• chiral analysis
• chiral solvating agents
• chirality
• enantiodiscrimination
• enantiomeric purity
• NMR spectroscopy

In the present PhD project new chiral solvating agents (CSAs) have been proposed for the differentiation of enantiomers of chiral substrates by 1H/19F NMR spectroscopy, aimed to enantiomeric purity determinations. In order to investigate the interaction mechanisms on which chiral recognition relies, the stereochemical, dynamic and thermodynamic features of the diastereomeric complexes formed by each chiral receptor and selected enantiomeric substrates have been deeply investigated by means of ROESY and DOSY experiments, as well as by analysing the dependence of observed NMR spectral parameters on total concentration or substrate-to-CSA molar ratios.
CSAs belonging to the classes of polyamide systems, natural products, and macrocyclic receptors were selected.
Among polyamide CSAs, mono- and bis-thioureas have been taken into consideration. Mono-thioureas BTMA, BTMA1, BTMA2, BTMA3 and TFTMA were obtained by reacting the corresponding amine platforms 2‑[(1R)‑1-aminoethyl]phenol (MA), (1S,2R)-(−)-cis-1-amino-2-indanol (MA1), (1R,2R)-(−)-trans-1-amino-2-indanol (MA2) and (R)-1-phenylethylamine (MA3) with benzoyl isothiocyanate (BI) or 3,5-bis(trifluoromethyl)phenyl isothiocyanate (TFI). For the preparation of C2 symmetrical bis-thioureas BTDA and TFTDA, (1R,2R)-1,2-bis(2-hydroxyphenyl)ethylenediamine (DA) has been selected as amine precursor.
The two mono-thiourea CSAs with highest enantiodiscriminating efficiency have proven to be BTMA for the separation of N-dinitrobenzoyl (N-DNB) amino acid derivatives and TFTMA for N-acetyl (N-Ac) and N-trifluoroacetyl (N-TFA) amino acid derivatives. However, the corresponding bis-thiourea BTDA and TFTDA turned out to be increasingly more effective than their monomeric counterpart, leading to even higher differentiations of enantiomeric signals. For both dimeric systems, a cooperation between the two thiourea arms in the stabilization of diastereomeric solvates was pointed out.
In all cases, a strong base (DABCO/DMAP) was needed as solubilizer for amino acid derivatives with underivatized carboxyl functions. NMR investigations demonstrated that the base plays a key role also in the stabilization of diastereomeric complexes, by acting as a bridge in the interaction between CSA and two enantiomers. As well as it was ascertained that the phenolic hydroxyl of BTMA, TFTMA, BTDA and TFTDA participated to the tight network of hydrogen bond interactions substrate to CSA, favouring a syn/anti arrangement of the two thiourea NHs. Attractive π-π interactions involving the 2-hydroxyphenyl moiety of BTMA or BTDA and the 3,5-dinitrophenyl groups of N-DNB derivatives of amino acids contributed to the stabilization of the diastereomeric complexes formed in solution. 3,5-Bis(trifluoromethyl)phenyl moieties of TFTDA mainly acted on the acidity of the adjacent thiourea NH, boosting the hydrogen bond interactions between the CSA and enantiomers of N-Ac or N-TFA derivatives of amino acids, thus accounting for the different enantiodiscriminating features of the two kinds of thiourea CSAs.
In the field of natural products, we considered quinine (Qui) as possible multireceptorial chiral auxiliary endowed with a strong basic site, i.e. its quinuclidine nitrogen, in the NMR enantiodiscrimination of N-TFA amino acid derivatives. Qui produced particularly high 1H and 19F enantiomers differentiation in C6D6 (or CDCl3 for amino acids scarcely soluble in other solvents) also in sub-stoichiometric conditions of CSA. Qui was effective in the analysis of complex multicomponent mixtures and allowed very accurate enantiomeric purity determinations.
Derivatized cyclodextrins (CDs) were considered among macrocyclic receptor, for the NMR differentiation both of lipophilic and hydrophilic chiral substrates of pharmaceutical relevance. The three acetylated-silylated (α, β- and γ-AcSiCD) CDs effectively enantiodiscriminated chiral fluorinated compound B (COMP B) and methyl 2-chloropropanoate (MCP).
The greatest NMR enantioseparations were observed for COMP B and MCP in C6D12 by employing γ-AcSiCD and β-AcSiCD as chiral solvating agents, respectively. NMR mechanistic investigations demonstrated that, in the case of fluorinated chiral substrate, the enantiodiscrimination efficiency depends on deviations from truncated-cone shape structure of CDs, favouring attractive Si-F interactions at the external surface, rather than on the optimal fitting between the cavity of the cyclodextrin and the guest, leading to a non-deep inclusion of the guest. In that way the cyclodextrin having the greater number of glucopyranose rings, i.e. γ-AcSiCD, produces better enantiodiscrimination in comparison with β-AcSiCD, which forms the more stable diastereomeric solvates in virtue of the better size-fitting. On the contrary, MCP devoid of fluorinated groups is deeply included into the cyclodextrin and β-AcSiCD, giving the best fitting between the size of the cavity and the guest, shows greater enantiodiscriminating efficiency in comparison to γ-AcSiCD.
Among CD derivatives, also the partially and exhaustively methylated β-cyclodextrins were considered and compared in the enantiodiscrimination and in the hydrolysis of oxazepam hemisuccinate (OXM). The chemical stability of OXM over time as a pure compound and in the presence of β-CD, (2-methyl)-β-CD (MCD), heptakis(2,6-di-O-methyl)-β-CD (DIMEB) and heptakis(2,3,6-tri-O-methyl)-β-CD (TRIMEB) was evaluated, by monitoring the hydrolysis of the hemisuccinate chain. Among the methylated cyclodextrins, DIMEB has proven to be the most suitable to differentiate the two enantiomers of OXM, with high nonequivalences and complexation shifts, without producing hydrolysis of the hemisuccinate chain, which is mainly due to the presence of the cyclodextrin hydroxyls at the 2 and 6 positions.