Synthesis and study of novel pH-independent fluorescent mitochondrial labels based on Rhodamine B

Abstract

Several Rhodamine B derivatives based on a tri-substituted pyrimidine core were prepared using solid-phase chemistry with a combinatorial approach. These compounds were screened for their basic fluorescence properties and their ability to penetrate through eukaryotic cell membranes. Most can penetrate through the cell membrane and specifically accumulate in mitochondria. Notably, some of our new rhodamine derivatives showed partially sustained mitochondrial localization and exhibited fluorescence at high pH, making them promising candidates for molecular probes to elucidate mitochondrial biology and pathology.

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Introduction

One of the most important organelles studied with respect to cell viability and function is mitochondria. The ability to influence mitochondrial processes is motivated by efforts to understand their role(s) in various metabolic pathways, such as those linked to apoptosis or drug transformation. Among the various methods used to study mitochondrial processes, fluorescence visualization techniques are among those most widely applied. To date, several fluorescent systems have been developed to visualize mitochondria in living cells. A common drawback of the currently available approaches is their washing out during cell fixation procedures that are routinely used to preserve cell morphology. The commercially available MitoTracker dyes, which are used to stain mitochondria and have the ability to persist in these organelles after fixation, are based on structures that contain a mildly thiol-reactive chloromethyl moiety. The main disadvantages of the MitoTrackers include the following: (i) staining of mitochondria in cultured neurons and astrocytes is limited and data interpretation is difficult due to the sensitivity of these dyes to mitochondrial transmembrane potential and oxidation;¹ (ii) MitoTrackers can be strongly influenced by reactive oxygen species;² (iii) MitoTrackers are highly unstable and cannot be stored in solution or at room temperature; and (iv) these dyes are unsuitable for applications in cells that experience loss of plasma membrane integrity.³ Therefore, the development of alternative systems to visualize mitochondria is desirable.

One of the drawbacks to the development of new systems for organelle staining is the low-throughput synthesis that is often accompanied by complicated reaction steps. The supplementation of various derivatives for the systematic study of fluorescent properties as well as intracellular behaviour is thus often undesirably time consuming and ineffective. Additionally, some labels represent a mixture of isomers. Another general drawback is the accessibility of the fluorescent dye itself for the desired modification. Staining mitochondria is also challenging because of the wide pH range inside this organelle, a feature that can influence the fluorescence properties of some dyes.

This report focuses on the development of a high-throughput protocol to prepare pH-independent fluorescent systems suitable for staining mitochondria using inexpensive and easily accessible Rhodamine B.

Results and discussion

The systems prepared and characterized in this study are based on the structure drawn in Fig. 1. It is formed by a central core, a unit to control permeability or solubility and a fluorescent dye bound via an appropriate linker.

Fig. 1 Structure of a three-arm system.

We chose Rhodamine B as the fluorescent dye because of its good water solubility and excellent photophysical properties, such as a high molar absorptivity in the visible wavelength region, high fluorescence quantum yield, high photostability and relatively long emission wavelength.^4,5 Rhodamines are also often used in systems for pH detection^6–24 due to their ability to open and close a spirolactam ring depending on the pH, thereby inducing a colour change and enhancing fluorescence. Rhodamine derivatives are also suited for the detection of nitrite ions at very low concentrations via nitrosation of the dye resulting in altered fluorescence.²⁵

The permeability/solubility control unit was designed using amino acids, which are fully biocompatible. Amino acids can generally regulate hydrophobicity or hydrophilicity of the systems, and peptides are commonly used for penetration regulation or targeted delivery.²⁶ For the development of our synthetic protocol, we chose simple β-Ala and the dipeptide Phe-β-Ala as model substituents.

The linker for rhodamine binding was designed as o-, m- and p-phenylenediamine forming the amide bond with the dye. The aromatic amides derived from rhodamine should maintain the stability of the open form of the dye due to the lower basicity of the nitrogen in the amide group. Preventing the closure of spirolactam by steric hindrance can be expected with these three phenylenediamines as well.

Both of these units are bound to the pyrimidine scaffold as a central unit, which was chosen as a suitable synthetic intermediate (see Scheme 1).

Scheme 1 Retrosynthesis of the desired compounds.

The synthetic procedures are based on advantageous solid-phase synthesis using principles of combinatorial chemistry that enable the rapid and efficient synthesis of a larger number of derivatives for the subsequent investigation of their fluorescent properties.

Synthesis of the fluorescent systems

The retrosynthetic pathway of the designed systems is pictured in Scheme 1. The appropriate commercially accessible dichloronitro pyrimidines were substituted in two steps with suitable nucleophiles, one of which was immobilized on a solid support. Modification of the phenylenediamine linker with an appropriate dye possibly followed by the reduction of a nitro group afforded the final derivatives (see Scheme 1).

The building blocks/substituents applied in the synthesis of the final compounds are listed in Fig. 2. The numbering of the individual substituents used in the structural numbering follows a combinatorial manner.

Fig. 2 Structure and numbering of building blocks used for the solid-phase synthesis.

Synthesis of the desired compounds is described in the following schemes. First, we synthesized two different types of amine linkers (Scheme 2). Briefly, resin-bound amines 1(1) and 1(2) were prepared from Wang resin acylated with Fmoc-protected β-alanine. Subsequently, deprotection and acylation with Fmoc-phenylalanine was carried out (for resin 1(1)).

Scheme 2 Solid-phase synthesis of the linkers used for derivative immobilization^a. ^aReagents and conditions: (i) Fmoc-β-Ala, N-hydroxybenzotriazole (HOBt), DMAP, DIC, DMF/DCM (1 : 1), 16 h; (ii) 50% piperidine, DMF, 15 min; (iii) Fmoc-Phe, HOBt, DIC, DMF/DCM (1 : 1), 16 h.

The resin-bound amine 1 reacted with 4,6-dichloro-5-nitropyrimidine to obtain resin 2 (Scheme 3). Interestingly, the chlorine atoms of the resins prepared in this way were very reactive and could be easily hydrolysed to give unreactive hydroxy derivatives. This behaviour complicated not only the isolation and characterization of these intermediates but also the long-term storage of the resin. For this reason, an immediate subsequent substitution was required. The chlorine atom of derivative 2 was further replaced with three types of phenylenediamines (o-, m- or p-) to afford resin 3. The terminal amino group of 3 attached to the phenylenediamine was acylated with Rhodamine B via the N-hydroxybenzotriazole (HOBt) activated ester.

Scheme 3 Solid-phase synthesis of model compounds 6 and 7^a. ^aReagents and conditions: (i) 4,6-dichloro-5-nitro-pyrimidine, DIEA, dry DMF, 2 h; (ii) phenylenediamine, DIEA, dry DMF, 16 h; (iii) Rhodamine B, HOBt, DIC, DMF/DCM (1 : 1), 16 h; (iv) SnCl₂·2H₂O, DIEA, DMF, N₂, rt, 16 h or Na₂S₂O₄, K₂CO₃, TBAHS, H₂O/DCM, rt, 16 h; (v) 50% TFA in DCM, rt, 1 h.

The resulting nitro derivative 4 was isolated by standard cleavage and HPLC purification. Part of the resin was used to prepare the appropriate amino derivative 5. The nitro group of 4 was reduced using two different types of reducing agents to obtain the amino derivative 5 immobilized on the solid support. The first strategy used a common tin(II) chloride-based reduction method. Due to the appearance of starting material 4 after an overnight reaction, the reduction using tin(II) chloride had to be repeated twice and even three times in some cases. In addition, tin(II) salts further complicated the HPLC purification of the final product 6 after its cleavage from resin 5 by clogging the column. The application of a relatively new method²⁷ in the solid-phase synthesis using Na₂S₂O₄ provided a suitable solution to the problem caused by the tin(II) salts. This method consisted of using sodium dithionite under phase-transfer catalysis conditions in a DCM–water system. Finally, product 6 was obtained via cleavage with 50% TFA in DCM from the nitro 4 and amino 5 precursors and is depicted in Table 1.

Table 1 Synthesized Rhodamine B derivative 6(R¹,R²,R³) with 4,5,6-trisubstituted-pyrimidine skeleton

Entry	R^1	R^2	R^3	Purity^a [%]	Yield^b [%]
a Purity of crude products estimated from LC traces@210–500 nm.b Isolated yield after HPLC purification.
6(1,1,1)	1(1)	NO2	p-	82	61
6(1,2,1)	1(1)	NH2	p-	77	36
6(1,1,2)	1(1)	NO2	m-	82	86
6(1,2,2)	1(1)	NH2	m-	45	72
6(1,1,3)	1(1)	NO2	o-	85	83
6(1,2,3)	1(1)	NH2	o-	67	70
6(2,1,1)	1(2)	NO2	p-	70	25
6(2,2,1)	1(2)	NH2	p-	60	32
6(2,1,2)	1(2)	NO2	m-	65	37
6(2,2,2)	1(2)	NH2	m-	45	23
6(2,1,3)	1(2)	NO2	o-	65	50
6(2,2,3)	1(2)	NH2	o-	40	11

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Analogously, we applied the above-mentioned synthetic procedure for the 2,4,5-trisubstituted pyrimidine derivative 11 (Scheme 4). Resin 1 was reacted with 2,4-dichloro-5-nitropyrimidine, subsequently modified with phenylenediamines (o-, m- or p-) and acylated with Rhodamine B. Reduction of the nitro group of resin 9 resulted in the resin-bound amino derivative 10. Final 50% TFA cleavage afforded product 11 depicted in Table 2.

Scheme 4 Solid-phase synthesis of model compound 11^a. ^aReagents and conditions: (i) 2,4-dichloro-5-nitro-pyrimidine, DIEA, dry DMF, 2 h; (ii) phenylenediamine, DIEA, dry DMF, 16 h; (iii) Rhodamine B, HOBt, DIC, DMF/DCM (1 : 1), 16 h; (iv) SnCl₂·2H₂O, DIEA, DMF, N₂, rt, 16 h; Na₂S₂O₄, K₂CO₃, TBAHS, H₂O/DCM, rt, 16 h; (v) 50% TFA in DCM, rt, 1 h.

Table 2 Synthesized Rhodamine B derivative 11(R¹,R²,R³) with 2,4,5-trisubstituted-pyrimidine skeleton

Entry	R^1	R^2	R^3	Purity^a [%]	Yield^b [%]
a Purity of crude products estimated from LC traces@210–500 nm.b Isolated yield after HPLC purification.
11(1,1,1)	1(1)	NO2	p-	78	70
11(1,2,1)	1(1)	NH2	p-	80	23
11(1,1,2)	1(1)	NO2	m-	89	92
11(1,2,2)	1(1)	NH2	m-	56	14
11(1,1,3)	1(1)	NO2	o-	72	76
11(1,2,3)	1(1)	NH2	o-	77	67
11(2,1,1)	1(2)	NO2	p-	75	23
11(2,1,2)	1(2)	NO2	m-	65	15
11(2,1,3)	1(2)	NO2	o-	70	13
11(2,2,1)	1(2)	NH2	p-	50	12
11(2,2,2)	1(2)	NH2	m-	65	15
11(2,2,3)	1(2)	NH2	o-	40	10

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In general, difficulties with the purification of the cleaved amino products 6 and 11 were encountered due to the formation of a trifluoroacetyl derivative during cleavage, which decreased the total yield and complicated the purification of the final compounds. For this reason, we decided to protect the amino group of 5 using the Fmoc protecting group (Scheme 5). Resins 5 and 10 were treated with N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-Osu) to give rise to resins 12 and 13. After we obtained the cleaved compounds 12 and 13, we removed the Fmoc group using piperidine in methanol to afford the amino derivatives 6 and 11, followed directly by HPLC purification.

Scheme 5 Solid-phase protection of resins 5 and 10 for cleavage^a. ^aReagents and conditions: (i) Fmoc-OSu, DCM, rt, 16 h; (ii) TFA/DCM, rt, 1 h; (iii) piperidine/MeOH (1 : 1), rt, 15 min.

NMR studies

During identification of the final compounds by NMR, we observed that, although all prepared compounds were isolated in a pure form according to HPLC and identity was proven by HRMS, they exhibited unexpected isomery in their NMR spectra. Thus, some compounds were identified as one isomer, whereas others were identified as a mixture of two isomers.

Such isomery is apparent from the fact that the signals for the methyl groups of the rhodamine skeleton unambiguously differ in some cases. For some compounds, we observed a clear triplet around 1 ppm representing the 12 protons of the rhodamine skeleton (e.g., for derivatives 6(1,1,1), 6(2,1,1), 6(1,2,1)). However, other compounds exhibited a pair of signals, each for 6 protons around 1 ppm, indicating a difference of two ethylamino groups on the rhodamine dye (e.g., for derivative 6(1,1,3); Fig. 3).

Fig. 3 Portions of the NMR spectra of compound 6(1,2,1) (on the left) and compound 6(1,1,3) (on the right).

Accordingly, the ¹³C-NMR spectrum showed the corresponding signal or pair of signals around 13 ppm for the methyl groups (Fig. 4).

Fig. 4 Portions of the NMR spectra of compound 6(1,2,1) (on the left) and compound 6(1,1,2) (on the right).

We attempted to find a relationship between the isomery of the pyrimidine skeleton (2,4,6- and 4,5,6-substituted pyrimidine) and the type of linker R¹ and phenylenediamine R³ used (Table 3).

Table 3 Overview of compounds 6(R¹,R²,R³) and 11(R¹,R²,R³) isolated in the form of one or two isomers

Entry	R^1	R^2	R^3	Number of isomers
6(1,1,1)	1(1)	NO2	p-	1
6(1,2,1)	1(1)	NH2	p-	1
6(1,1,2)	1(1)	NO2	m-	2
6(1,2,2)	1(1)	NH2	m-	2
6(1,1,3)	1(1)	NO2	o-	2
6(1,2,3)	1(1)	NH2	o-	2
6(2,1,1)	1(2)	NO2	p-	1
6(2,2,1)	1(2)	NH2	p-	1
6(2,1,2)	1(2)	NO2	m-	1
6(2,2,2)	1(2)	NH2	m-	1
6(2,1,3)	1(2)	NO2	o-	2
6(2,2,3)	1(2)	NH2	o-	2
11(1,1,1)	1(1)	NO2	p-	2
11(1,2,1)	1(1)	NH2	p-	2
11(1,1,2)	1(1)	NO2	m-	2
11(1,2,2)	1(1)	NH2	m-	2
11(1,1,3)	1(1)	NO2	o-	2
11(1,2,3)	1(1)	NH2	o-	2
11(2,1,1)	1(2)	NO2	p-	1
11(2,2,1)	1(2)	NH2	p-	1
11(2,1,2)	1(2)	NO2	m-	1
11(2,2,2)	1(2)	NH2	m-	1
11(2,1,3)	1(2)	NO2	o-	2
11(2,2,3)	1(2)	NH2	o-	2

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¹H-NMR and ¹³C-NMR showed that the isomery of the isolated form of the final structure was independent of the R² substitution (nitro or amino group) and the pyrimidine skeleton used. However, the observed isomery depended on the phenylenediamine aromatic ring and linker R¹. When the data on the 4,5,6-pyrimidine-substituted compounds (derivatives 6) were compared, we observed that all of the para derivatives were isolated in the form of one isomer and all of the ortho compounds were isolated in the form of two isomers (independent of linker R¹ or R² substitution). Furthermore, these data showed that meta derivatives were isolated in the form of two isomers for linker 1(1), in contrast to one isomer for linker 1(2).

In the case of 2,4,5-trisubstituted pyrimidine (derivative 11), we observed the same isomery for ortho and meta derivatives as for derivatives 6, but for para derivatives the formation of two isomers was observed in combination with linker 1(1) (Table 3).

Moreover, despite using sufficient amounts of certain compounds, the NMR measurements contained broad signals with very low intensities. Therefore, we performed several experiments at 25–80 °C. Interestingly, we observed that a temperature increase generally resulted in the improvement of NMR signals especially in aromatic part (see example in Fig. 5 and 6). Furthermore, higher temperatures lead to fusion of the two signals for the rhodamine methyl groups into one broad signal.

Fig. 5 ¹H-NMR spectrum of compound 11(1,1,3) at 25 °C with magnification of the 6.8–7.8 ppm area.

Fig. 6 ¹H-NMR spectrum of compound 11(1,1,3) at 80 °C with magnification of the 6.8–7.8 ppm area.

These experiments provide evidence for conformation differentiation depending on the structure. The hindered rotation mainly observed for o-phenylenediamines is responsible for the broadening of the signals and the differentiation of the methyl group shifts. The energetic barrier is overcome by heat supplementation, resulting in sharper signals and unified methyl signals.

Fluorescence measurements

(a) Basic fluorescence properties. The prepared chemical library was first assessed for the compounds' fluorescent properties in pure DMSO as a rapid screening method from the solvent used for NMR characterization. This was compared to 10% DMSO in water (only “water” hereafter) as a mixture suitable for biological experiments to determine whether there was a significant difference.

In pure DMSO, we found that the various substitutions of the central group, as well as different linkers, significantly affected the fluorescence intensity (the fluorescence quantum yields increased from 0.1% for 6(1,1,2) to 69% for 6(1,2,1)). High quantum yields were mostly observed for compounds with a 4,5,6-trisubstituted-pyrimidine skeleton (see Table 4), but compounds 11(1,1,1), 11(1,1,2) and 11(1,2,1) derived from 2,4,5 trisubstituted pyrimidine also exhibited relatively high quantum yields. From the measured spectral data, an unambiguous structure-fluorescence quantum yield trend is not evident. Substituent 2 in the R¹ position and, surprisingly, an amino group in the R² position negatively affected the fluorescence properties of the molecule in most cases, in spite of the fact that nitro group substitution typically causes fluorescence quenching. The excitation and emission spectra of the prepared rhodamine-based fluorescent systems mostly had similar shapes and maxima as Rhodamine B (excitation and emission maxima shifted in the range from 550 to 570 nm and 572 to 592 nm, respectively). To compare fluorescent efficiency with commercially available Mitotrackers, we measured quantum yield of Mitotracker Red having similar excitation/emission wavelengths and determined it as 0.1916.

Table 4 Fluorescence properties of the studied compounds

Entry	λex/λem (nm) (DMSO)	QY^a (DMSO)	λex/λem (nm) (water)	QY^a (water)	Penetration
a QY, fluorescence quantum yield (determined with quinine sulphate in 0.5 M sulphuric acid taken as a reference fluorescence standard).^28
6(1,1,1)	556/580	0.2132	556/580	0.0478	No
6(1,2,1)	550/572	0.6912	550/572	0.4258	No
6(1,1,2)	566/591	0.0011	566/591	0.0076	Yes
6(1,2,2)	393/459	0.0313	560/584	0.1511	Yes
6(1,1,3)	557/581	0.2254	557/581	0.2997	Yes
6(1,2,3)	386/463	0.0871	559/588	0.1456	No
6(2,1,1)	559/580	0.1809	559/580	0.1989	Yes
6(2,2,1)	350/475	0.0067	560/581	0.1741	Yes
6(2,1,2)	558/582	0.3845	558/582	0.2231	Yes
6(2,2,2)	550/568	0.1238	550/568	0.1862	Yes
6(2,1,3)	560/582	0.0373	560/582	0.1024	Yes
6(2,2,3)	336/463	0.0052	560/584	0.3421	Yes
11(1,1,1)	555/580	0.2712	555/580	0.2511	Yes
11(1,2,1)	553/583	0.3019	553/583	0.2112	No
11(1,1,2)	560/586	0.1841	560/586	0.1912	Yes
11(1,2,2)	557/582	0.2149	557/582	0.0768	No
11(1,1,3)	568/592	0.0528	568/592	0.1065	Yes
11(1,2,3)	568/590	0.0284	568/590	0.1554	Yes
11(2,1,1)	560/590	0.2562	560/590	0.2438	Yes
11(2,1,2)	566/590	0.0995	566/590	0.0286	Yes
11(2,1,3)	350/489	0.0064	560/584	0.1928	Yes
11(2,2,1)	568/590	0.2549	568/590	0.2574	No
11(2,2,2)	373/439	0.0233	560/584	0.2115	Yes
11(2,2,3)	562/586	0.3125	562/586	0.3603	Yes

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Notably, we found that compounds 6(1,2,2), 6(1,2,3), 6(2,2,1), 6(2,2,3), 11(2,1,3) and 11(2,2,2) exhibited only one maximum between 336–393 nm and very low quantum yields (see Table 4). For these compounds, the situation dramatically changed when the spectra were measured in water. The excitation and emission wavelengths reached the values of Rhodamine B and the quantum yields significantly increased. Improved quantum yields were also characteristic of some other compounds (Table 4).

The relationship between the water content and fluorescence intensity was studied in detail for compound 11(2,1,3) and 6(1,1,3). As examplified by the data shown in the graph in Fig. 7, compound 11(2,1,3) exhibited low emission when the excitation 560 nm was used with solutions up to 50% water. Then, the intensity increases rapidly up to 70% water content, followed by a sustained maximum value. In the other hand, the fluorescence intensity of the 6(1,1,3) remained the same whether DMSO or water were used.

Fig. 7 Fluorescence intensity of prepared derivatives 6(1,1,3) and 11(2,1,3) as function of water/DMSO ratio at excitation wavelength 560 nm. 6(1,1,3) ♦; 11(2,1,3) ■.

From these experiments, it was concluded that the screening of compounds in DMSO does not provide data comparable to water and that prior dilution is necessary.

The other well-known feature of rhodamine derivatives is their ability to be switched on at low pH and switched off at neutral and basic pH values. Because the quantum yields were relatively high in some cases in neutral water (see Table 4), we predicted that our system could also preserve significant fluorescence at alkaline pH. To validate this prediction, we measured the fluorescence of all compounds in 10% DMSO in water at physiological pH and found them detectable by fluorescence microscopy at 561 nm, a feature that allowed us to perform subsequent cell culture penetration experiments.

(b) Cell penetration experiments. The set of compounds studied for their ability to penetrate into living cells is listed in Table 4. As a model cell line, we chose U-2-OS human osteosarcoma cells because they facilitate microscopic analysis due to their relatively flat morphologies and adherent growth in cell culture. These experiments were performed in 24-well plates with 0.17 mm-thick glass bottoms, which are compatible with most immersion objectives and wavelengths above 300 nm. Among the 24 studied compounds, 18 were able to penetrate into the cell interior (see Table 4). According to the fluorescence microscopy, all penetrating compounds stained mitochondria, as confirmed by concomitant visualization of the mitochondria by MitoTracker® Green FM (example in Fig. 8).

Fig. 8 Fluorescence images of the compound 11(2,1,3) able to penetrate into live U-2-OS cells with the detail of a single mitochondria mapping exact localization: (a) cells after 30 min of treatment with 11(2,1,3) excited at 561 nm; (b) live mitochondria visualized by MitoTracker® Green FM (c) overlay of (a) and (b).

In order to exclude the accumulation of the compounds in lysosomes the U-2-OS cells were treated with LysoTracker® Green DND – 26 and analysed. The obtained microscopic data showed that the studied compounds did not stain lysosomes (see ESI†).

The derivatives capable of cell penetration were subsequently tested in cells upon fixation. We fixed cells with formaldehyde and compared the fluorescence intensity before and after fixation. Compounds 6(1,1,2), 6(1,1,3), 6(2,1,3) and 11(2,1,3) were retained inside the cell, although their fluorescence intensities decreased (see Table 5).

Table 5 Comparison of fluorescence intensity before and after cell fixation

Entry	Fluorescence intensity^a (%)	σ (%)
a The intensity before fixation = 100%. The fluorescence was measured for three different regions of observed objects and averaged. The standard deviation σ was calculated for each measured compound.
6(1,1,2)	5.7	0.9
6(1,1,3)	14.7	0.9
6(2,1,3)	2.8	0.9
11(2,1,3)	2.9	1.0

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For these derivatives we also verified their ability to label astrocytes mitochondria. We found out that the compounds similarly to Mitotracker Green are able to stain these mitochondria (see ESI†).

(c) pH stability. Given that mitochondrial pH values vary over a wide range (typically 4–8), we examined the fluorescence properties of the compounds under various pH values, focusing on compounds 6(1,1,2), 6(1,1,3), 6(2,1,3) and 11(2,1,3) selected from the cell penetration study.

The fluorescence emission measured under various pH values is presented in Fig. 9.

Fig. 9 Dependence of fluorescence intensity on pH for compounds 6(1,1,2) ▲, 6(1,1,3) ♦, 6(2,1,3) ● and 11(2,1,3) ■.

Although the emission fluorescence intensity decreased from pH ca. 3 to 6 for most of our compounds, it then remained nearly constant at pH 6 and above. Such independence of the spectral properties from pH was also evident from the UV/VIS measurements. The spectral profile was stable from pH 6.40 to 8.57 for all four compounds (Fig. 10), and thus, the pK value was out of this range.

Fig. 10 UV/VIS spectra of compounds 6(1,1,2) (left top), 6(1,1,3) (right top), 6(2,1,3) (left bottom) and 11(2,1,3) (right bottom) at pH 6.40 (red), 7.14 (green), 7.53 (blue) and 8.57 (black).

Surprisingly, the absorption at 565 nm, which is typical for the open form of Rhodamine B, is missing in this pH region. We found that this absorption is barely observable in the acidic pH range (see Fig. 11).

Fig. 11 UV/VIS spectra of compounds 6(1,1,2) (left top), 6(1,1,3) (right top), 6(2,1,3) (left bottom) and 11(2,1,3) (right bottom) at pH 2.70 (red), 3.53 (green), 4.45 (blue) and 5.51 (black).

Additional evidence for the spirolactam system of these rhodamine derivatives is the existence of a signal at 70 ppm in the ¹³C NMR, corresponding to its central atom. This result further supports the notion that, contrary to our presumption, the combination of substituents in the system preserves the fluorescence despite the closed rhodamine dye.

An important property of the fluorescent labels is photostability. We measured the intensity of fluorescence in water of all four compounds continuously for one hour and found no evidence of decreasing intensity in any case (see Fig. 12).

Fig. 12 Intensity of fluorescence of derivatives 6(1,1,2), 6(1,1,3), 6(2,1,3), 11(2,1,3) and MitoTracker® Red FM during one hour measurement. 6(1,1,2), black; 6(1,1,3), blue; 6(2,1,3), green; 11(2,1,3), red; MitoTracker® Red FM, purple.

Conclusions

We developed a high-throughput protocol for the synthesis of pH-independent fluorescent systems based on Rhodamine B that are capable of penetrating into the cell interior. These compounds are able to stain mitochondria, and some exhibited partially sustained localization in the mitochondria even after formaldehyde fixation of the cells.

The synthetic method presented here enables rapid development of novel fluorescent systems based on a widely used and inexpensive fluorescent dye. Future modifications can be used to develop specific labels for the visualization of other organelles, e.g., using cargo proteins instead of our model dipeptide moiety. Fluorescence properties can be modified by substituting any other dye possessing a carboxylic group and amino group on the pyrimidine ring for Rhodamine B to achieve the desired properties.

Experimental section

Synthesis of linker 1(1)

Wang resin (loading 1.0 mmol g⁻¹), ∼1 g, was washed three times with DCM. A solution of Fmoc-β-Ala-OH (2 mmol), HOBt (2 mmol), DMAP (0.5 mmol) and DIC (2 mmol) in DMF/DCM (1 : 1, v/v, 10 mL) was added to the resin. The resin slurry was shaken at rt for 16 h. The resin was washed three times with DMF and three times with DCM. Further, the Fmoc protecting group was removed by 15 min of exposure to 50% piperidine in DMF (v/v 10 mL), and then, the resin was washed three times with DMF and three times with DCM. A solution of Fmoc-Phe-OH (2 mmol), HOBt (2 mmol), DMAP (0.5 mmol) and DIC (2 mmol) in DMF/DCM (1 : 1, v/v, 10 mL) was added to the resin. The resin slurry was shaken at rt for 16 h. The resin was washed three times with DMF and three times with DCM. The Fmoc protecting group was removed as described above.

Synthesis of linker 1(2)

Wang resin (loading 1.0 mmol g⁻¹), ∼1 g, was washed three times with DCM. A solution of Fmoc-β-Ala-OH (2 mmol), HOBt (2 mmol), DMAP (0.5 mmol) and DIC (2 mmol) in DMF/DCM (1 : 1, v/v, 10 mL) was added to the resin. The resin slurry was shaken at rt for 16 h. The resin was washed three times with DMF and three times with DCM. The Fmoc protecting group was removed as described above.

Reaction with dichloro-5-nitropyrimidines (resins 2, 7)

Resin 1, ∼1 g, was washed three times with dry DMF and reacted with a solution of dichloro-5-nitropyrimidines (5 mmol) and DIEA (5 mmol) in dry DMF (10 mL) at rt for 2 h. The resin was washed five times with DMF and three times with DCM.

Reaction with phenylenediamines (resins 3, 8)

Resins 2 and 7, ∼0.5 g, were washed three times with dry DMF and reacted with a solution of (p-, m- or o-) phenylenediamines (2.5 mmol) and DIEA (2.5 mmol) in dry DMF (5 mL) at rt for 16 h. The resin was washed three times with DMF and three times with DCM.

Acylation with Rhodamine B (resins 4, 9)

Resins 3 and 8, ∼0.5 g, were washed three times with DCM. A solution of Rhodamine B (1.5 mmol), HOBt (1.5 mmol) and DIC (1.5 mmol) in DMF/DCM (1 : 1, v/v, 5 mL) was added to the resin. The resin slurry was shaken at rt for 16 h. The resin was washed eight times with DMF and eight times with DCM.

Reduction of nitro group (resins 5, 10)

Resins 4 and 9, ∼250 mg, were washed three times with degassed DMF. A solution of SnCl₂·2H₂O (2.5 mmol) and DIEA (2.5 mmol) in degassed DMF (2.5 mL) was added to the resin. The resin slurry was shaken at rt for 16 h. The resin was washed five times with DMF and three times with DCM.

Resin 4 and 9, ∼0.5 g, was washed three times with DCM. A solution of Na₂S₂O₄ (5 mmol), K₂CO₃ (7 mmol) and TBAHS (0.5 mmol) in water (5 mL) and DCM (5 mL) was added to the resin. The resin slurry was shaken at rt for 16 h. The resin was washed three times with each solvent: DCM/water (1 : 1, v/v), DMF, MeOH and DCM.

Fmoc protection (resins 12, 13)

Resins 5 and 10, ∼0.5 g, were washed three times with DCM. A solution of Fmoc-Osu (0.5 M) in DCM (5 mL) was added to the resin. The resin slurry was shaken at rt for 16 h. The resin was washed five times with DCM.

Cleavage from resin with TFA (compounds 6, 11, 14, 15)

Resins 4, 5, 9, 10, 12 and 13, ∼250 mg, were treated with 2 mL of a solution of TFA/DCM (1 : 1, v/v) for 1 h. The cleavage cocktail was collected, and the resin was washed three times with 50% TFA in DCM. The combined extracts were evaporated under a stream of nitrogen, and the crude products were purified by reversed-phase HPLC.

Deprotection of compounds 14, 15 (compounds 6, 11)

Crude 14 and 15, ∼300 mg, was dissolved in MeOH (2 mL), and 2 mL of piperidine were added. The mixture was stirred for 15 min, and the crude products were purified by reversed-phase HPLC.

Fluorescence measurement

Fluorescence emission spectra were measured on spectrophotometer Cary Eclipse (FL1009M015, Varian) in DMSO or 10% DMSO in water. UV/VIS spectra were measured on spectrophotometer Cary 300 UV/VIS (UV111M031, Agilent) in DMSO or 10% DMSO in water.

Microscopy of compounds' penetration into live cells

Human U-2-OS osteosarcoma cell line was obtained from ATCC, SNB-75 glioblastoma cells were obtained from NCI and cultivated according to the supplier's recommendations. Cells were seeded 24 h before analysis into a 24-well plate with a 0.170 mm glass bottom (In Vitro Scientific) at 7 × 10⁴ cells per well. Compounds were added directly into the culture medium 30 min before the microscopic analysis at a final concentration of 1–2 μM from 300× DMSO solution. Cells were further examined at various time points using Zeiss Axioimager Z.1 platform equipped with the Elyra PS.1 super-resolution system for SR SIM and the LSM780 module for CLSM. Light source for SR SIM included diode lasers 488 nm and 561 nm and appropriate band pass filters (495–575 nm for the green channel and 576–650 for the red channel). Images were captured with an EM-CCD camera (Andor iXON EM+; 1004 × 1002 px, cooled at −64 °C, 16-bit) at typical exposure times varying around 100 ms and with gain values between 20–25. SR-SIM setup included Zeiss Plan Apochromat 100×/NA1.46 oil objective (tot. mag. 1600×), 5 rotations and 5 phases of the grated pattern for each image layer. Gratings for patterned illumination were spaced by 42 μm for the green channel and 51 μm for the red channel. Up to 7 (usually 3) 110 nm Z-stacks were acquired. Selected images were displayed as multiple Z-stacks in 3-D projections. Light source for CLSM included Argon–Neon Laser (458, 488 and 514 nm) and solid state laser 561 nm. In the LSM mode the samples were acquired through Zeiss objective C-Apochromat 40×/NA1.20 water immersion. The tested compounds were excited primarily by a 561 nm laser, which produced the best signal/noise ratio, but other lasers of 458 nm, 488 nm, and 514 nm were also tested. Compounds' emissions were collected by LSM780 embedded 32-channel spectral detector spanning the wavelength range 371–740 nm. All the images acquisitions and basic image processing were performed using Zen software (Zeiss).

Acknowledgements

The authors are grateful to the Ministry of Education, Youth and Sport of the Czech Republic, for the grant IGA_PrF_2015_007, to the European Social Fund for grants CZ.1.07/2.3.00/20.0009, CZ.1.07/2.3.00/30.0041 and Technology Agency of the Czech Republic for grant TE02000058. The infrastructural portion of this project (Institute of Molecular and Translational Medicine) was supported by the National Program of Sustainability (Project LO1304).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra20183g

‡ These authors contributed equally to the work.

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