Chromatography and mass spectrometry in the study of structure and dynamics of chiral molecules

Part A Dynamic high performance liquid chromatography on chiral stationary phases is a consolidated technique that allows the investigation of chiral molecules with labile stereogenic elements that interconvert very quickly at room temperature and result in stereoinversion processes occurring on the time scale of the separation process. Kinetic parameters for on-column interconversions can be extracted from exchange-deformed experimental peak profiles by computer simulation. The technique has been used in a wide range of temperatures and is complementary in scope to dynamic nuclear magnetic resonance spectroscopy. Here we report, in the first part, the separation of the enantiomers of benzodiazepines, a class of molecules whose conformational enantiomers interconvert not only through a single bond rotation but also via “ring-flip” inversion. The second part concerns the first HPLC resolution of the conformational enantiomers of tri-O-thymotide (TOT) a macrocyclic trilactone existing in fast-exchanging multiple chiral conformations. Variable chromatography on brush type stationary phases showed dynamic features due to on-column interconversions of TOT. Part B Chiral recognition is a branch of chemistry aimed at understanding the reactivity as well as the size- and shape-specificity of non-covalent interactions between molecular aggregates formed by chiral species. Mass spectrometry (MS) is a powerful tool for investigating chiral recognition in the gas phase in the absence of perturbing environmental phenomena and discriminating and even quantifying chiral species by interaction with chiral reference molecules. A small library of synthetic receptors was prepared by macrocyclization of complementary A and B fragments to yield A2B2 macrocycles, where A are activated forms of isophthalic acid derivatives and B are chiral, C2 symmetric 1,2-diamines derived from 1,2-diphenylethylendiamine. Their enantioselectivity was investigated by ESI-MS and revealed large enantioselectivities towards the enantiomers of aminoacids and peptide guests. The stability of the complexes increases with the size of guests and with large aromatic portions on the guest. Part C This part of my academic program was developed at the University of California, Davis, under the supervision of prof. Carlito Lebrilla, during the last period of my Ph.D. activity. An important goal in proteomic is to quantify the profile changes of protein abundances in biological systems. Quantifying these changes is a key to understand changes in cell state at a molecular level. In the last years label-free quantitation using MRM (multiple reaction monitoring), which correlates the mass spectrometric signal of intact proteolytic peptides with the relative or absolute protein quantity, is become a powerful tool for accurate quantitation. Here we report a label-free method to quantify proteins and their glycoforms in biologic fluids (milk, feces and urine) by MRM, including the selection of proteotryptic peptides and the optimization and validation of transitions

Effect of destoned olive cake on the aromatic profile of cows’ milk and dairy products: comparison of two techniques for the headspace aroma profile analysis

The aim of this study, carried out within the NOVOROD project (PSR Campania 2007-2013 Misura 124 HC), was to evaluate the aromatic profile of milk and dairy products of dairy cows supplemented with destoned olive cake (DOC). Two techniques for the headspace aroma profile analysis: the thermal desorption by gas chromatography with a mass selective (GC/MS) detector and the electronic nose were compared. The trial was performed into a dairy cow farm. Ten dairy cows were allotted into two homogenous groups: control and experimental. Animals received the same diet, while in the experimental group the concentrate supplementation (15% dry matter) was replaced with DOC. After two weeks of adaptation, animals were fed with the experimental diet for other 15 days. During the experimental period, the milk was collected and cumulatively processed in Caciotta cheese (a soft cheese, 25 days ripened) and in Semicotto cheese (hard cheese, 3 months ripened) for 13 times for each group. Milk and cheese volatile organic compounds (VOCs) were extracted and concentrated with dynamic headspace method and determined by GC/MS. Statistical analysis of data, expressed in arbitrary units (a.u.=peak area × 10–6) and allotted in compound classes, was carried out by ANOVA. All data collected of VOCs were processed with principal component analysis (PCA). Milk and cheese samples were comparatively analysed with the electronic nose, fitted with a tenmetal oxide sensors electronic device. PCA has been used for the evaluation of the pattern data. Our results show that the use of DOC, as an unconventional feed for livestock, has no effect on the aromatic profile of both milk and dairy products. In fact, no significant differences were found in both milk and dairy products between the two groups using the GC/MS method. Also the discriminating analysis carried out by electronic nose did not show significant differences between the two groups. The two compared techniques seem to lead to the same result

Dynamic behavior of clobazam on high-performance liquid chromatography chiral stationary phases

Clobazam, a 1,5-benzodiazepin-2,4-dione, is a chiral molecule because its ground state conformation features a nonplanar seven-membered ring lacking reflection symmetry elements. The two conformational enantiomers of clobazam interconvert at room temperature by a simple ring-flipping process. Variable temperature HPLC on the Pirkle type (R)-N-(3,5-dinitronenzoyl)phenylglycine and (R,R)-Whelk-O1 chiral stationary phases (CSPs) allowed us to separate for the first time the conformational enantiomers of clobazam and to observe peak coalescence-decoalescence phenomena due to concomitant separation and interconversion processes occurring on the same time scale. Clobazam showed temperature dependent dynamic high-performance liquid chromatography (HPLC) profiles with interconversion plateaus on the two CSPs indicative of on-column enantiomer interconversion. (enantiomerization) in the column temperature range between Tcol = 10°C and Tcol = 30°C, whereas on-column interconversion was absent at temperature close to or lower than Tcol = 5°C. Computer simulation of exchange-deformed HPLC profiles using a program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At Tcol = 20°C the averaged enantiomerization barriers, ΔG(‡) , for clobazam were found in the range 21.08-21.53 kcal mol(-1) on the two CSPs. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this article are consistent with the literature data measured by DNMR at higher temperatures and in different solvents

Dynamic high performance liquid chromatography on chiral stationary phases. Low temperature separation of the interconverting enantiomers of diazepam, flunitrazepam, prazepam and tetrazepam

Diazepam and the structurally related 1,4-benzodiazepin-2-ones tetrazepam, prazepam and flunitrazepam are chiral molecules because they adopt a ground state conformation featuring a non-planar seven membered ring devoid of any reflection-symmetry element. The two conformational enantiomers of this class of benzodiazepines interconvert rapidly at room temperature by a simple ring flipping process. Low temperature HPLC on the Whelk-O1 chiral stationary phase allowed us to separate the conformational enantiomers of diazepam and of the related 1,4-benzodiazepin-2-ones, under conditions where the interconversion rate is sufficiently low, compared to the chromatographic separation rate. Diazepam, tetrazepam and prazepam showed temperature dependent dynamic HPLC profiles with interconversion plateaus indicative of on-column enantiomer interconversion (enantiomerization) in the temperature range between −10 °C and −35 °C, whereas for flunitrazepam on-column interconversion was observed at temperatures between −40 °C and −66 °C. Simulation of exchange-deformed HPLC profiles using a computer program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At −20 °C the enantiomerization barriers, ΔG≠, for diazepam, prazepam and tetrazepam were determined to be in the range 17.6–18.7 kcal/mol. At −55 °C ΔG≠ for flunitrazepam was determined to be in the 15.6–15.7 kcal/mol range. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this paper call for a reinterpretation of previously published results on the HPLC behavior of diazepam on chiral stationary phases

The dynamic chromatographic behavior of tri-o-thymotide on HPLC chiral stationary phases

The interconverting stereoisomers of tri-o-thymotide have been separated by HPLC on chiral stationary phases and the temperature dependence of the dynamic chromatographic patterns has been interpreted in terms of exchange between enantiomeric propeller and helical conformations. Computed low energy structures and CD spectra were used in absolute configuration assignment

Degradation of post-consumer PLA. Hydrolysis of polymeric matrix and oligomers stabilization in aqueous phase

Degradation of post-consumer PLA to lactic acid was analysed in order to assess the economic feasibility of the PLA chemical recycling process. Hydrolysis of PLA, in batch reactor, was analysed in the temperature range of 443-473K, under autogenous pressure and a constant PLA to water ratio (equal to approximately 0.11 by weight), without the use of a catalyst. The experimental results suggest that the complete degradation of PLA can be obtained using relatively low reaction-times with the production of a mixture containing the monomer and traces of the dimer of lactic acid. The overall process was modelled using a two-step process: bulk degradation of PLA (in the solid or molten phase) with the solubilisation of low molecular weight oligomers, and their subsequent hydrolysis in water (stabilization). The model describes the trend of oligomer concentrations in the aqueous phase and PLA conversion as a function of time with both high accuracy and agreement with experimental results

Total Synthesis of (±)-Kuwanol E

The total synthesis of the Diels–Alder-type adducts (±)-kuwanol E and the heptamethyl ether derivative of (±)-kuwanon Y has been accomplished via a convergent strategy involving 2′-hydroxychalcone 6 or 9 and dehydroprenylstilbene 7, in nine steps. The synthesis features, as a key step, a Lewis acid-mediated biomimetic intermolecular Diels–Alder [4+2] cycloaddition for the construction of the cyclohexene skeleton with three stereogenic centers. Notably, the endo/exo diastereoselectivity of the reaction proved to be temperature-controlled

3,5-Dinitrobenzoyl-9-amino-9-deoxy-9-epiquinine as Pirkle-Anion Exchange Hybrid-Type Chiral Selector in High-Performance Liquid Chromatography

A new chiral stationary phase was designed by introducing 9-amino-9-deoxy-9-epiquinine, one of the most versatile organocatalysts in asymmetric synthesis, as chiral scaffold. The derivatization of its amino group with the 3,5-dinitrobenzoyl (DNB) fragment provided hydrogen bonding and π–π donor/acceptor systems in addition to the quinoline and quinuclidine moieties having two nitrogen atoms with different basicities. The selector offers multiple interaction sites in both typical of the Pirkle-type phases and classical of weak-anion-exchanger phases. The immobilization step took place through thiol-ene addition onto 3-mercaptopropyl-silica gel and gave a grafting density of 180 μmol of chiral selector per gram of silica. A silica with reduced particle size (Daisogel silica, pore size 120 Å, particle size 2.5 μm, and specific surface area 343 m2 g−1) has been employed to improve the efficiency and the speed of separations. The chiral stationary phase was packed in a small format column (50 × 4.6 mm) that allowed, by van Deemter analysis, 180,000 plates/m and approximately 5.1 μm of plate height. The ability of chiral discrimination was then studied with more than 30 test compounds using both polar-organic and normal phase conditions. In polar-organic mode, N-protected amino acids, α-aryloxy carboxylic acids, as well the non-steroidal anti-inflammatory profens were analyzed. Interesting results were obtained in normal phase elution, where the chiral selector behaves like a Pirkle-type stationary phase. Aryl amides, esterified DNB-amino acids, benzodiazepines, and binaphthol were well resolved with a very good peak symmetry and in short analysis time (mainly in less than 5 min)