Aggregation Induced Emission from a new Naphthyridine- ethynyl-Gold(I) Complex as a potential tool for sensing Guanosine Nucleotides in Aqueous Media

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. 13 C NMR spectrum of 3 in DMSO.   4.45/4.28** 72.27 *These quaternary carbons were not observed, possibly due to (1) low relaxation times or (2) low sample concentration; **Unequivocal assignment of these signals was not possible on the acquired spectra.              Table S5. Atomic coordinates ( x 10 4 ) and equivalent isotropic displacement parameters (Å 2 x 10 3 ) for complex 3. U(eq) is defined as one third of the trace of the orthogonalized U ij tensor.

Introduction
Over the past years, research on alkynyl-gold(I) complexes (AGCs) has experienced a significant growth, mainly due to their luminescent properties, which confers them with outstanding potential for several applications, including molecular electronics and materials science. [1][2][3] AGCs are linear complexes, with a central Au(I) bound to two axial ligands, one of which is a terminal alkyne. The general strategy for obtaining luminescent AGCs relies on the fact that one of the ligands is, in fact, a fluorophore by itself whose triplet emission is strongly increased due to the strong heavy atom effect induced by Au(I) which favors Inter-System Crossing (ISC). 1,4 A particular feature from AGCs that has attracted significant attention from several research groups is their ability to self-assemble in aqueous solution through the formation of intermolecular aurophilic bonds, i.e. between two Au(I) atoms 5 , which comparable to strong hydrogen bonds. 6,7 This self-assembly into larger supramolecular aggregates can promote additional interactions between alkynyl ligands from neighboring complexes, such as π-π stacking, which can originate new luminescence bands, resulting in Aggregation Induced Emission (AIE). 8,9 The structure of the alkynyl ligands strongly influences both the luminescence as well as the size and shape of the obtained supramolecules. 10 Furthermore, factors such as concentration, solvent polarity, temperature are known to have a significant impact on the kinetics and thermodynamics of aggregation process, which hinders the full control of this phenomenon. 11 Recently, our group has reported a series of AGCs bearing fluorophores capable of coordinating divalent metal ions. 12 AGCs were found to aggregate in aqueous medium, producing luminescent structures with red emission. However, upon addition of Zn(II), the aggregates were disrupted and recovered their non-aggregated optical properties. Moreover, further addition of a stronger chelating agent (cryptand) allowed for the aggregation to take place once more, thus proving the control of this phenomenon by external stimuli.
Taking this into account, we aim to use the same strategy, i.e. aggregation/de-aggregation as However, these sensor systems generally present poor water solubility and/or low sensitivity for detecting nucleotides in aqueous media, due to the competition of solvent molecules for the naphthyridine binding sites. 13,14 Only few systems capable of performing in water have been reported, and these require complex architectures (nanoparticles with embedded reference dyes/quantum dots) for obtaining a reproducible response. 15,16 In this work, we designed an ethynyl-naphthyridine ligand and coupled it with a gold(I)

General
All solvents used were of spectroscopic grade. All reagents were purchased from Sigma-Aldrich and used as such without further purification. NMR spectra were recorded on a Bruker Advance III 400 spectrometer (400 MHz for 1 H, 101 MHz for 13 C, and 161.9 MHz for 31 P) at 298 K.

Synthesis
The

UV/Vis and fluorescence spectroscopies
Absorption spectra were obtained in a 1 cm quartz cuvette in acetonitrile on a Varian Cary 100 Bio UV-spectrophotometer. Emission spectra in solution were obtained in fluorescence quartz cuvette 1 cm, using a Horiba-Jobin-Yvon SPEX Fluorolog 3.22 spectrofluorimeter.
Solid state emission spectra were acquired in the same apparatus, and the samples were obtained by dropcasting a acetonitrile solution onto a quartz plate.
Luminescence measurements at 77K were performed in the same spectrofluorimeter, equipped with a cryogenic support with liquid nitrogen, in a glass tube. Association constants 7 were determined by fitting the experimental data to a 1:1 Henderson-Hasselbalch model using the Solver Add-In from Microsoft Excel.

Small-Angle X-Ray Scattering (SAXS)
SAXS data have been performed on the NCD beamline at the synchrotron ALBA at 12.4 keV and the distance sample/detector was 2.2m to cover the range of momentum transfer 0.09 < q <5.6 nm -1 . The data were collected on an ImXPad S1400 detector with a pixel size of 130.0x130.0m 2 . The exposure time was 10s. The q-axis calibration was obtained by measuring silver behenate. 18 The program pyFAI 19 was used to integrate the 2D SAXS data into 1D data. The data were then subtracted by the background using PRIMUS software. 20 The maximum particle dimension D max and the pair-distance distribution function P(r) were determined with GNOM. 21 The low-resolution structure of the aggregates was reconstructed ab initio from the initial portions of the scattering patterns using the program DAMMIN. 22,23 1·10 -4 M and 1·10 -5 M solutions of complex 3 were prepared in two different water/DMSO mixtures: 90:10 and 0:100.

Computational Methods
All calculations have been performed using the Amsterdam Density Functional (ADF) program and dispersion-corrected density functional theory (DFT-D3-BJ) at BLYP/TZ2P in gasphase. No symmetry constraint has been imposed. For interactions between ligand 2 and Guanine, only two matches have been reported since Guanine is characterized by a certain structural rigidity and therefore no other association could reasonably be probed.
ΔE Bond , the energetic parameter hereby used to estimate the strength of the bonding between chemical structures is expressed as: where E(adduct) is the total bonding energy of the optimized complex and, in turn, E(structure 1) and E(structure 2) represents the energy of each of the chemical structures that compose the adduct, both individually optimized.

Synthesis
The structure of Au(I) complex for sensing Guanosine nucleotides was designed to present (1) Aggregation Induced Emission (AIE) and (2)  The starting 2-acetamido-7-chloro-1,8-naphthyridine was synthesized according to previously established procedures. 17 Addition of an ethynyl moiety was performed via Sonogashira coupling with ethynyl-(trimethyl)silane (1), with subsequent removal of TMS with tetrabutylammonium fluoride, yielding crystals of compound 2 from chloroform (see Supporting Information for crystal data and NMR, figs. S1-S5 and tables S1, S4-S7). The obtained crystal structure shows a nearly perfect planarity of the naphthyridine moiety, ideal for the required complementarity with Guanine.

Converging synthesis with PTA-Au(I)-Cl in basic conditions 25 yielded the final complex 3
(NMR and HR-MS data is shown in figs. S6-S12 and table S2 of the Supplementary Information).

3.2.
Aggregation studies in water

UV-Vis and fluorescence spectroscopy
Given that detection of nucleotides is to be performed in aqueous media, the influence of concentration increase in the optical properties of 3 in water was assessed.
As can be seen in the UV-Vis spectra ( fig. S13), upon increasing the concentration of 3, an increase in the optical density at longer wavelengths (above 500 nm) is observed, which indicates a higher turbidity of the sample, a typical observation when precipitation or aggregation takes place. 25 Moreover, a band at around 400 nm becomes more prominent. The absorption from this band follows the same linear trend as the maximum absorption, and may be related to a metal-to-ligand-or ligand-to-metal-charge-transfer transition.
When the sample is excited at λ exc = 400 nm, we obtain the previously observed band at 450 nm ( fig. 3C), with a similar trend as the one shown in fig. 3B. Moreover, an increase in the intensity between 550 to 600 nm occurs along the studied concentration range, in two incremental steps ( fig. 3D). Intersecting the linear trendlines from the two steps yields the Critical Aggregation Concentration (CAC), i.e., the concentration at which aggregation starts to occur, corresponding to 25.1 µM. For this reason, this band was attributed to AIE from complex 3.  To better mimic the aggregation of 3 in aqueous solution observed in the emission spectra (see fig. 3C above), further experiments at 77K were performed in water. Figure S15

Small-Angle X-Ray Scattering (SAXS)
The formation of aggregates was analyzed by SAXS. Measurements were performed for 1 ×  For the UV-Vis spectra, an overall decrease in the absorption is observed, including a decrease in the baseline, which may be an indication that some of the higher aggregates are being disrupted ( fig. S22).
In the emission spectra, an increase in intensity at 450 nm occurs with increasing concentrations of analyte (this is also observed when exciting the samples at 343 nm, fig.   S23). Moreover, a concomitant decrease in the emission band of the aggregates (~600 nm) is observed ( fig. 5). This effect becomes weaker as we increase the number of phosphates in the nucleotide, indicating that electrostatic repulsion may play a role on the loss of sensitivity of 3 towards more charged Guanosines ( fig. S24).
If we plot the ratio of luminescence between the bands at 600 nm and 450 nm, we obtain a system that behaves as a 1:1 association equilibrium between 3 and the analyte, yielding association constants in the order of 10 5 M -1 (Table 1), which are, to the best of our knowledge, among the highest recorded for chemosensors based on hydrogen bonding capable of working in aqueous environment (see table S3 for comparison with the literature).
Furthermore, the strong signal changes upon analyte binding reach up to 63% variation when compared to the absence thereof, which further highlights the unprecedented sensitivity of this system in aqueous environment. The values for association constants are within the same order of magnitude for the three studied Guanosine derivatives, being higher for GDP. However, the percentage in signal change (Δ[%]) varies with increasing number of phosphate groups (GMP > GDP > GTP), which suggests that the negative charges or bulkiness/sterical effects have a significant influence on the overall emission of the aggregates.

Computational studies
Naphthyridines have already been reported in the literature as fluorescent units capable of forming stable bonds with guanine derivatives. [11][12][13][14] To better understand the specific mode with which our ligand binds to guanine, a series of computational experiments were performed.
We have analyzed two different interaction modes (depicted A and B) by which ligand 2 and Guanine could interact via hydrogen bonding ( fig. 6).  The schematic representation of model A was found to be significantly more stable, since it involves three hydrogen bonding interactions, yielding a complex adduct with nearly perfect planarity, while B comprises only 2 hydrogen bonds (see Table S2, in the Supp. Information). In order to gain further insight on why the association of 3 and phosphorylated Guanosine nucleotides is higher, additional energy calculations were also conducted for the adduct formed from 3 and GMP ( fig. 7)

Conclusions
A new Naphthyridine-ethynyl-Gold(I) complex 3 was synthesized and its structure fully characterized. UV-Vis spectra recorded in water at higher concentrations showed an increase in the baseline, indicating the presence of aggregation. The corresponding emission spectra revealed the appearance of a band with maximum at 550-600 nm, which was absent when the spectra were acquired in a good solvent (DMSO). This Aggregation Induced Emission (AIE) was probably favored by intermolecular (1) π-π interactions and (2)