CN116478240A - LPETG derivative and application thereof - Google Patents

LPETG derivative and application thereof Download PDF

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CN116478240A
CN116478240A CN202310466906.1A CN202310466906A CN116478240A CN 116478240 A CN116478240 A CN 116478240A CN 202310466906 A CN202310466906 A CN 202310466906A CN 116478240 A CN116478240 A CN 116478240A
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韩守法
江锋
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Xiamen University
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Abstract

The invention discloses an LPETG derivative and application thereof, the structural formula isWherein R is a fluorescent emitting group capable of being quenched by Dabcyl. The invention can efficiently and rapidly specifically and covalently label SrtA-expressing bacteria, and has good selectivity and high signal-to-noise ratio; can effectively distinguish the adsorption signal of vancomycin and the SrtA-mediated covalent labeling signal; the bacteria-targeted photodynamic therapy can be achieved by covalently labelling the photosensitizer with bacteria.

Description

LPETG derivative and application thereof
Technical Field
The invention belongs to the technical field of pathogenic bacteria labeling, and particularly relates to an LPETG derivative and application thereof.
Background
Pathogenic bacteria are used as pathogens and pose a great threat to human health. However, not all bacteria are pathogenic to humans, and some bacteria act as symbiotic bacteria, playing a vital role in host immunity and maintaining metabolic homeostasis. On the basis of no harm to symbiotic bacteria, the development of a method for selectively targeting pathogenic bacteria has important value in the aspect of treating bacteria-related diseases.
SortaseA (SrtA) is a transpeptidase which binds to the surface of the bacterial cell membrane and is able to catalyse the attachment of surface proteins to peptidoglycans. The Leu-Pro-X-Thr-Gly (LPXTG) polypeptide sequence can be broken to obtain a thioester intermediate containing a SrtA substrate. The nucleophilic attack of the exposed amine groups on the peptidoglycan then results in covalent attachment of the protein to the peptidoglycan. Therefore, the SrtA-based fluorescent labeling can realize selective labeling of bacteria expressing SrtA, but the fluorescent labeling method has the defects of high incubation concentration, long labeling time, difficulty in distinguishing adsorption signals from covalent labeling signals and the like due to the fact that the SrtA is subjected to fluorescent labeling in the prior art, and limits the application of the fluorescent labeling method.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an LPETG derivative.
It is a further object of the present invention to provide the use of the above LPETG derivative.
The technical scheme of the invention is as follows:
an LPETG derivative, characterized by: the structure is as followsWherein R is a fluorescent emitting group capable of being quenched by Dabcyl.
In a preferred embodiment of the invention, the R is a tetrabromofluorescein group or a tetraiodofluorescein group.
Further preferably, the structural formula is
The LPETG derivative is applied to a fluorescent probe for marking gram-positive bacteria with the surface of a cell membrane for expressing Sortase A.
A fluorescence labeling method of gram-positive bacteria adopts the LPETG derivative as a fluorescence probe, and the gram-positive bacteria are gram-positive bacteria with a cell membrane surface expressing Sortase A.
Use of the LPETG derivative described above for the preparation of a photodynamic therapeutic composition for bacterial diseases.
In a preferred embodiment of the invention, the bacteria causing the bacterial disease are gram positive bacteria expressing SortaseA on the cell membrane surface.
A photodynamic therapeutic composition for bacterial diseases comprises the above LPETG derivative as effective component.
In a preferred embodiment of the present invention, the active ingredient is the above-mentioned LPETG derivative.
The beneficial effects of the invention are as follows:
1. the invention can efficiently and rapidly specifically and covalently label SrtA-expressing bacteria, and has good selectivity and high signal to noise ratio.
2. The invention can effectively distinguish the adsorption signal of vancomycin and the SrtA-mediated covalent labeling signal.
3. The invention can realize bacteria targeted photodynamic therapy by covalently marking the photosensitizer by bacteria.
Drawings
FIG. 1 is a schematic diagram of the action of Van-Sub-proPS.
FIG. 2 shows the synthetic route of Compound 18 in example 1 of the present invention.
FIG. 3 shows the synthetic route for Van-Sub-proPS and Sub-PS in example 1 of the present invention.
FIG. 4 is a diagram showing Compound 4 obtained in example 1 of the present invention 1 H NMR spectrum (CDCl) 3 )。
FIG. 5 shows the compound 4 produced in example 1 of the present invention 13 C NMR spectrum (CDCl) 3 )；
FIG. 6 shows a compound 7 prepared in example 1 of the present invention 1 H NMR spectrum (DMSO-D) 6 )；
FIG. 7 is a diagram showing the compound 7 produced in example 1 of the present invention 13 C NMR spectrum (DMSO-D) 6 )；
FIG. 8 is a diagram ofCompound 11 obtained in example 1 of the present invention 1 H NMR spectrum (CDCl) 3 )；
FIG. 9 is a diagram showing a compound 11 produced in example 1 of the present invention 13 C NMR spectrum (CDCl) 3 )。
FIG. 10 shows a compound 14 prepared in example 1 of the present invention 1 H NMR spectrum (CDCl) 3 )。
FIG. 11 shows a compound 14 prepared in example 1 of the present invention 13 C NMR spectrum (CDCl) 3 )。
FIG. 12 shows a compound 15 prepared in example 1 of the present invention 1 H NMR spectrum (CDCl) 3 )。
FIG. 13 shows a compound 15 prepared in example 1 of the present invention 13 C NMR spectrum (CDCl) 3 )。
FIG. 14 shows a compound 18 prepared in example 1 of the present invention 1 H NMR spectrum (CDCl) 3 )。
FIG. 15 shows a compound 18 prepared in example 1 of the present invention 13 C NMR spectrum (CDCl) 3 )。
FIG. 16 shows a compound 21 prepared in example 1 of the present invention 1 H NMR spectrum (DMSO-D) 6 )。
FIG. 17 shows a compound 21 prepared in example 1 of the present invention 13 C NMR spectrum (DMSO-D) 6 )。
FIG. 18 shows a Sub-PS obtained in example 1 of the present invention 1 H NMR spectrum (DMSO-D) 6 )。
FIG. 19 is a Sub-PS obtained in example 1 of the present invention 13 C NMR spectrum (DMSO-D) 6 )。
FIG. 20 shows a Sub-proPS obtained in example 1 of the present invention 1 H NMR spectrum (DMSO-D) 6 )。
FIG. 21 shows the Sub-proPS obtained in example 1 of the present invention 13 C NMR spectrum (DMSO-D) 6 )。
FIG. 22 is an HRMS analysis of Sub-PS prepared in example 1 of the present invention.
FIG. 23 is an HRMS analysis of Sub-proPS prepared in example 1 of the present invention.
FIG. 24 shows HPLC and HRMS analysis of Van-Sub-proPS obtained in example 1 of the present invention.
FIG. 25 is a graph showing the emission spectrum of Van-Sub-proPS in example 4 in which fluorescence is suppressed by the fluorescence quencher Dabcyl.
FIG. 26 is a graph showing the chemiluminescence spectrum of Van-Sub-proPS in example 5 of the present invention in which the photodynamic properties are inhibited by the fluorescence quencher Dabcyl.
FIG. 27 is a graph showing the results of selective covalent labelling of SrtA-expressing bacteria by Van-Sub-proPS in example 6 of the present invention.
FIG. 28 is a graph showing the results of photodynamic treatment of SrtA-expressing bacteria by Van-Sub-proPS in example 7 of the present invention.
Detailed Description
The technical scheme of the invention is further illustrated and described below by the specific embodiments in combination with the accompanying drawings.
Example 1: synthesis of fluorescent probe Van-Sub-proPS and reference probe Sub-PS
The following steps (1) to (6) are as shown in fig. 2:
(1) To CH containing Boc-glycine (4.3 g,24.6 mmol) 2 Cl 2 To a solution (30 mL) was added 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, 7.06g,35.1 mmol), 3-azidopropylamine (2.34 g,23.4 mmol), N-diisopropylethylamine (DIPEA, 4.52g,35.0 mmol) and 1-hydroxybenzotriazole (HOBt, 4.74g,35.1 mmol). The reaction mixture was stirred for 4h, then extracted 2 times with aqueous hydrochloric acid (1 m,30.0 ml). The organic phase was separated and extracted 2 times with saturated aqueous sodium bicarbonate (30.0 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product compound 1. Compound 1 was dissolved in a solution of trifluoroacetic acid (TFA, 4.0 mL) in dichloromethane (12.0 mL). The reaction mixture was stirred at room temperature for 1h, then concentrated under reduced pressure to remove the solvent. By CH 3 OH (10.0 mL) dissolved the residue and the pH was adjusted to neutral with DIPEA. Then concentrated under reduced pressure to give the crude product compound 2. In CH containing Boc-L-threonine (5.5 g,25.1 mmol) 2 Cl 2 To a solution (20 mL) was added N-hydroxysuccinimide (NHS, 4.33g,37.7 mmol) and EDC (9.62 g,50.2 mmol). The reaction mixture was stirred for 2h with aqueous hydrochloric acid solution @1m,50.0 ml) was extracted 1 time, an organic phase was separated, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product compound 3. In CH containing Compound 3 2 Cl 2 To a solution (15.0 mL) was added Compound 2 and DIPEA (15.1 g,117.1 mmol). The reaction solution was stirred for 4 hours, then extracted 2 times with saturated aqueous sodium bicarbonate (50.0 mL), and the separated organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH 2 Cl 2 /CH 3 Oh=40:1) to give compound 4 (6.77 g, 78%) as shown in fig. 4 and 5. 1 H NMR(500MHz,CDCl 3 )δ7.46(s,1H),7.12(s,1H),5.76(d,J＝6.1Hz,1H),4.30(d,J＝3.9Hz,1H),4.10(d,J＝4.6Hz,1H),3.92(s,2H),3.33(t,J＝6.5Hz,4H),1.80–1.72(m,2H),1.44(s,9H),1.24–1.19(m,3H). 13 C NMR(125MHz,CDCl 3 )δ172.13,169.46,156.43,80.62,67.29,59.68,48.99,43.10,36.97,28.51,28.29,18.94.
(2) Compound 4 (6.4 g,17.9 mmol) was dissolved in CH containing trifluoroacetic acid (TFA, 5 mL) 2 Cl 2 (15.0 mL) in solution. The reaction mixture was stirred at room temperature for 1h, then concentrated under reduced pressure. The residue was taken up in CH 3 OH (10.0 mL) was dissolved, followed by adjusting the pH of the organic solution to neutral with DIPEA and concentrating under reduced pressure to give crude compound 5. To a solution of N-Fmoc-L-glutamic acid-5-tert-butyl ester (7 g,16.5 mmol) in methylene chloride was added NHS (2.84 g,24.7 mmol) and EDC (6.3 g,32.9 mmol). The reaction mixture was stirred for 2h, extracted 1 time with 1M aqueous hydrochloric acid (50.0 mL), the organic phase separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude compound 6. In CH containing Compound 6 2 Cl 2 To the solution (15.0 mL) was added compound 5 and DIPEA (3.45 g,26.7 mmol). The reaction mixture was stirred for 1h, then extracted 2 times with saturated aqueous sodium bicarbonate (50.0 mL), and the separated organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH 2 Cl 2 /CH 3 Oh=50:1) to give compound 7 (10.1 g, 85%) as shown in fig. 6 and 7. 1 H NMR(500MHz,DMSO-d6)δ8.12(s,1H),7.89(d,J＝7.3Hz,2H),7.77(s,1H),7.73(d,J＝6.8Hz,3H),7.63(d,J＝7.9Hz,1H),7.41(t,J＝7.2Hz,2H),7.33(t,J＝7.2Hz,2H),5.07(d,J＝4.2Hz,1H),4.28(ddd,J＝25.2,16.1,8.1Hz,3H),4.14(dd,J＝17.7,9.1Hz,2H),4.00(d,J＝4.8Hz,1H),3.69(s,2H),3.12(d,J＝5.8Hz,2H),2.25(s,2H),1.94(d,J＝6.8Hz,1H),1.75(dt,J＝14.7,8.4Hz,1H),1.69–1.56(m,2H),1.39(s,9H),1.36–1.16(m,2H),1.04(d,J＝6.0Hz,3H). 13 C NMR(125MHz,DMSO-d6)δ171.69,170.15,168.68,156.00,143.84,143.68,140.69,127.60,127.04,125.23,120.06,79.62,66.44,65.69,58.43,53.86,48.27,46.64,42.20,35.84,31.37,28.25,27.72,27.05,19.41.
(3) Boc-L-leucine (9 g,39.0 mmol) was dissolved in 50mL of CH 2 Cl 2 To this was added L-proline methyl ester (7.07 g,42.8 mmol), EDC (11.2 g,58.4 mmol), DIPEA (20.1 g,148.7 mmol) and HOBt (7.89 g,58.4 mmol). The reaction mixture was stirred for 4h, extracted 2 times with 1M aqueous hydrochloric acid (100.0 mL), the organic phase separated and the organic phase extracted 1 more times with saturated sodium bicarbonate (100.0 mL). The separated organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product compound 9. Compound 9 (2.7 g,7.89 mmol) and LiOH H 2 O (1 g,23.8 mmol) was dissolved in CH 3 OH (10 mL). The reaction solution was stirred at room temperature for 1h, the pH of the reaction solution was adjusted to neutrality with aqueous hydrochloric acid (1M), and then concentrated under reduced pressure to give crude product compound 10. Compound 7 (5.1 g,7.7 mmol) was dissolved in CH containing diethylamine (DEA, 4 mL) 2 Cl 2 (16.0 mL) in solution. The reaction mixture was stirred at room temperature for 1h, and then concentrated under reduced pressure to give crude product compound 8. Compound 8 was dissolved in 15mL of CH 2 Cl 2 To this was added compound 10, EDC (2.5 g,13.0 mmol), DIPEA (1 g,7.75 mmol) and HOBt (1.45 g,10.7 mmol). The reaction solution was stirred for 4h, and then extracted 2 times with aqueous hydrochloric acid (1 m,30.0 ml). The organic phase was separated, extracted 2 times with saturated aqueous sodium bicarbonate (30.0 mL), then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. By CH 2 Cl 2 /CH 3 OH (50:1) as eluent and the residue was purified by silica gel column chromatography to give compound 11 (4.74 g, 82%) as shown in FIGS. 8 and 9. 1 H NMR(500MHz,CDCl 3 )δ8.27(d,J＝3.5Hz,1H),7.49(d,J＝7.9Hz,1H),7.43(t,J＝6.1Hz,1H),7.08(t,J＝5.3Hz,1H),5.09(d,J＝8.0Hz,1H),4.66–4.41(m,2H),4.35–4.20(m,2H),4.13–3.91(m,3H),3.82(td,J＝17.4,6.8Hz,2H),3.34–3.15(m,4H),2.68(dd,J＝18.4,6.4Hz,1H),2.38(dd,J＝17.0,9.4Hz,2H),2.27–2.15(m,1H),2.13–1.94(m,2H),1.93–1.78(m,2H),1.73(tt,J＝13.7,6.8Hz,3H),1.66–1.50(m,1H),1.46(s,9H),1.42(s,9H),1.26(d,J＝6.7Hz,3H),1.24(s,1H),0.99(d,J＝6.2Hz,3H),0.95(d,J＝6.3Hz,3H). 13 C NMR(125MHz,CDCl 3 )δ175.43,174.55,173.53,172.13,171.19,169.56,155.69,82.26,80.46,67.72,62.90,60.07,57.13,51.30,48.82,47.71,43.30,40.69,36.38,33.38,29.27,28.45,28.28,28.07,25.52,25.39,24.69,23.27,21.59,19.67.
(4) N-alpha-Fmoc-N-epsilon-Boc-L-lysine (400 mg,0.85 mmol) was dissolved in CH 2 Cl 2 To (10 mL) was added 3-azidopropylamine (100 mg,1 mmol), EDC (245 mg,1.28 mmol), DIPEA (441 mg,3.41 mmol) and NHS (118 mg,1.03 mmol). After stirring the reaction mixture for 2h, it was then extracted 2 times with aqueous hydrochloric acid (1M, 30.0 mL). The separated organic phase was extracted 2 times with saturated aqueous sodium bicarbonate (30.0 mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude product compound 13. Compound 13 was dissolved in CH containing diethylamine (DEA, 3 mL) 2 Cl 2 (10.0 mL). The reaction solution was stirred at room temperature for 2 hours, and then concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography on silica gel (CH 2 Cl 2 /CH 3 Oh=20:1) to give compound 14 (225 mg, 80%) as shown in fig. 10 and 11. 1 H NMR(500MHz,CDCl 3 )δ7.52(s,1H),4.61(s,1H),3.38–3.35(m,2H),3.34(dd,J＝8.3,4.7Hz,2H),3.12(d,J＝6.2Hz,2H),1.89–1.83(m,1H),1.83–1.79(m,2H),1.78(d,J＝3.5Hz,2H),1.58–1.47(m,3H),1.43(d,J＝11.2Hz,9H),1.39(dd,J＝9.9,7.2Hz,1H). 13 C NMR(125MHz,CDCl 3 )δ175.07,156.11,79.13,54.99,49.33,40.12,36.58,34.53,29.89,28.92,28.42,22.85.
(5) To a solution of compound 14 (800 mg,2.44 mmol) in pyridine (6 mL) was added Dabcyl-COOH (550 mg,2.04 mmol) and EDC (700 mg,3.65 mmol). The reaction solution was stirred at room temperature for 3 hours, and then concentrated under reduced pressure. The residue was treated with 50.0mL of CH 2 Cl 2 Dissolving. The organic solution was extracted 2 times with hydrochloric acid (1M, 30.0 mL) and then saturated carbonic acidAqueous sodium hydrogen (30.0 mL) was extracted 2 times, the organic phase was separated, dried and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography on silica gel (CH 2 Cl 2 /CH 3 Oh=50:1) to give compound 15 (1.03 g, 73%) as shown in fig. 12 and 13. 1 H NMR(500MHz,CDCl 3 )δ7.95–7.82(m,6H),7.25(s,1H),7.14(s,1H),6.74(d,J＝8.5Hz,2H),4.79(s,1H),4.70(d,J＝6.2Hz,1H),3.34(d,J＝6.2Hz,4H),3.09(s,6H),2.03–1.93(m,1H),1.91–1.82(m,1H),1.81–1.74(m,2H),1.55(d,J＝5.6Hz,2H),1.46(d,J＝6.8Hz,2H),1.41(s,9H),1.27(d,J＝14.4Hz,1H). 13 C NMR(125MHz,CDCl 3 )δ172.02,167.23,156.22,155.25,152.88,143.69,133.64,128.09,125.53,122.27,111.51,79.13,53.64,49.16,40.28,40.06,37.07,32.27,29.69,28.71,28.43,22.83.
(6) Compound 15 (600 mg,1.04 mmol) was dissolved in CH with trifluoroacetic acid (TFA, 5 mL) 2 Cl 2 (15.0 mL). The reaction mixture was stirred for 2h, concentrated under reduced pressure, and the residue was dissolved in CH 2 Cl 2 (30.0 mL). Saturated NaHCO for organic solution 3 Aqueous (30.0 mL) was extracted 1 time and the separated organic phase was taken up in anhydrous Na 2 SO 4 Drying and concentrating to obtain a crude product compound 16. Compound 16 was dissolved in N, N-dimethylformamide (DMF, 15 mL), and succinic anhydride (114 mg,1.14 mmol), 4-dimethylaminopyridine (DMAP, 63mg,0.52 mmol) and TEA (150 mg,1.49 mmol) were added. The reaction mixture was stirred at room temperature for 2h, then concentrated under reduced pressure. The residue was treated with 50.0mL of CH 2 Cl 2 Dissolving. The organic solution was extracted 2 times with saturated aqueous ammonium chloride (30.0 mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude product compound 17. To CH in which Compound 11 (1 g,1.32 mmol) was dissolved 3 Pd/C (400 mg) was added to the OH solution (20 mL). The reaction mixture is under H 2 Stirred under atmosphere for 24h and Pd/C was removed by filtration. The filtrate was concentrated under reduced pressure to give the crude product compound 12. Compound 12 was dissolved in a pyridine (10.0 mL) solution containing EDC (300 mg,1.56 mmol) and compound 17. The reaction mixture was stirred at room temperature for 4h, then concentrated under reduced pressure. The residue was dissolved with 50.0mL of dichloromethane. The organic solution was extracted 2 times with hydrochloric acid (1M, 30.0 mL) and then with saturated sodium bicarbonateThe aqueous solution (30.0 mL) was extracted 2 times, the organic phase was separated, dried and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography on silica gel (CH 2 Cl 2 /CH 3 Oh=30:1) to give compound 18 (1.07 g, 80%) as shown in fig. 14 and 15. 1 H NMR(500MHz,CDCl 3 )δ8.18(s,1H),7.94(d,J＝8.0Hz,2H),7.86(dd,J＝17.8,8.3Hz,4H),7.67(d,J＝19.2Hz,2H),7.51(dd,J＝21.1,7.5Hz,2H),7.38(s,1H),7.16(s,1H),7.03(s,1H),6.74(d,J＝8.7Hz,2H),5.48(d,J＝8.0Hz,1H),4.63(d,J＝5.4Hz,1H),4.58–4.44(m,2H),4.39(d,J＝7.4Hz,1H),4.32(t,J＝7.0Hz,1H),4.19–4.01(m,2H),3.91(s,1H),3.75(d,J＝9.4Hz,2H),3.34(s,4H),3.20(s,4H),3.09(s,6H),2.59(dd,J＝17.4,5.7Hz,2H),2.49(s,3H),2.38(dd,J＝16.1,9.2Hz,2H),2.29(d,J＝6.2Hz,1H),2.18(s,1H),2.02(s,2H),1.89(d,J＝4.3Hz,3H),1.84–1.71(m,4H),1.61(d,J＝28.7Hz,4H),1.57–1.48(m,2H),1.44(s,9H),1.38(s,9H),1.26(d,J＝14.8Hz,3H),1.19(d,J＝5.4Hz,3H),0.98(d,J＝6.0Hz,3H),0.94(d,J＝6.2Hz,3H). 13 C NMR(125MHz,CDCl 3 )δ174.79,174.19,173.53,173.11,172.69,172.37,172.26,171.62,169.85,167.23,155.82,155.12,152.86,143.63,133.85,128.23,125.47,122.14,111.49,81.92,80.01,67.12,62.14,59.38,56.28,53.68,51.07,49.08,47.46,43.48,40.87,40.25,38.94,36.88,36.63,36.52,32.81,32.40,32.09,31.49,30.13,29.66,29.01,28.91,28.69,28.32,28.06,25.87,25.37,24.65,23.33,22.92,21.56,19.70.
The following steps (7) to (10) are shown in fig. 3:
(7) To a solution of tetrabromofluorescein (1 g,1.45 mmol) in DMF (10 mL) was added 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM, 0.8g,2.89 mmol), 1-Boc-piperazine (0.4 g,2.15 mmol) and TEA (0.145 g,1.44 mmol). The reaction solution was stirred at room temperature for 3 hours, and then concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 (30 mL). The resulting organic solution was extracted 2 times with aqueous hydrochloric acid (1M, 30.0 mL) and then 1 time with saturated aqueous sodium bicarbonate (20.0 mL), and the separated organic phase was taken up in anhydrous Na 2 SO 4 Drying and concentrating to obtain a residue. The residue was purified by silica gel chromatography (CH 2 Cl 2 /CH 3 Oh=10:1) to give compound 21 (950 mg,75%)。 1 H NMR(500MHz,DMSO-d6)δ7.71(dd,J＝5.4,3.4Hz,2H),7.66–7.59(m,1H),7.54–7.48(m,1H),7.11(s,2H),3.32(d,J＝23.4Hz,4H),3.19(d,J＝22.9Hz,4H),1.37(s,9H). 13 CNMR(125MHz,DMSO-d6)δ167.15,166.49,153.64,152.53,148.74,135.09,130.77,130.42,130.18,129.68,129.61,127.32,117.85,111.43,99.62,79.26,48.57,46.63,41.01,27.95.
(8) Compound 18 (235 mg,0.18 mmol) was dissolved in CH containing trifluoroacetic acid (TFA, 2.0 mL) 2 Cl 2 (8.0 mL) in solution. The reaction solution was stirred at room temperature for 1h, and then concentrated under reduced pressure. Residue with CH 3 OH (10.0 mL) was dissolved and then the pH of the organic solution was adjusted to neutral with saturated sodium bicarbonate solution. The organic solution was concentrated under reduced pressure to give crude product compound 19. Compound 21 (150 mg,0.18 mmol) was dissolved in dichloromethane (6.0 mL) containing trifluoroacetic acid (TFA, 2 mL). The reaction solution was stirred at room temperature for 1h, and then concentrated under reduced pressure. Residue with CH 3 OH (10.0 mL) was dissolved and the pH of the organic solution was adjusted to neutral with TEA. And then concentrated under reduced pressure to give the crude product compound 22. Compound 22 was dissolved in DMF (5 mL) and succinic anhydride (20 mg,0.2 mmol), DMAP (5 mg,0.04 mmol) and TEA (30 mg,0.30 mmol) were added. The reaction mixture was stirred for 2h and then concentrated under reduced pressure. The residue was treated with 15.0mL of CH 2 Cl 2 Dissolving. The organic solution was extracted 1 time with hydrochloric acid (1 m,10.0 ml), the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated by rotary evaporator to give crude product compound 23. EDC (140 mg,0.73 mmol) and NHS (42 mg,0.36 mmol) were added to a DMF solution (5 mL) containing compound 23. The reaction mixture was stirred for 1h and then concentrated under reduced pressure. The residue was treated with 15.0mL of CH 2 Cl 2 Dissolving. The organic solution was extracted 2 times with hydrochloric acid (1M, 10.0 mL), dried, and concentrated under reduced pressure to give crude compound 24. Compound 24 was dissolved in pyridine (5 mL) and all of compound 19 above was added. The reaction solution was stirred at room temperature for 2 hours, and then concentrated under reduced pressure. Purification by HPLC gave Sub-proPS as a dark red solid (206 mg, 58%) as in FIGS. 20, 21 and 23. HPLC conditions: under 254nm ultraviolet detection, acetonitrile/H at 5mL/min 2 O is mobile phase elution. 10% acetonitrile lasted 5min and then gradually increased to 100% over 30min, 100%For 10min (t) R ＝18min)。 1 H NMR(500MHz,DMSO-d6)δ8.48(s,1H),8.04(s,6H),7.81–7.62(m,12H),7.51(s,1H),7.17(s,2H),6.85(s,2H),4.51(s,2H),4.34(s,6H),4.28(s,4H),4.16(s,3H),4.01(s,2H),3.68(s,2H),3.48(s,1H),3.40(s,1H),3.35(s,4H),3.14(s,2H),3.07(s,6H),3.03(s,3H),2.28(s,6H),2.01(s,1H),1.93(s,2H),1.84(s,2H),1.74(s,2H),1.67(s,2H),1.63–1.57(m,1H),1.51(s,3H),1.40(s,5H),1.29–1.23(m,4H),1.04(s,3H),0.86(s,6H). 13 C NMR(200MHz,DMSO-d6)δ174.61,173.46,172.45,172.30,171.96,171.79,171.33,171.25,170.81,170.77,170.50,169.15,166.92,166.33,166.16,154.34,153.30,152.69,152.65,149.35,143.05,142.98,135.48,134.77,130.99,130.90,130.41,130.37,129.22,127.98,125.67,121.80,117.83,114.51,112.17,100.43,66.99,59.80,58.99,54.30,52.60,51.75,49.24,48.86,47.18,42.74,41.59,36.93,36.81,36.37,31.72,31.42,30.61,30.30,29.45,29.29,28.86,28.14,27.38,27.32,25.00,24.47,23.64,22.03,19.95.HRMS(ESI)calc’d for C 81 H 98 Br 4 N 18 O 18 Na + (M+Na + )m/z 1953.3892,found 1953.3965.
(9) Sub-proPS (60 mg,0.03 mmol) and DMF (2 mL) were added DBCO Van (55 mg,0.03 mmol). The reaction solution was stirred at room temperature for 2 hours, and then concentrated under reduced pressure. The residue was dissolved in a small amount of DMF. Adding CH to the above solution 3 OH is recrystallized. It was allowed to stand for 0.5h and then filtered to give a dark red solid. The solid was dissolved in DMF and then purified by HPLC to give Van-Sub-proPS (58 mg, 50%) as shown in FIG. 24. HPLC conditions: under 254nm ultraviolet detection, acetonitrile/H at 5mL/min 2 O is mobile phase elution. 10% acetonitrile lasted 5min and then gradually increased to 100% (t) over 30min R ＝13min)。MALDI-TOF MS calcd for C 166 H 187 Br 4 Cl 2 N 29 O 43 SNa + (M+Na + )m/z 3719.9054,found 3719.9014.
(10) Compound 11 (125 mg,0.17 mmol) was dissolved in CH containing trifluoroacetic acid (TFA, 2.0 mL) 2 Cl 2 (8.0 mL) in solution. The reaction mixture was stirred at room temperature for 1h, then concentrated under reduced pressure. Residues ofBy CH 3 OH (10.0 mL) was dissolved. The pH of the organic solution was adjusted to neutral with TEA, and the organic phase was concentrated under reduced pressure to give the crude product compound 20. All compound 20 was dissolved in DMF (5 mL) and compound 24 (150 mg,0.17 mmol) and TEA (35 mg,0.35 mmol) were added. The reaction solution was stirred at room temperature for 2 hours, and then concentrated under reduced pressure. Purification of the residue by HPLC gave Sub-PS (116 mg, 50%) as an orange-red solid as shown in fig. 18, 19 and 22. 1 H NMR(500MHz,DMSO-d6)δ8.51–7.89(m,4H),7.76–7.64(m,5H),7.49(s,1H),7.06(s,2H),4.51(s,1H),4.33(s,1H),4.26(s,1H),4.13(s,1H),4.00(s,1H),3.68(s,3H),3.49(s,1H),3.40–3.29(m,8H),3.12(s,2H),2.41–2.22(m,4H),2.21–2.08(m,1H),2.01(s,1H),1.93(s,2H),1.84(s,2H),1.78(dd,J＝9.3,5.8Hz,1H),1.69–1.58(m,3H),1.44–1.35(m,3H),1.30–1.12(m,3H),1.04(s,3H),0.86(s,6H). 13 C NMR(125MHz,DMSO-d6)δ174.13,171.90,171.61,171.48,171.43,171.03,170.41,170.11,168.89,168.02,152.76,130.14,129.68,127.38,118.20,110.49,104.75,99.67,99.60,99.12,66.58,59.56,58.74,52.35,48.99,48.36,46.80,42.33,41.20,35.94,30.44,30.21,30.03,28.87,28.28,27.79,27.11,26.85,24.61,24.08,23.15,22.11,21.59,19.50.HRMS(ESI)calc’d for C 53 H 61 Br 4 N 11 O 14 Na + (M+Na + )m/z 1418.0985,found1418.1028.
The Van-Sub-proPS synthesized in this example has the structural formula
The structural formula of Sub-PS synthesized in this example is
Example 2: preparing a standard solution of a fluorescent probe Van-Sub-proPS with the concentration of 10mM
36.9mg of Van-Sub-proPS prepared in example 1 are weighed out and dissolved in 1mL of dimethyl sulfoxide. Thus, 10mmol/L (10 mM) of Van-Sub-proPS was obtained as a standard solution.
Example 3: preparing a standard solution of reference probe Sub-PS with the concentration of 10mM
13.9mg of Sub-PS prepared in example 1 was weighed out and dissolved in 1mL of dimethyl sulfoxide. Thus, a standard solution of 10mmol/L (10 mM) of the reference probe was obtained.
Example 4: the fluorescence of Van-Sub-proPS is inhibited by the fluorescence quencher Dabcyl
The standard solution of Sub-PS obtained in example 3 and the standard solution of Van-Sub-proPS obtained in example 2 were added to a final concentration of 10. Mu.M in phosphate buffered saline (PBS, 10 mM), respectively, and the fluorescence emission spectrum of the fluorescent probe at an excitation wavelength of 525nm was detected. The experimental results are shown in FIG. 25, where the fluorescence of Van-Sub-proPS is quenched by the fluorescence quencher Dabcyl.
Example 5: the photodynamic properties of Van-Sub-proPS are inhibited by the fluorescence quencher Dabcyl
The Sub-PS obtained in example 3 and Van-Sub-proPS obtained in example 2 were each added to a final concentration of 100. Mu.M in phosphate buffered saline (PBS, 10 mM), chemiluminescent probes were added to a final concentration of 100. Mu.M, the solutions were placed in a centrifuge tube, irradiated for 1min at 520nm monochromatic light, and chemiluminescence was detected. A PBS solution containing chemiluminescent probe (100. Mu.M) was used as a reference. The experimental results are shown in FIG. 26, van-Sub-proPS under light conditions 1 O 2 The yield was about 20% of Sub-PS, indicating that the photodynamic properties of Van-Sub-proPS are inhibited, i.e.the Dabcyl group thereon is also capable of quenching the photodynamic properties of tetrabromofluorescein. The structure of the chemiluminescent probe is
Example 6: van-Sub-proPS selective marker SrtA expressing bacteria
Staphylococcus aureus and Escherichia coli were grown in LB medium at 37℃until OD 600 Reaching 0.6. Diluting the bacterial liquid to OD by using fresh LB culture medium 600 About 0.05. Mu.L of a standard solution containing Van-Sub-proPS obtained in example 2 was added to998. Mu.L of the above-mentioned bacterial liquid was allowed to grow for 4 hours. Bacteria were collected by centrifugation, washed three times with PBS and confocal fluorescent signal acquisition was performed. The experimental results are shown in FIG. 27, which illustrates that Van-Sub-proPS can label SrtA-expressing Staphylococcus aureus, and that E.coli is difficult to label by Van-Sub-proPS due to the lack of SrtA.
Example 7: photodynamic treatment of bacteria expressing SrtA by Van-Sub-proPS
Staphylococcus aureus was incubated with LB medium to OD at 37 ℃ 600 Reaching 0.6. Diluting the bacterial liquid to OD by using fresh LB culture medium 600 0.05. Bacterial solutions incubated without compound served as reference. Staphylococcus aureus and incubated with LB medium containing Van-Sub-proPS (20. Mu.M) prepared in example 1, respectively, for 4h. Next, the Staphylococcus aureus bacterial liquid was diluted in PBS gradient 10 5 Multiple times. The diluted bacterial solutions were subjected to light of 520nm (40 mW/cm 2 ) Irradiating for 30min. Then 10 mu L of the bacterial liquid is uniformly smeared on LB solid medium, and then incubated for 18-24h at 37 ℃. All experiments were performed in triplicate. The bacteriostatic activity of the compounds against bacteria was evaluated by the number of colony forming units of staphylococcus aureus. The experimental results are shown in FIG. 28, which illustrates that Van-Sub-proPS can achieve photodynamic therapy targeting Staphylococcus aureus.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, i.e., the invention is not to be limited to the details of the invention.

Claims (9)

1. An LPETG derivative, characterized by: the structure is as followsWherein R is a fluorescent emitting group capable of being quenched by Dabcyl.
2. An LPETG derivative as defined in claim 1, wherein: and R is a tetrabromofluorescein group or a tetraiodofluorescein group.
3. An LPETG derivative as defined in claim 2, wherein: the structure is as follows
4. Use of the LPETG derivative of any one of claims 1 to 3 as a fluorescent probe for labeling gram positive bacteria expressing Sortase a on the cell membrane surface.
5. A method for fluorescent labelling of gram-positive bacteria, characterised by: use of the LPETG derivative of any one of claims 1 to 3 as a fluorescent probe, and the gram-positive bacterium is a gram-positive bacterium whose cell membrane surface expresses SortaseA.
6. Use of an LPETG derivative as defined in any one of claims 1 to 3 for the preparation of a photodynamic therapeutic composition for bacterial disease.
7. The use according to claim 6, wherein: the bacteria causing the bacterial disease are gram positive bacteria with SortaseA expressed on the surface of the cell membrane.
8. A photodynamic therapeutic composition for bacterial disease, characterized by: an effective ingredient thereof comprising the LPETG derivative as defined in any one of claims 1 to 3.
9. A bacterial disease photodynamic therapy composition as claimed in claim 8, wherein: an LPETG derivative as claimed in any one of claims 1 to 3 as an active ingredient.
CN202310466906.1A 2023-04-27 2023-04-27 LPETG derivative and application thereof Pending CN116478240A (en)

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