X-ray crystallographic studies on the structure and interactions of L-ascorbic acids

Abstract

It is well known that non?covalent interactions endow biological systems with extreme subtlety and complexity. In fact, the structure and assembly of proteins, and the dynamical processes involved in their action, are largely dependent on these interactions. Very useful information regarding their geometrical features has been derived from single?crystal X?ray analysis of globular proteins. But these are often imprecise on account of the limited resolution of protein electron?density maps. A programme of single?crystal X?ray studies on crystalline complexes involving amino acids and other biomolecules is being carried out in this laboratory in an attempt to elucidate, at atomic resolution, the geometrical features of the possible non?covalent interactions important in the structure and function of proteins. The work carried out by the author as a part of this programme, on complexes involving L?ascorbic acid, forms a major part of this thesis. A brief survey of the chemical, physico?chemical, biochemical, theoretical and crystallographic studies on L?ascorbic acid, preceded by a short description of vitamin C, is therefore presented in Chapter 1. Preparation of single crystals suitable for X?ray analysis of complexes between L?ascorbic acid on the one hand and amino acids or other biomolecules on the other was the most difficult and the rate?limiting step in the whole investigation, especially because of the spontaneous oxidation and degradation of ascorbic acid in aqueous solution. The details of the crystallisation experiments carried out and the results thereof are presented in Chapter 2. The structure analysis of L?arginine L?ascorbate is presented in Chapter 3. The structure was solved by the non?centrosymmetric symbolic?addition procedure and refined to an R of 0.067 for 1501 photographically observed reflections. The conformation of the arginine molecule in the structure is different from those reported previously. This structure provides the first description of the ascorbate molecule unaffected by the constraints and disturbances imposed by the requirements of metal coordination. The arginine molecules and the ascorbate ions aggregate separately into alternating layers. The molecules in the arginine layer are held together by interactions involving the ??amino and the ??carboxylate groups, a situation analogous to that found in peptides. The two layers of unlike molecules are interconnected primarily through interactions of the side?chain guanidyl group of arginine with the ascorbate anion. These include two specific interactions: one involving two convergent hydrogen bonds and the other a pair of nearly parallel hydrogen bonds. The structure of a 1:1 complex between L?serine and L?ascorbic acid, described in Chapter 4, was solved by direct methods and refined to an R of 0.036 for 951 observed reflections. Both L?serine and L?ascorbic acid are neutral in the structure. The conformation of L?serine is different from those observed so far in crystal structures containing serine. The conformation of ascorbic acid is similar to that found in arginine ascorbate. The unlike molecules aggregate into separate columns in the crystal structure, held together by hydrogen bonds. Among these, a pair of hydrogen bonds between the enediol group of ascorbic acid and the carboxylate group of serine provides a possible model for specific interaction between ascorbic acid and a carboxylate ion. The two structures mentioned above provided examples of ionic and purely hydrogen?bonded interactions involving ascorbic acid. As ascorbic acid is known to be involved in charge?transfer interaction with nicotinamide, the structure analysis of a complex between the two compounds was taken up (Chapter 5), although crystals were of very poor quality. The triclinic crystals contained four molecules each of ascorbic acid and nicotinamide. Intensity data existed only up to a Bragg angle of 30° and contained only 322 observed reflections. As the resolution was less than 1.5 Å, the structure was not amenable to conventional crystallographic techniques. However, using special features noted in the diffraction data—such as pseudo?monoclinic symmetry and relatively high intensity of a few reflections—a possible model for the distribution of scattering matter in the unit cell was worked out. Subsequently, a plausible electron?density distribution was arrived at by a combination of direct methods, Buerger’s minimum function, electron?density modification, and Fourier transformation. Four out of the eight molecules appeared to be disordered, while the other four were related by an approximate 2? screw axis. The five?membered ring of ascorbic acid is stacked against the planar nicotinamide molecule in the crystal. The molecular geometry of ascorbic acid observed in crystal structures indicated some degree of flexibility. The nature and extent of this conformational flexibility were investigated through energy calculations employing classical semi?empirical potential functions. The technical details and results are presented in Chapter 6. The crystal structure of N?acetyl?L?histidine N?methylamide is reported in an Appendix. The structure was solved by direct methods and refined to an R of 0.036 for 1018 observed reflections. The molecule has an extended conformation. The histidyl side chain assumes the sterically most favourable conformation, with the imidazole group trans to the carbonyl group. The crystal structure is stabilised by an interesting network of hydrogen bonds. As the structure was solved simultaneously and independently in another laboratory, an inter?experimental comparison is also provided.