Fluorescent Tools for Imaging Oxidative Stress in Biology

265 Pages·2018·10.925 MB·English

by  Amandeep Kaur (auth.)

Springer Theses
Recognizing Outstanding Ph.D. Research
Amandeep Kaur

Fluorescent Tools for
Imaging Oxidative
Stress in Biology
Springer Theses
Recognizing Outstanding Ph.D. Research
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Amandeep Kaur
Fluorescent Tools
for Imaging Oxidative Stress
in Biology
Doctoral Thesis accepted by
the University of Sydney, Australia
123
Author Supervisor
Dr. Amandeep Kaur Dr. ElizabethJ. New
Schoolof Chemistry Schoolof Chemistry
University of Sydney University of Sydney
Sydney,NSW Sydney,NSW
Australia Australia
ISSN 2190-5053 ISSN 2190-5061 (electronic)
SpringerTheses
ISBN978-3-319-73404-0 ISBN978-3-319-73405-7 (eBook)
https://doi.org/10.1007/978-3-319-73405-7
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’
Supervisor s Foreword
The ability to visualise cellular processes in real time is crucial to understand
diseasedevelopmentandstreamlinetreatment.Thiscanbeachievedbyfluorescent
chemical tools that can reversibly sense disturbances in cellular environments
duringpathogenesis.Inparticular,perturbationsincellularredoxstateareofcurrent
interestinmedicalresearch,withoxidativestressnowimplicatedinpathogenesisof
many diseases.
The work described here addressed the lack of tools to study redox changes
withincellswithtemporalresolution:despitetheplethoraofreaction-basedprobes
for reactive oxygen species, there were very few reversible sensors. Importantly,
thisworkrepresentsthefirstcomprehensivestudiesoffluorescentsensorsbasedon
theendogenousflavinredoxswitch.Strategieswerealsoinvestigatedandappliedto
achieve ratiometric fluorescence output of the reversible redox probes, which
nullify concentration effects associated with intensity-based probes.
The work also describes suitable strategies to target these probes to specific
cellular organelles, thereby enabling medical researchers to visualise subcellular
oxidative stress levels and addressing the typically poor uptake of chemical tools
into biological studies. In total, four new redox probes are reported in this thesis,
whichhavealreadyfounduseinovertwentyresearchgroupsworldwide.Thefinal
chaptersofthethesisdemonstratesuccessfulapplicationsofthesensorsinavariety
of biological systems ranging from prokaryotes to mammalian cells, to whole
organisms, highlighting the application of the developed small-molecule chemical
sensors in the diagnosis of diverse pathophysiological conditions.
Significant progress in the field of chemical biology requires genuine,
cross-disciplinary collaboration, to drive development of the most useful tools and
to ensure their impactful application. This work involved the direct input of six
other research groups in Australia, with varied expertise from electrochemistry to
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vi Supervisor’sForeword
immunology.Inparticular,theinvolvementofmedicalresearchers,whowillbethe
end users of the probes described herein, ensured that target design focussed on
probesthatarealreadyhavingsignificantimpactintheunderstandingofhealthand
disease.
Sydney, Australia Dr. Elizabeth J. New
September 2017
Abstract
Oxidative stress has been implicated in a myriad of pathological conditions, but
measuringitanddeciphering theunderlying mechanismshasbeenalong-standing
challenge.Mostoftheexistingredoxprobesarereaction-basedandirreversible.In
contrast, reversible redox probes with biologically tuned redox potential can dis-
tinguish between transient and chronic elevations in oxidative capacity.
In this work, a series of novel fluorescent redox probes based on flavins and
nicotinamides have been developed and tested. The first redox-responsive probe,
NpFR1,localisesinthecytoplasmandexhibitsrobustreversibilityofoxidationand
reduction and a clear fluorescence increase upon oxidation. Based on these
promising results, attention turned to developing analogues of NpFR1, with two
mainaims:tosendtheprobetothespecificsubcellularorganellesandtodevelopa
ratiometric probe, in which a change in redox state was signalled by a change in
colour rather than fluorescence intensity.
To this end, six further probes were developed. A mitochondrially localising
derivative NpFR2 was developed by incorporating a lipophilic cationic tag. The
designoftheemission-ratiometricredoxprobeFCR1,whosefluorescenceemission
changesfrombluetogreenuponoxidation,isbasedontheFRETprocessbetween
coumarin donor and flavin acceptor. Furthermore, a set of excitation-ratiometric
FRET probes FRR1 and FRR2 were developed, in which mitochondrial targeting
wasachievedbyemployingrhodamineBwithinherentmitochondrialaccumulation
properties. Finally, nicotinamide-based redox probes NCR3 and NCR4 demon-
strate the use of ICT as a strategy to attain ratiometric fluorescence properties.
Following the successful utilisation of ratiometric and targeting strategies, the
developedprobeswereappliedinavarietyofbiological investigations,whetherin
cellulo, ex vivo or non-mammalian systems. The probes demonstrated excellent
abilities to report on the differences in oxidative capacities under different condi-
tions within each system. This work therefore demonstrates that reversible redox
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viii Abstract
probes based on flavins and nicotinamides exhibit suitable properties for use as
cellular redox probes. The developed probes can be modulated to give ratiometric
output and targeted to specific subcellular compartments. These probes therefore
exhibit potential to aid in deciphering the role of oxidative stress in pathogenesis
and disease progression.
Parts of this thesis have been published in the following journal articles:
1. JonathanYeow, AmandeepKaur,MatthewD.Anscomb andElizabethJ.New,
2014,Anovelflavinderivativerevealstheimpactofglucoseonoxidativestress
in adipocytes. Chemical Communications, 50, 8181–8184. DOI:
10.1039/C4CC03464C
2. Amandeep Kaur, Mohammad A. Haghighatbin, Conor F. Hogan and
Elizabeth J. New, 2015, A FRET-based ratiometric redox probe for detecting
oxidative stress by confocal microscopy, FLIM and flow cytometry. Chemical
Communications, 52, 10510–10513. DOI: 10.1039/C5CC03394B
3. Amandeep Kaur, Kurt W. L. Brigden, Timothy F. Cashman, Stuart T. Fraser
and Elizabeth J. New, 2015, Mitochondrially targeted redox probe reveals the
variations in oxidative capacity of the haematopoietic cells. Organic &
Biomolecular Chemistry, 24, 6686–6689. DOI: 10.1039/C5OB00928F
4. Amandeep Kaur, Jacek L. Kolanowski and Elizabeth J. New, 2016, Reversible
fluorescent probes of biological redox state. Angewandte Chemie International
Edition, 55, 1602–1613. DOI: 10.1002/anie.201506353
5. Jacek L. Kolanowski, Amandeep Kaur and Elizabeth J. New, 2016, Selective
and reversible approaches towards imaging redox signalling using small
molecule probes. Antioxidants and Redox Signaling. DOI: 10.1089/
ars.2015.6588
6. Amandeep Kaur, Karolina Jankowska, Chelsea Pilgrim, Stuart T. Fraser and
Elizabeth J. New, 2016, Studies of haematopoietic cell differentiation with a
ratiometric and reversible sensor of mitochondrial oxidative capacity.
Antioxidants and Redox Signaling. DOI: 10.1089/ars.2015.6495
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