Exploring the substitution of the C21-OAc moiety in [11]Cytochalasans: Synthesis and Evaluation of Fluorophore Conjugates

The actin cytoskeleton plays a central role in cellular organization and dynamics, yet the tools available for targeting actin function remain limited. Cytochalasans represent a large class of actin-targeting natural products that are readily cell-permeable with varying cytotoxicity and actin-targeting capabilities in mammalian cells. Their pharmacologic exploitation not only requires an understanding of their mode of action but also exact knowledge of how they can be derivatized without affecting their bioactivity. Here, we report the design, synthesis, and evaluation of five fluorescently labeled [11]cytochalasan derivatives generated from pyrichalasin H (PyriH) and 19,20-epoxycytochalasin C (EpoxyCytoC). Guided by molecular docking, we identified the C21-OAc moiety as a promising site for tag attachment without disrupting actin binding. Semi-synthetic modifications enabled the conjugation of different dyes to PyriH and EpoxyCytoC scaffolds. Moreover, the effect of linkers separating cytochalasans and dyes was analyzed. Biological evaluation supplemented by in vitro assays to additionally interrogate their activities on the assembly of pure actin filaments revealed distinct activity profiles. Together, this study compares permeability, cytotoxicity, and actin binding of five novel [11]cytochalasan probes bearing substitutions of the C21-OAc moiety. Our findings establish the C21-OAc position as a versatile functionalization site and provide a framework for developing next generation cytochalasan-based actin targeting probes.