Heterocycle-modified 2'-Deoxyguanosine Nucleolipid Analogs Stabilize Guanosine Gels and Self-assemble to Form Green Fluorescent Gels.

Nucleoside-lipid conjugates are very useful supramolecular building blocks to construct self-assembled architectures suited for biomedical and material applications. Such nucleoside derivatives can be further synthetically manipulated to endow additional functionalities that could augment the assembling process and impart interesting properties. Here, we report the design, synthesis and self-assembling process of multifunctional supramolecular nucleolipid synthons containing an environment-sensitive fluorescent guanine. The amphiphilic synthons are composed of an 8-(2-(benzofuran-2-yl)vinyl)-guanine core and alkyl chains attached to 3'-O and 5'-O-positions of 2'-deoxyguanosine. The 2-(benzofuran-2-yl)vinyl (BFV) moiety attached at the C8 position of the nucleobase adopted a syn conformation about the glycosidic bond, which facilitated the self-assembly process through the formation of a G-tetrad as the basic unit. While 3',5'-diacylated BFV-modified dG analog stabilized the guanosine hydrogel by hampering the crystallization process and imparted fluorescence, BFV-modified dGs containing longer alkyl chains formed a green fluorescent organogel, which transformed into a yellow fluorescent gel in the presence of a complementary non-fluorescent cytidine nucleolipid. The ability of the dG analog containing short alkyl chains to modulate the mechanical property of a gel, and interesting fluorescence properties and self-assembling behavior exhibited by the dG analogs containing long alkyl chains in response to heat and complementary base underscore the potential use of these new supramolecular synthons in material applications.

Nucleic Acid Conformation Influences Postsynthetic Suzuki-Miyaura Labeling of Oligonucleotides.

Chemoselective transformations that work under physiological conditions have emerged as powerful tools to label nucleic acids in cell-free and cellular environments. However, detailed studies investigating the influence of nucleic acid conformation on the performance of such chemoselective nucleic labeling methods are less explored. Given that nucleic acids adopt complex structures, it is highly important to study the scope of the chemical modification method in the context of nucleic acid conformations. Here we report a systematic study on the effect of local conformation on the postsynthetic Suzuki-Miyaura functionalization of human telomeric (H-Telo) DNA repeat oligonucleotide (ON) sequences, which form multiple G-quadruplex (GQ) structures. 5-Iodo-2'-deoxyuridine (IdU)-modified H-Telo ONs were synthesized by the solid-phase method, and when subjected to Suzuki-Miyaura cross-coupling reaction, its efficiency was found to depend on the type of conformation and the position of IdU label in different loops of the GQ structure. IdU-labeled GQs gave better yields as compared to single-stranded random coil structures. However, the IdU-labeled duplex under different ionic conditions did not undergo the coupling reaction. Further, using this method, we directly installed an environment-sensitive fluorescent probe, which photophysically reported the formation as well as distinguished different GQ topologies of telomeric repeat. Collectively, this systematic study underscores the influence of nucleic acid conformation, which has to be taken into account when establishing postsynthetic chemoselective functionalization strategies.

Posttranscriptional Suzuki-Miyaura Cross-Coupling Yields Labeled RNA for Conformational Analysis and Imaging.

Chemical labeling of RNA by using chemoselective reactions that work under biologically benign conditions is increasingly becoming valuable in the in vitro and in vivo analysis of RNA. Here, we describe a modular RNA labeling method based on a posttranscriptional Suzuki-Miyaura coupling reaction, which works under mild conditions and enables the direct installation of various biophysical reporters and tags. This two-part procedure involves the incorporation of a halogen-modified UTP analog (5-iodouridine-5'-triphosphate) by a transcription reaction. Subsequent posttranscriptional coupling with boronic acid/ester substrates in the presence of a palladium catalyst provides access to RNA labeled with (a) fluorogenic environment-sensitive nucleosides for probing nucleic acid structure and recognition, (b) fluorescent probes for microscopy, and (3) affinity tags for pull-down and immunoassays. It is expected that this method could also become useful for imaging nascent RNA transcripts in cells if the nucleotide analog can be metabolically incorporated and coupled with reporters by metal-assisted cross-coupling reactions.

Self-assemblies of nucleolipid supramolecular synthons show unique self-sorting and cooperative assembling process.

The inherent control of the self-sorting and co-assembling process that has evolved in multi-component biological systems is not easy to emulate in vitro using synthetic supramolecular synthons. Here, using the basic component of nucleic acids and lipids, we describe a simple platform to build hierarchical assemblies of two component systems, which show an interesting self-sorting and co-assembling behavior. The assembling systems are made of a combination of amphiphilic purine and pyrimidine ribonucleoside-fatty acid conjugates (nucleolipids), which were prepared by coupling fatty acid acyl chains of different lengths at the 2'-O- and 3'-O-positions of the ribose sugar. Individually, the purine and pyrimidine nucleolipids adopt a distinct morphology, which either supports or does not support the gelation process. Interestingly, due to the subtle difference in the order of formation and stability of individual assemblies, different mixtures of supramolecular synthons and complementary ribonucleosides exhibit a cooperative and disruptive self-sorting and co-assembling behavior. A systematic morphological analysis combined with single crystal X-ray crystallography, powder X-ray diffraction (PXRD), NMR, CD, rheological and 3D X-ray microtomography studies provided insights into the mechanism of the self-sorting and co-assembling process. Taken together, this approach has enabled the construction of assemblies with unique higher ordered architectures and gels with remarkably enhanced mechanical strength that cannot be derived from the respective single component systems.

Post-transcriptional labeling by using Suzuki-Miyaura cross-coupling generates functional RNA probes.

Pd-catalyzed C-C bond formation, an important vertebra in the spine of synthetic chemistry, is emerging as a valuable chemoselective transformation for post-synthetic functionalization of biomacromolecules. While methods are available for labeling protein and DNA, development of an analogous procedure to label RNA by cross-coupling reactions remains a major challenge. Herein, we describe a new Pd-mediated RNA oligonucleotide (ON) labeling method that involves post-transcriptional functionalization of iodouridine-labeled RNA transcripts by using Suzuki-Miyaura cross-coupling reaction. 5-Iodouridine triphosphate (IUTP) is efficiently incorporated into RNA ONs at one or more sites by T7 RNA polymerase. Further, using a catalytic system made of Pd(OAc)2 and 2-aminopyrimidine-4,6-diol (ADHP) or dimethylamino-substituted ADHP (DMADHP), we established a modular method to functionalize iodouridine-labeled RNA ONs in the presence of various boronic acid and ester substrates under very mild conditions (37°C and pH 8.5). This method is highly chemoselective, and offers direct access to RNA ONs labeled with commonly used fluorescent and affinity tags and new fluorogenic environment-sensitive nucleoside probes in a ligand-controlled stereoselective fashion. Taken together, this simple approach of generating functional RNA ON probes by Suzuki-Miyaura coupling will be a very important addition to the resources and tools available for analyzing RNA motifs.

Standard manufacturing procedure for Laghu Malini Vasanta Rasa in context of Bhavana (levigation).

INTRODUCTION: Laghu Malini Vasanta (LMV) Rasa is a well-known Vasanta Kalpa (formulation). As per reference of Yoga Ratnakara, Rasaka and Maricha are chief ingredients in 2:1 proportion. Bhavana (levigation) is said to be given first with Navaneeta (freshly prepared cow's butter) and then with Nimbu Swarasa (lemon juice) until Ghrita Vimukta stage is reached. Quantity of Bhavana Dravya (levigating media) and duration of levigation are not mentioned. AIMS: To develop standard manufacturing procedure of LMV Rasa. MATERIALS AND METHODS: The study was carried out in two stages - preparation of Yashada Bhasma and preparation of LMV Rasa and its tablet. A pilot study was carried out to fix quantity of cow's butter as levigation media. Based on results of the pilot study, LMV Rasa was prepared in two groups, that is, LMV 50 (LMV Rasa - prepared with weight of freshly prepared butter in 50% quantity of total ingredients) and LMV 75 (LMV Rasa prepared with weight of freshly prepared butter in 75% quantity of total ingredients). Complete drying of levigated mass and minimal spreading of fatty portion on filter paper was considered as the end point of levigation. Tablets of both samples were prepared by adding Pippali and honey in it and analyzed for their quality control parameters. RESULTS: Twenty-eight hours duration of repeated levigation was required in LMV 50 which was prolonged up to 48 h in batch carried out in rainy season. In LMV 75, comparatively maximum duration of 54 h was required for levigation which was prolonged in the rainy season to 88 h. In both groups, lemon juice required for repeated levigation was 10 times of quantity of butter added initially. CONCLUSION: From pharmaceutical point of view, preparation of LMV Rasa tablets with quantity of butter in 50% of total ingredients is more convenient.