Towards combining backbone and sugar constraint in 3'-3' bis-phosphonate tethered 2'-4' bridged LNA oligonucleotide trimers.

Therapeutic oligonucleotides are chemically modified to enhance their drug-like properties - including binding affinity for target RNA. Many nucleic acid analogs that enhance RNA binding affinity constrain the furanose sugar in an RNA-like sugar pucker. The improvements in binding affinity result primarily from increased off-rates with minimal effects on on-rates for hybridization. To identify alternate chemical modification strategies that can modulate on- and off-rates for oligonucleotide hybridization, we hypothesized that extending conformational restraint across multiple nucleotides could modulate hybridization kinetics by restricting rotational freedom of the sugar-phosphate backbone. As part of that effort, we recently reported that using hydrocarbon tethers to bridge adjacent phosphodiester linkages as phosphonate tethered bridges can pre-organize nucleic acids in conformations conducive for Watson-Crick base-pairing and modulate hybridization kinetics. In this report, we describe the synthesis of locked nucleic acid (LNA) trimers linked through alkylphosphonate tethers which restrict conformation of the furanose sugar in addition to restricting conformational mobility of the sugar-phosphate backbone across three nucleotide units.

Metabolomics during the spontaneous fermentation in cocoa (Theobroma cacao L.): An exploraty review.

Spontaneous fermentation is a process that depends on substrates' physical characteristics, crop variety, and postharvest practices; it induces variations in the metabolites that are responsible for the taste, aroma, and quality. Metabolomics makes it possible to detect key metabolites using chemometrics and makes it possible to establish patterns or identify biomarker behaviors under certain conditions at a given time. Therefore, sensitive and highly efficient analytical techniques allow for studying the metabolomic fingerprint changes during fermentation; which identify and quantify metabolites related to taste and aroma formation of an adequate processing time. This review shows that studying metabolomics in spontaneous fermentation permits the characterization of spontaneous fermentation in different stages. Also, it demonstrates the possibility of modulating the quality of cocoa by improving the spontaneous fermentation time (because of volatile aromatic compounds formation), thus standardizing the process to obtain attributes and quality that will later impact the chocolate quality.

Evaluation of Phosphorus and Non-Phosphorus Neutral Oligonucleotide Backbones for Enhancing Therapeutic Index of Gapmer Antisense Oligonucleotides.

The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O-isopropyl and O-tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.

Rheological, bioactive properties and sensory preferences of dark chocolates with partial incorporation of Sacha Inchi (Plukenetia volubilis L.) oil.

We studied the effect of substituting partially, cocoa butter (CB) with Sacha Inchi (Plukenetia volubilis L.) oil (SIO) on rheology, bioactive properties, and sensory preferences in potentially functional chocolate. For this 70% dark chocolates were prepared and the CB was substituted with 1.5%, 3%, and 4.5% of SIO. Hardness and viscosity of the SIO-chocolates were significantly reduced compared to the control (5451 ± 658 g; 17.01 ± 0.94 Pa s, respectively). Total phenolic content remained constant while the antioxidant capacity increased up to IC50 of 2.48 ± 0.10 as the content of SIO increased. The Casson yield stress and Casson plastic viscosity decreased as the amount of SIO increased. Chocolates with 4.5% SIO had a similar color, better glossiness, preferable snap attributes, and were more accepted (7.50 ± 0.08) compared to the control (p < 0.05), measured with a hedonic scale. Then, SIO can improve the bioactive properties of dark chocolates obtaining a potentially functional food with acceptable physicochemical characteristics. SIO can be considered as a new cocoa butter equivalent.

Site-specific incorporation of 5'-methyl DNA enhances the therapeutic profile of gapmer ASOs.

We recently showed that site-specific incorporation of 2'-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5'-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5'-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2'-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5'-Me and R-5'-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5'-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.

Flux Synthesis of a Metal Carbide Hydride Using Anthracene As a Reactant.

La15(FeC6)4H was synthesized from the reaction of iron and anthracene in La/Ni eutectic flux. Anthracene was the source of both the carbon and hydrogen in the product. The structure of this metal carbide hydride features hydride ions in tetrahedral interstitial sites surrounded by lanthanum ions, which was confirmed by single-crystal neutron diffraction studies. The trigonal planar FeC6 units in which the central iron atom is coordinated by three ethylenide groups are similar to those found in La3.67FeC6, a previously reported compound that is formed in the absence of a hydride source. Magnetic susceptibility data confirm that the iron sites do not have magnetic moments. Density of states calculations indicate that La15(FeC6)4H is metallic and is stabilized by the incorporation of hydride anions.

Understanding the effect of controlling phosphorothioate chirality in the DNA gap on the potency and safety of gapmer antisense oligonucleotides.

Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically modulate interactions with RNase H1 as evidenced by changes in RNA cleavage patterns, these were insufficient to improve the overall therapeutic profile. We also found that controlling PS chirality of only two PS linkages in the DNA gap was sufficient to modulate RNase H1 cleavage patterns and combining these designs with simple modifications such as 2'-OMe to the DNA gap resulted in dramatic improvements in therapeutic index. However, we were unable to demonstrate improved potency relative to the stereorandom parent ASO or improved safety over the 2'-OMe gap-modified stereorandom parent ASO. Overall, our work shows that while controlling PS chirality can modulate RNase H1 cleavage patterns, ASO sequence and design are the primary drivers which determine the pharmacological and toxicological properties of gapmer ASOs.

Synthesis and Antitumor Activity Evaluation of Compounds Based on Toluquinol.

Encouraged by the promising antitumoral, antiangiogenic, and antilymphangiogenic properties of toluquinol, a set of analogues of this natural product of marine origin was synthesized to explore and evaluate the effects of structural modifications on their cytotoxic activity. We decided to investigate the effects of the substitution of the methyl group by other groups, the introduction of a second substituent, the relative position of the substituents, and the oxidation state. A set of analogues of 2-substituted, 2,3-disubstituted, and 2,6-disubstituted derived from hydroquinone were synthesized. The results revealed that the cytotoxic activity of this family of compounds could rely on the hydroquinone/benzoquinone part of the molecule, whereas the substituents might modulate the interaction of the molecule with their targets, changing either its activity or its selectivity. The methyl group is relevant for the cytotoxicity of toluquinol, since its replacement by other groups resulted in a significant loss of activity, and in general the introduction of a second substituent, preferentially in the para position with respect to the methyl group, was well tolerated. These findings provide guidance for the design of new toluquinol analogues with potentially better pharmacological properties.

In Situ Neutron Diffraction Studies of the Metal Flux Growth of Ba/Yb/Mg/Si Intermetallics.

The Ba/Yb/Mg/Si intermetallic system is extremely complex, with four competing structurally related compounds forming from reactions of barium, ytterbium, and silicon in magnesium-rich Mg/Al flux. In addition to the previously reported Ba2Yb0.9Mg11.1Si7, Ba5Yb2Mg17Si12, and Ba20Yb5Mg61Si43, a new compound has been found. Ba6Yb1.84Mg18.16Si13 crystallizes in the P6̅ space group, with the Zr6Ni20P13 structure type. Quenching experiments and in situ neutron powder diffraction studies were carried out to determine the reaction parameters that favor particular products. Under slow-cooling conditions, Ba5Yb2Mg17Si12 precipitates from the flux at 800 °C. A faster cooling rate of an identical reaction results in the formation of single crystals of Ba20Yb5Mg61Si43 in the flux at 640 °C. This indicates that the crystallization of products in this metal flux reaction does not involve precipitation and interconversion of different phases but instead depends on the rate of cooling across the supersaturated metastable zone in this system.

Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins.

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.