# Lanthanide Luminescence Modulation by Cation-π Interaction in a Bioinspired Scaffold: Selective Detection of Copper(I)

Manon Isaac, Sergey A. Denisov, Amandine Roux, Daniel Imbert, Gediminas

Jonusauskas, Nathan Mc Clenaghan, Olivier Sénèque

► To cite this version:

Manon Isaac, Sergey A. Denisov, Amandine Roux, Daniel Imbert, Gediminas Jonusauskas, et al. "Lanthanide Luminescence Modulation by Cation-π Interaction in a Bioinspired Scaffold: Selective Detection of Copper(I). Angewandte Chemie International Edition, 2015, 54 (39), pp.11453-11456. 10.1002/anie.201505733. hal-01211550

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# Lanthanide Luminescence Modulation by Cation-π Interaction in a Bioinspired Scaffold: Selective Detection of Copper(I)

Manon Isaac, Sergey A. Denisov, Amandine Roux, Daniel Imbert, Gediminas Jonusauskas, Nathan D. McClenaghan,* and Olivier Sénèque*

**Abstract:** A prototype luminescent turn on probe for Cu⁺ (and Ag⁺) is described, harnessing a selective binding site (log K<sub>ass</sub> = 9.4 and 7.3 for Cu⁺ and Ag⁺, respectively) based on the coordinating environment of the bacterial metallo chaperone CusF, integrated with a terbium ion signaling moiety. Cation π interactions were shown to enhance tryptophan triplet population, which subsequently sensitized, on the microsecond timescale, the long lived terbium emission, offering a novel approach in bioinspired chemosensor design.

Copper is an essential element for life.[1] It is required for various biological processes and its homeostasis is finely regulated in living organisms.[2] Misregulation of copper can lead to various diseases (e.g., Menkes, Wilson, and Parkinson diseases).[3] To better understand the biology of copper, techniques are required to detect and quantify it, knowing that extracellular copper is in the + II oxidation state, whereas mobile copper is in the reduced + I state in cells. Generally, fluorescence detection is considered to be one of the cheapest and easiest techniques.[4] However, the design of fluorescent probes for Cu⁺ is more challenging than many other cations, such as Ca²⁺ or Zn²⁺, because Cu⁺ is an effective quencher of fluorescence through charge transfer and intersystem crossing (ISC) mechanisms.[5] As turn on emission is preferred for detecting an analyte, Cu⁺ selective fluorescent probes were designed in which the fluorophore is spatially disconnected from the chelate.[5,6] These probes rely on a photoinduced electron transfer (PET) mechanism in which the chelator, in its unbound form only, acts as an electron donor to the excited state of the fluorophore and quenches its emission.[5,6] In this communication, we report a new type of turn on Cu⁺ responsive probe based on a lanthanide ion (Ln³⁺) emitter, that has a long luminescence lifetime (in the millisecond range) compared to classical organic fluorophores (nano

<figure><img src="image_2.png" /><figcaption>**Figure 1.** A) Amino acid sequence of LCC1<sup>Tb</sup>, chelating moieties are underlined. B) Principle of the probe design based on the X ray structure of the Cu<sup>+</sup> binding loop of CusF.[14] C) Simplified Jablonski Perrin diagram of LCC1<sup>Tb</sup> probe and pertinent photophysical process es.</figcaption></figure>

second range) and that allows time gated detection to suppress background fluorescence contributions.[7–10]

Our probe structure (Figure 1A) is inspired by the metal binding site of the metallo chaperone CusF,[11] which is part of the CusCFBA system responsible for copper or silver detoxification in gram negative bacteria.[12] CusF binds either Cu⁺ or Ag⁺ by the side chains of four amino acids: two methionines (M), a histidine (H), and a tryptophan (W) as shown in Figure 1B (right).[13,14] Indeed, the indole ring of the tryptophan establishes a cation π interaction with the metal ion that red shifts the π π* transition of the indole and fully quenches its fluorescence.[14] Metal cation π interactions are known to efficiently enhance ISC and increase the population of the excited triplet state of a fluorophore, thereby quenching the fluorescence.[15]

Ln³⁺ ions have desirable luminescence properties that make them prime candidates for biological applica- tions.[8,9,16,17] Direct lanthanide excitation is inefficient because 4f 4f transitions are Laporte forbidden. However, indirect excitation of Ln³⁺ ions is possible in complexes incorporating a chromophore that, once excited, transfers its energy to the lanthanide (this photosensitization process has been deemed an antenna effect).[18] One of the main pathways for lanthanide sensitization involves electronic energy trans- fer (EET) from the excited triplet state of the antenna to the

<footnote>[*] M. Isaac, Dr. A. Roux, Dr. O. Sénèque
Univ. Grenoble Alpes, LCBM/PMB, and CEA, IRTSV/CBM/PMB and CNRS, LCBM UMR 5249, PMB
38000 Grenoble (France)
E mail: olivier.seneque@cea.fr
Dr. S. A. Denisov, Dr. G. Jonusauskas, Dr. N. D. McClenaghan
Univ. Bordeaux and CNRS, ISM (UMR 5255) and LOMA (UMR 5798)
33405 Talence (France)
E mail: nathan.mcclenaghan@u.bordeaux.fr
Dr. D. Imbert
Univ. Grenoble Alpes and CEA, INAC/SCIB/RICC
38000 Grenoble (France)</footnote>
emissive Ln³⁺ ion (Figure 1 C).[7,18] Among natural amino acids, tryptophan is an efficient antenna for Tb³⁺ sensitization.[19] Therefore, we designed a probe based, on the one hand, on a peptide mimicking the Cu⁺ binding site of CusF providing high affinity and selectivity and, on the other hand, on a Tb³⁺ complex as signaling unit. We reasoned that we could benefit from an ISC enhancement due to a cation π interaction between Cu⁺ and the tryptophan to increase the population of the tryptophan excited triplet state and, subsequently, increase also the population of Tb³⁺ excited states to transduce the copper binding event into an increased Tb³⁺ emission.

The peptidic probe, namely LCC1 Tb (Figure 1 A and B), comprises 1) the 16 amino acid sequence of the Cu⁺ binding loop of CusF, which includes the four metal binding amino acids (see above), 2) an Aib DPro dipeptide[20] to cyclize the loop and preorganize it, and 3) a DOTA macrocycle grafted on the amine side chain of a lysine to bind a Tb³⁺ ion. LCC1 Tb was synthesized by a combination of solid phase and solution reactions (Supporting Information, SI). The metal binding properties of LCC1 Tb were investigated under argon by circular dichroism (CD) spectroscopy (Figure 2). The titra

<figure><img src="image_3.png" /><figcaption>**Figure 2.** A) CD titration of LCC1 Tb (16 µM) in phosphate buffer (10 mM, pH 7.5) by Cu⁺ generated in situ by reduction of CuSO₄ by NH₂OH (2 mM). The inset shows the evolution of the CD signal at 200 nm (○) and 225 nm (●). B) CD spectra of LCC1 Tb (18 µM) before and after addition of various metal ions.</figcaption></figure>

tion of LCC1 Tb in phosphate buffer (10 mM, pH 7.5) by Cu⁺, generated in situ by reduction of Cu²⁺ by NH₂OH, shows a linear evolution of the CD signal which reaches a plateau in the presence of 1.0 equiv Cu⁺, indicating the formation of a 1:1 complex, Cu¹·LCC1 Tb, which was confirmed by ESI MS analysis (SI). The same behavior is observed with Ag⁺ due to the similarity between these two ions. LCC1 Tb is not able to bind any of the other physiologically relevant metal ions [Na⁺, K⁺ (100 mM), Ca²⁺, Mg²⁺ (10 mM), Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ (30 µM)] as demonstrated by the absence of change in the CD spectrum (Figure 2 B). It is noteworthy that LCC1 Tb can bind Cu⁺ but not Cu²⁺.

The coordination of Cu⁺ or Ag⁺ was further investigated by electronic absorption spectroscopy and photoluminescence to gain further insight into the establishment and effect of a cation π interaction. Concerning the UV/Vis absorption and the fluorescence of tryptophan, the binding of Cu⁺ or Ag⁺ is associated with a red shift of the indole π π* transition absorption band (Figure 3 A) and a partial quenching of its

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Steady state spectroscopic characterization of LCC1 Tb (solid line), Cu¹·LCC1 Tb (dashed line), and Ag¹·LCC1 Tb (dotted line). A) Electronic absorption spectra. B) Representation of possible fluorescent and non fluorescent forms of tryptophan in Cu¹·LCC1 Tb. C,D) Tryptophan fluorescence excitation (C, λem = 355 nm) and emission (D, λex = 280 nm) spectra. E,F) Time gated Tb³⁺ luminescence excitation (E, λem = 545 nm) and emission (F, λex = 280 nm) spectra. G) Time gated emission spectra with excitation at 310 nm. H) Selectivity diagram showing the time gated Tb³⁺ emission at 545 nm (λex = 310 nm) of LCC1 Tb (5 µM) before (black) and after (grey) addition of 1.5 equiv Cu⁺ in the presence of various cations (from left to right: none, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺ (10 µM), Na⁺, K⁺ (100 mM), Ca²⁺, and Mg²⁺ (10 mM)). Spectra were recorded in HEPES buffer (10 mM, pH 7.5) under argon.</figcaption></figure>

fluorescence (Figure 3 D). This suggests the presence of a cation π interaction in Cu¹·LCC1 Tb and Ag¹·LCC1 Tb as observed for CusF.

The Tb³⁺ luminescence properties were investigated by exciting the tryptophan antenna at 280 nm, which corresponds to the maximum absorption of the tryptophan indole π π* transition in LCC1 Tb. Titrations of LCC1 Tb by Cu⁺ or Ag⁺ show that the formation of Cu¹·LCC1 Tb and Ag¹·LCC1 Tb is associated with an increase of the Tb³⁺ emission. The Tb³⁺ luminescence excitation spectra of LCC1 Tb, Cu¹·LCC1 Tb, and Ag¹·LCC1 Tb (Figure 3 E) correspond to the π π* transition observed in the electronic absorption spectra, indicating that the tryptophan acts as an antenna for Tb³⁺ in LCC1 Tb and its Cu⁺ or Ag⁺ complexes. Interestingly, the Tb³⁺ excitation spectra (λem = 545 nm) of Cu¹·LCC1 Tb and Ag¹·LCC1 Tb are red shifted compared to LCC1 Tb (Figure 3 E), but the tryptophan fluorescence excitation spectra (λem = 355 nm) are not
(Figure 3C). This is consistent with two kinds of tryptophan indole that are present in solution when Cu⁺ or Ag⁺ are bound to LCC1 Tb: one corresponding to an indole that is fluorescent and has an unshifted π π* transition and the other one corresponding to a non fluorescent indole with a red shifted π π* transition and a higher Tb³⁺ luminescence. As the cation π interaction in CusF totally quenches the tryptophan fluorescence, we can propose that two forms of the 1:1 complex co exist in solution, one with the tryptophan indole establishing a cation π interaction and the other not (Figure 3B). Figure 3F compares the time gated Tb³⁺ emission spectra of LCC1 Tb, Cu¹·LCC1 Tb, and Ag¹·LCC1 Tb with excitation at 280 nm. Cu⁺ and Ag⁺ enhance the Tb³⁺ emission six times with respect to LCC1 Tb and thus, LCC1 Tb acts as a turn on luminescent probe for these cations. Moreover, the red shift of the indole π π* transition can be used to increase the contrast of the probe: Tb³⁺ luminescence enhancement factors of 58 and 52 were obtained for Cu⁺ and Ag⁺, respectively, by exciting the probe at 310 nm (see SI for rationalization of this wavelength choice) instead of 280 nm (Figure 3G). Furthermore, the Tb³⁺ emission of LCC1 Tb and Cu¹·LCC1 Tb is not affected by the presence of physiological cations (Figure 3H). Overall, LCC1 Tb is a high contrast turn on luminescent probe for the time gated detection of Cu⁺ among physiological cations. It is also able to detect Ag⁺. The binding constants for Cu⁺ and Ag⁺, determined by competition experiments with imidazole are 10⁹.⁴ M⁻¹ and 10⁷.³ M⁻¹, respectively (SI). The K_M for other physiological cations is estimated to be below 10³ M⁻¹.

The enhancement of Tb³⁺ luminescence upon Cu⁺ or Ag⁺ binding may originate from 1) a reduction of the number of water molecules bound to Tb³⁺, 2) a change in photophysical processes caused by the cation π interaction, or 3) a conformational change, that is, a shortening of the distance between the antenna and the Tb³⁺ ion and/or a change in the orientation of the antenna with respect to Tb³⁺. Concerning the latter point, changes in CD upon Cu⁺ or Ag⁺ binding may arise from conformational changes but also from the contribution of ligand metal charge transfer transitions. The NMR spectra of LCC1<sup>La</sup>, the diamagnetic homologous probe in which the Tb³⁺ ion is replaced by a La³⁺ ion, and of its Cu⁺ or Ag⁺ complexes display broad resonances that preclude any structural analysis, unfortunately. To elucidate the mechanism of the Tb³⁺ luminescence enhancement and quantify fast processes, the emission of the probe was characterized in detail. Regarding Tb³⁺ emission, Cu⁺ or Ag⁺ binding has almost no effect on the luminescence lifetime (τ ≈ 1.9 ms). Measurements of luminescence lifetime values in H₂O and D₂O additionally showed that only one water molecule is coordinated to the Tb³⁺ ion in LCC1 Tb and its Cu⁺ and Ag⁺ complexes (SI).<sup>[7,18,21]</sup> Therefore, the enhancement of Tb³⁺ emission is not due to a change in the Tb³⁺ primary coordination sphere. Emission was further investigated at the ns and µs timescale by time resolved emission spectroscopy with streak camera detection. The fluorescence of LCC1 Tb is characterized by a bi exponential decay (τ₁ = 0.9 ns and τ₂ = 4.8 ns, Table 1), which is common for trypto phan.<sup>[22]</sup> The lifetimes of the fluorescence of Cu¹·LCC1 Tb and Ag¹·LCC1 Tb, which accounts for the species with the indole

<table><caption>**Table 1:** Decay lifetimes of tryptophan emission and rise time of Tb³⁺ emission for LCC1 Tb, Cu¹·LCC1 Tb, and Ag¹·LCC1 Tb. Error on τ values is estimated at 10%.</caption><thead><tr><th>Compound</th><th>Tryptophan fluo<br>rescence decay<br>(ns)</th><th>Tryptophan phos<br>phorescence decay<br>(µs)</th><th>Tb³⁺ lumines<br>cence rise (µs)</th></tr></thead><tbody><tr><td>LCC1<sup>Tb</sup></td><td>0.9 (13%),<br>4.8 (87%)</td><td>not detected</td><td>23</td></tr><tr><td>Cu¹·LCC1<sup>Tb</sup></td><td>0.7 (16%),<br>3.9 (84%)</td><td>16</td><td>16</td></tr><tr><td>Ag¹·LCC1<sup>Tb</sup></td><td>0.9 (17%),<br>4.2 (83%)</td><td>19</td><td>18</td></tr><tr><td>Cu¹·LCC1<sup>La</sup></td><td></td><td>18</td><td></td></tr><tr><td>Ag¹·LCC1<sup>La</sup></td><td></td><td>20</td><td></td></tr></tbody></table>

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Time resolved emission spectroscopy in degassed buffer solutions. Spectra were recorded in time gated mode using a streak camera (λ<sub>ex</sub> 266 nm). Time resolved emission spectra of A) LCC1 Tb and B) Cu¹·LCC1 Tb recorded several µs after the laser pulse (2 µs integration time). C) Evolution of the tryptophan phosphorescence emission at 440 nm (dots) and of the Tb³⁺ emission at 545 nm (square; the tryptophan phosphorescence has been subtracted) for Cu¹·LCC1 Tb. The solid and dashed lines correspond to the respective fits which yielded τ = 16 ± 2 µs for both phosphorescence decay and Tb³⁺ emission grow in. D) Phosphorescence emission spectra of Cu¹·LCC1<sup>La</sup> (dashed line) and Ag¹·LCC1<sup>La</sup> (dotted line).</figcaption></figure>

not involved in a cation π interaction, are similar. Emission on the µs timescale was investigated in a time gated mode to eliminate the tryptophan fluorescence signal (SI).

Figures 4A and 4B compare the emission spectra of LCC1 Tb and Cu¹·LCC1 Tb recorded several µs after the laser pulse (λ<sub>ex</sub> = 266 nm, 2 µs integration time). For LCC1 Tb, the rise of Tb³⁺ luminescence is the only observed emission with a rise time of 23 µs. This rise time on the µs scale is in agreement with a sensitization of the Tb³⁺ taking place by energy transfer from the triplet state of the tryptophan. However, tryptophan triplet emission could not be detected for LCC1 Tb or for LCC1<sup>La</sup>, the homologous probe with the non luminescent La³⁺ ion. Conversely, for Cu¹·LCC1 Tb, the growing Tb³⁺ emission overlaps with a broad emission band that decays with a lifetime of 16 µs, which is synchronous with
the rise of Tb³⁺ emission (τ = 16 µs, Figure 4C). The broad decaying emission band is clearly seen with the Cu⁺ complex of LCC1<sup>La</sup> (Figure 4D). Due to its lifetime in the µs scale and spectrum, this band can be attributed to the triplet emission of the tryptophan. This confirms that Tb³⁺ sensitization occurs through a tryptophan(T₁) to Tb³⁺(⁵D₄) energy transfer. Ag¹·LCC1¹ⁿ (Ln = Tb or La) behaves in the same way as Cu¹·LCC1¹ⁿ but with blue shifted tryptophan phosphorescence emission compared to the analogous copper complex (Figure 4D). The above results show that both Cu⁺ and Ag⁺ binding to LCC1Tb increase tryptophan triplet state emission as well as Tb³⁺ emission. Together with the loss of tryptophan fluorescence for the 1:1 complex conformer that establishes a cation π interaction, this is compatible with an ISC enhancement promoted by the cation π interaction.[15] There fore, the binding of Cu⁺ or Ag⁺ to LCC1Tb through a cation π interaction favors ISC and increases the population of the excited triplet state of the tryptophan. Hence, more energy can be transferred to the Tb³⁺·⁵D₄ excited state, which in turn emits more. Although it cannot be excluded that conformational changes may be, in part, responsible for Tb³⁺ luminescence enhancement, the spectroscopic data presented here point to a major role of the cation π interaction that is established between the metal ion and the tryptophan indole. In addition to the global ISC enhancement, the cation π interaction with Cu⁺ and Ag⁺ shifts the tryptophan triplet excited state emission but to a different extent. Indeed, comparison of the room temperature phosphorescence spec tra of Cu¹·LCC1¹ᵃ and Ag¹·LCC1¹ᵃ (SI) with those reported in the literature for proteins[23,24] show that Cu⁺ and Ag⁺ lower the energy of the excited triplet state of tryptophan by ca. 2300 cm⁻¹ and 500 cm⁻¹, respectively.

Here we describe a new luminescent probe for selective Cu⁺ detection among physiological cations. This probe is characterized by a high contrast and long lived emission of its Tb³⁺ ion, which allows time gated detection. Additionally, detailed spectroscopic characterization shows that the cation π interaction established between the metal ion and the tryptophan indole plays a major role in modulating the Tb³⁺ luminescence in this prototype by modulation of the photo physical properties of the tryptophan antenna. As cation π interactions may be formed with several cations (e.g., Cu⁺, Ag⁺, Cd²⁺, Hg²⁺, and Pb²⁺), this work paves the way for the design of lanthanide based luminescent probes for Cu⁺ or toxic cations with desirable emission properties relying on a mechanism other than metal induced PET quenching.

## Acknowledgements

O.S. acknowledges the support of the Agence Nationale de la Recherche (ANR 12 BS07 0012 01) and Labex ARCANE (ANR 11 LABX 0003 01).

**Keywords:** cation π interactions · copper · luminescence · peptides · terbium

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