title: Kinetic studies of redox and related reactions of some new isoalloxazines

authors: Gumbley, Stewart John

abstract: This thesis describes the synthesis of three new isoalloxazines with special structural characteristics which we hoped would, and in fact did, modify their chemical reactivity so as to improve our understanding of mechanisms of flavin reactions generally: kinetic studies of their reactions with a dithiol, reduced nicotinamide and sulphite were made.

A new general synthesis is described for the preparation of o-nitro-(N-alkylamino) aromatic compounds via nucleophilic displacement by alkylamine of a methoxy group ortho to a nitro group. Two new compounds, the isomers 1-(2'-hydroxyethylamino)-2-nitronaphthalene and 2-(2'-hydroxyethylamino)-1-nitronaphthalene, were prepared by this method.

These compounds were used to synthesize two isoalloxazines which each have an additional benzene ring fused either at the 6,7-bond of the isoalloxazine nucleus (7-(2'-hydroxyethyl)-10-methylnaphtho[1,2-g]pteridine- 9,11(7H,10H)-dione; isoalloxazine A) or at the 8,9-bond (12-(2'-hydroxyethyl)-9-methylnaphtho[2,1-g]pteridine- 8,10(9H,12H)-dione; isoalloxazine B). The third new isoalloxazine (10-(2'-hydroxyethyl)-3-methylbenzo[g]pteridine- 2,4(3H,10H)-dione; isoalloxazine C) was prepared by a conventional route.

Kinetic studies, the first recorded for isoalloxazines of the type of A and B, were undertaken for reactions with 1,3-dithio-2-propanol, with NADH and with sulphite.

In the case of the dithiopropanol reaction, a linear free-energy relationship was observed between the logarithm of the second order rate constant and the polarographic half-wave potential, E⅟₂ (slope 26 ± 1 V⁻¹). The conclusion drawn from this is that, if the redox reaction occurs through an addition-elimination sequence, the 5-, 6- and 8-positions are not essential centres for thiol oxidation.

In the case of the reaction with NADH, direct evidence was obtained for the formation of a kinetically important complex between the isoalloxazine and NADH from the observation of saturation kinetics. This is the first reported instance of the observation of such direct evidence for complex formation with NADH. The rate constants for the three isoalloxazines follow the reverse order expected from the E⅟₂ values and from the rates of reaction with DTP. The equilibrium constants (Kₑ) for the complexing of tryptophan with the isoalloxazines were measured by fluorescence quenching. A linear free-energy relationship was not observed between the logarithm of Kₑ and the logarithm of the rate constant for NADH oxidation. It was concluded that the unexpectedly high rate of reaction of A relative to that of B was due to the formation by A of a complex with NADH closer to the hydride acceptor site, the 5-position, than is the case for isoalloxazine B; and that the productivity of the complex therefore is markedly determined by the orientation of the NADH molecule with respect to the isoalloxazine within the complex.

The anaerobic nucleophilic addition reaction of sulphite with isoalloxazines A and C is a two step process of the form X⇌Y→Z, whereas the reaction with B is a single equilibrium step. The reduced isoalloxazine products from A and C are oxidized by molecular oxygen to new isoalloxazines but that from B is not. The new isoalloxazine from C reacts further with sulphite to yield a reduced species different from that of the anaerobic reaction, while the product from A does not react further. These observations provide support for the mechanism previously suggested in the literature for the reaction of sulphite with isoalloxazines. A small rate depression was observed for A relative to B and C in a plot of logarithm of rate constant against E ⅟₂. This is interpreted as being due to steric crowding at the 5-position caused by the α-hydrogen on the naphthalene moiety, an effect not observed in the dithiol reaction which more likely involves nucleophilic addition at the 4a- rather than the 5-position.

text: Kinetic studies of redox and related reactions of some new isoalloxazines This thesis describes the synthesis of three new isoalloxazines with special structural characteristics which we hoped would, and in fact did, modify their chemical reactivity so as to improve our understanding of mechanisms of flavin reactions generally: kinetic studies of their reactions with a dithiol, reduced nicotinamide and sulphite were made.

A new general synthesis is described for the preparation of o-nitro-(N-alkylamino) aromatic compounds via nucleophilic displacement by alkylamine of a methoxy group ortho to a nitro group. Two new compounds, the isomers 1-(2'-hydroxyethylamino)-2-nitronaphthalene and 2-(2'-hydroxyethylamino)-1-nitronaphthalene, were prepared by this method.

These compounds were used to synthesize two isoalloxazines which each have an additional benzene ring fused either at the 6,7-bond of the isoalloxazine nucleus (7-(2'-hydroxyethyl)-10-methylnaphtho[1,2-g]pteridine- 9,11(7H,10H)-dione; isoalloxazine A) or at the 8,9-bond (12-(2'-hydroxyethyl)-9-methylnaphtho[2,1-g]pteridine- 8,10(9H,12H)-dione; isoalloxazine B). The third new isoalloxazine (10-(2'-hydroxyethyl)-3-methylbenzo[g]pteridine- 2,4(3H,10H)-dione; isoalloxazine C) was prepared by a conventional route.

Kinetic studies, the first recorded for isoalloxazines of the type of A and B, were undertaken for reactions with 1,3-dithio-2-propanol, with NADH and with sulphite.

In the case of the dithiopropanol reaction, a linear free-energy relationship was observed between the logarithm of the second order rate constant and the polarographic half-wave potential, E⅟₂ (slope 26 ± 1 V⁻¹). The conclusion drawn from this is that, if the redox reaction occurs through an addition-elimination sequence, the 5-, 6- and 8-positions are not essential centres for thiol oxidation.

In the case of the reaction with NADH, direct evidence was obtained for the formation of a kinetically important complex between the isoalloxazine and NADH from the observation of saturation kinetics. This is the first reported instance of the observation of such direct evidence for complex formation with NADH. The rate constants for the three isoalloxazines follow the reverse order expected from the E⅟₂ values and from the rates of reaction with DTP. The equilibrium constants (Kₑ) for the complexing of tryptophan with the isoalloxazines were measured by fluorescence quenching. A linear free-energy relationship was not observed between the logarithm of Kₑ and the logarithm of the rate constant for NADH oxidation. It was concluded that the unexpectedly high rate of reaction of A relative to that of B was due to the formation by A of a complex with NADH closer to the hydride acceptor site, the 5-position, than is the case for isoalloxazine B; and that the productivity of the complex therefore is markedly determined by the orientation of the NADH molecule with respect to the isoalloxazine within the complex.

The anaerobic nucleophilic addition reaction of sulphite with isoalloxazines A and C is a two step process of the form X⇌Y→Z, whereas the reaction with B is a single equilibrium step. The reduced isoalloxazine products from A and C are oxidized by molecular oxygen to new isoalloxazines but that from B is not. The new isoalloxazine from C reacts further with sulphite to yield a reduced species different from that of the anaerobic reaction, while the product from A does not react further. These observations provide support for the mechanism previously suggested in the literature for the reaction of sulphite with isoalloxazines. A small rate depression was observed for A relative to B and C in a plot of logarithm of rate constant against E ⅟₂. This is interpreted as being due to steric crowding at the 5-position caused by the α-hydrogen on the naphthalene moiety, an effect not observed in the dithiol reaction which more likely involves nucleophilic addition at the 4a- rather than the 5-position.

year: 1976

title: Assessing stability in body weight in the brushtail possum (Trichosurus vulpecular)

authors: Cameron, Kristie Elizabeth

subjects: brushtail possum

abstract: When conducting controlled laboratory studies with non-traditional laboratory animals it is important that methods for determining body weight stability are reliable. This helps ensure the health and welfare of animals when they are maintained during periods of free feeding or food restriction. This study compared different methods for determining body weight stability in six common brushtail possums (Trichosurus vulpecula) maintained on a free-feeding diet under laboratory conditions. A criterion of five consecutive weighings with less than ±2.5% change across days and no more than two consecutive days of weight loss or weight gain was judged to be the most suitable criteria for determining stability. It is important to study non-traditional animals, especially endangered or pest species, under controlled laboratory conditions and to have robust methods for establishing body weight stability.

text: Assessing stability in body weight in the brushtail possum (Trichosurus vulpecular) When conducting controlled laboratory studies with non-traditional laboratory animals it is important that methods for determining body weight stability are reliable. This helps ensure the health and welfare of animals when they are maintained during periods of free feeding or food restriction. This study compared different methods for determining body weight stability in six common brushtail possums (Trichosurus vulpecula) maintained on a free-feeding diet under laboratory conditions. A criterion of five consecutive weighings with less than ±2.5% change across days and no more than two consecutive days of weight loss or weight gain was judged to be the most suitable criteria for determining stability. It is important to study non-traditional animals, especially endangered or pest species, under controlled laboratory conditions and to have robust methods for establishing body weight stability.

year: 2014

title: Bilingual/immersion education in Aotearoa/New Zealand: Setting the context.

authors: May, Stephen

subjects: bilingual education

abstract: Introduction to a special issue of IJBEB, focusing on recent developments in bilingual/
immersion education in Aotearoa/New Zealand, particularly, but not solely,
Maori-medium education.

text: Bilingual/immersion education in Aotearoa/New Zealand: Setting the context. Introduction to a special issue of IJBEB, focusing on recent developments in bilingual/
immersion education in Aotearoa/New Zealand, particularly, but not solely,
Maori-medium education.

year: 2005-01-01