of overnight culture was centrifuged, and the cells were washed 2 times with PBS buffer and then resuspended in 1mL of PBS buffer. 100. mu.L of the resuspended suspension was inoculated into 10mL CDM medium and cultured at 37 ℃ under shaking at 200rpm for 12 hours. P-FL (I-1) and equimolar ZnSO4Incubate at 37 ℃ for 15 minutes. Taking 1mL of cultured pseudomonas aeruginosa bacterial liquid, centrifuging, discarding supernatant, suspending into 1mL of PBS, adding ZnSO and ZnSO with final concentration of 10 mu M4P-FL/Zn after incubation, without ZnSO4Incubated P-FL or FL (5-carboxyfluoroscein) was incubated for 15 minutes at 37 ℃. After incubation, the cells were collected by centrifugation, washed 3 times with PBS, and then resuspended in PBS. 10 mu L of the mixture is dripped on a glass slide, a cover glass is covered, and a fluorescence signal of the thallus is observed by a Nikon A1R-si confocal microscope under the conditions of 60 times of oil lens, 485 plus or minus 20nm excitation/516 plus or minus 20nm emission.
As shown in FIG. 2, P-FL/Zn was added to the cells, whereas P-FL or FL was added to the cells, whereas Pseudomonas aeruginosa showed a clear fluorescent signal, indicating that only Zn was present2+After incubation, P-FL (I-1) was able to label Pseudomonas aeruginosa.
4. Pseudomonas aeruginosa identification by Pseudomonas aeruginosa transport system through Pseudomonas fluorescein couplet P-FL (I-1)
The specific method comprises the following steps: pseudomonas aeruginosa was inoculated on LB solid medium plate and cultured overnight at 37 ℃. Single colonies were picked from the plate, inoculated into LB liquid medium, and cultured overnight at 37 ℃ with shaking at 200 rpm. 1mL of overnight culture was centrifuged, and the cells were washed 2 times with PBS buffer and then resuspended in 1mL of PBS buffer. Inoculating 100 μ L of the resuspended bacterial solution into 10mL CDM and CDM+5μM ZnSO4LB or LB + 5. mu.M ZnSO4In (c), the cells were cultured at 37 ℃ for 12 hours with shaking at 200 rpm. The pseudoplaline transport system is in CDM +5 mu M ZnSO4LB or LB + 5. mu.M ZnSO4Is not expressed under the conditions of (1).
P-FL (I-1) and equimolar ZnSO4Incubation was carried out at 37 ℃ for 15 minutes to obtain P-FL/Zn. 1mL of cultured pseudomonas aeruginosa bacterial liquid is taken, centrifuged, the supernatant is discarded, and the pseudomonas aeruginosa bacterial liquid is resuspended in 1mLPBS, P-FL/Zn with the final concentration of 10 MuM is added, and the pseudomonas aeruginosa bacterial liquid is incubated for 15 minutes at 37 ℃. After incubation, the cells were collected by centrifugation, washed 3 times with PBS, and then resuspended in PBS. The labeling effect of P-FL (I-1) on Pseudomonas aeruginosa was detected with a TECAN F200 PRO microplate reader at 485nm/535nm (excitation/emission).
The results are shown in FIG. 3A, at CDM +5 μ M ZnSO4LB or LB + 5. mu.M ZnSO4After the cultured pseudomonas aeruginosa is incubated with P-FL/Zn, the bacteria can not detect obvious fluorescent signals basically, and the signal intensity is obviously lower than that of the pseudomonas aeruginosa cultured by CDM.
Meanwhile, a P-FL (I-1) is detected by adopting a competition experiment to mark the Pseudomonas aeruginosa-dependent Pseudomonas aeruginosa transport system. The specific method comprises the following steps: pseudomonas aeruginosa was inoculated on LB solid medium plate and cultured overnight at 37 ℃. Single colonies were picked from the plate, inoculated into LB liquid medium, and cultured overnight at 37 ℃ with shaking at 200 rpm. 1mL of overnight culture was centrifuged, and the cells were washed 2 times with PBS buffer and then resuspended in 1mL of PBS buffer. 100 μ L of the resuspended suspension was inoculated into 10mLCDM medium and cultured at 37 ℃ for 12 hours with shaking at 200 rpm.
P-FL (I-1) and equimolar ZnSO4Incubation was carried out at 37 ℃ for 15 minutes to obtain P-FL/Zn. 1mL of cultured pseudomonas aeruginosa bacterial liquid is taken, centrifuged, the supernatant is discarded, and the pseudomonas aeruginosa bacterial liquid is resuspended in 1mLPBS, P-FL/Zn with the final concentration of 10 MuM and Pseudopaline molecules with the final concentration of 10 MuM, 50 MuM or 100 MuM are added, and the pseudomonas aeruginosa bacterial liquid is incubated for 15 minutes at 37 ℃. After incubation, the cells were collected by centrifugation, washed 3 times with PBS, and then resuspended in PBS. The labeling effect of P-FL (I-1) on Pseudomonas aeruginosa was detected with a TECAN F200 PRO microplate reader at 485nm/535nm (excitation/emission).
As shown in FIG. 3B, after the Pseudomonas aeruginosa molecules are exogenously added, the fluorescence intensity of P-FL/Zn labeled Pseudomonas aeruginosa is reduced along with the increase of the concentration of Pseudomonas aeruginosa.
The above results show that P-FL (I-1) enters the pseudomonas aeruginosa cells through the pseudoplaine transport system to realize the fluorescence labeling of the pseudomonas aeruginosa.
5. Marking effect of pseudoplaine fluorescein couplet P-FL (I-1) on different bacteria and cells
The labeling effect of the pseudoplaine fluorescein couplet P-FL (I-1) on different bacteria. The specific method comprises the following steps: inoculating pseudomonas aeruginosa, acinetobacter baumannii, enterobacter cloacae and klebsiella pneumoniae to an LB solid culture medium plate, inoculating staphylococcus aureus to a TSB solid culture medium plate, inoculating enterococcus faecium to an M.R.S. solid culture medium plate, and standing and culturing at 37 ℃ overnight. Single colonies were picked from the plate, inoculated into the corresponding liquid medium, and cultured overnight at 37 ℃ with shaking at 200 rpm. 1mL of overnight culture was centrifuged, and the cells were washed 2 times with PBS buffer and then resuspended in 1mL of PBS buffer. 100 μ L of the resuspended suspension was inoculated into 10mLCDM medium and cultured at 37 ℃ for 12 hours with shaking at 200 rpm. P-FL (I-1) and equimolar ZnSO4Incubation was carried out at 37 ℃ for 15 minutes to obtain P-FL/Zn. 1mL of the cultured bacterial solution was centrifuged, the supernatant was discarded, and the suspension was resuspended in 1mL of PBS, and P-FL/Zn was added to the suspension at a final concentration of 10. mu.M, and incubated at 37 ℃ for 15 minutes. After incubation, the cells were collected by centrifugation, washed 3 times with PBS, and then resuspended in PBS. 10 mu L of the mixture is dripped on a glass slide, a cover glass is covered, and a fluorescence signal of the thallus is observed by a Nikon A1R-si confocal microscope under the conditions of 60 times of oil lens, 485 plus or minus 20nm excitation/516 plus or minus 20nm emission.
The labeling effect of the pseudoplaine fluorescein couplet P-FL (I-1) on mammalian cells. The specific method comprises the following steps: raw264.7 cells and Hela cells are cultured in DMEM medium containing 10% fetal calf serum and placed in CO 25% CO in the incubator2And then, the culture is carried out at 37 ℃. When 80% coverage was achieved, cells were trypsinized and fresh medium was added to make the cell number approximately 1X 105cells/mL, transferred to an 8-well plate with sterile coverslips on the bottom (200. mu.L/well), placed in CO 25% CO in the incubator2And standing and culturing at 37 ℃ for 24 h. The medium was aspirated off with a pipette and the cells were washed 2 times with PBS. P-FL (I-1) and equimolar ZnSO4After incubation at 37 ℃ for 15 minutes, P-FL/Zn was diluted to 10. mu.M with PBS, added to 8-well plates (200. mu.L/well), and incubated at 37 ℃ for 30 minutes. After the supernatant was aspirated by a pipette gun, the cells were washed 3 times with PBS, and 200. mu.L of PBS was added to each well. The cell fluorescence signal was observed with a Leica TCS SP 8X confocal microscope under 60 times oil lens, 485 + -20 nm excitation/516 + -20 nm emission conditions.
As shown in FIG. 4, only P-FL/Zn labeled Pseudomonas aeruginosa showed a clear fluorescent signal, whereas Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus aureus, enterococcus faecium, Raw264.7 cells and Hela cells were not labeled with P-FL/Zn.
6. Marking effect of pseudoplaine fluorescein couplet P-FL (I-1) on pseudomonas aeruginosa under mixed bacteria condition
The specific method comprises the following steps: inoculating pseudomonas aeruginosa, acinetobacter baumannii, enterobacter cloacae and klebsiella pneumoniae to an LB solid culture medium plate, inoculating staphylococcus aureus to a TSB solid culture medium plate, inoculating enterococcus faecium to an M.R.S. solid culture medium plate, and standing and culturing at 37 ℃ overnight. Single colonies were picked from the plate, inoculated into the corresponding liquid medium, and cultured overnight at 37 ℃ with shaking at 200 rpm. 1mL of overnight culture was centrifuged, and the cells were washed 2 times with PBS buffer and then resuspended in 1mL of PBS buffer. 100. mu.L of the resuspended suspension was inoculated into 10mL CDM medium and cultured at 37 ℃ under shaking at 200rpm for 12 hours. Centrifugally suspending the cultured bacterial liquid into PBS with the same volume, and respectively mixing the pseudomonas aeruginosa with staphylococcus aureus, enterobacter cloacae, enterococcus faecium, acinetobacter baumannii and klebsiella pneumoniae. P-FL (I-1) and equimolar ZnSO4Incubation was carried out at 37 ℃ for 15 minutes to obtain P-FL/Zn. 1mL of the mixed bacterial solution was added with P-FL/Zn at a final concentration of 10. mu.M, and incubated at 37 ℃ for 15 minutes. After incubation, the cells were collected by centrifugation, washed 3 times with PBS, and then resuspended in PBS. Dripping 10 μ L onto glass slide, covering with cover glass, observing thallus fluorescence under 60 times oil lens, 485 + -20 nm excitation/516 + -20 nm emission condition with Nikon A1R-si confocal microscopeA signal.
As shown in FIG. 5, only P-FL/Zn labeled Pseudomonas aeruginosa showed a significant fluorescence signal in the mixed bacterial solution of Pseudomonas aeruginosa and other bacteria.
7. Marking effect of pseudoplaine fluorescein couplet P-FL (I-1) on pseudomonas aeruginosa in cell infection model
The specific method comprises the following steps: culturing Raw264.7 cells and Hela cells in DMEM medium containing 10% fetal calf serum, and placing in CO 25% CO in the incubator2And then, the culture is carried out at 37 ℃. When 80% coverage was achieved, cells were trypsinized and fresh medium was added to make the cell number approximately 1X 105cells/mL, transferred to an 8-well plate with sterile coverslips on the bottom (200. mu.L/well), placed in CO 25% CO in the incubator2And standing and culturing at 37 ℃ for 24 h. The medium was aspirated off with a pipette and the cells were washed 2 times with PBS. Pseudomonas aeruginosa was inoculated on LB solid medium plate and cultured overnight at 37 ℃. Single colonies were picked from the plate, inoculated into LB liquid medium, and cultured overnight at 37 ℃ with shaking at 200 rpm. 1mL of overnight culture was centrifuged, and the cells were washed 2 times with PBS buffer and then resuspended in 1mL of PBS buffer. 100. mu.L of the resuspended suspension was inoculated into 10mL CDM medium and cultured at 37 ℃ under shaking at 200rpm for 12 hours. 1mL of the cultured bacterial solution was centrifuged, and the cells were washed 2 times with PBS buffer and then resuspended in 1mL of PBS buffer. P-FL (I-1) and equimolar ZnSO4Incubation was carried out at 37 ℃ for 15 minutes to obtain P-FL/Zn. Adding P-FL/Zn into the Pseudomonas aeruginosa PBS suspension with the final concentration of 10 μ M, adding 200 μ L into an 8-well plate for culturing cells, and incubating at 37 ℃ for 30 min. The cells were washed 3 times with PBS after aspirating the bacterial solution with a pipette gun, and 200. mu.L of PBS was added to each well. The fluorescence signals of the thalli are observed by a Leica TCS SP 8X confocal microscope under the conditions of 60 times of oil lens and 485 plus or minus 20nm excitation/516 plus or minus 20nm emission.
As shown in FIG. 6, P-FL/Zn specifically identified P-FL/Zn in the P-FL-S-H infected cell model, while P-FL adhered to the cell surface and showed a clear fluorescent signal, but neither Raw264.7 cells nor Hela cells detected a fluorescent signal.
8. Marking effect of pseudoplaine fluorescein couplet P-FL (I-1) on pseudomonas aeruginosa in tissue section infection model
The specific method comprises the following steps: and (3) taking 20-25 g C57BL/6J mouse stomach tissues to prepare slices. Animal-related procedures were conducted according to the guidelines of the animal committee of the institute of pharmacy, the institute of medicine, and the Beijing coordination, the Chinese academy of medicine. After mice were sacrificed, gastric tissues were collected, embedded in tissue cryogens (OCT) and frozen sections of 8 μm thickness were prepared by the company Solarbio technologies, beijing for imaging analysis. 1mL of the overnight culture of Pseudomonas aeruginosa was centrifuged, washed with PBS buffer for 2 times, and then resuspended in 1mL of PBS buffer. 100. mu.L of the resuspended suspension was inoculated into 10mL CDM medium and cultured at 37 ℃ under shaking at 200rpm for 12 hours. 1mL of the cultured bacterial solution was centrifuged, and the cells were washed 2 times with PBS buffer and then resuspended in 1mL of PBS buffer. P-FL (I-1) and equimolar ZnSO4Incubation was carried out at 37 ℃ for 15 minutes to obtain P-FL/Zn. P-FL/Zn was added to the Pseudomonas aeruginosa PBS suspension at a final concentration of 10. mu.M. 400 μ L of the suspension was added to the stomach tissue sections and incubated at 37 ℃ for 1 h. The stomach tissue was then washed three times with PBS, dried, and then 5. mu.L of a fluorescent anti-quencher was added dropwise and the coverslip was applied. Fluorescent signals were observed with a Leica TCS SP 8X confocal microscope under 60 times oil lens, 485 + -20 nm excitation/516 + -20 nm emission conditions.
As shown in FIG. 7, the red dotted line shows the P.aeruginosa thallus, showing a distinct fluorescence signal, while the stomach tissue does not detect a fluorescence signal, indicating that P-FL/Zn can specifically identify P.aeruginosa in the P.aeruginosa tissue infection model.
9. Pseudoplaline fluorescein couplet P-FL (I-1) cytotoxicity assay
The specific method comprises the following steps: culturing Raw264.7 cells and Hela cells in DMEM medium containing 10% fetal calf serum, and placing in CO 25% CO in the incubator2And then, the culture is carried out at 37 ℃. When 80% coverage was achieved, cells were trypsinized and fresh medium was added to make the cell number approximately 1X 104cells/mL, transferred to 96-well plates (100. mu.L/well), placed in CO 25% CO in the incubator2And standing and culturing at 37 ℃ for 24 h. mu.L of MTS solution was added and incubated at 37 ℃ for 3 h. The 490nm absorbance was measured by a microplate reader (Tecan) and the cell viability (%) was 100 × (A-A)0)/(As-A0) (A: absorbance of experimental group, As: absorbance of control group, A0: blank absorbance).
The results show (FIG. 8) that in the experimental concentration range (0-50. mu.M), P-FL/Zn has no effect on the survival of cells, indicating that P-FL/Zn has no toxicity to mammalian cells and high safety.
Meanwhile, the I-2, I-3 and I-4 also have the similar test results as the I-1.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (12)
1. A pseudocaline fluorescein couplet, characterized in that it is selected from one of the following structural formulas, m is an integer of 1-10; n is an integer of 1 to 10:
2. the method for synthesizing a fluorescein conjugate as claimed in claim 1, wherein the fluorescein conjugate is obtained by addition or substitution reaction of pseudoephedrine derivative and fluorescein;
the pseudoplaline derivative has the following structure:
wherein m is as defined in claim 1.
3. The synthetic method according to claim 2, wherein when the fluorescein is compound 4, the fluorescein couplet I-2 is obtained by substitution reaction of the fluorescein and the pseudoephedrine derivative under alkaline conditions;
the synthetic route is as follows:
wherein n is an integer of 1 to 10.
4. The method of synthesizing according to claim 3, wherein the fluorescein couplet I-3 is obtained by substituting the compound 4.
5. Use of the fluorescein conjugate of claim 1 in the preparation of a probe for the identification of bacterial fluorescein, wherein the bacterium is pseudomonas aeruginosa.
6. Use according to claim 5, wherein the fluorescein conjugate is conjugated with Zn2+The complex is formed to realize the identification of the pseudomonas aeruginosa.
7. The use of claim 6, wherein the fluorescein conjugate is conjugated to the Zn2+The molar ratio of (a) to (b) is 1:1 to 1: 5.
8. The use of claim 7, wherein the fluorescein conjugate is conjugated to the Zn2+Is 1: 1.
9. The use according to any one of claims 5 to 8, wherein the fluorescein conjugate is used for identifying P.aeruginosa by means of an exogenous pseudocaline molecule transport system.
10. The use according to claim 9, wherein the concentration of the Pseudopaline molecule is 10-100 μ Μ.
11. The use according to claim 10, wherein the concentration of the Pseudopaline molecule is 10-50 μ Μ.
12. The use of claim 9, wherein the fluorescein couplet is used at a concentration of no more than 50 μ Μ.
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| De novo synthesis, structural assignment and biological evaluation of pseudopaline,a metallophore produced by Pseudomonas aeruginosa;Jian Zhang,et al.;《Chem. Sci.》;20190530;第10卷;6635-6641 * |
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