Diagnostic Tools: Molecular Probes for the Detection of Biological Aldehydes Open Access
Wills, Rachel (Spring 2024)
Abstract
Aldehydes, pervasive in various environments, pose health risks at elevated levels due to their collective toxicity. Monitoring aldehyde content in living systems is paramount because of their cumulative impact and proven roles in human pathogenesis. Current methods for detecting cellular aldehydes are limited by their reversible nature, leading to inaccurate content analysis. This work emphasizes utilizing turn-on BODIPY fluorescence sensors for the elucidation of the physiological and pathological functions of aldehydes in human health. Herein, we report three classes of chemical sensor systems that accurately illuminate aldehydes in biological settings.
Our initial experimentation examined diamine BODIPY sensors that generate a benzimidazole moiety upon reaction with various aliphatic aldehydes (C1–C10), resulting in turn-on fluorescence. These sensors exhibit high quantum yields, high dynamic range, and enable the quantification of changes in exogenous and endogenous aliphatic aldehydes in live mammalian cells. This tool has great potential to transform aldehyde research by illuminating cellular metabolites that remain elusive in living systems.
Furthermore, we generated an innovative reaction-based trigger that leverages the exceptional selectivity of 2-aminothiophenol for all aldehyde types, leading to the production of dihydrobenzothiazole and activation of a fluorescence response. Using this trigger, we developed a series of fluorescent sensors for aldehydes by altering the fluorophore, allowing for excitation and emission wavelengths across the visible to near-infrared spectral regions without compromising the reactivity of the bioorthogonal moiety. These sensors exhibit remarkable chemoselectivity, rapid kinetics, and high quantum yields, enabling the detection of diverse aldehyde types, both exogenous and endogenous, within complex biological contexts. Our sensors provide valuable tools for exploring the multifaceted roles of aldehydes in living systems, providing groundwork for further investigations.
Additionally, we developed a FLIM-FRET system with our diamine BODIPY sensor as a donor and boronic acid functionalized-rhodamine dyes as acceptors for the selective study of 3,4-dihydroxyphenylacetaldehyde (DOPAL). We utilized the fluctuations in donor lifetime upon conjugation with the acceptor to probe the DOPAL concentrations in living neuronal cells. These sensors displayed excellent selectivity and rapid reaction rates when introduced to biological systems, allowing for future analysis of the role of DOPAL in pathological events.
Table of Contents
Table of Contents:
Introduction……...…1
Common Biological Aldehydes - 1
Aldehyde Pathogenesis - 2
Propagation of Aldehydes by ROS - 3
Aldehyde DNA Adduct Detection - 4
Aza-Cope Sensors for the Detection of Formaldehyde - 6
Hydrazine Sensors for the Detection of Aldehydes - 7
Hydrazide Sensors for the Detection of Aldehydes - 9
Chapter 1: Chemical Sensors for Detection of Cellular Aliphatic Aldehydes ……...… 10
Turn-on Fluorescent Sensors for Aliphatic Aldehyde Detection - 12
Computationally Derived HOMO and LUMO of Sensor 1a and 1b -13
Scheme 1: Synthesis of Sensor 1a - 14
Scheme 2: Synthesis of Sensor 1b - 14
Quantum Yield of Sensor 1a - 15
Quantum Yield of Sensor 1b - 16
Scheme 3: Synthesis of Sensor Products 3a and 4a - 17
Fluorescence Characterization of Sensor 1a with Various Aldehydes -17
Rate of Aliphatic Aldehyde Detection be Sensor 1a - 18
Flow Cytometry Analysis of Cell Death by Aldehyde Sponges - 20
Flow Cytometry Analysis of Cell Death by Sensor 1a - 21
Flow Cytometry Analysis of Cell Death by Aliphatic Aldehydes - 21
Detection of Exogenous Aliphatic Aldehydes by Sensor 1a in Live Cells - 23
Confocal Analysis of LNCaP Cells Treated with Aliphatic Aldehydes - 24
Confocal Analysis of Sensor 1a Limit of Detection - 25
Detection of Aliphatic Aldehydes by Sensor 1a in Live Cells - 27
Confocal Microscopy Detection of Propanal, MGO and NO Levels - 28
Live Cell Imaging of Endogenous Aldehyde Production by Sensor 1a - 29
Live Cell Imaging of Endogenous Aldehyde Levels by Sensor 1a in the Presence of ALDH2 Activator and Inhibitor - 31
Quantification of Live Cell Imaging of Endogenous Aldehyde Levels by Sensor 1a in the Presence of ALDH2 Activator and Inhibitor - 32
Chapter 1 Supplementary Information - 34-53
Chapter 2: Tunable Fluorescent Probes for Detecting Aldehydes in Living Systems……...…54
Tunable 2-aminophenol BODIPY for the Detection of Aldehydes - 55
Scheme 4: Synthesis of Sensor 1c - 57
Quantum Yield of Sensor 1c - 58
Computationally Derived HOMO and LUMO of Sensor 1a and 1c - 59
Characterization of Sensor 1c - 60
Flow Cytometry Analysis of Cell Death by Sensor 1c - 61
Live Cell Detection of Aldehydes by Sensor 1c - 62
Live Cell Imaging of Endogenous Aldehyde Levels by Sensor 1c in the Presence of ALDH2 Activator and Inhibitor - 63
Scheme 5: Synthesis of Sensor 1e - 64
Scheme 6: Synthesis of Sensor 1f - 65
Fluorescence Characterization of Sensor 1f - 66
Flow Cytometry Analysis of Cell Death by Sensor 1e - 66
Confocal Analysis of Sensor 1e in T-47D Cells - 67
Detection of Aldehydes in Live Organoids by Sensor 1e - 68
Detection of Aldehydes in Mouse Tissue by Sensor 1e - 70
Chapter 2 Supplementary Information - 72-98
Chapter 3: Selective Detection of DOPAL in Neuronal Systems……...…99
Biosynthetic Pathway of DOPAL Production and αSyn Accumulation in the Midbrain - 101
Jablonski Diagram of FRET Pairs - 102
Scheme 7: Synthesis of Rho-BA - 103
Fluorescence Characterization of Sensor 1a with DOPAL - 104
FLIM-FRET Characterization of Sensor 1a-DOPAL with Rho-BA - 105
Flow Cytometry Analysis of Cell Death by Sensor 1a and Rho-BA - 106
FLIM-FRET Comparison of Sensor 1a-DOPAL Complex with Sensor 1a-propanal Complex, with and without the Presence of Rho-BA - 107
Flow Cytometry Analysis of Cell Death by Activators and Inhibitors - 108
Detection of DOPAL by Sensor 1a-Rho-BA Complex due to Exogenous Stimulation - 109
Detection of Endogenous DOPAL by Sensor 1a-Rho-BA Complex - 111
Chapter 3 Supplementary Information - 113-120
References……...…121-139
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