CN114591345B - Rhodamine derivative RH-GP-X, preparation method thereof and application thereof in gram positive bacteria detection - Google Patents
Rhodamine derivative RH-GP-X, preparation method thereof and application thereof in gram positive bacteria detection Download PDFInfo
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical class [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 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 241000192125 Firmicutes Species 0.000 title claims abstract description 16
- 238000001514 detection method Methods 0.000 title claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract 6
- 241000894006 Bacteria Species 0.000 claims abstract 2
- 238000006243 chemical reaction Methods 0.000 claims description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 18
- DILRJUIACXKSQE-UHFFFAOYSA-N n',n'-dimethylethane-1,2-diamine Chemical compound CN(C)CCN DILRJUIACXKSQE-UHFFFAOYSA-N 0.000 claims description 12
- 238000001816 cooling Methods 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 8
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims description 7
- -1 rhodamine compound Chemical class 0.000 claims description 7
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 6
- 238000004440 column chromatography Methods 0.000 claims description 6
- 238000010791 quenching Methods 0.000 claims description 6
- 230000000171 quenching effect Effects 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- MUSLHCJRTRQOSP-UHFFFAOYSA-N rhodamine 101 Chemical compound [O-]C(=O)C1=CC=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MUSLHCJRTRQOSP-UHFFFAOYSA-N 0.000 claims description 5
- 229940043267 rhodamine b Drugs 0.000 claims description 4
- RLOWWWKZYUNIDI-UHFFFAOYSA-N phosphinic chloride Chemical compound ClP=O RLOWWWKZYUNIDI-UHFFFAOYSA-N 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- WGTODYJZXSJIAG-UHFFFAOYSA-N tetramethylrhodamine chloride Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C(O)=O WGTODYJZXSJIAG-UHFFFAOYSA-N 0.000 claims description 3
- 230000008685 targeting Effects 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 238000010186 staining Methods 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 238000010521 absorption reaction Methods 0.000 description 7
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000007850 fluorescent dye Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000012295 chemical reaction liquid Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000007363 ring formation reaction Methods 0.000 description 2
- 238000007142 ring opening reaction Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000000990 laser dye Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000004557 single molecule detection Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/10—Spiro-condensed systems
- C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
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- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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Abstract
The invention relates to the field of chemical synthesis, in particular to rhodamine derivatives RH-GP-X, a preparation method thereof and application thereof in gram-positive bacteria detection. The rhodamine derivative RH-GP-X has a structural general formula shown in the specification (I). Wherein R is 1 ＝R 2 ＝R 3 ＝R 4 =h; or R is 1 ＝R 4 ＝H，R 2 ＝‑CH 2 CH 3 ，R 3 ＝‑CH 3 The method comprises the steps of carrying out a first treatment on the surface of the Or R is 1 ＝R 2 ＝‑CH 3 ，R 3 ＝R 4 =h; or R is 1 ＝R 2 ＝‑CH 2 CH 3 ，R 3 ＝R 4 =h; or R is 1 And R is 4 Together form- (CH) 2 ) 3 ‑，R 2 And R is 3 Together form- (CH) 2 ) 3 -. The rhodamine derivative RH-GP-X is constructed, and the positive charge targeting effect is utilized to target and gather gram-positive staining bacteria, so that the method has the advantages of rapid detection, simplicity and convenience in operation and the like.
Description
Technical Field
The invention relates to the field of chemical synthesis, in particular to rhodamine derivatives RH-GP-X, a preparation method thereof and application thereof in gram-positive bacteria detection.
Background
Rhodamine is a dye with excellent optical properties, and compared with other commonly used fluorescent dyes, the rhodamine fluorescent dye has the advantages of good light stability, long wavelength absorption, large absorption coefficient, high light stability in a ring-opened form, insensitivity to pH, wider wavelength range, higher fluorescence quantum yield, long fluorescence lifetime and the like. Therefore, the fluorescent dye is widely applied to the aspects of pharmacology, physiology, molecular biology, cell biology, molecular genetics, environmental chemistry, single molecule detection, information science, fluorescent marking, laser dye and the like, and is the most commonly used fluorescent dye in the biotechnology field of analytical chemistry and biological medicine science.
Due to the existence of the targeting group, the reagent can be targeted to gather on gram-positive bacteria, and rhodamine derivative RH-GP-X is utilized, and after photo-induced ring opening and thermal-induced ring closing reaction are carried out, the ring closing state is non-fluorescent in a visible light region. The resulting open-loop state has intense fluorescence when irradiated with ultraviolet light, and the molecules show absorption "on" and fluorescence "off" responses under ultraviolet light irradiation in both solution and solid matrices. The response is visual, and can be used for rapidly detecting gram-positive bacteria. In the prior art, no report on the detection of gram-positive bacteria by the rhodamine derivative RH-GP-X is yet seen.
Disclosure of Invention
The invention aims to provide a preparation method of a series of rhodamine derivatives RH-GP-X and a detection technology for gram-positive bacteria.
The technical scheme adopted by the invention is as follows: rhodamine derivatives RH-GP-X, wherein the rhodamine derivatives RH-GP-X have a structural general formula shown in (I):
wherein,
R 1 ＝R 2 ＝R 3 ＝R 4 ＝H；
or R is 1 ＝R 4 ＝H，R 2 ＝-CH 2 CH 3 ，R 3 ＝-CH 3 ；
Or R is 1 ＝R 2 ＝-CH 3 ，R 3 ＝R 4 ＝H；
Or R is 1 ＝R 2 ＝-CH 2 CH 3 ，R 3 ＝R 4 ＝H；
Or R is 1 And R is 4 Together form- (CH) 2 ) 3 -，R 2 And R is 3 Together form- (CH) 2 ) 3 -。
The preparation method of the rhodamine derivative RH-GP-X comprises the following steps: adding rhodamine compound into dehydrated and dried 1, 2-dichloroethane, and dropwise adding POCl under stirring 3 Heating and refluxing, cooling to room temperature, removing a solvent, adding dry acetonitrile, adding sodium azide for reaction at room temperature, heating to high temperature for reaction, cooling to room temperature, adding N, N-dimethyl ethylenediamine for reaction, adding water for quenching, extracting, and purifying by column chromatography to obtain RH-GP-X.
Preferably, in the preparation method of the rhodamine derivative RH-GP-X, the rhodamine compound is rhodamine B, tetramethyl rhodamine TMR or rhodamine 101.
Preferably, in the preparation method of the rhodamine derivative RH-GP-X, the heating reflux is performed for 4 hours at 90 ℃.
Preferably, the preparation method of rhodamine derivative RH-GP-X is carried out for 8-12 hours at room temperature.
Preferably, in the preparation method of rhodamine derivatives RH-GP-X, the high-temperature reaction temperature is 82 ℃ and the time is 40min.
Preferably, in the preparation method of rhodamine derivative RH-GP-X, the reaction time of adding N, N-dimethyl ethylenediamine is 30min.
Preferably, the preparation method of the rhodamine derivative RH-GP-X comprises the following steps of: sodium azide: n, N-dimethylethylenediamine=1 (2-3): (2-3).
Application of rhodamine derivatives RH-GP-X in detection of gram-positive bacteria.
Preferably, for the above application, the gram positive bacteria is bacillus.
The beneficial effects of the invention are as follows: due to the existence of the targeting group, the reagent can be targeted to gather in gram-positive bacteria, and the rhodamine derivative is utilized to perform photo-induced ring opening and thermal-induced ring closing reaction, so that the ring closing state is non-fluorescent in the visible light region. The resulting open-loop state has intense fluorescence when irradiated with ultraviolet light, and the molecules show absorption "on" and fluorescence "off" responses under ultraviolet light irradiation in both solution and solid matrices. The response is visual, and can be used for rapidly detecting gram-positive bacteria.
Drawings
FIG. 1 is a graph showing the trend of the absorption strength of RH-GP-1 prepared in example 1 with pH.
FIG. 2 shows the trend of fluorescence intensity of RH-GP-1 prepared in example 1 with pH.
FIG. 3 is a bright field image under a Bacillus microscope.
FIG. 4 is a fluorescent staining image of bacillus for RH-GP-1 prepared in example 1.
Detailed Description
The reaction general formula of rhodamine derivative RH-GP-X is as follows:
wherein,
R 1 ＝R 2 ＝R 3 ＝R 4 ＝H；
or R is 1 ＝R 4 ＝H，R 2 ＝-CH 2 CH 3 ，R 3 ＝-CH 3 ；
Or R is 1 ＝R 2 ＝-CH 3 ，R 3 ＝R 4 ＝H；
Or R is 1 ＝R 2 ＝-CH 2 CH 3 ，R 3 ＝R 4 ＝H；
Or R is 1 And R is 4 Together form- (CH) 2 ) 3 -，R 2 And R is 3 Together form- (CH) 2 ) 3 -。
The preparation method of the rhodamine derivative RH-GP-X comprises the following steps: adding rhodamine compound into dehydrated and dried 1, 2-dichloroethane, and dropwise adding POCl under stirring 3 Heating and refluxing, cooling to room temperature, removing a solvent, adding dry acetonitrile, adding sodium azide for reaction at room temperature, heating to high temperature for reaction, cooling to room temperature, adding N, N-dimethyl ethylenediamine for reaction, adding water for quenching, extracting, and purifying by column chromatography to obtain RH-GP-X.
Preferably, in the preparation method of the rhodamine derivative RH-GP-X, the rhodamine compound is rhodamine B, tetramethyl rhodamine TMR or rhodamine 101.
Preferably, in the preparation method of the rhodamine derivative RH-GP-X, the heating reflux is performed for 4 hours at 90 ℃.
Preferably, the preparation method of rhodamine derivative RH-GP-X is carried out for 8-12 hours at room temperature.
Preferably, in the preparation method of rhodamine derivatives RH-GP-X, the high-temperature reaction temperature is 82 ℃ and the time is 40min.
Preferably, in the preparation method of rhodamine derivative RH-GP-X, the reaction time of adding N, N-dimethyl ethylenediamine is 30min.
Preferably, the preparation method of the rhodamine derivative RH-GP-X comprises the following steps of: sodium azide: n, N-dimethylethylenediamine=1 (2-3): (2-3).
Application of rhodamine derivatives RH-GP-X in detection of gram-positive bacteria.
Preferably, for the above application, the gram positive bacteria is bacillus.
EXAMPLE 1 rhodamine B derivative RH-GP-1
The reaction formula is as follows:
1mol of rhodamine B is weighed, 120mL of dehydrated and dried 1, 2-dichloroethane is added, 0.18mL of phosphorus oxychloride is added dropwise under stirring, and the mixture is heated and refluxed at 90 ℃ for 4 hours. The reaction liquid is cooled to room temperature, the solvent is removed, 120mL of dry acetonitrile is added, 2-3mol of sodium azide is added for reaction at room temperature for 8-12h, the temperature is raised to 82 ℃ for 40min, 2-3mol of N, N-dimethylethylenediamine is added for reaction for 30min after cooling to room temperature, water quenching and dichloromethane extraction are carried out, and RH-GP-1 is obtained through column chromatography purification. HRMS:527.3241.
EXAMPLE 2 TMR rhodamine derivative RH-GP-2
1mol of rhodamine TMR was added to the mixture, 120mL of dehydrated and dried 1, 2-dichloroethane was added dropwise with stirring to the mixture, 0.18mL of phosphorus oxychloride was added dropwise, and the mixture was heated under reflux at 90℃for 4 hours. The reaction liquid is cooled to room temperature, the solvent is removed, 120mL of dry acetonitrile is added, 2-3mol of sodium azide is added for reaction at room temperature for 8-12h, the temperature is raised to 82 ℃ for 40min, 2-3mol of N, N-dimethylethylenediamine is added for reaction for 30min after cooling to room temperature, water quenching and dichloromethane extraction are carried out, and RH-GP-2 is obtained through column chromatography purification. HRMS:471.2590.
EXAMPLE 3 rhodamine 101 derivative RH-GP-3
1mol of rhodamine 101 was added to 120mL of dehydrated and dried 1, 2-dichloroethane, 0.18mL of phosphorus oxychloride was added dropwise with stirring, and the mixture was heated under reflux at 90℃for 4 hours. The reaction liquid is cooled to room temperature, the solvent is removed, 120mL of dry acetonitrile is added, 2-3mol of sodium azide is added for reaction at room temperature for 8-12h, the temperature is raised to 82 ℃ for 40min, 2-3mol of N, N-dimethylethylenediamine is added for reaction for 30min after cooling to room temperature, water quenching and dichloromethane extraction are carried out, and RH-GP-3 is obtained through column chromatography purification. HRMS:575.3300.
example 4 Spectrum testing and imaging experiments
1. And (5) measuring fluorescence spectrum.
The concentration of the preparation is 2 multiplied by 10 -5 The pH of the probe solution is respectively regulated by sodium hydroxide and hydrochloric acid, solutions with different pH values ranging from 2.5 to 13 are respectively prepared by mol/L RH-GP-1 probe aqueous solution containing 60% acetonitrile, absorption and fluorescence spectra are respectively tested and recorded by an ultraviolet spectrophotometer and a fluorescence spectrophotometer, the maximum absorption and the maximum fluorescence intensity are selected to be plotted against the pH, as shown in figures 1 and 2, each point in figures 1 and 2 represents the maximum absorption and the maximum fluorescence intensity of different pH values, and the result shows that the fluorescence intensity is continuously enhanced along with the decrease of the pH value. The probe is insensitive to pH in the range of 6.0-13.
2. Fluorescence microscopy imaging.
To dishes containing live cells of Staphylococcus, bacillus, and Streptococcus, 2×10 concentration were added - 5 The solution of RH-GP-1 of M was mixed with this culture solution uniformly, stained for 5min, washed three times with phosphate buffer solution at ph=7.4, and finally the dish was observed under confocal microscope. As shown in figure 4, the bacillus infected with RH-GP-1 shows obvious red fluorescence, and experimental results show that rhodamine derivative RH-GP-1 has the advantages of high sensitivity, high selectivity, simple operation, light-operated fluorescence sensitivity and the like, and can show good reversible conversion and optical activity by combining the advantages and disadvantages of the existing fluorescence immunoassay method, and can carry out microscopic imaging on gram-positive bacteria.
Claims (9)
1. The application of rhodamine derivative RH-GP-X in gram-positive bacteria detection is characterized in that the rhodamine derivative RH-GP-X has a structural general formula shown in the following (I):
wherein,
R 1 ＝R 2 ＝R 3 ＝R 4 ＝H；
or R is 1 ＝R 4 ＝H，R 2 ＝-CH 2 CH 3 ，R 3 ＝-CH 3 ；
Or R is 1 ＝R 2 ＝-CH 3 ，R 3 ＝R 4 ＝H；
Or R is 1 ＝R 2 ＝-CH 2 CH 3 ，R 3 ＝R 4 ＝H；
Or R is 1 And R is 4 Together form- (CH) 2 ) 3 -，R 2 And R is 3 Together form- (CH) 2 ) 3 -。
2. The use according to claim 1, characterized in that the preparation method of a rhodamine derivative RH-GP-X comprises the following steps: adding rhodamine compound into dehydrated and dried 1, 2-dichloroethane, and dropwise adding POCl under stirring 3 Heating and refluxing, cooling to room temperature, removing a solvent, adding dry acetonitrile, adding sodium azide for reaction at room temperature, heating to high temperature for reaction, cooling to room temperature, adding N, N-dimethyl ethylenediamine for reaction, adding water for quenching, extracting, and purifying by column chromatography to obtain RH-GP-X.
3. The use according to claim 2, wherein the rhodamine compound is rhodamine B, tetramethylrhodamine TMR, rhodamine 101.
4. The method according to claim 3, wherein the heat-reflow is performed at 90℃for 4 hours.
5. The process according to claim 4, wherein the reaction is carried out at room temperature for a period of from 8 to 12 hours.
6. The method according to claim 5, wherein the reaction is carried out at a temperature of 82℃for a period of 40 minutes.
7. The method according to claim 6, wherein the reaction time for adding N, N-dimethylethylenediamine is 30 minutes.
8. The use according to claim 7, wherein the rhodamine compound is present in a molar ratio: sodium azide: n, N-dimethylethylenediamine=1:2-3: 2-3.
9. The use according to claim 1, wherein the gram positive bacterium is bacillus.
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