OLIGONUCLEOTIDE-HIRULOG18 CONJUGATED BY CLICK CHEMISTRY AND ITS INHIBITION ON THROMBIN

We have shown previously that oligonucleotide conjugated hirulog (bivalirudin) has a higher inhibitory activityagainst thrombin compared with hirulog. The negative charged oligonucleotide was considered to be responsible for theadditive activity. To further investigate the related effect of oligonucleotide, the oligonucleotide conjugated hirulog18 wasprepared in the present work and the activity of the conjugate against thrombin was measured. Hirulog18, a peptide lack oftwo N-terminal amino acid residues of hirulog, has little inhibitory activity against thrombin compared with hirulog. Theconjugate oligonucleotide-hiruglog18 was successfully synthesized using click chemistry and validated by MALDI-MS andgel electrophoresis. The activity of the conjugate against thrombin was measured by the chromogenic assay usingChromozym TH as substrate. It was found surprisingly that oligonucleotide-hirulog18 had an inhibitory effect better thanhirulog18 and hirulog. Strong negative charged heparin was used to study the binding mode betweenoligonucleotide-hirulog18 and thrombin. The results suggested that the negative charged oligonucleotide could be helpful forthe conjugate’s binding to the anion-binding exosite of thrombin via the Coulomb force. The highly inhibitory effect of theoligonucleotide-hirulog18 conjugate against thrombin may present a new strategy to generate a novel class of directthrombin inhibitors.