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

A prototype luminescent turn on probe for Cu (and Ag) is described, harnessing a selective binding site (log Kass = 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 p 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. It is required for various biological processes and its homeostasis is finely regulated in living organisms. Misregulation of copper can lead to various diseases (e.g., Menkes, Wilson, and Parkinson diseases). 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. However, the design of fluorescent probes for Cu is more challenging than many other cations, such as Ca or Zn, because Cu is an effective quencher of fluorescence through charge transfer and intersystem crossing (ISC) mechanisms. 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. 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. 6] In this communication, we report a new type of turn on Cu responsive probe based on a lanthanide ion (Ln) emitter, that has a long luminescence lifetime (in the millisecond range) compared to classical organic fluorophores (nano second range) and that allows time gated detection to suppress background fluorescence contributions. 10] Our probe structure (Figure 1A) is inspired by the metal binding site of the metallo chaperone CusF, which is part of the CusCFBA system responsible for copper or silver detoxification in gram negative bacteria. 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). Indeed, the indole ring of the tryptophan establishes a cation p interaction with the metal ion that red shifts the p p* transition of the indole and fully quenches its fluorescence. Metal cation p interactions are known to efficiently enhance ISC and increase the population of the excited triplet state of a fluorophore, thereby quenching the fluorescence. Ln ions have desirable luminescence properties that make them prime candidates for biological applica tions. 9,16, 17] Direct lanthanide excitation is inefficient because 4f 4f transitions are Laporte forbidden. However, indirect excitation of Ln 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). One of the main pathways for lanthanide sensitization involves electronic energy trans fer (EET) from the excited triplet state of the antenna to the Figure 1. A) Amino acid sequence of LCC1, chelating moieties are underlined. B) Principle of the probe design based on the X ray structure of the Cu binding loop of CusF. C) Simplified Jablonski Perrin diagram of LCC1 probe and pertinent photophysical process

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 + Is tate in cells.G enerally, fluorescence detection is considered to be one of the cheapest and easiest techniques. [4] However,t he design of fluorescent probes for Cu + is more challenging than many other cations, such as Ca 2+ or Zn 2+ ,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,C u + selective fluorescent probes were designed in which the fluorophore is spatially disconnected from the chelate. [5,6] These probes rely on ap hotoinduced 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 an ew type of turn on Cu + responsive probe based on al anthanide ion (Ln 3+ )e mitter, that has al ong luminescence lifetime (in the millisecond range) compared to classical organic fluorophores (nano second range) and that allows time gated detection to suppress background fluorescence contributions. [7 10] Our probe structure ( Figure 1A)isinspired by the metal binding site of the metallo chaperone CusF, [11] which is part of the CusCFBAs ystem 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), ah istidine (H), and at ryptophan (W) as shown in Figure 1B (right). [13,14] Indeed, the indole ring of the tryptophan establishes ac ation p interaction with the metal ion that red shifts the p p*transition of the indole and fully quenches its fluorescence. [14] Metal cation p interactions are known to efficiently enhance ISC and increase the population of the excited triplet state of af luorophore, thereby quenching the fluorescence. [15] Ln 3+ 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 4ft ransitions are Laporte forbidden. However, indirect excitation of Ln 3+ ions is possible in complexes incorporating ac hromophore 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 Figure 1. A) Amino acid sequence of LCC1 Tb ,chelating moieties are underlined. B) Principle of the probe design based on the X ray structure of the Cu + binding loop of CusF. [14] C) Simplified Jablonski Perrin diagram of LCC1 Tb probe and pertinent photophysical process es.
[ 1 emissive Ln 3+ ion ( Figure 1C). [7,18] Among natural amino acids,t ryptophan is an efficient antenna for Tb 3+ sensitiza tion. [19] Therefore,w ed esigned ap robe based, on the one hand, on ap eptide mimicking the Cu + binding site of CusF providing high affinity and selectivity and, on the other hand, on aT b 3+ complex as signaling unit. We reasoned that we could benefit from an ISC enhancement due to ac ation p interaction between Cu + and the tryptophan to increase the population of the tryptophan excited triplet state and, subsequently,i ncrease also the population of Tb 3+ excited states to transduce the copper binding event into an increased Tb 3+ emission. Thep eptidic probe,n amely LCC1 Tb ( Figure 1A and B), comprises 1) the 16 amino acid sequence of the Cu + binding loop of CusF,w hich includes the four metal binding amino acids (see above), 2) an Aib D Pro dipeptide [20] to cyclize the loop and preorganize it, and 3) aD OTAm acrocycle grafted on the amine side chain of alysine to bind aTb 3+ ion. LCC1 Tb was synthesized by acombination of solid phase and solution reactions (Supporting Information, SI). Them etal binding properties of LCC1 Tb were investigated under argon by circular dichroism (CD) spectroscopy ( Figure 2). Thet itra tion of LCC1 Tb in phosphate buffer (10 mm,p H7.5) by Cu + , generated in situ by reduction of Cu 2+ by NH 2 OH, shows alinear evolution of the CD signal which reaches aplateau in the presence of 1.0 equiv Cu + ,i ndicating the formation of a1:1 complex, Cu I ·LCC1 Tb ,which was confirmed by ESI MS analysis (SI). Thesame behavior is observed with Ag + due to the similarity between these two ions.L CC1 Tb is not able to bind any of the other physiologically relevant metal ions [Na + , K + (100 mm), Ca 2+ ,M g 2+ (10 mm), Mn 2+ ,F e 2+ ,C o 2+ ,N i 2+ , Cu 2+ ,a nd Zn 2+ (30 mm)] as demonstrated by the absence of change in the CD spectrum ( Figure 2B). It is noteworthy that LCC1 Tb can bind Cu + but not Cu 2+ .
Thec oordination of Cu + or Ag + was further investigated by electronic absorption spectroscopy and photolumines cence to gain further insight into the establishment and effect of acation p interaction. Concerning the UV/Vis absorption and the fluorescence of tryptophan, the binding of Cu + or Ag + is associated with ar ed shift of the indole p p*t ransition absorption band ( Figure 3A)a nd ap artial quenching of its fluorescence ( Figure 3D). This suggests the presence of ac ation p interaction in Cu I ·LCC1 Tb and Ag I ·LCC1 Tb as observed for CusF.
TheT b 3+ luminescence properties were investigated by exciting the tryptophan antenna at 280 nm, which corre sponds to the maximum absorption of the tryptophan indole p p*t ransition in LCC1 Tb .T itrations of LCC1 Tb by Cu + or Ag + show that the formation of Cu I ·LCC1 Tb and Ag I ·LCC1 Tb is associated with an increase of the Tb 3+ emission. TheTb 3+ luminescence excitation spectra of LCC1 Tb ,C u I ·LCC1 Tb ,a nd Ag I ·LCC1 Tb ( Figure 3E)c orrespond to the p p*t ransition observed in the electronic absorption spectra, indicating that the tryptophan acts as an antenna for Tb 3+ in LCC1 Tb and its Cu + or Ag + complexes.I nterestingly,t he Tb 3+ excitation spectra (l em = 545 nm) of Cu I ·LCC1 Tb and Ag I ·LCC1 Tb are red shifted compared to LCC1 Tb ( Figure 3E), but the trypto phan fluorescence excitation spectra (l em = 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 :o ne corresponding to an indole that is fluorescent and has an unshifted p p*transition and the other one corresponding to an on fluorescent indole with ar ed shifted p p*t ransition and ah igher Tb 3+ luminescence.A s the cation p interaction in CusF totally quenches the tryptophan fluorescence,w ecanp ropose that two forms of the 1:1complex co exist in solution, one with the tryptophan indole establishing ac ation p interaction and the other not ( Figure 3B). Figure 3F compares the time gated Tb 3+ emis sion spectra of LCC1 Tb ,C u I ·LCC1 Tb ,a nd Ag I ·LCC1 Tb with excitation at 280 nm. Cu + and Ag + enhance the Tb 3+ emission six times with respect to LCC1 Tb and thus,L CC1 Tb acts as aturn on luminescent probe for these cations.Moreover,the red shift of the indole p p*transition can be used to increase the contrast of the probe:T b 3+ luminescence enhancement factors of 58 and 52 were obtained for Cu + and Ag + , respectively,b ye xciting the probe at 310 nm (see SI for rationalization of this wavelength choice) instead of 280 nm ( Figure 3G). Furthermore,t he Tb 3+ emission of LCC1 Tb and Cu I ·LCC1 Tb is not affected by the presence of physiological cations ( Figure 3H). Overall, LCC1 Tb is ahigh contrast turn on luminescent probe for the time gated detection of Cu + among physiological cations.Itisalso able to detect Ag + .The binding constants for Cu + and Ag + ,d etermined by competi tion experiments with imidazole are 10 9.4 m À1 and 10 7.3 m À1 , respectively (SI). The K M for other physiological cations is estimated to be below 10 3 m À1 .
Theenhancement of Tb 3+ luminescence upon Cu + or Ag + binding may originate from 1) ar eduction of the number of water molecules bound to Tb 3+ ,2)achange in photophysical processes caused by the cation p interaction, or 3) ac on formational change,t hat is,ashortening of the distance between the antenna and the Tb 3+ ion and/or achange in the orientation of the antenna with respect to Tb 3+ .C oncerning the latter point, changes in CD upon Cu + or Ag + binding may arise from conformational changes but also from the con tribution of ligand metal charge transfer transitions.T he NMR spectra of LCC1 La ,the diamagnetic homologous probe in which the Tb 3+ ion is replaced by aLa 3+ ion, and of its Cu + or Ag + complexes display broad resonances that preclude any structural analysis,unfortunately.T oelucidate the mechanism of the Tb 3+ luminescence enhancement and quantify fast processes,t he emission of the probe was characterized in detail. Regarding Tb 3+ emission, Cu + or Ag + binding has almost no effect on the luminescence lifetime (t % 1.9 ms). Measurements of luminescence lifetime values in H 2 Oa nd D 2 Oa dditionally showed that only one water molecule is coordinated to the Tb 3+ ion in LCC1 Tb and its Cu + and Ag + complexes (SI). [7,18,21] Therefore,t he enhancement of Tb 3+ emission is not due to ac hange in the Tb 3+ primary coordination sphere.E mission was further investigated at the ns and mst imescale by time resolved emission spectros copy with streak camera detection. Thef luorescence of LCC1 Tb is characterized by ab i exponential decay (t 1 = 0.9 ns and t 2 = 4.8 ns,T able 1), which is common for trypto phan. [22] Thelifetimes of the fluorescence of Cu I ·LCC1 Tb and Ag I ·LCC1 Tb ,w hich accounts for the species with the indole not involved in ac ation p interaction, are similar.E mission on the mstimescale was investigated in atime gated mode to eliminate the tryptophan fluorescence signal (SI). Figures 4A and 4B compare the emission spectra of LCC1 Tb and Cu I ·LCC1 Tb recorded several msa fter the laser pulse (l ex = 266 nm, 2 msi ntegration time). ForL CC1 Tb ,t he rise of Tb 3+ luminescence is the only observed emission with ar ise time of 23 ms. This rise time on the mss cale is in agreement with as ensitization of the Tb 3+ taking place by energy transfer from the triplet state of the tryptophan. However,t ryptophan triplet emission could not be detected for LCC1 Tb or for LCC1 La ,t he homologous probe with the non luminescent La 3+ ion. Conversely,f or Cu I ·LCC1 Tb ,t he growing Tb 3+ emission overlaps with ab road emission band that decays with alifetime of 16 ms, which is synchronous with  the rise of Tb 3+ emission (t = 16 ms, Figure 4C). Theb road decaying emission band is clearly seen with the Cu + complex of LCC1 La ( Figure 4D). Due to its lifetime in the msscale and spectrum, this band can be attributed to the triplet emission of the tryptophan. This confirms that Tb 3+ sensitization occurs through at ryptophan(T 1 )t oT b 3+ ( 5 D 4 )e nergy transfer. Ag I ·LCC1 Ln (Ln = Tb or La) behaves in the same way as Cu I ·LCC1 Ln but with blue shifted tryptophan phosphores cence emission compared to the analogous copper complex ( Figure 4D). Thea bove results show that both Cu + and Ag + binding to LCC1 Tb increase tryptophan triplet state emission as well as Tb 3+ emission. Together with the loss of tryptophan fluorescence for the 1:1c omplex conformer that establishes ac ation p interaction, this is compatible with an ISC enhancement promoted by the cation p interaction. [15] There fore,the binding of Cu + or Ag + to LCC1 Tb through acation p interaction favors ISC and increases the population of the excited triplet state of the tryptophan. Hence,m ore energy can be transferred to the Tb 3+ 5 D 4 excited state,which in turn emits more.A lthough it cannot be excluded that conforma tional changes may be,inpart, responsible for Tb 3+ lumines cence enhancement, the spectroscopic data presented here point to am ajor role of the cation p interaction that is established between the metal ion and the tryptophan indole.
In addition to the global ISC enhancement, the cation p interaction with Cu + and Ag + shifts the tryptophan triplet excited state emission but to ad ifferent extent. Indeed, comparison of the room temperature phosphorescence spec tra of Cu I ·LCC1 La and Ag I ·LCC1 La (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 À1 and 500 cm À1 ,respectively.
Here we describe an ew luminescent probe for selective Cu + detection among physiological cations.T his probe is characterized by ahigh contrast and long lived emission of its Tb 3+ ion, which allows time gated detection. Additionally, detailed spectroscopic characterization shows that the cation p interaction established between the metal ion and the tryptophan indole plays am ajor role in modulating the Tb 3+ luminescence in this prototype by modulation of the photo physical properties of the tryptophan antenna. As cation p interactions may be formed with several cations (e.g., Cu + , Ag + ,C d 2+ ,H g 2+ ,a nd Pb 2+ ), this work paves the way for the design of lanthanide based luminescent probes for Cu + or toxic cations with desirable emission properties relying on amechanism other than metal induced PET quenching.