Chirality matters: stereo-defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo.

A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5' end and Sp diastereomers at the 3' end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5' end and Sp isomer at the 3' end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner.

siRNAs containing 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2'-F-NMC NTPs.

We recently reported the synthesis of 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2'-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2'-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5' phosphate, suggesting that the 2'-F-NMC is a poor substrate for 5' kinases. In mice, the 2'-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2'-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5'-phosphate mimic 5'-(E)-vinylphosphonate was attached to the 2'-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2'-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2'-F-NMC. Finally, the 5'-triphosphate of 2'-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.

Kinetic study of the binding of triplex-forming oligonucleotides containing partial cationic modifications to double-stranded DNA.

Several triplex-forming oligonucleotides (TFOs) partially modified with 2'-O-(2-aminoethyl)- or 2'-O-(2-guanidinoethyl)-nucleotides were synthesized and their association rate constants (kon) with double-stranded DNA were estimated by UV spectrophotometry. Introduction of cationic modifications in the 5'-region of the TFOs significantly increased the kon values compared to that of natural TFO, while no enhancement in the rate of triplex DNA formation was observed when the modifications were in the middle and at the 3'-region. The kon value of a TFO with three adjacent cationic modifications at the 5'-region was found to be 3.4 times larger than that of a natural one. These results provide useful information for overcoming the inherent sluggishness of triplex DNA formation.

Synthesis of oligonucleotides containing N,N-disubstituted 3-deazacytosine nucleobases by post-elongation modification and their triplex-forming ability with double-stranded DNA.

A phosphoramidite of a 2'-O,4'-C-methylene-bridged nucleoside, bearing 4-(2,4,6-triisopropylbenzenesulfonyloxy)pyridin-2-one as a nucleobase precursor, was synthesized and introduced into an oligonucleotide. Treatment with various secondary amines after elongating the oligonucleotide on an automated DNA synthesizer enabled facile and mild conversion of the precursor into the corresponding N,N-disubstituted 3-deazacytosine nucleobases. The evaluation of the triplex-forming ability of the synthesized oligonucleotides with double-stranded DNA showed that the nucleobase possessing the (3S)-3-guanidinopyrrolidine moiety can recognize a CG base pair with high sequence-selectivity and binding-affinity.

Synthesis and Biophysical Properties of Phosphorodiamidate Piperidino Oligomers.

We report the synthesis of piperidino nucleoside phosphoramidates functionalized with uracil, cytosine, guanine, and adenine and their incorporation into oligomers. High-performance liquid chromatography analyses demonstrated that a phosphorodiamidate piperidino oligomer (PPO) is more lipophilic than a phosphorodiamidate morpholino oligomer (PMO) of the same tetrameric sequence. A PMO containing piperidino residues formed duplexes with both DNA and RNA, and the PPO had higher stability at endosomolytic pH and higher hydrophobicity than the PMO.

RNAs Containing Carbocyclic Ribonucleotides.

Toward the goal of evaluation of carbocyclic ribonucleoside-containing oligonucleotide therapeutics, we developed convenient, scalable syntheses of all four carbocyclic ribonucleotide phosphoramidites and the uridine solid-support building block. Crystallographic analysis confirmed configuration and stereochemistry of these building blocks. Duplexes with carbocyclic RNA (car-RNA) modifications in one strand were less thermodynamically stable than duplexes with unmodified RNA. However, circular dichroism spectroscopy indicated that global conformations of the duplexes containing car-RNAs were similar to those in the unmodified duplexes.

Synthesis of 2'-Fluorinated Northern Methanocarbacyclic (2'-F-NMC) Nucleosides and Their Incorporation Into Oligonucleotides.

This article describes chemical synthesis of 2'-fluorinated Northern methanocarbacyclic (2'-F-NMC) nucleosides and phosphoramidites, based on a bicyclo[3.1.0]hexane scaffold bearing all four natural nucleobases (U, C, A, and G), and their incorporation into oligonucleotides by solid-supported synthesis. This synthesis starts from commercially available cyclopent-2-en-1-one to obtain the fluorinated carbocyclic pseudosugar intermediate (S.13), which can be converted to the uridine intermediate by condensation with isocyanate, followed by cyclization, and to adenine and guanine precursors by microwave-assisted reactions. All four 2'-F-NMC phosphoramidites are synthesized from S.13 in a convergent approach, and the monomers are used for synthesis of 2'-F-NMC-modified oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of fluorinated carbocyclic pseudosugar intermediate Basic Protocol 2: Preparation of 2'-F-NMC uridine and cytidine phosphoramidites Basic Protocol 3: Preparation of 2'-F-NMC adenosine phosphoramidite Basic Protocol 4: Preparation of 2'-F-NMC guanosine phosphoramidite Basic Protocol 5: Synthesis of oligonucleotides containing 2'-F-NMC.

Synthesis and Biophysical Characterization of RNAs Containing 2'-Fluorinated Northern Methanocarbacyclic Nucleotides.

2'-Fluorinated Northern methanocarbacyclic (2'-F-NMC) nucleosides and phosphoramidites, based on a bicyclo[3.1.0]hexane scaffold bearing all four natural nucleobases (U, C, A, and G), were synthesized to enable exploration of this novel nucleotide modification related to the clinically validated 2'-deoxy-2'-fluororibonucleotides (2'-F-RNA). Biophysical properties of the 2'-F-NMC-containing oligonucleotides were evaluated. A duplex of 2'-F-NMC-modified oligonucleotide with RNA exhibited thermal stability similar to that of the parent RNA duplex, 2'-F-NMC-modified oligonucleotides had higher stability against 5'- and 3'-exonucleolytic degradation than the corresponding oligonucleotides modified with 2'-F-RNA, and 2'-F-NMC-modified oligonucleotides exhibited higher lipophilicity than the corresponding RNA oligonucleotides as well as those modified with 2'-F-RNA.

Total Syntheses of Bulgecins A, B, and C and Their Bactericidal Potentiation of the ß-Lactam Antibiotics.

The bulgecins are iminosaccharide secondary metabolites of the Gram-negative bacterium Paraburkholderia acidophila and inhibitors of lytic transglycosylases of bacterial cell-wall biosynthesis and remodeling. The activities of the bulgecins are intimately intertwined with the mechanism of a cobiosynthesized ß-lactam antibiotic. ß-Lactams inhibit the penicillin-binding proteins, enzymes also critical to cell-wall biosynthesis. The simultaneous loss of the lytic transglycosylase (by bulgecin) and penicillin-binding protein (by ß-lactams) activities results in deformation of the septal cell wall, observed microscopically as a bulge preceding bacterial cell lysis. We describe a practical synthesis of the three naturally occurring bulgecin iminosaccharides and their mechanistic evaluation in a series of microbiological studies. These studies identify potentiation by the bulgecin at subminimum inhibitory concentrations of the ß-lactam against three pathogenic Gram-negative bacteria and establish for the first time that this potentiation results in a significant increase in the bactericidal efficacy of a clinical ß-lactam.