Systematic Investigation of Tether Length and Phosphorus Configuration in Backbone Constrained Macrocyclic Nucleic Acids to Modulate Binding Kinetics for RNA

Abstract

We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson–Crick base pairing for modulating the binding kinetics of single-stranded oligonucleotides (ONs) using bis-phosphonate esters bridging hydrocarbon tethers to provide 11- and 15-membered macrocyclic analogues. In this manuscript, we describe the synthesis of all eight P-stereoisomers of macrocyclic 12-, 13-, 14-, and 16-membered hydrocarbon-bridged nucleotide trimers, their incorporation into ONs, and biophysical characterization of the modified ONs. The size of the macrocyclic tether and configuration at phosphorus had profound effects on hybridization kinetics. ONs containing 12- and 13-membered rings exhibited faster on-rates (up to 5-fold) and off-rates (up to 161-fold). In contrast, ONs using the larger ring size macrocycles generally exhibited smaller changes in binding kinetics relative to unmodified DNA. Interestingly, several of the analogues retained significant binding affinity for RNA based on their dissociation constants, despite being modestly destabilizing in the thermal denaturation experiments, highlighting the potential utility of measuring dissociation constants versus duplex thermal stability when evaluating novel nucleic acid analogues. Overall, our results provide additional insights into the ability of backbone-constrained macrocyclic nucleic acid analogues to modulate hybridization kinetics of modified ONs with RNA.

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

“…Cleavage of the DMT with p-toluenesulfonic acid hydrate gave both dimers 6a/6b in high yield aer separation by silica gel chromatography without deprotection of the silyl ether group. The assignment of stereochemistry at phosphorus in each isomer followed conventional methods described in our previous work, 22,23 and correlating with data reported by Baran. 26 A coupling reaction of the S-6a isomer with an excess of 1-heptenyl phosphoramidite 7 and tetrazole as the activator was performed on a small scale in batches (Scheme 2).…”

“…1B). 23 However, these macrocyclic analogues showed reduced RNA-binding affinity and duplex stability, because of increased off-rates relative to unmodified DNA.…”

My 50-Plus Years of Academic Research Collaborations with Industry. A Retrospective

“…We followed up the study by a systematic investigation of 12-, 13-, 14-, and 16-membered bisphosphonate macrocycles to modulate binding kinetics for RNA. 343 Insights into the on/off rates of this extended series of macrocyclic ONs were extremely useful to plan and improve further modifications. The highly academic nature of this project is another example of a long-standing productive collaboration to advance the science of RNA therapeutics.…”

“…These results agreed with the predictions of molecular dynamics simulations. We followed up the study by a systematic investigation of 12-, 13-, 14-, and 16-membered bis-phosphonate macrocycles to modulate binding kinetics for RNA . Insights into the on/off rates of this extended series of macrocyclic ONs were extremely useful to plan and improve further modifications.…”