Luminescent and Chiroptical Properties of 1:1 Eu (III) : Tetracycline Species Probed by Circularly Polarized Luminescence

Abstract

This study investigates the significantly different luminescent and chiroptical properties of tetracycline (TC) when coordinated to Eu(III). The approach involves understanding the 1) speciation of TC and 2) conformation and species formed between Eu(III) and TC in a ratio of 1:1 in a dimethylformamide (DMF) solution and as a function of the pH value. By identifying the conformational changes of the various 1:1 Eu(III) :TC species, the results from this study explain information on the local microenvironment about the Eu(III) metal center. In particular, ⁵D₀←⁷F₀ Eu(III) laser excitation spectroscopy was employed to distinguish the different types of species found in solution in order to understand the interaction between Eu(III) and TC. On the other hand, circularly polarized luminescence (CPL) spectroscopy was used to understand the structural changes within the 1:1 Eu(III) :TC complex that could be related to the chirality of the Eu(III)-containing species. The CPL spectrum serves as a “fingerprint” to indicate the conformational changes within the 1:1 Eu(III) :TC complex as a result of the chiroptical signal arising from the various Eu(III) :TC species.

1. Introduction

The phenomenon of chirality and spatial anisotropy plays a fundamental role in regulating the interactions of metal ions with biologically active molecules, and as such, great emphasis has been placed in the development of sensitive analytical techniques able to distinguish the structural biology of these substances. For this reason, advancements have been made related to creating smart imaging probes that are truly quantitative, highly sensitive, and readily understood.^[1]

Biomolecular design of fluorescent probes offers a practical method towards addressing fundamental bioinorganic questions at a molecular level.^[2] In particular, a better knowledge of the metal complex reactivity facilitates the understanding of biomolecular phenomenon observed and, also, the design of new molecular systems with anticipated functional properties. The availability of a variety of metal ions and ligands allows the construction of such biomolecular systems with specific reactive or structural roles in mind.^[3]

One such spectroscopic method utilizes lanthanide(III), Ln (III), ions for their ability to luminesce and form highly coordinated compounds possessing chiral properties. This technique involves the formation of a chiral structure due to the arrangement of the chiral biomolecules (or ligands) surrounding the luminescent Ln(III) center and studying the light emitted from the resulting complex. Whereas normal chiral molecules rotate linearly polarized light, these complexes emit light that is circularly polarized, meaning the passing electromagnetic wave changes direction in a rotary manner within the electric field.^[4] The advantage of this phenomenon has been exploited using various Ln(III) metals including Pr(III), Dy(III), Yb(III), Eu(III), or Tb(III); however, most spectroscopic techniques utilize the most emissive Ln(III) ions, europium (Eu) and terbium (Tb), because each Ln(III) metal emits light at distinct visible wavelengths (Eu emitting red and Tb emitting green), which can be used to elucidate the chemical environment and chiral structure of the Ln(III)-containing compound.^[5,6]

In practice, lanthanides form stable trivalent ions without any known essential role in biology, thus by embedding the Ln (III) ion with a bioactive molecule, a rigid and extended molecular framework can be defined as a three-dimensional structured^[7] The coordination of various carbonyl and amide-containing ligands or solvent molecules provides a microenvironment to the Ln(III) metal ion, in which the luminescence is sensitive to the structural conformation of its surrounding environment.^[8] This characteristic has been exploited in various biological studies for the (a) development of a lanthanide-complex molecular beacon used as an allosteric effector to allow for the split-probing of specific genes,^[8] (b) use of lanthanide-binding peptides with DNA intercalating units for the detection of double-stranded DNA,^[9] (c) sensing of phosphate or other anions to assess the body’s energy source, ATP, to understand how it causes ischemia, Parkinson’s disease, and hyphoglycaemia,^[10] (d) conformational changes in calcium and iron binding proteins to assess their binding to the active site, coordination sphere of the metal, and distinct classes of binding sites,^[11] or (e) chiral discrimination of bioactive molecules to recognize their interactions with Ln(III) complexes.^[12]

It is noteworthy to mention that the choice of the complementary spectroscopic techniques is dependent on the type and complexity of the metal-containing system under investigation. In addition to the ESI-MS spectrometry, NMR spectroscopy is often used to elucidate the presence of various species as a function of, e.g., ligand, metal, temperature, pressure, or pH. However, it is common that the NMR spectrum that results is often convoluted and difficult to interpret without additional methods to further identify the concentration variation of the different species in solution, especially in the presence of paramagnetic metal centers. That is the reason why a preference was made for the use of an optical method, the circularly polarized luminescence (CPL) spectroscopy.^[4] CPL has the capability of enabling a direct and distinct visualization of the microenvironment changes altering the Eu(III) luminescence properties. Unlike the circular dichroism spectroscopy (CD), CPL is only dependent on the active CPL species and free of potentially interfering background signals. However, the information gained from CD is often from all chromophoric species present in solution without being specific to one target. On the other hand, the use of CD has led to the development of modified techniques including the spectroscopic approach of comparing observed and calculated vibrational circular dichroism (VCD), exciton chirality, Raman optical activity (ROA), or optical rotation values.^[13] It is worth noting that each technique may have their own limitations (e.g., time consuming, large sample preparation, high cost, limited sensitivity…). For instance, Abbate et al., among other CPL experts,^[4] stressed out the importance of the experimental aspects as well as the potential density functional theory-based theoretical and computational approaches potentially available to help with a better interpretation of CPL signals.^[14] It is of interest to note that Polavaraplu commented on the importance of the use of several optical tools to obtain chiral molecular structural information (e.g., independent verification since single methods may only provide partial or ambiguous information…).^[15] More recently, Wu et al. demonstrated the use of ROA has a suitable technique to probe, e.g., secondary and tertiary structures of peptides and proteins through the binding of lanthanide(III) complexes used as spectroscopic probes.^[16a] Another of their studies concerned the chiral sensing of amino acids and proteins leading to the detection of very weak CPL-active signals from the systems investigated, thus demonstrating the relevance of the ROA tool.^[16b]

Inspired with the importance of using Eu(III) CPL spectroscopy to investigate the metal-ion environments and the associated chiral structures of metal-containing biological systems,^[4,6] we hereby report as a starting point of this project on the species formed by tetracycline (TC) and its interaction with the metal ion (Eu(III)), solvent (DMF), and pH was envisaged with an emphasis on the luminescent and chiroptical properties. One important feature for the CPL spectroscopy was to show that one could understand the relationships between the chiral structures of biomolecules (such as proteins or drugs) and their ability to bind metal ions.^[4,6] It is worth noting that the use of TC as our ligand molecule coordinated to Eu(III) was a deliberated choice to demonstrate that the “fingerprint” CPL signal can be matched with the multiple binding conformations that may exist in vivo. Indeed, the observed CPL signal is dependent of the TC conformation and, then, would allow one to investigate the binding interaction between the Eu:TC-like probe and targeted biomolecular systems. As such, it is important to primarily demonstrate the potential of CPL in the current study using well-known systems (e.g., TC) prior to applying it to unknown biological problems related to human health when one is interested in developing a new methodology.

2. Results and Discussion

We investigated TC-like Eu(III) complexes as potential chiral luminescent probes.^[17] TC is a commonly used antibiotic that prevents the growth and spread of bacterial infections, treats acne breakouts, and most recently rosacea. In fact, the TC class of antibiotics has achieved importance beyond their therapeutic use as broad-spectrum antibiotics because many metal complexes utilizing TC have been used as luminescent probes for the investigation of biomolecular interactions.^[18] However, the chemical-structural properties of TC are under extensive study. It has been shown that the existence of the different conformations of TC depend on the experimental conditions used (e.g., substituents, metal chelation, and solvent).^[19]

In particular, TC adopts a twisted conformation in acidic to neutral conditions. It can be seen that the dimethylamino group lies above the BCD ring system in order to relieve the steric crowding between the protonated nitrogen of the dimethylamino group and its neighboring hydroxyl group (Scheme 1). On the other hand, a shift of the equilibrium to the extended conformation is observed when basic and nonaqueous solution conditions are used.^[3a] In this extended conformation, the dimethylamino group lies below the plane of the BCD ring system. TC also exhibits three macroscopic acidity constants (pK₁~3–4, pK₂~7.3–8.1, and pK₃~8.8–9.8) allowing its chemical structure to dictate its conformation in solution as seen in Scheme 1. Several studies using NMR, CD, absorption and fluorescence spectroscopy, as well as electroanalytical methods, were devoted to the investigation on the relatively complicated decomplexation pattern of TC and its numerous possible chelation sites. These studies were conducted using various metal ions in aqueous and organic media.^[3a] The structure of TC defines the complexation behavior with metal ions that, ultimately, determines largely their biological action and pharmacokinetic properties. Clearly, the physical-chemical properties and associated functionality can be affected by various factors ranging from the solvent, pH, and buffer’s ionic strength to metal:ligand ratio. It is worth noting that various metal:TC stoichiometries can be observed due to the presence of numerous possible chelation sites of TC.

Scheme 1.

Deprotonation scheme (bottom) and conformation change (top) of TC.

Thus, a complete study of the species formed by TC and its interaction with the metal ion (Eu(III)), solvent (DMF), and pH was conducted with an emphasis on the luminescent and chiroptical properties. In this work, we report the use of TC coordinated to Eu(III) in a 1:1 ratio with the understanding that the ligand of interest is coordinated based on its several protonation sites and complexation behavior of the BCD ring system (Scheme 1).

2.1. Steady-State and Time-Resolved Luminescence

The photophysical characterization of the 1:1 Eu(III) :TC complexes over a broad range of pH values was completed using steady-state and time-resolved luminescence. Utilizing this type of spectroscopy allows for the study of the complexation of the lanthanide ion, Eu(III), and the ligand, TC, in order to examine the absorption of light by the ligand and the energy transfer to the lanthanide(III) ion.

Under the study of steady-state luminescence, excitation and emission scans were performed to see the transition experienced by TC from its free-uncoordinated to coordinated forms within the 1:1 Eu(III) :TC species. The excitation spectrum of the uncomplexed TC in DMF solution at 295 K displays one broad band defined by a maximum around 439.0–445.0 nm as well as a much weaker broad band in the range of 264.0–290.0 nm. A slight bathochromic shift is observed for these two distinct broad bands as pH increases from 2.0 to 10.7 (see left top of Figure 1). The effects of pH on the emission spectrum of TC are more pronounced in comparison to the excitation spectrum. The former spectra showed a larger blue-shift of the band maximum around 520.0-575.0 nm when the pH increased from 2.0 to 10.7 and irradiated in the range of 264.0-290.0 nm (see right top of Figure 1).

Figure 1.

Steady-state excitation (left) and emission (right) spectra of 0.01 M TC (top, λ_em = 520.0-575.0 nm, λ_exc = 264.0-290.0 nm) and 1:1 Eu(III) :TC (bottom) in DMF at 295 K (λ_em, = 617.0 nm, λ_exc = 287.0-295.0 nm).

Upon complexation to Eu(III), the excitation and emission profiles of the steady-state luminescence spectra vary over each change in pH (see bottom of Figure 1). When the pH increases, there is a visible decrease in the peak width of the broad band around 439.0–445.0 nm in the excitation spectra, suggesting an increase in the Eu(III) :TC complexation and the presence of various 1:1 Eu(III) :TC species formed (see left bottom of Figure 1). Moreover, the excitation spectra also show a new band centered around 390.0 nm appearing, whereas the much weaker broad band in the range of 264.0–290.0 nm in the free-uncoordinated TC significantly increases in peak width. In summary, at low pH (acidic conditions), TC complexation to Eu(III) is not favored; however, subsequent deprotonation of TC (basic conditions) favors complexation with Eu(III), allowing for the change in the profile of the bands present in the excitation spectra, and resulting from the formation of the various 1:1 Eu(III) :TC species. This phenomenon is clearly corroborated by the emission profile of the steady-state luminescence spectra (see right bottom of Figure 1). The latter ones illustrate a clear increase in intensity (or peak area to be more correct) of the emitted light from the 1:1 Eu(III) :TC species and originating from the transitions ⁵D₀→⁷F₂, ⁵D₀→⁷F₂ ⁵D₀→⁷F₃ and ⁵D₀→⁷F₄ of the Eu(III) ion at their corresponding wavelengths of 590.0, 615.0, 650.0, and 690.0 nm, respectively. As shown in Figure 1 (right bottom), the peak observed around 264.0–290.0 nm is the Rayleigh of the wavelength to which each solution was irradiated with.

At pH 2.0, the characteristic emission bands of Eu(III) luminescence at 615.0 and 690.0 nm are barely visible, but cannot be distinguished from the broad emission band of TC. At pH 4.0, the ⁵D₀→⁷F₂ and ⁵D₀→⁷F₄ transitions of Eu(III) are expressed and visible along with the broad emission peak of TC, indicative that the first deprotonation of TC has occurred and complexation with Eu(III) can occur. Upon complexation to Eu(III), the broad fluorescence band of uncomplexed TC is red-shifted, confirming the complexation of TC to Eu(III). From pH 6.0 to 10.7, the broad TC-centered emission band is eliminated, and only the emission bands of Eu(III) from the various 1:1 Eu(III) :TC species at its characteristic peaks of 590.0, 615.0, 650.0, and 690.0 nm are observed. These four characteristic emission bands increase in peak height as the pH of the solution increases. This change in the Eu(III)-centered emission-band-peak areas suggests that, as the pH increases, the deprotonation of the TC ligand favors its complexation to the Eu(III) ion, thus the energy absorbed from the excitation of the ligand begins to steadily be transferred to the lanthanide(III) ion. This results in the change of the broad TC-centered emission band to the sharp-line emission bands characteristic of the luminescence of Eu(III).

Under the study of time-resolved luminescence, excitation and emission scans were performed to see the transition experienced by TC within the various 1:1 Eu(III) :TC species formed at room temperature. As observed in Figure 2 (left), the time-resolved luminescence excitation spectra exhibit a similar pH-dependent profile to the steady-state luminescence excitation one (see left bottom of Figure 1) upon increasing the pH from 2.0 to 10.7. The changes observed in the excitation profile imply the progressive coordination of TC to Eu(III) in various 1:1 Eu(III) :TC species, resulting from the successive deprotonation of TC as the pH increases. This is because TC has several possible metal-binding sites, depending on the pH, to interact with the Eu(III) ion present in solution. On the other hand, the formation of only one 1 :1 Eu(III) :TC species would have been supported by an excitation profile independent of the pH changes once the species was formed.

Figure 2.

Time-resolved excitation (left) and emission (right) spectra of 0.01 M 1:1 Eu(III) :TC in DMF at 295 K and recorded with a time delay of 0.1 ms (λ_em = 617.0 nm, λ_exc = 285.0-296.0 nm).

It must be noted that the emission profile of the time-resolved luminescence spectra also illustrates a clear increase in intensity (or peak area to be more correct) of the emitted light from the 1:1 Eu(III) :TC species and originating from the transitions ⁵D₀→⁷F₁, ⁵D₀→⁷F₂, ⁵D₀→⁷F₃, and ⁵D₀→⁷F₄ of the Eu(III) ion at their corresponding wavelengths of 590.0, 615.0, 650.0, and 690.0 nm, respectively (see right of Figure 2). As the pH of the solution increases, the Eu(III) luminescence bands become predominant, and each transition peak enlarges to suggest that, the ligand to metal energy transfer increases and light is emitted showing an increase in the Eu(III) luminescence. This is a consequence of the fact that the complexation is favored under basic conditions. In addition, the conformational change induced upon the third deprotonation (pK₃ ~ 8.8—9.8) and the orientation of the multiple chelation sites on TC alters the photophysical properties of all 1:1 Eu(III) :TC species found in solution.

2.2. ⁵D₀←⁷F₀ Eu(III) Laser Excitation Spectroscopy

An important feature is the use of the unique excitation transition of Eu(III) from the non-degenerate ⁷F₀ ground level to the non-degenerate ⁵D₀ emissive level that can be monitored by observing the usually strong emissive transition ⁵D₀→⁷F₂ at around 612.0–615.0 nm. This former transition appears as a single sharp line and is very sensitive to small differences in the metal ion-ligand environment. It is often the case that a single resolvable peak is observed for each Eu(III)-containing species present if they can be distinguished from each other on the luminescence time scale.^[20] Additionally, the use of the dependence of the nephelauxetic effect, a well-known concept developed by Choppin et al. and deW. Horrocks et al.,^[21] may potentially lead to a deeper understanding of the interaction between TC and Eu(III). However, such an interpretation is not trivial, as other significant factors need to be taken into account. Wagner et al. highlighted the complexity of such an interpretation when they demonstrated that the proposed equation failed for Eu(III) complexes with heterocyclic N-donor ligands (the number of coordinating donors was highly overestimated).^[21a] The variation observed was the consequence of a significantly larger share of covalence in the Eu(III)-N-donor bonds compared to hard oxygen donor ligands previously used. As a result, Wagner et al. proposed the development of a different equation correlating the bathochromic shift and the complex stoichiometry of Eu(III) N-donor systems.

Due to the complexity of the applicability of the nephelauxetic effect and to avoid misinterpretation, the speciation of the 1:1 Eu(III) :TC species over a broad range of pH values was only completed using the ⁵D₀←⁷F₀ Eu(III) laser excitation spectroscopy. Using this type of spectroscopy allows for the study of how the subsequent deprotonation of TC changes its speciation in solution and ultimately, its complexation with Eu(III) ion.

As Figure 3 illustrates, there is an apparent change within the observed spectra as the pH of the solution increases. This observation supports the idea that the speciation of TC changes with respect to its complexation to Eu(III) ion as its conformation alters at the various observed pHs.

Figure 3.

⁵D₀←⁷F₀ Eu(III) excitation spectra of 0.01 M 1:1 Eu(III) :TC in DMF at 295 K and upon monitoring the Eu(III) luminescence at 615.0 nm.

At pH 2.0, there is one visible peak corresponding to the predominant Eu(III) :TC species (λ_exc = 579.0 nm) in solution since TCH⁺ species of TC lacks the ability to complex with the Eu(III) ion. At pH 3.0 to 4.0, there is a slight shoulder expanding from the right side of this peak, which indicates the presence of another species (λ_exc = 579.3–579.6 nm), Eu:TC⁻, within the solution. Since both Eu(III) :TC and Eu(III) :TC⁻ species complex to the twisted conformation of TC, this resolves as one major peak at 579.0 nm in the spectra that broadens to two at 579.3 to 579.6 nm as the pH of the solution increases. At pH 5.0 to 10.7, the peak height (or peak area to be more correct) of the perceived peak decreases as a new peak for the Eu(III) :TC²⁻ species (λ_exc = 580.2 nm) is introduced, indicating the continual transformation of TC in solution.

To understand the complete transition of the speciation of TC, an in-depth assessment of the conversion of this ligand to lanthanide complex was performed. Indeed, the key point of the data analysis is the determination of the concentration of each species present in the equilibrium solution studied. For this purpose, the ⁵D₀←⁷F₀ Eu(III) excitation spectra were analyzed using the deconvolution software Jandel Peak Fit. The setup of such a curve fit analysis has already been published in previous work,^[22] where it was shown that the total concentration of each Eu(III) species is related to the area of the peak associated with the transition. Thus, we can fit the ⁵D₀←⁷F₀ Eu (III) laser excitation spectra plotted in Figure 3 to a sum of two or three pure Lorentzian peaks, as appropriate, and relate the concentration of each species to the corresponding peak area as a function of the pH. As in previous work,^[22] all peaks were assumed to be pure Lorentzian in shape, so only the peak position and Lorentzian width were varied (see experimental section for further details). The curve fit analysis of the observed spectra, as one shown in Figure 4, allowed for the separation of the overlapping peaks to determine the percent area of each peak (or concentration) corresponding to a different conformation of the 1:1 Eu(III) :TC species present in solution.

Figure 4.

⁵D₀←⁷F₀ Eu(III) excitation spectrum of 0.01 M 1:1 Eu(III) :TC in DMF for a selected pH of 4.0 at 295 K (see Figure 3). (A) The blue dashed line is the resulting spectrum from the curve fitting of the experimental spectrum (black solid line). (B) The gray, green, and cyan dashed lines in this plot are the results of curve fitting the experimental spectrum (black solid line in (A)) to a sum of three Lorentzian peaks.

The plots in Figure 5 are the results of curve fitting the ⁵D₀←⁷F₀ Eu(III) excitation spectra to a sum of two or three pure Lorentzian peaks corresponding to the presence of the different 1:1 Eu(III) :TC species at various pHs ranging from 2.0 to 10.7. As Figure 5 illustrates, the transformation of each pertaining conformation of TC, with respect to its complexation to Eu(III), shows an ideal conversion as predicted by the deprotonation scheme and the conformational change of TC (see Scheme 1). From Figure 5, the conformational change TC undergoes at the amino-deprotonation from the twisted to extended form can clearly be seen as there is an abrupt increase and decrease shown from Peak 2 and Peak 3 corresponding to the structural changes of TC⁻ to TC²⁻ (pK₃ ~ 8.8–9.8, see Scheme 1). The data from the ⁵D₀←⁷F₀ Eu(III) excitation spectra plainly propose the idea that the conversion of Eu(III) :TC complex into different species and conformations does occur as the pH of the solution is changed. Since there is variability in the conformation of TC in solution, Eu(III) can bind at different positions to TC, as the deprotonation and conformation change of TC play an important factor in defining the coordinating groups to Eu(III).

Figure 5.

Plot of %Area of Eu(III) species present in solution versus pH as determined from curve fit analysis of the ⁵D₀←⁷F₀ Eu(III) excitation spectra of 0.01 M 1:1 Eu(III) :TC in DMF at 295 K and upon monitoring the luminescence at 617.0 nm (see Figure 3).

2.3. Circularly Polarized Luminescence

The chiroptical characterization of 1 :1 Eu(III) :TC complexes over a broad range of pH values was investigated using circularly polarized luminescence (CPL). CPL has become an increasingly useful tool as a probe for luminescent Ln(III) complexes in fields like biochemistry, biology, medicine, and related biomedical disciplines, where recognizing the chirality associated with biological and synthetic chemicals is crucial for understanding their specific activity and functionality.^[4,6] Using CPL, researchers are able to gain useful information regarding the metal-ion environments and the associated chiral structures of metal-containing biological systems. The degree of CPL is commonly reported in terms of the luminescence dissymmetry factor, g_lum (λ) = 2ΔI/I = 2(I_L − I_R)/(I_L + I_R), where I_L and I_R refer to the intensity of left and right circularly polarized light of well-resolved f-f emission lines and each line splitting into several Stark levels.^{[4,6, 23]} The spectrum of CPL measures a g_lum value for each wavelength, and is reported as change in intensity (ΔI) versus the wavelength. A combination of positive and negative CPL signs ensures the splitting of narrow emission lines of the Ln(III) ions which provide unique chiroptical properties that can be used to probe for target proteins and biomolecules.^[23a] Thus, the CPL activity serves as a “fingerprint” to indicate any structural changes within the Ln(III)-containing system and/or around the local environment of the Ln(III) metal.

Utilizing this type of spectroscopy allows for the understanding of how the conformational speciation of TC within its complexation to Eu(III) ion reflects the chiral structural changes with the emitting lanthanide(III) metal. The study of the microenvironment about the lanthanide(III) ion using CPL gives insight to understanding the chirality associated to metal-containing compounds. Being able to analyze and understand the binding interactions around the local environment of the lanthanide(III) metal or complex will allow us to use 1 :1 Eu(III) :TC as a luminescent molecular probe that can be designed in diverse solutions at targeted pH values for the chiral recognition/sensing of biomolecules.

It must be emphasized that the use of a selective excitation for getting individual emission currently presents challenges.^[3,20] Unlike the ⁵D₀→⁷F₁ emission transition (seen at −592.0 nm on Figure 6), the use of the species-selective ⁵D₀←⁷F₀ excitation transition (seen at −579.0–580.0 nm on Figure 3) does not meet the requirement of the Laporte selection rules (⁵D₀→⁷F₁ is the only emission transition of Eu(III) that meets the appropriate selection rules) and the small gap between these two transitions would require the use of very sharp and accurate optical filters. However, the cut-off filters available have a bandwidth range that will also cut the light emitted by the sample, thus tampering the results. The absence of such filters would allow the detection of scattered light from the laser absorption in the emission. Therefore, the experimental CPL signal detected would result from the circular polarization light, but mainly from the laser’s linear polarized light. Thus, the outcome of this choice would not be conclusive in an attempt to ameliorate the measure of CPL and was not pursued in this work. However, the use of the ⁵D₁←⁷F₀ transition at about nm in absorption and the ⁵D₀→⁷F₁ transition in emission (at −592.0 nm), as both are magnetic-dipole allowed transitions, will certainly considerably enhance the structural information gained of targeted systems.^[20d] Nevertheless, the feasibility of using the CPL spectroscopy as currently shown in this work is already a proof of concept of the sensitivity and flexibility of this tool (observation of “fingerprint” CPL signals related to the microenvironment changes around the Eu(III) center as a function of the pH and as shown in Figures 6 and 7).

Figure 6.

CPL (top) and total luminescence spectra (bottom) of the ⁵D₀→⁷F₁ transition of 0.01 M 1:1 Eu(III) :TC species in DMF at 295 K and upon excitation at 414.0-422.0 nm.

Figure 7.

CPL (top) and total luminescence spectra (bottom) of the ⁵D₀→⁷F₁ transition of 0.01 M 1:1 Eu(III) :TC species in DMF at pH 5.0 (solid lines), 7.0 (dashed lines), and 10.9 (dotted lines) at 295 K, following excitation at 424.0 nm.

Under the study of CPL (Figure 6), each total luminescence spectrum for the ⁵D₀→⁷F₁ transition shows two distinct peaks at 590.0 (at pH < 8.0) and 594.0 nm (at pH >8.0); however, at pH 8.8, the total luminescence spectrum shows mainly one peak centered at 592.0 nm which is indicative of the initial conversion of the twisted to extended conformation of TC (see middle bottom of Figure 6).

Furthermore, the CPL spectra show an increased variability of the proposed chiral structural changes of the 1:1 Eu(III) :TC species (see top of Figure 6). At pH <6.0, the major peak centered at 591.5 nm begins to decrease and broaden as the solution reaches the pH of the second deprotonation of TC (pK₂~ 7.3—8.1, see Scheme 1). At pH 7.0, there are two prominent peaks at 591.5 and 597.0 nm; however at pH 8.0, there is an inversion of these two peaks as the peak centered at 597.0 nm begins to increase in height (or peak area to be more correct). This peak reaches its maximum height at pH 8.8, which is the pK_a of the third deprotonation of TC (pK₃ ~8.8–9.8, see Scheme 1). As the pH of the solution is increased, the peak height of this peak decreases steadily and a mixture of multiple peaks to the left of 597.0 nm appears. The chirality changes associated with TC at its third deprotonation suggest that the energy being emitted from the local microenvironment about the Eu(III) ion center is greatly altered by the presence of strong chelating groups and the conformational change of TC. Within this pH range, the deprotonation of TC forms a deprotonated amine group with lone pair electrons that can act as a donating group to coordinate to the Eu(III) ion, while the conformational change allows the TC molecule to extend along the Eu(III), thus forging new coordinations to be made between the ligand and metal. As the pH of the solution increases past 10.0, the CPL signal strengthens at two peaks centered at 592.2 and 596.0 nm. Thus, the coordination of each TC species at targeted pH values provides a unique microenvironment to the Eu(III) ion, in which the luminescence is sensitive to the structural conformation of its surrounding environment, causing the polarization of the emitted light to serve as a “fingerprint” to indicate any structural changes to the chiroptical properties of each 1:1 Eu(III) :TC species present in solution.

Using the information received from the ⁵D₀←⁷F₀ Eu(III) excitation spectra, we can predict that one Eu(III) :TC species may have the obtained CPL spectrum for any given pH, since each CPL spectrum is a signature of one 1 :1 Eu(III) :TC species or a transitional phase of different species present in solution. This is illustrated in Figure 7 where the CPL spectra of the ⁵D₀→⁷F₁ transition of 0.01 M 1 :1 Eu(III) :TC species in DMF at pHs of 5.0, 7.0, and 10.9 are plotted. The evolution of the total luminescence and CPL spectra as the pH increases from 5.0 to 10.9 can be clearly evidenced, corroborating a change in the speciation of the TC forms present at various pHs and thus complexing Eu(III). The g_lum values amount to −0.062, −0.020, +0.019, and +0.003, −0.018, −0.065 nm at about the two maximum emission wavelengths for the pHs of 5.0, 7.0, and 10.9, respectively. From the ⁵D₀←⁷F₀ Eu(III) excitation spectra shown on Figure 3, it can be correlated that the CPL spectrum of the 0.01 M 1 :1 Eu(III) :TC species in DMF at pH of 5.0 is the result of the predominance of Eu(III) :TC in solution. On the other hand, Eu(III) :TC²⁻ is the main species responsible for the CPL spectrum at a pH of 10.9. It must be pointed out that the CPL spectrum measured for the aforementioned 0.01 M 1:1 Eu(III) :TC complex solution in DMF at pH 7.0 clearly shows the transition TC is undergoing as the pH increases from 5.0 to 10.9. It shows an “intermediate” CPL resulting from the presence in solution of both Eu(III) :TC and Eu(III) :TC⁻ species at pH 7.0. Upon increasing the pH from 7.0 to 10.9, the CPL spectra features evolve with a gradual increase of the TC forms coordinated to Eu(III) from TC to TC⁻ and finally TC²⁻ (see Figure 6).

3. Conclusion

The preliminary findings indicated that (a) uncomplexed TC fluorescence increased in the course of deprotonation and decreased upon metal ion chelation, (b) TC speciation with Eu(III) in a ratio of 1:1 gave rise to three different Eu(III)-TC species in DMF at various pHs, and (c) multiple chelation sites and conformational change of TC altered the luminescence of all Eu(III)-containing species. We also applied the CPL spectroscopy to understand how the conformational speciation of TC reflects the chiral structural changes with the emitting lanthanide(III) cation, Eu(III). It was observed that the polarization of emitted light served as a “fingerprint” to indicate any structural changes to the chiroptical properties of each 1:1 Eu(III)-TC species in solution. Research in this direction is currently underway.

In summary, the applications of CPL outlined above and involving carefully constructed chiral luminescent complexes designed to probe specific aspects of biomolecular structure appeared to be a fruitful area of research.^[4,9] These results support our proposed goal to demonstrate the utility of CPL to provide unique information concerning the chiral structure or structural changes to a range of molecular and biomolecular systems (e.g., an effective and reliable analytical tool to investigate biomolecules such as amino acids and/or the speciation of luminescent chiral Ln(III) complexes developed for use as potential chiral CPL probes in biomedical applications).

Experimental Section

Materials and Methods

All reagents and solvents were commercially available and used as received. The Eu(III) content of stock solutions was determined by titrations with a standardized solution of EDTA in the presence of 0.1 M ammonium acetate and aqueous arsenazo(III). All spectroscopic measurements were done in DMF solutions. The final concentration for Eu(III) :TC complex solutions were 0.01 M with a 1:1 ratio of Eu(III) :TC. These Eu(III) :TC complex solutions were prepared in situ from stock solutions of Eu(III) and TC in DMF. The pH of the solutions was adjusted with aliquot amounts of 0.01 M HCl or trimethylamine accordingly. The pH range was reached using the respective aliquot amounts of HCl and trimethylamine. The solutions were then allowed to sit stirring for a 24 h period. The pH was tested again before running all spectroscopic measurements to ensure the pH was stabilized within the range desired.^[20]

Circularly polarized luminescence (CPL) and total luminescence spectra were recorded on an instrumentation described previously.^[20] In short, the instrumentation is equipped with a 1000 W xenon arc lamp from a Spex FluoroLog-2 spectrofluorometer, with excitation and emission monochromators of dispersions 4 nm/mm (SPEX, 1681B). The maximum excitation wavelength was determined by running an excitation scan monitoring at λ_em = 615.0 nm and corresponding to the ⁵D₀←⁷F₂ (Eu) transition. The maximum emission wavelength was then determined by running an emission scan at the maximum λ_exc. CPL spectra were measured at the maximum λ_exc value, with λ_em ranging from 582.0 to 605.0 nm. ⁵D₀←⁷F₀ (Eu) laser excitation measurements for 0.01 M 1:1 Eu(III) :TC complex solutions in DMF at 295 K were measured using a Coherent-599 tunable dye laser (0.03 nm resolution) with a Coherent Innova Sabre TMS 15 as a pump source. The laser dye used in the measurements was rhodamine 6G dissolved in ethylene glycol. The calibration of the emission monochromator (and subsequently the dye laser wavelength) was performed by passing scattered light from a low power He–Ne laser through the detection system. The error in the dye-laser wavelength is assumed to correspond to the resolution of the emission monochromator (0.1 nm). The optical detection system consisted of a focusing lens long-pass filter and 0.22 m monochromator. A cooled EMI-9558B photomultiplier tube operating in photon-counting mode detected the emitted light. All measurements were performed in a quartz cuvette with a path length of 1.0 cm. ⁵D₀←⁷F₀ (Eu) laser excitation spectra were analyzed using the deconvolution software Jandel Peak Fit. As in previous work,^[22] the ⁵D₀←⁷F₀ (Eu) laser excitation spectra plotted in Figure 3 were fitted to a sum of two or three Lorentzian peaks as appropriate. All peaks were assumed to be pure Lorentzian in shape, so only the peak position and Lorentzian width were varied. For the case where there are two Eu(III) species (α and β) in equilibrium, the equilibrium constant, K_eq, may be expressed as depicted below. Therefore, the total concentration of each species will be related to the area, A, of the peak associated with the transition, where k is a proportionality factor. It must be noted that it is assumed that the proportionality constant, k, are independent of pressure (or temperature).^[22] A similar procedure was used when three Eu(III) species were in equilibrium instead of two [Eqs. (1)-(3)].

$$\text{Eu}(\alpha )\rightleftarrows \text{Eu}(\beta )$$ (1)

$$\begin{gathered} C_{\alpha }=k_{\alpha }{A}_{\alpha } \\ \text{and} \\ {C}_{\beta }=k_{\beta }{A}_{\beta } \end{gathered}$$ (2)

$$K_{e{q}^{\prime }}=\frac{C_{\beta }}{C_{\alpha }}=\frac{k_{\beta }{A}_{\beta }}{k_{\alpha }{A}_{\alpha }}$$ (3)