Enantiomeric Recognition of α-Aminoacids by a Uranyl Salen-Bis-Porphyrin Complex

A novel uranyl salen-bis-porphyrin complex, in which two porphyrin subunits and salen moiety were directly linked, was synthesized for the recognition of tetrabutylammonium (TBA) amino acids. This uranyl salen complex, due to the presence of porphyrins with their fluorescence properties, represents the first example of a luminescence of uranyl salen complexes. UV/Vis measurements indicate the formation of 1:1 host–guest complexes, whereas UV-vis and fluorescence studies revealed that this complex acts as a receptor for the enantiomeric recognition of α-aminoacids derivatives, with high association constants and an excellent enantiomeric discrimination between the two enantiomers of phenylalanine–TBA.


INTRODUCTION
Salen ligands are a class of molecules that have been widely explored in the field of supramolecular chemistry. The most fascinating and promising use of salen derivatives is due to their chiral complexes with numerous metals. The salen structure, due to two contiguous stereogenic carbon atoms in the diimine bridge, creates a chiral pocket which can coordinate a metal cation (via imine nitrogen and phenolic oxygen atoms). Salen ligands rose to prominence thanks to the pioneering work of Jacobsen and Katsuki that paved the way to one of the most well-designed protocol for the enantioselective epoxidation of unfunctionalized alkenes catalyzed by chiral manganese salen complexes (Jacobsen, 1993;Katsuki, 2000;Yoon and Jacobsen, 2003;La Paglia Fragola et al., 2012;Trusso Sfrazzetto et al., 2015;Ballistreri et al., 2016Ballistreri et al., , 2018Zammataro et al., 2019). Furthermore, salen ligands are structures of great value in homogeneous catalysis (Katsuki, 1995;Jacobsen, 2000;Cozzi, 2004;McGarrigle and Gilheany, 2005;Baleizao and Garcia, 2006;Wezenberg and Kleij, 2008;Whiteoak et al., 2012).
In recent years, our research group exploited these chiral salen-metal complexes as enantiomeric receptors for chiral guests. In fact, depending on the metal ion and different substituents in the aromatic ring of salen framework, these salen-metal complexes can be used as efficient enantioselective catalysts and highly sensitive chemosensors (D'Urso et al., 2014;Puglisi et al., 2017Puglisi et al., , 2018Puglisi et al., , 2019. In particular, chiral uranyl salen complexes have proved to be excellent receptors for amino acid salts (Amato et al., 2007(Amato et al., , 2010(Amato et al., , 2011Ballistreri et al., 2010;Pappalardo et al., 2012a), since the uranyl metal center, acting as a Lewis acid, possesses an equatorial fifth position able to coordinate one molecule of carboxylate anion Brancatelli et al., 2013). These synthetic enantioselective receptors could help to better understand the mechanisms of drugs action; processes that are involved in immunological responses and processes of the storage of genetic information. Besides, slightly modification of their structures could lead to chemosensors, which due to their simple use, relatively low cost and high sensitivity are particularly significant in the chemical analysis.
Taking into account the salen ligand applications in the field of catalysis and enantiomeric recognition, previous studies have inspired us to extend current research on the synthesis of salen receptors comprising porphyrin macrocycles which, with their high stability and fluorescence properties, could greatly extend the use of salen ligands as chemosensors. Porphyrins, due to their rigid molecular structure, tunable substituents, large skeleton dimensions, and additional metallation sites in the core, are very attractive macrocycles for their applications in many technological fields (Beletskaya et al., 2009;Drain et al., 2009). In our strategy, the rational combination of porphyrin derivatives with chiral uranyl-salen ligands in one structure, would lead to chemosensors that possess unprecedented luminescence properties, that till now were precluded in uranyl-salen complexes due to the presence of uranyl metal center. Here we report on the synthesis of a novel uranyl salen-bis-porphyrin complex, in which two porphyrin subunits and the salen ligand are directly connected, and the enantiomeric recognition properties of this receptor toward selected αaminoacids derivatives assessed by UV-vis and fluorescence measurements (Figure 1).

General Experimental Methods
The NMR experiments were carried out at 27 • C on a Varian UNITY Inova 500 MHz spectrometer ( 1 H at 499.88 MHz, 13 C NMR at 125.7 MHz) equipped with pulse field gradient module (Z axis) and a tuneable 5 mm Varian inverse detection probe (ID-PFG). ESI mass spectra were acquired on a API 2000-ABSciex using CH 3 OH (positive ion mode). A JASCO V-560 UV-Vis spectrophotometer equipped with a 1 cm path-length cell was used for the UV-Vis measurements. Luminescence measurements were carried out using a Cary Eclipse Fluorescence spectrophotometer with resolution of 0.5 nm, at room temperature. The emission was recorded at 90 • with respect to the exciting line beam using 10:10 slit-widths for all measurements. All chemicals were reagent grade and were used without further purification.
General Procedure for the Synthesis of TBA Amino Acid Derivates  An aqueous solution of tetrabutylammonium hydroxide (40% w/w, 13 mmol) was added to an aqueous suspension of the desired amino acid (13 mmol). The resultant reaction mixture was heated at 60 • C for 2 h. Water was removed in vacuo at 80 • C. The residue was dissolved in CH 2 Cl 2 (10 mL), filtered and the solvent was evaporated in vacuo to afford in high yield the desired product.

Procedure for UV-vis and Fluorescence Titrations
Two stock solutions of host and guest (1.0 × 10 −3 M) in dry chloroform were prepared. From these, different solutions with different ratio receptor/guest (host concentration = 1.0 × 10 −6 M) were prepared, and UV-vis and emission spectra were recorded at 25 • C. Fluorescence titrations were carried out using λ ex = 350 nm in dry chloroform, recording at λ em = 650 and 715 nm. With this data treatment, the apparent binding affinities of receptor with amino acid guests were estimated using HypSpec (version 1.1.33) (Pappalardo et al., 2012b), a software designed to extract equilibrium constants from potentiometric and/or spectrophotometric titration data. HypSpec starts with an assumed complex formation scheme and uses a least-squares approach to derive the spectra of the complexes and the stability constants. χ 2 -test (chi-square) was applied, where the residuals follow a normal distribution (for a distribution approximately normal, the χ 2 -test value is around 12 or less). In all of the cases, χ 2 ≤ 10 were found, as obtained by 3 independent measurement sets.
After proving the luminescent properties of uranyl salenbis-porphyrin complex 1, enantioselective recognition properties were evaluated by UV-vis and fluorescence measurements in chloroform, in particular for fluorescence titrations, following the emission changes at these two emission wavelengths of porphyrin moiety (650 nm and 715 nm, by using λ ex 350 nm). Unfortunately, the fluorescence titrations showed a small intensity variation with all the selected amino acid guests except for the L-tryptophan derivative, which led to a poor data fitting. For that reasons, binding constant values between uranyl salen-bis-porphyrin complex 1 and amino acid derivatives were determined by UV-vis titrations, following a decrease of the absorption at 419 nm upon addition of increasing aliquots of guests. A representative example of UV-vis titration and the fluorescence titration of uranyl salen-bis-porphyrin complex 1 with L-Trp-TBA are shown in Figure 3. Table 1 reports the pertinent binding constant values with selected amino acid derivatives, the detection limit observed (DL) and the corresponding enantiomeric excess. In all cases, binding constant values have been calculated using 1:1 stoichiometry, suggested by Job's plots (see Supplementary Material).
Notably, due to the presence of the porphyrin moieties, receptor 1 is able to detect the amino acid guests at low concentration. In fact, 1 µM solution of uranyl salen-bisporphyrin complex 1 is able to detect amino acid derivatives at very low concentrations (ppb).
Enantiomeric recognition is very efficient with the L-Phe-TBA, which is recognized more than 8 times respect to the D-enantiomer (K L /K D = 8.51). A good enantioselectivity is also observed for the D-and L-Trp-TBA pair (K D /K L = 4.04). Moreover, for the L-Trp-TBA we were able to determine the binding constant value by fluorescence titration (K (M −1 = 2.63 ± 0.03 × 10 6 ), in good agreement with the value calculated by UV-vis titration. In particular, a decrease of the emission intensity has been observed, probably due to a photoinduced electron transfer mechanism (PET) (Trusso Sfrazzetto et al.,  2016). Only the D-and L-Ala-TBA (K D /K L = 1.70) pair shows a slight selective recognition. With the smaller amino acid guest, coordination to the uranyl metal center appears less susceptible to the different configurations of the carbon atom stereocenter and then the molecular recognition is less selective. With aromatic amino acid derivatives, the possibility for the carboxylate anion to bind the fifth equatorial coordination site of the uranyl metal and, at the same time, to exploit CH-π interactions with the salen moiety and the porphyrin macrocycles of receptor 1 might be responsible for the strong observed enantioselectivity. Moreover, the strong recognition for the L-Phe-TBA enantiomer is in contrast with respect to those observed with our previous receptors having the same configuration of the chiral diamine bridge (R, R) Amato et al., 2011;Pappalardo et al., 2012a;Forte et al., 2015). Probably, the presence of the two porphyrin arms not only increase the limit of detection by the presence of a fluorescence signal, but also plays a fundamental role in the recognition event. ) with selected amino acid derivatives, detection limit observed (DL), and enantiomeric excess calculated by UV-vis titrations in dry chloroform at 25 • C.

CONCLUSION
We have synthesized a new chiral uranyl salen complex bearing two porphyrin macrocycles and evaluated the enantiomeric recognition properties of this complex toward α-amino acid derivatives by UV-vis titrations. The presence of porphyrin arms lead to a receptor that possesses luminescence properties that are not quenched by the coordination with the uranyl cation, which decreases the fluorescence intensity in uranyl salen complexes. UV/Vis measurements and Job plots indicate the formation of 1:1 host-guest complexes. This receptor displays a very high selectivity toward amino acid derivatives, in particular for the two enantiomers of Phe-TBA. The two porphyrin macrocycles play a key role in the enantioselectivity interacting through CH-π with the aromatic moiety of aminoacids, leading to high binding affinities. Work is in progress in our laboratory to better understand the rules governing the interactions of this salen receptor with amino acid guests in order to design new hostguest systems that possess luminescence properties.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.