Kinetic model studies on the chemical ligation of oligonucleotides via hydrazone formation

We report on the suitability of hydrazone formation for activator-free ligation of oligonucleotides. 5′-Acyl hydrazides were synthesized using a previously described phosphoramidite modifier, whereas 3′-hydrazides resulted from a hydrazinolysis of an ester group serving as a linker to the solid support. Aromatic aldehydes could be directly introduced on the 5′-terminus via the respective phosphoramidates. Aliphatic aldehydes were generated by periodate cleavage of the corresponding 3′- and 5′-modified diol precursors. Ligation of a 3′-hydrazide-modified oligonucleotide with oligonucleotides bearing an aromatic aldehyde in 5′-position showed a fast reaction kinetics (k1 about 10−1) and irreversible hydrazone formation. The ligation of a 5′-hydrazide-modified oligonucleotide and a 3′-ribobisaldehyde appeared to proceed reversibly at the beginning, but became irreversible with increasing reaction time. Hydrazide-modified oligonucleotides were found to be somewhat unstable in aqueous solutions.