Carbazole could be recrystallised from ethyl acetate to prov
Carbazole could be recrystallised from ethyl acetate to provide bright red crystals suitable for single crystal X-ray diffraction (see ). The first crop showed had crystallised preferentially as the ()-isomer (A). Examining the single crystal X-ray data of it was interesting to find that the N2C15 bond was much shorter than the C15C16 bond and C9N2 bonds [1.3201(17)Å vs. 1.3914(18)Å vs. 1.4242(17)Å respectively]. These data are consistent with extensive delocalisation of the nitrogen lone pair into the enamine unit in the ()-isomer (B), and could explain its deep red colour. After Neochlorogenic acid receptor of the mother liquors and another recrystallization, the quality of the red crystals diminished and an orange powder was obtained upon evaporation of the mother liquors. Optical microscopy of this material showed two microcrystalline forms, one red and one orange, with the latter in large excess. We surmised the orange material may be the more soluble ()-isomer. A portion of the orange powder was examined by X-ray powder diffraction using a PANAlytical Empyrean diffractometer operating with Cu Kα radiation. It was shown to be highly crystalline, but the diffraction pattern of the orange powder did not match that calculated from the structure of the red crystals obtained (). It was possible to index the powder diffraction pattern of the orange powder using a large triclinic cell. The volume of this cell (2506Å) is approximately three times that of the cell found for the red crystals. It seems likely that the bulk of the orange sample contains the () isomer of , and the tripling of the volume suggests that there are three molecules in the asymmetric unit, perhaps two of one isomeric form and one of the other. Importantly, H NMR of either the red or orange crystalline forms showed, in CDCl solution, the same 1:1 mixture of the two geometric isomers. We were able to select a small single crystal of the orange material from among the original red crystals. The small crystal only diffracted weakly but we were able to obtain a crystal structure, albeit of lower quality than that of the red form. The structure is not too dissimilar to that of the red crystals, however it had a different orientation of both the -ethyl and ethyl ester groups (). It also showed that the nitro group was not co-planar with the NH, as in the red crystals, but was inclined at an angle of 25(1) °. The crystal structure of this orange single crystal did not, however, match the powder diffraction pattern of the bulk orange material (). In order to probe the details of the interaction of these inhibitors with DHODH, docking (with both eHiTS and Autodock) of the - and -forms of and into the putative ubiquinione binding channel of DHODH was performed using the DHODH crystal structure (PDB code; 1TV5). The data produced suggest several possibilities for the binding of each isomer of and into DHODH. However all of the suggested poses are predicted to bind in the same general space of the enzyme, with the aromatic ‘tail’ group of each ligand binding deep within the large hydrophobic pocket of the binding site (A and B). For each of the four structures (i.e. both - and - of both and ) two equally favourable rotamers were observed with the tail group seen to adopt the orientations shown in A and B. For both molecules and the polar ‘head group’ of the molecule is predicted to bind in the polar region of the binding site. Both isomers of each molecule can adopt two distinct hydrogen bonding networks (A and B). This is again due to a rotation of 180° around ArNH bond causing the polar ‘head’ of the molecule to flip. Although this ‘flipping’ forces a change in the hydrogen bonds made by the molecule and the enzyme, the residues involved in binding in DHODH are preserved, i.e. histidine (H185), tyrosine (Y528) and arginine (R265). All suggested binding poses have hydrogen bonds to at least two of these residues. The results from the docking studies suggest that the ligands may bind with multiple poses to the ubiquinone channel and that there is no clear preference for any one arrangement.