Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. In this study, we evaluated how the size of the linker, site of its attachment and nature of the fluorophore, affect the localization and trafficking properties of fluorescent sterol probes. Varying lengths of linker using the same fluorophore affected cell penetration and retention in specific cell compartments. A C-4 linker was confirmed as optimal. Derivatives of heterocyclic sterol precursors attached with identical C-4 linker to different fluorophores at diverse positions also showed significant differences in their binding properties to various intracellular compartments and kinetics of trafficking. Two novel red-emitting probes with good cell permeability, fast intracellular labelling and slightly different distribution displayed very promising characteristics for sterol probes. These probes also strongly labelled endo/lysosomal compartment in cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, that overlapped with filipin staining of cholesterol. Overall, the present study demonstrates that the physicochemical properties of the fluorophore/linker pairing determine the kinetics of uptake and distribution and subsequently influence the applicability of final probes.

CZ OPENSCREEN Institute of Molecular Genetics of the Czech Academy of Sciences v v i Vídeňská 1083 142 20 Prague 4 Czech Republic

University of Chemistry and Technology Technická 5 166 28 Prague 6 Czech Republic

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Baker JG, et al. Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A1 receptor ligands. Br. J. Pharmacol. 2010;159:772–786. doi: 10.1111/j.1476-5381.2009.00488.x. PubMed DOI PMC

Hao M, et al. Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle. J. Biol. Chem. 2002;277:609–617. doi: 10.1074/jbc.M108861200. PubMed DOI

Solanko KA, Modzel M, Solanko LM, Wustner D. Fluorescent sterols and cholesteryl esters as probes for intracellular cholesterol transport. Lipid Insights. 2015;8:95–114. doi: 10.4137/LPI.S31617. PubMed DOI PMC

Marks DL, Bittman R, Pagano RE. Use of BODIPY-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells. Histochem. Cell Biol. 2008;130:819–832. doi: 10.1007/s00418-008-0509-5. PubMed DOI PMC

Gimpl G, Gehrig-Burger K. Probes for studying cholesterol binding and cell biology. Steroids. 2011;76:216–231. doi: 10.1016/j.steroids.2010.11.001. PubMed DOI

Sezgin E, et al. A comparative study on fluorescent cholesterol analogs as versatile cellular reporters. J. Lipid Res. 2016;57:299–309. doi: 10.1194/jlr.M065326. PubMed DOI PMC

Maxfield FR, Wustner D. Intracellular cholesterol transport. J. Clin. Invest. 2002;110:891–898. doi: 10.1172/JCI16500. PubMed DOI PMC

Králová J, Jurášek M, et al. Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders. Sci. Rep. 2018;8:14428. doi: 10.1038/s41598-018-32776-6. PubMed DOI PMC

Bajaj A, Kondiah P, Bhattacharya S. Design, synthesis, and in vitro gene delivery efficacies of novel cholesterol-based gemini cationic lipids and their serum compatibility: a structure-activity investigation. J. Med. Chem. 2007;50:2432–2442. doi: 10.1021/jm0611253. PubMed DOI

Cooper AG, et al. Alkyl indole-based cannabinoid type 2 receptor tools: exploration of linker and fluorophore attachment. Eur. J. Med. Chem. 2018;145:770–789. doi: 10.1016/j.ejmech.2017.11.076. PubMed DOI

Kubankova M, et al. Linker length affects photostability of protein-targeted sensor of cellular microviscosity. Methods Appl. Fluoresc. 2019;7:044004. doi: 10.1088/2050-6120/ab481f. PubMed DOI

Murphy MP. Targeting lipophilic cations to mitochondria. Biochim. Biophys. Acta. 2008;1777:1028–1031. doi: 10.1016/j.bbabio.2008.03.029. PubMed DOI

Krejcir R, et al. A Cyclic pentamethinium salt induces cancer cell cytotoxicity through mitochondrial disintegration and metabolic collapse. Int. J. Mol. Sci. 2019 doi: 10.3390/ijms20174208. PubMed DOI PMC

O’Connor D, Byrne A, Keyes TE. Linker length in fluorophore-cholesterol conjugates directs phase selectivity and cellular localisation in GUVs and live cells. RSC Adv. 2019;9:22805–22816. doi: 10.1039/c9ra03905h. PubMed DOI PMC

Li Z, Mintzer E, Bittman R. First synthesis of free cholesterol-BODIPY conjugates. J. Org. Chem. 2006;71:1718–1721. doi: 10.1021/jo052029x. PubMed DOI

Li ZG, Bittman R. Synthesis and spectral properties of cholesterol- and FTY720-containing boron dipyrromethene dyes. J. Org. Chem. 2007;72:8376–8382. doi: 10.1021/jo701475q. PubMed DOI PMC

Rihn S, Retailleau P, Bugsaliewicz N, De Nicola A, Ziessel R. Versatile synthetic methods for the engineering of thiophene-substituted BODIPY dyes. Tetrahedron Lett. 2009;50:7008–7013. doi: 10.1016/j.tetlet.2009.09.163. DOI

Zhang M, et al. One-pot efficient synthesis of pyrrolyl BODIPY dyes from pyrrole and acyl chloride. RSC Adv. 2012;2:11215–11218. doi: 10.1039/c2ra22203e. DOI

Awuah SG, Das SK, D'Souza F, You Y. Thieno-pyrrole-fused BODIPY intermediate as a platform to multifunctional NIR agents. Chem. Asian J. 2013;8:3123–3132. doi: 10.1002/asia.201300855. PubMed DOI PMC

Loudet A, Burgess K. BODIPY dyes and their derivatives: syntheses and spectroscopic properties. Chem. Rev. 2007;107:4891–4932. doi: 10.1021/cr078381n. PubMed DOI

Liu Z, et al. Synthesis of cholesterol analogues bearing BODIPY fluorophores by Suzuki or Liebeskind-Srogl cross-coupling and evaluation of their potential for visualization of cholesterol pools. ChemBioChem. 2014;15:2087–2096. doi: 10.1002/cbic.201402042. PubMed DOI PMC

Bergstrom F, et al. Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology. J. Am. Chem. Soc. 2002;124:196–204. doi: 10.1021/ja010983f. PubMed DOI

Ohsaki Y, Shinohara Y, Suzuki M, Fujimoto T. A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy. Histochem. Cell Biol. 2010;133:477–480. doi: 10.1007/s00418-010-0678-x. PubMed DOI

Andrade LD. Understanding the role of cholesterol in cellular biomechanics and regulation of vesicular trafficking: the power of imaging. Biomed. Spectrosc. Ima. 2016;5:S101–S117. doi: 10.3233/Bsi-160157. DOI

Ikonen E. Cellular cholesterol trafficking and compartmentalization. Nat. Rev. Mol. Cell Biol. 2008;9:125–138. doi: 10.1038/nrm2336. PubMed DOI

Elustondo P, Martin LA, Karten B. Mitochondrial cholesterol import. Biochim. Biophys. Acta Mol. Cell. Biol. Lipids. 1862;90–101:2017. doi: 10.1016/j.bbalip.2016.08.012. PubMed DOI

Mesmin B, Kovacs D, D'Angelo G. Lipid exchange and signaling at ER-Golgi contact sites. Curr. Opin. Cell. Biol. 2019;57:8–15. doi: 10.1016/j.ceb.2018.10.002. PubMed DOI

Thelen AM, Zoncu R. Emerging roles for the lysosome in lipid metabolism. Trends Cell Biol. 2017;27:843–860. doi: 10.1016/j.tcb.2017.07.006. PubMed DOI PMC

Meng Y, Heybrock S, Neculai D, Saftig P. Cholesterol handling in lysosomes and beyond. Trends Cell Biol. 2020;30:452–466. doi: 10.1016/j.tcb.2020.02.007. PubMed DOI

van Meer G. Caveolin, cholesterol, and lipid droplets? J. Cell Biol. 2001;152:F29–34. doi: 10.1083/jcb.152.5.f29. PubMed DOI PMC