RESUMEN
Ecdysteroids represent a large class of polyhydroxylated steroids which, due to their anabolic properties, are marketed as dietary supplements. Some ecdysteroids also act as important hormones in arthropods, where they regulate molting, development, and reproduction and many of these insects are miniature organisms that contain submicroliter levels of circulating biofluids Analysis of ecdysteroids is further complicated by their very low abundance, large fluctuations during development, and difficult access to a pooled sample, which is important for quantitative measurements. In this work, we propose a new method that overcomes the described difficulties and allows validated quantification of four ecdysteroids in minimal amounts of biological material. After methanolic extraction, detectability of the ecdysteroids is increased 16- to 20-fold by conversion to their 14,15-anhydrooximes. These are further purified by pipette tip solid phase extraction (PT-SPE) on a three-layer sorbent and subjected to HPLC-MS/MS analysis. Full validation was achieved using hemolymph from larvae of the firebug Pyrrhocoris apterus as a blank matrix and by the determination of ecdysteroids in a single Drosophila larva. The LLOQs for the four target ecdysteroids (20-hydroxyecdysone, ecdysone, makisterone A, and 2-deoxyecdysone) were 0.01; 0.1; 0.05; 0.025 pg·mL-1 (20; 200; 100; 50 fmol·mL-1) respectively, with very good accuracy, precision (RSD < 15%) and recoveries (96% - 119.9%).The general suitability of the new method was demonstrated by quantification of ecdysteroids in various biological materials including human serum.
RESUMEN
Fluorine labeling of ribonucleic acids (RNA) in conjunction with 19 Fâ NMR spectroscopy has emerged as a powerful strategy for spectroscopic analysis of RNA structure and dynamics, and RNA-ligand interactions. This study presents the first syntheses of 2'-OCF3 guanosine and uridine phosphoramidites, their incorporation into oligoribonucleotides by solid-phase synthesis and a comprehensive study of their properties. NMR spectroscopic analysis showed that the 2'-OCF3 modification is associated with preferential C2'-endo conformation of the U and G ribose in single-stranded RNA. When paired to the complementary strand, slight destabilization of the duplex caused by the modification was revealed by UV melting curve analysis. Moreover, the power of the 2'-OCF3 label for NMR spectroscopy is demonstrated by dissecting RNA pseudoknot folding and its binding to a small molecule. Furthermore, the 2'-OCF3 modification has potential for applications in therapeutic oligonucleotides. To this end, three 2'-OCF3 modified siRNAs were tested in silencing of the BASP1 gene which indicated enhanced performance for one of them. Importantly, together with earlier work, the present study completes the set of 2'-OCF3 nucleoside phosphoramidites to all four standard nucleobases (A, U, C, G) and hence enables applications that utilize the favorable properties of the 2'-OCF3 group without any restrictions in placing the modification into the RNA target sequence.