The use of proton transfer reaction mass spectrometry for high throughput screening of terpene synthases

In this work, we introduce the application of proton transfer reaction mass spectrometry (PTR-MS) for the selection of improved terpene synthase mutants. In comparison with gas chromatography mass spectrometry (GC-MS)-based methods, PTR-MS could offer advantages by reduction of sample preparation steps and analysis time. The method we propose here allows for minimal sample preparation and analysis time and provides a promising platform for the high throughput screening (HTS) of large enzyme mutant libraries. To investigate the feasibility of a PTR-MS-based screening method, we employed a small library of Callitropsis nootkatensis valencene synthase (CnVS) mutants. Bacterial cultures expressing enzyme mutants were subjected to different growth formats, and headspace terpenes concentrations measured by PTR-Qi-ToF-MS were compared with GC-MS, to rank the activity of the enzyme mutants. For all cultivation formats, including 96 deep well plates, PTR-Qi-ToF-MS resulted in the same ranking of the enzyme variants, compared with the canonical format using 100 mL flasks and GC-MS analysis. This study provides a first basis for the application of rapid PTR-Qi-ToF-MS detection, in combination with multi-well formats, in HTS screening methods for the selection of highly productive terpene synthases.     

Heptagon-Containing Nanographene Embedded into [10]Cycloparaphenylene

We report the synthesis and characterization of a novel type of nanohoop, consisting of a cycloparaphenylene derivative incorporating a curved heptagon-containing π-extended polycyclic aromatic hydrocarbon (PAH) unit. We demonstrate that this new macrocycle behaves as a supramolecular receptor of curved π-systems such as fullerenes C₆₀ and C₇₀, with remarkably large binding constants (ca. 10⁷ M⁻¹), as estimated by fluorescence measurements. Nanosecond and femtosecond spectroscopic analysis show that these host-guest complexes are capable of quasi-instantaneous charge separation upon photoexcitation, due to the ultrafast charge transfer from the macrocycle to the complexed fullerene. These results demonstrate saddle-shaped PAHs with dibenzocycloheptatrienone motifs as structural components for new macrocycles displaying molecular receptor abilities and versatile photochemical responses with promising electron-donor properties in host-guest complexes.     

Gadolinium chelate coated gold nanoparticles as contrast agents for both X-ray computed tomography and magnetic resonance imaging

Functionalized gold nanoparticles were applied as contrast agents for both in vivo X-ray and magnetic resonance imaging. These particles were obtained by encapsulating gold cores within a multilayered organic shell which is composed of gadolinium chelates bound to each other through disulfide bonds. The contrast enhancement in MRI stems from the presence of gadolinium ions which are entrapped in the organic shell, whereas the gold core provides a strong X-ray absorption. This study revealed that these particles suited for dual modality imaging freely circulate in the blood vessels without undesirable accumulation in the lungs, spleen, and liver.     

Transient ordering in a quasi-two-dimensional liquid near freezing

We report the results of a theoretical study of locally ordered fluctuations in a quasi-two-dimensional colloid fluid. The fluctuations in the equilibrium state are monitored by the aperture cross-correlation function of radiation scattered by the fluid, as calculated from molecular dynamics simulations of near hard spheres with diameter sigma confined between smooth hard walls. These locally ordered fluctuations are transient; their decay can be monitored as a function of the time between the cross-correlated scattered radiation signals, but only the single-time cross-correlated signals are discussed in this paper. Systems with thicknesses less than two hard sphere diameters were studied. For wall separation H in the range 1 sigma/=1.57 sigma, hexagonal fluctuations persist in the dense liquid up to H=1.75 sigma, and fluctuations with square ordered symmetry, that of the solid to which the liquid freezes, only emerge at densities approximately 2% below freezing. For H=1.8 sigma and 1.85 sigma, hexagonal ordered flucuations are no longer found, and the square ordered fluctuations dominate the dense liquid region as the system freezes into a two layer square solid. For H=1.9 sigma and 1.95 sigma, where the liquid freezes into a two layer hexagonal solid, both square and hexagonal ordered fluctuations are observed. At lower densities, the ordered fluctuations only exhibit square symmetry. Hexagonal ordered fluctuations appear at densities approximately 7% below freezing and become more dominant as the density is increased, but the square ordered fluctuations persist until the system is converted into the solid.     

Analysis of protamine peptides in insulin pharmaceutical formulations by capillary electrophoresis

Protamines are a group of highly basic peptides that are sometimes added to insulin formulations to prolong the pharmacological action. In this study, different methods were investigated to identify protamine in insulin formulations. Capillary electrophoresis in aqueous and non‐aqueous media was tested to separate these peptides with very close amino acid sequences. Different buffers (phosphate or formate, both acidified) and various additives (principally negatively charged and neutral surfactants) were investigated to optimize peptide separation. Finally, a micellar electrokinetic capillary chromatography method using a capillary of 120 cm effective length and an aqueous background electrolyte made up of 100 mM phosphate buffer (pH 2) and 50 mM Thesit® gave the best results, providing the separation of the four major protamine peptides within 25 min.     

Synthesis and evaluation of caged Garcinia xanthones

Inspired by the combination of unique structure and potent bioactivities exhibited by several family members of the caged Garcinia xanthones, we developed a synthesis of simplified analogues that maintain the overall caged motif. The caged structure of these compounds was constructed via a site-selective Claisen/Diels-Alder reaction cascade. We found that the fully substituted caged structure, in which are included the C18 and C23 geminal methyl groups, is necessary to maintain bioactivity. Analogue had comparable activity to the natural products of this family, such as gambogic acid. These compounds exhibit cytotoxicity in a variety of tumor cell lines at low micromolar concentrations and were found to induce apoptosis in HUVE cells. In addition, studies with HL-60 and HL-60/ADR cells indicate that these compounds are not affected by the mechanisms of multidrug resistance, conferred by P glycoprotein expression, typical of relapsed cancers and thus represent a new and potent pharmacophore.