Dark Hydrazone Fluorescence Labeling Agents Enable Imaging of Cellular Aldehydic Load

Abstract

Aldehydes are key intermediates in many cellular processes, from endogenous metabolic pathways like glycolysis to undesired exogenously induced processes such as lipid peroxidation and DNA interstrand cross-linking. Alkyl aldehydes are well documented to be cytotoxic, affecting the functions of DNA and protein, and their levels are tightly regulated by the oxidative enzyme ALDH2. Mutations in this enzyme are associated with cardiac damage, diseases such as Fanconi anemia (FA), and cancer. Many attempts have been made to identify and quantify the overall level of these alkyl aldehydes inside cells, yet there are few practical methods available to detect and monitor these volatile aldehydes in real time. Here, we describe a multicolor fluorogenic hydrazone transfer (“DarkZone”) system to label alkyl aldehydes, yielding up to 30-fold light-up response in vitro. A cell-permeant DarkZone dye design was applied to detect small-molecule aldehydes in the cellular environment. The new dye design also enabled the monitoring of cellular acetaldehyde production from ethanol over time by flow cytometry, demonstrating the utility of the DarkZone dyes for measuring and imaging the aldehydic load related to human disease.

Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis

“…44–46 Furthermore, numerous aldehydes and ketones (e.g., ketone bodies) 47 are generated through normal and pathogenic metabolism. 48 Specific conditions, like oxidative stress, 49–51 are also known to trigger the production of a variety of aldehydes. Consequently, the concentration of cellular carbonyl compounds can span several orders of magnitude, from low nanomolar to high micromolar.…”

“…As expected, the hydrolysis and transimination of hydrazones and oximes are significantly faster under acidic conditions and at elevated temperature. 48,128–131 Senter and co-workers studied the release of the antimitotic agent auristatin E from a monoclonal antibody conjugate and found that the half-life time t 1/2 of the respective hydrazone linkage dropped from t 1/2 = 183 h at pH 7.2 to t 1/2 = 4.4 h at pH 5. 132 This can be a very useful feature for targeted drug delivery.…”

“…Notably, 2,4-DMA (131, 10 mM) showed little toxicity to human cells and was employed by Yuen et al to perform intracellular hydrazone exchange reactions, using a dark hydrazone (“DarkZone”) dye to label and image cellular aldehydes (Figure 15). 48 …”

Aldehyde dehydrogenase 2 in aplastic anemia, Fanconi anemia and hematopoietic stem cells

“…Therapeutic efforts to decrease exposure to toxic reactive aldehydes are likely to be advanced by developing methods for measurement of these substrates Recently, a general sensor of reactive aldehydes and specific sensors of formaldehyde have been developed [94–96]. The challenges for use of these sensors include the volatility of the aldehydic substrates, and the need for reactions to occur at physiological pH and temperature if they are to be applied to viable cells.…”

“…The challenges for use of these sensors include the volatility of the aldehydic substrates, and the need for reactions to occur at physiological pH and temperature if they are to be applied to viable cells. We are presently validating the dark hydrazone sensor [96] for measurement of overall reactive aldehydes in HSC, with the goal of using this technology to detect differences in intracellular aldehyde levels in HSC or other cells from ALDH2*1 and ALDH2*2 individuals and to monitor the effects of ALDH2 activators on these levels, in anticipation of clinical trials.…”