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Mary Angelia Alfred, Kamil Lang, Kaplan Kirakci, Pavel Stuzhin, Petr Zimcik, [Jan Labuta](https://orcid.org/0000-0002-8329-0634), Veronika Novakova

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[Subphthalocyanines as fluorescence sensors for metal cations](https://mdr.nims.go.jp/datasets/b9ff981b-3512-46e4-85a7-a006dcf5ecf6)

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Subphthalocyanines as fluorescence sensors for metal cationsDaltonTransactionsPAPERCite this: Dalton Trans., 2024, 53,2635Received 16th November 2023,Accepted 3rd January 2024DOI: 10.1039/d3dt03839drsc.li/daltonSubphthalocyanines as fluorescence sensors formetal cations†Mary Angelia Alfred, a Kamil Lang, b Kaplan Kirakci, b Pavel Stuzhin, cPetr Zimcik, a Jan Labuta *d and Veronika Novakova *aSubphthalocyanines (SubPcs) and their aza-analogues (SubTPyzPzs) are fluorophores with strong orangefluorescence emission; however, their sensing ability towards metal cations remains uncharted. To fill thisgap, we have developed an efficient method for introducing aza-crown moieties at the axial position ofSubPcs and SubTPyzPzs to investigate the structure–activity relationship for sensing alkali (Li+, Na+, K+)and alkaline earth metal (Ca2+, Mg2+, Ba2+) cations. SubPcs showed better photostability than SubTPyzPzsand even a commonly utilized dye, 6-carboxyfluorescein. Selectivity toward metal cations was driven bythe size of the aza-crown, irrespective of the counter anion. The stoichiometry of binding was found tobe 1 : 1 in all cases, and the interaction between SubPcs and cations was characterized by the corres-ponding apparent binding constants (Ka). Notably, an unusually strong interaction of all sensoric SubPcswith Ba2+ compared to other studied cations was demonstrated. The role of the surrounding environ-ment, i.e. the addition of water or methanol, in sensing cations is shown in detail as well. Selectivitytowards K+ over Na+ was demonstrated in aqueous media with SubPcs bearing the 1-aza-6-crown-18 moiety in Tween 80 micelles. In this case, a 5-fold increase of the fluorescence quantum yield wasobserved upon binding K+ ions. The high brightness, photostability, and sensing activity in aqueous mediamake SubPc macrocycles promising fluorophores for metal cation sensing.IntroductionMetal cations are fundamental elements for sustaining plant,animal, and human life. Their absence or increase to toxiclevels can cause growth disorders, severe malfunction, or evendeath.1,2 Various fluorescent probes are, therefore, being devel-oped to enable their monitoring. Switching such probes onand off is typically driven by photo-induced electron transfer,charge transfer, fluorescence resonance energy transfer,excimer formation or metal-coordination-induced chemicalreactions. The details of these switching mechanisms may befound in many reviews.3–5For the recognition of alkali and alkaline earth metalcations, crowns are probably the most suitable hosts, sincetheir selectivity and sensitivity of recognition may be easilytuned by their size, exchange of oxygen for other heteroatomsor attachment of additional binding sites either in the form ofa supporting ligand or as a cryptand.6–10 Wide diversity may beon the other hand found in signalling moieties, since manystructurally different fluorophores can be found in theliterature.11,12 Phthalocyanines and especially their aza-ana-logues have been shown to possess advantageous properties insensing applications due to their spectral and photophysicalproperties.13,14 The lower homologues of phthalocyanines (i.e.,subphthalocyanines, SubPcs) and of tetrapyrazinoporphyra-zines (SubTPyzPzs), have been used as fluorophores for photo-chromic switches,15,16 and as photosensitizers for photo-dynamic therapy of cancer.17–21 The great application potentialof these macrocycles has been summarized in recentreviews.22,23 To the best of our knowledge, no aza-crown con-taining SubPcs and only a limited number of crown containingSubPcs have been successfully prepared with a crown at theperipheral positions24 or a benzocrown21 at the axial position.Surprisingly, SubPcs have not yet been studied as fluorescencesensors for cation recognition. Thanks to promising literature†Electronic supplementary information (ESI) available: NMR spectra, effect ofthe counter anion, stoichiometry studies, summary of obtained apparentbinding constants, binding isotherms from fluorescence measurements, effectof water, characterization of micelles, and sensing in water. See DOI: https://doi.org/10.1039/d3dt03839daFaculty of Pharmacy in Hradec Kralove, Charles University, Ak. Heyrovskeho 1203,Hradec Kralove, 500 05 Czech Republic. E-mail: veronika.novakova@faf.cuni.czbInstitute of Inorganic Chemistry of the Czech Academy of Sciences, 250 68 Husinec-Řez, Czech RepubliccInstitute of Macroheterocycles, Ivanovo State University of Chemistry andTechnology, Sheremetevskij Pr-t 7, 153000 Ivanovo, RussiadResearch Center for Materials Nanoarchitectonics (MANA), National Institute forMaterials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.E-mail: labuta.Jan@nims.go.jpThis journal is © The Royal Society of Chemistry 2024 Dalton Trans., 2024, 53, 2635–2644 | 2635Open Access Article. Published on 04 January 2024. Downloaded on 2/7/2024 6:47:45 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineView Journal  | View Issuehttp://rsc.li/daltonhttp://orcid.org/0000-0002-2139-418Xhttp://orcid.org/0000-0002-4151-8805http://orcid.org/0000-0002-1068-5133http://orcid.org/0000-0002-1347-3654http://orcid.org/0000-0002-3533-3601http://orcid.org/0000-0002-8329-0634http://orcid.org/0000-0002-2183-1220https://doi.org/10.1039/d3dt03839dhttps://doi.org/10.1039/d3dt03839dhttps://doi.org/10.1039/d3dt03839dhttp://crossmark.crossref.org/dialog/?doi=10.1039/d3dt03839d&domain=pdf&date_stamp=2024-02-02http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3dt03839dhttps://pubs.rsc.org/en/journals/journal/DThttps://pubs.rsc.org/en/journals/journal/DT?issueid=DT053006notes about these fluorophores in the sensing of pH25 and flu-orides,26 we decided to investigate the suitability of thesemacrocycles for cation recognition. We believe that they mayextend the range of fluorophores for the visible area (around590 nm) and that they may be superior to some of the fluoro-phores that suffer from notable photobleaching (e.g., cyaninedyes). Moreover, the great advantage of these compounds isthe possibility of introducing a recognition moiety into theaxial position, whereas peripheral sites may be used for tuningthe spectral and physicochemical properties.In this project, we, therefore, aimed to investigate for thefirst time SubPcs and SubTPyzPzs as signalling moieties forrecognition of alkali and alkaline earth metal cations and toprove the sensing principle even in water employing Tween80 micelles.Results and discussionDesign and synthesisThe target compounds were designed to possess SubPc orSubTPyzPz as a signalling moiety, which ensures strong fluo-rescence emission at about 590 and 560 nm, respectively.Phenyl groups attached to the periphery of macrocycles enrichthe π-system of the macrocycle, thus shifting the absorptionmaximum toward red. Both SubPc and SubTPyzPz cores wereincluded to study the effect of aza-substitution. As axialligands, they carry either 1-aza-4-crown-12, 1-aza-5-crown-15 or1-aza-6-crown-18 capable of cation recognition. These reco-gnition moieties were attached with a macrocycle through aphenylene linker to minimize the basicity of nitrogen respon-sible for switching and thus limit undesirable pH sensitivity(see also the discussion below). A control compound (always-ON) with phenol as the axial ligand was also introduced in thestudy.Ligands intended to be attached to the axial positions ofmacrocycles were prepared by a three-step procedure begin-ning from p-bromophenol (Scheme 1, i–iii). Its hydroxy groupwas protected with a tetrahydropyranyl ether followed byBuchwald–Hartwig coupling with an appropriate aza-crownether. Finally, the protecting group was removed by acidichydrolysis. Noteworthily, similar synthetic pathways withmethyl as a protecting group and using BBr3 or HBr for de-protection were successful in our hands as well, but the yieldsdiffered significantly at each batch. By the above-mentionedprocedure, we obtained the desired ligands 3a–c repeatedly inoverall yields of about 80%.Precursors 4 and 5 were prepared according to theliterature27–29 but crystallization from benzene and hexaneinstead of sublimation as a final purification step of 4 wasused to get this compound in an improved yield of 78%.Cyclotrimerization of 4,5-diphenylphthalonitrile (4) fol-lowed by direct substitution with the respective ligands as aone-pot reaction25 led to the target macrocycles 6a–d in yieldsof about 23% (Scheme 2). Toluene and p-xylene were chosen assolvents to avoid chlorination by Cl2 evolved from BCl3. Thesimilar conditions applied for 5,6-diphenylpyrazine-2,3-dicar-bonitrile (5) and ligand 3b produced the desired SubTPyzPz7b, which was, however, found to be less stable than the SubPcanalogue 6b. The decomposition products made the purifi-cation difficult, so we obtained 7b in a yield of only 11%.Based on this experience, we decided to study the (photo)stabi-lity of the target derivatives by UV–vis spectroscopy by observ-ing the decrease in the Q band in THF both in the dark orupon light exposure (100 W Xe-ozone free lamp, Newport)(Fig. 1). Sensors 6b and 7b, and controls 6d and 7d werechosen as model compounds and compared with unsubsti-tuted zinc(II) phthalocyanine (ZnPc) and 6-carboxyfluorescein(FAM) under the same experimental conditions. All thestudied derivatives were stable in the dark but decomposed invarious degrees under light irradiation. As expected, all thestudied derivatives were less stable than ZnPc, which is knownScheme 1 Synthesis of ligands and precursors – (i) pyridinium 4-tolue-nesulfonate, anhydrous dichloromethane, argon, room temperature,2 h, 98%; (ii) Pd2(dba)2, NaOtBu, Dave Phos, anhydr. toluene, argon,80 °C, 12 h, 80–92%; (iii) 4-toluenesulfonic acid, methanol, argon atmo-sphere, room temperature, 2 h, 75–90%; (iv) phenylboronic acid,PdCl2(PPh3)2, NaBr, saturated K2CO3, dioxane, argon, reflux, 6 h, 78%; (v)acetic acid, reflux, 2 h, 75%.Scheme 2 Synthesis of target macrocycles – (i) BCl3, p-xylene, argon,reflux, 2 h; followed by 3a–c or phenol, anhydrous toluene, argon,reflux, 12 h, 20–25%.Paper Dalton Transactions2636 | Dalton Trans., 2024, 53, 2635–2644 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 04 January 2024. Downloaded on 2/7/2024 6:47:45 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3dt03839dfor great stability,30,31 but had better photostability than FAM,which is a common commercially available fluorophore widelyused in practice. Obviously, the isosteric replacement of ben-zenes in SubPcs with pyrazines decreased the stability of themacrocycle. Decomposition produced no distinct spots on theTLC, so the decomposition products could not be determined.From the fact that only a decrease of all absorption bands wasobserved without the formation of new bands (Fig. S11†), wecan only assume that the macrocycle decomposes into low-molecular-weight substances. Many factors influence thephotostability of SubPcs and their analogues;32 thus, a clearexplanation is difficult. It is, however, worth noting that longirradiation times were used to clearly observe the differencesbetween compounds. Such light doses are unnecessary in prac-tical applications and were used to test that the target sensorcan be considered stable under biologically relevant con-ditions. Nevertheless, these experiments clearly demonstratedthat 6b is superior to its aza-analogue 7b in photostability.UV–vis spectral propertiesThe characteristic Q band was observed for SubPcs andSubTPyzPzs at about 582 nm and 550 nm, respectively(Table 1). Peripheral phenyls contributed to the π-system of theparent macrocycle, resulting in an ∼20 nm red shift in com-parison with nonsubstituted macrocycles.16,25,33 The identicalpositions of Q bands of 6a–d indicated no involvement of theaxial ligands, which is in accordance with the literature.25Since 6b has a more straightforward purification procedure,better (photo)stability and a red-shifted absorption band (incomparison with 7b), which is advantageous for any biologicalexperiments, we decided to exclude aza-analogue 7b fromfurther fluorescence studies and focus on SubPc derivativesonly, which seem to have better potential in this application.Metal cation sensing in tetrahydrofuranSwitching fluorescence between ON and OFF states is typicallybased on blocking and facilitating a quenching process,respectively. Highly efficient quenching in the OFF state isnecessary for good signal-to-noise ratios during analyte detec-tion. In our case, quenching in the OFF state was enabled byphoto-induced electron transfer (PET) occurring between anitrogen in the aza-crown (serving as a donor of electrons) andthe SubPc macrocycle (serving as an acceptor). By coordinatingthe cation to the recognition site of the aza-crown, the loneelectron pair of the nitrogen of the aza-crown cannot provideelectrons for the PET process, which results in the restorationof strong fluorescence emission. The selectivity of recognitionof a given cation should be driven by the size of the aza-crowncavity while the increase in fluorescence (fluorescenceenhancement factor, FEF) should be driven by the strength ofthe coordination of the lone pair to the cation. To prove thismechanism, extensive fluorescence studies were performed.Upon excitation, SubPcs 6a–c in THF emitted only weakfluorescence at 593 nm with a Stokes shift of 10 nm.19,25Fluorescence quantum yields (ΦF) were below 0.003 due tohighly efficient quenching by PET from the axial nitrogen tothe macrocyclic core. The always-ON control 6d possessed, onthe other hand, strong fluorescence emission with themaximum at 592 nm and ΦF = 0.12 (THF), which correspondto the values published for SubPcs and their analogues (forexample ΦF in acetone for unsubstituted SubPc with thephenoxy group at the axial position, 6d and 7d were reportedto be 0.18, 0.28 and 0.13, respectively25).Before studying the cation binding to azacrown SubPcs, wefocused first on the potential effect of the counter anion. Inour previous project with zinc(II) azaphthalocyanines,9 wedescribed that the sensitivity toward cations is strongly depen-dent on the counter anion used and increased as follows:NO3− < Br− < CF3SO3− < ClO4− ≪ SCN−. Extraordinary highsensitivity toward SCN− resulted from coordination of theseanions to the central zinc(II) atom. To select the most suitablecounter anion for the current series of SubPcs, we first titrated6c as a model compound with different potassium salts(Fig. S12†). Since only negligible differences were observedbetween thiocyanates and other analytes, we further confirmedthe role of zinc(II) in the coordination of the counter aniondescribed in our previous project (because such an interactionis impossible with the central boron atom of 6c). Overall, theeffect of the counter anion was rather limited; therefore, tri-flate (CF3SO3−) was selected as a counter anion for the follow-ing experiments due to high ΦF in the ON state.The ability of 6a–c to serve as fluorescence sensors by theabove-described mechanism was studied in detail by titrationswith Li+, Na+, K+, Mg2+, Ca2+ and Ba2+ triflates. In the case ofsensitive analytes, the stepwise addition of the salt (in aMeOH stock solution) to the THF solution of the studiedSubPcs led to a steep increase in fluorescence emission(Table 1, FEF values; Fig. 2c, d and Fig. S16†), reaching a valueof ΦF ∼0.1. This ΦF is close to the value of the always-ONcontrol 6d, i.e. the maximum possible value, which suggestsstrong binding of particular cations with efficient blocking ofPET. Expectedly, no changes were observed in the absorptionspectra (Fig. 2a) since electrons of the aza-crown are notinvolved in the π-conjugated system of the macrocycle.The stoichiometry of binding of sensitive analytes wasstudied by both NMR titration studies (Fig. 3, S13 and S14†)and Job’s method of continuous variations with fluorescenceFig. 1 (a) Changes in the absorption spectra of 6b (1 μM, THF) uponlight irradiation. (b) Normalized decrease of absorption at Q bandmaximum upon light irradiation (100 W Xe-ozone free lamp, Newport).ZnPc and FAM states for unsubstituted zinc(II) phthalocyanine and 6-car-boxyfluorescein, respectively.Dalton Transactions PaperThis journal is © The Royal Society of Chemistry 2024 Dalton Trans., 2024, 53, 2635–2644 | 2637Open Access Article. Published on 04 January 2024. Downloaded on 2/7/2024 6:47:45 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3dt03839dmonitoring (Fig. S15†). In the 1H NMR spectra of 6c, protonson the axial phenylene linker at 5.33 and 6.18 ppm shifteddownfield upon the addition of K+ up to 5.46 and 6.62 at a 1 : 1ratio of 6c : KOTf with Δδ = 0.13 and 0.44 ppm, respectively(Fig. 3). Further addition of K+ did not alter the position of theresonances. The shift can be explained by the fact that coordi-nation of the cation leads to a lower ability of nitrogen todonate electrons to phenylene, which leads to a magneticdeshielding effect. Similarly, the signal at approximately3.27 ppm corresponding to the protons of –CH2– next to thenitrogen in the aza-crown shifted upfield to 2.97 ppm (Δδ =−0.30 ppm) at a 1 : 1 ratio of 6c : KOTf and did not movefurther at higher salt concentrations. This opposite shift of 6cresonances was probably caused by the reduction of aza-crownconformational flexibility in conjunction with changes in theelectron density on the nitrogen atom upon cation encapsula-tion. The interaction of 6c with Ba2+ (Fig. S13†) showed asimilar pattern with chemical shift differences of Δδ = 0.16and 0.61 ppm for phenylene and Δδ = −0.19 ppm for aliphaticsignals of aza-crown protons. Similar cation-induced NMRshifts were also observed in the literature for aza-crown con-taining benzyl sidearms.35 No changes in the 1H NMR of thealways-ON control 6d were noticed (Fig. S14†). NMR studieswith 6c and its negative control 6d, thus, unequivocally proved1 : 1 binding stoichiometry in the case of 6c/K+ and 6c/Ba2+.This is in agreement with the results from Job’s plot (i.e.,method of continuous variations, Fig. S15†), where the 1 : 1stoichiometry was further confirmed independently for 6b/Na+, 6b/Ba2+, 6c/K+ and 6c/Ba2+ using the fluorescence emis-sion intensity.The binding strength between SubPcs 6a–c and individualcations was then analyzed using a 1 : 1 host–guest bindingmodel,36,37 with the apparent binding constant Ka. The con-stant is apparent due to the presence of MeOH in the cationstock solution, which also interacts with SubPc’s aza-crown38unit (more details are given below). The binding isotherms areplotted in Fig. 2c, d and Fig. S16† and the actual values aregiven in Table 1 and Table S1.†Size-driven recognition of cations took place only for alkalimetal cations, whereas limited size preference was observed inthe group of alkaline earth metal cations. For example, 6cbearing a 1-aza-6-crown-18 moiety is switched ON by K+ andnot by smaller cations, such as Na+ and Li+ (where the bindingconstants are small). Although the FEF increases in the pres-ence of both K+ and Li+, in the case of Li+ this occurs only athigh concentrations since Ka = 5.7 M−1.SubPc 6b with a 1-aza-5-crown-15 moiety prefers, on theother hand, Na+ over K+, which is documented by higher FEFand Ka values for 6b/Na+ interaction than those for 6b/K+. Thesmallest analogue 6a with a 1-aza-4-crown-12 moiety recog-Table 1 Spectral and photophysical properties of target derivatives in THFaCompound Axial ligand λA (nm) λF (nm) ΦF (OFF) ΦF (ON) FEF Kab (M−1)6a 1-Aza-4-crown-12 583 593 0.0023 0.097 Li+ 42 9.90.0023 Na+ —c —c0.0023 K+ —c —c0.10 Ca2+ 43 5000.013 Mg2+ 5.7 90800.14 Ba2+ 61 192 0006b 1-Aza-5-crown-15 582 593 0.0014 0.0117 Li+ 8.4 5.50.019 Na+ 14 6150.0053 K+ 3.8 760.0006 Ca2+ 11 580.0041 Mg2+ 2.9 5200.15 Ba2+ 107 13 9006c 1-Aza-6-crown-18 583 593 0.0022 0.089 Li+ 40 5.70.012 Na+ 5.5 19400.097 K+ 44 36 100 d0.10 Ca2+ 45 11 3000.094 Mg2+ 43 6 2000.090 Ba2+ 41 384 000 e6d Phenol 582 592 0.12 0.11 Li+ —c —c0.12 Na+ — c —c0.12 K+ —c —c0.12 Ca2+ —c —c0.12 Mg2+ —c —c0.13 Ba2+ —c —c7b 1-Aza-5-crown-15 550 5637d Phenol 550 562a Absorption maximum (λA), fluorescence emission maximum (λF), fluorescence quantum yield (ΦF) – either without any analyte (ΦF (OFF)) or atthe plateau phase of titration in excess of appropriate salt (in the form of triflates), fluorescence enhancement factor (FEF, i.e. the ratio betweenΦF (ON) and ΦF (OFF)), and apparent association constant (Ka) determined using 1 : 1 host–guest binding model. ΦF values were determined by acomparison method using rhodamine 6G as the reference (ΦF = 0.94, EtOH34). For the details of titration experiments, see the Experimental part.b Values determined from fluorescence emission data. All values have a relative error <15%. cNo changes in ΦF of statistical significance wereobserved upon salt addition. d Value determined from NMR is Ka = 34 800 M−1. e Value determined from NMR is Ka > 230 000 M−1 (axial phenyllinker resonances at 5.33 and 6.18 ppm were used for analyses).Paper Dalton Transactions2638 | Dalton Trans., 2024, 53, 2635–2644 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 04 January 2024. Downloaded on 2/7/2024 6:47:45 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3dt03839dnized Li+ over the others; however, Ka was 9.9 M−1 only, indi-cating that this sensor is selective for Li+ but can be switchedON only at high Li+ concentrations.Alkaline earth metal cations were coordinated to 6a–c moretightly than alkali metal cations, which was obvious from bothhigh FEF values reaching the value of the control compound6d and the high Ka values (approximately in thousands of M−1)(see Table 1 and Table S1†). A similar trend has been pub-lished before using different methods (i.e., absorption39 andfluorescence8 spectroscopy, polarography40 or laser infraredmultiple photon dissociation spectroscopy41). This can beexplained by the fact that alkaline earth metal cations areroughly similar in size to alkali metals, but being divalentcations, they possess a larger positive charge. As a result, theirinteraction with the aza-crown moiety is stronger, resulting inhigher FEF and Ka values. Surprisingly, the binding of Ba2+was, in our case, abnormally strong since the Ka values weredetermined to be 192 000, 13 900 and 384 000 M−1 for 6a, 6band 6c, respectively. This gives us a great starting point forfollow-up studies on Ba2+ sensors.Importantly, there was a decrease in the fluorescence emis-sion upon the addition of water or MeOH into the solution ofthe already switched-ON SubPc 6c (1 μM) by Ba2+ (10.0 μM), asseen in Fig. 2e. This effect was moderate for MeOH and moresubstantial for water. This indicates that more polar moleculesbind more strongly to the aza-crown unit38 and knock out theBa2+ cation, which enables fluorescence quenching via PET.This is further depicted by the weakened fluorescenceresponse (and lower sensitivity) of a 6c solution containing adefined amount of water when titrated with Ba2+ cations(Fig. S17a†). Similar behaviour can also be observed for K+(Fig. S17b†). These results suggest that the ambient environ-ment plays a very important role in cation sensing, and it isessential to investigate the sensing capability of the sensors inaqueous media to assess their impact as fluorescence sensors.Metal cation sensing in waterSubPcs are too hydrophobic sensor molecules to be used inwater without losing their monomeric character and fluo-rescence properties. To overcome these limitations, compound6c, chosen as a model compound of the series, and 6d, acontrol compound always in the ON state, were integrated into∼10 nm-sized Tween 80 micelles. Tween 80 is a common non-ionic surfactant and emulsifier, and the characteristics of pre-pared micelles in water are presented in Table S2 and Fig. S18and S19.† The absorption spectra of 6c and 6d in micelles cor-responded well with their absorption spectra in THF (Fig. 4a),which proves the monomeric character of both compoundsunder aqueous conditions. Surprisingly, the fluorescenceintensity of 6c/Tween 80 was not efficiently quenched (ΦF =0.04), indicating a specific interaction of the 1-aza-6-crown-18 moiety with the solvent. As a result of the high initial ΦFvalue, the addition of KCl led only to a small increase in thefluorescence intensity (data not shown). Explanation consistsof the fact that aza-crown nitrogen has the character of weaklybasic aromatic amine and can be partially protonated in useddeionized water of approximately pH 6. We assumed that nitro-gen protonation leads to the reduction of the efficacy of PETeven in the OFF state and, therefore, to a high initial ΦF value.To prove that, we investigated the fluorescence intensities ofthe 6c/Tween 80 and 6d/Tween80 at different pH values, andFig. 2 Changes in the (a) absorption and (b) fluorescence emissionspectra of 6c (1 μM) upon titration with KOTf. (c) Increase in ΦF of 6c(1 μM) upon the addition of an analyte (in the form of triflates). (d)Enlarged part of the graph up to 0.1 μM of triflate salt. (e) Increase in ΦFof 6c (1 μM) upon the addition of Ba2+ triflate (titration done to reach theplateau phase where c(Ba2+) = 10 μM), followed by titration by water ormethanol (c(Ba2+) = 10 μM). The excitation wavelength of 505 nm wasused for all experiments shown in this figure.Fig. 3 1H NMR spectra (600 MHz, THF-d8/MeOH-d4 5 : 2) of 6c withKOTf at different SubPc : salt ratios.Dalton Transactions PaperThis journal is © The Royal Society of Chemistry 2024 Dalton Trans., 2024, 53, 2635–2644 | 2639Open Access Article. Published on 04 January 2024. Downloaded on 2/7/2024 6:47:45 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3dt03839dindeed, the a considerable decrease of fluorescence intensities,i.e., high efficacy of PET, of 6c/Tween80 was restored at pHvalues above 7 (Fig. 4b). Since the physiological pH is 7.4, theeffect of protonation should not affect the sensor’s propertiesin the biological environment.Based on the above results, the sensing ability of 6c/Tween80 was investigated in TRIS buffer at approximately pH 8 toeliminate protonation of aza-crown nitrogens. In agreementwith the fluorescence behaviour of 6c in THF, the fluorescenceemission of 6c/Tween 80 in Tris buffer was weak (the OFFstate, ΦF = 0.014), and responded to the addition of KCl (up toΦF = 0.072) (Fig. 4c and d), but not to the addition of NaCl (ΦF= 0.017) (Fig. S20†). In contrast, the always-ON control 6d/Tween 80 exhibited constant fluorescence emission (ΦF = 0.10)irrespective of the pH value (Fig. 4b), and slight fluorescencequenching with increased concentrations of NaCl (ΦF = 0.089)or KCl (ΦF = 0.095) (Fig. 4d, Fig. S20†). The results of 6c/Tween80, showing approximately a 5-time increase of ΦF in the pres-ence of KCl, proved that prepared derivatives can be trans-ferred to aqueous media using micelles as delivery systemswithout losing their sensing abilities.ConclusionsA straightforward synthetic method for introducing aza-crownmoieties at the axial position of SubPcs or SubAzaPcs wasdeveloped. Since the synthesized SubAzaPcs had blue-shiftedabsorption and emission spectra, were less stable during puri-fication processes, and were less photostable when comparedwith the corresponding SubPc derivatives, the sensing abilitytowards alkali and alkaline earth metal cations was studied indetail only in a series of SubPc aza-crown derivatives 6a–6c.SubPc sensors were switched ON in the presence of sensitiveanalytes, that was monitored as an increase in ΦF. Size prefer-ence was observed only in the case of alkali metal cations,while alkaline earth metal cations were bonded regardless oftheir size. High molar absorption coefficients of the studiedSubPc derivatives (log ε ∼4.7 dm3 mol−1 cm−1), high fluo-rescence enhancement factors of selective anions (FEF up to107), and high fluorescence quantum yields in the ON state(ΦF up to 0.15) rank these derivatives among promising fluo-rescence sensors. Interestingly, the binding of Ba2+ was extra-ordinarily strong, making some of these derivatives promisingtargets for follow-up studies aimed to monitor Ba2+ in theenvironment.Importantly, we have shown that assessment of the selecti-vity of a sensor towards an analyte requires collecting charac-teristics such as the FEF, ΦF in the ON state, and the corres-ponding Ka values and evaluating them together. It can bedocumented by the behavior of 6c. This derivative showed astrong fluorescence emission increase upon meeting Li+, K+,Ca2+, Mg2+ and Ba2+; however, only K+ and Ba2+ ions exhibitedreasonable Ka values. Since the levels of Ba2+ in plasma arenegligible,42 6c can be considered as a promising fluorescencesensor candidate for the selective recognition of K+ levels inhuman fluids.This work also highlighted the need to study the sensingability of sensors in aqueous environments, since the presenceof water (and other polar molecules such as MeOH) signifi-cantly altered the binding of cations to aza-crowns. Finally, thepresent study opened new directions for broader applicationsof SubPcs in fluorescence sensing, since these derivativesprovide photostable fluorophores with advantageous spectralproperties, and the sensing ability can be transposed intoaqueous media. Their great advantage is that recognition moi-eties can be attached at the axial position, whereas their peri-pheral sites on signalling moieties may be used to tune thespectral and physico-chemical properties.ExperimentalGeneralAll chemical reagents were purchased from certified suppliers(Sigma-Aldrich, TCI, Acros Organic, Fluorochem) and used asreceived. All the organic solvents used in the synthesis were ofanalytical grade. Thin-layer chromatography was performed onMerck aluminum sheets coated with silica gel 60 F254. MerckKieselgel 60 (0.040–0.063 mm) was used for column chromato-graphy. The infrared spectra were measured on a Nicolet 6700spectrometer in the ATR mode. The 1H and 13C NMR spectrawere recorded on a VNMR S500 NMR spectrometer or a JeolJNM-ECZ600R. The chemical shifts are reported as δ values inppm and are indirectly referenced to Si(CH3)4 via the signalfrom the solvent. J values are given in Hz. The UV–vis spectrawere recorded using a Shimadzu UV-2600 spectrophotometer(Shimadzu, Kyoto, Japan). Fluorescence spectra were recordedFig. 4 (a) Normalized absorption spectra of 6c in THF and 6c/Tween80 in deionized water (pH∼6). (b) Changes in the fluorescence intensityin aqueous media of different pH values. (c) Changes in the fluorescenceemission spectra of 6c/Tween 80 upon the addition of KCl (Tris buffer,pH ∼8). (d) Changes in fluorescence quantum yields during titration withKCl or NaCl (d). The excitation wavelength of 530 nm was used for allexperiments.Paper Dalton Transactions2640 | Dalton Trans., 2024, 53, 2635–2644 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 04 January 2024. Downloaded on 2/7/2024 6:47:45 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3dt03839dusing an FS5 spectrofluorometer (Edinburgh Instruments,Edinburgh, UK). A UHPLC Acquity UPLC I-class system(Waters, Millford, USA) coupled to a high-resolution massspectrometer (HRMS) Synapt G2Si (Waters, Manchester, UK)based on Q-TOF were used for HRMS spectra measurement.Chromatography was carried out using an Acquity UPLC BEHC18 column (2.1 × 50 mm, 1.7 µm) or an Acquity UPLC BEHC4 (2.1 × 50 mm, 1.7 µm, 300 Å) columns for molecules up toor above 1200 m/z, respectively, using gradient elution withacetonitrile and 0.1% formic acid at a flow rate of 0.4 mlmin−1. Electrospray ionization was operated in positive mode.The ESI spectra were recorded using leucine-enkephalin as alock mass reference and sodium formate (in the range50–1200 m/z) or sodium iodide (in the range 50–2000 m/z) formass calibration.The compounds 1,43 4,27,28 5,29 6d25 and 7d25 were syn-thesized according to the previously reported procedures.SynthesisGeneral procedure for 2a–c. The conditions were adoptedfrom the literature.44 A mixture of 2-(4-bromophenoxy)tetrahy-dro-2H-pyran (1) (256 mg, 1 mmol), Pd2(dba)2 (9.2 mg,0.01 mmol), NaOtBu (135 mg, 1.4 mmol), and DavePhos(11.8 mg, 0.03 mmol) under an argon atmosphere was pre-heated to approximately 60 °C. A solution of the aza-crownether (1.14 mmol) in 2 mL of anhydrous toluene was added tothe mixture and stirred at 80 °C for 12 h. The reaction mixturewas then cooled to room temperature, diluted with dichloro-methane (60 mL) and filtered through Celite. The crudeproduct was purified using column chromatography on silicausing ethyl acetate as an eluent.10-(4-((Tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1,4,7-trioxa-10-azacyclododecane (2a). Reaction time, 8 hours; yield 285 mg,(81%) as pale yellow-coloured oil. 1H NMR (600 MHz, metha-nol-d4) δ 6.89 (d, J = 9.6 Hz, 2H), 6.70 (d, J = 9.6 Hz, 2H), 5.20(t, J = 3.6 Hz, 1H), 3.97–3.91 (m, 1H), 3.77 (t, J = 4.9 Hz, 4H),3.67–3.62 (m, 4H), 3.62–3.58 (m, 4H), 3.59–3.53 (m, 1H), 3.46(t, J = 4.9 Hz, 4H), 2.01–1.92 (m, 1H), 1.87–1.79 (m, 1H),1.79–1.73 (m, 1H), 1.69–1.61 (m, 2H), 1.63–1.54 (m, 1H). 13CNMR (151 MHz, methanol-d4) δ 149.97, 145.81, 119.05, 115.10,99.20, 72.33, 71.03, 70.89, 63.30, 53.99, 31.75, 26.43, 20.24. IR(ATR): νmax (cm−1) 2949, 2857, 2360, 1227, 1123, 1111, 1022,921, 823. HRMS (ESI) calculated for C19H29NO5 + H+ 352.2119,found 352.2116 [M + H]+.13-(4-((Tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1,4,7,10-tetra-oxa-13-azacyclopentadecane (2b). Reaction time, 12 hours;yield, 364 mg (92%) as pale yellow-coloured oil. 1H NMR(600 MHz, methanol-d4) δ 6.90 (d, J = 9.4 Hz, 2H), 6.62 (d, J =9.0 Hz, 2H), 5.19 (t, J = 3.6 Hz, 1H), 3.97–3.90 (m, 1H), 3.70 (t, J= 6.1 Hz, 4H), 3.65–3.60 (m, 12H), 3.58–3.54 (m, 1H), 3.50 (t, J= 6.1 Hz, 4H), 2.01–1.93 (m, 1H), 1.86–1.80 (m, 1H), 1.79–1.73(m, 1H), 1.68–1.61 (m, 2H), 1.60–1.54 (m, 1H). 13C NMR(151 MHz, methanol-d4) δ 149.68, 144.84, 119.32, 113.95,99.27, 72.05, 71.21, 71.02, 70.13, 63.28, 53.70, 31.76, 26.45,20.25. IR (ATR): νmax (cm−1) 2939, 2867, 1512, 1355, 1235,1125, 1036, 971, 921. HRMS (ESI) calculated for C21H33NO6 +H+ 396.2381, found 396.2377 [M + H]+, 418.2195 [M + Na]+.16-(4-((Tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1,4,7,10,13-pen-taoxa-16-azacyclooctadecane (2c). Reaction time, 12 hours;yield, 339 mg (77%) as pale yellow-coloured oil. 1H NMR(600 MHz, methanol-d4) δ 6.90 (d, J = 9.2 Hz, 2H), 6.68 (d, J =9.0 Hz, 2H), 5.21 (t, J = 3.6 Hz, 1H), 3.98–3.91 (m, 1H),3.69–3.60 (m, 20H), 3.62–3.54 (m, 1H), 3.52 (t, J = 6.0 Hz, 4H),2.02–1.92 (m, 1H), 1.88–1.80 (m, 1H), 1.80–1.73 (m, 1H),1.67–1.62 (m, 2H), 1.61–1.54 (m, 1H). 13C NMR (151 MHz,methanol-d4) δ 149.99, 145.08, 119.23, 114.91, 99.22, 71.76,71.74, 71.70, 71.64, 70.11, 63.31, 52.99, 31.74, 26.43, 20.24. IR(ATR): νmax (cm−1) 2940, 2869, 2557, 1512, 1234, 1109, 1036,971, 921, 816. HRMS (ESI) calculated for C23H37NO7 + H+440.2643, found 440.2647 [M + H]+, 462.2462 [M + Na]+.General procedure for 3a–c. Compound 2a–c (0.075 mmol),and p-toluenesulfonic acid (0.075 mmol) in methanol (1.2 mL)were stirred under an argon atmosphere for 2 hours at roomtemperature. On completion, the product was extracted withchloroform (30 mL) and a saturated aqueous solution ofNaHCO3 (30 mL). The organic layer was collected, and thecrude product was purified using column chromatography onsilica. The yields and mobile phases are mentioned at eachcompound below.4-(1,4,7-Trioxa-10-azacyclododecan-10-yl)phenol (3a). Mobilephase: triethyl acetate/ethyl acetate 1 : 45; yield 15 mg (74%) asa light pink oil. 1H NMR (500 MHz, DMSO-d6) δ 8.48 (s, 1H),6.61–6.57 (m, 2H), 6.57–6.54 (m, 2H), 3.67 (t, J = 5.0 Hz, 4H),3.56–3.50 (m, 8H), 3.36 (t, J = 5.0 Hz, 4H). 13C NMR (126 MHz,DMSO-d6) δ 148.26, 141.92, 115.69, 113.77, 70.83, 69.30, 69.04,52.07. IR (ATR): νmax (cm−1) 2924, 2856, 1514, 1360, 1226,1125, 1089, 1067, 817, 677, 646. HRMS (ESI) calculated forC14H21NO4 + H+ 268.1544, found 268.1547 [M + H]+, 290.1363[M + Na]+.4-(1,4,7,10-Tetraoxa-13-azacyclopentadecan-13-yl)phenol (3b).Mobile phase: chloroform/ethyl acetate 3 : 2; yield 21 mg (87%)as a light pink oil. 1H NMR (500 MHz, DMSO-d6) δ 8.45 (s, 1H),6.64–6.57 (m, 2H), 6.51–6.44 (m, 2H), 3.58 (t, J = 6.1 Hz, 4H),3.55–3.47 (m, 12H), 3.37 (t, J = 6.1 Hz, 4H). 13C NMR(126 MHz, DMSO-d6) δ 148.01, 140.99, 115.87, 112.78, 70.35,69.44, 69.19, 68.36, 52.21. IR (ATR): νmax (cm−1) 2865, 1734,1514, 1355, 1222, 1121, 938, 814, 734. HRMS (ESI) calculatedfor C16H25NO5 + H+ 312.1806, found 312.1810 [M + H]+,334.1629 [M + Na]+.4-(1,4,7,10,13-Pentaoxa-16-azacyclooctadecane-16-yl)phenol(3c). Mobile phase: ethyl acetate/methanol 95 : 15; yield 24 mg(89%) as a light pink oil. 1H NMR (600 MHz, DMSO-d6) δ 8.50(s, 1H), 6.64–6.58 (m, 2H), 6.57–6.51 (m, 2H), 3.55–3.52 (m,20H), 3.38 (t, J = 6.1 Hz, 4H). 13C NMR (151 MHz, DMSO-d6) δ148.36, 141.27, 115.82, 113.67, 70.07, 70.06, 69.98, 69.92,68.39, 51.40. IR (ATR): νmax (cm−1) 2866, 1514, 1351, 1249,1112, 945, 816. HRMS (ESI) calculated for C18H29NO6 + H+356.2068, found 356.2065 [M + H]+, 378.1887 [M + Na]+.General procedure for the synthesis of target macrocycles.The procedure was adopted from the literature25 using a one-pot reaction procedure. Briefly, an equimolar amount of a 1 MDalton Transactions PaperThis journal is © The Royal Society of Chemistry 2024 Dalton Trans., 2024, 53, 2635–2644 | 2641Open Access Article. Published on 04 January 2024. Downloaded on 2/7/2024 6:47:45 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3dt03839dsolution of BCl3 in p-xylene was added to 4 (or 5) upon stirringunder an argon atmosphere. The reaction mixture was refluxedfor 2 hours. Afterward, the solvent was removed under reducedpressure. The residue was then mixed with 3a–c (3 equivalents)dissolved in anhydrous toluene, and the resulting mixture wasrefluxed under an argon atmosphere overnight. The crudeproduct was purified using column chromatography on silica.The amounts of reactants, as well as mobile phases, are listedfor each compound below.SubPcs 6a. Compounds 4 (56 mg, 0.2 mmol) and 3a(160 mg, 0.6 mmol), reaction time 10 h, mobile phase: di-chloromethane/acetone 90 : 4, yield 18 mg (23%) of violetsolid. 1H NMR (600 MHz, CDCl3) δ 8.89 (s, 6H), 7.32–7.29 (m,12H), 7.27–7.25 (m, 18H), 6.21–6.18 (m, 2H), 5.42–5.39 (m,2H), 3.68–3.66 (m, 4H), 3.60 (dd, J = 4.3, 2.3 Hz, 4H), 3.55 (td, J= 3.9, 2.4 Hz, 4H), 3.32 (t, J = 4.9 Hz, 4H). 13C NMR (151 MHz,CDCl3) δ 151.64, 143.04, 141.25, 130.23, 130.11, 128.15, 127.14,124.21, 119.79, 113.50, 71.60, 70.03, 69.94, 52.99. IR (ATR):νmax (cm−1) 3648, 3545, 3524, 3502, 2926, 2854, 1731, 1511,1455, 1439, 1163, 1068, 762, 724, 656. HRMS (ESI) calculatedfor C78H64BN7O6 + H+, 1118.4560 found 1118.4568 [M + H]+1118.4388 [M + Na]+. λmax (THF, log ε) 581 (4.762), 525 (sh),and 323 nm (4.441 dm3 mol−1 cm−1).SubPc 6b. Compound 4 (56 mg, 0.2 mmol) and 3b (190 mg,0.6 mmol), reaction time 12 h, mobile phase: dichloro-methane/acetone 9 : 1, yield 17 mg (22%) of violet solid. 1HNMR (600 MHz, THF-d8) δ 8.85 (s, 6H), 7.30–7.28 (m, 12H),7.23–7.19 (m, 18H), 6.11–6.09 (m, 2H), 5.37–5.34 (2H, m),3.51–3.49 (m, 8H), 3.47–3.44 (m, 8H), 3.27 (t, J = 6.1 Hz, 4H).13C NMR (151 MHz, THF-d8) δ 152.926, 143.94, 142.474,131.281, 131.142, 128.987, 127.948, 124.846, 120.676, 112.924,72.377, 71.266, 71.118, 70.007, 53.599. IR (ATR): νmax (cm−1)2927, 2854, 1732, 1454, 1439, 1242, 1163, 1123, 1069, 763, 701,670. HRMS (ESI) calculated for C78H64BN7O6 + H+, 1162.4822found 1162.4833 [M + H]+ 1184.4651 [M + Na]+. λmax (THF,log ε) 581 (4.732), 525 (sh), and 328 nm (4.334 dm3 mol−1cm−1).SubPc 6c. Compounds 4 (56 mg, 0.2 mmol) and 3c (213 mg,0.6 mmol), reaction time 12 h, mobile phase: dichloro-methane/acetone 3 : 2, yield 21 mg (26%) of violet solid. 1HNMR (600 MHz, THF-d8) δ 8.84 (s, 6H), 7.33–7.30 (m, 12H),7.26–7.22 (m, 18H), 6.17–6.14 (m, 2H), 5.37–5.33 (m, 2H),3.53–3.50 (m, 12H), 3.48–3.45 (m, 8H), 3.31 (t, J = 6.1 Hz, 4H).13C NMR (151 MHz, THF-d8) δ 152.93, 143.96, 142.48, 131.28,131.16, 129.02, 127.98, 124.83, 120.65, 113.62, 71.96, 71.91,71.89, 71.70, 70.15, 52.82. IR (ATR): νmax (cm−1) 3409, 2925,2853, 1732, 1576, 1454, 1438, 1163, 1068, 1015, 762, 701, 612.HRMS (ESI) calculated for C78H64BN7O6 + H+, 1206.5084 found1206.5094 [M + H]+ 1228.4918 [M + Na]+. λmax (THF, log ε) 582(4.665), 525 (sh), and 328 nm (4.332 dm3 mol−1 cm−1).SubAzaPc 7b. Compounds 5 (56.4 mg, 0.2 mmol) and 3b(190 mg, 0.6 mmol) reaction time 8 h, mobile phase: dichloro-methane/acetone 9 : 1, yield 9 mg (11%) of violet solid. 1H NMR(600 MHz, THF-d8) δ 7.79–7.75 (m, 12H), 7.43–7.37 (m, 18H),6.14–6.10 (m, 2H), 5.38–5.33 (2H, m), 3.51–3.47 (m, 12H),3.46–3.44 (m, 4H), 3.28 (t, J = 6.2 Hz, 4H). 13C NMR (151 MHz,THF-d8) δ 155.78, 149.92, 141.86, 140.22, 131.54, 130.21, 129.22,122.57, 120.55, 113.06, 72.39, 71.29, 71.13, 69.96, 53.59. HRMS(ESI) calculated for C78H64BN7O6 + H+, 1168.4537 found1168.4543 [M + H]+ 1190.4364 [M + Na]+. λmax (THF, log ε) 549(4.557), 500 (sh) and 377 nm (4.420 dm3 mol−1 cm−1).Spectral and photophysical studies in the presence of ana-lytes. A volume of 25 μL of a stock solution of a SubPc (100 μM,THF) was transferred to 2.475 mL of THF in the cuvette (i.e., c= 1 μM). Both absorption and emission spectra were measured(λexc = 505 nm). Appropriate analytes were added stepwise inthe form of a triflate (stock solution in MeOH), and absorptionand emission spectra were measured after each addition. Theobtained data were analysed by the determination of ΦF viathe reference method using rhodamine 6G as the reference (ΦF= 0.94 in EtOH34).Photobleaching studies. Samples (1 μM, in THF) were pre-pared in the dark by the addition of 25 μL of the stock solution(100 μM, THF) to 2.45 mL of THF in the cuvette. The solutionwas then irradiated for specific times (100 W Xe-ozone freelamp, Newport) while stirring, using a water filter to removeheat. The absorption spectrum was measured after eachirradiation step. Decomposition was monitored as a decreasein absorption intensity at Q band maximum (550 nm and582 nm, for 7b and 7d, and for 6b and 6d, respectively, and665 and 489 nm for ZnPc and FAM, respectively) as a functionof irradiation time.Job’s method of continuous variationStock solutions of a SubPc (10 μM) and salt (10 μM) in amixture of THF/MeOH 8 : 2 were prepared. A series of fluo-rescence measurements with different SubPc/salt ratios(18 measurements ranging between 1 : 4 and 4 : 1 ratios) with aconstant total volume of 2.5 mL and total concentration ofcomponents of 10 μM ([SubPc] + [salt] = 10 μM) was per-formed. The final stoichiometry was determined from theJob’s plot constructed from the dependence of Fx − F0 on[analyte]/([analyte] + [SubPc]), where Fx refers to the fluo-rescence emission intensity of the SubPc/salt mixture and F0 tothe mixture where THF/MeOH 8 : 2 (without dissolved salt) wasused instead of a salt stock solution.Preparation of micelles. 0.1 mg of SubPc 6c (or 6d) and200 mg of Tween 80 in DMSO (200 µL) were added dropwise todeionized water (5 mL). The resulting solution was left stirringfor 18 hours. The size and zeta potential distributions of themicellar solutions in deionized water (pH ∼6) were deter-mined using dynamic light scattering (DLS) on a particle sizeanalyzer Zetasizer Nano ZS (Malvern, UK). Data are shown inthe ESI.†The effect of pH value on the fluorescence properties ofSubPc/Tween 80. Several aqueous solutions adjusted to pHvalues from 3 to 9 either by acetic acid or pyridine were pre-pared. A volume of 1 mL of those solutions was mixed with0.1 mL of a micelle 6c/Tween 80 (or 6d/Tween 80), stirred for30 seconds, and the fluorescence emission spectrum wasmeasured (excitation wavelength 530 nm). The pH value ofeach sample was determined directly in the cuvette using a pHPaper Dalton Transactions2642 | Dalton Trans., 2024, 53, 2635–2644 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 04 January 2024. Downloaded on 2/7/2024 6:47:45 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3dt03839dmeter Sentron SI 400 equipped with a MicroFET pH probe(Sentron, Leek, The Netherlands). The results were analysed asF/Fmax as a function of the pH value, where F refers to the fluo-rescence intensity at a given pH value and Fmax to the highestfluorescence intensity within the measurement (i.e., at pH =3.98).Titration of SubPc/Tween 80 with KCl and NaCl. 1 mL of theprepared SubPc/Tween 80 micelle was diluted with 4 mL ofTris buffer (pH = 8.5) to prepare a stock solution of the dye.For titration, 2 mL of the stock solution was taken in a cuvette,to which increments of 1 M aqueous KCl and NaCl solutionswere added, and their absorption and emission spectra weremeasured until maximum fluorescence was observed. Bothcontrol and sensor compounds were subjected to thisprocedure.Author contributionsMAA – conducted the research and investigation process,specifically performed the experiments; JL – performed the cal-culations (association constants, etc.) and wrote the manu-script, KL and KK – prepared and characterized the micelles,and edited the manuscript; PS and PZ – conduced a criticalreview of the research hypothesis and wrote the draft of themanuscript; VN – formulated the research goals, providedmentorship, and wrote the manuscript.Conflicts of interestThere are no conflicts to declare.AcknowledgementsThe financial support from the Czech Science Foundation (21-14919J), Russian Foundation for Basic Research (20-53-26004)and Charles University (SVV 260 666, PRIMUS/20/SCI/013) isgratefully acknowledged.References1 A. Sigel, H. Sigel and R. K. O. Sigel, in The Alkali Metal Ions:Their Role for Life, Springer, Cham, 2016, vol. 16, p. 628,DOI: 10.1007/978-3-319-21756-7.2 P. Pathak, R. R. 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