Field effects induce bathochromic shifts in xanthene dyes

Abstract

There is ongoing interest in near infrared (NIR) absorbing and emitting dyes for a variety of biomedical and materials applications. Simple and efficient synthetic procedures enable the judicious tuning of through-space polar (field) effects as well as low barrier hydrogen bonding to modulate the HOMO-LUMO gap in xanthene dyes. This affords unique NIR-absorbing xanthene chromophores.

INTRODUCTION

Near infrared (NIR) dyes are of great interest in biomedicine due to diminished interfering absorption and fluorescence from biological samples, inexpensive laser diode excitation, reduced scattering and enhanced tissue penetration depth.¹ However, only one NIR probe, indocyanine green (ICG), is approved by the FDA.² ICG is challenging to use for many applications due to its amphiphilicity and limited functionalizability. Moreover it displays high affinity for proteins with concomitant quenching upon binding.³ It displays a small Stokes shift and low quantum yield and is known to dimerize in aqueous solution at low concentrations which further lowers its quantum yield.⁴ Designing alternatives to ICG is thus an ongoing goal.

Several halogenated and non-halogenated xanthene dyes are sufficiently non-toxic to be used as food dyes.⁵ However, xanthene dyes typically possess small Stokes shifts and are not active in the NIR. Thus, strategies for red-shifting xanthene dyes for biodiagnostics and imaging applications have been an active area of investigation for many years. Strategies employed have included replacement of the phenyl ring with electron withdrawing groups such as cyano or trifluoromehtyl,⁶ extending the π-conjugation,⁷ replacement of the bridging O-atom by tatetrahedral carbon,⁸ and annulation.⁹ As a result, there are currently a large number of excellent long wavelength xanthene-based dyes. They mainly find utility in cellular imaging applications, as their spectral properties are outside the useful NIR (ca. 700 nm – 800 nm) range.

In 2008 it was demonstrated that when an internal xanthene ether oxygen is replaced with a dimethylsilyl moiety, a bathochromic shift of of 90 nm is obtained.¹⁰ This is due to the well-known polar effect of heavy atoms on the HOMO-LUMO energy gap observed in many related classes of chromophores.¹¹ This useful design concept has been recently applied to other scaffolds including anthracene,¹² fluorescein,¹³ and a variety rhodamine derivatives.¹⁴ Si-rhodamines with NIR emission as great as 740 nm have been created. However, their Stokes shifts are typically less than 20 nm and they are obtained in low overall yields from multistep procedures.^14d

Experimentally observable intramolecular coulombic interactions resulting from direct action through space rather than through bonds are known as field effects.¹⁵ In 2008 we reported that altering the known regiochemical functionalization of annulated xanthene dyes led to large (> 100 nm) Stokes shifts and NIR emission.¹⁶ This prior study has led us to propose that by tuning through-space interactions of polar groups (field effects)¹⁵ in the xanthene ring system, one could also favorably alter the electronic properties of the chromophore.

Benzannulated xanthenes typically possess polar groups on C-3 and C-11, as in 1 or C-3 and C-10 as in 3 (Figure 1). Positioning a polar group on C-1 (as in 4) or C-1 and C-3 (as in 2) can potentially modulate the HOMO-LUMO gap. This embodies a relatively facile means to reduce the energy gap without the need to replace the internal oxygen with heavy atoms.

Figure 1.

Structures of benzannulated xanthenes.

RESULTS AND DISCUSSION

Synthesis of symmetric and asymmetric naphthoxanthenes

In order to test this hypothesis, a series of symmetric and asymmetric naphthoxanthenes 1–4 were synthesized and evaluated. Naphthofluorescein 1a is commercially available. Known SNAFL-1 (herein 3a) was prepared as previously reported.^9b The rest of the symmetric and asymmetric naphthoxantene fluorophores were synthesized by condensation of 1,8-dihydroxynaphthalene with phthalic anhydride, 3,5-dimethylbenzaldehyde, and 2-(2,4-dihydroxybenzoyl)benzoic acid respectively; using a mixture of CH₃SO₃H:TFA 1:1 at 80 °C for 16–24 h. The corresponding methyl ester derivatives were obtained by Fischer esterification in MeOH catalyzed by H₂SO₄; further methyl alkylation was furnished by treatment of either the carboxylate or methyl ester intermediate with methyl iodide in the presence of K₂CO₃ in DMF. The required 1,8-dihydroxynaphthalene and 2-(2,4-dihydroxybenzoyl)-benzoic acid derivatives were synthesized according to described or modified literature protocols.^9a,16,17 All compounds were isolated by flash column chromatography and characterized by NMR and MS. The structure of compound 4a as the lactone form (Figure S1, SI) was also confirmed by X-ray single crystal structure. The target compounds were obtained in overall yields ranging from 17 to 86% as described in the supporting information.

Spectroscopic Characterization

The study of 1a–4a in MeOH, DMSO and aqueous acid is complicated by the carboxylate-lactone equilibrium. (SI Figures S27–S30, Scheme S2) This prompted us to focus our initial investigations on esters 1b–4b and their corresponding anions1c–4c. It is noteworthy, however, that 2a exists as the corresponding colorless lactone in 1:9 DMSO:buffer below physiological pH of 7.4. It thus embodies a unique pH probe, in stark contrast to known intracellular pH sensors¹⁸ such as 1a and congeners that generally function as turn-on indicators > pH = 5 (SI, Figure S29).

Figure 2a displays the absorption spectra and a room light photograph of 1b–4b in MeOH, a summary of the experimental and calculated absorption and emission maxima values are given in Table 1. The placement of R₂ proximal to the xanthene internal oxygen in 2b and 4b affords bathochromic shifts of 103 and 15 nm as compared to 1b and 3b, respectively. The formal transposition of R₂ also imparts similar corresponding shifts in the excitation spectra (Figure 2b) and large bathochromic shifts in the fluorescence emission (Figure 2c) of 2b and 4b (128 nm and 32 nm as compared to 1b and 3b, respectively). Importantly, 2b absorbs and emits in the near infra-red with absorption and emission maxima of 701 nm and 816 nm.¹⁹ Analogous trends are observed in other solvent systems (see below and SI, Figure S34). Spectral properties of these and other compounds from families 1–4 in other solvents are tabulated in the supporting information (SI, Table S1).

Figure 2.

Absorption and fluorescence spectra of fully annulated 1b, 2b, and semiannulated 3b and 4b in MeOH. (a) Absorption. Inset: Room light Photograph of solutions in MeOH (left to right: 3b, 4b, 1b and 2b). (b) Normalized fluorescence excitation spectra. (c) Normalized fluorescence emission spectra. Formal transposition of R₂ (Figure 1) modulates the HOMO-LUMO gap and shifts the absorption and fluorescence in both the symmetric (1 and 2) and asymmetric (3 and 4) pairs. Additional spectra in MeOH and DMSO are provided (SI, Figures S31–S33).

Table 1.

Experimental and calculated spectral properties of 1b–4b in MeOH.

Comp.	λmax abs (nm)	λmax em (nm)	HOMO (eV)	LUMO (eV)	Gap (eV)	λmax abs calc (nm)
1b	598	688	−5.011	−2.719	−2.292	541
2b	701	816	−4.781	−2.688	−2.093	592
3b	487, 521	550	5.442	2.367	3.075	403
4b	501, 536	582	5.606	2.667	2.939	422

Induced through-space interactions and intramolecular hydrogen bonding

Although the red shift in the absorption of 4b in MeOH appears modest, its corresponding anion 4c in DMSO:aqueous base 1:9 is shifted more substantially by 50 nm to 599 nm as compared to anion 3c under the same condition.²⁰ Its fluorescence emission is shifted by an even larger 130 nm, emitting at 760 nm (see below). The comparatively large shift of the anion 4c further supports a polar effect as playing a key role in modulating the HOMO-LUMO gap. The oxoanion of asymmetric 4c should have a more significant field effect on the polarity of the proximal ether oxygen as compared to the corresponding oxoanion 3c.

There are through-space polar interactions between R₂ and the internal bridge oxygen in compounds 4a–d. In the case of symmetric compounds 2a–d the field effect is further enhanced by the oxygen on C-13 (Figure 1). The relatively larger difference in absorption maxima of 2b as compared to 1b (Figure 2a) is also attributable at least in part to the unique intramolecular hydrogen bonding network present in 2b (Figures 3 and 4). This network results in enhanced negative charge density on the ionizable oxygen atoms, thereby promoting a relatively large bathochromic shift, as in the analogous case of anionic 4c described above.

Figure 3.

¹H NMR spectra in DMSO-d₆ of compounds 1b, 2b, 3b, and 4b showing the different chemical shifts observed for the hydroxyl protons.

Figure 4.

Hydrogen bond network of 2 and energy-minimized structure of 2b showing how this enhances coplanarity of the fused ring system.

Hydrogen bonding is evidenced in the ¹H NMR in DMSO-d₆ of 2b (Figure 3) and absorption spectra (SI, Figure S35). The shared proton resonates significantly downfield (14.45 ppm) as compared to the corresponding phenolic proton of 1b (10.05 ppm). This strong deshielding can be attributed to a redistribution of the electronic density around the hydrogen atom that occurs upon hydrogen bond formation between the hydroxyl proton, the carbonyl and the central oxygen of 2b. Evidence of hydrogen bonding is also observed in 4b, but to a lesser degree. The hydrogen bonded proton is slightly downfield (10.75 ppm) as compared to the phenolic proton of 3b (10.58 ppm). The strong deshielding observed for the hydroxyl proton of 2b indicates that it is involved in the formation of a low barrier hydrogen bond, (LBHB).²¹ Low barrier hydrogen bonds play an important role in enzymatic processes ^21a,22 as well as in supramolecular assemblies.²³

In general, low barrier hydrogen bonds have been observed in systems were the donor acceptor distances are shorter than normal hydrogen bonds.^22c,24 In 2b, simulations show that the total hydrogen bond distance O···H-O between the carbonyl and the hydroxyl at the C-1 position is 2.52 Å, which is lower than the typical hydrogen bonding distance O···H-O (≥ 2.8 Å) and close to the average distance observed for low barrier hydrogen bonds (2.55 Å). In 4b, the total hydrogen bond distance O···H-O between the hydroxyl at the C-1 position and the xanthene central oxygen is 2.95 Å, which falls in range of common hydrogen bonds.

As further evidence of the effect of the hydrogen bonding network on the characteristics of 2, removal of the shared proton (under harsh conditions, 12.5 mM NaOH, to form 2c) does not result in a large bathochromic shift in the absorption as is observed when 1b is deprotonated to 1c (SI, Figure S36). In addition, unlike conventional naphthofluorescein compounds in which the fluorescence is minimal for the protic species in aqueous solvents^9a,9f the protic species of family 2 is more fluorescent than the corresponding ionized chromophore. For example, NIR fluorescence was observed in family 2 compounds up to pH 9; while removal of the shared proton was observed to quench the fluorescence (SI, Figure S37–38). Molecular simulations demonstrate that the hydrogen bonding network enhances coplanarity of the fused ring system of compounds in family 2 (Figure 4 and SI, Figure S39).

Molecular modeling

The bathochromic shifts in the absorption spectra induced through formal transposition (Figure 1, from C3 to C1) of the polar ionizable oxygen groups were evaluated by calculating the HOMO-LUMO energies of both the protic and anionic species and comparing the calculated absorbance maxima with experimental data in MeOH or DMSO:aqueous 1:9, respectively.²⁵ Molecular surfaces and energies are provided in the supporting information (SI, Figures S40–S50) and included for comparison with the spectral data in Table S1. Modeling generally systematically underestimated the HOMO-LUMO gap (i.e. overestimated the absorption wavelength) of deprotonated species and overestimated the gap (i.e. underestimated the absorption wavelength) for protic species. However, in each case, bathochomic shifts were both predicted and observed for compounds with formally transposed R groups (i.e. families 2 and 4 as compared to 1 and 3 respectively). Figure 5 shows the calculated molecular surfaces anions 1c and 2c. The results indicate that formal transposition effectively modulates the HOMO-LUMO gap. Molecular modeling most adequately predicts the magnitude of transposition-induced shifts for the deprotonated fully annulated compounds. When comparing 1c and 2c, the 73 nm calculated bathochromic shift is in agreement with the 79 nm observed experimental shift of 2c as compared to 1c. An experimental bathochromic shift of ~50 nm (calculated shift of ~90 nm) was observed for semiannulated anion 4c as compared to 3c. The modeling also reveals an unusual through-space overlap of the C-1 and C-13 oxygen orbitals in the HOMO of 2c that result in homoconjugation of the π-system in the chromophore. For 2c, the distance between the oxygens of C-1 and C-13 is 3.2 Å. Since the typical distance for a O-O bond is about 1.5 Å, this interaction could be involving variations of the molecular dipole moment through space. The five-membered fused-ring system in the 2 family is less planar in the anionic form due to the electrostatic repulsion of the oxygens of C-1 and C-13, and this allows the through-space overlapping of the oxygens’ orbitals of the HOMO that would not be possible if the anionic form was planar (Figure 6).

Figure 5.

Calculated molecular surfaces of the HOMO and LUMO of 1c and 2c (basis set DFT B3LYP/6-31G).²⁹ Additional molecular surfaces for other compounds are provided in the supporting information.

Figure 6.

Overlapping of the molecular orbitals of the oxygens at C-1 and C-13 in compound 2c.

Examples of other molecular architectures (restricted coplanarity featuring oxygen atoms relatively close in space) similar to 2 include 1,1′-binaphthalene-2.2′-diol (binol)²⁶ and more recently, HelMe, a [4]heterohelicene cation.²⁷ In binol the positions of the oxygens are not restricted and therefore a direct comparison with 2c is not possible. In the case of the [4]heterohelicene cation, HelMe (Figure 7) the oxygen atoms seem to be forced to be close but the calculated molecular surfaces of the published HOMO do not show a through-space overlapping of the oxygens’ orbitals as in the case of 2c.²⁸

Figure 7.

Known architectures positioning oxygen atoms relatively close in space.

The NIR absorbing properties of 2 render these compounds attractive. NIR absorbing compounds are widely used in areas such as optical recording, thermal-writing displays, laser printers, laser filters, infrared photography, photodynamic therapy and many other applications including solar cells and heat absorbers.³⁰ A current limitation in the field of solar cells is the mismatch between the absorbing dye and the solar emission spectrum.^30b Compound 2b is NIR active in the solid state (SI, Figure S51) and the absorption spectra of 2b and 2c match well the 690 nm maximum photon flux of sunlight.^30b

The long-wavelength absorptions of the dyes in the 2 family suggest that they could be useful as dyes for solar cells, given that a major goal for improvement of organic solar cells is improved absorption in the far red and NIR region.³¹ In addition, the best dyes for dye-sensitized solar cells (DSSCs) use phenylcarboxylates as anchoring groups and have a donor-acceptor structure in which the HOMO is localized on the donor portion while the LUMO is localized on the acceptor portion; this facilitates injection of the excited electron into the semiconductor (e.g., TiO₂) on which the dye is adsorbed.³²

The electron density of the HOMO of 2c (Figure 5) and 4c is located nearly exclusively on the chromophore, whereas in the LUMO nearly all of the electron density is located on the lower ring containing the ester moiety. These striking electronic properties are unique to chromophores 2 and 4, except, as expected, for 2d, which contains electron donating groups on the lower arene. Enhanced electron density in the LUMO at the sight of attachment to the metal surface of a solar cell is critical for efficient electron transfer.

Spectroscopic Characterization in Aqueous Media

The unique absorption and fluorescence properties of protic species 2b that arise from through space field effects and intramolecular hydrogen bonding, involving all three oxygens of the xanthene chromophore, were described above. Compounds 4a–d possess enhanced spectral properties for applications including biological imaging and diagnostics wherein long wavelength activity is advantageous. Positioning the hydroxyl group on C-1, to afford a field effect between two polar atoms, the bridging xanthene oxygen and the C-1 oxygen in the case of 4c, was found to have a greater effect on the emission wavelength of 4c than on the absorption as compared to 3c (~130 nm bathochromic shift in emission vs. ~50 nm shift in absorption, described above). The difference between these shifts results in NIR emission at 760 nm with a significantly enhanced Stokes shift of 160 nm. For comparison, the analogous long wavelength anionic form of commercial SNAFL-1 (3a) is reported to absorb and emit at 537 nm and 624 nm, respectively with a comparatively modest Stokes shift of 87 nm and a quantum yield of 0.093.^9b The Stokes shift of other xanthene dyes, i.e. fluorescein and rhodamine derivatives, is typically ~ 20 nm. Note that in the 1 and 3 series, field effects are not promoted due to the distal positioning of all three oxygens of the chromophore moiety.

Like many other NIR emitting compounds, 4c displays a relatively low quantum yield (less than 1%) in aqueous solution. The fluorescence quantum yield of ICG is limited by internal conversion to only a few percent.⁴ The brightness of 4c is comparable to that of other novel NIR probes including carbon nanotubes³³ which exhibit extinction coefficient³⁴ of approximately 4,400 and low fluorescence efficiencies typically < 0.1%.³⁵ It is well known that the absence of interfering background increases the sensitivity of NIR fluorophores far beyond other visible dyes with much higher quantum yields.³⁶ Efforts to increase the quantum yield beyond that of other NIR emitting probes are underway.

We find that straightforward functionalization through methylation of the C-3 oxygen to afford methyl ether 4d dramatically improves the quantum yield. The quantum yield of yellow-orange emitting 4d is 0.4649, 40 times greater than the corresponding neutral 4b. It is slightly higher than yellow-green emitting neutral 3b and > 2 times higher than orange-red emitting anion 3c (Figure 8). Formal transposition of the hydroxyl moiety (Figure 1) shifts the spectral properties such that the short wavelength forms of these compounds (4b and 4d) are redder than then short wavelength forms and approach the long wavelength forms of known seminaphthofluorescein probes (for example 3a and its analogues, i.e. carboxy SNAFL-1 including an additional carboxylate group on the lower ring). The emission enhancing properties afforded through the combination of formal transposition with methylation is in contrast to previously reported methyl ethers of seminaphthofluorescein compounds lacking such enhancement.^9b Interestingly, methylation of 4b results in only 4d (exhaustive attempts to synthesize the corresponding isomer of 4d, where the substituents on C-1 and C-10 are interchanged were unsuccessful, see Scheme S1). The quantum yield of 4d is ~1.5 times greater than the short wavelength emitting methyl ether of carboxy SNAFL-1 in which the C-3 oxygen was methylated, and ~15 times greater than the analogous red-emitting C-10 methyl ether.^9b Thus, methylation of 4b affords pH independent and relatively brighter red fluorescence compared to known seminaphthofluorescein probes.

Figure 8.

Fluorescence emission spectra of 3b, 3c and methyl ether 4d in 10:90 DMSO:aqueous. Solutions were prepared such that the absorbances at the 500 nm excitation wavelength were equal for all solutions. Inset: Fluorescence emission in a darkened room of 7.5 μM solutions (3b, 4d, and 3c from left to right) excited from below with 3-watt megaMAX 505 nm ALS system. Neutral compound 3b is in pH 6 phosphate buffer, 12.5 mM while anion 3c is in pH 12 NaOH. Compound 4d is in pH 7.4 phosphate buffer, 12.5 mM.

CONCLUSIONS

In summary, the results described herein demonstrate that xanthene dye absorption and emission can be shifted to the NIR via relatively simple structural (e.g., regioisomeric) changes involving field effects or low barrier hydrogen bonds, depending on the ionization of the chromphore. This design principle, involving the environment of the bridging ether oxygen, apparently allows for favorable modulation of the HOMO-LUMO gap. Large Stokes shifts are observed as well as an enhancement in quantum yield and brightness as compared to analogous known long wavelength emitting dyes. Ongoing efforts in our laboratory include the study of related field effects on rhodols, rhodamines, and other xanthenes, as well as other chromophores.

EXPERIMENTAL

Chemistry

Unless otherwise indicated, all commercially available starting materials were used directly without further purification. Silica gel Sorbent Technologies 32–63 μm was used for flash column chromatography. ¹H- and ¹³C NMR were obtained on either a ARX-400 or ARX 600 Advance Bruker spectrometer. Chemical shifts (δ) are given in ppm relative to DMSO-d₆ (2.50 ppm, ¹H, 39.52 ¹³C) unless otherwise indicated. MS (HRMS, ESI) spectra were obtained at the PSU Bioanalytical Mass Spectrometry Facility on a ThermoElectron LTQ-Orbitrap high resolution mass spectrometer with a dedicated Accela HPLC system. 1,8-dihydroxynaphthalene and 2-(2,4-dihydroxybenzoyl)-benzoic acid were synthesized as described in the literature.^16,9a,17

Crystallographic data for compound 4a

C₂₄H₁₄O₅, monoclinic space group C2/c, a = 19.5457(15), b = 12.2406(10), c = 15.4763(14) Å, β = 114.233(5)°, V = 3376.5(5) ų, Z = 8, ρ_calcd= 1.504 g cm⁻³, μ(CuKα) = 0.87 mm⁻¹, R = 0.033 for 2667 data with Fo²>2σ(Fo²) of 3070 unique data and 269 refined parameters. A total of 13,499 data (R_int = 0.031) was collected at T = 90.0(5) K to θ = 68.9° with CuKα radiation (λ = 1.54178 Å) on a Bruker Kappa Apex II diffractometer, using an amber crystal of dimensions 0.11 × 0.13 × 0.17 mm. Refinement was by full-matrix least squares using SHELXL, with H atoms in idealized positions, except for OH hydrogen atoms, which were located by difference maps and their positions refined.

UV-vis and Fluorescence measurements

Absorbance spectra were collected on a Cary 50 UV-Vis spectrophotometer. Fluorescence spectra were collected on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies). All absorbance spectra were reference corrected. Fluorescence spectra were corrected for the wavelength dependent response of the R928 photomultiplier tube using a manufacturer generated correction file. Quantum yields are reported as the average of multiple measurements using multiple references. Red emitting probes were referenced to rhodamine 101 (0.913)³⁷ and sulforhodamine 101 (0.900)³⁸ in EtOH. NIR emitting probes were referenced to Nile Blue (0.27)³⁹, IR-125 (0.132)³⁷, and HEDITCP (0.195)³⁷ in EtOH. HEDITCP was prepared as reported by Descalzo.⁴⁰ Concentrations were adjusted such that absorbance values were ~0.05 or lower. Differences in refractive index between sample and solution were accounted for.

Molecular Modeling

molecular orbital calculations were performed using the Density functional theory (DFT) at B3LYP/6-31G level using Gaussian 03W.

ABBREVIATIONS

DMF

dimethyl formamide

DMSO

dimethylsulfoxide

DSSCs

dye-sensitized solar cells

ESI

electrospray ionization

FDA

Food and Drug Administration

HEDITCP

1,10,3,3,30,30-hexamethyl-3,5-ethylene-4-(dimethylamino)-2,20-indotricarbocyanine perchlorate

HelMe

1,13-dimethoxy-5,9-dimethyl-5,9-dihydroquinolino[2,3,4-kl]acridin-13b-ylium

HPDITCP

1,10,3,3,30,30-hexamethyl-3,5-propylene-4-(dimethylamino)-2,20-indotricarbocyanine perchlorate

HOMO

highest occupied molecular orbital

HRMS

high resolution mass spectrometry

ICG

indocyanine green

LBHB

low-barrier hydrogen bond

LUMO

lowest unoccupied molecular orbital

NIR

near-infrared

SNAFL

seminaphthofluorescein

TFA

trifluoroacetic acid