Recognition of the cell-wall binding site of the vancomycin-group antibiotics by unnatural structural motifs: 1H NMR studies of the effects of ligand binding on antibiotic dimerisation

Abstract

The synthetic compound N-(9-oxofluoren-2-yl)oxamic acid 2 is shown by ¹H NMR spectroscopy to bind to the peptide cell-wall recognition site of the vancomycin-group antibiotic, ristocetin A, through hydrogen bonding interactions analogous to those of the cell-wall analogue di-N-Ac-L-Lys-D-Ala-D-Ala 1. The synthetic ligand 2 binds in a highly anti-cooperative manner to the ristocetin dimer (K_dim for ristocetin A alone ≈ 500 dm³ mol^–1, K_dim for ristocetin–2 complex < 1 dm³ mol^–1, at 300 K), while the natural substrate 1 has a relatively small anti-cooperative effect on dimerisation (K_dim≈ 350 dm³mol^–1 at 300 K). Structural and thermodynamic aspects of cooperative/anti-cooperative binding, relevant to the formation of biological aggregates, are examined in this study. ¹H NOE data show that the pendant sugars of the antibiotic are ‘organised’ into a hydrophobic ‘wall’ with which compound 2 interacts, and which is evidently incompatible with the complementarity required for back-to-back dimerisation. An investigation of the effects on antibiotic dimerisation of a number of substrate analogues, and several ligands which approximate to stepwise truncation of compound 2, reveals that ligands which introduce unnatural binding interactions with the ring 4 tetrasaccharide and ring 6 ristosamine sugar (particularly hydrophobic interactions with ligand aryl rings), appear to bind highly anti-cooperatively to the dimer, perturbing the subtle complementarity that appears to be synonymous with the formation of the extended aggregate. A number of other vancomycin group antibiotics exhibit positive cooperative effects, suggesting a possible functional role for dimerisation in promoting antibacterial action.