A lpetg derivative and its use

Abstract

The application discloses an LPETG derivative and application thereof, and a structural formula of the LPETG derivative is shown in the following formula: wherein R is a fluorescent emission group capable of being quenched by Dabcyl. The application can efficiently and rapidly covalently label bacteria expressing SrtA, has good selectivity, and has high signal-to-noise ratio; the application can effectively distinguish an adsorption signal of vancomycin and a covalent labeling signal mediated by SrtA; and the application can covalently label a photosensitizer through the bacteria, so that target photodynamic therapy of the bacteria is realized.

Description

Technical Field

[0001] This invention belongs to the field of pathogenic bacteria labeling technology, specifically relating to an LPETG derivative and its applications. Background Technology

[0002] Pathogenic bacteria, as pathogens, pose a significant threat to human health. However, not all bacteria are pathogenic to humans; some, as symbiotic organisms, play a crucial role in the host's immunity and maintaining metabolic homeostasis. Developing a method to selectively target pathogenic bacteria without harming symbiotic organisms is of great value in treating bacterial diseases.

[0003] Sortase A (SrtA) is a transpeptidase bound to the bacterial cell membrane surface that catalyzes the attachment of surface proteins to peptidoglycan. It can cleave the Leu-Pro-X-Thr-Gly (LPXTG) polypeptide sequence, yielding a thioester intermediate containing the SrtA substrate. Subsequently, the exposed amino groups on the peptidoglycan undergo nucleophilic attack, leading to covalent bonding between the protein and the peptidoglycan. Therefore, SrtA-based fluorescent labeling can selectively label bacteria expressing SrtA. However, current SrtA fluorescent labeling techniques suffer from drawbacks such as high incubation concentrations, long labeling times, and difficulty in distinguishing between adsorption and covalent labeling signals, limiting the application of this fluorescent labeling method. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an LPETG derivative.

[0005] Another object of the present invention is to provide applications of the above-mentioned LPETG derivatives.

[0006] The technical solution of the present invention is as follows:

[0007] An LPETG derivative, characterized by the following structural formula: Wherein, R is a fluorescent emitting group that can be quenched by Dabcyl.

[0008] In a preferred embodiment of the present invention, R is a tetrabromofluorescein group or a tetraiodofluorescein group.

[0009] A further preferred embodiment has the following structural formula:

[0010]

[0011] The above-mentioned LPETG derivatives are used as fluorescent probes to label Gram-positive bacteria expressing Sortase A on their cell membrane surface.

[0012] A fluorescent labeling method for Gram-positive bacteria, using the above-mentioned LPETG derivative as a fluorescent probe, wherein the Gram-positive bacteria are Gram-positive bacteria that express Sortase A on the cell membrane surface.

[0013] The application of the above-mentioned LPETG derivatives in the preparation of photodynamic therapy compositions for bacterial diseases.

[0014] In a preferred embodiment of the present invention, the bacteria causing the bacterial disease are Gram-positive bacteria that express Sortase A on their cell membrane surface.

[0015] A photodynamic therapy composition for bacterial diseases, the active ingredient of which includes the above-mentioned LPETG derivative.

[0016] In a preferred embodiment of the present invention, the active ingredient is the LPETG derivative described above.

[0017] The beneficial effects of this invention are:

[0018] 1. This invention can efficiently and rapidly and specifically covalently label bacteria expressing SrtA with good selectivity and high signal-to-noise ratio.

[0019] 2. This invention can effectively distinguish between the adsorption signal of vancomycin and the SrtA-mediated covalent labeling signal.

[0020] 3. This invention enables bacterial-targeted photodynamic therapy by covalently labeling photosensitizers with bacteria. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the working principle of Van-Sub-proPS.

[0022] Figure 2 This is the synthetic route for compound 18 in Example 1 of the present invention.

[0023] Figure 3 This is the synthesis route of Van-Sub-proPS and Sub-PS in Embodiment 1 of the present invention.

[0024] Figure 4 Compound 4 obtained in Example 1 of this invention 1 1H NMR spectrum (CDCl3).

[0025] Figure 5 Compound 4 obtained in Example 1 of this invention 13 C10 NMR spectrum (CDCl3);

[0026] Figure 6 Compound 7 obtained in Example 1 of this invention 11H NMR spectrum (DMSO-D6);

[0027] Figure 7 Compound 7 obtained in Example 1 of this invention 13 C10 NMR spectrum (DMSO-D6);

[0028] Figure 8 Compound 11 obtained in Example 1 of this invention 1 1H NMR spectrum (CDCl3);

[0029] Figure 9 Compound 11 obtained in Example 1 of this invention 13 C10 NMR spectrum (CDCl3).

[0030] Figure 10 Compound 14 obtained in Example 1 of this invention 1 1H NMR spectrum (CDCl3).

[0031] Figure 11 Compound 14 obtained in Example 1 of this invention 13 C10 NMR spectrum (CDCl3).

[0032] Figure 12 Compound 15 obtained in Example 1 of this invention 1 1H NMR spectrum (CDCl3).

[0033] Figure 13 Compound 15 obtained in Example 1 of this invention 13 C10 NMR spectrum (CDCl3).

[0034] Figure 14 Compound 18 obtained in Example 1 of this invention 1 1H NMR spectrum (CDCl3).

[0035] Figure 15 Compound 18 obtained in Example 1 of this invention 13 C10 NMR spectrum (CDCl3).

[0036] Figure 16 Compound 21 obtained in Example 1 of this invention 1 1H NMR spectrum (DMSO-D6).

[0037] Figure 17 Compound 21 obtained in Example 1 of this invention 13 C10 NMR spectrum (DMSO-D6).

[0038] Figure 18The Sub-PS obtained in Example 1 of this invention 1 1H NMR spectrum (DMSO-D6).

[0039] Figure 19 The Sub-PS obtained in Example 1 of this invention 13 C10 NMR spectrum (DMSO-D6).

[0040] Figure 20 The Sub-proPS obtained in Example 1 of this invention 1 1H NMR spectrum (DMSO-D6).

[0041] Figure 21 The Sub-proPS obtained in Example 1 of this invention 13 C10 NMR spectrum (DMSO-D6).

[0042] Figure 22 HRMS analysis of the Sub-PS prepared in Example 1 of this invention.

[0043] Figure 23 HRMS analysis of the Sub-proPS prepared in Example 1 of this invention.

[0044] Figure 24 HPLC and HRMS analysis of Van-Sub-proPS prepared in Example 1 of this invention.

[0045] Figure 25 This is the emission spectrum of Van-Sub-proPS in Example 4 of the present invention, where the fluorescence is suppressed by the fluorescence quencher Dabcyl.

[0046] Figure 26 This is the chemiluminescence spectrum of Van-Sub-proPS in Example 5 of the present invention, showing the photodynamic properties suppressed by the fluorescence quencher Dabcyl.

[0047] Figure 27 This is a diagram showing the results of bacteria expressing SrtA using Van-Sub-proPS selective covalent labeling in Example 6 of the present invention.

[0048] Figure 28 This is a diagram showing the results of Van-Sub-proPS photodynamic therapy on bacteria expressing SrtA in Example 7 of the present invention. Detailed Implementation

[0049] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0050] Example 1: Synthesis of fluorescent probe Van-Sub-proPS and reference probe Sub-PS

[0051] The following steps (1) to (6) are as follows Figure 2 As shown:

[0052] (1) To a CH2Cl2 (30 mL) solution containing Boc-glycine (4.3 g, 24.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 7.06 g, 35.1 mmol), 3-azidopropylamine (2.34 g, 23.4 mmol), N,N-diisopropylethylamine (DIPEA, 4.52 g, 35.0 mmol), and 1-hydroxybenzotriazole (HOBt, 4.74 g, 35.1 mmol) were added. The reaction mixture was stirred for 4 h and then extracted twice with aqueous hydrochloric acid (1 M, 30.0 mL). The organic phase was separated and then extracted twice with saturated sodium bicarbonate aqueous solution (30.0 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude compound 1. Compound 1 was dissolved in a solution of dichloromethane (12.0 mL) containing trifluoroacetic acid (TFA, 4.0 mL). The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure to remove the solvent. The residue was dissolved in CH3OH (10.0 mL), and the pH was adjusted to neutral with DIPEA. The mixture was then concentrated under reduced pressure to give crude compound 2. N-hydroxysuccinimide (NHS, 4.33 g, 37.7 mmol) and EDC (9.62 g, 50.2 mmol) were added to a CH2Cl2 (20 mL) solution containing Boc-L-threonine (5.5 g, 25.1 mmol). The reaction mixture was stirred for 2 h, extracted once with aqueous hydrochloric acid (1 M, 50.0 mL), and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude compound 3. Compound 2 and DIPEA (15.1 g, 117.1 mmol) were added to a CH2Cl2 (15.0 mL) solution containing compound 3. The reaction mixture was stirred for 4 hours, then extracted twice with saturated sodium bicarbonate aqueous solution (50.0 mL). 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 (CH2Cl2 / CH3OH = 40:1) to obtain the following... Figure 4 and Figure 5 Compound 4 (6.77 g, 78%) is shown. 1H NMR (500MHz, CDCl3) 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, CDCl3) δ172.13,169.46,156.43,80.62,67.29,59.68,48.99,43.10,36.97,28.51,28.29,18.94.

[0053] (2) Compound 4 (6.4 g, 17.9 mmol) was dissolved in a CH2Cl2 solution (15.0 mL) containing trifluoroacetic acid (TFA, 5 mL). The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The residue was dissolved in CH3OH (10.0 mL), and the pH of the organic solution was adjusted to neutral with DIPEA and concentrated under reduced pressure to give crude product compound 5. NHS (2.84 g, 24.7 mmol) and EDC (6.3 g, 32.9 mmol) were added to a dichloromethane solution containing N-Fmoc-L-glutamic acid-5-tert-butyl ester (7 g, 16.5 mmol). The reaction mixture was stirred for 2 h, extracted once with 1 M hydrochloric acid aqueous solution (50.0 mL), and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude product compound 6. Compound 5 and DIPEA (3.45 g, 26.7 mmol) were added to a CH2Cl2 solution (15.0 mL) containing compound 6. The reaction mixture was stirred for 1 h, then extracted twice with a saturated sodium bicarbonate aqueous solution (50.0 mL). The separated organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2 / CH3OH = 50:1) to give the following... Figure 6 and Figure 7 Compound 7 (10.1 g, 85%) is shown. 1H 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.

[0054] (3) Boc-L-leucine (9 g, 39.0 mmol) was dissolved in 50 mL of CH2Cl2, and 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) were added. The reaction mixture was stirred for 4 h, and extracted twice with 1 M hydrochloric acid aqueous solution (100.0 mL) to separate the organic phase. The organic phase was then extracted once with saturated sodium bicarbonate (100.0 mL). The separated organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product compound 9. Compound 9 (2.7 g, 7.89 mmol) and LiOH·H2O (1 g, 23.8 mmol) were dissolved in CH3OH (10 mL). The reaction mixture was stirred at room temperature for 1 h, and the pH was adjusted to neutral with hydrochloric acid aqueous solution (1M). The mixture was then concentrated under reduced pressure to give crude product compound 10. Compound 7 (5.1 g, 7.7 mmol) was dissolved in 16.0 mL of CH2Cl2 containing diethylamine (DEA, 4 mL). The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure to give crude product compound 8. Compound 8 was dissolved in 15 mL of CH2Cl2, and compound 10, EDC (2.5 g, 13.0 mmol), DIPEA (1 g, 7.75 mmol), and HOBt (1.45 g, 10.7 mmol) were added. The reaction mixture was stirred for 4 h, and then extracted twice with hydrochloric acid aqueous solution (1M, 30.0 mL). The organic phase was separated, extracted twice with saturated sodium bicarbonate aqueous solution (30.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Using CH2Cl2 / CH3OH (50:1) as the eluent, the residue was purified by silica gel column chromatography to obtain the following... Figure 8 and 9 Compound 11 (4.74 g, 82%) is shown. 1H NMR(500MHz,CDCl3)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,CDCl3)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.

[0055] (4) N-α-Fmoc-N-ε-Boc-L-lysine (400 mg, 0.85 mmol) was dissolved in CH2Cl2 (10 mL), and 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) were added. The reaction mixture was stirred for 2 h, and then extracted twice with hydrochloric acid aqueous solution (1 M, 30.0 mL). The separated organic phase was extracted twice with saturated sodium bicarbonate aqueous solution (30.0 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product compound 13. Compound 13 was dissolved in CH2Cl2 (10.0 mL) containing diethylamine (DEA, 3 mL). The reaction mixture was stirred at room temperature for 2 h, and then concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (CH2Cl2 / CH3OH = 20:1) to obtain the following... Figure 10 and 11 Compound 14 (225 mg, 80%) is shown. 1 H NMR (500MHz, CDCl3) 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, CDCl3) δ175.07,156.11,79.13,54.99,49.33,40.12,36.58,34.53,29.89,28.92,28.42,22.85.

[0056] (5) Add Dabcyl-COOH (550 mg, 2.04 mmol) and EDC (700 mg, 3.65 mmol) to a pyridine (6 mL) solution containing compound 14 (800 mg, 2.44 mmol). Stir the reaction mixture at room temperature for 3 h, then concentrate under reduced pressure. Dissolve the residue in 50.0 mL of CH2Cl2. Extract the organic solution twice with hydrochloric acid (1 M, 30.0 mL), then twice with saturated sodium bicarbonate aqueous solution (30.0 mL) to separate the organic phase. Dry and concentrate under reduced pressure to obtain the crude product. Purify the crude product by silica gel column chromatography (CH2Cl2 / CH3OH = 50:1) to obtain the following... Figure 12 and 13Compound 15 (1.03 g, 73%) is shown. 1 H NMR (500MHz, CDCl3) 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, CDCl3) 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.

[0057] (6) Compound 15 (600 mg, 1.04 mmol) was dissolved in 15.0 mL of CH2Cl2 containing trifluoroacetic acid (TFA, 5 mL). The reaction mixture was stirred for 2 h, concentrated under reduced pressure, and the residue was then dissolved in 30.0 mL of CH2Cl2. The organic solution was extracted once with 30.0 mL of saturated NaHCO3 aqueous solution, and the separated organic phase was dried over anhydrous Na2SO4 and concentrated to give 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, 63 mg, 0.52 mmol), and TEA (150 mg, 1.49 mmol) were added. The reaction mixture was stirred at room temperature for 2 h, and then concentrated under reduced pressure. The residue was dissolved in 50.0 mL of CH2Cl2. The organic solution was extracted twice with saturated ammonium chloride aqueous solution (30.0 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude product compound 17. Pd / C (400 mg) was added to a CH3OH solution (20 mL) containing compound 11 (1 g, 1.32 mmol). The reaction mixture was stirred for 24 h under H2 atmosphere and filtered to remove Pd / C. The filtrate was concentrated under reduced pressure to give crude product compound 12. Compound 12 was dissolved in a solution of pyridine (10.0 mL) containing EDC (300 mg, 1.56 mmol) and compound 17. The reaction mixture was stirred at room temperature for 4 h and then concentrated under reduced pressure. The residue was dissolved in 50.0 mL of dichloromethane. The organic solution was extracted twice with hydrochloric acid (1 M, 30.0 mL) and then twice with saturated sodium bicarbonate aqueous solution (30.0 mL) to separate the organic phase, which was dried and concentrated under reduced pressure to give crude product. The crude product was purified by silica gel column chromatography (CH2Cl2 / CH3OH = 30:1) to obtain the following: Figure 14 and 15 Compound 18 (1.07 g, 80%) is shown. 1H NMR(500MHz,CDCl3)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,CDCl3)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.

[0058] The following steps (7) to (10) are as follows Figure 3 As shown:

[0059] (7) 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 0.8 g, 2.89 mmol), 1-Boc-piperazine (0.4 g, 2.15 mmol), and TEA (0.145 g, 1.44 mmol) were added to a DMF (10 mL) solution containing tetrabromofluorescein (1 g, 1.45 mmol). The reaction mixture was stirred at room temperature for 3 h, and then concentrated under reduced pressure. The residue was dissolved in CH2Cl2 (30 mL). The resulting organic solution was extracted twice with aqueous hydrochloric acid (1 M, 30.0 mL), and then once with saturated sodium bicarbonate aqueous solution (20.0 mL). The separated organic phase was dried over anhydrous Na2SO4 and concentrated to obtain the residue. The residue was purified by silica gel chromatography (CH2Cl2 / CH3OH = 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.

[0060] (8) Compound 18 (235 mg, 0.18 mmol) was dissolved in a CH2Cl2 solution (8.0 mL) containing trifluoroacetic acid (TFA, 2.0 mL). The reaction solution was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The residue was dissolved in CH3OH (10.0 mL), and 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 a dichloromethane solution (6.0 mL) containing trifluoroacetic acid (TFA, 2 mL). The reaction solution was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The residue was dissolved in CH3OH (10.0 mL), and the pH of the organic solution was adjusted to neutral with TEA. The solution was then concentrated under reduced pressure to give 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 2 h, and then concentrated under reduced pressure. The residue was dissolved in 15.0 mL of CH2Cl2. The organic solution was extracted once with hydrochloric acid (1 M, 10.0 mL), and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate and concentrated by rotary evaporation to obtain the crude product compound 23. EDC (140 mg, 0.73 mmol) and NHS (42 mg, 0.36 mmol) were added to the DMF solution (5 mL) containing compound 23. The reaction mixture was stirred for 1 h, and then concentrated under reduced pressure. The residue was dissolved in 15.0 mL of CH2Cl2. The organic solution was extracted twice with hydrochloric acid (1 M, 10.0 mL), dried, and concentrated under reduced pressure to obtain the crude product compound 24. Compound 24 was dissolved in pyridine (5 mL), and all of the above-mentioned compounds 19 were added. The reaction mixture was stirred at room temperature for 2 h, and then concentrated under reduced pressure. The solution was purified by HPLC to obtain the following... Figure 20 , 21 And 23 dark red solid Sub-proPS (206 mg, 58%). HPLC conditions: Elution with acetonitrile / H2O at 5 mL / min under UV detection at 254 nm. 10% acetonitrile was eluent for 5 min, then gradually increased to 100% over 30 min, and 100% acetonitrile was eluent for 10 min (t). R =18min). 1H 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 Br4N 18 O 18 Na + (M+Na + )m / z 1953.3892,found 1953.3965.

[0061] (9) Add Sub-proPS (60 mg, 0.03 mmol) to DMF (2 mL) and DBCO Van (55 mg, 0.03 mmol). The reaction solution was stirred at room temperature for 2 h, then concentrated under reduced pressure. The residue was dissolved in a small amount of DMF. CH3OH was added to the above solution for recrystallization. After standing for 0.5 h, the solution was filtered to obtain a dark red solid. The solid was dissolved in DMF and then purified by HPLC to obtain the following... Figure 24 The Van-Sub-proPS (58 mg, 50%) shown was eluted under the following HPLC conditions: acetonitrile / H₂O was used as the mobile phase at 5 mL / min under UV detection at 254 nm. 10% acetonitrile was used for 5 min, then gradually increased to 100% (t) over 30 min. R =13min). MALDI-TOF MS calcd for C 166 H 187 Br4Cl2N 29 O 43 SNa + (M+Na + )m / z 3719.9054,found3719.9014.

[0062] (10) Compound 11 (125 mg, 0.17 mmol) was dissolved in a CH2Cl2 solution (8.0 mL) containing trifluoroacetic acid (TFA, 2.0 mL). The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The residue was dissolved in CH3OH (10.0 mL). The pH of the organic solution was adjusted to neutral with TEA, and the organic phase was concentrated under reduced pressure to give crude product compound 20. All of 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 mixture was stirred at room temperature for 2 h, and then concentrated under reduced pressure. The residue was purified by HPLC to obtain the following product: Figure 18 , 19 And the orange-red solid Sub-PS shown in Figure 22 (116 mg, 50%). 1H 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 .89,168.02,152.76,130.14,129.68,127.38,118.20,110.49,104.75,99.67,99.60,99.12,6 6.58,59.56,58.74,52.35,48.99,48.36,46.80,42.33,41.20,35.94,30.44,30.21,30.03,28 .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 Br4N 11 O 14 Na + (M+Na + )m / z 1418.0985,found1418.1028.

[0063] The structural formula of the Van-Sub-proPS synthesized in this embodiment is:

[0064]

[0065] The structural formula of the Sub-PS synthesized in this embodiment is as follows:

[0066]

[0067] Example 2: Preparation of a standard solution with a concentration of 10 mM fluorescent probe Van-Sub-proPS

[0068] Weigh 36.9 mg of Van-Sub-proPS prepared in Example 1 and dissolve it in 1 mL of dimethyl sulfoxide. This yields a 10 mmol / L (10 mM) standard solution of Van-Sub-proPS.

[0069] Example 3: Preparation of a standard solution with a concentration of 10 mM reference probe Sub-PS

[0070] Weigh 13.9 mg of the Sub-PS prepared in Example 1 and dissolve it in 1 mL of dimethyl sulfoxide. This yields a standard solution of the reference probe at 10 mmol / L (10 mM).

[0071] Example 4: Fluorescence of Van-Sub-proPS was inhibited by the fluorescence quencher Dabcyl.

[0072] The Sub-PS standard solution obtained in Example 3 and the Van-Sub-proPS standard solution obtained in Example 2 were added to phosphate buffer solution (PBS, 10 mM) to a final concentration of 10 μM, respectively. The fluorescence emission spectrum of the fluorescent probe at an excitation wavelength of 525 nm was then detected. The experimental results are as follows: Figure 25 The fluorescence of Van-Sub-proPS is quenched by the fluorescence quencher Dabcyl.

[0073] Example 5: The photodynamic properties of Van-Sub-proPS were inhibited by the fluorescence quencher Dabcyl.

[0074] The standard solutions of Sub-PS obtained in Example 3 and Van-Sub-proPS obtained in Example 2 were added to phosphate buffered saline (PBS, 10 mM) to a final concentration of 100 μM, respectively. Then, the chemiluminescent probe was added to a final concentration of 100 μM. The solutions were placed in centrifuge tubes and irradiated under 520 nm monochromatic light for 1 min, followed by detection of chemiluminescence. A PBS solution containing the chemiluminescent probe (100 μM) was used as a reference. The experimental results are as follows: Figure 26 Under illumination conditions, Van-Sub-proPS 1 The O2 yield is approximately 20% of that of Sub-PS, indicating that the photodynamic properties of Van-Sub-proPS are suppressed, meaning that the Dabcyl group on it can also quench the photodynamic properties of tetrabromofluorescein. The structural formula of the above-mentioned chemiluminescent probe is as follows:

[0075] Example 6: Van-Sub-proPS selective labeling of bacteria expressing SrtA

[0076] Staphylococcus aureus and Escherichia coli were grown in LB medium at 37°C until OD (Oxygen Demand). 600 The concentration reached 0.6. The bacterial culture was diluted to OD using fresh LB medium. 600 ~0.05, 2 μL of a standard solution containing Van-Sub-proPS obtained in Example 2 was added to 998 μL of the above bacterial culture, and the culture was allowed to grow for 4 h. Bacteria were collected by centrifugation, washed three times with PBS, and then confocal fluorescence signal was acquired. Experimental results are shown below. Figure 27 This indicates that Van-Sub-proPS can label Staphylococcus aureus expressing SrtA, while Escherichia coli is difficult to label with Van-Sub-proPS due to the lack of SrtA.

[0077] Example 7: Van-Sub-proPS photodynamic therapy for bacteria expressing SrtA

[0078] Staphylococcus aureus was incubated in LB medium at 37°C until OD. 600 The concentration reached 0.6. Dilute the bacterial culture with fresh LB medium to an OD value of 0.6. 600 ~0.05. The bacterial culture without compound incubation was used as a control. Staphylococcus aureus was incubated for 4 h with LB medium containing Van-Sub-proPS (20 μM) prepared in Example 1. Next, the Staphylococcus aureus culture was serially diluted 10 μM with PBS. 5 The diluted bacterial solutions were then tested under a 520nm light source (40mW / cm²). 2 Irradiate for 30 min. Then, spread 10 μL of bacterial suspension evenly on LB solid medium and incubate at 37°C for 18–24 h. All experiments were performed in triplicate. The antibacterial activity of the compound against the bacteria was evaluated by the number of Staphylococcus aureus colony-forming units. Experimental results are shown below. Figure 28 This demonstrates that Van-Sub-proPS can achieve photodynamic therapy targeting Staphylococcus aureus.

[0079] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. An LPETG derivative, characterized in that: Its structural formula is 。 2. Use of the LPETG derivative of claim 1 for the preparation of a fluorescent probe for labeling the cell membrane surface of Gram-positive bacteria expressing Sortase A, characterized in that: The Gram-positive bacteria is Staphylococcus aureus.

3. The use of the LPETG derivative according to claim 1 in the preparation of a photodynamic therapy composition for bacterial diseases, characterized in that: The bacteria that causes the aforementioned bacterial disease is Staphylococcus aureus.

4. A photodynamic therapy composition for bacterial diseases, characterized in that: Its active ingredient includes the LPETG derivative as described in claim 1, and the bacteria causing the bacterial disease is Staphylococcus aureus.

5. The photodynamic therapy composition for bacterial diseases as described in claim 4, characterized in that: Its active ingredient is the LPETG derivative as described in claim 1.

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