A Turn-ON Near-Infrared Fluorescent Probe for the Detection of Dipeptidyl Peptidase IV Activity in Liver Cancers

doi:10.6023/cjoc202404028

Chinese Journal of Organic Chemistry ›› 2024, Vol. 44 ›› Issue (11): 3417-3426.DOI: 10.6023/cjoc202404028 Previous Articles Next Articles

ARTICLE

激活型近红外荧光探针用于肝癌中二肽基肽酶活性检测

张飞, 梅煜, 丁雪峰, 李再峰^*(), 张鹏^*(), 丁彩凤^*()

青岛科技大学化学与分子工程学院光电传感与生命分析教育部重点实验室山东省生化分析重点实验室山东省生命分析高校重点实验室山东青岛 266042

收稿日期:2024-04-19 修回日期:2024-05-20 发布日期:2024-06-13
基金资助:
国家自然科学基金(21904077); 国家自然科学基金(22074074); 山东省高校优秀青年创新团队项目(2022KJC004); 山东省泰山学者计划(tstp20231227); 青岛市自然科学基金(23-2-1-175-zyyd-jch)

A Turn-ON Near-Infrared Fluorescent Probe for the Detection of Dipeptidyl Peptidase IV Activity in Liver Cancers

Fei Zhang, Yu Mei, Xuefeng Ding, Zaifeng Li^*(), Peng Zhang^*(), Caifeng Ding^*()

Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science (Ministry of Education), College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, Shandong 266042

Received:2024-04-19 Revised:2024-05-20 Published:2024-06-13
Contact: *E-mail:lizfengphd@126.com;pengzhang@qust.edu.cn;dingcaifeng@qust.edu.cn
Supported by:
National Natural Science Foundation of China(21904077); National Natural Science Foundation of China(22074074); Youth Innovation Team Development Program of Shandong Higher Education Institutions(2022KJC004); Taishan Scholar Program of Shandong Province(tstp20231227); Natural Science Foundation of Qingdao City(23-2-1-175-zyyd-jch)

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Abstract

Dipeptidyl peptidase IV (DPP-IV), a transmembrane glycoprotein overexpressed in cancer cells, is often used as an important biomarker for early detection of tumors. A near-infrared turn ON fluorescence probe (MB-DPP) was developed for the sensitively detection of DPP-IV in liver cancer cells. MB-DPP employs methylene blue as fluorophore and p-aminobenzyl alcohol as the connecting arm to bond glycyl-L-proline as the specific cleavage site of DPP-IV through dehydration condensation reaction, thereby achieving the fluorescence “turn on” reaction triggered by DPP-IV. Probe MB-DPP emits strong near-infrared fluorescence at 691 nm after the incubation with DPP-IV. Spectral experiments showed that MB-DPP exhibited high selectivity and detection sensitivity (with a detection limit of 1.12 ng/mL) for DPP-VI. Bio-imaging studies showed that MB-DPP could not only achieve fluorescence imaging analysis of DPP-IV activity in liver cancer cells, but also be used for imaging of DPP-IV in liver cancer tumors. Therefore, the development of probe MB-DPP will contribute to the early diagnosis of liver cancer.

Key words: dipeptidyl peptidase, near infrared fluorescent probe, methylene blue, liver cancer, living cell imaging, in vivo imaging

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Fei Zhang, Yu Mei, Xuefeng Ding, Zaifeng Li, Peng Zhang, Caifeng Ding. A Turn-ON Near-Infrared Fluorescent Probe for the Detection of Dipeptidyl Peptidase IV Activity in Liver Cancers[J]. Chinese Journal of Organic Chemistry, 2024, 44(11): 3417-3426.

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Fig. & Tab. 11

Scheme 1. Sensing mechanism of MB-DPP to DPP-IV

Scheme 2. Synthetic route of probe MB-DPP

Figure 1. (a) Absorption and (b) fluorescence spectra of MB-DPP before (black line) and after (red line) the incubation with 80 ng/mL DPP-IV in PBS (pH=7.4, containing 0.5% DMSO) at 37 ℃for 90 min ([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 2. (a) Fluorescence spectra of MB-DPP upon incubated with 80 ng/mL DPP-IV at 37 ℃ for 60 min in PBS of different pH from 4.0~9.0 and (b) fluorescence intensity of MB-DPP at 691 nm before (blue line) and after (red line) the incubation with DPP-IV in PBS of different pH values (4.0~9.0) ([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 3. (a) Fluorescence spectra of MB-DPP incubated with 80 ng/mL DPP-IV in PBS of pH 7.4 at different temperatures for 60 min and (b) fluorescence intensity of MB-DPP at 691 nm before (blue line) and after (red line) the incubation with DPP-IV in PBS at 5~60 ℃ ([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 4. (a) Fluorescence spectra of MB-DPP incubated with 80 ng/mL DPP-IV at 37 ℃ in PBS of pH 7.4 for 0~120 min and (b) fluorescence intensity of MB-DPP at 691 nm before (blue line) and after (red line) the incubation with DPP-IV in PBS of pH 7.4 at 37 ℃ ([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 5. (a) Fluorescence spectra of MB-DPP after the incubation with different concentrations of DPP-IV (0~80 ng/mL) in 10 mmol/L PBS of pH 7.4 at 37 ℃ for 90 min and (b) linear relationship between the fluorescence intensity of MB-DPP at 691 nm and the concentrations of DPP-IV from 0 to 80 ng/mL ([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 6. (a) Inhibition efficiency of different concentrations of sitagliptin (0 to 0.01 μmol/L) for 80 ng/mL DPP-IV activity and (b) linear relationship between the logarithm of concentrations of sitagliptin (0 to 0.01 μmol/L) and the inhibition efficiency ([MB-DPP]=10 μmol/L, λex=600 nm)

Figure 7. (a~d) Real-time fluorescence imaging of Hep G2 cells after incubated with MB-DPP (0~15 μmol/L) for 30 min and (e) relative fluorescence intensities of figures a~d (λex=628 nm, λem: 660~720 nm, ruler bar=100 μm)

Figure 8. (a~d) Real-time fluorescence imaging of Hep G2 cells incubated with 10 μmol/L MB-DPP for 0~90 min, (e) fluorescence imaging of Hep G2 cells pre-treated with 0.1 μmol/L sitagliptin for 30 min and then incubated with MB-DPP for 30 min, (f) fluorescence imaging of HEK 293 cells incubated with 10 μmol/L MB-DPP for 30 min, and (g) relative fluorescence intensities of figures a~e (λex=628 nm, λem: 660~720 nm, ruler bar=100 μm)

Figure 9. (a~h) Fluorescence images of subcutaneous DPP-IV activity in tumor-bearing mice (pseudo-color image) and (i) relative fluorescence intensities of figures a~h The mice were incubated with MB-DPP (2.5 mmol/L, 100 μL) for (a) 0, (b) 0.5, (c) 1.0, (d) 1.5, (e) 2.0, (f) 2.5, (g) 3.0 and (h) 24 h. For the inhibition experiments, the mice were pretreated with sitagliptin (0.1 mmol/L, 100 μL) for 30 min, and then treated with MB-DPP. λex=600 nm, λem: 680~720 nm

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激活型近红外荧光探针用于肝癌中二肽基肽酶活性检测

A Turn-ON Near-Infrared Fluorescent Probe for the Detection of Dipeptidyl Peptidase IV Activity in Liver Cancers

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