title: Fungal metabolite chemistry: Sporidesmin, crepidotine

authors: Ronaldson, John William

abstract: This thesis was concerned with the chemistry of two fungal products, sporidesmin and crepidotine. The structure of sporidesmin was known but aspects of its chemistry, such as how it could be modified to function as an antigen, were not. The structure of crepidotine was unknown but was elucidated in the course of this work.
Since only 50 mg of crepidotine was available, non-destructive methods were used to arrive at the structure, 2-phenyl-1,6-naphthyridin-4-one. Of these methods, ¹H nuclear magnetic resonance spectroscopy was the most important. This spectrum (¹H n.m.r.) of crepidotine was compared with known or expected spectra of possible isomers most of which were unknown.
Sporidesmin is the toxin which causes Facial Eczema. One approach for combating the problem of Facial Eczema is to develop antibodies in the animals against sporidesmin. Sporidesmin is too small a molecule per se to induce antibody formation so one of the aims of this thesis was to produce derivatives of sporidesmin and to complex them to proteins. Proteins with many molecules of sporidesmin covalently bound to them were produced to be used as antigens. When animals were treated with these antigens, antibodies were detected in their sera. The titre was low for sporidesmin complexed to albumins but four times higher when sporidesmin was complexed to bovine thyroglobulin. 
The method of complexing sporidesmin to the proteins was to open the -S-S- bridge (in sporidesmin) and alkylate the sulphur atoms with iodoacetic esters. When protein was treated with these sporidesmin derivatives the ε-amino-groups of the lysyl residues (in the protein) were transacylated yielding modified sporidesmin covalently bound to the protein, synthetic or natural.
The new compounds formed from sporidesmin in these syntheses were: diethyl and dimethyl secosporidesmin-S,S'-diacetate, ethyl and methyl 11a-mercaptosecosporidesmin-3-S-acetate, methyl 3-mercaptosecosporidesmin-11a-S-acetate; and one of the antigens was cross-linked poly(ethoxy)-poly(secosporidesmin-S,S'-diacetyl)poly-(L-lysine). Of other new derivatives of sporidesmin that were synthesized, 3,11a-dimercaptosecosporidesmin, S,S'-homosporidesmin, and sporidesmin 11-(methyl glutarate), the first was the most difficult to prepare. When attempted in the normal way (opening the -S-S- bridge as for the above esters) only sporidesmin was recovered. But the dimercapto-compound was obtained when oxygen was eliminated in the reaction. 
Because the toxicity of the sporidesmin molecule resides in the epidithiodioxopiperazine ring, means of covalently linking sporidesmin to protein without altering the -S-S- bridge were sought, e.g. acylation or diazonium coupling with suitable bridging groups. Having successfully acylated with the chloroacetyl group (forming sporidesmin di(chloroacetate) it may be possible to complex this ester to protein through the halogen atom.
In the diazonium coupling reaction, the sole aromatic hydrogen resisted coupling. Diazonium salts eliminated the epidithiodioxopiperazine ring by rupturing the pyrrolidine ring: the products were the strongly coloured substituted indoleazobenzenes, 5-chloro-2-hydroxy-6,7- dimethoxy-1-methyl-4'-nitroindole-3-azobenzene and the 2'-chloro- and 2'-nitro-analogues. From anhydrodethiosporidesmin 5-chloro-6,7-dimethoxy-8-methyl-2' ,4'-dinitropyrrolo[2,3-b]indole-2-azobenzene was obtained. These indoleazobenzenes are new compounds.
In the search for methods of diazonium coupling to o-dihydroxy-benzene, veratrole, the following new compounds were synthesized: 2-chloro-3', 4'-dimethoxy-4-nitrobiphenyl, 2-chloro-4'-hydroxy-3'-methoxy-4-nitroazobenzene, 4'-hydroxy-3'-methoxy-2, 4-dinitroazobenzene, 3', 4'-dimethoxy-2,4-dinitroazobenzene, 2-chloro-3', 4'-dimethoxy-4-nitroazobenzene and 2-chloro-4, 5-dimethoxy-2', 4'-dinitroazobenzene. 
In the ¹³C nuclear magnetic resonance spectra of sporidesmin and sporidesmin-D (dimethylthiosecosporidesmin) (decoupled and undecoupled) the peaks were assigned to the appropriate carbons. Where a resonance seemed anomalous, a reasonable explanation of the anomaly has been put forward. Comparison of the spectra of the two compounds indicated the resonances of those carbons which were involved in the strained epidithiodioxopiperazine ring system. Having assigned the peaks for sporidesmin, the structure of unknown analogues may now be more readily elucidated. 
An analogue of sporidesmin, sporidesmin-E, of comparable toxicity, having an -S-S-S- bridge was synthesized from sporidesmin for field studies. It was an unstable molecule readily decomposing to sporidesmin plus sulphur, e.g. pure sporidesmin-E always showed three spots in thin layer chromatography. The mixed melting point between these two compounds was undepressed (they melted within 3.5° of each other). That sporidesmin-E had been formed was confirmed by the M⁺ being 32 mass units greater than sporidesmin but this was still no criterion of purity. In the hydrogen-bonding investigation of the two compounds sporidesmin absorbed in the νOH region where sporidesmin-E did not so the degree that sporidesmin-E was contaminated with sporidesmin could be determined. Comparison of the infra-red spectra of sporidesmin and sporidesmin-E prepared in different ways (in KBr from MeOH or EtOEt, in nujol or in halocarbon oil) showed there were only minor differences between them. 
The hydrogen bonding analysis revealed that the C-S bonds of 3,11a-di-mercaptosecosporidesmin and sporidesmin-E were similarly orientated (i.e. diverging) and not more or less parallel as in sporidesmin-D. The mono-mercapto-derivative (methyl 11a-mercaptosecosporidesmin-3-S-acetate) showed no νSH peak: the elements of the thiol group are present (m.s.) but the hydrogen is ionic and with the adjacent amido-group is apparently in hyperconjugation.

text: Fungal metabolite chemistry: Sporidesmin, crepidotine This thesis was concerned with the chemistry of two fungal products, sporidesmin and crepidotine. The structure of sporidesmin was known but aspects of its chemistry, such as how it could be modified to function as an antigen, were not. The structure of crepidotine was unknown but was elucidated in the course of this work.
Since only 50 mg of crepidotine was available, non-destructive methods were used to arrive at the structure, 2-phenyl-1,6-naphthyridin-4-one. Of these methods, ¹H nuclear magnetic resonance spectroscopy was the most important. This spectrum (¹H n.m.r.) of crepidotine was compared with known or expected spectra of possible isomers most of which were unknown.
Sporidesmin is the toxin which causes Facial Eczema. One approach for combating the problem of Facial Eczema is to develop antibodies in the animals against sporidesmin. Sporidesmin is too small a molecule per se to induce antibody formation so one of the aims of this thesis was to produce derivatives of sporidesmin and to complex them to proteins. Proteins with many molecules of sporidesmin covalently bound to them were produced to be used as antigens. When animals were treated with these antigens, antibodies were detected in their sera. The titre was low for sporidesmin complexed to albumins but four times higher when sporidesmin was complexed to bovine thyroglobulin. 
The method of complexing sporidesmin to the proteins was to open the -S-S- bridge (in sporidesmin) and alkylate the sulphur atoms with iodoacetic esters. When protein was treated with these sporidesmin derivatives the ε-amino-groups of the lysyl residues (in the protein) were transacylated yielding modified sporidesmin covalently bound to the protein, synthetic or natural.
The new compounds formed from sporidesmin in these syntheses were: diethyl and dimethyl secosporidesmin-S,S'-diacetate, ethyl and methyl 11a-mercaptosecosporidesmin-3-S-acetate, methyl 3-mercaptosecosporidesmin-11a-S-acetate; and one of the antigens was cross-linked poly(ethoxy)-poly(secosporidesmin-S,S'-diacetyl)poly-(L-lysine). Of other new derivatives of sporidesmin that were synthesized, 3,11a-dimercaptosecosporidesmin, S,S'-homosporidesmin, and sporidesmin 11-(methyl glutarate), the first was the most difficult to prepare. When attempted in the normal way (opening the -S-S- bridge as for the above esters) only sporidesmin was recovered. But the dimercapto-compound was obtained when oxygen was eliminated in the reaction. 
Because the toxicity of the sporidesmin molecule resides in the epidithiodioxopiperazine ring, means of covalently linking sporidesmin to protein without altering the -S-S- bridge were sought, e.g. acylation or diazonium coupling with suitable bridging groups. Having successfully acylated with the chloroacetyl group (forming sporidesmin di(chloroacetate) it may be possible to complex this ester to protein through the halogen atom.
In the diazonium coupling reaction, the sole aromatic hydrogen resisted coupling. Diazonium salts eliminated the epidithiodioxopiperazine ring by rupturing the pyrrolidine ring: the products were the strongly coloured substituted indoleazobenzenes, 5-chloro-2-hydroxy-6,7- dimethoxy-1-methyl-4'-nitroindole-3-azobenzene and the 2'-chloro- and 2'-nitro-analogues. From anhydrodethiosporidesmin 5-chloro-6,7-dimethoxy-8-methyl-2' ,4'-dinitropyrrolo[2,3-b]indole-2-azobenzene was obtained. These indoleazobenzenes are new compounds.
In the search for methods of diazonium coupling to o-dihydroxy-benzene, veratrole, the following new compounds were synthesized: 2-chloro-3', 4'-dimethoxy-4-nitrobiphenyl, 2-chloro-4'-hydroxy-3'-methoxy-4-nitroazobenzene, 4'-hydroxy-3'-methoxy-2, 4-dinitroazobenzene, 3', 4'-dimethoxy-2,4-dinitroazobenzene, 2-chloro-3', 4'-dimethoxy-4-nitroazobenzene and 2-chloro-4, 5-dimethoxy-2', 4'-dinitroazobenzene. 
In the ¹³C nuclear magnetic resonance spectra of sporidesmin and sporidesmin-D (dimethylthiosecosporidesmin) (decoupled and undecoupled) the peaks were assigned to the appropriate carbons. Where a resonance seemed anomalous, a reasonable explanation of the anomaly has been put forward. Comparison of the spectra of the two compounds indicated the resonances of those carbons which were involved in the strained epidithiodioxopiperazine ring system. Having assigned the peaks for sporidesmin, the structure of unknown analogues may now be more readily elucidated. 
An analogue of sporidesmin, sporidesmin-E, of comparable toxicity, having an -S-S-S- bridge was synthesized from sporidesmin for field studies. It was an unstable molecule readily decomposing to sporidesmin plus sulphur, e.g. pure sporidesmin-E always showed three spots in thin layer chromatography. The mixed melting point between these two compounds was undepressed (they melted within 3.5° of each other). That sporidesmin-E had been formed was confirmed by the M⁺ being 32 mass units greater than sporidesmin but this was still no criterion of purity. In the hydrogen-bonding investigation of the two compounds sporidesmin absorbed in the νOH region where sporidesmin-E did not so the degree that sporidesmin-E was contaminated with sporidesmin could be determined. Comparison of the infra-red spectra of sporidesmin and sporidesmin-E prepared in different ways (in KBr from MeOH or EtOEt, in nujol or in halocarbon oil) showed there were only minor differences between them. 
The hydrogen bonding analysis revealed that the C-S bonds of 3,11a-di-mercaptosecosporidesmin and sporidesmin-E were similarly orientated (i.e. diverging) and not more or less parallel as in sporidesmin-D. The mono-mercapto-derivative (methyl 11a-mercaptosecosporidesmin-3-S-acetate) showed no νSH peak: the elements of the thiol group are present (m.s.) but the hydrogen is ionic and with the adjacent amido-group is apparently in hyperconjugation.

year: 1978

title: A low frequency supercapacitor circulation technique to improve the efficiency of linear regulators based on LDO ICs

authors: Kularatna, Nihal

subjects: converters

abstract: Linear regulators have output specifications far superior to switch-mode techniques, except for the overall efficiency. This efficiency limitation can be overcome by applying a very low frequency supercapacitor circulation technique at the input side of a low dropout regulator IC. The technique was proven in 12V to 5 V versions, and, can be easily applied to other power supplies such as 5 to 3.3V or 5 to 1.5V versions required by various processors. The paper outlines the concepts and experimental results related to this technique. With the commercial LDO chips available with output current ratings up to 10A, and, thin profile supercapacitors available are with DC voltage ratings from 2.3V to 5.5V, the technique assists developing medium current linear regulators which could compete with present day switch-mode power supplies in efficiency and compactness, maintaining the superior output specifications of a linear regulator.

text: A low frequency supercapacitor circulation technique to improve the efficiency of linear regulators based on LDO ICs Linear regulators have output specifications far superior to switch-mode techniques, except for the overall efficiency. This efficiency limitation can be overcome by applying a very low frequency supercapacitor circulation technique at the input side of a low dropout regulator IC. The technique was proven in 12V to 5 V versions, and, can be easily applied to other power supplies such as 5 to 3.3V or 5 to 1.5V versions required by various processors. The paper outlines the concepts and experimental results related to this technique. With the commercial LDO chips available with output current ratings up to 10A, and, thin profile supercapacitors available are with DC voltage ratings from 2.3V to 5.5V, the technique assists developing medium current linear regulators which could compete with present day switch-mode power supplies in efficiency and compactness, maintaining the superior output specifications of a linear regulator.

year: 2011

title: Pōuri: Two original screenplays

authors: Kohere, Anthony

subjects: Screenplays

abstract: Pōuri, as defined on Te Aka Māori Dictionary, means “regret, mournful, gloomy, in the dark.” It is an apt description of the following stories: Unprivate Moments, and Haytham; or, The Māori Gothic. A common thematic tie between the characters is that they regret past actions, have become disheartened, and experience severe distress. As a thesis, the aim is to expand on what a “Māori story” is beyond the familiar, or dare I say, expected.

'Unprivate Moments' is about an estranged father and son reconciling their differences, disguised as a low-level spy film. I see this as an opportunity to take a well-defined genre and treat it in a realistic fashion in terms of location and character, although not necessarily in believability. Set in Whanganui it can play with many tropes and preconceptions, almost with self-awareness without falling into parody or metacommentary. The formatting does not follow industry standards but inverts the layout of the scene descriptions and dialogue. While I cannot say for certain, Stanley Kubrick is the only one I am aware of that has done this when he adapted 'A Clockwork Orange'. His rationale for this break in style was to draw the eye to the visual storytelling rather than the dialogue.

'Haytham' is centred on a young Byronic Māori man during the late 1880s. Like the archetype he is derived from, Haytham is a tall, dark, and mysterious man. As he reveals himself both to the audience and to the other characters, we come to find someone who has been  damaged and corrupted. This issue of childhood trauma is the central crux of the story and is explored through his relationships with the women in his life. And finally, despite the script’s setting and intentional use of familial names, this is not a biographical work.

text: Pōuri: Two original screenplays Pōuri, as defined on Te Aka Māori Dictionary, means “regret, mournful, gloomy, in the dark.” It is an apt description of the following stories: Unprivate Moments, and Haytham; or, The Māori Gothic. A common thematic tie between the characters is that they regret past actions, have become disheartened, and experience severe distress. As a thesis, the aim is to expand on what a “Māori story” is beyond the familiar, or dare I say, expected.

'Unprivate Moments' is about an estranged father and son reconciling their differences, disguised as a low-level spy film. I see this as an opportunity to take a well-defined genre and treat it in a realistic fashion in terms of location and character, although not necessarily in believability. Set in Whanganui it can play with many tropes and preconceptions, almost with self-awareness without falling into parody or metacommentary. The formatting does not follow industry standards but inverts the layout of the scene descriptions and dialogue. While I cannot say for certain, Stanley Kubrick is the only one I am aware of that has done this when he adapted 'A Clockwork Orange'. His rationale for this break in style was to draw the eye to the visual storytelling rather than the dialogue.

'Haytham' is centred on a young Byronic Māori man during the late 1880s. Like the archetype he is derived from, Haytham is a tall, dark, and mysterious man. As he reveals himself both to the audience and to the other characters, we come to find someone who has been  damaged and corrupted. This issue of childhood trauma is the central crux of the story and is explored through his relationships with the women in his life. And finally, despite the script’s setting and intentional use of familial names, this is not a biographical work.

year: 2022