molecules Article Solid-Phase Synthesis of Fluorescent Probes for Plasma Membrane Labelling Shuo Zhang, Annamaria Lilienkampf and Mark Bradley * EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, Edinburgh EH8 9YL, UK; [email protected] (S.Z.); [email protected] (A.L.) * Correspondence: [email protected] Abstract: The cellular plasma membrane plays a fundamental role in biological processes, including cell growth, signaling and transport. The labelling of the plasma membrane with targeted fluorescent probes offers a convenient and non-invasive way to image the morphological changes and dynamics of a membrane in real-time and, despite many examples of fluorescent plasma membrane probes, a “universal targeting/anchoring moiety” is still required. In this study, a small number of stearic acid-based probes labelled with 6-carboxyfluorescein was designed and fabricated via solid-phase synthesis in which variations in both charge and hydrophobicity were explored. To ease the synthesis process, a gram-scale synthesis of the Fmoc-Lys(6-carboxyfluoresein diacetate)-OH building block was developed, allowing the discovery of optimal probes that carried a positively charged amino group and a stearic acid tail that exhibited intense plasma membrane brightness and robust retention. Keywords: solid-phase synthesis; cell membrane staining; fluorescent probe 1. Introduction Citation: Zhang, S.; Lilienkampf, A.; Fluorescent imaging of various cellular systems and components enables the detailed Bradley, M. Solid-Phase Synthesis of analysis of cellular behaviour and modulation. Amongst these is the plasma membrane, Fluorescent Probes for Plasma which provides both a natural barrier and a door for communicating with the extracellular en- Membrane Labelling. Molecules 2021, vironment. Imaging the cell membrane while monitoring its dynamics by fluorescent probes 26, 354. https://doi.org/10.3390/ offers a non-invasive, highly sensitive and semi-quantitative mean of exploring and unravel- molecules26020354 ling various cellular processes and cell signaling pathways. A variety of fluorescent probes Academic Editors: Péter Kele and targeting the plasma membrane exist, with an efficient membrane anchoring moiety typically Takuya Terai needed to achieve robust and stable membrane labelling. The cellular membrane can be Received: 24 November 2020 targeted by probes that interact with a variety of moieties, and this includes membrane glyco- Accepted: 8 January 2021 proteins (e.g., wheat germ agglutinin-based lectin probes) [1] or antibody-linked fluorophores Published: 12 January 2021 that recognise specific cell-surface markers [2]. Probes that interact with the cell surface via hydrophobic and/or electrostatic interactions are commonly used, including the long-chain (C- 0 0 0 Publisher’s Note: MDPI stays neu- 18) dialkylcarbocyanine dye (DiR, 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindotricarbocyanine tral with regard to jurisdictional clai- iodide) [3], though poor water solubility can limit its application. Cholesterol [4] and ms in published maps and institutio- phospholipid [5] conjugates are good candidates for the plasma membrane, targeting nal affiliations. natural lipids and interacting and entering the membrane phospholipid bilayer via hy- drophobic interactions. Additionally, many plasma membrane probes rely on electrostatic interactions that promote prolonged membrane staining, with the choice of charged moiety varying between probes. The outer leaflet of a cell surface is rich in negatively charged Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. groups due to sialic acid-rich glycoproteins and phospholipids [6], and the incorporation This article is an open access article of positive-charged moieties can help to drive anchoring efficiency. Thus, the commer- distributed under the terms and con- cial lipophilic membrane dye DiR and fluorescent styryl pyridinium dyes, e.g., FM1-43 ditions of the Creative Commons At- (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide [7,8]) tribution (CC BY) license (https:// are all positively charged at physiological pH. Similarly, the conjugation of a quaternary creativecommons.org/licenses/by/ ammonium group onto the hydrophobic dye Nile Red produces an efficient cell membrane 4.0/). stain, which remains attached on/in the cell membrane for over 90 min [9]. Molecules 2021, 26, 354. https://doi.org/10.3390/molecules26020354 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 354 2 of 12 On the other hand, formally negatively charged moieties can result in a poor inter- nalisation of the probes and, consequently, enhanced membrane labelling perhaps not unexpected, in view of a cell surface rich in negatively charged groups. Indeed, the fluo- rophore bis-sulphonate aryleneethynylene gives an efficient labelling of cell membranes, with the lipophilic fluorene ring embedding into the membrane, while the negatively charged sulfonates are solvent-exposed [10]. Indeed, the phenols of fluorescein are typ- ically capped as cleavable esters to allow cellular entry, e.g., the dye Calcein used in live/dead cell assays is made cell-permeable by esterification. Zwitterionic groups have shown good performance in anchoring and fixing fluo- rophores onto the cell membrane, with the suggestion of minimising the probes’ “flip-flop” between the leaflets, and their attachment onto/into the outer interface of the lipid bi- layer [7,11,12]. Overall, as has been discussed, many plasma membrane dyes/probes exist, and all have individual advantages/disadvantages and specific applications and spectral ranges. As such, considering the broad chemical space these probes occupy, there is a lack of well-defined structural criteria for the design of membrane labelling probes, beyond the need for a hydrophobic anchor. Solid-phase synthesis is an efficient and convenient way to generate compounds, allowing a parallel, step-wise assembly of building blocks (e.g., amino acids) [13]. Herein, we report a solid-phase strategy for the parallel synthesis of fluorescently-labelled probes 1–5 (Figure1), bearing stearic acid chain(s) in combination with charged amino acids, with the compounds evaluated for their plasma membrane labelling efficiency and duration. Stearic acid was pragmatically chosen, being widely found in lipids, inert to peroxidation and readily available and highly affordable. It provided a site of commonality while other changes were made across the compound series. 6-Carboxyfluorescein (λex/em 492/517 nm) was used as the fluorophore due to its biocompatibility, high extinction coefficient (70,000 M−1 cm−1) and high fluorescence quantum yield (φ = 0.85) [14,15] with the building block, Fmoc-Lys(6-Carboxyfluorescein-diacetate)-OH, developed to allow an efficient/clean probe synthesis. Carboxyfluorescein was also chosen, rather Molecules 2021, 26, x FOR PEER REVIEW 3 of 13 than rhodamine-based dyes, as it is negatively charged and as such known not to be cell-permeable. O O O O O O HN HN CO2 OH CO2 OH O O NH2 H N N NH H N 2 1 O H O O 2 O NH3 O O HN O CO2 OH O O O H N NH N 2 HN H 3 O O O O CO2 OH O O O O H N NH N N 2 H H O O O 4 NH3 O O O HN NH CO2 OH O O N NH2 N N H H O O 5 FigureFigure 1. 1.Chemical Chemical structuresstructures of of the the fluorescent fluorescent probes probes 1–51.– 5. 2. Results 2.1. Synthesis of Fmoc-Lys(6-Carboxyfluoresein diacetate)-OH To allow for the efficient synthesis of fluorescently labelled probes on solid-phase, without the need for orthogonal protecting group strategies, Fmoc-Lys(6-carboxyfluores- cein-diacetate)-OH 6 was synthesised on a gram scale (Scheme 1). 5(6)-carboxyfluorescein diacetate 7 was activated as an N-hydroxysuccinimide (NHS) ester, with the two regioi- somers 8a/b readily separated by column chromatography [16] (8a 28% yield and 8b 48% yield). A single regioisomer (6-Carboxyfluorescein diacetate NHS ester 8b (2.0 g)) was coupled onto Fmoc-Lys-OH·HCl, in the presence of DIPEA (to quench the HCl salt and NHS generated during the reaction), giving, after column chromatography, Fmoc-L- Lys(6-Carboxyfluorescein-diacetate)-OH 6 in 50% yield. The reaction time (20 min) re- quired a careful control, with extended reaction times resulting in deacetylation of the phenolic hydroxy groups and Fmoc deprotection. Molecules 2021, 26, 354 3 of 12 2. Results 2.1. Synthesis of Fmoc-Lys(6-carboxyfluoresein diacetate)-OH To allow for the efficient synthesis of fluorescently labelled probes on solid-phase, with- out the need for orthogonal protecting group strategies, Fmoc-Lys(6-carboxyfluorescein- diacetate)-OH 6 was synthesised on a gram scale (Scheme1). 5(6)-carboxyfluorescein diacetate 7 was activated as an N-hydroxysuccinimide (NHS) ester, with the two regioiso- mers 8a/b readily separated by column chromatography [16](8a 28% yield and 8b 48% yield). A single regioisomer (6-Carboxyfluorescein diacetate NHS ester 8b (2.0 g)) was coupled onto Fmoc-Lys-OH·HCl, in the presence of DIPEA (to quench the HCl salt and NHS generated during the reaction), giving, after column chromatography, Fmoc-L-Lys(6- Carboxyfluorescein-diacetate)-OH 6 in 50% yield. The reaction time (20 min) required a Molecules 2021, 26, x FOR PEER REVIEWcareful control, with extended reaction times resulting in deacetylation of the phenolic4 of 13 hydroxy groups and Fmoc deprotection. HO O OH AcO O OAc AcO O OAc AcO O OAc O a b O O O O + O O O N O O O O O O OH OH O 7 O O 8a 8b N O c AcO O O OAc HN O O OH FmocHN 6 O ◦ Scheme 1. Synthesis of Fmoc-Lys(6-carboxyfluorescein-diacetate)-OHScheme 1. Synthesis of Fmoc-Lys(6-carboxyfluorescein-diacetate)-OH6. Reaction condition (a) Ac2 O,6. Reaction Pyridine, condition 1 h, 110 C,(a) yield 95%; (b) DIC/NHS, DCM,Ac2O, Pyridine, 30 min, and1 h, column110 °C, yield chromatography 95%; (b) DIC/NH to separateS, DCM, the 30 min, isomers and (column8a 28% chromatography and 8b 42%); (c) Fmoc-Lys-OH·HCl, DIPEA,to separate anhydrous the DMF;isomers yield (8a 50%.28% and 8b 42%); (c) Fmoc-Lys-OH· HCl, DIPEA, anhydrous DMF; yield 50%.