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.

Post-synthetic and site-specific modification of endocyclic nitrogen atoms of purines in DNA and its potential for biological and structural studies

“…The resulting pentamer was found by HPLC analysis to be exactly the same as one produced with concentrated ammonia. This confirmed that ethylenediamine did not degrade the normal oligomers and that ethylenediamine can be safely used for cleavage and deprotection (27,28). …”

Efficient synthesis of an (aminooxy) acetylated‐somatostatin derivative using (aminooxy)acetic acid as a ‘carbonyl capture’ reagent

“…Oxime ligation is one of the most preferred procedures, due to the high chemoselectivity between an aminooxy function and a carbonyl group1, 2. Formation of chemoselective oxime bonds has been successfully applied for the synthesis of various peptide conjugates3, 4, glycopeptides5, conjugates of DNA with different biomolecules6, oligonucleotide–peptide conjugates7, 8, and proteins from peptide fragments9, 10. Moreover, oxime ligation has recently been investigated for the preparation of 18 F‐labeled peptide conjugates11, 12 and drug‐peptide conjugates13–15.…”

First Poly(2‐oxazoline)s with Pendant Amino Groups

“…Our interest in the reactivity of this group is focused on the possibility of synthesizing polymer‐peptide conjugates. Through the linking groups introduced in the side chain or as end functionalization, the polymers can be coupled to peptides by means of standard amide coupling reactions or, alternatively, via chemoselective ligation with isothiocyanate groups to the corresponding thiourea 16,47–54…”