Improved Metabolic Stability and Silencing Efficacy of siRNAs with Phosphorothioate Linkage-Free, GalNAc-Conjugated Sense Strands Containing Morpholino-LNA Modifications.

To ensure specificity, loading of the sense strand of small interfering RNAs (siRNAs) into RISC must be inhibited. We show here that siRNAs with 5'- and 6'-morpholino LNA residues or 6'-OH-LNA at the 5' terminus of a fully phosphodiester sense strand resulted in metabolically stable siRNAs with a potency and a duration of action in mice that were greater than those of an siRNA in which the 5' terminus of the sense strand has two terminal phosphorothioate linkages and regular LNA.

Conformation matters: siRNAs with antisense strands with 5'-()-vinyl-phosphonate-α-L-LNA elicit stronger RNAi-mediated gene silencing than those with 5'-()-vinyl-phosphonate-LNA.

Conformationally constrained nucleotides, LNA or α-L-LNA, at the 5' terminus of the antisense strand impeded gene silencing of small interfering RNA (siRNA) by hindering phosphorylation, thereby deterring loading into the RNA-induced silencing complex. Installation of a phosphate mimic, ()-vinyl phosphonate (VP), improved activity considerably. Gene silencing was more efficient when the antisense strand of the siRNA was modified with 5'-VP-α-L-LNA, which adopts a C3'- (south) conformation, than when the antisense strand was modified with 5'-VP-LNA, which adopts a C3'- (north) pucker.

Single-Stranded Hairpin Loop RNAs (loopmeRNAs) Potently Induce Gene Silencing through the RNA Interference Pathway.

Synthetic small interfering RNAs conjugated to trivalent -acetylgalactosamine (GalNAc) are clinically validated drugs for treatment of liver diseases. Incorporation of phosphorothioate linkages and ribose modifications are necessary for stability, potency, and duration of pharmacology. Although multiple alternative siRNA designs such as Dicer-substrate RNA, shRNA, and circular RNA have been evaluated in vitro and in preclinical studies with some success, clinical applications of these designs are limited as it is difficult to incorporate chemical modifications in these designs.

Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs.

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'--methyl or 2'-fluoro ribose modifications.

Rational optimization of siRNA to ensure strand bias in the interaction with the RNA-induced silencing complex.

To ensure specificity of small interfering RNAs (siRNAs), the antisense strand must be selected by the RNA-induced silencing complex (RISC). We have previously demonstrated that a 5'-morpholino-modified nucleotide at the 5'-end of the sense strand inhibits its interaction with RISC ensuring selection of the desired antisense strand. To improve this antagonizing binding property even further, a new set of morpholino-based analogues, Mo2 and Mo3, and a piperidine analogue, Pip, were designed based on the known structure of Argonaute2, the slicer enzyme component of RISC.

Targeted siRNA lipid nanoparticles for the treatment of KRAS-mutant tumors.

K-RAS is a highly relevant oncogene that is mutated in approximately 90% of pancreatic cancers and 20-25% of lung adenocarcinomas. The aim of this work was to develop a new anti-KRAS siRNA therapeutic strategy through the engineering of functionalized lipid nanoparticles (LNPs). To do this, first, a potent pan anti-KRAS siRNA sequence was chosen from the literature and different chemical modifications of siRNA were tested for their transfection efficacy (KRAS knockdown) and anti-proliferative effects on various cancer cell lines.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary -acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural β-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with -, and -glycosides with both α- and β-anomeric linkages, -glycosides with β-anomeric linkage, and the glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method.

Gemdimethyl Peptide Nucleic Acids (α/β/γ-PNA): E/Z-Rotamers Influence the Selectivity in the Formation of Parallel/Antiparallel -PNA:DNA/RNA Duplexes.

Peptide nucleic acids (PNAs) consist of an aminoethylglycine () backbone to which the nucleobases are linked through a tertiary amide group and bind to complementary DNA/RNA in a sequence-specific manner. The flexible backbone has been the target for several chemical modifications of the PNA to improve its properties such as specificity, solubility, etc. PNA monomers exhibit a mixture of two rotamers (Z/E) arising from the restricted rotation around the tertiary amide N-CO bond.

A Chemical Approach to Introduce 2,6-Diaminopurine and 2-Aminoadenine Conjugates into Oligonucleotides without Need for Protecting Groups.

We report a simple, postsynthetic strategy for synthesis of oligonucleotides containing 2,6-diaminopurine nucleotides and 2-aminoadenine conjugates using 2-fluoro-6-amino-adenosine. The strategy allows introduction of 2,6-diaminopurine and other 2-amino group-containing ligands. The strongly electronegative 2-fluoro deactivates 6-NH obviating the need for any protecting group on adenine, and simple aromatic nucleophilic substitution of fluorine makes reaction with aqueous NH or R-NH feasible at the 2-position.

Aminooxy Click Chemistry as a Tool for Bis-homo and Bis-hetero Ligand Conjugation to Nucleic Acids.

An aminooxy click chemistry (AOCC) strategy was used to synthesize nucleoside building blocks for incorporation during solid-support synthesis of oligonucleotides to enable bis-homo and bis-hetero conjugation of various biologically relevant ligands. The bis-homo aminooxy conjugation leads to bivalent ligand presentation, whereas the bis-hetero conjugation allows the placement of different ligands with either the same or different chemical linkages. This facile synthetic methodology allows introduction of two different ligands with different biological functions simultaneously.

Gem-dimethyl peptide nucleic acid (α/β/γ--PNA) monomers: synthesis and the role of -substituents in preferential stabilisation of /-rotamers.

The flexible backbone of aminoethylglycine (aeg) PNA upon substitution becomes sterically constrained to enable conformational pre-organization for preferential binding to DNA or RNA. The bulky gem-dimethyl (gdm) substituent on carbons adjacent to the t-amide sidechain either at Cα (glycyl) or Cβ/Cγ (aminoethylene) sides may influence the Z/E rotamer ratio arising from a restricted rotation around the t-amide bond. Employing 2D NMR (NOESY), it is shown here that the Cα-gdm-PNA-T monomer exhibits exclusively the Z-rotamer, while the Cβ-gdm-PNA-T monomer shows only the E-rotamer.

1,3-Oxazine-2-one derived dual-targeted molecules against replicating and non-replicating forms of Mycobacterium tuberculosis.

The high mortality rate and increasing prevalence of resistant Mtb are the major concerns for the Tuberculosis (TB) treatment in this century. To curtail the prevalence of resistant Mtb, we have prepared 1,3-oxazine-2-one based dual targeted molecules. Compound 67 and 68 were found to be equally active against replicating and non-replicatiing form of Mtb (MIC 3.

2-Aryl benzazole derived new class of anti-tubercular compounds: Endowed to eradicate mycobacterium tuberculosis in replicating and non-replicating forms.

The high mortality rate and the increasing prevalence of Mtb resistance are the major concerns for the Tuberculosis (TB) treatment in this century. To counteract the prevalence of Mtb resistance, we have synthesized 2-aryl benzazole based dual targeted molecules. Compound 9m and 9n were found to be equally active against replicating and non-replicating form of Mtb (MIC 1.

Structural Design and Synthesis of Bimodal PNA That Simultaneously Binds Two Complementary DNAs To Form Fused Double Duplexes.

Bimodal PNAs are new PNA constructs designed to bind two different cDNA sequences synchronously to form double duplexes. They are synthesized on solid phase using sequential coupling and click reaction to introduce a second base in each monomer at C via alkyltriazole linker. The ternary bimodal PNA:DNA complexes show stability higher than that of individual duplexes.

A convenient route to synthesize -(isobutyryl)-9-(carboxymethyl)guanine for -PNA backbone.

Synthesis of exclusive -(isobutyryl)-9-(carboxymethyl)guanine, an important moiety for peptide nucleic acid synthesis has been reported through a high-yielding reaction scheme starting from 6-chloro-2-amino purine. Crystal structures of two intermediates confirmed the formation of 9-regioisomer. This new synthetic route can potentially replace the conventional tedious method with moderate overall yield.

Self-Assembly of Diphenylalanine-Peptide Nucleic Acid Conjugates.

The synthesis and self-assembled nanostructures of a series of nucleopeptides (NPs) derived from the dipeptide Phe-Phe and the peptide nucleic acid unit which are covalently attached through an amide or a triazole linker are described. Depending on the variables such as protecting groups, linkers, and nucleobases, spherical nanoparticles were observed through scanning electron microscopy and high-resolution transmission electron microscopy images, and the porous nature of representative NPs was corroborated by carboxyfluorescein entrapment. Hydrophobic substituents on different sites of NPs and solvents employed for peptide self-assembly played a crucial role for corresponding morphologies.

Sulfonic nucleic acids (SNAs): a new class of substrate mimics for ribonuclease A inhibition.

Sulfonic nucleic acids were identified as inhibitors of ribonuclease A (RNase A). The incorporation of a strongly acidic group (sulfonic, -SOH) at the 3'-end of pyrimidine nucleosides thymidine and uridine was prompted by the low inhibition constant (K) values recorded for carboxymethylsulfonyl (-SOCHCOH) and -COH functionalized nucleosides. It was envisaged that the sulfonic acid-modified pyrimidines would bind effectively with the positively charged P site of ribonuclease A.

New archetypes in self-assembled Phe-Phe motif induced nanostructures from nucleoside conjugated-diphenylalanines.

During the last two decades, the molecular self-assembly of the short peptide diphenylalanine (Phe-Phe) motif has attracted increasing focus due to its unique morphological structure and utility for potential applications in biomaterial chemistry, sensors and bioelectronics. Due to the ease of their synthetic modifications and a plethora of available experimental tools, the self-assembly of free and protected diphenylalanine scaffolds (H-Phe-Phe-OH, Boc-Phe-Phe-OH and Boc-Phe-Phe-OMe) has unfurled interesting tubular, vesicular or fibrillar morphologies. Developing on this theme, here we attempt to examine the effect of structure and properties (hydrophobic and H-bonding) modifying the functional C-terminus conjugated substituents on Boc-Phe-Phe on its self-assembly process.

1,5-Disubstituted 1,2,3-triazole linked disaccharides: Metal-free syntheses and screening of a new class of ribonuclease A inhibitors.

1,5-Regioisomeric triazole linked disaccharides have been synthesized and screened for their inhibitory properties against ribonuclease A (RNase A). The angular constraint-driven 'crescent shaped' inhibitors accommodated themselves into the enzyme active site. An improved enzyme inhibition was observed with increased H-bonding ability of polar functional groups in the modified disaccharides.

Approaches towards the development of chimeric DPP4/ACE inhibitors for treating metabolic syndrome.

Designing drug candidates exhibiting polypharmacology is one of the strategies adopted by medicinal chemists to address multifactorial diseases. Metabolic disease is one such multifactorial disorder characterized by hyperglycaemia, hypertension and dyslipidaemia among others. In this paper we report a new class of molecular framework combining the pharmacophoric features of DPP4 inhibitors with those of ACE inhibitors to afford potent dual inhibitors of DPP4 and ACE.

Inhibition of copper-mediated aggregation of human γD-crystallin by Schiff bases.

Protein aggregation, due to the imbalance in the concentration of Cu and Zn ions is found to be allied with various physiological disorders. Copper is known to promote the oxidative damage of β/γ-crystallins in aged eye lens and causes their aggregation leading to cataract. Therefore, synthesis of a small-molecule 'chelator' for Cu with complementary antioxidant effect will find potential applications against aggregation of β/γ-crystallins.

Attenuation of Mycobacterium species through direct and macrophage mediated pathway by unsymmetrical diaryl urea.

Tuberculosis is a major threat for mankind and the emergence of resistance strain of Mycobacterium tuberculosis (Mtb) against first line antibiotics makes it lethal for human civilization. In this study, we have synthesized different diaryl urea derivatives targeting the inhibition of mycolic acid biosynthesis. Among the 39 synthesized molecules, compounds 46, 57, 58 and 86 showed MIC values ≤ 10 μg/ml against H37Rv and mc6030 strains.

Carboxymethylsulfonylated Ribopyrimidines: Rational Design of Ribonuclease A Inhibitors Employing Chemical Logic.

Hydrolysis of RNA by ribonuclease A crucially depends on the participation of the 2'-OH group as well as the positioning of the internucleotide bond at two different sites of the enzyme. Therefore, ribopyrimidines were modified with -SO2CH2CO2H, an acidic functional group, which was expected to interact with the phosphate binding site. These ribonucleosides were designed to understand the influence of the 2'-OH group of these inhibitors on ribonuclease A inhibition along with the effect of the -SO2CH2CO2H group.

A Rationally Designed Thymidine-Based Self-Assembled Monolayer on a Gold Electrode for Electroanalytical Applications.

A self-assembled monolayer (SAM) of 1-(3,5-epidithio-2,3,5-trideoxy-β-D-threo-pentofuranosyl)thymine (EFT) on a gold electrode was prepared and characterized by Raman spectral and electrochemical measurements. Voltammetric and electrochemical impedance measurements show that the SAM of EFT on a Au electrode impedes the electron-transfer reaction. The SAM of EFT was successfully used for the voltammetric sensing of urate in neutral solution.

Ribonuclease A inhibition by carboxymethylsulfonyl-modified xylo- and arabinopyrimidines.

A group of acidic nucleosides were synthesized to develop a new class of ribonuclease A (RNase A) inhibitors. Our recent study on carboxymethylsulfonyl-modified nucleosides revealed some interesting results in RNase A inhibition. This positive outcome triggered an investigation of the role played by secondary sugar hydroxy groups in inhibiting RNase A activity.