New 1,2-Dihydropyridine-Based Fluorophores and Their Applications as Fluorescent Probes.

New 1,2-dihydropyridine (1,2-DHP)-based fluorophores 1a-1h were designed and synthesized by a one-pot four-component condensation reaction using dienaminodioate, aldehydes, and an in situ-generated hydrazone mediated by trifluoroacetic acid. The photophysical properties of 1,2-DHPs were studied in detail, and a few of them exhibited selective mitochondrial staining ability in HeLa cell lines (cervical cancer cells). A detailed photophysical investigation led to the design of 1,2-DHP 1h as an optimal fluorophore suitable for its potential application as a small molecule probe in the aqueous medium. Also, 1,2-DHP 1h exhibited sixfold enhanced emission intensity than its phosphorylated analogue 1h' in the long wavelength region (λem ≈ 600 nm), which makes 1,2-DHP 1h' meet the requirement as a bioprobe for protein tyrosine phosphatases, shown in L6 muscle cell lysate.

Introduction

Small molecule-based organic fluorophores are essential for sensing and imaging of biological specimens with high sensitivity and fast response.[1] Even though a large variety of fluorophores are known, only a few have optimal performance because a majority of them often suffer from photobleaching, autofluorescence, and cytotoxic behavior that limit their further applications in biology.[2] A number of heterocyclic fluorophores were reported for fluorescent labeling of biomolecules, sensing, and bioimaging applications; however, for most of these molecules, the emission maxima were observed in the green window of less than 500 nm.[3] Consequently, the discovery of new heterocyclic fluorophore scaffolds with improved photophysical properties is highly warranted. Fluorescent properties exhibited by 1,4-dihydropyridines (1,4-DHPs)[4] and our recent interest in 1,2-dihydropyridines (1,2-DHPs)[5] have inspired us to develop new 1,2-DHP-based fluorophores with improved photophysical features.

1,4-DHPs are known to exhibit blue fluorescence with appropriately substituted electron-donating groups at the 1-position and electron-withdrawing groups at 3- and 5-positions (Figure ).[6] Furthermore, a higher Stokes shift was observed by the presence of an electron-donating aryl system in the 4-position of 1,4-DHP, which is attributed to an internal charge transfer in the excited state between the two π-systems.[7] The 4-aryl-substituted 1,4-DHP comprising two different chromophores separated by an sp3 carbon served as a tunable photoactivated dyad involving energy and electron transfer processes between them (Figure ).[8] The fluorophore ability of 1,4-DHP was further extended as a chemosensor where a water-soluble glucopyranosyl 1,4-DHP is used in the detection of 2,4,6-trinitrophenol.[9] 1,2-DHPs, however, were not explored in detail for their photophysical properties to an extent as that of 1,4-DHPs, but 2-pyridones, which are structural analogues of 1,2-DHPs, were recently reported as fluorescent probes.[10] Recently, ylidenemalononitrile enamines were reported as fluorescent “turn-on” indicators for their ability to undergo cyclization with 1° amines to produce fluorescent 1,2-DHP products.[11]

Figure 1

Design strategy of the N-benzylideneamine-appended 1,2-DHP-based fluorophore.

In the quest for developing new fluorophores with improved photophysical properties, herein, we have explored 1,2-DHPs with an extended π-conjugation as novel fluorophores. As N-phenyl-1,2-DHPs absorb in the near UV region (Table S1), the corresponding derivatives with absorption in the visible region would be preferred for biological applications. Hence, the present 1,2-DHP design (Figure ) involves a push–pull system with different electron-rich N-benzylideneamine substitutions that offer tuning of their photophysical behavior.[12] This new N-benzylideneamine-appended 1,2-DHP offered a remarkable bathochromic shift in the absorption and emission profiles with large Stokes shifts (Table ). The application of these fluorophores was demonstrated as selective mitochondrial staining agents in HeLa cells. Furthermore, the design offers different sites for appendage to bioactives or functionalities required for conjugation, and such applicability has been demonstrated here as a probe for protein tyrosine phosphatase (PTP) enzymes in L6 muscle cell lysate.

Table 1

Photophysical Characterization of 1,2-DHPs 1a–1h′

entry	λmax (nm)a	ε (M–1 cm–1)b	λem (nm)	Δc	Φd	τe (ns)
1a	419	10 454	522	4709	0.059	1.41
1b	422	15 461	524	4613	0.084	1.41
1c	419	16 293	534	5140	0.067	1.06
1d	422	12 261	527	4721	0.098	1.28
1e	396	5388	507	5529	0.032	0.43
1f	420	13 741	530	4942	0.077	0.99
1g	436	23 292	582	5753	0.125	1.12
1h	448	27 300	586	5256	0.122	1.20
	g455	27 400	583	4825	0.127	1.95
	h454	25 000	609	5606	0.012	1.94
1h′	448	15 500	594	5486	0.094	1.07
	g458	16 723	611	5467	0.025	n.d.f
	h452	14 938	610	5730	0.007	n.d.f

Measured in methanol at room temperature.

Molar extinction coefficient.

Stokes shift (cm–1).

Quantum yield determined at room temperature relative to Coumarin 153 in MeOH (Φ = 0.46).

Fluorescence lifetime (λex = 418 nm) was measured using time-correlated single photon counting (TCSPC) and monitoring at the respective emission maximum.

n.d. = not determined due to weak fluorescence.

Measured in Tris buffer [25 mM, pH 7.4, 0.3% dimethyl sulfoxide (DMSO)].

Measured in Hepes buffer (25 mM, pH 7.4, 0.3% DMSO).

Results and Discussions

Synthesis

We have recently reported a one-pot multicomponent synthesis of 1,2-DHPs from dienaminodioate and imines, generated from aromatic aldehydes and amines, mediated by trifluoroacetic acid at room temperature.[5] As an extension of this methodology, the aromatic amine is replaced with an in situ-generated hydrazone, and by condensation with other components, the expected N-benzylideneamine-appended 1,2-DHP was observed under mild conditions, thus serving as a facile one-pot four-component reaction (Scheme ). A series of 1,2-DHPs 1a–1g were synthesized in moderate to good yields (20–60%) by utilizing hydrazones of differing electronic properties to decipher their photophysical properties (Scheme ). 1,2-DHPs 1a–1b were prepared to assess the role of phenyl substitution in the 6-position. The remaining 1,2-DHPs 1c–1g were synthesized with acetaldehyde to evaluate the effect of the phenyl group as a contributing factor behind 1,2-DHP’s fluorophore ability. This methodology offers a choice of appending any aliphatic or aromatic group at the 6-position, thus, a suitable place for conjugation with bioactives or biomolecules. In addition, these 1,2-DHPs can undergo regioselective hydrolysis of 5-CO2Me, which was supported by its single crystal X-ray structure (Figure S1). This selectivity can be realized by difference in nitrogen lone pair delocalization with 3- and 5-CO2Me, thus offering another site for conjugation via an amide linkage. Furthermore, we have designed and synthesized a water-soluble fluorophore 1,2-DHP 1h by utilizing an aldehyde generated from triethylene glycol monomethyl ether and N,N-diethyl salicylaldehyde, which further offers an appropriate hydroxyl group substituent for appending any cleavable targeting group such as phosphate for in vitro phosphatase-sensing applications (Scheme ).

Scheme 1

Synthesis of 1,2-DHP Fluorophores 1a–1h′ by the Four-Component Condensation Reaction

Reagents and conditions: (a) TFA (1 equiv), CH3CN, rt, overnight; (b) (OMe)2P(O)Cl (1.5 equiv), NaH (1.5 equiv), THF, rt, 3 h; and (c) (i) TMSBr (10 equiv), CH2Cl2 and (ii) MeOH.

Photophysical Properties

The photophysical properties of 1,2-DHPs 1a–1h, viz., absorption, emission, quantum yields, and emission lifetime measurements, are provided in Table and Supporting Information (Figures S2 and S3). The present design involves a D−π–A or push–pull type system; thus, the nature and position of the substituents on the 1,2-DHP moiety are crucial to tune their intramolecular charge transfer (ICT) properties, which leads to different photophysical properties. 1,2-DHPs 1a–1h exhibit maximum absorption wavelengths (λmax) between 396 and 448 nm in methanol with strong molar extinction coefficients (5388–27 300 M–1 cm–1) and emit in the long wavelength region of 500–600 nm. 1,2-DHPs 1a and 1b exhibited similar photophysical properties; however, replacement of the phenyl group at the sixth position with a methyl group did not offer any change in the properties of 1,2-DHPs 1c, 1d, and 1f when compared to the former. These results indicate that the tuning of fluorophoric properties of these 1,2-DHPs can be made by variations in the N-benzylideneamine moiety. Thus, the sixth position of 1,2-DHP is an ideal position for conjugation with other biomolecules for fluorophore tagging. To assess the role of N-benzylideneamine in 1,2-DHP, the N-ethanimine-appended 1,2-DHP 1e was also synthesized, and indeed, it was found poorly emissive when compared to all other 1,2-DHPs because of the reduced ICT character with the lowest molar extinction coefficients (ε = 5388 M–1 cm–1). As expected, 1,2-DHP 1g with a strong donating group led to a significant bathochromic shift of λmax (ca. 20 nm) and λem (ca. 50 nm) with a higher molar extinction coefficient (ε = 23 292 M–1 cm–1).

The fluorescence quantum yields for 1,2-DHPs 1a–1g were determined by a relative comparison method using coumarin 153[13] as a standard and were found to be in the range of 0.032–0.125 with 1,2-DHP 1g being the highest. These compounds exhibited remarkable Stokes shift values, which can help in obtaining better fluorescence imaging with minimum self-absorption of the fluorophore. It is already established that for better cellular imaging, compounds should have absorption in the visible region and high fluorescence quantum yield. In this regard, on the basis of the observed photophysical properties, the present design of 1,2-DHPs possesses the potential for their application as bioprobes.

Applications

The mitochondrial membrane has a negative potential of −180 mV; therefore, it is typical to use cationic dyes for imaging these organelles.[14] The push–pull system in 1,2-DHPs (Figure ) makes the ring nitrogen of 1,2-DHP to attain a sufficient positive charge; thus, 1,2-DHPs may have an ability to serve as mitochondrial staining agents. Further, to assess the potential of 1,2-DHPs for specific mitochondrial staining, 1,2-DHPs 1a–1g were studied in HeLa cells. Initially, cytotoxicity of 1,2-DHPs was evaluated using MTT assay, and it was found that 1,2-DHPs exhibit greater than 80% cell viability at 30 μM (Figure S4). HeLa cells were incubated with 30 μM of 1,2-DHPs for 10 min, and the excess compound was washed with Hanks’ balanced salt solution (HBSS) buffer solution. As shown in Figures and S5, 1,2-DHPs were localized mostly in the cytoplasm, specifically  stained mitochondria in HeLa cells, and no nuclear uptake was observed. Additionally, the co-staining experiment with MitoTracker red chloromethyl-X-rosamine (CMXRos), a commercially available mitochondria-imaging dye, confirmed the localization of 1,2-DHPs in the mitochondria supported by Pearson’s correlation coefficient in the range of 0.75–0.89. Among all the 1,2-DHPs under study, 1,2-DHP 1b, 1d, and 1g were found to exhibit high fluorescence intensity compared to others.

Figure 2

Fluorescent images of HeLa cells (a) treated with 1,2-DHPs 1b, 1d, and 1g (30 μM) for 10 min, (b) MitoTracker red CMXRos (50 nM) for 30 min, (c) merged image of (a,b) with a bright-field image (60× magnification). Excitation wavelength: 440 nm (for 1,2-DHP) and 540 nm (for CMXRos) and emission wavelength: 515 nm (for 1,2-DHP) and 645 nm (for CMXRos). Pearson’s correlation coefficients were obtained as 0.79, 0.86, and 0.75 for 1,2-DHPs 1b, 1d, and 1g, respectively.

As a proof of concept, to justify the importance of the new 1,2-DHP as a fluorescent probe, we have synthesized a phosphorylated analogue 1h′ from 1,2-DHP 1h (Scheme ). It is well-known that direct and rapid analysis of the crude lysate for endogenous phosphatase enzymes such as PTPs are of prime interest owing to their significant role in insulin-signaling pathways[15] and a variety of disease states[16] including hepatocellular carcinoma[17] as well as metabolic disorders.[18] PTPs are significant targets in many diseases, and there is a growing need for direct determination of endogenous protein phosphatase activity.[19] The UV–vis absorption spectrum of the 1,2-DHP 1h′ in methanol exhibited absorption maximum at 448 nm, and the corresponding emission spectrum shows a peak at 594 nm, whereas in aqueous buffer medium (25 mM Hepes buffer, pH 7.4), a small bathochromic shift was observed both in absorption and emission spectra (Figure ). The quantum yield of 1,2-DHP 1h′ in Hepes buffer medium is reduced to 0.007, which can be rationalized by differences in the electron density involved in conjugation with phosphate and phenoxide groups. This difference of electronic distribution also reflected in the fluorescence lifetime profile. 1,2-DHP 1h in Hepes buffer exhibited a fluorescence lifetime of 1.94 ns, which is good enough for imaging experiments,[20] whereas its phosphorylated analogue 1,2-DHP 1h′ did not show any decay profile because of its weak fluorescence property (Table ).

Figure 3

Absorption and emission spectral profile (normalized) of (a) 1,2-DHP 1h and (b) 1,2-DHP 1h′ in methanol and Hepes buffer (25 mM, pH 7.4, 0.3% DMSO) at room temperature.

To get the structural details of 1,2-DHPs 1h and 1h′, both the structures in their ground state were optimized using density functional theory (DFT) with the B3LYP[21] exchange correlation functional and the 6-31G** basis set[22] with a Gaussian G09 package,[23] and the corresponding structures have been given in Figure a,b. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of 1,2-DHP 1h have been given in Figure c,d, respectively, which shows that the HOMO of 1,2-DHP 1h is largely localized on the diethylaniline group, whereas the LUMO is predominantly confined on the 1,2-DHP core, thus supporting our concept of the push–pull system. In aqueous medium, at physiological pH (Hepes buffer, pH 7.4), the fluorescence emission properties of 1,2-DHPs 1h and 1h′ showed a distinct change. 1,2-DHP 1h with a free hydroxyl group exhibited a sixfold higher orange fluorescence than that of 1,2-DHP 1h′ appended with a phosphate group (Figure e). The corresponding fluorescence changes were also reflected in visual appearance of both the solutions (Figure e, inset).

Figure 4

Energy-minimized structures of (a) 1,2-DHP 1h and (b) 1,2-DHP 1h′ at the DFT level and corresponding (c) HOMO and (d) LUMO of 1,2-DHP 1h. (e) Absorption and emission (λex = 450 nm) spectra of 1,2-DHPs 1h and 1h′ at room temperature (25 mM Hepes buffer, pH 7.4, 0.3% DMSO) and corresponding visual fluorescence change (inset).

This significant difference in emission intensity inspired us to explore 1,2-DHP 1h′ as a phosphatase sensor. As it is well-known that blinking and photobleaching of the fluorophores may cause problems for the imaging experiments,[24] the photostability of 1,2-DHP 1h′ was first tested by monitoring the fluorescence intensity as a function of time upon continuous irradiation (λ = 445 nm) in Hepes buffer solution (25 mM, pH 7.4, 0.3% DMSO) over a period of 20 min under aerobic conditions and was found to be quite stable (Figure S6). Encouraged by the fluorescence features of 1,2-DHPs 1h and 1h′, we further investigated their suitability as a probe for biological systems. The cytotoxicity of 1,2-DHPs 1h and 1h′ was determined by MTT assay in L6 cell lines. Cells were treated with different concentrations of 1,2-DHPs 1h and 1h′ ranging from 1 to 30 μM, and after 2 h of treatment, we found that both 1,2-DHPs 1h and 1h′ were less than 20% toxic up to 30 μM (Figure a).

Figure 5

(a) Cytotoxicity of 1,2-DHPs 1h and 1h′ in L6 myoblast by MTT assay at different concentrations. Values are the mean ± SD of three different experiments. (b) Comparative emission intensity for the direct assessment of PTP activity of 1,2-DHP 1h′ from cell lysate.

In our next attempt, we investigated the applicability of 1,2-DHP 1h′ as a chemosensor in the presence of PTPs from L6 muscle cell lysate as a preliminary study. This enzymatic reaction was performed in a 96 micro-well plate by the addition of cell lysate (5 μL, 0.8 μg/μL) to a 100 μL aqueous solution of 1,2-DHP 1h′ (30 μM) in Hepes buffer (25 mM, pH 7.4, 0.3% DMSO). After incubation at room temperature for 15 min, the fluorescence intensities were measured at an excitation wavelength of 450 nm and emission at 590 nm. The increase in fluorescence intensity with time clearly indicated cleavage of the phosphate group, which is a result of conversion of 1,2-DHP 1h′ to 1,2-DHP 1h (Figure b), thus indicating the suitability of 1,2-DHP 1h′ as a fluorescent bioprobe useful for monitoring the activity of PTPs. Further, to demonstrate the interference of 1,2-DHP 1h′ with other biologically relevant analytes, we measured the change in fluorescence intensity of 1h′ in the presence of various metal ions, reactive oxygen species, and under different pH conditions. Interestingly, there was no influence of these analytes in varying the fluorescence intensity of 1,2-DHP 1h′ (Figures S7 and S8).

Conclusions

In summary, we have designed and synthesized a new class of 1,2-DHP-based fluorophores by a facile one-step multicomponent protocol, and their photophysical properties were studied in detail. The results indicate that 1,2-DHPs with an extended N-benzylideneamine appendage have an absorption and emission maxima around 420 and 600 nm, respectively, having prominent Stokes shift. In particular, 1,2-DHPs 1g and 1h showed remarkable photophysical properties with high fluorescence. Furthermore, 1,2-DHPs 1b, 1d, and 1g are recognized as well-suited mitochondrial staining agents in HeLa cells. The potential of fluorophore 1,2-DHP 1h′ as a fluorescent probe in tyrosine phosphatase activity on the cell lysate was also explored. Synthetic accessibility and scope for conjugation warrants the utility of 1,2-DHP as a potential fluorescent probe for biological applications.

Experimental Section

General Experimental Methods

All the reactions were conducted using undistilled solvents, whereas CH2Cl2 was distilled over CaH2, which was used for the demethylation of the phosphate ester of 1,2-DHP 1h. Silica gel 60 F254 aluminum thin-layer chromatography (TLC) plates were used to monitor the reactions with short and long wavelength UV and visible lights to visualize the spots. Column chromatography was performed on the silica gel 100–200 and 230–400 mesh. The Shimadzu HPLC instrument with C18-phenomenex reversed-phase column (250 × 21.2 mm, 5 μm) was used for the purification of 1,2-DHP 1h′ using methanol and water. 1H, 13C, and 31P NMR spectra were recorded on a Bruker AVANCE II spectrometer at 500, 125, and 202 MHz, respectively. Chemical shifts are given in ppm using solvent residual peaks of chloroform (δ 7.26) and methanol (δ 3.31 ppm) as reference, and coupling constants in hertz. High-resolution electrospray ionization mass spectrometry analysis was recorded on a Thermo Scientific Exactive-LCMS instrument with ions given in the m/z ratio. Absorption spectra were recorded using a Shimadzu UV-2450, UV–visible spectrophotometer using a quartz cuvette with a 1 cm path length. The fluorescence spectrum of the 1,2-DHPs were recorded on a FluoroLog-322 (Horiba) instrument, which was equipped with a 450 W Xe arc lamp as the excitation source. The fluorescence quantum yields were determined with the relative method, employing an optically matched solution of coumarin 153 in MeOH as the reference (ΦR = 0.46). The following equation was used for calculating the quantum yieldwhere the subscripts R and S refer to the reference and samples, respectively. Abs, area, and n are the absorbance at the excitation wavelength, area under the fluorescence spectrum, and refractive index of the solvent, respectively. Fluorescence lifetimes were measured using an IBH (FluoroCube) TCSPC system. L6 myoblast and HeLa cells were obtained from the National Centre for Cell Sciences, Pune, India. Tris buffer (25 mM, pH 7.4, 0.3% DMSO), Hepes buffer (25 mM, pH 7.4, 0.3% DMSO) and HBSS (pH 7.4) buffers were used for the cell culture studies. The cells were visualized using a fluorescent microscope (Pathway 855, BD Bioscience, USA). Pearson’s correlation coefficients were calculated using ImageJ software with a JACoP plugin.

General Procedure for the Synthesis of Hydrazone

To a solution of hydrazine hydrate (10 equiv) in ethanol (10 mL) was added pertinent aldehyde (1 equiv), and the resulting mixture was stirred under reflux overnight. After complete consumption of the aldehyde, as indicated by 1H NMR, the reaction mixture was diluted with water and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4, and concentrated, and the resulting residue was used directly for the next step without further purification.

General Procedure for the Synthesis of 1,2-DHPs 1a–1g

To a solution of dienaminodioate (0.77 mmol, 1 equiv) in CH3CN (3 mL) were added aldehyde (1.15 mmol, 1.5 equiv), hydrazone (1.15 mmol, 1.5 equiv), and trifluoroacetic acid (0.77 mmol, 1 equiv) at room temperature. The reaction mixture usually develops a yellow to dark red coloration immediately, which is an indication of the formation of 1,2-DHP. After complete consumption of dienaminodioate, as observed on TLC, the reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was dried over anhydrous Na2SO4 and concentrated, and the resulting residue was purified by column chromatography to afford the desired 1,2-DHP.

Cellular Studies

Cell Culture and Treatment

Rat skeletal muscle cell lines (L6 myoblasts) and cervical cancer cell lines (HeLa) were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic mix at 37 °C under 5% CO2 atmosphere.

Cell Viability Study of 1,2-DHPs 1h and 1h′ on L6 Myoblast

MTT assay was performed to check the cytotoxicity of the compounds. The viability of L6 myoblast was measured by means of MTT assay. Cytotoxicities of 1,2-DHPs 1h and 1h′ (1, 5, 10, 20, and 30 μM) were standardized based on the concentration. Briefly, cells after incubation with the compound were washed, and MTT (0.5 g/L), dissolved in DMEM, was added to each well for the estimation of mitochondrial dehydrogenase activity, as described previously by Mosmann.[25] After an additional 2 h of incubation at 37 °C in a CO2 incubator, 10% of SDS in DMSO was added to each well, and the absorbance at 570 nm of solubilized MTT formazan products was measured after 45 min using a microplate reader (BioTek-USA). Results were expressed as percentage of cytotoxicity.

Preparation of Cell Lysate

Cells were grown in T25 flasks and after attaining 60% confluency, cells were differentiated in DMEM containing 2% horse serum for 5 days. Differentiated cells were then washed three times with Hepes buffer (25 mM, pH 7.4). Cells were scraped off from the plates using a cell scrapper and centrifuged, and the proteins were extracted from the cell pellet using 0.15 M KCl (4 °C for 30 min). The protein content of the lysate was then measured using a BCA protein assay kit.

Cell Viability on the HeLa Cell

Viability of the HeLa cell was measured by means of MTT assay, as explained before for the L6 myoblast. Cytotoxicities of 1a, 1b, 1c, 1d, 1f, 1g, 1h, and 1h′ (10, 20, 30, 50, 75, and 100 μM) were carried out based on the concentrations.

Colocalization Study of 1,2-DHPs with MitoTracker CMXRos

Cells were grown in a 96-well black clear bottom plates (BD Biosciences, Franklin Lakes, BJ), and after attaining 90% confluency, the cells were taken for the experiments. HeLa cells were incubated with MitoTracker CMXRos (50 nM) for 20 min at 37 °C, followed by the addition of the corresponding 1,2-DHPs (30 μM) and incubated for 10 min. This was followed by washing the cells twice with HBSS to remove the unbound dye. The cells were visualized under a fluorescent microscope (Pathway 855, BD Bioscience, USA).