Green synthesis and two-step chromatographic separation of thiocanthal and thiocanthol: Two novel biologically active sulfur derivatives of oleocanthal and oleacein from extra virgin olive oil.

Oleocanthal and oleacein are the two major secoiridoids exclusively present in extra virgin olive oil (EVOO). Both compounds exert important pharmacological activities, including anti-inflammatory, anti-tumoral, neuro- and cardiovascular protective effects. Due to their enormous potential as possible drugs the extraction of these two bioactive natural products from EVOO has been extensively investigated in the last years and is generally supported by the use of organic chemistry. It is quite difficult to produce large quantities of these two compounds, either by organic solvent extraction and purification or by chemical synthesis, and furthermore organic processes such as cleaning, defatting, and extraction of EVOO pose a threat to the environment and are potentially harmful to workers. In this work we set up a novel aqueous extraction and isolation method from EVOO by transforming oleocanthal and oleacein into two water-soluble sulfonated products. The two derived compounds, here named thiocanthal and thiocanthol, were isolated by a two-step organic free chromatographic strategy, chemically characterized, and evaluated for their inhibitory activity on cyclooxygenase (COX). The results demonstrate that thiocanthal and thiocanthol possess anti-inflammatory effect, which is comparable to their precursors and higher than the well-known non-steroidal anti-inflammatory drug ibuprofen. Computational docking studies were performed to obtain and analyse putative models of the interaction of thiocanthal and thiocanthol with COX-1 and COX-2 binding sites. Predicted binding energy values suggested that both compounds might preferentially bind COX-2, which may have a significant pharmacological impact. Therefore, thiocanthal and thiocanthol, obtained by this novel green process, are extremely interesting both as new bioactive compounds per se and as lead compounds for the development of novel non-steroidal anti-inflammatory drugs (NSAIDs).

Assessing the performance of new chromatographic technologies for the separation of peptide epimeric impurities: the case of Icatibant.

The biopharmaceutical industry faces the challenge of efficiently characterising impurity profiles of therapeutical peptides, also due to their complex polar and ionisable attributes. This research explores the potential of advanced chromatographic techniques to address this challenge. The study compares dynamic electrostatic repulsion reversed phase (d-ERRP) to its counterparts (static ERRP and ion pair reversed phase IP-RP) in analysing Icatibant and its elusive epimeric impurity, [L-Arg]1-Icatibant and highlights its exceptional capabilities in generating symmetric peaks, mitigating the common tailing phenomenon, and serving as a steadfast guardian of column longevity. The result highlights d-ERRP as a pioneering tool in the domain of liquid chromatography, fostering its role as a reference technique for the analysis of therapeutic peptides.

Preparation of a high-density vinyl silica gel to anchor cysteine via photo-click reaction and its applications in hydrophilic interaction chromatography.

Modification of surface silanols is a topic of interest in the preparation of organo-functionalized silica particles. Herein, two novel contributions, mainly focused on separation science, were presented: i) the horizontal polymerization on silica surface by using the vinyl-triacetoxy silane and ii) a preparation of cysteine-based stationary phase via photo-click thiol-ene coupling. In the first derivatization step, the vinyl-triacetoxy silane was employed instead of conventional trichloro vinyl one. The one-step synthetic procedure needed imidazole as an activating agent in addition to the silica hydration. Modified silica particles offer a high loading of vinyl fragments and an extensive passivation of silanols such as to not require a subsequent end-capping procedure. The structural morphology of media was deeper characterized by combining infrared spectroscopy, solid-state nuclear magnetic resonance, and elemental analysis. A first application, the photo-click cysteine-based material was prepared by photo-click reaction and the stationary phase was employed in the separation of some conventional targets by hydrophilic interaction chromatography.

Expanding the Use of Dynamic Electrostatic Repulsion Reversed-Phase Chromatography: An Effective Elution Mode for Peptides Control and Analysis.

Bioactive peptides are increasingly used in clinical practice. Reversed-phase chromatography using formic or trifluoroacetic acid in the mobile phase is the most widely used technique for their analytical control. However, sometimes it does not prove sufficient to solve challenging chromatographic problems. In the search for alternative elution modes, the dynamic electrostatic repulsion reversed-phase was evaluated to separate eight probe peptides characterised by different molecular weights and isoelectric points. This technique, which involves TBAHSO4 in the mobile phase, provided the lowest asymmetry and peak width at half height values and the highest in peak capacity (about 200 for a gradient of 30 min) and resolution concerning the classic reversed-phase. All analyses were performed using cutting-edge columns developed for peptide separation, and the comparison of the chromatograms obtained shows how the dynamic electrostatic repulsion reversed-phase is an attractive alternative to the classic reversed-phase.

Online comprehensive hydrophilic interaction chromatography × reversed phase liquid chromatography coupled to mass spectrometry for in depth peptidomic profile of microalgae gastro-intestinal digests.

In this study, a comprehensive hydrophilic interaction chromatography × reversed phase coupled to high resolution mass spectrometry was developed for the peptide profile of microalgae formulations subjected to gastro-intestinal digestion. A BEH Amide column was employed in the first dimension, while a BIOshell ES-C18 Peptide in the second. As modulation interface, two trapping columns, in house packed with 1.9 µm fully porous monodisperse C18 particles characterized by high retention and efficiency, were tested and compared with SecurityGuard C18 cartridges, together with a dilution flow, to reduce first dimension mobile phase strength. The platform was coupled to both diode array detector and Orbitrap mass spectrometry. The developed setup provided high peak capacity (nc: 957) in only 60 min and a good orthogonality (A0: 0.70). The employment of the custom made C18 traps resulted in improved sensitivity (signal enhancement = 4) and a higher number of peptides detected (+58) especially of short lenght (≤ 6 aminoacids), with respect to the setup based on the security guard C18 traps. 184 phycocyanin-derived peptides were detected in Klamath and Spirulina gastro-intestinal digests, whose sequence and protein origin has been elucidated in detail by mass spectrometry. The results show the potential of the developed HILIC × RP-MS platform for in depth peptide mapping of microalgae and its possible application to highlight the products of gastro-intestinal digestion of other microalgae species.

A multidimensional liquid chromatography-tandem mass spectrometry platform to improve protein identification in high-throughput shotgun proteomics.

A new on-line multidimensional system for sequential trapping and individual elution and separation of peptides based on their molecular weight is described. By sequentially using two chemically different trapping columns, a polymethacrylate monolith and a packed C18 one, peptides from complex samples can be on-line trapped and divided into two fractions, containing respectively mainly medium-large peptides and smaller peptides. Then, by means of two switching valves working in parallel, the two fractions were individually separated by reversed phase chromatography. The whole gradient consisted of two subgradients, with the first one dedicated to the separation of smaller peptides and the second one to the separation of larger peptides. Such configuration allowed to identify up to 1476 proteins in a standard E. coli tryptic digest, with improved performance, increased average sequence coverage and reduced single unique peptide identifications compared to a conventional shotgun proteomics configuration comprising only the C18 trapping column and the analytical column.

Development of an improved online comprehensive hydrophilic interaction chromatography × reversed-phase ultra-high-pressure liquid chromatography platform for complex multiclass polyphenolic sample analysis.

In this study, an improved online comprehensive two-dimensional liquid chromatography platform coupled to tandem mass spectrometry was developed for the analysis of complex polyphenolic samples. A narrowbore hydrophilic interaction chromatography column (150 × 2.0 mm, 3.0 µm, cross-linked diol) was employed in the first dimension, while a reversed-phase column based on monodisperse sub-2 µm fully porous particles (50 × 3.0 mm, 1.9 µm d.p.) with high surface area (410 m2 /g) was employed in the second dimension. The combination of a trapping column modulation interface with the high retentive fully porous monodisperse reversed-phase column in the second dimension resulted in higher peak capacity values (1146 versus 867), increased sensitivity, sharper and more symmetrical peaks in comparison with a conventional loop-based method, with the same analysis time (70 min). The system was challenged against a complex polyphenolic extract of a typical Italian apple cultivar, enabling the simultaneous separation of multiple polyphenolic classes, including oligomeric procyanidins, up to degree of polymerization of 10. Hyphenation with an ion trap time-of-flight mass spectrometer led to the tentative identification of 121 analytes, showing how this platform could be a powerful analytical tool for the accurate profiling of complex polyphenolic samples.

Capillary methacrylate-based monoliths by grafting from/to γ-ray polymerization on a tentacle-type reactive surface for the liquid chromatographic separations of small molecules and intact proteins.

Capillary methacrylate-based monoliths were prepared for the high performance liquid chromatography (HPLC) separation of both small molecules and large biomolecules. An efficient grafting from/to synthetic approach was adopted introducing a network of activated sites in the inner wall surface using the new silanization agent (N-trimethoxysilylpropyl)-polyethylenimine. Copolymerization of lauryl methacrylate monomer and 1,6-hexanediol dimethacrylate cross-linker in the presence of porogenic solvents was obtained under continuous γ-ray exposure with high conversion yield. The morphology and porous structure of the resulting monoliths have been investigated by Scanning Electron Microscopy (SEM) and 1H NMR cryoporosimetry. By chromatographic investigation, the new capillary columns attested high kinetic performance (with efficiency larger than 100,000 theoretical plate/m for small molecules at optimum mobile phase linear velocity of about 0.5mm/s) and also excellent mechanical stability and repeatability. The new methacrylate-based monolithic capillary columns have been successfully employed for efficient reversed-phase separation of intact proteins and peptides.

Efficient organic monoliths prepared by γ-radiation induced polymerization in the evaluation of histone deacetylase inhibitors by capillary(nano)-high performance liquid chromatography and ion trap mass spectrometry.

New monolithic HPLC columns were prepared by γ-radiation-triggered polymerization of hexyl methacrylate and ethylene glycol dimethacrylate monomers in the presence of porogenic solvents. Polymerization was carried out directly within capillary (250-200 µm I.D.) and nano (100-75 µm I.D.) fused-silica tubes yielding highly efficient columns for cap(nano)-LC applications. The columns were applied in the complete separation of core (H2A, H2B, H3, and H4) and linker (H1) histones under gradient elution with UV and/or electrospray ionization (ESI) ion trap mass spectrometry (MS) detections. Large selectivity towards H1, H2A-1, H2A-2, H2B, H3-1, H3-2 and H4 histones and complete separation were obtained within 8 min time windows, using fast gradients and very high linear flow velocities, up to 11 mm/s for high throughput applications. The method developed was the basis of a simple and efficient protocol for the evaluation of post-translational modifications (PTMs) of histones from NCI-H460 human non-small-cell lung cancer (NSCLC) and HCT-116 human colorectal carcinoma cells. The study was extended to monitoring the level of histone acetylation after inhibition of Histone DeACetylase (HDAC) enzymes with suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved by the FDA for cancer therapy. Attractive features of our cap(nano)-LC/MS approach are the short analysis time, the minute amount of sample required to complete the whole procedure and the stability of the polymethacrylate-based columns. A lab-made software package ClustMass was ad hoc developed and used to elaborate deconvoluted mass spectral data (aligning, averaging, clustering) and calculate the potency of HDAC inhibitors, expressed through a Relative half maximal Inhibitory Concentration parameter, namely R_IC(50) and an averaged acetylation degree.

Extending the use of "Inverted Chirality Columns Approach" for enantiomeric excess determination in absence of reference samples: Application to a water-soluble camptothecin derivative.

The aim of the present study was to extend the use of the "Inverted Chirality Columns Approach (ICCA)" previously developed for the identification and quantitation of the trace enantiomer in highly enriched samples of the camptothecin (CPT) family of drugs to a novel water-soluble CPT derivative, namely namitecan (ST1968), currently undergoing phase I clinical trials as anticancer agent. Namitecan, identified from a series of hydrophilic 7-oxyiminomethyl derivatives, contains a free terminal amino group, which traditionally hampers the analysis under normal-phase HPLC conditions. Nevertheless, commercially available Pirkle-type chiral stationary phases (CSPs) available in both the enantiomeric forms (i.e., having the same bound selector with opposite configuration) mainly operate under normal-phase HPLC conditions. For this reason, namitecan was pre-column N-protected with isocyanates A-D and their sulfur analogues E-H to reduce its polarity by converting the amino group into a fragment compatible with the chiral recognition mechanism (i.e., ureido and thioureido groups). Once the optimal columns system and derivatizing agents were selected, an original enantioselective HPLC-MS/MS technique was developed on the Whelk-O1 CSPs.

Towards enzyme-like enantioselectivity in the gas phase: conformational control of selectivity in chiral macrocyclic dimers.

Conformational factors in self-assembled chiral tetraamide macrocycles control their gas-phase enantioselectivity towards the ethyl ester of naphthylalanine to levels typical of enzymes.

Gas-phase enantioselective reactions in noncovalent ion-molecule complexes.

Noncovalent diastereomeric ion-molecule complexes are produced in the gas phase and are ideal for the study of chiral recognition in the absence of complicating solvent and counterion effects. This review article describes the state-of-art in this field with special emphasis on the most recent mass spectrometric studies of the structure, dynamics, and reactivity of diastereomeric ion/molecule aggregates.

Dynamic HPLC of stereolabile iron(II) complexes on chiral stationary phases.

A series of chiral tris-(1,10)-phenanthroline iron(II) complexes have been resolved by HPLC on chiral stationary phases based on either cellulose tris-(3,5-dimethylphenylcarbamate) or teicoplanin. At sub ambient temperatures, baseline separation of the enantiomers was observed for five different iron(II) complexes featuring substituted phenanthroline ligands. Dynamic HPLC profiles were observed near or above room temperature, indicating on-column Delta/Lambda enantiomerization. Rate constants for the Delta/Lambda interconversion in free solution and during chromatography were obtained by thermal racemization experiments and by computer simulation of the HPLC dynamic plots, respectively.

Induced-fit in the gas phase: conformational effects on the enantioselectivity of chiral tetra-amide macrocycles.

The structure, stability, and reactivity of proton-bound diastereomeric [M x H x A]+ complexes between some amino acid derivatives (A) and several chiral tetra-amide macrocycles (M) have been investigated in the gas phase by ESI-FT-ICR and ESI-ITMS-CID mass spectrometry. The displacement of the A guest from the diastereomeric [M x H x A]+ complexes by reaction with the 2-aminobutane enantiomers (B) exhibits a distinct enantioselectivity with regards to the leaving amino acid A and, to a minor extent, to the amine reactant B. The emerging selectivity picture, discussed in the light of molecular mechanics calculations, provides compelling evidence that the most stable conformers of the selected chiral tetra-amide macrocycles M may acquire in the gas phase a different conformation by induced fit on complexation with some representative amino acid derivatives A. This leads to the coexistence in the gas phase of stable diastereomeric [M x H x A]+ eq-eq and ax-ax structures, in proportions depending on the configuration of A and M and characterized by different stability and reactivity toward the 2-aminobutane enantiomers. The enantioselectivity of the gas-phase A-to-B displacement in the diastereomeric [M x H x A]+ complexes essentially reflects the free energy gap between the homo- and heterochiral [M x H x A]+ complexes, except when the tetra-amidic host presents an additional macrocycle generated by a decamethylene chain. In this case, the measured enantioselectivity mostly reflects the stability difference between the relevant diastereomeric transition structures.

Synthesis and characterization of novel internal surface reversed-phase silica supports for high-performance liquid chromatography.

An original synthetic method was developed for the preparation of a family of six novel deactivated restricted-access materials (RAMs), belonging to the group of the internal surface reversed-phase (ISRP) supports. The supports (ISRP-RAM phases A-F) have an alkyl-chain (14 methylenes) with two embedded ureido groups bound only to the internal surfaces of the porous silica, and polyvinyl alcoholic groups (PVA, 100,000-->22,000 molecular weight) chemically bound to the external surfaces. The average pore diameters of the prepared ISRP-RAM supports, calculated by inverse size-exclusion chromatography, ranged between 49 A and 88 A, and were able to exclude macromolecules heavier than about 24000 Da (such as serum proteins) from the pores. The novel supports were designed for the determination of a semi-synthetic anticancer drug of the camptothecin family in human plasma, but they represent universal ISRP-RAM supports not limited to such class of compounds.

Combination of HPLC "inverted chirality columns approach" and MS/MS detection for extreme enantiomeric excess determination even in absence of reference samples. Application to camptothecin derivatives.

An original, extremely sensitive and selective HPLC-MS/MS technique for the identification and determination of the minor enantiomer in nonracemic mixtures, even when only one enantiomer is available as reference, is described. The method is based on the so-called "inverted chirality columns approach" (ICCA) and consists of the use of chiral stationary phases (CSPs) available in both enantiomeric forms: in fact, inversion of the elution order for a pair of enantiomers is observed in response to the change in column chirality. This offers two key advantages: first, it is possible to demonstrate the potential enantioselectivity of the system by generating a virtual racemate, and second, it permits the choosing of the right column chirality for trace determination. Combination with MS/MS detection affords high specificity allowing not only high sensitivity (down to 0.0025% of the minor enantiomer) but also unequivocal peak identification in complex mixtures. Applications to semisynthetic derivatives of camptothecin, endowed with antitumor activity, are reported. Moreover, applicability of ICCA is not limited to this class of molecules but generates universal support. Its use might also be extended to other classes of compounds by using other CSPs, available in both enantiomeric forms.

Exceptional gas-phase enantioselectivity of chiral tetramide macrocycles.

Diastereomeric proton-bound complexes between some phenylalanine derivatives (A) and chiral tetramide macrocycles (M) exhibit an uncommon enantioselectivity when reacting with the enantiomers of 2-aminobutanes in the gas phase (B). The measured enantioselectivity depends mainly on two distinct factors: (i) the configuration of the A guest; and (ii) the structure and the relative stability of isomeric [MHA]+ complexes. No significant effects of the B configuration are observed. The diastereomeric [MHA]+ complexes with A = 1-naphthylalanine ethyl ester exhibit the largest enantioselectivity factor ever measured in the gas phase (khomo/khetero = 0.046). The origin of such an exceptional enantioselectivity is mainly attributed to the relative stability of the diastereomeric [MHA]+ complexes, as demonstrated by the comparison of the kinetic results with those from collision-induced dissociation of the trimeric [M2HA]+ adducts and with computational evidence.

Enantiomerization study of some alpha-nitroketones by dynamic high-resolution gas chromatography.

The kinetics of the reversible enantiomer interconversion of 3-nitrobutan-2-one (1), 3-nitropentan-2-one (2), and 2-nitropentan-3-one (3) have been studied by dynamic high-resolution gas chromatography (DHRGC) by using a beta-cyclodextrin derivative chiral stationary phase; the process occurs via enolization of the keto forms. The DHRGC experiments involving the studied nitroketones and the chiral stationary phase show chromatographic profiles with a typical interconversion plateau in the temperature range between 130 and 160 degrees C. Computer simulation of the experimental chromatographic elution profiles was employed for the determination of rate constants and the corresponding enantiomerization barriers (k, DeltaG#(T), DeltaH#, and DeltaS#). The highly negative entropy of activation (DeltaS# values from -19 to -37 cal mol(-1) K(-1)) points to a transition state (TS) with large charge separation. The obtained results for 1-3 show the dramatic effect of an alpha-nitro-substituent on the rate of enolization of simple ketones, when compared with those for 3-chloro-2-butanone and 3-methyl-2-pentanone. To get some information on the separate contributions of the stationary liquid phase and the mobile gas phase on the studied process, some DFT ab initio calculations have been performed for the same compounds.

The first gas chromatographic resolution of carnitine enantiomers.

The enantiomers of carnitine are converted on-line in the injection port of a gas chromatograph into beta-hydroxy-gamma-butyrolactones and are separated on a derivatized beta-cyclodextrin chiral stationary phase.