Reaction-based probe for enzyme activity assay and bioimaging
효소 활성 측정과 생물 영상화를 위한 반응기반 탐침의 개발
- 자연과학대학 화학부
- Issue Date
- 서울대학교 대학원
- bioimaging; reaction-based probe; fluorescence; optical properties enzyme activity assay; spectral responses
- 학위논문 (박사)-- 서울대학교 대학원 자연과학대학 화학부, 2017. 8. 홍종인.
- The detection of enzyme activity provides many clues to the nature of living systems and the interaction between biomolecules. Enzyme reaction triggers chemical transformation of substrate to regulate its function relating to many biological events. The most general approach for enzyme activity assay is a reaction-based strategy using the chemical transformation of substrates. Numerous probes have been developed based on the enzymatic reaction. However, only a few strategies to control a signal have been developed, and most of them have not been applied practically. Herein, several probes based on two basic strategies are proposed.
The chemical transformation of a probe by enzymatic reaction triggers intramolecular cyclization to form fluorescent N-methylisoindole, which is a basic strategy to detect the enzyme activity.
Sulfatases catalyze the hydrolysis of sulfate esters that are present in a range of biomolecules. We have developed a new activity-based sulfatase probe (probe 1) that generates a fluorescent N-methylisoindole upon hydrolysis by sulfatase. Because of the autoxidation of N-methylisoindole, the sulfatase activity was also tested under reducing conditions containing either glutathione (GSH) or tris(2-carboxyethyl)phosphine (TCEP), which exhibited little change in the kinetic parameters, but a stronger emission than non-reducing conditions. Probe 1 also showed stronger intensity upon treatment with sulfatase under neutral conditions than acidic conditions, but it still has limitations in the selectivity of a specific sulfatase. Nevertheless, the fluorescent signal of the released N-methylisoindole provides a new assay for measuring sulfatase activity that could be applied for high-throughput screening (HTS).
Bacterial arylsulfatases are crucial to biosynthesis in many microorganisms, as they cleave aromatic sulfate esters for use as a sulfur source. They are associated with pathogenesis and are applied in many areas, such as industry and agriculture. The hydrolysis of probe 1 by sulfatases induced fluorescence intensity enhancement and the generation of colored precipitates through the polymerization of N-methylisoindole, which enabled the monitoring of bacterial arylsulfatase activity and the discrimination of periplasmic sulfatases from cytosolic sulfatases through liquid- and solid-phase colony-based assays.
For imaging analysis, the fluorescence of N-methylisoindole, the product of probe 1 from the cleavage upon sulfatase activity emitted a signal at the shorter wavelength. To overcome this limitation, fluorescence resonance energy transfer (FRET), aggregation-induced emission (AIE), and the excimer formation process were introduced. The FRET-based probe showed no fluorescence changes upon the treatment of sulfatase because the distance between N-methylisoindole and naphthalimide, an FRET acceptor, was 6.6 Å, which was out of range of the Förster distance. The AIE-based probe exhibited decreasing fluorescence and color changes, meaning that N-methylisoindole generation and polymerization did not intensify the AIE process. The excimer formation-based probe showed decreasing excimer emission, but increasing monomer and N-methylisoindole emissions. It also generated colored precipitates. When the probe was applied to bacteria species, the tendency of the emission was different from that in the results of the test in the homogenous condition, but precipitates were still formed. Thus, the excimer formation-based probe was only applicable to staining periplasmic sulfatase-expressing bacteria.
The next strategy to design a probe was based on intramolecular charge transfer (ICT). The basic scaffold was 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) unit conjugated to a modified electron-donating group.
Leucine aminopeptidases (LAPs) are widely distributed in organisms, from bacteria to humans, and play crucial roles in cell maintenance and growth. Thus, assays for LAP are necessary for measuring its activity and inhibitor potency. In this paper, we report a small-molecule probe, DCDHF conjugated to a leucine residue, which exhibits colorimetric and fluorogenic changes according to LAP activity.
Human steroid sulfatase (STS) plays a pivotal role in the regulation of biologically active steroid to bind to the estrogen receptor (ER), which is related to hormone-dependent diseases. DCDHF conjugated with phenyl sulfate ester exhibits pH-dependent emission, which was appropriate for discriminating STS, whose optimal pH is 7
other arylsulfatases usually exhibit the maximal activity at pH 5. DCDHF conjugated with phenyl sulfate interposing a self-immolative spacer between them is a ratiometric probe that could detect and monitor sulfatase activity in a complex cellular context.