New Red-Shifted 4-Styrylcoumarin Derivatives as Potential Fluorescent Labels for Biomolecules

Important scientific areas, such as cellular biology, medicine, pharmacy, and environmental sciences, are dependent on very sensitive analytical techniques to track and detect biomolecules. In this work, we develop a simple, low-cost and effective synthetic strategy to produce new red-shifted 4-styrylcoumarin derivatives as promising inexpensive fluorescent labels for biomolecules. The extension of the delocalized π-electron system results in bathochromic shifts in these new coumarin derivatives, which also present large Stokes shifts. In addition, density functional theory and time-dependent density functional theory calculations helped to rationalize the photophysical properties observed by the experimental results.


Introduction
Cellular biology, medicine, pharmacy and environmental sciences require highly sensitive analytical techniques to track and detect nucleic acids, oligonucleotides, antibodies, amino acids, proteins, lipids, carbohydrates, and other biomolecules [1][2][3][4][5][6]. Most available techniques generally require labelling with a sensor, as fluorescent labels [7,8] electrochemical sensors [9], photochromic compounds [10], photo switchable biomaterials [11], colorimetric biosensors [12], radioactive tracers [13] and isotope markers [14]. Of all sensitive analytical techniques, fluorescent labelling, taking into account the high sensitivity of the fluorescence technique and its non-destructive nature, presents numerous advantages as it allows the use of small sample quantities as well as the respective fluorescent labels [15]. The availability and the development of new fluorophores are now enabling previously impossible studies of cellular processes and the detection of specific components of complex biomolecular assemblies with selectivity and exquisite sensitivity, in vitro and in vivo, as well the analysis of their interactions [16][17][18]. Fluorescent labels offer many advantages as they are highly sensitive, even at very low concentrations, and can form covalent linkages with the sample to be analyzed, producing stable bioconjugates [19]. They should be chemically stable and small in size, with insignificant interference on the biological functions and structure of the unlabeled biomolecules, producing high fluorescence quantum yield bioconjugates. The amine-reactive fluorescent labels, since amino groups are either abundant or easily introduced into biomolecules, are the most frequently used to prepare numerous bioconjugates for direct or indirect immunochemistry, fluorescence in situ hybridization (FISH), histochemistry, cell tracing, receptor binding and other biological applications [20][21][22]. In this context, due to the high cost of the available commercial fluorescent labels, coumarin derivatives can be a solution to develop low-cost new fluorophores with absorption and emission at long wavelengths, combined with large Stokes shifts.

Synthesis
The main synthetic strategy to obtain 4-styrylcoumarin derivatives was based on the high acidity of the methyl protons present at position 4 in 2-(7-(diethylamino)-4-methyl-2Hchromen-2-ylidene)malononitrile (3), which enable aldol condensation reactions. The mentioned dicyanomethylene coumarinylmethyl derivative, presenting a higher bathochromic shift (more than 100 nm) when compared with its precursor (2), was obtained by the incorporation of two cyano groups in position 2 after the thionation of (1) [35,36]. We reasoned that the incorporation of one 4-styryl group containing electron-donating groups (EDGs) or electron-withdrawing groups (EWGs) at the para position could increase the π delocalization and the push−pull character of the chromophore. These modifications can promote large bathochromic shifts in the absorption and emission bands, as well as improve other photophysical properties. According to the above, we describe the design, synthesis, and spectroscopic characterization of a small library of 4-styrylcoumarin derivatives to explore the effect of EWGs and EDGs on the photophysical properties of the new chromophores. Moreover, one of the best candidates considering the binomial cost/photophysical properties has been functionalized through a reactive succinimidyl ester as an effective fluorescent label for biomolecules. The synthetic routes followed for the preparation of the novel 4-styrylcoumarin derivatives are shown in Scheme 1. The stereoselective and highly efficient aldol condensation reaction between the intermediate (3) and the aromatic aldehydes (methyl 4-formylbenzoate, benzaldehyde, 4-methoxybenzaldehyde, and 4-(dimethylamino)benzaldehyde), afforded the 4-styrylcoumarin derivatives (4 to 7) in very-good-to-high yields.
Unexpectedly, despite needing stronger reaction conditions, aldehydes containing electron-donating groups gave better yields than the one with electron-withdrawing group at the para position, which may be due to the superior stabilization of the intermediate alcohol, by the first ones, in this kind of molecule. The aforementioned new π-extended coumarins ( Figure 1) are easily isolated after silica column chromatography. All spectral data were consistent with the proposed structures (SI). Unexpectedly, despite needing stronger reaction conditions, aldehydes containing electron-donating groups gave better yields than the one with electron-withdrawing group at the para position, which may be due to the superior stabilization of the intermediate alcohol, by the first ones, in this kind of molecule. The aforementioned new π-extended coumarins (Figure 1) are easily isolated after silica column chromatography. All spectral data were consistent with the proposed structures (SI). Considering the good photophysical properties induced by the methoxy group in the 4-styrylcoumarin 4, we selected this compound to synthesize the amine-reactive fluorescent label 9 (Scheme 2). The referred fluorescent label can be obtained through five effective and linear synthetic steps from cheap commercially available precursors and, as it is later in the paper, its photophysical properties are very similar to those of derivative 7. The aldol condensation of 3 with the 6-(4-formylphenoxy)hexanoic acid afforded compound 8, which was further reacted with N-hydroxysuccinimide to attain the fluorescent label 9 (scheme 2).

Photophysical Properties
The photophysical properties of the synthesized coumarin derivatives were studied, and their absorption and emission properties, as well as fluorescence quantum yields, are summarized in Table 1.  Unexpectedly, despite needing stronger reaction conditions, aldehydes containing electron-donating groups gave better yields than the one with electron-withdrawing group at the para position, which may be due to the superior stabilization of the intermediate alcohol, by the first ones, in this kind of molecule. The aforementioned new π-extended coumarins (Figure 1) are easily isolated after silica column chromatography. All spectral data were consistent with the proposed structures (SI). Considering the good photophysical properties induced by the methoxy group in the 4-styrylcoumarin 4, we selected this compound to synthesize the amine-reactive fluorescent label 9 (Scheme 2). The referred fluorescent label can be obtained through five effective and linear synthetic steps from cheap commercially available precursors and, as it is later in the paper, its photophysical properties are very similar to those of derivative 7. The aldol condensation of 3 with the 6-(4-formylphenoxy)hexanoic acid afforded compound 8, which was further reacted with N-hydroxysuccinimide to attain the fluorescent label 9 (scheme 2).

Photophysical Properties
The photophysical properties of the synthesized coumarin derivatives were studied, and their absorption and emission properties, as well as fluorescence quantum yields, are summarized in Table 1. Considering the good photophysical properties induced by the methoxy group in the 4-styrylcoumarin 4, we selected this compound to synthesize the amine-reactive fluorescent label 9 (Scheme 2). The referred fluorescent label can be obtained through five effective and linear synthetic steps from cheap commercially available precursors and, as it is later in the paper, its photophysical properties are very similar to those of derivative 7. The aldol condensation of 3 with the 6-(4-formylphenoxy)hexanoic acid afforded compound 8, which was further reacted with N-hydroxysuccinimide to attain the fluorescent label 9 (Scheme 2).  Unexpectedly, despite needing stronger reaction conditions, aldehydes containing electron-donating groups gave better yields than the one with electron-withdrawing group at the para position, which may be due to the superior stabilization of the intermediate alcohol, by the first ones, in this kind of molecule. The aforementioned new π-extended coumarins (Figure 1) are easily isolated after silica column chromatography. All spectral data were consistent with the proposed structures (SI). Considering the good photophysical properties induced by the methoxy group in the 4-styrylcoumarin 4, we selected this compound to synthesize the amine-reactive fluorescent label 9 (Scheme 2). The referred fluorescent label can be obtained through five effective and linear synthetic steps from cheap commercially available precursors and, as it is later in the paper, its photophysical properties are very similar to those of derivative 7. The aldol condensation of 3 with the 6-(4-formylphenoxy)hexanoic acid afforded compound 8, which was further reacted with N-hydroxysuccinimide to attain the fluorescent label 9 (scheme 2).

Photophysical Properties
The photophysical properties of the synthesized coumarin derivatives were studied, and their absorption and emission properties, as well as fluorescence quantum yields, are summarized in Table 1.
Synthesis of the amine-reactive fluorescent label 9 for biomolecules.

Photophysical Properties
The photophysical properties of the synthesized coumarin derivatives were studied, and their absorption and emission properties, as well as fluorescence quantum yields, are summarized in Table 1.
The absorption and emission spectra of the new 4-styrylcoumarin derivatives (4 to 7) are displayed in Figure 2. All previous mentioned coumarin derivatives exhibit absorption and emission maxima at longer wavelengths, when compared to the intermediate, dicyanomethylene coumarin methyl derivative 3, due to the intramolecular charge transfer (ICT) effect by the conjugation of both the electron-donating NEt 2 group and the styryl groups in position 4 with the electron-withdrawing dicyanomethylene group in position 2. The absorption and emission spectra of the new 4-styrylcoumarin derivatives (4 to 7) are displayed in Figure 2. All previous mentioned coumarin derivatives exhibit absorption and emission maxima at longer wavelengths, when compared to the intermediate, dicyanomethylene coumarin methyl derivative 3, due to the intramolecular charge transfer (ICT) effect by the conjugation of both the electron-donating NEt2 group and the styryl groups in position 4 with the electron-withdrawing dicyanomethylene group in position 2. Generally, the presence of electron-withdrawing groups at position 4 promotes higher bathochromic shifts than the electron-donating groups [37], but this effect is not significantly pronounced in the 4-styrylcoumarin derivatives (4 to 7), possibly due to the strong electron-withdrawing effect of the dicyanomethylene group in position 2. On the other hand, the molar extinction coefficients were strongly affected by the nature of EDGs or EWGs in the para position at the 4-styryl group. The analysis of Table 1 allows the verification of the fact that, in the case of the 4-styrylcoumarin derivatives (4 to 7), EDGs promote high coefficients (e.g., ε (6) = 34000 cm −1 M −1 vs. ε (4) = 19000 cm −1 M −1 ) and also high fluorescence quantum yields (e.g., ΦF (6) = 0.95 vs. ΦF (4) = 0.04).
All coumarin derivatives exhibit large Stokes shifts due the extension of π-conjugated system in the molecule, which is essential to the effective intramolecular charge transfer process of emissive excited state. The coumarin derivative 6 presents the higher fluorescence quantum yield, possibly due the presence of a strong electron donating amino substituent, but the decrease observed in the derivative 7 might be attributed to a substantial reduction in the oxygen atom electronic density in the presence of the dicyanomethylene electron-withdrawing substituent [38]. Generally, the presence of electron-withdrawing groups at position 4 promotes higher bathochromic shifts than the electron-donating groups [37], but this effect is not significantly pronounced in the 4-styrylcoumarin derivatives (4 to 7), possibly due to the strong electronwithdrawing effect of the dicyanomethylene group in position 2. On the other hand, the molar extinction coefficients were strongly affected by the nature of EDGs or EWGs in the para position at the 4-styryl group. The analysis of Table 1 allows the verification of the fact that, in the case of the 4-styrylcoumarin derivatives (4 to 7), EDGs promote high coefficients (e.g., ε (6) = 34,000 cm −1 M −1 vs. ε (4) = 19,000 cm −1 M −1 ) and also high fluorescence quantum yields (e.g., Φ F (6) = 0.95 vs. Φ F (4) = 0.04).
All coumarin derivatives exhibit large Stokes shifts due the extension of π-conjugated system in the molecule, which is essential to the effective intramolecular charge transfer process of emissive excited state. The coumarin derivative 6 presents the higher fluorescence quantum yield, possibly due the presence of a strong electron donating amino substituent, but the decrease observed in the derivative 7 might be attributed to a substantial reduction in the oxygen atom electronic density in the presence of the dicyanomethylene electronwithdrawing substituent [38].
Fluorescent labels with large Stokes shifts values offer an advantage due the possible elimination of spectral overlap between absorption and emission, which reduces interference and also eliminates the quenching process, providing a very simple detection of the fluorescence emission. Fluorescent labels with large Stokes shifts are very important for Förster-type resonance energy-transfer (FRET) applications [39] and optical microscopy based on stimulated emission depletion (STED) [40].

Theoretical Calculations
The optimized molecular geometry of the most relevant coumarin derivatives in both the ground and the first excited singlet state computed at PBE0/6-31G(d, p) level in acetonitrile (see the SI for computational details) are depicted in Figure S1 (Supplementary Material) and detailed in Table S1. In the electronic ground state, all compounds present a deviation from planarity between the mean plane of the coumarin rings moiety and the 4-styryl group plane, restraining the π conjugation that links the donor and acceptor groups in the molecules. The ethylene bridge stays nearly in the plane of the benzyl group and the torsion occurs on the bond with the coumarin moiety. This dihedral angle is smaller for compound 6 (19.8 • ), 19.8 • and 19.7 • for 7 and 9, respectively, and presents higher values for compounds 4 (24.5 • ) and 5 (23.0 • ). For the excited state S1, however, a completely different picture emerges, with all the molecules becoming nearly planar with a minimum of −0.9 • for compounds 4 and maximum of 0.7 • for compound 9. Another important geometrical property that is pertinent to access the electronic delocalization throughout the π-conjugation framework is the bond length alternation (BLA), the average length difference between a single and adjacent double bond. While the BLA of the compounds varies between 0.08 Å (compound 6) and 0.11 Å (compounds 4 and 5) in the ground S0 state, it considerably reduces to 0.04 Å for all compound in the S1 excited state. These significant differences in geometry between the relaxed S0 and S1 states, contributing for an enhancement of electronic delocalization and a decrease in the HOMO-LUMO energy gap, might justify a large Stokes shift, where, after a vertical excitation, a significant structural relaxation of the excited state follows prior to emission [41,42].
The absorption wavelengths, oscillator strength (f ) and the main components of lowest energy transitions of the compounds were calculated by time-dependent density functional theory (TDDFT) methods ( Figure 3, Table S1). The lowest-energy excitations S1 and S2, despite presenting different oscillator strengths, share the same composition for all the compounds and are mainly of HOMO→LUMO and HOMO-1→LUMO character, respectively (more than 98% in all cases).  The shape and spatial location of the states, however, is very different, which imply different characteristics of the intramolecular charge transfer that occurs upon excitation. In most cases, the HOMO and HOMO-1 are more localized either in the coumarin or the 4-styryl group, while the LUMO are less localized, extending over the bridging zone, which facilitates low energy internal charge transfer absorptions [43]. The lowest energy excitation for compound 6, which exhibit the transitions with the higher oscillator strength, correspond to charge transfers from the 4-styryl group to the coumarin moiety, reflecting the strong donor capability of the amine group attached to the 4-styryl group. For the other compounds, the S0→S1 excitation corresponds to the opposite charge transfers, with the HOMO state mainly located on the coumarin moiety and the LUMO spreading to the 4-styryl group. For these derivatives, however, this is not the most intense excitation, with the S0→S2 transition presenting much higher f values and thus dominating The shape and spatial location of the states, however, is very different, which imply different characteristics of the intramolecular charge transfer that occurs upon excitation. In most cases, the HOMO and HOMO-1 are more localized either in the coumarin or the 4-styryl group, while the LUMO are less localized, extending over the bridging zone, which facilitates low energy internal charge transfer absorptions [43]. The lowest energy excitation for compound 6, which exhibit the transitions with the higher oscillator strength, correspond to charge transfers from the 4-styryl group to the coumarin moiety, reflecting the strong donor capability of the amine group attached to the 4-styryl group. For the other compounds, the S0→S1 excitation corresponds to the opposite charge transfers, with the HOMO state mainly located on the coumarin moiety and the LUMO spreading to the 4-styryl group. For these derivatives, however, this is not the most intense excitation, with the S0→S2 transition presenting much higher f values and thus dominating the absorption spectra. The donor character of compounds 7 and 9 attached units is evidenced by this transition that takes place between the HOMO-1 state, mostly located on the 4-styryl group, and the LUMO, mostly located on the coumarin moiety nitrile groups.
Comparing the MOs of 7 and 9, they are comparable since the attached reactive group does not take part on the π-conjugation framework, thus resulting in very close spectroscopic properties. For most of the compounds, the calculated lowest energy transitions present good agreement with the experimental absorption maxima of the coumarin derivatives absorption (Tables 1 and S2) with the exception of compound 6. For this compound, the experimental maximum at 496 nm can be assigned to the S0→S2 transition, while the lowest energy S0→S1 transition corresponds to a visible shoulder located around 536 nm.

Conclusions
With the objective to extend the delocalization of the π-electron system, we designed and synthesized new 4-styrylcoumarin derivatives, with absorption and emission at long wavelengths, combined with large Stokes shifts, using a simple, low-cost and efficient synthetic strategy. The results obtained from the UV/Vis spectra allow to conclude that the 2-(7-(diethylamino)-4-methyl-2H-chromen-2-ylidene)malononitrile (3) is a useful intermediate to the synthesis of promising new fluorescent labels for biomolecules. DFT and TDDFT calculations helped to rationalize the observed photophysical properties, particularly the large Stokes shifts, ascribed to a significant structure relaxation found in the molecules' excited states. The development of further red-shifted coumarin fluorescent labels for biomolecules with improved features is currently in progress in our laboratory and the results will be reported briefly.
Supplementary Materials: The following supporting information can be downloaded online. 1 H and 13 C NMR and HRMS spectra of the 4-styrylcoumarin derivatives (4 to 9). Figures S1 and S2 and Tables S1, S2 and S3 of Quantum chemical calculations.
Funding: This research received no external funding.

Data Availability Statement:
The data presented in this study are available in this article and in the Supporting Information.