Design, Synthesis and Application of catalyCEST MRI Agents for Enzyme Detection

Persistent Link:
http://hdl.handle.net/10150/626152
Title:
Design, Synthesis and Application of catalyCEST MRI Agents for Enzyme Detection
Author:
Fernández-Cuervo Velasco, Gabriela
Issue Date:
2017
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Embargo:
Release after 04-Oct-2018
Abstract:
A notable need exists for noninvasive tools to increase our mechanistic understanding of disease progression at a cellular and molecular level. Studying the functions of proteins in their innate in vivo tissue environment can provide useful information about pathology enabling appropriate treatment and early diagnosis. Chemical exchange saturation transfer MRI contrast provides real-time functional characterization of the biological landscape and can be used to detect multiple enzyme biomarker activities. A dual-enzyme catalyCEST contrast agent was developed as a proof-of-concept to demonstrate the potential of using a salicylic acid scaffold and control the CEST signal through enzyme activation. In addition, a straightforward route was designed to synthesize a diamagnetic catalyCEST MRI agent that is a substrate for β-galactosidase and β-glucuronidase enzymes. The synthesized agents generated two peaks in the CEST spectrum, at 4.25 ppm corresponding to a carbamate moiety and at 9.25 ppm corresponding to the salicylic acid moiety. Chemical exchange rates of liable protons were determined from a QUESP Hanes-Woolf plot. In the presence of the corresponding enzymes, the catalyCEST agent was activated via saccharide hydrolysis followed by a spontaneous disassembly to produce 4-aminosalicylic acid. This reaction converted the carbamate moiety into a free primary amine, and caused a loss of CEST signal at 4.25 ppm. The CEST signal at 9.25 ppm was unaffected by the enzyme catalysis, and therefore used as an internal control signal. Michaelis-Menten enzyme kinetics studies were performed with CEST MRI to verify that catalyCEST MRI could truly detect enzyme activity. The Michaelis-Menten kinetics constants from MRI studies were compared to the kinetics constants measured with UVvis results from the same contrast agent, demonstrating the quantitative potential of catalyCEST MRI with both contrast agents. These findings demonstrate that the newly synthesized modular agents have the potential to become reliable catalyCEST MRI imaging probes. In addition, the modular design of these agents facilitates the conjugation of other enzyme substrates to the carbamate spacer, so that this approach constitutes a platform technology for the detection of enzyme activity.
Type:
text; Electronic Dissertation
Keywords:
Chemical Biology; Enzyme Detection; Glycosidase; Molecular Imaging; MRI
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Pharmaceutical Sciences
Degree Grantor:
University of Arizona
Advisor:
Pagel, Mark D.; Wondrak, Georg

Full metadata record

DC FieldValue Language
dc.language.isoen_USen
dc.titleDesign, Synthesis and Application of catalyCEST MRI Agents for Enzyme Detectionen_US
dc.creatorFernández-Cuervo Velasco, Gabrielaen
dc.contributor.authorFernández-Cuervo Velasco, Gabrielaen
dc.date.issued2017-
dc.publisherThe University of Arizona.en
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en
dc.description.releaseRelease after 04-Oct-2018en
dc.description.abstractA notable need exists for noninvasive tools to increase our mechanistic understanding of disease progression at a cellular and molecular level. Studying the functions of proteins in their innate in vivo tissue environment can provide useful information about pathology enabling appropriate treatment and early diagnosis. Chemical exchange saturation transfer MRI contrast provides real-time functional characterization of the biological landscape and can be used to detect multiple enzyme biomarker activities. A dual-enzyme catalyCEST contrast agent was developed as a proof-of-concept to demonstrate the potential of using a salicylic acid scaffold and control the CEST signal through enzyme activation. In addition, a straightforward route was designed to synthesize a diamagnetic catalyCEST MRI agent that is a substrate for β-galactosidase and β-glucuronidase enzymes. The synthesized agents generated two peaks in the CEST spectrum, at 4.25 ppm corresponding to a carbamate moiety and at 9.25 ppm corresponding to the salicylic acid moiety. Chemical exchange rates of liable protons were determined from a QUESP Hanes-Woolf plot. In the presence of the corresponding enzymes, the catalyCEST agent was activated via saccharide hydrolysis followed by a spontaneous disassembly to produce 4-aminosalicylic acid. This reaction converted the carbamate moiety into a free primary amine, and caused a loss of CEST signal at 4.25 ppm. The CEST signal at 9.25 ppm was unaffected by the enzyme catalysis, and therefore used as an internal control signal. Michaelis-Menten enzyme kinetics studies were performed with CEST MRI to verify that catalyCEST MRI could truly detect enzyme activity. The Michaelis-Menten kinetics constants from MRI studies were compared to the kinetics constants measured with UVvis results from the same contrast agent, demonstrating the quantitative potential of catalyCEST MRI with both contrast agents. These findings demonstrate that the newly synthesized modular agents have the potential to become reliable catalyCEST MRI imaging probes. In addition, the modular design of these agents facilitates the conjugation of other enzyme substrates to the carbamate spacer, so that this approach constitutes a platform technology for the detection of enzyme activity.en
dc.typetexten
dc.typeElectronic Dissertationen
dc.subjectChemical Biologyen
dc.subjectEnzyme Detectionen
dc.subjectGlycosidaseen
dc.subjectMolecular Imagingen
dc.subjectMRIen
thesis.degree.namePh.D.en
thesis.degree.leveldoctoralen
thesis.degree.disciplineGraduate Collegeen
thesis.degree.disciplinePharmaceutical Sciencesen
thesis.degree.grantorUniversity of Arizonaen
dc.contributor.advisorPagel, Mark D.en
dc.contributor.advisorWondrak, Georgen
dc.contributor.committeememberPagel, Mark D.en
dc.contributor.committeememberWondrak, Georgen
dc.contributor.committeememberJewett, John J.en
dc.contributor.committeememberSun, Daekyuen
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