Template-independent enzymatic synthesis of RNA oligonucleotides.

RNA oligonucleotides have emerged as a powerful therapeutic modality to treat disease, yet current manufacturing methods may not be able to deliver on anticipated future demand. Here, we report the development and optimization of an aqueous-based, template-independent enzymatic RNA oligonucleotide synthesis platform as an alternative to traditional chemical methods. The enzymatic synthesis of RNA oligonucleotides is made possible by controlled incorporation of reversible terminator nucleotides with a common 3'-O-allyl ether blocking group using new CID1 poly(U) polymerase mutant variants. We achieved an average coupling efficiency of 95% and demonstrated ten full cycles of liquid phase synthesis to produce natural and therapeutically relevant modified sequences. We then qualitatively assessed the platform on a solid phase, performing enzymatic synthesis of several N + 5 oligonucleotides on a controlled-pore glass support. Adoption of an aqueous-based process will offer key advantages including the reduction of solvent use and sustainable therapeutic oligonucleotide manufacturing.

Chiral Resolution of the Enantiomers of the Slow-Onset Dopamine Reuptake Inhibitor CTDP-32476 and Their Activities.

An improved synthesis was developed for CDTP-32476, a potent slow-onset dopamine reuptake blocker that may have utility as a treatment for cocaine abuse. The enantiomers of the compound were separated by fractional crystallization with N-acetylleucine enantiomers. An X-ray crystal structure was obtained of the RR enantiomer paired with N-acetyl-d-leucine. Chiral chromatography showed that the resolved enantiomers were pure with little contamination by the other enantiomer. The enantiomers were tested for their ability to block the reuptake of monoamines at their respective transporters and to stimulate locomotor activity in mice. Both enantiomers potently blocked the reuptake of dopamine and stimulated locomotor activity in mice. The RR enantiomer that corresponds to the active RR enantiomer of methylphenidate was slightly more potent at the dopamine reuptake site. The RR enantiomer also was found to be about twice as selective for the dopamine transporter relative to the norepinephrine transporter, which may have clinical implications. A method for designing slow-onset stimulants is proposed since there is increasing evidence that such activity is an important factor in stimulants that may have limited abuse potential.