Biogenetically-Inspired Total Synthesis of Epidithiodiketopiperazines and Related Alkaloids

Natural products chemistry has historically been the prime arena for the discovery of new chemical transformations and the fountain of insights into key biological processes. It remains a fervent incubator of progress in the fields of chemistry and biology and an exchange mediating the flow of ideas between these allied fields of science. It is with this ethos that our group has taken an interest in and pursued the synthesis of a complex family of natural products termed the dimeric epipolythiodiketopiperazine (ETP) alkaloids. We present here an Account of the highly complex target molecules to which we pegged our ambitions, our systematic and relentless efforts toward those goals, the chemistry we developed in their pursuit, and the insight we have gained for their translational potential as potent anticancer molecules.National Institute of General Medical Sciences (U.S.) (Grant GM089732)Amgen Inc

Utjecaj medija i temperature na tvorbu gliotoksina u sojeva Aspergillus fumigatus

Gliotoxin is a secondary metabolite of the epipolythiodioxopiperazine family with biologically active internal disulfide bridge. It is produced by many fungal species, including Aspergillus fumigatus and A. terreus. A. fumigatus, which produces gliotoxin and more than twenty other secondary metabolites, is the leading cause of invasive aspergillosis. Gliotoxin production in situ influence the development of aspergillosis. This study investigated the in vitro production of gliotoxin in nine A. fumigatus isolates from the upper respiratory tract of immunocompromised patients. The effects of media composition and incubation temperature were studied. Gliotoxin was extracted from biomass and its concentration was semi-quantitatively analysed using thin-layer chromatography. Gliotoxin production was higher in the yeast-extract liquid medium (YES) than in the synthetic Czapek-Dox liquid medium (CZA). Incubation at 37 °C resulted in higher gliotoxin production than at 25 °C, probably because higher temperatures favour expansive growth of the mycelium. Gliotoxin could be detected after three days of incubation at concentrations 4.06 mg mL-1 (in YES at 37 °C) and 1.07 mg mL-1 (in CZA at 25 °C). YES broth as a medium containing 4 % sucrose and 2 % of yeast extract is a very rich substrate for the production of gliotoxin in vitro.Gliotoksin je sekundarni metabolit iz skupine epipolitiodioksipiperazina s biološki aktivnim internim disulfidnim mostom u molekuli, koji tvore razne plijesni i gljivica Candida albicans. Plijesan Aspergillus fumigatus vodeći je uzročnik invazivnih aspergiloza i također može tvoriti gliotoksin. Pretpostavlja se da in situ tvorba gliotoksina utječe na patogenezu aspergiloze. U ovom radu ispitali smo in vitro tvorbu gliotoksina u devet sojeva A. fumigatus vrste, izoliranih iz imunokompromitiranih pacijenata. Praćen je utjecaj medija i temperature inkubacije na tvorbu gliotoksina. Gliotoksin je ekstrahiran iz biomase i koncentracija mu je utvrđena polukvantitativno tankoslojnom kromatografijom. Tvorba gliotoksina uočena je već nakon trodnevne inkubacije u koncentracijama 4,06 mg mL-1 (u bujonu s kvaščevim ekstraktom – YES na 37 °C) i 1,07 mg mL-1 (u sintetskom Czapek-Dox bujonu – CZA na 25 °C). Tvorba gliotoksina bila je veća u YES bujonu, za razliku od sintetskog CZA bujona. Viša temperatura inkubacije (37 °C) također utječe na jaču tvorbu gliotoksina nego niža temperatura (25 °C). YES bujon s dodatkom 4 % saharoze i 2 % kvaščeva ekstrakta vrlo je bogat supstrat za in vitro tvorbu gliotoksina, kao jednog od lipofilnih sekundardnih metabolita plijesni vrste A. fumigatus

Towards understanding the gliotoxin detoxification mechanism: in vivo thiomethylation protects yeast from gliotoxin cytotoxicity

Gliotoxin (GT) is a mycotoxin produced by some species of ascomycete fungi including the opportunistic human pathogen Aspergillus fumigatus . In order to produce GT the host organism needs to have evolved a selfprotection mechanism. GT contains a redox-cycling disulfide bridge that is important in mediating toxicity. Recently is has been demonstrated that A. fumigatus possesses a novel thiomethyltransferase protein called GtmA that has the ability to thiomethylate GT in vivo , which aids the organism in regulating GT biosynthesis. It has been suggested that thiomethylation of GT and similar sulfur-containing toxins may play a role in providing self-protection in host organisms. In this work we have engineered Saccharomyces cerevisiae , a GT-naïve organism, to express A. fumigatus GtmA. We demonstrate that GtmA can readily thiomethylate GT in yeast, which results in protection of the organism from exogenous GT. Our work has implications for understanding the evolution of GT self-protection mechanisms in organisms that are GT producers and non-producers

Karakterizacija sirodezmina izolovanih iz fitopatogene gljive Leptosphaeria maculans

The pathogenicity of phytopathogenic fungi is associated with phytotoxins, especially with their chemical nature and quantity. Sirodesmins are phytotoxins from the epipolythiodioxopiperazines group, produced by the fungus Leptosphaeria maculans, which are a cause of blackleg and stem canker in oilseed rape (Brassica napus L.). The aim of this work was to obtain a detailed chemical profile of sirodesmins in five fungal isolates (four from Vojvodina, Serbia, and one from the Centre for Agricultural Research, Rothamsted, UK). Sirodesmins showing different phytotoxicity on treated cotyledons of cv. Quinta were separated and detected by thin layer chromatography in all analysed isolates (L.m, C-3, St-5 and S-11) except K-113, which neither contained sirodesmin congeners nor did it exhibit activity. By use of high performance liquid chromatography coupled with tandem mass spectrometer, it was possible to identify total of 10 sirodesmins, together with their precursor-phomamide. It was found that the dominant epipolythiodioxopiperazines of the investigated L. maculans isolates were sirodesmin PL, sirodesmin C, and their de-acetylated derivatives.Patogenost fitopatogenih gljiva povezana je sa fitotoksinima, a naročito sa njihovom hemijskom prirodom i količinom. Sirodezmini su fitotoksini iz grupe epipolitiodioksopiperazina, koje proizvodi gljiva Leptosphaeria maculans, uzročnik suve truleži korena i raka stabla uljane repice. Cilj ovog rada bila je detaljna hemijska karakterizacija sirodezmina u pet izolata gljiva (četiri iz Vojvodine i jedan iz Velike Britanije, Centar za poljoprivredna istraživanja, Rothamsted). Kod svih ispitivanih izolata (L. maculans, C-3, St-3, S-11), osim K-113 (koji nije sadržao sirodezmine niti pokazivao aktivnost) tankoslojnom hromatografijom su razdvojeni i detektovani sirodezmini koji su pokazali različitu fitotoksičnost na tretiranim kotiledonima sorte Quinta. Primenom tečne hromatografije visoke efikasnosti, kuplovane sa tandemskim masenim spektrometrom, bilo je moguće identifikovati ukupno 10 sirodezmina, kao i njihov prekursor - fomamid. Utvrđeno je da su dominantni epipolitio-dioksopiperazini ispitivanih izolata L. maculans sirodezmin PL, sirodezmin C i njihovi deacetilovani derivati

Total synthesis of (+/-)-aspirochlorine, The

1993 Fall.Includes bibliographical references.Aspirochlorine is a unique epidithiodioxopiperazine isolated from Aspergillus oryzae, Asp. tamarii and Asp. flavus. The molecule contains a highly unusual bicyclo [3.2.2]disulfide ring system which has previously never been prepared. The first total synthesis of (±)-Aspirochlorine was achieved from commercially available 5-chlororesorcinol 324 in 16 steps. The key step in the synthesis was an efficient intramolecular cycloaddition reaction of hydroxamic ester 344 to form the parent spiro [benzofuran-2(3H),2'-piperazine] ring system 345 as a single stereoisomer. In addition the synthesis employed a 2-nitrobenzyl moiety as a novel amide protecting group. The 2-nitrobenzyl group could be removed in 72% yield under photolytic conditions. Synthetic aspirochlorine was identical to natural material in comparison by 1H NMR, IR and HPLC. Comparison of the biological activity of aspirochlorine versus other epidithiodioxopiperazines was investigated as a function of superoxide production. Although aspirochlorine was shown to be capable of producing superoxide as evidenced in DNA plasmid nicking and NBT reduction assays, the observed activity was less than the 6-membered epidithiodioxopiperazines

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

<p>Abstract</p> <p>Background</p> <p>Genes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.</p> <p>Results</p> <p>Putative ETP gene clusters are present in 14 ascomycete taxa, but absent in numerous other ascomycetes examined. These clusters are discontinuously distributed in ascomycete lineages. Gene content is not absolutely fixed, however, common genes are identified and phylogenies of six of these are separately inferred. In each phylogeny almost all cluster genes form monophyletic clades with non-cluster fungal paralogues being the nearest outgroups. This relatedness of cluster genes suggests that a progenitor ETP gene cluster assembled within an ancestral taxon. Within each of the cluster clades, the cluster genes group together in consistent subclades, however, these relationships do not always reflect the phylogeny of ascomycetes. Micro-synteny of several of the genes within the clusters provides further support for these subclades.</p> <p>Conclusion</p> <p>ETP gene clusters appear to have a single origin and have been inherited relatively intact rather than assembling independently in the different ascomycete lineages. This progenitor cluster has given rise to a small number of distinct phylogenetic classes of clusters that are represented in a discontinuous pattern throughout ascomycetes. The disjunct heredity of these clusters is discussed with consideration to multiple instances of independent cluster loss and lateral transfer of gene clusters between lineages.</p

Gliotoxin and Bis-methyl-glitoxin production by Trichoderma spp. as biocontrol agents running title: human risk potential by using Trichoderma spp. metabolites

Os produtos derivados do Trichoderma são utilizados a nível mundialcomo agentes de controlo biológico, pelas suas propriedades e benefícios, em relação aos pesticidas sintéticos. Algumas das espécies de Trichoderma podem produzir metabolitos secundários, que podem ser toxinas, medicamentos, ou ambos. As propriedades desses metabolitos parecem ser responsáveis pelo uso extensivo dessas espécies contra fitopatógenos, sendo o potencial biotecnológico de Trichoderma e dos seus metabolitos também estendido à Indústria e à Saúde Humana. A gliotoxina pertence à classe epipolitiodioxopiperazina e é um metabolito de Trichoderma spp.. É tóxico para humanos e animais, mas tem efeitos sobre células tumorais humanas e contra patógenos humanos. O objetivo deste estudo foi avaliar o efeito de fitopatógenos, como Alternaria tomatophila e Rhizoctonia solani, na produção de gliotoxina e bis-metil-gliotoxina por Trichoderma spp. Com o objetivo de explorar o potencial uso desses metabolitos como agentes de biocontrolo, os resultados mostraram que a produção de metabolitos secundários por T. virens e por T. afroharzianum pode afetar o desenvolvimento do fitopatógeno A. tomatophila. Também foi realçado que T. reesei não é capaz de inibir o crescimento de A. tomatophila. As lacunas de informação sobre o estudo dos metabolitos secundários é de grande importância, devido à sua ampla presença no meio ambiente. Apesar dos reconhecidos benefícios a nível ambiental, a exposição dos trabalhadores à gliotoxina e bis-metil-gliotoxina poderá ter efeitos na saúde, pela exposição crónica, pelo que sugerese o seu estudo, no sentido de compreender a extensão e consequências na saúde resultantes da exposição humana ocupacional a Trichoderma spp. e seus metabolitos. Os riscos e benefícios do uso de Trichoderma spp. como um agente de biocontrolo devem ser criteriosamente medidos, no sentido de desenvolver regulamentação

Gliotoxin Production in Aspergillus fumigatus

Aspergillus fumigalus is a ubiquitous fungus that causes an infection called aspergillosis in immune-compromised surgery patients (Latge, 2001, 384; Bok et al. 2006). Aspergillosis cases have increased in the past decade due to the increasing number of patients undergoing immunosuppressive therapy and receiving organ transplants (Bok et al. 2006). Although there is still disagreement as to the causative agent of the symptoms accompanying aspergillosis, many scientists suspect that the compound, gliotoxin, plays a major role, hi this study, we compared the amounts of gliotoxin produced from clinical and environmental isolates of A. fumigatus. We hypothesized that the amount of gliotoxin produced would be higher in the clinical isolates in comparison to the environmental isolates. Cell assay results showed that the clinical isolates produced more gliotoxin than the environmental isolates while the HPLC results showed no significant difference between gliotoxin production in clinical and environmental isolates