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To do so, it needs to interact with and permeate through cell membranes, despite its high overall charge at physiological pH values. In this work, we address the effect of pH in the association of H33342 with lipid bilayers using a combined experimental and computational approach. The partition of H33342 to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes was experimentally quantified using fluorescence spectroscopy and isothermal titration calorimetry (ITC) measurements. Quantum chemical calculations were performed to select the most stable isomer of H33342 for the overall charges 0, +1, and +2, expected to predominate across the 5 &lt; pH &lt; 10 range. The interaction of these isomers with POPC bilayers was then studied by both unrestrained and umbrella sampling molecular dynamics (MD) simulations. Both experimental results and computational free energy profiles indicate that the partition coefficient of H33342 displays a small variation over a wide pH range, not exceeding one order of magnitude. The enthalpy variation upon partition to the membrane suggests efficient hydrogen bonding between the probe and the lipid, namely, for the protonated +2 form, which was confirmed in the MD simulation studies. The relatively high lipophilicity obtained for the charged species contrasts with the decrease in their general hydrophobicity as estimated from octanol\/water partition. This highlights the distinction between lipophilicity and hydrophobicity, as well as the importance of considering the association with lipid bilayers when predicting the affinity for biomembranes.<\/jats:p>","DOI":"10.3390\/molecules28155640","type":"journal-article","created":{"date-parts":[[2023,7,26]],"date-time":"2023-07-26T01:09:01Z","timestamp":1690333741000},"page":"5640","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Interaction of Hoechst 33342 with POPC Membranes at Different pH Values"],"prefix":"10.3390","volume":"28","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9404-831X","authenticated-orcid":false,"given":"Margarida M.","family":"Cordeiro","sequence":"first","affiliation":[{"name":"Coimbra Chemistry Center, Institute of Molecular Sciences (CQC-IMS), University of Coimbra, 3004-535 Coimbra, Portugal"},{"name":"Department of Chemistry, Faculty of Sciences and Technology, University of Coimbra, 3004-535 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6935-4307","authenticated-orcid":false,"given":"Hugo A. L.","family":"Filipe","sequence":"additional","affiliation":[{"name":"Coimbra Chemistry Center, Institute of Molecular Sciences (CQC-IMS), University of Coimbra, 3004-535 Coimbra, Portugal"},{"name":"Polytechnic of Guarda, CPIRN-IPG\u2014Center of Potential and Innovation of Natural Resources, 6300-559 Guarda, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8747-5396","authenticated-orcid":false,"given":"Patr\u00edcia dos","family":"Santos","sequence":"additional","affiliation":[{"name":"Coimbra Chemistry Center, Institute of Molecular Sciences (CQC-IMS), University of Coimbra, 3004-535 Coimbra, Portugal"},{"name":"Department of Chemistry, Faculty of Sciences and Technology, University of Coimbra, 3004-535 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6561-9320","authenticated-orcid":false,"given":"Jaime","family":"Samelo","sequence":"additional","affiliation":[{"name":"Coimbra Chemistry Center, Institute of Molecular Sciences (CQC-IMS), University of Coimbra, 3004-535 Coimbra, Portugal"},{"name":"Department of Chemistry, Faculty of Sciences and Technology, University of Coimbra, 3004-535 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4419-6431","authenticated-orcid":false,"given":"Jo\u00e3o P. Prates","family":"Ramalho","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Hercules Laboratory, Department of Chemistry, School of Science and Technology, University of E\u0301vora, 7000-671 E\u0301vora, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1051-2312","authenticated-orcid":false,"given":"Lu\u00eds M. S.","family":"Loura","sequence":"additional","affiliation":[{"name":"Coimbra Chemistry Center, Institute of Molecular Sciences (CQC-IMS), University of Coimbra, 3004-535 Coimbra, Portugal"},{"name":"CNC\u2014Center for Neuroscience and Cell Biology, University of Coimbra, 3004-535 Coimbra, Portugal"},{"name":"Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3076-9905","authenticated-orcid":false,"given":"Maria J.","family":"Moreno","sequence":"additional","affiliation":[{"name":"Coimbra Chemistry Center, Institute of Molecular Sciences (CQC-IMS), University of Coimbra, 3004-535 Coimbra, Portugal"},{"name":"Department of Chemistry, Faculty of Sciences and Technology, University of Coimbra, 3004-535 Coimbra, Portugal"},{"name":"CNC\u2014Center for Neuroscience and Cell Biology, University of Coimbra, 3004-535 Coimbra, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2023,7,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"701","DOI":"10.1080\/07391102.2009.10507283","article-title":"V Binding of Hoechst 33258 and Its Derivatives to DNA","volume":"26","author":"Bazhulina","year":"2009","journal-title":"J. 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