¹H/¹⁵N HSQC NMR Studies of Ligand Carboxylate Group Interactions with Arginine Residues in Complexes of Brodimoprim Analogues and Lactobacilluscasei Dihydrofolate Reductase^†,‡

¹H and ¹⁵N NMR studies have been undertaken on complexes of Lactobacilluscasei dihydrofolate reductase (DHFR) formed with analogues of the antibacterial drug brodimoprim (2,4-diamino-5-(3‘,5‘-dimethoxy-4‘-bromobenzyl)pyrimidine) in order to monitor interactions between carboxylate groups on the ligands and basic residues in the protein. These analogues had been designed by computer modeling with carboxylated alkyl chains introduced at the 3‘-O position in order to improve their binding properties by making additional interactions with basic groups in the protein. Specific interactions between ligand carboxylate groups and the conserved Arg57 residue have been detected in studies of ¹H/¹⁵N HSQC spectra of complexes of DHFR with both the 4-carboxylate and the 4,6-dicarboxylate brodimoprim analogues. The spectra from both complexes showed four resolved signals for the four NH^η protons of the guanidino group of Arg57, and this is consistent with hindered rotation in the guanidino group resulting from interactions with the 4-carboxylate group in each analogue. In the spectra of each complex, one of the protons from each of the two NH₂ groups and both nitrogens are considerably deshielded compared to the shielding values normally observed for such nuclei. This pattern of deshielding is that expected for a symmetrical end-on interaction of the carboxylate oxygens with the NH^η12 and NH^η22 guanidino protons. The differences in the degree of deshielding between the complexes of the two structurally similar brodimoprim analogues and the methotrexate indicates that the shielding is very sensitive to geometry, most probably to hydrogen bond lengths. The ¹H/¹⁵N HSQC spectrum of the DHFR complex with the brodimoprim-6-carboxylate analogue does not feature any deshielded Arg NH^η protons and this argues against a similar interaction with the Arg57 in this case. It has not proved possible to determine whether the 6-carboxylate in this analogue is interacting directly with any residue in the protein. ¹H/¹⁵N HSQC spectra have been fully assigned for the complexes with the three brodimoprim analogues and chemical shift mapping used to explore interactions in the binding site. The ¹H signals of the bound ligands for all three brodimoprim analogues have been assigned. Their ¹H chemical shifts were found to be fairly similar in the different complexes indicating that the 2,4-diaminopyrimidine and the benzyl ring are binding in essentially the same binding sites and with the same overall conformation in the different complexes. The rotation rate about the N^εC^ζ bond in the brodimoprim-4,6-dicarboxylate complex with DHFR has been determined from a zz-HSQC exchange experiment, and its value is quite similar to that observed in the DHFR·methotrexate complex (24 ± 10 s^-1 at 8 °C and 50 ± 10 s^-1 at 15 °C, respectively). The ¹H and ¹⁵N chemical shift differences of selected amide and guanidino NH groups, measured between the DHFR complexes, provided further evidence about the interactions involving Arg57 with the 4-carboxylate and 4,6-dicarboxylate brodimoprim analogues.