For drugs to be active they have to reach their targets. Within cells this requires crossing the cell membrane, and then free diffusion, distribution, and availability. Here, we explored the in-cell diffusion rates and distribution of a series of small molecular fluorescent drugs, in comparison to proteins, by microscopy and fluorescence recovery after photobleaching (FRAP). While all proteins diffused freely, we found a strong correlation between pKa and the intracellular diffusion and distribution of small molecule drugs. Weakly basic, small-molecule drugs displayed lower fractional recovery after photobleaching and 10- to-20-fold slower diffusion rates in cells than in aqueous solutions. As, more than half of pharmaceutical drugs are weakly basic, they, are protonated in the cell cytoplasm. Protonation, facilitates the formation of membrane impermeable ionic form of the weak base small molecules. This results in ion trapping, further reducing diffusion rates of weakly basic small molecule drugs under macromolecular crowding conditions where other nonspecific interactions become more relevant and dominant. Our imaging studies showed that acidic organelles, particularly the lysosome, captured these molecules. Surprisingly, blocking lysosomal import only slightly increased diffusion rates and fractional recovery. Conversely, blocking protonation by N-acetylated analogues, greatly enhanced their diffusion and fractional recovery after FRAP. Based on these results, N-acetylation of small molecule drugs may improve the intracellular availability and distribution of weakly basic, small molecule drugs within cells.

• Keywords
• biochemistry, chemical biology, diffusion, human, in-cell, lysosome, physics of living systems, small molecule drugs,
• MeSH
• Diffusion MeSH
• Fluorescence Recovery After Photobleaching * MeSH
• Hydrogen-Ion Concentration MeSH
• Pharmaceutical Preparations metabolism   chemistry   MeSH
• Humans MeSH
• Lysosomes * metabolism   MeSH
• Protons * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Pharmaceutical Preparations MeSH
• Protons * MeSH

While bioorthogonal reactions are routinely employed in living cells and organisms, their application within individual organelles remains limited. In this review, we highlight diverse examples of bioorthogonal reactions used to investigate the roles of biomolecules and biological processes as well as advanced imaging techniques within cellular organelles. These innovations hold great promise for therapeutic interventions in personalized medicine and precision therapies. We also address existing challenges related to the selectivity and trafficking of subcellular dynamics. Organelle-targeted bioorthogonal reactions have the potential to significantly advance our understanding of cellular organization and function, provide new pathways for basic research and clinical applications, and shape the direction of cell biology and medical research.

• Keywords
• Bioconjugations, Bioorthogonal reactions, Cellular organelles, Click chemistry, Targeting,
• MeSH
• Cell Biology MeSH
• Organelles * chemistry   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

meso-Methyl BODIPY photocages stand out for their absorption properties and easy chromophore derivatization. However, their low uncaging efficiencies often hinder applications requiring release of protected substrates in high amounts. In this study, we demonstrate that the sulfonothioated BODIPY group photocleaves a sulfonylthio group from the meso-methyl position with a 10-fold higher quantum yield than the most efficient leaving groups studied to date. Photocleavage, observed in solution and in cells, is accompanied by the spatiotemporally controlled photorelease of H2Sn. For this reason, sulfonothioated BODIPY may be applied in cell signaling, redox homeostasis, and metabolic regulation studies.

• MeSH
• Signal Transduction * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene MeSH Browser