Rational Design of Cost-Effective 4-Styrylcoumarin Fluorescent Derivatives for Biomolecule Labeling

Fluorescent labels are key tools in a wide range of modern scientific applications, such as fluorescence microscopy, flow cytometry, histochemistry, direct and indirect immunochemistry, and fluorescence in situ hybridization (FISH). Small fluorescent labels have important practical advantages as they allow maximizing the fluorescence signal by binding multiple fluorophores to a single biomolecule. At present, the most widely used fluorescent labels available present small Stokes shifts and are too costly to be used in routine applications. In this work we present four new coumarin derivatives, as promising and inexpensive fluorescent labels for biomolecules, obtained through a cost-effective, efficient, and straightforward synthetic strategy. Density functional theory and time-dependent density functional theory calculations of the electronic ground and lowest-lying singlet excited states were carried out in order to gain insights into the observed photophysical properties.


Introduction
Fluorescent labels, also called fluorescent markers or probes, have become elementary in a broad range of modern scientific practices.The referred fluorophores provide fluorescence when it is absent or inadequate in molecules of interest in many applications, such as fluorescence microscopy, flow cytometry, histochemistry, direct and indirect immunochemistry, and fluorescence in situ hybridization (FISH) [1][2][3][4][5][6][7].
Generally, fluorescent labels are classified into three major groups, i.e., organic dyes, proteins, and nanoparticles, but the vast majority of fluorophores belong to the organic dyes group.Organic dyes have important practical advantages over the other dyes as they allow maximizing the fluorescence signal by binding multiple fluorophores to a single biomolecule, due to their small size.Also, these small fluorescent molecules can be chemically modified through simple and effective organic synthetic methods, which allows the synthesis of labels with a wide range of photophysical properties [8][9][10][11].
Considering the efficiency and sensitivity of the detection process, the photophysical properties of fluorescent labels (wavelength of maximum absorption, molar extinction coefficient, wavelength of maximum emission, Stokes shift, and fluorescence quantum yield) are determinant for the wide range of specific applications available [12].
The combination of fluorescence imaging with fluorophores based on organic molecules has become a powerful tool as it has enabled the understanding of biological events at the molecular level due to its high sensitivity or selectivity, non-invasive character, easy implementation, rapid response rate, real-time detection, spatiotemporal resolution, facile visualization, and in situ detection [13][14][15].
Due to the abundance of amino groups, or their easy incorporation into biomolecules, the amine-reactive fluorescent labels are most commonly used to prepare stable bioconjugates for a wide range of biological applications.However, for super-resolution multi-color imaging experiments, such as optical microscopy of biomolecules based on stimulated emission depletion (STED) and in Förster-type resonance energy transfer (FRET) applications, the development of new fluorescent labels with large Stokes shifts is essential and indispensable [16][17][18][19][20][21].
Currently, the most widely used fluorescent labels available present small Stokes shifts, many of which are under 30 nm and are too costly to be used in routine applications, which is their major handicap.In this context, coumarin derivatives can be a real solution to develop new cost-effective brightness fluorophores, with large Stokes shifts [12,22,23].
Coumarins, found in many plant species [24] and often classified as classical fluorescent dyes, have been modified to obtain coumarin-based fluorescent dyes with adjustable optical properties due to their excellent photophysical properties, good cell membrane permeability, nontoxicity, and cost-effectiveness [10,25,26].Recent developments have conclusively shown that the arrangement of substituents and the types of substituents present in coumarin rings significantly impact the electronic delocalization within the conjugated π-system.Additionally, it is widely recognized that the inclusion of such substituents, forming donor-π-bridge-acceptor (D-π-A) structures, can facilitate an efficient intramolecular charge transfer (ICT) process.This, in turn, leads to remarkable and varied photophysical properties in coumarin derivatives [10,[27][28][29][30][31][32][33][34].Several of these coumarin derivatives have been applied in many important areas such as solar cells, organic light-emitting diodes, and fluorescent probes [26,[35][36][37].
In this study, we aim to establish a cost-effective synthetic approach for producing four novel 4-styrylcoumarin derivatives (9, 14, 15, and 20) with red-shifted fluorescence properties, utilizing the readily available and affordable 7-diethylamino-4-methylcoumarin (1) as our starting material.These newly synthesized compounds hold potential as fluorescent labels for biomolecules.To further enhance our understanding, we have conducted theoretical analyses on the optical, electronic, and geometric characteristics of these fluorescent labels in both their ground and lowest-energy singlet excited states.This analysis was performed using density functional theory (DFT) and time-dependent density functional theory (TD-DFT), complementing the overall investigation.

Synthesis and Characterization
The synthetic routes followed for the preparation of the fluorescent labels for biomolecules 8, 9, 14, 15, 19, and 20 (Figure 1), using the inexpensive 7-diethylamino-4-methylcoumarin (1) as the starting material, are shown in Scheme 1.The main synthetic approach for producing 4-styrylcoumarin derivatives relied on the elevated acidity of the methyl protons found at position 4 within the coumarin ring.This acidity facilitated aldol condensation reactions, particularly when electron-withdrawing groups (EWGs) were introduced at position 2. We believed that introducing a 4-styryl group with electron-donating groups (EDGs) in the para position could enhance both the π-delocalization and the push-pull nature of the chromophore.These modifications can improve photophysical properties of the coumarin derivatives, such as high-fluorescence quantum yields, as well as higher bathochromic and large Stokes shifts.The intermediates mentioned, which had electron-withdrawing groups (EWGs) at position 2, specifically 3, 11, and 16, were readily synthesized with high yields (85% to 89%).This synthesis involved a thionation reaction on the carbonyl group of 7diethylamino-4-methylcoumarin (1), followed by the condensation of the resulting thionated coumarin (2) with malononitrile, 4-pyridylacetonitrile hydrochloride, or piperidine, depending on the case.
The lower yields observed in the pyridinyl derivatives 12 and 13 may have been caused by an acidity decrease in the methyl protons present at position 4 due to the superior electronic delocalization across the molecular structure caused by the pyridine group.The intermediates mentioned, which had electron-withdrawing groups (EWGs) at position 2, specifically 3, 11, and 16, were readily synthesized with high yields (85% to 89%).This synthesis involved a thionation reaction on the carbonyl group of 7diethylamino-4-methylcoumarin (1), followed by the condensation of the resulting thionated coumarin (2) with malononitrile, 4-pyridylacetonitrile hydrochloride, or piperidine, depending on the case.
The lower yields observed in the pyridinyl derivatives 12 and 13 may have been caused by an acidity decrease in the methyl protons present at position 4 due to the superior electronic delocalization across the molecular structure caused by the pyridine group.The intermediates mentioned, which had electron-withdrawing groups (EWGs) at position 2, specifically 3, 11, and 16, were readily synthesized with high yields (85% to 89%).This synthesis involved a thionation reaction on the carbonyl group of 7-diethylamino-4methylcoumarin (1), followed by the condensation of the resulting thionated coumarin (2) with malononitrile, 4-pyridylacetonitrile hydrochloride, or piperidine, depending on the case.
The lower yields observed in the pyridinyl derivatives 12 and 13 may have been caused by an acidity decrease in the methyl protons present at position 4 due to the superior electronic delocalization across the molecular structure caused by the pyridine group.
The differences between the electronegativity of the nitrogen or oxygen atom in the electron-donating groups present in the aromatic aldehydes may explain the small difference in reaction yields observed, with a small advantage for the amino aldehydes.
The esterification of coumarin carboxylic acids 6, 7, 12, 13, 17, and 18 produced the N-hydroxysuccinimide (NHS) esters 8, 9, 14, 15, 19, and 20 as promising reactive fluorescent labels for biomolecules, in high yields.The referred fluorescent labels can be obtained through four effective and linear synthetic steps from cheap commercially available precursors (five steps to 14 and 15).All new compounds (9, 14, 15, and 20) were fully characterized by 1D and 2D NMR spectroscopy and HRMS.The spectral data were consistent with the proposed structures.

Photophysical Characterization
We conducted a study on the photophysics of the newly synthesized coumarin derivatives, examining their absorption and emission characteristics, along with fluorescence quantum yields.Table 1 provides a summary of these properties, while Figure 2 illustrates the UV/Vis spectra of these coumarin derivatives.The inclusion of electron-withdrawing groups (EWGs) at position 2, in the coumarin ring, promoted effective bathochromic shifts of 66, 106, and 179 nm, in the intermediates 16, 3, and 11, respectively.These modifications increased the push-pull character from the electron-donating group NEt 2 , at position 7, by extending the π-system with the introduction of a new double bond that was conjugated with the aforementioned electron-withdrawing groups (EWGs).The combination between an effective positive charge and the electronic delocalization present in the pyridine moiety of the EWG intermediate 11 allowed the largest bathochromic shift observed.In contrast, the intermediate 16 that only presented a positive charge as EWG had the lowest bathochromic shift value.Unsurprisingly, these shifts were further increased to longer wavelengths with the inclusion of a 4-styryl moiety, containing one electron-donating nitrogen or oxygen group at the para position.As expected, the nitrogen group, when compared with the oxygen group, promoted higher bathochromic shifts (approximately more than 40%) due to the superior mesomeric electron-donating character of this substituent.
The molar absorption coefficients of the fluorescent labels (8, 9, 14, 15, 19, and 20) were also strongly influenced by the nature of the EWGs at position 2 and the EDGs at position 4 in the coumarin ring (e.g., ε (8) = 24,000 cm −1 M −1 vs. ε (15) = 113,500 cm −1 M −1 ).Considering only the 4-styryl moiety effect, the presence of the nitrogen group more than doubled the molar absorption coefficients when compared with the counterpart oxygen group.
The new fluorescent labels 14 and 15 have high molar absorption coefficients, approximately twice as much as the others, which may be due to the referred combination present in the pyridine moiety of the EWG.
All fluorescent labels exhibited large Stokes shifts, which were essential to the effective intramolecular charge transfer (ICT) process of the emissive excited state, due to the extension of the π-conjugated system in the molecule.Large Stokes shifts minimized the undesirable spectral overlap between absorption and emission, allowing the reduction in the interference and the quenching process, providing an effective detection of the fluorescence emission.
The decrease in fluorescence quantum yields in the fluorescent labels 14 and 15 could be related to the effective intramolecular charge transfer process due to the increase in the extension of π-conjugated system in these molecules [38].Obviously, this effect was not so pronounced in the fluorescent labels 19 and 20, and that was why they had high fluorescence quantum yields.In agreement with the above, due to the lower electron- Unsurprisingly, these shifts were further increased to longer wavelengths with the inclusion of a 4-styryl moiety, containing one electron-donating nitrogen or oxygen group at the para position.As expected, the nitrogen group, when compared with the oxygen group, promoted higher bathochromic shifts (approximately more than 40%) due to the superior mesomeric electron-donating character of this substituent.
The molar absorption coefficients of the fluorescent labels (8, 9, 14, 15, 19, and 20) were also strongly influenced by the nature of the EWGs at position 2 and the EDGs at position 4 in the coumarin ring (e.g., ε (8) = 24,000 cm −1 M −1 vs. ε (15) = 113,500 cm −1 M −1 ).Considering only the 4-styryl moiety effect, the presence of the nitrogen group more than doubled the molar absorption coefficients when compared with the counterpart oxygen group.
The new fluorescent labels 14 and 15 have high molar absorption coefficients, approximately twice as much as the others, which may be due to the referred combination present in the pyridine moiety of the EWG.
All fluorescent labels exhibited large Stokes shifts, which were essential to the effective intramolecular charge transfer (ICT) process of the emissive excited state, due to the extension of the π-conjugated system in the molecule.Large Stokes shifts minimized the undesirable spectral overlap between absorption and emission, allowing the reduction in the interference and the quenching process, providing an effective detection of the fluorescence emission.
The decrease in fluorescence quantum yields in the fluorescent labels 14 and 15 could be related to the effective intramolecular charge transfer process due to the increase in the extension of π-conjugated system in these molecules [38].Obviously, this effect was not so pronounced in the fluorescent labels 19 and 20, and that was why they had high fluorescence quantum yields.In agreement with the above, due to the lower electron-donating mesomeric character of the oxygen group in the 4-stryl moiety, compared with the nitrogen group, this substituent promoted higher-fluorescence quantum yields.

Theoretical Calculations
The lower-energy bands of the spectra were dominated by two excitations, S 1 and S 2 , as can be seen in Figure 3.The lowest-energy excitations S 1 and S 2 consisted mainly of HOMO → LUMO and HOMO-1 → LUMO one-electron contributions, respectively, for all the studied compounds, and their relative oscillator strengths depended on the compound possessing the oxygen or the nitrogen group (Table S1).For the oxygen group compounds 8, 14, and 19, the S 2 excitation was dominant, while the S 1 was the most intense for the nitrogen group compounds 9, 15, and 20.This accounted for the elevated molar absorption coefficients observed in the nitrogen group compounds in comparison with their oxygen group counterparts.Additionally, the higher oscillator strength value of compound 15 aligned with the greater molar absorption observed for this particular compound.
The shape and spatial distribution of the HOMO, HOMO-1, and LUMO states exhibited notable differences, indicating distinct characteristics of the intramolecular charge transfer that took place upon excitation.For all the compounds, neither the HOMO, the HOMO-1, nor the LUMO orbitals presented relevant contributions over the attached reactive group, indicating that it did not take part in the π-conjugation framework and did not interfere in the excitation process.This is an important feature since a minimal interference of the attachment of the dye labels to a biomolecule with their fluorescence properties is desirable.
Turning to the HOMO states, the main feature that emerged was that the HOMO orbitals were mostly localized in the coumarin for the oxygen group compounds, while for the nitrogen group compounds, they were located on the 4-styryl group.Conversely, the inverse occurred for the HOMO-1 orbitals, with the HOMO of the oxygen group compounds presenting great similarity with the HOMO-1 of the nitrogen group compounds and vice versa.
For all compounds, however, the LUMO orbitals showed less localization and extended over the bridging zone and did not present important differences between the two compound families.This extension facilitated low-energy internal charge transfer absorptions.The most intense transition, whether being that with the longest wavelength or not, corresponded in all cases to a transition from the state mainly located in the styryl group to the LUMO, which suggested those were the states that overlapped to a larger extent with the LUMO orbitals.The lowest-energy transition in compounds 9, 15, and 20 exhibiting the highest oscillator strength involved a charge transfer process from the 4-styryl group to the coumarin moiety, highlighting the significant donor ability of the amine group connected to the 4-styryl moiety.
The ∆r index [39], a measure of the charge transfer length, was employed to characterize the nature of electronic transitions.Transitions exhibiting a substantial ∆r index indicated a significant charge transfer (CT) character, with a commonly accepted threshold of 2.0 Å used as a criterion for assigning the CT character.Following this criterion, most of the S 1 and S 2 excitations could be classified as CT.The smaller values arose in derivative 8, and particularly high values of ∆r occurred for compound 19, indicating a large distance displacement of the charge upon excitation.
Figure 4 presents the optimized molecular geometry of the coumarin derivatives in both the ground and the first excited singlet states.In the ground state, all the coumarin derivatives exhibited a deviation from planarity between the average plane of the coumarin ring moiety and the plane of the 4-styryl group.This deviation limited the π-conjugation that connected the donor and acceptor groups within the molecules.The magnitude of this dihedral angle varied among the compounds with derivative 15 presenting the smaller dihedral angle of 8.4 • and compound 19 presenting the higher dihedral angles of 19.9 • (Table S2 and Figure S28).The shape and spatial distribution of the HOMO, HOMO-1, and LUMO states exhibited notable differences, indicating distinct characteristics of the intramolecular charge transfer that took place upon excitation.For all the compounds, neither the HOMO, the HOMO-1, nor the LUMO orbitals presented relevant contributions over the attached reactive group, indicating that it did not take part in the π-conjugation framework and did not interfere in the excitation process.This is an important feature since a minimal interference of the attachment of the dye labels to a biomolecule with their fluorescence properties is desirable.In contrast, the S 1 electronic excited state revealed a distinct scenario, where all the molecules became nearly planar.The deviations from planarity were minimal, ranging from a minimum of 0.1 • for compound 9 to a maximum of 7.4 • for compound 20.The transition from the S 0 state to the S 1 state was also characterized by a decrease in the bond length alternation (BLA), the average length difference between a single and adjacent double bond, which considerably reduces, for all compounds, in the excited S 1 state.In the S 0 state the compounds exhibited variations of BLA ranging from 0.08 Å for compounds 9 and 20 to 0.10 Å for compounds 8 and 15.However, in the excited state S 1 , the BLA significantly decreased to 0.04 Å (compounds 8, 9, and 19) to a maximum of 0.06 Å for dye 20.In contrast, the S1 electronic excited state revealed a distinct scenario, where all the molecules became nearly planar.The deviations from planarity were minimal, ranging from a minimum of 0.1° for compound 9 to a maximum of 7.4° for compound 20.The transition from the S0 state to the S1 state was also characterized by a decrease in the bond length alternation (BLA), the average length difference between a single and adjacent double bond, which considerably reduces, for all compounds, in the excited S1 state.In the S0 state the compounds exhibited variations of BLA ranging from 0.08 Å for compounds 9 and 20 to 0.10 Å for compounds 8 and 15.However, in the excited state S1, the BLA significantly decreased to 0.04 Å (compounds 8, 9, and 19) to a maximum of 0.06 Å for dye 20.
According to Kasha's rule [40], fluorescence in most cases is restricted to the lowestlying excited state with the higher electronic excited states typically not directly contributing to the emission of excited fluorophores.Therefore, the emissions of the studied dyes would arise from the S1 state.
The emission properties of the dyes were calculated from the optimized S1 state geometry and are depicted in Table S3.The emission wavelengths S1 → S0 were systematically smaller than those corresponding to the absorption S0 → S1 process based According to Kasha's rule [40], fluorescence in most cases is restricted to the lowestlying excited state with the higher electronic excited states typically not directly contributing to the emission of excited fluorophores.Therefore, the emissions of the studied dyes would arise from the S 1 state.
The emission properties of the dyes were calculated from the optimized S 1 state geometry and are depicted in Table S3.The emission wavelengths S 1 → S 0 were systematically smaller than those corresponding to the absorption S 0 → S 1 process based on the ground state optimized geometry.The observed geometry changes that occurred in the electronic excited state S 1 promoted enhanced electronic delocalization within the π-conjugation structure, resulting in a reduction in the HOMO and LUMO energy gap.Consequently, following a vertical excitation, there was a notable structural relaxation of the excited state that occurred before emission [41,42], which led to a smaller emission energy compared with the excitation energy, thus resulting in a substantial Stokes shift.
From the lowest-lying excited state S 1 geometry optimization performed, the theoretical fluorescent lifetime of the excited states can be calculated from Einstein's transition probabilities of the spontaneous transitions equation where τ is the fluorescent lifetime, c is the velocity of light in vacuum, E is the transition energy, and f is the corresponding oscillator strength of the S 1 →S 0 transition.The fluorescent lifetimes of the calculated fluorescent dyes are depicted in Table S3, and for all dyes, all the values were shorter than 10 ns, typical for emissive states of organic fluorophores, while larger radiative lifetimes corresponded to nonradiative states.However, since nitrogen compounds exhibited a greater oscillator strength in the S 1 → S 0 transition, their fluorescent lifetimes were shorter compared with their oxygen counterparts.

Materials and Equipment
Analytical grade starting materials and reagents were used, purchased from Aldrich (St. Louis, MO, USA).Organic solvents were dried over appropriate drying agents and distilled before use.UV-Vis absorption spectra were recorded using a Thermo Electron Spectrophotometer Corporation (Waltham, MA, USA), model Nicolet Evolution 300.Fluorescence spectra were recorded using a PerkinElmer Model LS 55 spectrophotometer (PerkinElmer, Waltham, MA, USA).Emission spectra were collected with a 5.0 nm slit bandwidth for both excitation and emission, with correction files.All spectroscopic measurements were performed in 3 mL quartz fluorescence cuvettes with a 1 cm optical path at a temperature of 21 • C. Acetonitrile (CH 3 CN) was used as the solvent for these measurements.FTMS-ESI mass spectra were obtained using a Thermo Scientific Q Exactive Orbitrap Mass Spectrometer (Thermo Scientific, Waltham, MA, USA).Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR, using a Brucker Advance III spectrometer (Billerica, MA, USA).Deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), or deuterated dimethyl sulfoxide (CD 3 ) 2 SO was used as the solvent for NMR experiments.The chemical shift (δ) was measured in parts per million (ppm), coupling constants (J) were provided in Hertz (Hz), relative intensity was indicated by the number of protons (H), and multiplicities were indicated by singlet (s), doublet (d), triplet (t), quadruplet (q), and multiplet (m).

Quantum Chemical Calculations
To shed light into the observed photophysical properties, density functional theory (DFT) and time-dependent density functional theory (TD-DFT) and calculations were performed by the Gaussian 16 package [43] with the hybrid PBE0 functional [44] and the standard 6-31G(d,p) basis set being used for geometry optimizations of both the ground and first excited states.The solvent effects were considered by the implicit polarized continuum model (PCM) [45,46].Following geometry optimizations, the vibration analysis was conducted presenting no imaginary frequencies, thus attesting the structures as true minima.For the TD-DFT spectra calculations, the larger 6-311+G(d,p) basis set was used.
We are currently working on synthesizing other red-shifted coumarin fluorescent labels for biomolecules, aiming to enhance their characteristics.We anticipate sharing a concise report on the outcomes in the near future.

Figure 3 .
Figure 3. Experimental spectra (gray) compared with the calculate excitations (red and blue) and the molecular orbitals involved in the two lowest-energy electronic transitions of the 8, 9, 14, 15, 19, and 20 dyes in MeCN.

Figure 3 .
Figure 3. Experimental spectra (gray) compared with the calculate excitations (red and blue) and the molecular orbitals involved in the two lowest-energy electronic transitions of the 8, 9, 14, 15, 19, and 20 dyes in MeCN.

Figure 4 .
Figure 4. Optimized molecular geometries computed at the PBE0/6-31G (d,p) level in MeCN of the coumarin derivatives, in both the ground and the first excited singlet states.

Figure 4 .
Figure 4. Optimized molecular geometries computed at the PBE0/6-31G (d,p) level in MeCN of the coumarin derivatives, in both the ground and the first excited singlet states.
a Absorption maxima in acetonitrile.b Emission maxima in acetonitrile.c Molar extinction coefficient at longest wavelength transition.d Fluorescence quantum yield in ethanol, determined using 7-diethylamino-4-methylcoumarin (Φ F = 0.73 in ethanol) as a standard.e Shoulder.