Novel Benzo[a]phenoxazinium Chlorides Functionalized with Sulfonamide Groups as NIR Fluorescent Probes for Vacuole, Endoplasmic Reticulum, and Plasma Membrane Staining

The demand for new fluorophores for different biological target imaging is increasing. Benzo[a]phenoxazine derivatives are fluorochromophores that show promising optical properties for bioimaging, namely fluorescent emission at the NIR of the visible region, where biological samples have minimal fluorescence emission. In this study, six new benzo[a]phenoxazinium chlorides possessing sulfonamide groups at 5-amino-positions were synthesized and their optical and biological properties were tested. Compared with previous probes evaluated using fluorescence microscopy, using different S. cerevisiae strains, these probes, with sulfonamide groups, stained the vacuole membrane and/or the perinuclear membrane of the endoplasmic reticulum with great specificity, with some fluorochromophores capable of even staining the plasma membrane. Thus, the addition of a sulfonamide group to the benzo[a]phenoxazinium core increases their specificity and attributes for the fluorescent labeling of cell applications and fractions, highlighting them as quite valid alternatives to commercially available dyes.


Introduction
The use of fluorescent probes for bioimaging has proved to be an essential and robust tool in the fields of biochemistry, medicine, and environmental sciences [1][2][3]. The proficient characteristics of fluorescent imaging has proved to be fundamental for the real-time monitoring of the localization, behavior, and biological roles of biomolecules in complex living systems [4,5]. In this field, fluorescent probes include synthetic organic dyes that can be used to interact and specifically label a biological target [6,7]. As such, it is not surprising that there is a demand for the development of new fluorophores to overcome the drawbacks of commercial dyes, such as their difficult synthesis, restricted availability, and biological interference [8][9][10]. A fluorescent probe must possess a set of photophysical characteristics that suits it as a robust dye, such as a high molar extinction coefficient, high quantum yield values, minimum photodamage, good stability against photobleaching, as well as high affinity for the specific labelling of biological targets [7,[11][12][13]. Ideally, it should absorb and emit fluorescence in the long-wavelength part of the visible range of the spectrum, preferably in the NIR region, as light minimizes damage to biological samples, and decreases the influence of background autofluorescence [14][15][16].
Among these compounds, Nile Blue has been recognized as a lead compound due to its desirable physicochemical and photophysical properties, and has been used as a scaffold for the synthesis of new molecules for fluorescent staining applications [27,31,32].
Keeping in mind the physicochemical characteristics, as well as the multiple applications of benzophenoxazinium salts mentioned above, we decided to synthesize a new set of benzophenoxazinium chlorides functionalized with sulfonamide groups. We took this approach because, at least to our knowledge, there are no reports in the literature of the synthesis and characterization of benzo[a]phenoxazinium chlorides possessing these functional groups. In fact, sulfonamide or sulfonyl functional groups are of great importance in medicinal chemistry as they exhibit a broad reactivity profile and are considered safe for drug development [45][46][47]. Fluorescent-labelled sulfonamides have also been successfully used to visualize, identify, and monitor intracellular targets [48,49]. In fact, sulfonamides have been reported to behave as a class of electron donors that can be incorporated into both fluorescent and bioluminescent probes [50].
In previous publications, we have reported that the behavior of benzo[a]phenoxazinium chlorides depends on the substituents at the 5-and 9-positions of the heterocycle [23,51]. In the present study, we report the synthesis, photophysical, antifungal, and staining characterizations of six new benzo[a]phenoxazinium chlorides, functionalized with sulfonamide groups. Our main objective was to evaluate if the introduction of the sulfonamide group into the scaffolds of precursor compounds would be beneficial to the overall biological activity and/or fluorescent labeling capacity.
All the tested compounds exhibited antiproliferative activity against S. cerevisiae. Compared with precursor compounds recently reported [23], the addition of the sulfonamide group had a high positive effect on the fluorescence staining pattern. Analysis of the intracellular distribution of the compounds showed that they stain the vacuolar membrane and/or the perinuclear membrane of the endoplasmic reticulum with great specificity, with compounds even capable of staining the plasma membrane, highlighting them as valid molecules to be explored as viable alternatives to the available commercial dyes.

Chemistry
With the aim of obtaining benzo[a]phenoxazinium chlorides capable of specifically staining cell organelles, and the purpose of exploring the effects of sulfonamide groups as terminals of 5-amino substituents in combination with different substituents at the 9-amino position of the polycyclic core, a set of new benzo[a]phenoxazinium chlorides 3a-f was designed.
Different groups were introduced along with the sulfonamide function, namely an alkyl chain (3a,d,f) and a phenyl group with the methyl substituent as an electron donor group (3b,e) or the nitro group as an electron withdrawing group (3c). These variations, along with the differences in the 9-position groups, may allow accessing the most interesting combinations for fluorescence staining.
The 1 H NMR spectra of compounds 3a-f show the aromatic proton signals in the range δ 6.61-8.96 ppm. Compounds 3a-e show the signal corresponding to H-10 (δ 7.11-7.36 ppm); on the other hand, compound 3f possess a methyl group in the 10-position, that appears in the form of a singlet at δ 2.35 ppm. Regarding the 9-position of compounds 3a-e, the protons of the terminal methyl group appear in the form of a triplet or multiplet (δ 1.02-1.12 ppm), the protons of the adjacent methylene group appear in the form of a quintet or multiplet (δ 1.74-1.90 ppm), and the protons of the methylene group directly linked to nitrogen appear in the form of a triplet or multiplet (δ 3.38-3.67 ppm). Compound 3f shows the protons of the methylene group directly linked to nitrogen at 3.50-3.60 ppm, in the form of a multiplet, and the terminal CH 3 group appears as a multiplet (δ 1.32-1.39 ppm).  FTIR spectra of compounds 3a-f showed the bands corresponding to amine groups (3437-3170 cm −1 ) and the C-N bond of the central oxazine ring (1641-1638 cm −1 ). Bands corresponding to the S=O bond also appear (1383-1385 and 1159-1187 cm −1 ).
The 1 H NMR spectra of compounds 3a-f show the aromatic proton signals in the range δ 6.61-8.96 ppm. Compounds 3a-e show the signal corresponding to H-10 (δ 7.11-7.36 ppm); on the other hand, compound 3f possess a methyl group in the 10-position, that appears in the form of a singlet at δ 2.35 ppm. Regarding the 9-position of compounds 3a-e, the protons of the terminal methyl group appear in the form of a triplet or multiplet (δ 1.02-1.12 ppm), the protons of the adjacent methylene group appear in the form of a quintet or multiplet (δ 1.74-1.90 ppm), and the protons of the methylene group directly Regarding the 5-position amine substituents, all the compounds possess a propyl group. The protons of the central methylene, NHCH 2 CH 2 CH 2 NH, appear in the form of a quintet, multiplet, or extended singlet (δ 2.00-2.32 ppm), while the adjacent NHCH 2 CH 2 CH 2 NH protons appear in the form of a triplet, multiplet, or extended singlet (δ 3.06-3.33 ppm) and NHCH 2 CH 2 CH 2 NH appear as an extended triplet, multiplet, or singlet (δ 3.74-3.87 ppm). Compounds 3a, 3d, and 3f have the sulfonyl group attached to the propyl group, so their spectra show the signals of the methyl protons in the form of a triplet or multiplet (δ 1.02-1.12 ppm), the methylene proton signals NHSO 2 CH 2 CH 2 CH 3 as a sextet or multiplet (δ 1.74-1.90 ppm), and NHSO 2 CH 2 CH 2 CH 3 as a triplet or multiplet (δ 3.02-3.10 ppm). Spectra of compounds 3b and 3e show the signal of the protons of the para-methyl group of the aromatic ring attached to the sulfonyl group in the form of a singlet (δ 2.36 and 2.38 ppm), as well as by the aromatic protons of the ring (δ 7.34 ppm, ortho-CH 3 and δ 7.71-7.76 ppm, meta-CH 3 ). Compound 3c has the nitro group instead of the methyl group present in the two previous compounds, whose electron withdrawing effect displaces aromatic protons from the ortho-position to a higher chemical shift (δ 8.33 ppm) relative to the protons at the meta position (δ 8.08 ppm).
The 13 C NMR spectra of compounds 3a-f show the aromatic carbon signals, characteristic of the benzo[a]phenoxazine moiety (δ 94.01-164.62 ppm). It is possible to observe the differences between compound 3f and the remaining from the change in the chemical shift of C-10 from 112.02-117.55 ppm to 149.64 ppm.

Photophysical Characterization
Benzo[a]phenoxazinium chlorides 3a-f were studied in different solvents to assess their photophysical properties. The solvents used were dry ethanol, water, and aqueous solutions of different biologically relevant pH values. The maximum absorption (λ abs ) and emission (λ emi ) wavelengths (Figures S1-S12), the logarithm of the molar extinction coefficient (log ε), the relative fluorescence quantum yield (Φ F ), using Oxazine 1 as a standard (Φ F = 0.11 in ethanol) at 590 nm excitation, and the Stokes shifts (∆λ) were obtained for each compound in each solvent. The results obtained are summarized in Table 1.
Photophysical data in ethanol shows that there is a clear difference between dialkylated compounds at the 9-position (3a-c) and mono-alkylated compounds in the same position (3d-f). A bathochromic shift is seen for compounds with two propyl groups at the 9-position, both in λ abs values (638-641 nm) and in λ emi values (669-671 nm), when compared with mono-alkylated compounds (λ abs 624-631 nm/λ emi 643-646 nm). In addition, a decrease in Φ F values is seen for 3a-c (0.15-0.17) compared with 3d-f (0.33-0.55). Among the latter group, 3e possesses a different sulfonamide substituent and shows a lower Φ F value than the other two compounds. The conjugation of one propyl group at the 9-position and a propyl group as a substituent on the sulfonamide function, at the 5-position, appears to be beneficial for the increase in the fluorescence quantum yield.
Ethanol acidified with trifluoroacetic acid (TFA) was tested to make sure benzophenoxazines were in their acidic forms. As a result, the second absorption band (450-550 nm) showed in dry ethanol spectra for some of the compounds, corresponding to the basic form, disappeared in acidified ethanol. Furthermore, a small increase in both log ε and Φ F values can be noticed. Compounds 3a,c-f show very similar results in this solvent, when compared with ethanol.
In water and aqueous solutions of different pHs, the acid form is less fluorescent and a decrease in Φ F values can be seen for all the compounds. A bathochromic shift is observed for all the compounds both in λ abs and λ emi values. At pH 7.4, the basic form of the three compounds appears. Compounds 3d and 3f show the most promising fluorescence properties to be applied as NIR bioimaging probes in different biological relevant pHs media.

Biological Activity Evaluation
The biological activity of the six new benzo[a]phenoxazinium chlorides 3a-f was evaluated through a broth microdilution method for testing the antifungal activity against yeasts (M27-A3,CLSI), using S. cerevisiae PYCC 4072 as a model organism [53]. The MIC (Minimum Inhibitory Concentration) values that represent the grown of the cultures are listed in Table 2. Lipophilicity of a molecule is an important physical parameter when considering biological activity as it is directly related to the ability of a compound to enter the cell and diffuse intracellularly. This property can be evaluated with the logarithm of its partition coefficient between n-octanol and water, i.e., log (c octanol /c water ) or log P [44]. Thus, the determination of the partition coefficient (log P) [54] was performed using online software ds and the results are shown in Table 2 [55]. Compounds with higher log P values have low affinity for an aqueous environment and a high affinity for membranous systems. As can be seen in Table 2, all compounds tested showed antiproliferative activity against the yeast S. cerevisiae, with MIC values ranging from 12.5 to >100 µM (compound 3e showed antifungal activity at 100 µM, but due to its limited solubility, we were unable to increase the concentration to achieve 80% inhibition of cell growth). Comparing these results with those of structurally similar benzo[a]phenoxazines, without the sulfonamide group [23], it can be noticed that the introduction of this functional group does not result in an improvement trend in the biological activity of the compounds.
Among the six tested compounds, the di-alkylated derivatives (3a-c) show the best biological activity; the most active, compound 3b, displays a MIC value inferior to the corresponding analogue without the sulfonamide group (MIC 12.5 µM instead of 25 µM) [23]. The conjugation of these results with the optical properties of the set shows that compounds 3d-f may be the most promising benzo[a]phenoxazines for the fluorescent staining of biological samples as they have low toxicity.

General Observations
To evaluate the fluorescent staining capacity of the compounds, the intracellular distribution of the benzo[a]phenoxazinium chlorides 3a-f was assessed using fluorescence microscopy. To obtain preliminary information on the staining ability of these compounds, MIC values were used as reference concentrations. S. cerevisiae W303-1A cells were incubated with MIC concentrations for 1 h and observed under the fluorescence microscope. This incubation resulted in the accumulation of the compounds in the cells, as NIR fluorescence emission was observed for all compounds upon excitation ( Figure 1). Looking closely at Figure 1, it can be observed that these compounds appear to accumulate particularly at the vacuolar membrane and/or at the perinuclear membrane of the endoplasmic reticulum, which is consistent with our previous observations for this class of compounds [23,35,36]. In fact, Compounds 3a and 3b have a high specific labeling for the vacuole membrane and perinuclear membrane of the endoplasmic reticulum. Compound 3c shows an identical labeling pattern, but with lower specificity. A more unspecific labeling pattern is observed for compounds 3d-f, probably due to the high concentration used for these compounds. However, the tendency to accumulate in the endoplasmic reticulum and in some cells in the vacuolar membrane is observed.
Furthermore, these preliminary observations allowed us to conclude that the addition of the sulfonamide group significantly improves the specificity of labeling compared with compounds of a similar structure that lack this functional group [23].

Vacuole, Perinuclear Membrane of the Endoplasmic Reticulum and Plasma Membrane Stain
To confirm our previous observations and to evaluate the fluorescence pattern of each compound individually, we analyzed the specific intracellular distribution of the compounds using S. cerevisiae cells expressing GFP-tagged proteins located at the vacuole (W303-1A Vba1-GFP-expressing a GFP-tagged form of the vacuole membrane transporter Vba1 [56]), at the endoplasmic reticulum (BY4741 Sec66-GFP-expressing a GFP-tagged non-essential subunit of Sec63 ER complex [57]) and at the plasma membrane (23344c jen1∆ Jen1-GFP-expressing a GFP-tagged functional lactate transporter Jen1 [58]). We also reduced the concentration of the compounds and stained the cells with 2.5 µM of 3a-f, as we had previously observed that this concentration increased the specificity and labeling ability of the compounds of this family [35].  As previously reported, reducing the concentration of the compounds proved to be beneficial for live-cell imaging experiments. More specific staining patterns and clearer phenotypes were obtained. Staining experiments with compound 3a demonstrated the specificity of this compound to accumulate and stain the vacuolar membrane, as seen from the co-localization of NIR fluorescence and green fluorescence of Vba1-GFP (Figures 2 and 3). In addition, perinuclear ER membrane labeling can also be observed in some cells. Compounds 3b, 3c, and 3e showed the ability to accumulate and stain both the vacuolar membrane and the perinuclear ER membrane, as shown by the co-localization between the NIR and green fluorescence in both strains W303-1A Vba1-GFP and BY4741 Sec66-GFP (Figures 2 and 3). However, the quality and resolution of the staining of compounds 3c and 3e were not as good as the other compounds. Compounds 3d and 3f, which have the propyl sulfonamide group, are those with the highest relative fluorescence quantum yields (Table 1). Consistent with this is their excellent performance in fluorescence staining. These compounds, which show strong structural similarities, being mono-alkylated in the 9-position, besides possessing the same substituent group (propyl sulfonamide), showed a very clear labeling pattern with specificity for the vacuole and the perinuclear ER membrane (Figures 2 and 3). Moreover, at a concentration of 2.5 µM, there was an accumulation of the compounds at the plasma membrane. This was evidenced with co-localization with the Jen1 transporter, which is mainly localized in the plasma membrane after expression induction ( Figure 4). In some cells, it is also possible to observe Jen1 inside the vacuole, as this phenotype was also reported for this transporter [58].    Overall, the compounds synthesized were found to show high resolution fluorescence staining patterns demonstrating the ability to strain relevant intracellular structures. Indeed, compounds 3d and 3f were the ones that exhibited the sharpest staining phenotypes and better photophysical values of the series. However, compared with their precursors, they did not show significant improvement in their photophysical properties [23]. Compound 3f even exhibited lower fluorescence quantum yields than its precursor, which in a way shows that the sharpest fluorescence staining phenotypes of these compounds do not depend only on their photophysical properties. In our view, one of the reasons for the better fluorescence staining patterns of the sulfonamide-functionalized compounds seems to be correlated to their higher lipophilicity (indicated by their higher partition coefficient (log P) values- Table 2), which is theoretically correlated to their higher accumulation at the intramembrane regions and thus subsequently leads to a sharper phenotype of the staining of these structures.
In fact, the compounds containing sulfonamide groups have proven to be the ones that exhibit the best fluorescence labeling patterns of our entire library. Moreover, the fact that these compounds have high MICs and retain their ability to stain cellular structures at concentrations well below their MICs is a positive aspect for their use as fluorescent probes, since it is desirable that a probe does not affect the viability of a given organism.

Synthesis General
Melting points were correct and were measured on a Stuart SMP3 melting point apparatus. TLC analyses were carried out on 0.20 mm thick precoated silica plates (Macherey-Nagel, Dueren, Germany), and spots were visualized under UV light on a CN-6 camera. Chromatography on silica gel was carried out on Acros Organics 60 (0.035-0.070 mm) (Geel, Belgium). IR spectra were determined on a BOMEM MB 104 spectrophotom-

Synthesis General
Melting points were correct and were measured on a Stuart SMP3 melting point apparatus. TLC analyses were carried out on 0.20 mm thick precoated silica plates (Macherey-Nagel, Dueren, Germany), and spots were visualized under UV light on a CN-6 camera. Chromatography on silica gel was carried out on Acros Organics 60 (0.035-0.070 mm) (Geel, Belgium). IR spectra were determined on a BOMEM MB 104 spectrophotometer, Quebec City, QC, Canada. Samples were prepared in 1% KBr pellets. UV-Vis-NIR absorption spectra (200-800 nm) were obtained using Shimadzu UV/3101PC, Kyoto, Japan, spectrophotometer and fluorescence spectra with Fluoromax-4 spectrofluorometer, Horiba, NJ, USA. NMR spectra were obtained on a Bruker Avance III 400 at an operating frequency of 400 MHz for 1 H and 100 MHz for 13 C using the solvent peak as internal reference at 25 • C, Billerica, MA, USA. All chemical shifts are given in ppm using δ H Me 4 Si = 0 ppm as reference and J values are given in Hz. Assignments were made through a comparison of chemical shifts, peak multiplicities, and J values, and were supported spin decouplingdouble resonance and bidimensional heteronuclear correlation techniques. Mass spectrometry analyses were performed at the "C.A.C.T.I.-Unidad de Espectrometria de Masas", at University of Vigo, Pontevedra, Spain. All commercial reagents and solvents were used as received.

General Procedure for the Synthesis of N-Substituted Naphthylamine Derivatives 2a-c
To a stirring solution of N 1 -(naphthalen-1-yl)propane-1,3-diamine hydrobromide [59] in dichloromethane (3 mL), at 0 • C, triethylamine was added followed by the corresponding sulfonyl chloride. The progress of the reaction was monitored with TLC (mixtures of dichloromethane/methanol). After evaporation of the solvent and column chromatography purification on silica gel (mixtures of increasing polarity of dichloromethane/methanol as the eluent), the corresponding N-substituted naphthylamine derivative 2a-c was obtained.

General Procedure for the Synthesis of Benzo[a]phenoxazinium Chlorides 3a-f
To a solution of the corresponding nitrosophenol hydrochloride 1a-c in methanol (3 mL), concentrated hydrochloric acid was added followed by N-substituted naphthylamine derivative (2a-c), and the resulting solution was refluxed. The progress of the reaction was monitored with TLC (mixtures of dichloromethane/methanol). After evaporation of the solvent and column chromatography purification on silica gel (mixtures of increasing polarity of dichloromethane/methanol as the eluent), the corresponding benzo[a]phenoxazinium chloride 3a-f was obtained.
Fluorescence was measured at an angle of 90 • to excitation incident radiation in quartz cells. The excitation wavelength was 590 nm for all compounds. The area below the fluorescence spectrum curve was determined, allowing the relative fluorescence quantum yield (Φ F ) of the test compound to be calculated using Oxazine 1 as standard (Φ F = 0.11 in ethanol [63]).

Antifungal Activity Assays
Minimum Inhibitory Concentration (MIC) of growth for compounds 3a-f was determined using a broth microdilution method for the antifungal susceptibility testing of yeasts (M27-A3, CLSI-Clinical and Laboratory Standards Institute, Wayne, PA, USA). The yeast S. cerevisiae PYCC 4072 was used as a model organism. Cells were incubated at 30 • C in RPMI 1640 medium, buffered to pH 7.0 with 0.165 M morpholenepropanesulfonic acid (MOPS) buffer. Initial cell concentration was 2.25 × 10 3 cells/mL. Stock solutions of the compounds were prepared in DMSO and a final dilution was carried out in an RPMI 1640 medium (DMSO concentrations of 0.5% per well). MIC values were determined using a microplate photometer, after 48 h of incubation, as the lowest concentration of drug that resulted in a growth inhibition over 80%, as compared with the growth observed in the control wells containing 0.5% DMSO. Each drug concentration was tested in triplicate and in three independent experiments.
For general observation, W303-1A cells were stained with MIC concentration of the compounds for one hour, rinsed two times in PBS, and mounted on a slide for observation. To increase compound specificity and co-localization, W303-1A Vba1-GFP, BY4741 Sec66-GFP, and 23344c Jen1-GFP were stained with a low concentration of the compounds, 2.5 µM for one hour, rinsed two times in PBS, and mounted on a slide for observation.

Conclusions
A set of six new benzo[a]phenoxazinium chlorides functionalized with different sulfonamide groups at the 5-position were synthesized. These compounds display fluorescence emission in the NIR region, with maximum emission wavelengths (λ emi ) between 642 and 687 nm, in the different tested solvents.
All compounds were found to exhibit inhibitory activity against S. cerevisiae PYCC 4072, with MIC values varying from 12.5 to >100 µM. The introduction of the sulfonamide group does not result in an improvement trend in the biological activity of the compounds in direct comparison with those previously described with identical core structures. From the point of view of the use of these compounds as fluorescent probes, this proves to be a positive aspect, as it is desirable that a probe does not affect the viability of a given organism. Indeed, the introduction of the sulfonamide group has quite positive effects on the fluorescent labeling compared with similar compounds [23,36].
Our results show that at a low concentration of 2.5 µM, these compounds are able to stain with high specificity the vacuolar membrane and/or the endoplasmic reticulum, and in the case of compounds 3d and 3f, even the plasma membrane. To the best of our knowledge, this is the first report in the literature of the synthesis of benzo[a]phenoxazinium chlorides capable of staining the plasma membrane. All compounds showed interesting fluorescent labeling patterns, but compounds 3d and 3f are remarkable for their labeling specificity. Their structural features, such as mono-alkylation at the 9-position and a propyl sulfonamide at the 5-position, are likely a key factor in this behavior. In addition, both compounds exhibit the highest fluorescence quantum yields of the set and are associated with the highest MIC values, making them promising bioimaging probes.
In this sense, this work contributes to the valorization of benzo[a]phenoxazinium chlorides as an alternative to commercial probes, as we were able to obtain very robust compounds that can be used in studies with fluorescence microscopy.