Structure activity relationship studies of ochratoxin A analogues

Gabrielli, William Fullard (2002-03)

Thesis (MSc)--Stellenbosch University, 2002.

Thesis

ENGLISH ABSTRACT: Mycotoxins have assumed worldwide importance due to the ubiquitous occurrence of toxigenic fungi, their infestation of plant-based foods and feeds and the subsequent economical and health impact it because of their contamination of commercial products. Ochratoxin A (OA) is a nephrotoxic mycotoxin produced by isolates of Aspergillus ochraceus and Penicillium verrucosum and occurs frequently in nature. The major target for toxicity of OA in mammalian species is the kidneys and it has been the major cause of Danish Porcine Nephropathy. OA has also been extensively implicated in the aetiology of Balkan Endemic Nephropathy and Chronic Interstitial Nephropathy in Northern-Africa. Furthermore, OA has been identified as a carcinogen, an immunosuppressant and a teratogen with respect to the foetal central nervous system. Although a large amount of research has been conducted into the chemical nature of the toxicity of OA, the exact molecular mechanism of action of OA is not yet conclusive. Numerous structure activity relationship studies have suggested that the toxicity of OA may be assigned to three major processes: (i) inhibition of ATP production; (ii) inhibition of protein synthesis; and (iii) the disruption of hepatic microsomal calcium homeostasis through the promotion of membrane lipid peroxidation. It is the aim of this thesis to gain a better understanding, through the synthesis ofOA analogues, of the chemical structure responsible for the toxic function of the ochratoxins. The halogen-group has extensively been implicated in the toxicity of the ochratoxins. This is evident in ochratoxin B (OB), the dechloro analogue of OA, which is approximately ten times less toxic than OA. Preliminary tests have indicated that bromo-ochratoxin B(BrOB), the bromo analogue of OA, is more toxic than ochratoxin A to renal cells. Fluoro-ochratoxin B and other analogues of OA, where other amino acids are incorporated, should provide invaluable information on the structure-activity relationships and the mode of action of the ochratoxins. Our research effort addresses both these aspects (i) fluorination of the dihydroisocoumarin moiety and (ii) the coupling of different amino acids and dipeptides to the non-toxic hydrolysed product of OA, ochratoxin a. Chapter one includes a review of the important biological aspects of OA that has served as a guideline to the synthesis of effective OA analogues. An overview of the relevant chemistry involved in the modification of OA will conclude the chapter. Chapter two entails a discussion of fluorine in bio-organic chemistry. This includes an overview of the impact that fluorine substitution has on the biological reactivity of molecules. A review on the synthesis of organofluorine compounds, which forms the emphasis of this study, concludes the chapter. Chapter three elaborates on the different methodologies used in our attempts to synthesise fluoro-ochratoxin B and other analogues. These included the direct electrophilic fluorination of OB and different analogous aromatic model compounds by xenon difluoride, N-fluorobenzenesulfonimides and Selectfluor™ as fluorinating agents. Also involved is an investigation into an alternative route for the synthesis of fluoro aromatic compounds from bromo and chloro analogues by means of palladium catalysed trimethyl- and tributylstannyl and trimethylsilylation which in tum may be substituted with fluorine by means of xenon difluoride. Efforts towards the direct catalytic fluorosubstitution of aryl halides are also investigated. The synthesis of a key intermediate, fluoroacetoacetaldehyde, in a de nova synthetic route to fluoroochratoxin B is also discussed. Furthermore, the synthesis of novel OA analogues with respect to the replacement of the L-phenylalanine moiety is addressed. This includes the conversion of OA to Oa, by acid hydrolysis, followed by the coupling of ortho-, meta- and para- substituted DL-fluorophenylalanine to the lactone acid. This is followed by the synthesis of histidylhistidine methyl ester and attempted coupling to Oa. The coupling of halosalicylic acids and salicylic acid to L-phenylalanine, for use as model aromatic substrates for fluorination, IS discussed. Peptide coupling by dicyclohexylcarbodiimide carboxyl activation, with reference to the protection of the phenolic hydroxyl group in 5-chlorosalicylic acid for application to Oa, concludes this work.

AFRIKAANSE OPSOMMING: Mikotoksiene is van wêreld-wye belang as gevolg van die alomteenwoordige voorkoms van toksigeniese fungi, hul besmetting van plantaardige kossoorte en voerstowwe en die gevolglike ekonomiese en gesondheidsimpak deur die besoedeling van kommersiële produkte. Ochratoksien A (OA) is 'n nefrotoksiese mikotoksien wat geproduseer word deur isolate van Aspergillus ochraceus en Penicillium verrucosum en kom algemeen in die natuur voor. Die niere is die hoof teiken vir vergifiting deur OA in soogdierspesies en is as die vername oorsaak van "Danish Porcine Nephropathy" aangewys. OA word verder aangedui as die oorsaak vir "Balkan Endemic Nephropathy" en "Chronic Interstitial Nephropathy" in Noord- Afrika. OA is verder geïdentifiseer as 'n karsinogeen, immuno-onderdrukker en is teratogenies ten opsigte van die sentrale senuweestelsel van fetusse. Alhoewel aansienlike navorsing alreeds gewei is aan die chemiese natuur van die toksisiteit van OA, is die presiese molekulêre meganisme van OA reaktiwiteit onbeslis. Verskeie struktuur-aktiwitweit verwantskaps studies dui daarop dat die toksisiteit van OA hoofsaaklik toegeskryf kan word aan drie hoof prosesse: (i) inhibisie van ATP produksie; (ii) inhibisie van proteïen sintese; en (iii) die ontwrigting van hepatiese mikrosomale kalsiumhomeostase deur die bevordering van membraanlipiedperoksidasie. Hierdie tesis het ten doel, deur die sintese van OA analoë, om 'n beter insig oor die chemiese struktuur wat verantwoordelik is vir die toksiese funksionaliteit van ochratoksiene te verkry. Die halogeen substituent is grootliks geïmpliseer in die toksisiteit van OA. 'n Bewys hiervan is ochratoksien B (OB), die dechlooranaloog van OA, wat ongeveer tien maal minder toksies is as OA. Voorlopige ondersoeke het aangetoon dat bromoochratoksien B (BrOB), die broomanaloog van OA, meer toksies is vir nierselle as OA. Fluoorochratoksien B en ander analoë van OA, waar ander aminosure geïnkorporeer word, behoort waardevolle inligting te voorsien met betrekking tot die struktuur-aktiwiteitsverwantskappe en die wyse waarop ochratoksiene funksioneer. Hierdie navorsingspoging spreek beide aspekte aan; (i) die fluorering van die dihidroïsokumarien gedeelte en, (ii) die koppeling van verskillende armnosure en dipeptiede aan die nie-toksiese hidrolieseproduk van OA, nl. ochratoksien a. Hoofstuk een vervat 'n oorsig van die belangrike biologiese aspekte van OA wat dien as riglyn vir die sintese van doeltreffende OA analoë. Die hoofstuk word afgesluit met 'n oorsig van die relevante chemie betrokke by die modifisering van die struktuur van OA. Hoofstuk twee bevat 'n bespreking van die aanwending van fluoor in bio-organiese chemie. Dit bevat 'n oorsig van die impak wat fluoorsubstitusie het op die biologiese reaktiwiteit van molekules. 'n Opsomming oor die sintese van organofluoorverbindings, wat die essensie van hierdie studie is, beëindig die hoofstuk. Hoofstuk drie handeloor die veskillende metodes wat toegepas is in pogings om fluoorochratoksien B en ander analoë te sintetiseer. Dit sluit in die direkte elektrofiliese fluorering van OB en ander verwante aromatiese modelverbindings deur gebruik te maak van xenondifluoried, N-fluoorbenseensulfonimied en Selectfluor™ as fluoreringsreagense. Dit behels verder ook 'n ondersoek na 'n alternatiewe roete tot die sintese van fluooraromatiese verbindings vanaf broom- en chlooranaloë. Vir die doel word palladiumgekataliseerde trimetiel- en tributielstannilering, en trimetielsililering wat vervolgens deur middel van xenondifluoried met fluoor gesubstitueer kan word, aangewend. Pogings tot die direkte katalitiese fluoorsubstitusie van arielhaliede word ook bespreek. Die sintese van 'n sleutelintermediêr, fluoroasetoasetaldehied, in 'n de nova sintese roete tot fluoorochratoksien B word bespreek. Die sintese van nuwe OA analoë, met betrekking to die vervangmg van die Lfenielalanien (L-Phe) groep word ondersoek. Dit bevat die omsetting van OA na Oa, deur suurhidrolise, gevolg deur die koppeling van orto-, meta- en paragesubstitueerde DL-fluoorfenielalanien aan die laktoonsuur, Oa. Daarna word die sintese van histidielhistidienmetielester en die verdere pogings aangaande koppeling met Oa bespreek. Die koppeling van halosalisielsure en salisielsuur aan L-Phe wat dien as model aromatiese verbindings vir fluorering, word behandel. Peptiedkoppeling met behulp van disikloheksielkarbodiimied-karboksielaktivering, met inbegrip van die beskerming van die fenoliese hidroksiel groep m 5-chloorsalisielsuur Vir die toepassing op Oa, beëindig hierdie werk.

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