Extracellular Aldehyde Sensing Probes for In Vivo Imaging

Carbonyl-ligation reactions are considered to be largely bioorthogonal due to the rarity of ketones and aldehydes in normal mammalian biology, especially in the extracellular space. However, during development, in wound healing, or in response to many disease conditions, certain extracellular matrix (ECM) proteins can be post-translationally modified by lysyl oxidases to contain aldehyde-bearing side chains. In many diseases, accelerated ECM production is a part of a process called fibrosis (scarring of tissue), and about half of the deaths in the industrialized world arise from disease with a fibrotic component. During fibrogenesis (active fibrosis), lysyl oxidases are upregulated, catalyzing the oxidation of lysine residues on ECM proteins to form lysine aldehyde (allysine, Lys^Ald). Lys^Ald undergoes condensation reactions with other Lys^Ald or Lys residues of adjacent collagens to cross-link proteins. Despite the centrality of fibrogenesis in development and in so many diseases, there is a general lack of tools to noninvasively detect and quantify fibrogenesis in humans or in animal models. Our group used rational design to develop molecular probes for Lys^Ald to enable the detection, staging, and treatment monitoring of fibrogenesis. In this Account, we summarize our design strategies and validation methods of Lys^Ald targeting probes for applications in a wide range of diseases with a fibroproliferative component.The Lys^Ald concentrations exhibit distinct organ- and tissue-specific variations in the progression of fibrogenesis. To increase the sensitivity of Lys^Ald probes, we systematically optimized the probe structures to modulate the kinetics of aldehyde condensation reactions and the reverse hydrolysis reaction, molecular hydrophilicity, pharmacokinetics, and elimination. Incorporating electron-withdrawing groups, acidic moieties, and dual-binding ligands significantly enhanced the condensation rates. Combining these strategies with signal amplification by designing "off-on" probes, we extended the probe applicability from organs of high Lys^Ald levels (lung) to low-concentration systems (liver, tumor, and cardiac tissues). Reducing the hydrolysis rate of the probe-Lys^Ald adduct extended the imaging window and permitted the specific detection of Lys^Ald in the kidneys. Importantly, our design strategies demonstrate multimodal compatibility, validated through magnetic resonance imaging, positron emission tomography, and fluorescence imaging platforms. The multiscale detection capability in different imaging modalities (cellular to in vivo) provides critical spatial-temporal insights into fibroproliferative disease dynamics in different species and tissues, including onset, progression, and therapeutic response. While this Account focuses on the design of molecular probes for Lys^Ald, the strategies employed here establish a generalizable framework for molecular probe development with broad applicability in chemical biology and diagnostic imaging.