Solution- and bound-state conformational study of N,N',N"-triacetyl chitotriose and other analogous potential inhibitors of hevamine: application of trNOESY and STD NMR spectroscopy.

The solution-state conformations of N,N',N"-triacetyl chitotriose (1) and other potential chitinase inhibitors 2-4 were studied using a combination of NMR spectroscopy (NOESY) and molecular mechanics calculations. Determination solely of the global energy minimum conformation was found to be insufficient for an agreement with the NMR results. An appropriate consistency between the NMR experimental data and theoretical calculations was only reached by assessing the structures as population-weighted average conformers based on Boltzmann distributions derived from the calculated relative energies. Analogies, but also particular differences, between the synthetic compounds 2-4 and the naturally-occurring N,N',N"-triacetyl chitotriose were found. Furthermore, the conformation of compounds 1 and 2 when bound to hevamine was also studied using transferred NOESY experiments and the binding process was found to impart a level of conformational restriction on the ligands. The preferred conformation as determined for 1 in the bound state to hevamine belonged to one of the conformational families found for the compound when free in solution, although full characterisation of the bound-state conformations was impeded due to severe signal overlap. Saturation transfer difference NMR experiments were also employed to analyse the binding epitopes of the bound compounds. We thus determined that it is mainly the acetyl amido groups of the trisaccharide and the heterocyclic moiety which are in close contact with hevamine.

NMR spectroscopic and theoretical study of the complexation of the inhibitor allosamidin in the binding pocket of the plant chitinase hevamine.

Based on NMR spectroscopic information about the allosamidin-hevamine complex, ab initio MO calculations of the ring current effect of the aromatic moieties of Trp255, Tyr183 and Tyr6 of hevamine were carried out to investigate the role of these amino acid residues in binding interactions with allosamidin in solution. In addition, the intermolecular steric compression effect on the 13C chemical shifts of the allosamizoline carbon atoms and the hydrogen bonding to Glu127 was identified. It can be inferred that the binding forces are strongest in the allosamizoline moiety of allosamidin.

Solution-state conformational study of the hevamine inhibitor allosamidin and six potential inhibitor analogues by NMR spectroscopy and molecular modeling.

The solution-state conformations of the hevamine inhibitor allosamidin and six potential inhibitor analogues were studied by various NMR spectroscopic techniques and molecular modeling using force field calculations. Determination solely of the global energy minimum conformation was found to be insufficient for consensus with the NMR results, and agreement between the NMR experimental data and the theoretical calculations was only reached by assessing the structures as population-weighted average conformers on the basis of Boltzmann distributions derived from the calculated relative energies. The conformations of the glycosidic linkages in the compounds were found to be similar when the sugar residues were the same, but differences were markedly evident otherwise and also for the various heterocyclic group linkages. The binding of the compounds to hevamine, which may also complex to chitinases in general, was assessed using HMQC, transfer-NOESY, and both 1-D and 2-D saturation transfer difference NMR experiments. Under the conditions employed, only allosamidin was implicated to be bound to hevamine, and then only by HMQC with the dipolar coupling-based experiments failing to substantiate the formation of the complex. However, the results are consistent with the biochemical activities of the compounds whereby only allosamidin has been shown to act as a competitive inhibitor.