Synthesis, structural behaviors and biological properties of thrombin binding aptamers singly modified with chiral m-nitro-benzene glycidoyl moiety

The thrombin binding aptamer (TBA) is a 15-mer oligonucleotide (ON) which folds into a typical chair-like G-quadruplex structure containing one TGT and two TT loops.(1) It binds thrombin acting as an anticoagulant agent.(2) In our ongoing investigation on the molecular bases of the anti-thrombin activity of TBA, (3,4) we expanded TBA structure-activity relationships (SARs) by synthesizing four new analogues, named TBA-7R(Ph-NO2), TBA-7S(Ph-NO2), TBA-12R(Ph-NO2) and TBA-12S(Ph-NO2), in which, the (R) or (S)-3-(3-nitrophenoxy)propane-1,2-diol moiety replaced, one at a time, the thymine residues at positions 7 and 12. The new derivatives were then tested in vitro using the prothrombin time (PT) coagulation assay. In order to rationalize acquired SARs, a multi‐disciplinary approach including biophysical and computational studies was applied. The evaluation of the anti-thrombin activity showed that all the tested analogues are able to increase the prothrombin time of human plasma at both 2 and 20 μM concentrations. Preliminary results obtained by CD and Electrophoretic Mobility Shift Assay experiments, showed that all the synthesized aptamers are able to fold into G-quadruplex structures in buffered solutions containing Na+ or K+ ions. Analyses of CD melting curves, as well as, preliminary results obtained by computational studies, evidenced that the R or S chirality of (3-nitrophenoxy)propane-1,2-diol at 12 position affected the thermal stability of the resulting TBA-G-quadruplex, while the same effect is not observed at 7 position. Similarly, the relation between G-quadruplex thermal stability and antithrombin activity of the new analogues depends on the position (7 or 12) of the introduced glycerol derivative.