The application of chiroptical spectroscopy (circular dichroism) in quantifying binding events in lanthanide directed synthesis of chiral luminescent self-assembly structures

The binding of asymmetrical and optically pure tridentate ligands containing one carboxylic group and 2-naphthyl as an antenna to lanthanide ions was studied in CH3CN.


Introduction
Due to the many unique spectroscopic properties that the lanthanides (Ln) possess, such as long-lived excited states and long emission wavelength, the synthesis of novel luminescent lanthanide based self-assemblies has led to the development of various novel optical and functional materials in recent times. 1 Examples of such developments are the formation of supramolecular self-assembly structures, such as helicates and interlocked lanthanide-based catenanes, 1e,f luminescent sensors for ions and molecules, probes for cellular imaging and for observing biological process and as supramolecular polymers. 2Chiral lanthanide complexes have been developed increasingly for such applications. 3,4We have developed numerous examples of chiral ligands that have been employed in lanthanide directed synthesis of self-assembled architectures.These have been based on the dipicolinic acid motive (H 2 dpa) 5 which has been shown to be an efficient sensitiser for Eu(III) and Tb(III) luminescence. 6][9][10][11][12] In related work, both Bünzli and De Cola have recently demonstrated that mono-anionic asymmetrical ligands containing three donor atoms can form stable charge neutral complexes with various lanthanides. 13erein we present ligands 1(S) and 1(R) (Scheme 1) based on the H 2 dpa core, each ligand possessing a single chiral (S)-and Scheme 1 Synthesis of the ligands 1(S) and 1(R), and their corresponding complexes Eu(1(S)) 3 and Eu(1(R)) 3 synthesized under microwave irradiation.
(R)-1-(2-naphthyl)-ethylamine antenna, respectively.Both Eu(III) complexes were formed from these ligands using a synthesis under microwave irradiation and the photophysical properties of the resulting complexes Eu(1(S)) 3 and Eu(1(R)) 3 studied in CH 3 CN and CH 3 OH solutions.Self-assembly of these ligands with Ln(III) was studied in CH 3 CN solution by monitoring the changes in both ground and excited states (Ln ¼ Eu(III)) as well as by NMR spectroscopy (Ln ¼ La(III)).Due to the chiral nature of the ligands and concomitant formation of chiral Lncomplexes in solution upon titration with Ln(III), we studied their chiroptical properties using both CD and CPL spectroscopies.The crystallographic analysis of Eu(1(R)) 3 conrmed that the chirality of the ligand was transferred to the Eu(III) centre.Adequately, the changes in CD spectra of either 1(S) and 1(R) were drastically affected upon binding to Eu(III); being equal magnitude but of opposite signs.This allowed us to identify both the different Ln x :L y stoichiometries in solution and to quantify their binding affinities using non-linear regression analysis, in a similar manner to that used to quantify the same in both the absorption and the luminescence titrations.While CD spectroscopy is commonly used to monitor the interaction, formation or folding of larger self-assembly structures in solution, 14 to the best of our knowledge, carrying out CD titrations to assess the different equilibrium processes for the formation of chiral self-assembly metal ion complexes and determination of the affinity constants for such processes is relatively unexplored in supramolecular chemistry.Here we demonstrate that indeed such analysis gives greater understanding of the different stoichiometric speciation in solution and that the binding constants determined for these matched comfortably well with that determined by traditional spectroscopic methods.

Synthesis of the ligands and Eu(III) complexes
The tridentate ligands 1(S) and 1(R) were synthesised in three steps starting from commercially available H 2 dpa.First the dipicolinic acid was monoprotected with benzylbromide to give 2 in 38% yields (see ESI †). 15 This was further coupled, Scheme 1, with either (S)-or (R)-1-(2-naphthyl)-ethylamine (3(S) or 3(R), respectively) using standard peptide-coupling methodology 9b in presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI$HCl) and 1-hydroxybenzotriazole hydrate (HOBt) to give the intermediates 4(S) and 4(R) in ca.80% yield for both.The deprotection of the benzyl esters 4(S) and 4(R) was rst attempted by catalytic hydrogenation using 10% Pd/C catalyst.However, this resulted in the product containing impurities that were difficult to isolate from the desired products.For this reason we turned to the use of Pd-C-induced catalytic transfer hydrogenation using triethylsilane 16 which gave desired products 1(S) and 1(R) in ca.78% yield for both.The 1 H NMR spectrum of 1(S) is shown in Fig. 1, demonstrating the successful deprotection of the benzyl ester (the same result observed for 1(R); see ESI †).High resolution electrospray massspectrometry (see ESI †) also conrmed the successful formation of both ligands with of m/z ¼ 319.1075 for 1(S) and 319.1080 for 1(R) corresponding to [M À H + ] À , while the IR spectra revealed the presence of characteristic C-H, N-H and C]O vibrations.The results of the elemental analysis also conrmed the formation of the pure compounds.We were also able to grow single crystals suitable for X-ray crystal structure analysis of both 4(S) and 4(R) by recrystallization of powder samples from methanol.Similarly, single crystals of 1(S) and 1(R) were grown from a mixture of CH 3 OH and CH 3 CN and the low temperature (100(2) K) X-ray structures collected.These will be discussed in the next section.
Having obtained both 1(S) and 1(R) we next synthesised the Eu(III) complexes Eu(1(S)) 3 and Eu(1(R)) 3 from these, where each of the ligand is expected to coordinate to the lanthanide via the central pyridine nitrogen, the carboxylic acid and the carboxylic amide, in a tri-dentate manner.Three of these ligands therefore full the high coordination requirements of the lanthanide.This was achieved by reacting Eu(CF 3 SO 3 ) 3 with 1(S) and 1(R) in a 1 : 3 metal to ligand stoichiometry in CH 3 CN solution under microwave irradiation at 95 C for 30 minutes.The resulting complexes were isolated as white powders by slow diffusion of diethyl ether into the CH 3 CN solutions, yielding the desired complexes in ca.35% yields.High resolution matrix-assisted laser desorption/ionization (MALDI) mass-spectrometry conrmed the formation of both tris-chelates with the presence of m/z ¼ 1147.2034 which was assigned to [Eu(1(S)-H + ) 3 + K + ] + and 1147.2039,assigned to [Eu(1(R)-H + ) 3 + K + ] + (see ESI †).The 1 H NMR (CD 3 CN, 600 MHz) spectra for both Eu(III) complexes were different to that of the free ligands showing signicant broadening and shi of the proton resonances (see ESI †), due to the paramagnetic nature of the lanthanide ion.Similarly, in the IR spectra, the C]O vibronic transitions were signicantly shied by 126 cm À1 upon complexation to Eu(III), further con-rming the complex formation.Elemental analysis also conrmed the formation of the desired products.However, the latter, along with the MALDI results, suggests that partial deprotonation of the ligands upon complexation occurs, which is not surprising since the carboxylic acid is directly bound to the lanthanide, making it more acidic.Both complexes were also shown to be luminescent, as upon placing a solution of both under a UV light irradiation red emission characteristic of Eu(III) was observed, conrming that the ligands functions as sensitising antennae for the 5 D 0 excited state of Eu(III).Clear colourless crystals of Eu(1(R)) 3 were obtained as described previously by Bünzli et al. 13a-c The coordination geometry of the Eu(III) centres was rst evaluated in solution by measuring the decay of the lanthanide excited state in both H 2 O and D 2 O, upon excitation at the naphthalene antennae (l ex ¼ 281 nm) allowing for the determination of the Eu(III) hydration state (q, the number of metalbound water molecules). 17The Eu(III) 5 D 0 excited state life-times of Eu(1(S)) 3 were best-tted to monoexponential decay with s H 2 O ¼ 1.54 AE 0.01 ms and s D 2 O ¼ 2.57 AE 0.01 ms giving a q value of zero.This is to be expected as the lanthanides have coordination requirements of 8-10, these being fullled by the 9 coordination environment of the 1 : 3 Eu:L stoichiometry in Eu(1(S)) 3 . 11Similarly, the Eu(III) excited state life-times of Eu(1(R)) 3 were best tted to monoexponential decay with s H 2 O ¼ 1.55 AE 0.01 ms and s D 2 O ¼ 2.50 AE 0.04 ms, again conrming that the ions were complexed with saturation of coordination environment.
Crystal and molecular structures of 4(S), 4(R), 1(S), 1(R) and Eu(1(R)) 3 As stated above, crystalline materials of both 1(R) and 1(S) were obtained that allowed for the X-ray analysis of these enantiomers.The resulting structures are shown in Fig. 2A and B, respectively.Each enantiomer crystallizes in the chiral space group P2 1 and displays two different independent molecules in the asymmetric unit.The chiral centres are C13 and C37, and although the chirality was known throughout the syntheses, it was conrmed by the rened Flack parameter for each enantiomer.The two independent molecules differ by rotation of the naphthyl group around the C13-C15 (C37-39) bond (torsion angle C14-C13-C15-C16, C38-C37-C39-C40; 1(R) ¼ À19.5(2), 9.3(2): 1(S) ¼ 19.84 (17), À9.50 (17) ).The structures of 4(S) and 4(R), again in the chiral space group P2 1 , were obtained from enantiopure crystals grown from methanol solutions, see Fig. 2C and D, respectively.Here the asymmetric unit consists of a single molecule.The additional substitution on the carboxyl group leads to a planar pyridine-C(O)O-CH 2 -Ph unit (4(S), 4(R) ¼ 0.04 Å deviation from plane).The chiral centre is C11 and was conrmed by the Flack parameter.The naphthyl groups are rotated about the C10-C11 bond with C9-C10-C11-C12 torsion angles of 4(R): À62.7(2) and 4(S): 62.8(2) .The chiral subunit (R-C(O)NHCMe-naphthyl) seen in 1(R), 1(S), 4(S) and 4(R) has been structurally characterized previously and displays a wide range of naphthyl group: chiral centre arrangements. 18n both 1(S) and 1(R), strong hydrogen bonding was observed between the carboxylic acid group and the carbonyl group of neighbouring ligand.This orientation results in other weak inter-and intra-molecular hydrogen bonding between the back-to-back molecules (see Table 1) and creates a weakly connected supramolecular ribbon motif parallel to the c-axis.In 4(S) and 4(R) the hydrogen bonding motif is disrupted by the substitution on the carboxyl group.In this case only weak C-H/O interactions prevail.
The complex Eu(1(R)) 3 (Fig. 3, ESI †) crystallized in an orthorhombic crystal system with chiral space group C222 1 (Table 1).The asymmetric unit contains two different Eu(1(R)) 3 molecules and some water molecules.The ligands are arranged around Eu(III) ion in a manner predicted by us previously for the complexes formed from the use of ligands with 1-naphthyl group as an antenna (assigned here as Eu(5(S)) 3 and Eu(5(R)) 3 , see ESI † for structure) 11 and observed by others 13 with three naphthyl antennae located on the same side (Fig. 3A).As observed previously (q ¼ 0) and conrmed here, the coordination environment of Eu(III) centre in either of the molecules is fully saturated with three molecules of deprotonated 1(R) contributing with three coordination bonds each being the pyridine nitrogen anked by the carboxylate oxygen on one side and the amido oxygen on the other side.Both europium atoms are positioned in a nine-coordinated tri-capped trigonal prismatic N 3 O 6 coordination environment formed by the three pyridine nitrogen atoms located in the equatorial plane arranging in quite regular triangle, and six oxygen atoms where three are placed above while the other three lay below the equatorial plane forming a triangular prism among them.Thus, the complex remains C 3 symmetry.The average bond distances are 2.37 Å for Eu-O(carboxylate) bonds, 2.44 Å for Eu-O(amido) and 2.57 Å for Eu-N(pyridine).The chirality of the ligand, known from the synthesis, is also conrmed in this structure by the Flack parameter.
The naphthalene moieties interact with each other and with the pyridine groups via p-stacking and they form hydrophobic pockets in the structure.Multiple intramolecular and intermolecular p-stacking interactions can be observed in the fragment containing the Eu1 atom, the naphthalene C48-C57 interacts face to face with pyridine N3-C21-C25 and edge to face with naphthalene C29-C38 but this pyridine also interacts face to face with the naphthalene C10-C19 (Fig. 3B, ESI †).Similar interactions can be seen in the other fragment (the one that contains the Eu2 atom) with the pyridine N9-C78-C82 in between the naphthalene group C67-C76 and C116-C125.Naphthalene groups have some conformational freedom and they can, at some extent rock and rotate and as a result high disorder is found in these moieties.In the case of the naphthalene group attached to C(103) it has been found in two different orientations with occupancy factor 1/2.  ).The excitation into the 270 nm transition did not result in any signicant ligand centred emission.However, upon complexation to the metal ion signicant Eu(III)-centred emission was observed due to the energy transfer processes occurring from the 2-naphthyl antennae to the 5 D 0 excited state which was followed by deactivation to the 7 F J bands of the lanthanide.The photophysical properties of Eu(1(S)) 3 and Eu(1(R)) 3 were also studied in CH 3 OH and compared to that observed for Eu(5(S)) 3 and Eu(5(R)) 3 (ESI †). 11The results are shown in Table 2 and it was demonstrated that the structure of Eu(III)-centred emission bands is similar for both, conrming that these systems have similar coordination environments of the metal centre.The photoluminescence quantum yields (F tot , %) were also measured by a relative method using Cs 3 [Eu(dpa) 3 ]$9H 2 O as a standard. 19In general, the quantum yields of the 1-naphthyl derivatives were ca. 4 times higher in CH 3 CN and 2 times higher in CH 3 OH than seen for the 2naphthyl analogues.Based on our previous work, where we investigated the symmetrical Eu(III) "Trinity Sliotar" complexes, 8 we believe that this difference is due to sensitisation efficiency of Eu(III) luminescence being more favourable for Eu(5(S)) 3 and Eu(5(R)) 3 than Eu(1(S)) 3 and Eu(1(R)) 3 .This is evident from calculating the antenna-to-ion energy transfer efficiencies (h sens ) for these complexes, which is determined on the basis of the emission spectrum, the observed luminescence life-time (s obs ) and the experimental overall luminescence quantum yield (F tot ) upon ligand excitation, Table 2.The quantum yield of the Eu(III) complexes in CH 3 OH solution was found to be signicantly lower than that seen in CH 3 CN, which we contribute to a dissociation of these complexes in more competitive protic media, where one of the ligands is removed from the EuL 3 complex to give EuL 2 + L. The latter now being affected by quenching of the lanthanide excited state by energy matching solvent O-H oscillators.The Eu(III)centred emission decays for both groups of complexes were found to be bi-exponential with the main species being EuL 3 ($80%) for the complexes with 1(S) and 1(R) while in case of 1naphthyl derivatives EuL 2 species ($80%) were prevalent (Table 2).Interestingly, the values of F tot and s obs between symmetrical "Trinity Sliotar" 8 and the asymmetrical Eu(III) complexes (Eu(1(S)) 3 and Eu(1(R)) 3 developed herein and that of Eu(5(S)) 3 and Eu(5(R)) 3 ) 11 in CH 3 CN are found to be very similar.However, if one compares the ratio between 5 D 0 / 7 F J transitions in the Eu(III)-centred emission spectra of these two sets of complexes it is possible to identify common differences which suggest discrepancy in the Eu(III) ion site symmetry within these two systems.In turn, s R of the symmetrical complexes was found to be higher as the shielding of the metal centre is better in this case compared to that seen for the asymmetrical structures.Hence, the values of F Ln Ln were found to be higher for asymmetrical molecules Eu(1(S)) 3 , Eu(1(R)) 3 and Eu(5(S)) 3 , Eu(5(R)) 3 , suggesting the larger inuence of non-radiative processes in the symmetrical complexes.Finally, h sens was higher for the symmetrical complexes as the asymmetrical lose one antenna per ligand.Having probed the photophysical properties of Eu(1(S)) 3 and Eu(1(R)) 3 we next turned our attention to analysing the role of the Eu(III) ion in the metal directed synthesis of these complexes under kinetic control at room temperature.

Monitoring self-assembly processes between 1(S) and 1(R) with Eu(III) in CH 3 CN by absorption and luminescence spectroscopy
The self-assembly studies between 1(S) or 1(R) and Eu(III) were rst performed in CH 3 CN solution.However, the analysis of the data using non-linear regression analysis program SPECFIT® did not result in data convergence.As the self-assembly Table 2 Antenna-to-ion energy transfer efficiencies (h sens ) of Eu(III) complexes calculated on the basis of the observed emission spectrum, the observed luminescence life-time (s obs ) and the experimental overall luminescence quantum yield (F tot ) upon ligand excitation (l ex ¼ 279 nm).s R is the radiative life-time calculated using eqn (3) (see EP processes are highly dependent on the solvent and ions in solution, it was decided to introduce ionic strength into the system, as it has been shown by Piguet et al. 20 that the introduction of tetrabutylammonia perchlorate favours the formation of highly charged complexes in lanthanide-directed selfassemblies formations in aprotic solvent systems.Hence, to overcome the data convergence problem we choose to use 0.05 M tetraethylammonium chloride ((C 2 H 5 ) 4 NCl) as ionic strength in our studies.
The changes observed in the absorption, uorescence and Eu(III)-centred emission spectra upon titrating 1(S) and 1(R) with Eu(III) were identical for both enantiomers and as such the discussion herein will focus only on one of these ligands.The changes seen upon titrating 1(S) are shown in Fig. 4 (see ESI † for 1(R)).As described previously, the absorption spectrum of 1(S) possesses two main maxima at 222 and 270 nm (Fig. 4A).The addition of Eu(III) resulted in hyperchromicity in these absorption bands, where the main changes were observed in the bands centred at $270 nm, as demonstrated in Fig. 5A, where the changes at three different transitions are plotted against added Eu(III) equivalents.Here, the absorbance increase was initially observed until addition of 0.30 equivalents of Eu(III), signifying the formation of the desired 1 : 3 stoichiometry, aer which much slower increase occurred until the addition of 1.00 equivalents before beginning to plateau.The experimental changes were analysed using non-linear regression analysis program SPECFIT® (ref.21) where factor analysis conrms the presence of four absorbing species.These were assigned to the ligand (L) and the Eu(III) species M:L, M:L 2 and M:L 3 .The data was satisfactorily tted to the following equilibria and the associated binding constants, expressed as log b x : y , are summarised in Table 3: ][10][11][12] The speciation distribution diagram for the titration is shown in Fig. 5B, which demonstrates the initial formation of 1 : 3 (M:L) species in ca.60% yield upon addition of 0.30 equivalents of Eu(III).However, almost simultaneously the formation of both the 1 : 2 and 1 : 1 stoichiometries also occurs.All of these species present in the solution until the end of the titration with the presence of the 1 : 1 complex in 58% yield, while the 1 : 2 and the 1 : 3 stoichiometry exist in 20% and 22% yield, respectively.The changes in the uorescence emission and Eu(III)-centred emission were also monitored in parallel and are shown in Fig. 4B and C, respectively.The ligand uorescence was weak and as such it was not possible to monitor the changes in the ligand-centred emission accurately over the cause of the titration.However, upon addition of the lanthanide to a solution of 1(S) the formation of Eu:L assemblies between the two was clear from the appearance of the red Eu(III)-centred emission bands due to the deactivation of 5 D 0 / 7 F J (J ¼ 0-4) upon excitation of the ligand at 270 nm, Fig. 4B.Analysis of these uorescence emission changes, showed that the luminescence intensity of the 5 D 0 / 7 F 1,3,4 bands increased up until the addition of 0.30 equivalents of Eu(III), which the emission plateau.In contrast, the changes in the 5 D 0 / 7 F 2 based transition were more stepwise where an initial increase was observed upon addition of 0.30 equivalents of Eu(III), followed by slower increase in the intensity until the addition of 0.50 equivalents where the saturation of the luminescence intensity occurred.
Similar luminescence behaviour was observed in the Eu(III)centred emission spectra by recording the delayed emission from the ion in phosphorescence mode.Here, the ne structure in the emission transitions was more pronounced as is evident from Fig. 4C, for the splitting of 5 D 0 / 7 F 2 band into two maxima at 612 and 616 nm.The intensity of the band at 616 nm reaches its maximum upon addition of 0.30 equivalents of Eu(III) (Fig. 4C, see ESI †) while the intensity of the band at 612 nm increases gradually until the addition of 1.00 equivalent of Eu(III) aer which the emission saturated (see ESI †).The presence of 5 D 0 / 7 F 0 band suggests the formation of 1 : 3 species with the arrangement of the ligands around Eu(III) ion in C 3 symmetry conrming the results found in the crystal structure of Eu(1(R)) 3 complex (see above).Analysis of the changes in the Eu(III)-centred emission spectra conrmed the formation of the expected 1 : 1, 1 : 2 and 1 : 3 species in the solution.
The changes in both the uorescence and the Eu(III)-centred emission spectra were analysed using non-linear regression analysis program SPECFIT®.As expected the factor analysis for the changes in the uorescence emission spectra suggests the presence of four emissive species, while in the case of Eu(III)-centred emission three emissive species were identied.The changes were tted to the same equilibrium used for analysis of the ground state data, showing similar binding constants as seen in Table 3.

Monitoring self-assembly formation between 1(S) or 1(R) and La(III) in CD 3 CN solution by 1 H NMR spectroscopy
The interaction between the ligands and lanthanide ions in CD 3 CN solution was also studied using NMR spectroscopy where the binding was monitored using diamagnetic lanthanum ions as 1 H NMR spectra of the Eu(III) complexes were too broadened and shied to be fully analysed.The overall changes are seen in Fig. 6, where the 1 H NMR of the free ligand can be seen and assigned (i.e.Scheme 1 for assignment and ESI †).
The changes in 1 H NMR spectra of 1(S) or 1(R) were followed upon addition of La(CF 3 SO 3 ) 3 and identical for both enantiomers (Fig. 6 and ESI †).This reected the formation of a single species in solution on the NMR time-scale.Generally, upon addition of La(III) the NMR spectra became both broadened and shied indicating complexes formed in the solution.More specically, the CH(2) protons were shown to be shied upeld, while N-H protons (NH(13)) were shied downeld.Clear binding of the La(III) to the pyrindine ring can be also conrmed by broadening of the proton resonances (CH(10-12)).Similarly the changes in the naphthyl group protons CH(3,4) occurred with very minor downeld shi while CH(5-9) experienced much more signicant changes resulting in an upeld shi and appearance of the new resonances, which is indicative of the recognition process being in slow exchange.Thus, even though the exact mode of binding cannot be established it is possible to conclude that the binding of La(III) to 1(S) or 1(R) occurs through the pyridine centre with further rearrangement of the naphthyl groups around metal centre.The evolution of the changes in the spectra suggest the possible occurrence of 1 : 1, 1 : 2 and 1 : 3 (M:L) species.

Circular dichroism and circularly polarised luminescence spectroscopy studies
In our previous work, [8][9][10][11][12] we have used circular dichorsim and circularly polarised luminescence to demonstrate the chirality associated with the formation of lanthanide self-assemblies such as in "Trinity Sliotar" complexes, as well as triple stranded dimetallic lanthanide helicates.In these, the Eu(III) CPL emission was recorded, demonstrating that the complexes and helicates were formed as pairs of enantiomers, where the chirality of the ligands was transferred to the complexes, giving D and L absolute stereochemistry.Similarly, we set out to probe the chiral nature and enantiomeric purity of 1(S) or 1(R) and their Eu(III) complexes using CD along with CPL spectroscopy.The CD spectra of both ligands and their corresponding Eu(III) complexes were recorded in both CH 3 OH and CH 3 CN solvent systems at 1 Â 10 À5 M and are shown in Fig. 7.All the structures showed clear Cotton effects and the expected mirror images for each pair of ligands (i.e.demonstrating that 1(S) or 1(R) are synthesised as enantiomers) and complexes in both solvent   emission in CH 3 OH, as summarised in Table 1.The presence of only EuL 3 species in aprotic solvent at c ¼ 1 Â 10 À5 M was conrmed by recording the Eu(III) 5 D 0 excited state life-times which were best tted to monoexponential decay with s z 1.99 AE 0.06 ms for both enantiomers.The occurrence of bisignate intense CD Cotton effects suggests possible coupling between naphthyl chromophores as previously was observed by Parker et al. (exciton coupling). 24In order to elucidate the effect of temperature on the interaction of the aromatic groups we recorded CD spectra of Eu(III) complexes in the temperature range varying from À10 to 60 C.However, we did not observe an enhancement in the CD signals or a change in Davydov splitting (see ESI †), but the shape of CD spectra suggests the presence of the coupling interactions between aromatic antennas.
As stated above, CD spectroscopy has been widely used for performing quantitative analysis of various supramolecular systems, but mainly focusing on the interaction of biological substrates with organic molecules, hydrogen-bonded and saltbridged complexes or chirality-sensing systems. 14,25However, to the best of our knowledge, only very few reports study the selfassembly between organic ligands and Ln(III) ions in solution.12b,26 Consequently, we studied the binding equilibrium processes of Eu(III) to both 1(S) and 1(R) ligands in CH 3 CN solution by monitoring the changes in the main CD bands, following a titration of these ligands with Eu(III) as shown in Fig. 8A (see also ESI †).As the binding constants for Eu:L n assemblies were previously determined in CH 3 CN solution in presence of 0.05 M (C 2 H 5 ) 4 NCl, we monitored the changes in the same ionic media.It should be stated that the observed changes for one enantiomer are mirror images of the other and this can be clearly seen from the binding isotherms of the main bands versus equivalents of Eu(III) added into the solution, as shown in ESI.† In order to monitor conformational changes that can possibly occur in the solution the CD spectra were recorded directly aer each addition and aer 24 hours equilibration.However, in this particular case, no signicant differences occurred upon equilibration.Similarly to our previous selfassembly studies the main changes in the spectra occur upon addition of 0 / 1 equivalents of Eu(III) to the solution and indicates the formation of 1 : 1, 1 : 2 and 1 : 3 species.In order to perform more detailed analysis about the equilibria occurring in the solution here we attempted to t the data obtained using non-linear regression analysis program SPECFIT® in a similar manner to that carried out for the changes in the ground state absorption and the emission above.For both of the enantiomers, the least square factor analysis of the titration results suggested the presence of four responding species in the CD spectra, which was in line with our previous ndings discussed above.Based on our previous results and current data we anticipated the successive formation of 1 : 1, 1 : 2 and 1 : 3 species.Indeed, our analysis showed the formation of all of these species, which were comparable to those obtained by tting the changes in the absorption and luminescence data.Hence, for example, in the case of 1(S) binding constants of log b 1 : 1 ¼ 6.6 AE 0.5, log b 1 : 2 ¼ 12.8 AE 0.6 and log b 1 : 3 ¼ 18.3 AE 0.6 were determined.Gratifyingly, the tting of the titration of 1(R) with Eu(III) gave almost indicial results (within experimental error) of log b 1 : 1 ¼ 6.5 AE 0.5, log b 1 : 2 ¼ 12.0 AE 0.8 and log b 1 : 3 ¼ 18.7 AE 0.6.These binding constant results are slightly higher than determined above, but this can be attributed to the presence of (C 2 H 5 ) 4 NCl in the solution which did not allow monitoring the CD changes in the 200-223 nm spectral range and as such we were only able to analyse the changes in 275 and 226 nm bands, both of which possess small amplitude.It is clear from these results that the self-assembly processes can be both monitored and quantied, as well as that the results are comparable to that observed using more classical tting of absorbance and emission data.Thus probing the chiroptical properties of the lanthanide directed self-assembly process in real time allows for additional information to be revealed that can help us in furthering quantication and revealing more understanding of such processes.As the overall changes in the circular dichroism spectra are quite signicant they can also be employed as a ngerprint or signature for each of the stochiometries in solution.This is commonly done in the treatment of absorption spectra titrations data, where the information from the data tting can also be employed to generate calculated spectra of each of the species in solution.The changes observed in the CD spectra of 1(S) shown in Fig. 8A cannot be accurately presented as a 'signature' for each of the three stochiometries.In comparison, the calculated spectra generated for the changes in the CD titrations in CH 3 CN are shown in Fig. 8C for 1(S) and these clearly demonstrate that each of the species can be assigned.For example, the experimental CD spectrum of Eu(1(S)) 3 (Fig. 7A) shows negative band centred at 273 nm similarly to the one observed for the calculated spectrum (Fig. 8C).Each of these calculated spectra can, therefore, be employed as a ngerprint, or a signature, for that given species.This again, demonstrates the potential use of CD spectroscopy in accessing vital information about equilibrium processes in metal directed synthesis of supramolecular structures.
Having emissive metal centre in a chiral environment we further investigated the chiroptical properties of our systems using CPL spectroscopy.As anticipated the excitation into the ligand absorption bands resulted in energy transfer to Eu(III) ion and thus generation of mirror-image CPL spectra showing the appearance of 5 D 0 / 7 F J (J ¼ 0-4) transition bands, as shown in Fig. 9.The luminescence dissymmetry factors g lum , were calculated for all of these transitions (see ESI †) and for 5 D 0 / 7 F 1 (589 nm) were found to be 0.16 and À0.15, while for 5 D 0 / 7 F 2 (614 nm) these values were equal to À0.09 for the Eu(III) complex with 1(S) and 0.10 for 1(R), respectively.These correspond well to values that we previously obtained for similar asymmetrical complexes with 1-naphthyl antennae complexes 11 reecting the similarities in the helical twists, nature of the ligand eld, donor group solvation and timeaveraged local helicity around Eu(III) of Eu(1(S)) 3 and Eu(1(R)) 3 .The values for Eu(III) complexes with asymmetrical ligands are lower than these obtained in our group previously 7,8,9b as well as these obtained by Muller et al. 4c for the complexes with symmetrical ligands.However, this can be simply explained by the decrease in the degree of conformational rigidity of the complex when reducing the symmetry of the ligand.Overall, obtained g lum values are high as enantiopure Eu(III) and Tb(III) complexes typically possess these values between 0.1 and 0.5 and much higher compared to the chiral uorescent organic molecules where g lum < 0.01.3a,b Based on our previous work, 7,8,9b where we have assigned the absolute conguration of lanthanide based self-assembly complexes and helices, formed from chiral ligands with known absolute stereochemistry, as D or L, then from the circularly polarised emission in Fig. 9, we were able to do the same for Eu(1(S)) 3 and Eu(1(R)) 3 by comparison.The CPL spectra of both Eu(1(S)) 3 and Eu(1(R)) 3 were structurally identical to that observed for "Trinity Sliotar" complexes.Hence, the CPL of Eu(1(R)) 3 consists of a negative CPL signal for the DJ ¼ 1, a positive band for the DJ ¼ 2, and a split CPL signal for the DJ ¼ 4 (into a positive and a negative band), and these are identical not only in their sign, but also in the intensity ratio to that seen for the RR isomer used in the synthesis of the L "Trinity Sliotar" complex. 7,8Similarly, the RR isomer used in the formation of structurally similar dimetallic Eu(III) triple stranded helicates, 9b gave also such identical CPL spectra.Consequently, we can with a degree of condence, assign the absolute stereochemistry of Eu(1(R)) 3 as L; and consequently the absolute stereochemistry of the Eu(1(S)) 3 complex as D.Moreover, we were able to assign the conguration of Eu(1(R)) 3 with absolute certainty since we have grown its crystals of good enough quality for crystallographic determination (see above) and as such clearly relate spectroscopical data to known solid state structures of several "Trinity Sliotar" complexes resolved in our laboratory.

Conclusions
Chiral asymmetrical R-and S-6-(1-(naphthalen-2-yl)ethylcarbamoyl)pyridine-2-carboxylic acids (1(S) and 1(R)) were obtained in three steps and high yield.These ligands were reacted with Eu(III) ions resulting in the formation of red emissive complexes, of Eu(1(S)) 3 and Eu(1(R)) 3 , respectively, with $2% luminescence quantum yields in CH 3 CN solution.Crystal structures of 1(S) and 1(R) along with their benzyl protected forms (4(S) and 4(R)) were obtained from CH 3 CN-CH 3 OH or CH 3 OH solvent systems and all crystallized in the chiral space group P2 1 , while Eu(1(R)) 3 crystallized in an orthorhombic crystal system with chiral space group C222 1 .The selfassembly formation between 1(S) and 1(R) with Eu(CF 3 SO 3 ) 3 in aprotic CH 3 CN polar media were analysed using 1 H NMR, absorption, luminescence and CD spectroscopies at room temperature.In all the cases the changes suggests successive formation of M:L, M:L 2 and M:L 3 assemblies with comparable values of the binding constants.As expected the excitation into the ligands absorption bands resulted in the transfer of the chirality from the ligand onto the metal centres showing characteristic Eu(III) CPL bands.This allowed us to tentatively assign the absolute stereochemistry of the self-assemblies as D and L for Eu(1(S)) 3 and Eu(1(R)) 3 , respectively, and absolutely conrm it for Eu(1(R)) 3 by comparing to the solid state crystallographic data obtained.Here, we represent one of the rare examples where the binding constants of supramolecular self-assemblies were determined by tting the changes in the chiroptical spectra (CD) using non-linear regression analysis.This allowed us to identify three species in solution as the 1 : 1, 1 : 2 and 1 : 3 metal to ligand stoichiometries and quantify their binding constants, all of which gave good correlation with those Fig. 9 The CPL spectra for EuL 3 in CH 3 OH (L ¼ 1(S), 1(R)).The total luminescence is also shown.
determined by tting the changes in the absorption and luminescence spectra.Moreover, the analysis of the CD spectra of the ligands and their Eu(III) complexes allowed us to suggest the presence of exciton coupling between the aromatic chromophores in these assemblies.Furthermore, using the information of the tting of the CD data, allowed us to calculate the CD spectra of each of the three stoichiometries, which we can use as ngerprints or signatures for each one.We are actively employing CD spectroscopy in greater detail for the analysis of metal-directed synthesis of supramolecular structures.

Materials and methods
All solvents and chemicals were purchased from commercial sources and used without further purication.Dichloromethane and methanol were freshly distilled under argon atmosphere prior to use.Water was puried using a Millipore Milli-Q water purication system (18.2MU cm).Hydrochloric acid, sodium bicarbonate, Na 2 SO 4 , MgSO 4 , H 2 dpa, benzyl bromide, HOBt, triethylamine (Et 3 N), triethylsilane (Et 3 SiH), palladium on carbon (10 wt% loading), tetraethylammonium chloride ((C 2 H 5 ) 4 NCl), Eu(CF 3 SO 3 ) 3 $6H 2 O were purchased from Sigma-Aldrich, while N,N-dimethylformamide, (S)-or (R)-1-(1naphthyl)-ethylamine and (S)-or (R)-1-(2-naphthyl)-ethylamine and EDCI$HCl from TCI Europe.Deuterated solvents used for NMR analysis (CDCl 3 , CD 3 OD, (CD 3 ) 2 SO) were purchased from Apollo Scientic.The 1 H NMR spectra were recorded at 400 MHz using an Agilent Technologies 400-MR NMR Spectrometer.The 13 C NMR spectra were recorded at 100 MHz using an Agilent Technologies 400-MR NMR Spectrometer.NMR spectra were also recorded using a Bruker AV-600 instrument operating at 600.1 MHz for 1 H NMR and 150.9 MHz for 13 C NMR. 1 H NMR titrations were recorded using Bruker Spectrospin DPX-400 instrument operating at 400.1 MHz.The titrations for both enantiomers were started with the ligands at c ¼ 4.26 Â 10 À4 M upon gradual addition of La(CF 3 SO 3 ) 3 solution in CD 3 CN.Chemical shis are reported in ppm with the deuterated solvent as the internal reference.All NMR spectra were carried out at 293 K. Mass-spectrometry was carried out using HPLC grade solvents.Electrospray mass spectra were determined on a Micromass LCT spectrometer and high resolution mass spectra were determined relative to a standard of leucine enkephaline.Maldi-Q-Tof mass spectra were carried out on a MALDI-Q-TOF-Premier (Waters Corporation, Micromass MS technologies, Manchester, UK) and high resolution mass spectrometry was performed using Glu-Fib with an internal reference peak of m/z 1570.6774.Melting points were determined using an Electrothermal IA9000 digital melting point apparatus.Infrared spectra were recorded on a Perkin Elmer Spectrun One FT-IE spectrometer equipped with universal ATR sampling accessory.Elemental analysis was conducted at the Microanalytical Laboratory, School of Chemistry and Chemical Biology, University College Dublin.
Complexation reactions were carried out in 2-5 mL Biotage Microwave Vials in a Biotage Initiator Eight EXP microwave reactor.

Crystallographic experimental section
Diffraction data for all compounds were collected on a Bruker APEX 2 DUO CCD diffractometer using graphite-monochromatized Mo-Ka (l ¼ 0.71073 Å) and Incoatec ImS Cu-Ka (l ¼ 1.54178 Å) radiation.Crystals were mounted in a cryoloop/ MiTeGen micromount and collected at 100(2) K using an Oxford Cryosystems Cobra low temperature device.Data were collected using omega and phi scans and were corrected for Lorentz and polarization effects.27a The structures 1(R), 1(S), 4(R) and 4(S) were solved by direct methods and rened by full-matrix least-squares procedures on F 2 using SHELXL-2013 soware.27b All non-hydrogen atoms were rened anisotropically.Hydrogen atoms were added geometrically in calculated positions and rened using a riding model.
The structure for complex Eu(1(R)) 3 was solved initially using SHELXS-97 which was further rened using SHELXL-97.Some of the aromatic moieties which showed high disorder were constrained to regular geometry.Low resolution and low data/ parameter ratio prevented in some cases full anisotropic renement.The thermal parameters were either restrained or rened isotropically.Hydrogen atoms were placed geometrically using suitable constraints except for water molecules in which case they were placed to form a coherent hydrogen bond network and their positions kept xed.
Details of the data collection and renement are given in Table 1.†

Photophysical measurements
Unless otherwise stated, all measurements were performed at 298 K in acetonitrile solutions (spectroscopy grade, Aldrich).UV-visible absorption spectra were measured in 1 cm quartz cuvettes on a Varian Cary 50 spectrophotometer.Baseline correction was applied for all spectra.Emission (uorescence, phosphorescence and excitation) spectra and life-times were recorded on a Varian Cary Eclipse Fluorimeter.Quartz cells with a 1 cm path length from Hellma were used for these measurements.The temperature was kept constant throughout the measurements at 298 K by using a thermostated unit block.Phosphorescence life-times of the Eu( 5 D 0 ) excited state were measured in both water/deuterated water solutions in timeresolved mode at 298 K.They are averages of three independent measurements, which were made by monitoring the emission decay at 616 nm, which corresponds to the maxima of the Eu(III) 5 D 0 / 7 F 2 transition, enforcing a 0.1 ms delay, and were analyzed using Origin 7.5®.The number of water molecules directly bonded to Eu(III) center (q value) was determined according to the equation developed by Parker et al.: where s O-H is the life-time water or methanol solutions, s O-D is the life-time measured in deuterated water or deuterated methanol solutions.The quantum yields (Q Eu,L rel ) were measured by relative method 28,29 using Cs 3 [Eu(dpa) 3 ]$9H 2 O complex in 0.1 M Tris buffer (pH ¼ 7.45) (Q Eu abs ¼ 24.0 AE 2.5%) 19 as a standard with known quantum yield, to which the absorbance and emission intensity of the sample are compared according to: where subscript rreference and xsample; Eintegrated luminescence intensity; Aabsorbance at the excitation wavelength; Iintensity of the excitation light at the same wavelength, nrefractive index of the solution.The estimated error for quantum yields is AE10%.s R life-time was obtained using eqn (3): where n is the refractive index of the solvent, A MD,0 is the spontaneous emission probability for the 5 D 0 / 7 F 1 transition in vacuo, and I tot /I MD is the ratio of the total area of the corrected Eu(III) emission spectrum to the area of the 5 D 0 / 7 F 1 band (A MD,0 ¼ 14.65 s À1 ). 30he quantum yield of the luminescence step (F Ln Ln ) expresses how well the radiative process complete with non-radiative processes.
The efficiency of lanthanide sensitization (h sens ) is the ratio between F tot (determined experimentally) and F Ln Ln (see eqn (4)): CD spectra were recorded in both acetonitrile and methanol solutions on a Jasco J-810-150S spectropolarimeter.CD titrations were performed in CH 3 CN media starting with the ligands at c ¼ 1 Â 10 À5 M upon gradual addition from 0 to 4 equivalents of Eu(CF 3 SO 3 ) 3 to the solution.CPL spectra were recorded by Dr R. Peacock at the University of Glasgow.Excitation of Eu(III) (560-581 nm) was accomplished by using a Coherent 599 tunable dye laser (0.03 nm resolution) with argon ion laser as a pump source.Calibration of the emission monochromator was accomplished by passing scattered light from a low power HeNe laser through the detection system.The optical detection system consisted of a photoelastic modulator (PEM, Hinds Int.) operating at 50 kHz and a linear polarizer, which together act as a circular analyzer, followed by a long pass lter, focusing lens and a 0.22 m double monochromator.The emitted light was detected by a cooled EM1-9558QB photomultiplier tube operating in photon counting mode.The 50 kHz reference signal from the photoelastic modulator was used to direct the incoming pulses into two separated counters.An up counter, which counts every photon pulse and thus is a measure of the total luminescence signal I ¼ I le + I right , and an up/down counter, which adds pulses when the analyzer is transmitting to the le circularly polarized light and subtracts pulses when the analyzer is transmitting right circularly polarized light.The second counter provides a measure of the differential emission intensity DI ¼ I le À I right .

Spectrophotometric titrations and binding constants
The formation of the luminescent 1 : 1, 1 : 2 and 1 : 3 (M:L, where M ¼ Eu(III) and L ¼ 1(S), 1(R)) species was ascertained by both UV-visible, luminescence and CD titrations of a solution of L (1 Â 10 À5 M) in CH 3 CN in presence of 0.05 M (C 2 H 5 ) 4 NCl with Eu(CF 3 SO 3 ) 3 $6H 2 O (0 / 4 equivalents).The data were tted using the non-linear regression analysis program, SPECFIT®. 21nthesis of 6-((benzyloxy)carbonyl)pyridine-2-carboxylic acid (2) Compound 2 was synthesised by stirring 2,6-pyridinedicarboxylic acid (H 2 dpa; 6.00 g, 3.59 Â 10 À2 mol, 1 equivalent) with NaHCO 3 (3.62 g, 4.31 Â 10 À2 mol, 1.2 equivalents) in N,Ndimethylformamide solution (100 mL) at 60 C under argon for 30 minutes.Benzyl bromide (5 mL, 4.31 Â 10 À2 mol, 1.2 equivalents) was then added to this white suspension and le to stir under argon at 60 C overnight.Resulting clear yellow solution was diluted with water and neutralised with saturated solution of NaHCO 3 (pH $ 8) and extracted with diethyl ether to remove diester side product.The aqueous layer was acidied with 2 M HCl and extracted with ethyl acetate.Then, the organic layer was dried over Na 2 SO 4 , ltered and aer the solvent was evaporated the resulting solid was solubilised in dichlorometane.This solution was washed with water and brine (3 Â 20 mL) aer which the organic layer was dried over MgSO 4 , ltered and aer subsequent evaporation of the solvent white powder was obtained (3.47 g, 38% yield).M.p.

Preparation of 4(S) and 4(R)
General procedure.To a stirred solution of (S or R)-2-(1aminoethyl)naphthalene (3(S) or 3(R), 1.0 equivalent) in 30 mL of freshly distilled dichloromethane, HOBt (1.0 equivalent) and 6-((benzyloxy)carbonyl)pyridine-2-carboxylic acid (2, 1.0 equivalent) were added.The solution was stirred for 30 minutes at 0 C under an inert atmosphere of argon before the solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI$HCl, 1.1 equivalents) and triethylamine (1.1 equivalents) in 30 mL of dichloromethane was added dropwise.The reaction mixture was then stirred for further 30 minutes at 0 C under argon atmosphere, then allowed to reach room temperature and le stirring for 48 hours.The insoluble residue was removed by suction ltration before reaction mixture was washed with 1 M HCl, a saturated solution of NaHCO 3 , and water (each 2 Â 20 mL).The organic layer was then dried over MgSO 4 , ltered, and the solvent removed under reduced pressure.The obtained yellow-white powders where puried using normal phase silica column chromatography eluting with 95% dichloromethane and 5% methanol (R f ¼ 0.9).

General synthesis of europium complexes
Eu(III) complexes were prepared by reuxing, under microwave radiation, the relevant ligand with Eu(CF 3 SO 3 ) 3 $6H 2 O (0.33 equiv.) in acetonitrile (15 mL) for 30 minutes.The solution was subsequently cooled to room temperature and then precipitated by slow evaporation of the solvent at ambient conditions.The resulting white solid was ltered off and dried under vacuum.

Fig. 3
Fig. 3 Space filling representation of Eu(1(R)) 3 complex showing (A) the position of carboxyl oxygen atoms (in red) relatively to naphthyl antennae and (B) stacking interaction between pyridine and naphthyl groups.

Fig. 5 (
Fig. 5 (A) Experimental binding isotherms and their corresponding fit obtained using non-linear regression analysis program SPECFIT®, (B) speciation-distribution diagram obtained from the fit of the changes in the absorption spectrum of 1(S) upon addition of Eu(CF 3 SO 3 ) 3 in CH 3 CN (25 C, 0.05 M (C 2 H 5 ) 4 NCl).