4-(2-Aminooxyethoxy)-2-(ethylureido)quinoline−Oligonucleotide Conjugates: Synthesis, Binding Interactions, and Derivatization with Peptides

Abstract

Oligo-2'-O-methylribonucleotides conjugated with 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) and 4-ethoxy-2-(ethylureido)quinoline (EOQ) were prepared by reaction of the AOQ or EOQ phosphoramidite with the protected oligonucleotide on a controlled pore glass support. Deprotection with ethylenediamine enabled successful isolation and purification of the highly reactive AOQ-conjugated oligomer. Polyacrylamide gel electrophoresis mobility shift experiments showed that the dissociation constants of complexes formed between an AOQ- or EOQ-conjugated 8-mer and complementary RNA or 2'-O-methyl-RNA targets (9- and 10-mers) were in the low nM concentration range at 37 degrees C, whereas no binding was observed for the corresponding nonconjugated oligomer, even at a concentration of 500 nM. Fluorescence studies suggested that this enhanced affinity is most likely due to the ability of the quinoline ring of the AOQ or EOQ group to stack on the last base pair formed between the oligomer and target, thus stabilizing the duplex. The binding affinity of a 2'-O-methyl RNA 15-mer, which contained an alternating methylphosphonate/phosphodiester backbone, for a 59-nucleotide stem-loop HIV TAR RNA target, increased 2.3 times as a consequence of conjugation with EOQ. The aminooxy group of AOQ-conjugated oligomers is a highly reactive nucleophile, which reacts readily with aldehydes and ketones to form stable oxime derivatives. This feature was used to couple an AOQ-oligomer with leupeptin, a tripeptide that contains a C-terminus aldehyde group. A simple method was developed to introduce a ketone functionality into peptides that contain a cysteine residue by reacting the peptide with bromoacetone. The resulting keto-peptide was then coupled to the AOQ-oligomer. This procedure was used to prepare oligonucleotide conjugates of a tetrapeptide, RGDC, and a derivative of HIV tat peptide having a C-terminus cysteine. The combination of the unique reactivity of the aminooxy group and enhanced binding affinity conferred by its quinoline ring suggests that AOQ may serve as a useful platform for the preparation of novel oligonucleotide conjugates.