Flow-based bioconjugation of coumarin phosphatidylethanolamine probes: Optimised synthesis and membrane molecular dynamics studies.

Autor: Varandas PAMM; LAQV, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal., Belinha R; LAQV, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal., Cobb AJA; Department of Chemistry, King's College London, 7 Trinity Street, London SE1 1DB, United Kingdom., Prates Ramalho JP; Department of Chemistry, School of Science and Technology, University of Évora, Rua Romão Ramalho 59, 7000-671 Évora, Portugal; LAQV, REQUIMTE, University of Évora, Rua Romão Ramalho 59, 7000-671 Évora, Portugal; Hercules Laboratory, University of Évora, Palácio do Vimioso, Largo Marquês de Marialva 8, 7000-809 Évora, Portugal., Segundo MA; LAQV, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Electronic address: msegundo@ff.up.pt., Loura LMS; Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal; Coimbra Chemistry Center - Institute of Molecular Sciences (CQC-IMS), Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal; CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-535 Coimbra, Portugal. Electronic address: loura@ff.uc.pt., Silva EMP; Associate Laboratory i4HB - Institute for Health and Bioeconomy, University Institute of Health Sciences - CESPU, 4585-116 Gandra, Portugal; UCIBIO - Applied Molecular Biosciences Unit, Translational Toxicology Research Laboratory, University Institute of Health Sciences (1H-TOXRUN, IUCS-CESPU), 4585-116 Gandra, Portugal.
Jazyk: angličtina
Zdroj: Biochimica et biophysica acta. Biomembranes [Biochim Biophys Acta Biomembr] 2024 Oct; Vol. 1866 (7), pp. 184335. Date of Electronic Publication: 2024 May 18.
DOI: 10.1016/j.bbamem.2024.184335
Abstrakt: A series of phosphatidylethanolamine fluorescent probes head-labelled with 3-carboxycoumarin was prepared by an improved bioconjugation approach through continuous flow synthesis. The established procedure, supported by a design of experiment (DoE) set-up, resulted in a significant reduction in the reaction time compared to the conventional batch method, in addition to a minor yield increase. The characterization of these probes was enhanced by an in-depth molecular dynamics (MD) study of the behaviour of a representative probe of this family, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine labelled with 3-carboxycoumarin (POPE-COUM), in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) 2:1, mimicking the composition of the egg yolk lecithin membranes recently used experimentally by our group to study POPE-COUM as a biomarker of the oxidation state and integrity of large unilamellar vesicles (LUVs). The MD simulations revealed that the coumarin group is oriented towards the bilayer interior, leading to a relatively internal location, in agreement with what is observed in the nitrobenzoxadiazole fluorophore of commercial head-labelled NBD-PE probes. This behaviour is consistent with the previously stated hypothesis that POPE-COUM is entirely located within the LUVs structure. Hence, the delay on the oxidation of the probe in the oxygen radical absorbance capacity (ORAC) assays performed is related with the inaccessibility of the probe until alteration of the LUV structure occurs. Furthermore, our simulations show that POPE-COUM exerts very little global and local perturbation on the host bilayer, as evaluated by key properties of the unlabelled lipids. Together, our findings establish PE-COUM as suitable fluorescent lipid analogue probes.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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