A kind of coumarin derivative ss-590 and its preparation method and application

Abstract

The present invention provides a coumarin derivative SS-590 and a preparation method and application thereof. The coumarin derivative SS-590, whose Chinese name is (E)-1,4-diethyl-8-( 3-(4-hydroxy-3-methoxyphenyl)acryloyl)-1,2,3,4-tetrahydro-7H-pyrano[2,3-g]quinoxaline-7-one, English Named (E)‑1,4‑diethyl‑8‑(3‑(4‑hydroxy‑3‑methoxyphenyl)acryloyl)‑1,2,3,4‑tetrahydro‑7H‑pyrano[2,3‑g]quinoxalin ‑7‑one, abbreviated as SS‑590. The invention provides a method for specific detection of human serum albumin, which is based on the coumarin derivative SS-590 to detect human serum albumin in PBS. The human serum albumin labeled with the coumarin derivative SS-590 in the present invention can be used for SDS-PAGE Gel analysis. It has broad application prospects in the field of biomolecular detection.

Description

technical field

[0001] The invention relates to the technical field of analytical chemistry, in particular to a method for synthesizing coumarin derivatives SS-590 and SS-590, and the application of SS-590 in specific detection and covalent labeling of human serum albumin. Background technique

[0002] Human serum albumin is the main protein in the human blood system. It is synthesized by the liver and enters the blood circulatory system. It plays a key role in maintaining blood osmotic pressure and transporting various endogenous and exogenous substances. In clinical practice, the decrease of human serum albumin concentration is indicative of various diseases such as gastrointestinal inflammation and liver cirrhosis. As a human endogenous substance, the circulation time of albumin-modified drug molecules in the body is significantly prolonged, and the corresponding drug allergic reaction is significantly weakened, which is an effective way for drug optimization. Therefore,...

Examples

Embodiment 1

[0024] Preparation and characterization of SS-590:

[0025]

[0026] 8-Acetyl-1,4-diethyl-1,2,3,4-tetrahydro-7H-pyrano[2,3-g]quinoxalin-7-one (0.5 mmol, 0.15 g ), vanillin (0.6mmol, 0.09g) were dissolved in 20mL of ethanol, 3 to 5 drops of piperidine were added, and refluxed for 70h until the reaction was complete; the system was evaporated to dryness under reduced pressure to obtain a crude product, which was then separated by column chromatography (eluent according to Volume ratio of ethyl acetate: petroleum ether=1:1) to obtain fluorescent probe SS-590.

[0027] 1 H NMR (600MHz, Chloroform-d) δ8.52(s, 1H), 8.07(d, J=15.6Hz, 1H), 7.78(d, J=15.6Hz, 1H), 7.23–7.18(m, 2H) ,6.92(d,J=8.6Hz,1H),6.54(s,1H),6.43(s,1H),5.89(s,1H),3.96(s,3H),3.59–3.55(m,2H), 3.45(q,J=7.1Hz,2H),3.36(q,J=7.1Hz,2H),3.27–3.23(m,2H),1.25(t,J=7.1Hz,3H),1.21(t,J =7.1Hz,3H)( figure 1 ). 13 C NMR (151MHz, CDCl 3 )δ186.73,161.52,153.39,148.12,147.96,146.70,143.38,132.50,128.26,124.20,122.83,116.37,114...

Embodiment 2

[0029] Prepare PBS solution, prepare 2mM SS-590 in DMSO, prepare 20mg / mL human serum albumin in water; take 1950μL of PBS, 5μL of SS-590 in DMSO solution and add it to a fluorescence cuvette, add 50μL of human serum albumin The aqueous solution of protein was mixed evenly and detected on a spectrofluorometer (excitation at 505 nm) after 10 min. The fluorescence intensity at 590nm gradually increases, and the fluorescence emission diagram is shown in Figure 4 .

Embodiment 3

[0031] Prepare PBS solution, prepare 2mM SS-590 DMSO solution, prepare 20mg / mL human serum albumin aqueous solution, prepare 20mg / mL bovine serum albumin aqueous solution, prepare 10mg / mL mouse serum albumin aqueous solution, prepare 8mM Glu, respectively, Ile, Lys, Leu, Met, Phe, Ser, Hyp, Gln, Pro, Tyr, Cystine, Arg, Thr, Asp, Na 2 SO 3 , Na 2 S, H 2 O 2 , NaClO, 4mM Hcy, 40mM Cys and 80mM GSH aqueous solution; in 27 fluorescence cuvettes, take 1950μL of PBS and 5μL of DMSO solution of SS-590 into the fluorescence cuvette respectively, add 50μL of PBS, Glu , Ile, Lys, Leu, Met, Phe, Ser, Hyp, Gln, Pro, Tyr, Cystine, Arg, Thr, Asp, Na 2 SO 3 , Na 2 S, Cys, Hcy, GSH, H 2 O 2 , NaClO, Gly, mouse serum albumin, bovine serum albumin, and human serum albumin solutions were mixed, and the fluorescence intensity was detected on a fluorescence spectrophotometer after 10 min (excitation at 505 nm). Only the human serum albumin added group showed a significant increase in fluo...

technical field

[0001] The invention relates to the technical field of analytical chemistry, in particular to a method for synthesizing coumarin derivatives SS-590 and SS-590, and the application of SS-590 in specific detection and covalent labeling of human serum albumin. Background technique

[0002] Human serum albumin is the main protein in the human blood system. It is synthesized by the liver and enters the blood circulatory system. It plays a key role in maintaining blood osmotic pressure and transporting various endogenous and exogenous substances. In clinical practice, the decrease of human serum albumin concentration is indicative of various diseases such as gastrointestinal inflammation and liver cirrhosis. As a human endogenous substance, the circulation time of albumin-modified drug molecules in the body is significantly prolonged, and the corresponding drug allergic reaction is significantly weakened, which is an effective way for drug optimization. Therefore,...

Examples

Embodiment 1

[0024] Preparation and characterization of SS-590:

[0025]

[0026] 8-Acetyl-1,4-diethyl-1,2,3,4-tetrahydro-7H-pyrano[2,3-g]quinoxalin-7-one (0.5 mmol, 0.15 g ), vanillin (0.6mmol, 0.09g) were dissolved in 20mL of ethanol, 3 to 5 drops of piperidine were added, and refluxed for 70h until the reaction was complete; the system was evaporated to dryness under reduced pressure to obtain a crude product, which was then separated by column chromatography (eluent according to Volume ratio of ethyl acetate: petroleum ether=1:1) to obtain fluorescent probe SS-590.

[0027] 1 H NMR (600MHz, Chloroform-d) δ8.52(s, 1H), 8.07(d, J=15.6Hz, 1H), 7.78(d, J=15.6Hz, 1H), 7.23–7.18(m, 2H) ,6.92(d,J=8.6Hz,1H),6.54(s,1H),6.43(s,1H),5.89(s,1H),3.96(s,3H),3.59–3.55(m,2H), 3.45(q,J=7.1Hz,2H),3.36(q,J=7.1Hz,2H),3.27–3.23(m,2H),1.25(t,J=7.1Hz,3H),1.21(t,J =7.1Hz,3H)( figure 1 ). 13 C NMR (151MHz, CDCl 3 )δ186.73,161.52,153.39,148.12,147.96,146.70,143.38,132.50,128.26,124.20,122.83,116.37,114...

Embodiment 2

[0029] Prepare PBS solution, prepare 2mM SS-590 in DMSO, prepare 20mg / mL human serum albumin in water; take 1950μL of PBS, 5μL of SS-590 in DMSO solution and add it to a fluorescence cuvette, add 50μL of human serum albumin The aqueous solution of protein was mixed evenly and detected on a spectrofluorometer (excitation at 505 nm) after 10 min. The fluorescence intensity at 590nm gradually increases, and the fluorescence emission diagram is shown in Figure 4 .

Embodiment 3

[0031] Prepare PBS solution, prepare 2mM SS-590 DMSO solution, prepare 20mg / mL human serum albumin aqueous solution, prepare 20mg / mL bovine serum albumin aqueous solution, prepare 10mg / mL mouse serum albumin aqueous solution, prepare 8mM Glu, respectively, Ile, Lys, Leu, Met, Phe, Ser, Hyp, Gln, Pro, Tyr, Cystine, Arg, Thr, Asp, Na 2 SO 3 , Na 2 S, H 2 O 2 , NaClO, 4mM Hcy, 40mM Cys and 80mM GSH aqueous solution; in 27 fluorescence cuvettes, take 1950μL of PBS and 5μL of DMSO solution of SS-590 into the fluorescence cuvette respectively, add 50μL of PBS, Glu , Ile, Lys, Leu, Met, Phe, Ser, Hyp, Gln, Pro, Tyr, Cystine, Arg, Thr, Asp, Na 2 SO 3 , Na 2 S, Cys, Hcy, GSH, H 2 O 2 , NaClO, Gly, mouse serum albumin, bovine serum albumin, and human serum albumin solutions were mixed, and the fluorescence intensity was detected on a fluorescence spectrophotometer after 10 min (excitation at 505 nm). Only the human serum albumin added group showed a significant increase in fluo...

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