Ilaria D'Acquarica

Synthesis of sugar-based silica gels by copper-catalysed azide-alkyne cycloaddition via a single-step azido-activated silica intermediate and the use of the gels in hydrophilic interaction chromatography

Novel sugar-based silica gels were prepared by exploiting the copper-catalysed azide-alkyne cycloaddition (CuAAC) of two different sugar alkynes, namely, ethynyl C-galactoside 1 and propargyl O-lactoside 2, with new single-step azido-activated silica gels. The fully characterised stationary phases were generally used for hydrophilic interaction chromatography (HILIC), with particular application in the stereoselective separation of monosaccharides. Dynamic HILIC (DHILIC) experiments were performed to evaluate the influence of mutarotation on the chromatographic peak shapes of two interconverting sugar anomers. The potential of such materials was shown in the separation of other highly polar compounds, including amino acids and flavonoids.

Direct chromatographic resolution of carnitine and O-acylcarnitine enantiomers on a teicoplanin-bonded chiral stationary phase

R-(-)-Carnitine (vitamin B(T)) plays an important role in human energy metabolism, by facilitating the transport of long-chained fatty acids across the mitochondrial membranes. Its (S)-enantiomer acts as a competitive inhibitor of carnitine acetyltransferase, causing depletion of the body R-(-)-carnitine stock. Consequently, the separation of carnitine enantiomers is very important both to study their biological activities and to control the enantiomeric purity of pharmaceutical formulations. In the present paper we describe an easy, fast and convenient procedure for the separation of the enantiomers of carnitine and O-acylcarnitines by enantioselective HPLC on a laboratory-made chiral column containing covalently bonded teicoplanin as selector. High enantioselectivity factors (alpha values ranging from 1.31 to 3.02) and short-time analyses characterize the analytical procedure; in addition, analytes are easily detected by evaporative light scattering with no need for preliminary derivatization. The effects of pH and ionic strength of the mobile phase and of the nature of the organic modifier on the enantioselective separations were also investigated.

Evaluation of the macrocyclic glycopeptide A-40,926 as a high- performance liquid chromatographic chiral selector and comparison with teicoplanin chiral stationary phase

A new macrocyclic antibiotic of the vancomycin family, referred to by its industrial designation as A-40,926, was bonded to 5 microm silica particles and utilised as a chiral stationary phase (CSP). Since A-40,926 is structurally related to teicoplanin, the A-40,926 CSP was compared to a commercially available teicoplanin CSP. A set of 28 chiral compounds, including amino-acids and related compounds, compounds with a ring containing the stereogenic centre, compounds bearing aromatic structures near their stereogenic centres and alcohols, was tested for enantioseparation on the two CSPs. The results are compared and discussed in terms of enantioselective Gibbs energy difference. The A-40,926 CSP was able to resolve one compound that was not resolved by the teicoplanin CSP. However, it could not separate four compounds that the teicoplanin CSP did separate. It is shown that the A-40,926 CSP is complementary to the teicoplanin CSP, thereby enlarging the number of enantiomers that can be separated by the macrocyclic glycopeptide based CSPs.

Isolation and structure elucidation of four new triterpenoid estersaponins from fruits of Pittosporum tobira AIT.

Abstract Four new triterpenoid estersaponins, tentatively designated as IIIA2 ( 1 ), IIIA3 ( 2 ), IIIB2 ( 3 ), and IIIC4 ( 4 ) have been isolated from fruits of Pittosporum tobira ait . and their structures elucidated by spectroscopic and chemical analyses. All the four compounds consist of an acylated pentacyclic triterpenoid aglycone which bears the same oligosaccharide portion. The crude saponin mixture (CIDI) was found to show significant in vitro and in vivo cytotoxicity in different human cancer cell lines.

3,5,3′‐Triiodo‐L‐thyronine enhances the differentiation of a human pancreatic duct cell line (hPANC‐1) towards a β‐cell‐Like phenotype

The thyroid hormone, 3,5,3′‐Triiodo‐L‐thyronine (T3), is essential for growth, differentiation, and regulation of metabolic functions in multicellular organisms, although the specific mechanisms of this control are still unknown. In this study, treatment of a human pancreatic duct cell line (hPANC‐1) with T3 blocks cell growth by an increase of cells in G0/G1 cell cycle phase and enhances morphological and functional changes as indicated by the marked increase in the synthesis of insulin and the parallel decrease of the ductal differentiation marker cytokeratin19. Expression analysis of some of the genes regulating pancreatic β‐cell differentiation revealed a time‐dependent increase in insulin and glut2 mRNA levels in response to T3. As last step of the acquisition of a β‐cell‐like phenotype, we present evidence that thyroid hormones are able to increase the release of insulin into the culture medium. In conclusion, our results suggest, for the first time, that thyroid hormones induce cell cycle perturbations and play an important role in the process of transdifferentiation of a human pancreatic duct line (hPANC‐1) into pancreatic‐β‐cell‐like cells. These findings have important implications in cell‐therapy based treatment of diabetes and may provide important insights in the designing of novel therapeutic agents to restore normal glycemia in subjects with diabetes.

Efficient enantiorecognition of ruthenium(II) complexes by silica-bound teicoplanin

Abstract A series of chiral tris-diimine ruthenium(II) complexes have been resolved by HPLC on a chiral stationary phase. The stationary phase ( CSP1 ) was prepared by covalent attachment of the glycopeptide antibiotic teicoplanin to isocyanate activated silica gel. CSP1 selectively retains the enantiomers of [Ru(L) 3 ] 2+ (L=2,2′-bypyridine (bpy), 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline), with a preference for the Δ isomer. For the mixed-ligand complexes [Ru(bpy) 2 pztr] + and [Ru(bpy) 2 pytr] + (Hpztr=3-(pyrazin-2-yl)-1,2,4-triazole, Hpytr=3-(pyridin-2-yl)-1,2,4-triazole), where the triazole unit is bound to the metal centre either through the N 2 or the N 4 nitrogen of the ring, CSP1 discriminates both the enantiomers and the regioisomers. Diastereo- and enantioselective association was also observed between CSP1 and the stereoisomers of the dinuclear complex ((Ru(bpy) 2 ) 2 bpt] 3+ (Hbpt=3,5-bis(pyridin-2-yl)-1,2,4-triazole), with differences in binding affinities of 1.4 kJ/mol between the homochiral enantiomers.

On-column epimerization of dihydroartemisinin: An effective analytical approach to overcome the shortcomings of the International Pharmacopoeia monograph

We developed a cryo-HPLC/UV method for the simultaneous determination of artemisinin (1), alpha-dihydroartemisinin (2alpha), beta-dihydroartemisinin (2beta), and a ubiquitous thermal decomposition product of 2 (designated as diketoaldehyde, 3), starting from the International Pharmacopoeia monograph on dihydroartemisinin. The method takes for the first time the on-column epimerization process of 2 into consideration. Chromatographic separation was obtained under reversed-phase conditions on a Symmetry C18 column (3.5 microm particle size) with a mobile phase consisting of acetonitrile-water 60:40 (v/v), delivered at 0.60-1.00 ml/min flow-rates, with ultraviolet detection at low wavelength (lambda = 210 nm). Low temperatures (T = 0-10 degrees C) were selected on the grounds of a diastereoselective dynamic HPLC (DHPLC) study performed at different temperatures, aimed at identifying the best experimental conditions capable of minimizing the on-column interconversion process.

Chromatographic resolution and enantiomerization barriers of axially chiral 1-naphthamides

The interconverting enantiomers of axially chiral 1-naphthamides have been resolved by HPLC on the Whelk-O1 chiral stationary phase, at temperatures ranging from +50 C to -60 C. For each compound, a combination of eluent flow rate and column temperature was chosen to generate exchange-broadened chromatographic profiles. Computer simulation of these profiles gave the rate constants for the on-column R-S interconversion process, occurring by C A R -CO bond rotation. For one 1-naphthamide with different N-alkyl substituents, both C Ar -CO and CO-N rotations were detected by low temperature HPLC on the same chiral stationary phase.

High yield and optical purity in biocatalysed acylation of trans-2-phenyl- 1-cyclohexanol with Candida rugosa lipase in non-conventional media

Abstract Candida rugosa lipase (CRL) shows high enantioselectivity toward (1 R ,2 S )-(−)- trans -2-phenyl-1-cyclohexanol enantiomer in acetylation reaction employing vinyl acetate as acyl donor. Attempts to improve reaction yields have pointed out that supercritical CO 2 is the best reaction medium in the studied biocatalytic process. In these conditions an immobilised lipase from Candida rugosa is able to quantitatively resolve racemate with e.e. p 100%.

Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype

Hedgehog (Hh) inhibitors have emerged as valid tools in the treatment of a wide range of cancers. Indeed, aberrant activation of the Hh pathway occurring either by ligand-dependent or -independent mechanisms is a key driver in tumorigenesis. The smoothened (Smo) receptor is one of the main upstream transducers of the Hh signaling and is a validated target for the development of anticancer compounds, as underlined by the FDA-approved Smo antagonist Vismodegib (GDC-0449/Erivedge) for the treatment of basal cell carcinoma. However, Smo mutations that confer constitutive activity and drug resistance have emerged during treatment with Vismodegib. For this reason, the development of new effective Hh inhibitors represents a major challenge for cancer therapy. Natural products have always represented a unique source of lead structures in drug discovery, and in recent years have been used to modulate the Hh pathway at multiple levels. Here, starting from an in house library of natural compounds and their derivatives, we discovered novel chemotypes of Hh inhibitors by mean of virtual screening against the crystallographic structure of Smo. Hh functional based assay identified the chalcone derivative 12 as the most effective Hh inhibitor within the test set. The chalcone 12 binds the Smo receptor and promotes the displacement of Bodipy-Cyclopamine in both Smo WT and drug-resistant Smo mutant. Our molecule stands as a promising Smo antagonist able to specifically impair the growth of Hh-dependent tumor cells in vitro and in vivo and medulloblastoma stem-like cells and potentially overcome the associated drug resistance.