Toward amide-modified RNA: synthesis of 3'-aminomethyl-5'-carboxy-3',5'-dideoxy nucleosides

Recent discovery of RNA interference has reinvigorated the interest in chemically modified RNA. Chemical approaches may be used to optimize properties of small interfering RNAs, such as thermal stability, cellular delivery, in vivo half-life, and pharmacokinetics. From this perspective, amides as neutral and hydrophobic internucleoside linkages in RNA are highly interesting modifications that so far have not been tested in RNA interference. Amides are remarkably good mimics of the phosphodiester backbone of RNA and can be prepared using a relatively straightforward peptide coupling chemistry. The synthetic challenge that has hampered the progress in this field has been preparation of monomeric building blocks for such couplings, the nucleoside amino acid equivalents. Herein, we report two synthetic routes to enantiomerically pure 3'-aminomethyl-5'-carboxy-3',5'-dideoxy nucleosides, monomers for preparation of amide-modified RNA. Modification of uridine, a representative of natural nucleosides, using nitroaldol chemistry gives the target amino acid in 16 steps and 9% overall yield. The alternative synthesis starting from glucose is somewhat less efficient (17 steps and 6% yield of 3'-azidomethyl-5'-carboxy-3',5'-dideoxy uridine), but provides easier access to modified nucleosides having other heterocyclic bases. The syntheses developed herein will allow preparation of amide-modified RNA analogues and exploration of their potential as tools and probes for RNA interference, fundamental biochemistry, and bio- and nanotechnology.     

Synthesis and properties of RNA analogues having amides as interuridine linkages at selected positions

Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3'-CH(2)CONH-5' and AM2: 3'-CH(2)NHCO-5') at selected positions have been synthesized and the thermal stability of duplexes formed by these analogues with complementary RNA fragments has been evaluated by UV melting experiments. Two series of oligomers with either 2'-OH or 2'-OMe vicinal to the amide linkages were studied. Monomeric synthons (3' and 5'-C amines and carboxylic acids) were synthesized as follows: For synthesis of the AM1 analogue, the known sequence of radical allylation followed by the cleavage of the double bond was adopted. For synthesis of the AM2 analogue, novel routes via addition of nitromethane followed by conversion of the nitro function to either amino or carboxyl groups were developed. Coupling of monomeric amines and carboxylic acids followed by protecting group manipulation and phosphonylation gave dimeric 3'-hydrogenphosphonate building blocks for oligonucleotide synthesis. Monomeric model compounds having 3'-amide and 2'-OH or 2'-OMe groups were also prepared and their conformational equilibrium was determined by (1)H NMR. The AM1 and AM2 models showed equal preferences for the North conformers (at 40 degrees C, 88-89% with 2'-OH, and 92-93% with 2'-OMe). At physiological salt concentration (0.1 M NaCl) the duplexes between AM1 modified oligonucleotides and RNA had stability similar to unmodified RNA-RNA duplexes (Delta t(m)= -0.2 to +0.7 degrees C per modification). However, the AM2 modification resulted in substantial stabilization of duplexes: Delta t(m)= +1 to +2.4 degrees C per modification compared to all RNA. A 2'-O-methyl vicinal to the AM2 linkage further increased the duplex stability. Our results suggest that RNA analogues having amide internucleoside bonds are very promising candidates for medicinal applications.     

The Potential Use of Herbal Fingerprints by Means of HPLC and TLC for Characterization and Identification of Herbal Extracts and the Distinction of Latvian Native Medicinal Plants

The growing market of herbal medicines, the increase in international trade in Latvia, and the lack of adequate analytical methods have raised the question of the potential use of herbal fingerprinting methods. In this study, high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) methods were developed for obtaining chromatographic fingerprints of four taxonomically and evolutionary different medicinal plants (Hibiscus sabdariffa L., Calendula officinalis L., Matricaria recutita L., Achillea millefolium L.). Retention time shifting, principal component analysis (PCA), hierarchical cluster analysis (HCA), and orthogonal projections to latent structures (OPLS) analysis were used to improve and analyze the obtained fingerprints. HPLC data detection at 270 nm was determined superior to 360 nm for the distinction of medicinal plants and used data alignment method significantly increased similarity between samples. Analyzed medicinal plant extracts formed separate, compact clusters in PCA, and the results of HCA correlated with the evolutionary relationships of the analyzed medicinal plants. Herbal fingerprinting using chromatographic analysis coupled with multivariate analysis has a great potential for the identification of medicinal plants as well as for the distinction of Latvian native medicinal plants.     

Determination of trimethylamine‐N‐oxide in combination with l‐carnitine and γ‐butyrobetaine in human plasma by UPLC/MS/MS

An ultra‐high‐performance liquid chromatography–mass spectrometry (UPLC/MS/MS) method was developed and validated for the quantification of trimethylamine‐N‐oxide (TMAO) simultaneously with TMAO‐related molecules l ‐carnitine and γ‐butyrobetaine (GBB) in human blood plasma. The separation of analytes was achieved using a Hydrophilic interaction liquid chromatography (HILIC)‐type column with ammonium acetate–acetonitrile as the mobile phase. TMAO determination was validated according to valid US Food and Drug Administration guidelines. The developed method was successfully applied to plasma samples from healthy volunteers. Copyright © 2015 John Wiley & Sons, Ltd.