Solid-phase synthesis of terminal oligonucleotide-phosphoramidate conjugates

A novel phosphoramidite, N,N-diisopropylamino-2-cyanoethyl-9-anthracenemethyl phosphoramidite 1, was prepared and coupled with the terminal 5'-hydroxyl of support-bound T10 and the putative phosphite triester intermediate was subsequently reacted with iodine in the presence of either water or a series of primary and secondary amines. The reactivity of 1 compared to a previously reported benzyl phosphoramidite 2 was also investigated: oxidation of the product of coupling 2 with CPG-T10-5'OH under aqueous conditions resulted in greater than 30% of the benzyl moiety being retained. In contrast, essentially complete loss of the 9-anthracenemethyl group was observed using 1 under the same conditions. Oligonucleotides modified with a terminal phosphate monoester, lipophilic, fluorescent or cationic groups were thus prepared.

Expedited Solid-Phase Synthesis of Fluorescently Labeled and Biotinylated Aminoalkane Diphenyl Phosphonate Affinity Probes for Chymotrypsin- and Elastase-Like Serine Proteases

In this study, we report on a novel, expedited solid-phase approach for the synthesis of biotinylated and fluorescently tagged irreversible affinity based probes for the chymotrypsin and elastase-like serine proteases. The novel solid-phase biotinylation or fluorescent labeling of the aminoalkane diphenyl phosphonate warhead using commercially available Biotin-PEG-NovaTag or EDANS NovaTag resin permits rapid, facile synthesis of these reagents. We demonstrate the kinetic evaluation and utilization of a number of these irreversible inactivators for chymotrypsin-like (chymotrypsin/human cathepsin G) and elastase-like serine proteases. Encouragingly, these compounds display comparable potency against their target proteases as their N-benzyloxycarbonyl (Cbz)-protected parent compounds, from which they were derived, and function as efficient active site-directed inactivators of their target proteases. We subsequently applied the biotinylated reagents for the sensitive detection of protease species via Western blot, showing that the inactivation of the protease was specifically mediated through the active site serine. Furthermore, we also demonstrate the successful detection of serine protease species with the fluorescently labeled derivatives “in-gel”, thus avoiding the need for downstream Western blotting. Finally, we also show the utility of biotinylated and pegylated affinity probes for the isolation/enrichment of serine protease species, via capture with immobilized streptavidin, and their subsequent identification via de novo sequencing. Given their selectivity of action against the serine proteases, we believe that these reagents can be exploited for the direct, rapid, and selective identification of these enzymes from biological milieu containing multiple protease subclasses.

Fast, copper-free click chemistry: a convenient solid-phase approach to oligonucleotide conjugation

Solid-phase oligonucleotide conjugation by nitrile oxide-alkyne click cycloaddition chemistry has been successfully demonstrated; the reaction, compatible with all nucleobases, requires no metal catalyst and proceeds under physiological conditions.