School of Chemistry, South China Normal University, Guangzhou, 510006, China.
Department of Urology, State Key Laboratory of Oncology in South China, Collaborative Innovation Center For Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, 510060, China.
Mikrochim Acta. 2020 May 6;187(6):313. doi: 10.1007/s00604-020-04296-6.
A low-molecular-weight molecule (4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl-benzoate, DDPB) has been developed. The organic framework possesses very weak fluorescence . The feasibility of the signal transduction has been performed via fluorometric titrations in solution. DDPB gives rise to responses to carboxylesterase 2 (CES2) based on "off-on" responses. The red emission at 670 nm has been derived from the enzyme-induced hydrolysis of ester linkages, thus suppressing the intramolecular charge transfer (ICT) effect and thereby generating the fluorescent segment. The optical excitation window for this probe is extended to the visible light range (λ = 516 nm), and it will induce less harmful influence on biological substances. The detection limit for the measurement of CES2 concentration is as low as 2.33 mU/mL. The conventional studies concerning the activation process are generally performed within only a single liveing cell system. In this study, it is the first time that expression of carboxylesterase 2 in five kinds of cell lines (HeLa > C1498 > active T cell > Jurkat > unactive T cell) has been clarified by flow cytometry, Western blotting, and confocal microscopy analysis. The elucidation of CES2 and its variability in a variety of cells will open new ways for drug metabolism and disease prevention. Graphical abstract We reported a new "substrate-mediated light-on" strategy based on an ester bond cleavage reaction. Most of prepared nanomaterials and organic fluorophores possessed short wavelength emissions in the blue or green region which will not be difficult for cellular imaging. In this study, a novel functional molecule (DDPB) was considered as the substrate for CES2 and the optical "off-on" response was realized. DDPB was cell permeable and possessed very low cytotoxicity. Moreover, the identification of CES2 and their subtle changes in five different cells afforded the sequence for carboxylesterase-2 as Hela > C1498 > Active T cell > Jurkat > Unactive T cell. Inhibition studies showed that the hydrolysis of DDPB was effectively suppressed by bis-p-nitrophenyl phosphate and the cellular tracking results firmly supported this point. To our knowledge, the inter-individual variability for the CES2 expressions in five different cell lines has never been reported via the substrate induced optical changes.
一种低分子量分子（4-（2-（3-（二氰甲基）-5,5-二甲基环己-1-烯-1-基）乙烯基）苯基-苯甲酸酯，DDPB）已被开发出来。该有机骨架具有非常弱的荧光。通过溶液中的荧光滴定已经实现了信号转导的可行性。DDPB 基于“关-开”响应产生对羧酸酯酶 2（CES2）的响应。670nm 的红色发射源于酯键的酶促水解，从而抑制分子内电荷转移（ICT）效应，从而产生荧光片段。该探针的光学激发窗口扩展到可见光范围（λ=516nm），并且它将对生物物质产生较小的有害影响。CES2 浓度测量的检测限低至 2.33mU/mL。关于激活过程的传统研究通常仅在单个活细胞系统中进行。在这项研究中，首次通过流式细胞术、Western 印迹和共聚焦显微镜分析阐明了五种细胞系（HeLa>C1498>活性 T 细胞>Jurkat>非活性 T 细胞）中羧基酯酶 2 的表达。阐明 CES2 及其在各种细胞中的变异性将为药物代谢和疾病预防开辟新途径。图表摘要我们报道了一种基于酯键断裂反应的新的“底物介导的光开启”策略。大多数制备的纳米材料和有机荧光团在蓝色或绿色区域具有短波长发射，这对于细胞成像来说并不困难。在这项研究中，一种新型功能分子（DDPB）被认为是 CES2 的底物，并且实现了光学“关-开”响应。DDPB 具有细胞通透性和极低的细胞毒性。此外，五种不同细胞中 CES2 的鉴定及其细微变化提供了羧基酯酶-2 的序列为 Hela>C1498>活性 T 细胞>Jurkat>非活性 T 细胞。抑制研究表明，DDPB 的水解被双对硝基苯基磷酸酯有效地抑制，并且细胞跟踪结果坚定地支持了这一点。据我们所知，通过底物诱导的光学变化，从未在五种不同细胞系中报告过 CES2 表达的个体间变异性。
