Synthesis and characterization of steroid glucuronides for the preparation of horseradish peroxidase conjugates via hemin modification : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University
Steroid glucuronides including estrone glucuronide 12, estriol 3-, 16α- and 17β-monoglucuronides 13-15 and pregnanediol glucuronide 16 have been successfully synthesised. In particular, a new scheme for the synthesis of estriol monoglucuronides 13-15 from estriol provides a simple procedure and good yields of pure products based on the protection and deprotection of hydroxyl groups of estriol, glucuronidation, and hydrolysis. The new synthetic route retains the original stereochemical integrity of the estriol, and thus produced the estriol monoglucuronides with the correct stereochemistry. The steroid glucuronides 12-16 were characterised by 1H-1H 2D-COSY, 2D-NOESY and 1H-13C HETCOR spectra and the results unambiguously showed the β-linkage of the glucuronide ring with the steroid moiety in all of the steroid glucuronides. The conjugation positions of the glucuronic acid to estriol, as in estriol 16- or 17-glucuronide 14-15, were distinguished from their 13C chemical shift values and the proton 2D-NOESY spectra. The crystal structure analysis of one estriol 17-glucuronide derivative 112 also confirmed that the absolute configuration at all stereocentres was maintained during synthesis. Subsequently some α-amino acids (DL- or L-phenylalanine, L-tryptophan and L-arginine), estrone glucuronide (E1G) 12 and pregnanediol glucuronide (PdG) 16 have been successfully linked to hemin IX 227 (the prosthetic group of horseradish peroxidase) either by selective mono-acylation of protoporphyrin IX 216 followed by insertion of Fe2+ or by direct mono-conjugation of hemin IX 227 with α-amino acids or steroids. The mono-coupling reactions provided good yields and simlple reaction conditions, which have established the feasibility of this new methodology. The mono-conjugated structures and the high purities of both hemin-phenylalanine mono-conjugate 230 and the hemin-estrone glucuronide mono-conjugate 232 were confirmed by their 1H NMR and mass spectra. Both purified conjugates (230, 232) showed no contamination by unreacted hemin IX 227 by HPLC analyses. The reconstitution of hemin-estrone glucuronide mono-conjugate 232 with apo-horseradish peroxidase has been successfully achieved to form a new enzyme. The new enzyme (estrone glucuronide-horseradish peroxidase conjugate) retains good peroxidase activity (76% relative to reconstituted horseradish peroxidase), which is sufficient for exploitation in immunoassays. A suitable molecular linker (L-lysine) between the hemin propionate side chain and the estrone glucuronide moiety is crucial for retaining good peroxidase activity. Without a molecular linker, reconstitution of hemin-phenylalanine monoconjugate 230 with the apo-horseradish peroxidase showed a very poor reconstitution yield and activity. The extra carboxyl group, introduced by L-lysine, probably also made a great contribution in retaining a high activity of the new enzyme. Therefore, this thesis has exploited a new methodology in the preparation of horseradish peroxidase-hapten conjugates via hemin-modification. The new methodology is generic and it can be extended to the synthesis of horseradish peroxidase-conjugates with any analytes of interest. It will be very useful, not only as markers of fertility in home assays, but also in many other areas such as food, agriculture, medicine and the environmental monitoring. Hence, wide commercial applications for this new technology are expected in the future.