New assays for biologically active quinones
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
This thesis investigates the development of an HPLC assay for vitamin K, and the synthesis of carboxylic acid functionalised analogues of vitamin K and coenzyme Q. These analogues were used to investigate the feasibility of an immunoassay based method for the measurement of vitamin K. Chapter one outlines the biochemistry of vitamin K and coenzyme Q. The biological functions of these quinones and their proposed role in many diverse disease states are discussed. Chapter two contains a review of literature methods for the assay of vitamin K in plasma, food and tissue samples. The development of an HPLC assay for vitamin K using a metal catalyst, in the presence of an alcohol, to effect reduction of the quinine to the fluorescent hydro quinone for detection is presented. An investigation of a .number of metal catalyst/alcohol reduction systems showed platinum black/methanol to give the largest fluorescent response. The generality of this system towards a series of quinones showed that naphthoquinones (including vitamin K1, vitamin K3, 1,4-naphthoquinone and 2-hydroxy-1 ,4-naphthoquinone) were efficiently reduced, while the reduction of various benzoquinones (including CoQ-10 and CoQ-0) was also achieved. This chemistry was then applied to the measurement of vitamin K in both standard solutions and plasma samples, via an HPLC system with fluorescence detection. The synthesis of two side chain functionalised analogues of vitamin K (3.70 and 3.80) is presented in chapter three. Two synthetic routes to side chain functionalised vitamin K analogues were investigated. The first involved the conjugation of a functionalised allylic bromide (3.60) to a protected naphthoquinone core (3.11). The second approach developed involved the functionalisation of the natural isoprene side chain of vitamin K. This method was applied to a K vitamin synthesised via cuprate coupling of 3.11 and geranyl bromide, and to vitamin K2 (MK-4). The results of initial immunological studies are reported in chapter four. The production of antibodies recognising the naphthoquinone core of vitamin K was unsuccessful, postulated to be due to the 'bridge effect' leading to antibodies that recognise the isoprenoid side chain of the vitamin K analogue. From the results of these studies three short chain vitamin K analogues (4.5, and 4.7) were designed. Synthesis of 4.5 proceeded via oxidation of an aldehyde intermediate from the synthesis of 3.70. Compound 4.7 was obtained from the cuprate coupling of core 3.11 with ethyl bromoacetate, and subsequent functional group interconversions and deprotection. The coupling of analogues 3.70, 4.5, and 4.7 to protected lysine and a protected gly-lys dipeptide was investigated to model the coupling of these compounds to a carrier protein for immunological study. The conjugates produced (4.2, 4.4, and 4.13-4.15) were subsequently studied by NMR spectroscopy and mass spectrometry. The above synthetic work is applied to the structurally related quinone coenzyme Q and is presented in Chapter five. Using the synthetic methods developed for the synthesis of vitamin K analogues two carboxylic acid functionalised analogues of coenzyme Q (5.37 and 5.38) were synthesised. These analogues were also conjugated to protected lysine and gly-lys dipeptide, and studied by NMR spectroscopy and mass spectrometry.