Enantioselective high-performance liquid chromatography combined with spectroscopic methods and theoretical calculations: A valid strategy to determine the absolute configuration of eugenol derivatives.

The absolute configuration of three chiral eugenol derivatives was assigned by a multi-step methodology based on enantioselective HPLC combined with spectroscopic and theoretical calculations. Milligram amounts of enantiopure forms used for stereochemical characterization were isolated by HPLC on the immobilized amylose-based chiral stationary phase Chiralpak IG using normal phase elution conditions. The absolute configuration was indirectly determined for one of the three compounds by 1H NMR via methoxy-α-trifluoromethyl-α-phenylacetic acid derivatization (Mosher's acid). Comparison of the experimental and predicted electronic circular dichroism spectra confirmed the stereochemical assignment by Mosher's method and extended the absolute configuration assignment to two other chiral compounds.

Resorc[4]arene Modifiers for Supramolecular Site-Directed Immobilization of Antibodies on Multi-Walled Carbon Nanotubes.

One of the main problems in developing immunosensors featuring carbon nanotubes (CNTs) is immobilizing antibodies (Abs) onto the CNT surface to afford selective binding to target antigens (Ags). In this work, we developed a practical supramolecular Ab conjugation strategy based on resorc[4]arene modifiers. To improve the Ab orientation on the CNTs surface and optimizing the Ab/Ag interaction, we exploited the host-guest approach by synthesizing two newly resorc[4]arene linkers R1 and R2 via well-established procedures. The upper rim was decorated with eight methoxyl groups to promote selective recognition of the fragment crystallizable (Fc ) region of the Ab. Moreover, the lower rim was functionalized with 3-bromopropyloxy or 3-azidopropiloxy substituents to bind the macrocycles on the multi-walled carbon nanotubes (MWCNTs) surface. Accordingly, several chemical modifications of MWCNTs were evaluated. After the morphological and electrochemical characterization of nanomaterials, the resorc[4]arene-modified MWCNTs were deposited onto a glassy carbon electrode surface to evaluate their potential applicability for label-free immunosensor development. The most promising system showed an improved electrode active area (AEL ) of almost 20 % and a site-oriented immobilization of the SARS-CoV-2 spike protein S1 antibody (Ab-SPS1). The developed immunosensor revealed a good sensitivity (23.64 µA mL ng-1 cm-2 ) towards the SPS1 antigen and a limit of detection (LOD) of 1.01 ng mL-1 .

Theoretically Predicted and Experimentally Detected Chirality of Dibenzocyclooctynes and Their Triazole Adducts with Azides.

Dibenzocyclooctynes have emerged as promising scaffolds for bioorthogonal ligation. An important structural aspect that has not been addressed so far relates to their chirality. Herein, we explore, by theoretical and experimental methods, this structural aspect that has been neglected so far. First, computational analysis is conducted, and the results are used as a guide for the experimental investigation. Next, an array of different experiments (high-performance liquid chromatography (HPLC) on chiral columns, chiroptical spectroscopy, and X-ray diffraction) for structure elucidation is scrutinized in concert. Finally, this work demonstrates the chirality and the stereodynamic behavior of dibenzocyclooctynes and their triazole derivatives with simple azides and also uncovers their conformational behavior.

Triptycene derivatives as chiral probes for studying the molecular enantiorecognition on sub-2-µm particle cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase.

Two chiral triptycene derivatives were analyzed on the Chiralpak IB-U column packed with cellulose tris(3,5-dimethylphenylcarbamate)-based sub-2-µm diameter particles. Under normal-phase conditions, sub-minute baseline enantioseparations were obtained. Differences in structural elements and chromatographic behavior of the investigated compounds were evaluated to identify the interactions that drive the chiral discrimination process. From the evaluation of the experimental chromatographic data, it was found that hydrogen bond formation is essential for the separation of enantiomers.

Exploring the Assembly of Resorc[4]arenes for the Construction of Supramolecular Nano-Aggregates.

Many biologically active compounds feature low solubility in aqueous media and, thus, poor bioavailability. The formation of the host-guest complex by using calixarene-based macrocycles (i.e., resorcinol-derived cyclic oligomers) with a good solubility profile can improve solubilization of hydrophobic drugs. Herein, we explore the ability of resorc[4]arenes to self-assemble in polar solutions, to form supramolecular aggregates, and to promote water-solubility of an isoflavone endowed with anti-cancer activity, namely Glabrescione B (GlaB). Accordingly, we synthesized several architectures featuring a different pattern of substitution on the upper rim including functional groups able to undergo acid dissociation (i.e., carboxyl and hydroxyl groups). The aggregation phenomenon of the amphiphilic resorc[4]arenes has been investigated in a THF/water solution by UV-visible spectroscopy, at different pH values. Based on their ionization properties, we demonstrated that the supramolecular assembly of resorc[4]arene-based systems can be modulated at given pH values, and thus promoting the solubility of GlaB.

Comparison of Coated and Immobilized Chiral Stationary Phases Based on Amylose tris-[(S)-α-Methylbenzylcarbamate] for the HPLC Enantiomer Separation of α-Lipoic Acid and Its Reduced Form.

The couple of chiral sulfur compounds α-lipoic acid (ALA)/α-dihydrolipoic acid (DHALA) has attracted considerable attention in recent years owing to its remarkable anti-inflammatory and antioxidant properties. It is well known that the chirality of the C6 plays a key role in determining the biological activity of ALA. The natural occurring (R)-ALA enantiomer is an essential cofactor for key oxidative metabolism enzyme complexes and, after oral administration of the racemic mixture, it shows higher plasma concentration than (S)-ALA. Differently, the in vivo enantioselective action difference between the enantiomers of DHALA has not yet been studied. This lacking is perhaps due to the unavailability of analytical methods capable of determining the enantiomeric composition of biological samples during pharmacokinetic and pharmacodynamic events. In the present work, the direct and baseline enantioresolution of both chiral acids by HPLC on two amylose-derived chiral stationary phases is presented. The proposed chiral enantioselective protocol, therefore, does not require pre- or on-column derivatization. The performance of the coated Chiralpak AS-H CSP and the new immobilized Chiralpak IH-3 CSP, which have the same chiral selector amylose tris-[(S)-α-methylbenzylcarbamate], were compared using conventional normal-phase mobile phases containing ethanol or 2-propanol as alcoholic solvents and a fixed percentage of trifluoroacetic acid. Nonconventional eluents containing dichloromethane, ethyl acetate, and 2-methyltetrahydrofuran as organic cosolvents were applied in the separation of the enantiomers of two carboxylic acids on the immobilized Chiralpak IH-3 CSP. The effect of the column temperature was carefully evaluated in order to improve enantioselectivity. Adequate amounts of enantiomers were isolated by an analytical-size Chiralpak IH-3 column and submitted to chiroptical measurements. The absolute configuration assignment of the isolated enantiomers was determined by a multidisciplinary procedure based on the comparison of the experimental and calculated chiroptical properties.

Site-Directed Antibody Immobilization by Resorc[4]arene-Based Immunosensors.

One of the main problems in the development of immunosensors is to overcome the complexity of binding antibodies to the sensor surface. Most immobilizing methods lead to a random orientation of antibodies with a lower binding site density and immunoaffinity. In order to control the orientation of antibody immobilization, several resorc[4]arene derivatives were designed and synthesized. After the spectroscopic characterization of resorc[4]arene self-assembled monolayers (SAMs) onto gold films, the surface coverage and the orientation of insulin antibody (Ab-Ins) were assessed by a surface plasmon resonance (SPR) technique and compared with a random immobilization method. Experimental results combined with theoretical studies confirmed the dipole-dipole interaction as an important factor in antibody orientation and demonstrated the importance of the upper rim functionalization of resorcarenes. Accordingly, resorcarene 5 showed a major binding force towards Ab-Ins thanks to the H-bond interactions with the amine protein groups. Based on these findings, the resorcarene-based immunosensor is a powerful system with improved sensitivity providing new insight into sensor development.

Molecular Recognition of the HPLC Whelk-O1 Selector towards the Conformational Enantiomers of Nevirapine and Oxcarbazepine.

The presence of stereogenic elements is a common feature in pharmaceutical compounds, and affording optically pure stereoisomers is a frequent issue in drug design. In this context, the study of the chiral molecular recognition mechanism fundamentally supports the understanding and optimization of chromatographic separations with chiral stationary phases. We investigated, with molecular docking, the interactions between the chiral HPLC selector Whelk-O1 and the stereoisomers of two bioactive compounds, the antiviral Nevirapine and the anticonvulsant Oxcarbazepine, both characterized by two stereolabile conformational enantiomers. The presence of fast-exchange enantiomers and the rate of the interconversion process were studied using low temperature enantioselective HPLC and VT-NMR with Whelk-O1 applied as chiral solvating agent. The values of the energetic barriers of interconversion indicate, for the single enantiomers of both compounds, half-lives sufficiently long enough to allow their separation only at critically sub-ambient temperatures. The chiral selector Whelk-O1 performed as a strongly selective discriminating agent both when applied as a chiral stationary phase (CSP) in HPLC and as CSA in NMR spectroscopy.

A chromatographic study on the retention behavior of the amylose tris(3-chloro-5-methylphenylcarbamate) chiral stationary phase under aqueous conditions.

In this study, the retention properties of the immobilized polysaccharide-derived Chiralpak IG-3 chiral stationary phase under aqueous-organic conditions were investigated. A systematic evaluation of the retention factors of the enantiomers of the chiral sulfoxide oxfendazole, endowed with anthelmintic activity and selected as test compound, was carried out changing progressively the water content in hydro-organic eluents containing methanol, ethanol or acetonitrile. From the results obtained with acetonitrile/water mobile phases and the associated retention plots, clear U-shape retention dependencies, indicative of the interplay of both hydrophilic interaction liquid chromatography and reversed-phase modes, were highlighted. A U-turn point of retention mechanism was recorded in correspondence of the acetonitrile/water 100:40 v/v mobile phase. Retention was significantly affected by small percentages of trifluoroacetic acid or diethylamine additives incorporated in the mobile phase. It is worth emphasizing that the basic additive was more effective in reducing retention in the reversed-phase region, while the action of acid additive was more pronounced in the hydrophilic interaction liquid chromatography region. Finally, either in the transition from hydrophilic interaction liquid chromatography to reversed-phase conditions or after additive addition, the enantioselectivity did not vary significantly.

Motorcycle That See: Multifocal Stereo Vision Sensor for Advanced Safety Systems in Tilting Vehicles.

Advanced driver assistance systems, ADAS, have shown the possibility to anticipate crash accidents and effectively assist road users in critical traffic situations. This is not the case for motorcyclists, in fact ADAS for motorcycles are still barely developed. Our aim was to study a camera-based sensor for the application of preventive safety in tilting vehicles. We identified two road conflict situations for which automotive remote sensors installed in a tilting vehicle are likely to fail in the identification of critical obstacles. Accordingly, we set two experiments conducted in real traffic conditions to test our stereo vision sensor. Our promising results support the application of this type of sensors for advanced motorcycle safety applications.

"Inherently Chiral" Ionic-Liquid Media: Effective Chiral Electroanalysis on Achiral Electrodes.

To achieve enantioselective electroanalysis either chiral electrodes or chiral media are needed. High enantiodiscrimination properties can be granted by the "inherent chirality" strategy of developing molecular materials in which the stereogenic element responsible for chirality coincides with the molecular portion responsible for their specific properties, an approach recently yielding outstanding performances as electrode surfaces. Inherently chiral ionic liquids (ICILs) have now been prepared starting from atropisomeric 3,3'-bicollidine, synthesized from inexpensive reagents, resolved into antipodes without need of chiral HPLC and converted into long-chain dialkyl salts with melting points below room temperature. Both the new ICILs and shorter family terms, solid at room temperature, employed as low-concentration additives in achiral ILs, afford impressive enantioselection for the enantiomers of different probes on achiral electrodes, regularly increasing with additive concentration.

The Anancomeric Character of the Pharmacophore 1,3,4-Thiadiazoline Framework in Chiral Spiro-Cyclohexyl Derivatives: Effects on Stereochemistry and Spiro-Junction Lability. Thermodynamic Aspects.

Three new and easily accessible chiral compounds, containing the pharmacophore 1,3,4-thiadiazoline nucleus joined by a spiro center to a monoalkyl (methyl or t-butyl) substituted cyclohexyl fragment, have been synthesized and fully characterized from the structural and stereochemical point of view. The formation of a spiro-cyclohexyl-thiadiazoline system (sCT) offered the rare opportunity to generate at room temperature both anancomeric structures, displaying alkyl groups bound to the cyclohexyl ring in equatorial position, and other quite stable stereoisomers in which the same alkyl moieties are, instead, inserted in axial position, even for the extreme case represented by the really bulky t-butyl group. DFT calculations led to a clear rationalization of such stereochemical behaviors, pointing out that in all cases they arise from the unexpected strong anancomeric character possessed by the sCT framework in its 4-acetyl substituted version. In consideration of the large number of substances in which the 1,3,4-thiadiazoline heterocycle has been found as the active pharmacophore, the results discussed in this work may provide solid bases to allow a rational design of new chiral bioactive spiro-thiadiazolines characterized by well-defined stereochemical structures and single anancomeric geometries.

Stereolability of chiral ruthenium catalysts with frozen NHC ligand conformations investigated by dynamic-HPLC.

The stereolability of chiral Hoveyda-Grubbs II type ruthenium complexes bearing N-heterocyclic carbene (NHC) ligands with Syn-phenyl groups on the backbone and Syn- or Anti-oriented o-tolyl N-substituents was studied by resorting to dynamic high-performance liquid chromatography (D-HPLC). A complete chromatographic picture of the involved stereoisomers (four for Anti- and two for Syn-complexes) was achieved at very low temperatures (-53°C and -40°C respectively), at which the NHC-Ru bond rotations were frozen out. Inspection of the chromatographic profiles recorded at higher temperatures revealed the presence of plateau zones between the couples of either Syn or Anti stereoisomers, attesting to the active interconversion between the eluted species. Such dynamic chromatograms were successfully simulated through procedures based on both theoretical plate and classical stochastic models. The good superimposition achieved between experimental and simulated chromatographic profiles allowed determination of the related isomerization energy barriers (ΔGisom (#) ), all derived by rotation around the NHC-Ru bond. The obtained diastereomerization barriers between the Anti isomers were found in very good agreement with those previously measured by experimental nuclear magnetic resonance (NMR) and assessed through Density Functional Theory (DFT) calculations. With the same approach, for the first time we also determined the enantiomerization barrier of the Syn isomer. Focused changes to the structure of complex Syn, studied by a molecular modeling approach, were found suitable to strongly reduce the stereolability arising from rotation around the NHC-Ru bond.

Determination of the Enantiomerization Barrier of the Residual Enantiomers of C3 -Symmetric Tris[3-(1-Methyl-2-Alkyl)Indolyl]Phosphane Oxides: Case Study of a Multitasking HPLC Investigation Based on an Immobilized Polysaccharide Stationary Phase.

The residual enantiomers of three tris-(3-indolyl)-phosphane oxides bearing different alkyl groups (methyl, ethyl or i-propyl) in position 2 of the indole rings constituting the blades were separated on the immobilized type Chiralpak IC column in polar organic and reversed-phase modes. The good enantioselectivity and versatility of the IC CSP allowed easy isolation of the enantiomerically highly enriched samples suitable for configurational stability studies. The enantiomerization barriers of residual phosphane oxides were evaluated both by off-column techniques (CD signal and enantiomeric purity decay kinetics) and by dynamic enantioselective high-performance liquid chromatography (HPLC).

Synthesis of a double-spanned resorc[4]arene via ring-closing metathesis and calculation of aggregation propensity.

Ring-closing metathesis (RCM) catalyzed by a second-generation Grubbs catalyst has been used to synthesize resorc[4]arenes 2b-5b starting from undecenyl resorc[4]arene 1b fixed in the cone conformation. X-ray diffraction analysis of the major metathesis product, 3b (50% yield), revealed a cavity-shaped architecture resembling a basket, endowed with a large intramolecular space (∼10 Å) and a strong propensity to self-assemble as a supramolecular trio of heterochiral dimers. This prompted us to investigate the aggregation propensity of basket 3b in THF/water solution by UV-visible spectroscopy. The cavitation Gibbs free-energy change (ΔΔGcav = 4.78 kcal mol(-1)) associated with the self-assembly of macrocycle 3b was calculated as a measure of the solvophobic interactions involved in the process.

Chirality in the absence of rigid stereogenic elements: steric and electronic effects on the configurational stability of C3 symmetric residual tris-aryl phosphanes.

Residual stereoisomers result whenever closed subsets of appropriately substituted interconverting isomers (the residual stereoisomers) are generated from a full set of stereoisomers under the operation of a favored stereomerization mechanism. In the case of the three-bladed propellers, differentiation of the edges of the blades and strict correlation in the motion of the rings are the prerequisites for the existence of residual stereoisomers. In these systems, the two-ring flip mechanism is the lowest energy process. It does not interconvert all possible conformational stereoisomers generated by helicity and the three-blade-hub rotors. In the case of C3 symmetric systems, two noninterconverting subgroups (the residual stereoisomers) are generated, each one constituted of quickly interconverting diastereoisomers. A series of tris-aryl phosphanes, structurally designed for existing as residual enantiomers or diastereoisomers, bearing substituents differing in size and electronic properties on the aryl rings, were synthesized and characterized. The configurational stability of residual phosphanes, evaluated by dynamic (1) H- and (31) P-NMR analysis and by dynamic enantioselective high-performance liquid chromatography (HPLC), was found 10 kcal mol(-1) lower than that shown by the corresponding phosphane-oxides. In accordance with the calculations, an unexpectedly low barrier for phosphorus pyramidal inversion was invoked as responsible for the scarce configurational stability of the residual tris-arylphosphanes.

Potential-driven chirality manifestations and impressive enantioselectivity by inherently chiral electroactive organic films.

The typical design of chiral electroactive materials involves attaching chiral pendants to an electroactive polyconjugated backbone and generally results in modest chirality manifestations. Discussed herein are electroactive chiral poly-heterocycles, where chirality is not external to the electroactive backbone but inherent to it, and results from a torsion generated by the periodic presence of atropisomeric, conjugatively active biheteroaromatic scaffolds, (3,3'-bithianaphthene). As the stereogenic element coincides with the electroactive one, films of impressive chiroptical activity and outstanding enantiodiscrimination properties are obtained. Moreover, chirality manifestations can be finely and reversibly tuned by the electric potential, as progressive injection of holes forces the two thianaphthene rings to co-planarize to favor delocalization. Such deformations, revealed by CD spectroelectrochemistry, are elastic and reversible, thus suggesting a breathing system.

Chiral Hydroxy Metabolite of Mebendazole: Analytical and Semi-Preparative High-Performance Liquid Chromatography Resolution and Chiroptical Properties.

Mebendazole (MBZ) is a benzimidazole carbamate anthelmintic used worldwide for the treatment and prevention of parasitic disorders in animals and humans. A large number of in vivo and in vitro studies have demonstrated that MBZ also has anticancer activity in multiple types of cancers. After oral administration, the phenylketone moiety of MBZ is rapidly reduced to the hydroxyl group to form the chiral hydroxy metabolite (MBZ-OH). To the best of our knowledge, there is no information in the literature on the stereochemical course of transformation and the anthelmintic and antitumor activity of individual enantiomers of MBZ-OH. In the present study, we describe in detail the direct HPLC resolution of MBZ-OH on a 100 mm × 4.6 mm Chirapak IG-3 column packed with 3 µm silica particles containing amylose (3-chloro-5-methylphenylcarbamate) as a selector. At 25 °C and using pure methanol as the mobile phase, the enantioseparation and resolution factors were 2.38 and 6.13, respectively. These conditions were scaled up at a semi-preparative scale using a 250 mm × 10 mm Chiralpak IG column to isolate multi-milligram amounts of both enantiomeric forms of the chiral metabolite. The chiroptical properties of the collected enantiomers were determined and, through a theoretical study, were related to the more stable conformations of MBZ-OH. The first and second eluted enantiomers were dextrorotatory and levorotatory, respectively, in dimethylformamide solution. Finally, by recording the retention factors of the enantiomers as the water content in the water-acetonitrile mobile phases was progressively varied, U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography retention mechanism on the Chirapak IG-3 chiral stationary phase.

Chiral supramolecular selectors for enantiomer differentiation in liquid chromatography.

Biochemical processes in living organisms rely on a plethora of molecular reactions and interactions involving chiral molecules, and these processes often show different responses to the enantiomers of exogenous or endogenous chemicals. The interaction of enantiomeric drugs with a target receptor is a paradigmatic example of chirality effects on general biological action and is directly related to the formation of a drug-receptor supramolecular complex. In particular the drug-receptor model can be used to explore the relation of chirality to at least three important issues encountered in supramolecular chemistry: complementarity, preorganization, and (enantio)selectivity. A detailed understanding of those factors governing enantioselectivity of biological receptors is facilitated by the study at molecular level of model systems with simplified structures that are amenable to physico-chemical investigations. Liquid chromatography on chiral stationary phases offers the opportunity to study enantioselective interactions between surface immobilized chiral selectors, viewed as minimalist mimics of macromolecular receptors, and a large set of chiral guests, under a variety of experimental conditions. Indeed, for a significant number of systems, close agreement has been found between retention data gathered by chromatography and association constants measured by spectroscopy in free solution. Chiral supramolecular selectors featuring highly preorganized, medium-sized macrocyclic structures are attractive in this context because they often afford high levels of enantioselectivity and yet have relatively low molecular complexity, thus facilitating the understanding of operative enantioselective recognition mechanisms from easily collected chromatographic data. In the present chapter we first illustrate the general principles of supramolecular chemistry and their integration into the design of liquid chromatographic systems, with particular focus on enantioselective variants based on chiral macrocyclic selectors of natural or synthetic origin. In the second part we discuss the elaboration of enantioselective recognition models from chromatographic data and how these models can be extended to and studied with non-chromatographic systems (free solution by nuclear magnetic resonance, gas phase by mass spectrometry).

Steric and electronic effects on the configurational stability of residual chiral phosphorus-centered three-bladed propellers: tris-aryl phosphane oxides.

A series of tris-aryl phosphane oxides existing as residual enantiomers or diastereoisomers with substituents on the aryl rings differing in size and electronic properties were synthesized and characterized. Their electronic properties were evaluated on the basis of their electrochemical oxidation and reduction potentials together with those of the corresponding "blade bromides" (i.e., the naphthalene derivatives displaying the same substitution pattern of the tris-naphthyl phosphane oxide blades, with a bromo substituent where the phosphorus atom is located) determined by CV. The residual stereoisomeric phosphane oxides were isolated in a stereochemically pure state and were found to be highly configurationally stable at room temperature (stereoisomerization barriers of about 27 kcal mol(-1)). The chiroptical properties of the residual stereoisomers and the assignments of absolute configuration are discussed. The configurational stability of residual tris-aryl phosphane oxides was found to be scarcely influenced by the electronic properties of the substituents present on the aromatic rings constituting the blades, while steric effects play the most relevant role. Detailed theoretical calculations are in agreement with the experimental results and also contribute to a rational interpretation of the stereodynamics of these systems.