Synthesis And Spectroscopic Study Of Lipase And H₂S Dual-response Fluorescent Probes

Lipase is a member of hydrolase,on one hand,lipase promotes the digestion and absorption of lipids in the body,and its interaction with related proteins may become a new idea for the treatment of obesity;on the other hand,it is considered to be an important marker in the diagnosis of acute pancreatitis,and is also associated with common inflammatory reactions and arteriosclerosis.As the third signal molecule in vivo,H₂S is mainly involved in physiological processes such as neural activity,cell growth,angiogenesis and expansion,and inhibition of inflammation and apoptosis.The imbalance of H₂S can cause various diseases such as gastric mucosal damage,diabetes,cirrhosis,inflammation and so on.Some studies have shown that lipase and H₂S are related in the physiological activities of the body.For example,lipase and H₂S have related expression and effect in blood vessels and liver;when the inflammation occurs,the concentration of lipase and H₂S rises simultaneously at the same time.The study of the correlation between them is very likely to provide a basis for exploring the complex physiological regulation process in the body.Based on the previous research background,two kinds of fluorescent probes with aggregation-induced luminescence effect（AIE）were designed and synthesized,and we study the response of these probes to lipase and H₂S.Firstly,we synthesize five kinds of probes,and then they are characterized by elemental analysis,infrared spectroscopy,nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry.Secondly,the probe 1-3 was studied based on 4-（Diethylamino）salicylaldehyde.（1）To study the AIE characteristics of the probe,it indicated that 1 and 3 were ACQ molecules and 2 was AIE molecules;（2）the selective and competitive experiment was studied: competitive ions or molecules（cationic K+,Na+,anionic CO32-,amino acid molecules GSH,Cys,protein molecular BCA,carbonic anhydrase and so on）was not interference;（3）The experimental conditions were optimized: the appropriate p H was 7.4,the temperature was 37℃,the response time was about 30 min or 10 min;（4）Fluorescence titration of lipase: The results showed that the detection limit of 1-2 for Lipase was 0.0046 U/m L and 0.21 U/m L respectively.Importantly the probe 3 accelerated the response to lipase with the stimulation of H₂S,and the detection limit of lipase was 0.078 U/m L;（5）The detection mechanism of probe 3 to lipase was discussed by ESI-MS,IR,computational chemistry and molecular docking.The results indicated that lipase can be recongnised effectively by probe 1 and 2 based on the disruption of ester bond.Reaction of probe 3 with lipase can be accelerated with the stimulation of H₂S for flexibility of probe 3 was increased with the disruption of double carbon bond by H₂S.（6）The recongintion of lipase in cell level was studied by fluorescence imaging.Thirdly,the probe 4-5 was studied based on 4-（Diphenylamino）benzaldehyde.（1）The AIE effect of 4-5 were studied,it indicated that 4 and 5 was AIE molecules;（2）The experimental conditions of probe 4 toward lipase were optimized,the appropriate temperature was 37 °C,the time was 20 min;The selective and competitive experiment demonstrated that competitive ions or molecules（cationic K+,Na+,anionic CO32-,amino acid molecules GSH,Cys,protein molecular BCA,carbonic anhydrase and so on）had no interference on the detection of lipase;Fluorescence titration of lipase and H₂S showed that the probe 4 accelerated the response to lipase with the stimulation of H₂S,and the detection limit of lipase was 0.033 U/m L.（3）The experimental conditions of probe 5 toward H₂S were optimized,the appropriate temperature was 37 °C,the time was 30 min;The selective and competitive experiment demonstrated that competitive ions or molecules（cationic K+,Na+,anionic CO32-,amino acid molecules GSH,Cys,protein molecular BCA,carbonic anhydrase and so on）had no interference on the detection of H2S;Fluorescence titration of H₂S showed that the detection limit of 5 for H₂S was 1.79 μM;（4）The detection mechanism of the experiment was discussed by ESI-MS,IR,computational chemistry and molecular docking.The results indicated that double carbon bond of 4 can be broken with the stimulation of H₂S,which enhance the molecular flexibility and induced an accelerate detection of lipase.Although 5 was unable to detect lipase,there are two active sites for H2S（C=C and maleimide group）.（5）Fluorescence microscopy imaging showed that 4-5 could be used for exogenous lipase and H₂S detection in cell.In a word,five fluorescent probes were synthesized and four of them can be used for the detection of lipase.The reaction of probe 3 and 4 with lipase can be stimulated by H₂S,5 could be and efficient double-site probe of H₂S.