Selective Activation of <i>N</i>,<i>N</i>′‐Diacyl Rhodamine Pro‐fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE)

Abney, Kristopher K.; Ramos-Hunter, Susan J.; Romaine, Ian; Goodwin, J. Shawn; Sulikowski, Gary A.; Weaver, C. David

doi:10.1002/chem.201801409

Cited by 5 publications

(6 citation statements)

References 27 publications

(43 reference statements)

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“…Moreover, the specificity of NEP was also investigated against various analytes. Considering the previous report that a rhodamine probe containing trifluoroacetamide can be hydrolyzed by porcine liver esterase (PLE), 26 we screened some commonly used esterases or amidases in aqueous buffer solution to figure out the specificity of NEP. As anticipated, NEP shows very high specificity for human NE, since the responses caused by other amidases (chymotrypsin, trypsin, carboxypeptidase A, carboxypeptidase B) were negligible (Figure 1d).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Activity-Based Near-Infrared Fluorogenic Probe for Enabling in Vitro and in Vivo Profiling of Neutrophil Elastase

Liu

Xiong

et al. 2019

Anal. Chem.

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Neutrophil elastase (NE), a typical hematopoietic serine protease, has significant roles in inflammatory and immune responses, and thus is highly associated with various diseases such as acute lung injury (ALI) and lung cancer. Rapid and accurate measurement of NE activity in biological systems is particularly important for understanding the role of NE in inflammatory diseases, as well as clinical diagnosis. However, the specific detection and noninvasive imaging of NE in vivo remains a challenge. To address this issue, a small-molecule substrate based near-infrared fluorogenic probe (NEP) for NE was constructed via incorporating pentafluoroethyl as the recognition group with a hemicyanine dye-based fluorophore. This initially quenched probe possesses more than 25-fold red fluorescence enhancement upon the catalysis of human NE, and the detection limit is about 29.6 ng/mL. In addition, the high specificity and the long emission wavelength (λ em max = 700 nm) of NEP allowed the direct monitoring of NE-trafficking, exogenous NE uptake, and endogenous NE upregulation at the cellular level. Moreover, the successful spatiotemporal imaging of NE in ALI model mice also made it a promising new tool in clinical diagnosis for ALI and other lung diseases.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Activity-Based Near-Infrared Fluorogenic Probe for Enabling in Vitro and in Vivo Profiling of Neutrophil Elastase

Liu

Xiong

et al. 2019

Anal. Chem.

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“…This is typically caused by the chemical alteration of a conjugated π-electron system within the proflorophore and requires tolerance for the profluorophore moiety at the enzyme active site. This method has been utilized with great effect in recent years, as seen in the use of coumarin profluorophores in the study of p450 enzymes and rhodamine scaffolds in the investigation of liver esterases. , …”

Section: Discussionmentioning

confidence: 99%

“…This method has been utilized with great effect in recent years, as seen in the use of coumarin profluorophores in the study of p450 enzymes and rhodamine scaffolds in the investigation of liver esterases. 20,21 The second general strategy employs both a fluorophore and a quencher within the substrate that becomes spectroscopically monitorable only when either moiety of the quenched system is cleaved and released by the enzyme. Seminal work by Greenberg and others first applied this principle to study alkaline phosphatase kinetics with a simple naphthalene/ phenylalanine quenching system.…”

Section: ■ Discussionmentioning

confidence: 99%

“Switching On” Enzyme Substrate Specificity Analysis with a Fluorescent Competitive Inhibitor

2021

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Enzymatically driven change to the spectroscopic properties of a chemical substrate or product has been a linchpin in the development of continuous enzyme kinetics assays. These assays inherently necessitate substrates or products that naturally comply with the constraints of the spectroscopic technique being used, or they require structural changes to the molecules involved to make them observable. Here we demonstrate a new analytical kinetics approach with enzyme histidine triad nucleotide binding protein 1 (HINT1) that allows us to extract both useful k cat values and a rank-ordered list of substrate specificities without the need to track substrates or products directly. Instead, this is accomplished indirectly using a "switch on" competitive inhibitor that fluoresces maximally only when bound to the HINT1 enzyme active site. Kinetic information is extracted from the duration of the diminished fluorescence when the monitorable inhibitor-bound enzyme is challenged with saturating concentrations of a nonfluorescent substrate. We refer to the loss of fluorescence, while the substrate competes for the fluorescent probe in the active site, as the substrate's residence transit time (RTT). The ability to assess k cat values and substrate specificity by monitoring the RTTs for a set of substrates with a competitive "switch on" inhibitor should be broadly applicable to other enzymatic reactions in which the "switch on" inhibitor has sufficient binding affinity over the enzymatic product.

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“…45,46 As a result, the emission peak was blue-shifted to 551 nm with the emergence of yellow fluorescence, which made DPP-AM a suitable ratiometric fluorescent probe for imaging of esterase activity. In addition, DPP-AM was successfully applied to evaluate the health status of cells and discriminate live and dead cells, which is based on the high esterase activity in live cells 47,48 and the insufficient esterase activity in injured or dead cells. 49,50 ■ RESULTS AND DISCUSSION Synthesis of DPP-AM.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Upon reaction with esterase, however, the cation disappeared because the acetyl ester bond is cleaved by enzymatic hydrolysis, thus breaking the ICT process. , As a result, the emission peak was blue-shifted to 551 nm with the emergence of yellow fluorescence, which made DPP-AM a suitable ratiometric fluorescent probe for imaging of esterase activity. In addition, DPP-AM was successfully applied to evaluate the health status of cells and discriminate live and dead cells, which is based on the high esterase activity in live cells , and the insufficient esterase activity in injured or dead cells. , …”

Section: Introductionmentioning

confidence: 99%

New Diketopyrrolopyrrole-Based Ratiometric Fluorescent Probe for Intracellular Esterase Detection and Discrimination of Live and Dead Cells in Different Fluorescence Channels

Wang

Yang

et al. 2018

ACS Appl. Mater. Interfaces

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A new diketopyrrolopyrrole-based fluorescent probe (DPP-AM) was designed and synthesized for ratiometric detection of esterase and for imaging of live and dead cells in different modes. DPP-AM showed red fluorescence because of the intramolecular charge transfer (ICT) process from the DPP moiety to the pyridinium cation and gave remarkable ratio changes (about 70 folds), with the fluorescence changing from red to yellow, after treating with esterase because of the broken ICT process. Besides, the detection limit of DPP-AM toward esterase in vitro was 9.51 × 10 U/mL. After pretreating with HO and ultraviolet light radiation, the health status of TPC1 cells was successfully imaged. More importantly, DPP-AM showed yellow fluorescence in live cells and a red fluorescent signal in dead cells, indicating that DPP-AM has great potential applications for assessing esterase activity as well as for discriminating live and dead cells.

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Selective Activation of N,N′‐Diacyl Rhodamine Pro‐fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE)

Cited by 5 publications

References 27 publications

Activity-Based Near-Infrared Fluorogenic Probe for Enabling in Vitro and in Vivo Profiling of Neutrophil Elastase

Activity-Based Near-Infrared Fluorogenic Probe for Enabling in Vitro and in Vivo Profiling of Neutrophil Elastase

“Switching On” Enzyme Substrate Specificity Analysis with a Fluorescent Competitive Inhibitor

New Diketopyrrolopyrrole-Based Ratiometric Fluorescent Probe for Intracellular Esterase Detection and Discrimination of Live and Dead Cells in Different Fluorescence Channels

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