Publications by authors named "Isidro G Collado"

66 Publications

Impairment of botrydial production in Botrytis cinerea allows the isolation of undescribed polyketides and reveals new insights into the botcinins biosynthetic pathway.

Phytochemistry 2021 Mar 8;183:112627. Epub 2021 Jan 8.

Departamento de Química Orgánica, Facultad de Ciencias, Campus Universitario Río San Pedro s/n, Torre sur, 4a planta, Universidad de Cádiz, 11510, Puerto Real, Cádiz, Spain. Electronic address:

Botrytis cinerea is a necrotrophic fungal pathogen that affects a total of 586 genera representing approximately 1400 plant species. This pathogen produces two families of phytotoxins involved in its infection process i.e. botrydial and its relatives, and botcinic and botcineric acids and their relatives, botcinins. The botrydial biosynthetic cluster consists of seven genes, where the gene BcBOT4 encodes a cytochrome P450 monooxygenase that was shown to catalyse regio- and stereospecific hydroxylation at position C-4 of the presilphiperfolan-8-β-ol skeleton. The null mutant bcbot4Δ halted the production of botrydial and its derivatives, and instead accumulated tricyclic presilphiperfolane alcohol and overproduced a significant number of polyketides. A detailed study of the bcbot4Δ mutant led us to the isolation and characterization of five undescribed polyketides, three derived from botcinic and botcineric acids (botcinins H, I, J), one derived from the initial pentaketide (botcinin K), and one cinbotolide derivative (cinbotolide D). Botcinins are tetra-methylated tetraketides biosynthesized by the sequential assembly of a pentaketide (C10) based on an acetate primer unit which is lost through a retro-Claisen type C-C bond cleavage. The structural characterization of botcinin K showed a basic chemical structure corresponding to a botcinin (C14) derivative obtained directly from the original per-methylated pentaketide leading to the biosynthesis of botrylactone and other botcinins, confirming the previously proposed biosynthetic route.
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http://dx.doi.org/10.1016/j.phytochem.2020.112627DOI Listing
March 2021

Biocatalytic Preparation of Chloroindanol Derivatives. Antifungal Activity and Detoxification by the Phytopathogenic Fungus .

Plants (Basel) 2020 Nov 25;9(12). Epub 2020 Nov 25.

Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, Puerto Real, 11510 Cádiz, Spain.

Indanols are a family of chemical compounds that have been widely studied due to their broad range of biological activity. They are also important intermediates used as synthetic precursors to other products with important applications in pharmacology. Enantiomerically pure chloroindanol derivatives exhibiting antifungal activity against the phytopathogenic fungus were prepared using biocatalytic methods. As a result of the biotransformation of racemic 6-chloroindanol () and 5-chloroindanol () by the fungus , the compounds -(+)-6-chloroindan-1,2-diol (-(+)-), -(+)-5-chloroindan-1,3-diol (-(+)-), -(+)-5-chloroindan-1,3-diol (-(+)-), -(-)-5-chloroindan-1,3-diol (-(-)-), and -(+)-5-chloroindan-1,2-diol (-(+)-) were isolated for the first time. These products were characterized by spectroscopic techniques and their enantiomeric excesses studied by chromatographic techniques. The results obtained in the biotransformation seem to suggest that the fungus uses oxidation reactions as a detoxification mechanism.
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http://dx.doi.org/10.3390/plants9121648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759767PMC
November 2020

Identification of the Sesquiterpene Cyclase Involved in the Biosynthesis of (+)-4-Epi-eremophil-9-en-11-ol Derivatives Isolated from .

ACS Chem Biol 2020 10 1;15(10):2775-2782. Epub 2020 Oct 1.

Facultad de Ciencias, Departamento de Química Orgánica, Universidad de Cádiz, Campus Universitario Río San Pedro s/n, Torre sur, 4a planta, Puerto Real, 11510 Cádiz, Spain.

Cultivation of the phytopathogenic fungus using sublethal amounts of copper sulfate yielded a cryptic sesquiterpenoids family, which displayed the basic chemical structure of (+)-4-epi-eremophil-9-ene. The biosynthesis pathway was established, and the route involved the likely transformation of the diphosphate of farnesyl (FDP), to give a -fused eudesmane cation, through ()-hedycaryol, finally yielding the (+)-4-epi-eremophil-9-enol derivatives. An expression study of genes that code for the sesquiterpene cyclases (STC), including the recently reported gene present in the genome, was performed in order to establish the STC involved in this biosynthesis. The results showed a higher expression level for the gene with respect to the other genes in both wild-type strains, B05.10 and UCA992. Deletion of the gene eliminated (+)-4-epi-eremophilenol biosynthesis, which could be re-established by complementing the null mutant with the gene. Chemical analysis suggested that STC7 is the principal enzyme responsible for the key step of cyclization of FDP to eremophil-9-en-11-ols. Furthermore, a thorough study of the two wild-types and the complemented mutant revealed four new eremophilenol derivatives whose structures are reported here.
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http://dx.doi.org/10.1021/acschembio.0c00561DOI Listing
October 2020

Botrydial confers Botrytis cinerea the ability to antagonize soil and phyllospheric bacteria.

Fungal Biol 2020 01 15;124(1):54-64. Epub 2019 Nov 15.

Instituto Tecnológico de Chascomús (INTECH), Universidad Nacional de General San Martín (UNSAM)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Chascomús, Argentina. Electronic address:

The role of the sesquiterpene botrydial in the interaction of the phytopathogenic fungus Botrytis cinerea and plant-associated bacteria was analyzed. From a collection of soil and phyllospheric bacteria, nine strains sensitive to growth-inhibition by B. cinerea were identified. B. cinerea mutants unable to produce botrydial caused no bacterial inhibition, thus demonstrating the inhibitory role of botrydial. A taxonomic analysis showed that these bacteria corresponded to different Bacillus species (six strains), Pseudomonas yamanorum (two strains) and Erwinia aphidicola (one strain). Inoculation of WT and botrydial non-producing mutants of B. cinerea along with Bacillusamyloliquefaciens strain MEP18 in soil demonstrated that both microorganisms exert reciprocal inhibitory effects; the inhibition caused by B. cinerea being dependent on botrydial production. Moreover, botrydial production was modulated by the presence of B. amyloliquefaciens MEP18 in confrontation assays in vitro. Purified botrydial in turn, inhibited growth of Bacillus strains in vitro and cyclic lipopeptide (surfactin) production by B. amyloliquefaciens MEP18. As a whole, results demonstrate that botrydial confers B. cinerea the ability to inhibit potential biocontrol bacteria of the genus Bacillus. We propose that resistance to botrydial could be used as an additional criterion for the selection of biocontrol agents of plant diseases caused by B. cinerea.
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http://dx.doi.org/10.1016/j.funbio.2019.11.003DOI Listing
January 2020

Synthesis of Trichodermin Derivatives and Their Antimicrobial and Cytotoxic Activities.

Molecules 2019 Oct 22;24(20). Epub 2019 Oct 22.

Department of Organic Chemistry, Campus of Puerto Real, Science Faculty, University of Cádiz, 11510 Puerto Real, Cádiz, Spain.

Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In , the first specific step in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes the conversion of farnesyl diphosphate to trichodiene and is encoded by the gene. Overexpression of resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with a short chain at the C-4 position displayed selective antimicrobial activity against and they showed MIC values similar to those displayed by trichodermin. It is important to highlight the cytotoxic selectivity observed for compounds , , and , which presented average IC values of 2 μg/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against Fa2N4 (non-tumoral immortalized human hepatocytes).
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http://dx.doi.org/10.3390/molecules24203811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6833013PMC
October 2019

Natural Compounds That Modulate the Development of the Fungus and Protect .

Plants (Basel) 2019 Apr 26;8(5). Epub 2019 Apr 26.

Química Orgánica de Productos Naturales, Instituto de Química, Universidad de Antioquia, Cl. 62 #52⁻59, Medellín, Colombia.

is the causal agent of gray mold disease and is responsible for the loss of millions of dollars in crops in worldwide. Currently, this pathogen exhibits increasing resistance to conventional fungicides; therefore, better control methods and novel compounds with a more specific mechanism of action but without biocidal effects, are required. In this work, several natural compounds to control were analyzed in vitro. Detected effects were dependent on the stage of fungus development, and 3-phenyl-1-propanol displayed the most potent inhibition of in vitro germination, germ tube development, and sporulation. However, it had lower protection of leaves and postharvest fruit in plant infection. Isoeugenol and 1-phenylethanol exhibited lower inhibition of in vitro germination and sporulation, but at the highest concentrations, they inhibited germ tube elongation. Although the lowest rates of foliage infection were recorded using isoeugenol and 3-phenyl-1-propanol, 1-phenylethanol significantly decreased the disease in postharvest tomato fruit, with an efficacy like Mancozeb, but at 18 times lower micromolar concentration. All compounds resulted in high cell viability after spores were removed from the treatment solution exhibited high cell viability, suggesting a non-biocidal effect. The diversity of and in-plant effects seems to indicate a different mechanism of action.
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http://dx.doi.org/10.3390/plants8050111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571876PMC
April 2019

Botcinic acid biosynthesis in Botrytis cinerea relies on a subtelomeric gene cluster surrounded by relics of transposons and is regulated by the ZnCys transcription factor BcBoa13.

Curr Genet 2019 Aug 8;65(4):965-980. Epub 2019 Mar 8.

UMR BIOGER, INRA, AgroParisTech, Université Paris-Saclay, 78850, Thiverval-Grignon, France.

Botcinic acid is a phytotoxic polyketide involved in the virulence of the gray mold fungus Botrytis cinerea. Here, we aimed to investigate the specific regulation of the cluster of Bcboa genes that is responsible for its biosynthesis. Our analysis showed that this cluster is located in a subtelomeric genomic region containing alternating G + C/A + T-balanced regions, and A + T-rich regions made from transposable elements that underwent RIP (Repeat-Induced Point mutation). Genetic analyses demonstrated that BcBoa13, a putative ZnCys transcription factor, is a nuclear protein with a major positive regulatory role on the expression of other Bcboa1-to-Bcboa12 genes, and botcinic acid production. In conclusion, the structure and the regulation of the botcinic acid gene cluster show similar features with the cluster responsible for the biosynthesis of the other known phytotoxin produced by B. cinerea, i.e., the sesquiterpene botrydial. Both clusters contain a gene encoding a pathway-specific ZnCys positive regulator, and both are surrounded by relics of transposons which raise some questions about the role of these repeated elements in the evolution and regulation of the secondary metabolism gene clusters in Botrytis.
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http://dx.doi.org/10.1007/s00294-019-00952-4DOI Listing
August 2019

Isotopic Labeling Studies Reveal the Patulin Detoxification Pathway by the Biocontrol Yeast Rhodotorula kratochvilovae LS11.

J Nat Prod 2018 12 21;81(12):2692-2699. Epub 2018 Nov 21.

Departamento de Química Orgánica, Facultad de Ciencias , Universidad de Cádiz , Campus Universitario Río San Pedro s/n, Torre sur, 4a planta, 11510 , Puerto Real , Cádiz , Spain.

Patulin (1) is a mycotoxin contaminant in fruit and vegetable products worldwide. Biocontrol agents, such as the yeast Rhodotorula kratochvilovae strain LS11, can reduce patulin (1) contamination in food. R. kratochvilovae LS11 converts patulin (1) into desoxypatulinic acid (DPA) (5), which is less cytotoxic than the mycotoxin (1) to in vitro human lymphocytes. In the present study, we report our investigations into the pathway of degradation of patulin (1) to DPA (5) by R. kratochvilovae. Isotopic labeling experiments revealed that 5 derives from patulin (1) through the hydrolysis of the γ-lactone ring and subsequent enzymatic modifications. The ability of patulin (1) and DPA (5) to cause genetic damage was also investigated by the cytokinesis-block micronucleus cytome assay on in vitro human lymphocytes. Patulin (1) was demonstrated to cause much higher chromosomal damage than DPA (5).
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http://dx.doi.org/10.1021/acs.jnatprod.8b00539DOI Listing
December 2018

A GC-MS untargeted metabolomics approach for the classification of chemical differences in grape juices based on fungal pathogen.

Food Chem 2019 Jan 10;270:375-384. Epub 2018 Jul 10.

National Wine and Grape Industry Centre, School of Agricultural and Wine Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, New South Wales 2678, Australia. Electronic address:

Fungal bunch rot of grapes leads to production of detrimental flavour compounds, some of which are well characterised but others remain unidentified. The current study uses an untargeted metabolomics approach to classify volatile profiles of grape juices based on the presence of different fungal pathogens. Individual grape berries were inoculated with Botrytis cinerea, Penicillium expansum, Aspergillus niger or A. carbonarius. Grape bunches were inoculated and blended with healthy fruit, to provide 10% (w/w) infected juice. Juices from the above sample batches were analysed by GC/MS. PLS-DA of the normalised summed mass ions indicated sample classification according to pathogen. Compounds identified from those mass ion matrices that had high discriminative value for classification included 1,5-dimethylnaphthalene and several unidentified sesquiterpenes that were relatively higher in B. cinerea infected samples. A. niger and A. carbonarius samples were relatively higher in 2-(4-hexyl-2,5-dioxo-2,5-dihydrofuran-3-yl)acetic acid, while P. expansum samples were higher in γ-nonalactone and m-cresol.
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http://dx.doi.org/10.1016/j.foodchem.2018.07.057DOI Listing
January 2019

Structural and biosynthetic studies on eremophilenols related to the phytoalexin capsidiol, produced by Botrytis cinerea.

Phytochemistry 2018 Oct 19;154:10-18. Epub 2018 Jun 19.

Departamento de Química Orgánica, Facultad de Ciencias, Campus Universitario Río San Pedro s/n, Torre sur, 4a planta, Universidad de Cádiz, 11510, Puerto Real, Cádiz, Spain. Electronic address:

A thorough study of the fermentation broth of three strains of Botrytis cinerea which were grown on a modified Czapek-Dox medium supplemented with 5 ppm copper sulphate, yielded five undescribed metabolites. These metabolites possessed a sesquiterpenoid (+)-4-epi-eremophil-9-ene carbon skeleton which was enantiomeric to that of the phytoalexin, capsidiol. The isolation of these metabolites when the fungus was stressed, suggests that they may be potential effectors used by B. cinerea to circumvent plant chemical defences against phytopathogenic fungi. The biosynthesis of these compounds has been studied using H and C labelled acetate.
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http://dx.doi.org/10.1016/j.phytochem.2018.06.010DOI Listing
October 2018

Relevance of the deletion of the Tatri4 gene in the secondary metabolome of Trichoderma arundinaceum.

Org Biomol Chem 2018 04;16(16):2955-2965

Departamento de Química Orgánica, Facultad de Ciencias, Campus Universitario Río San Pedro s/n, Torre sur, 4° planta, Universidad de Cádiz, 11510, Puerto Real, Cádiz, Spain.

The fungus Trichoderma arundinaceum (Ta37) has a significant biocontrol application which has been related to the production of the trichothecene, harzianum A (2). Previous studies with a strain of T. arundinaceum which was blocked for the production of 2, revealed the existence of a chemical cross-regulation between the biocontrol fungus and its target organism. A study of the secondary metabolome of a single culture of a mutant of T. arundinaceum TaΔTri4 which produces trichothecene biosynthetic intermediates, has now been carried out. The production of secondary metabolites in a co-culture with the phytopathogen, Botrytis cinerea, was then analyzed. The mutant produced a larger quantity of the aspinolides B (6) and C (7) and other derivatives when compared to the wild type Ta37. Ten new metabolites were isolated: three aspinolides 12-14, the γ-lactones 15 and 16, two hemi-ketals 17 and 18 and three aspinolide degradation products, 19, 21 and 22. In the confrontation cultures involving the TaΔTri4 and the B. cinerea B05.10 strains, there was a higher production of the aspinolides B and C by the TaΔTri4 mutant while the production of the botryanes and botcinins by B. cinerea was reduced in the area of interaction between the cultures. These results shed light on the chemical cross-talk and ecological interactions between these fungi.
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http://dx.doi.org/10.1039/c8ob00338fDOI Listing
April 2018

Metabolism of Antifungal Thiochroman-4-ones by Trichoderma viride and Botrytis cinerea.

J Nat Prod 2018 04 2;81(4):1036-1040. Epub 2018 Apr 2.

Departamento de Química Orgánica, Facultad de Ciencias , Universidad de Cádiz , Campus Río S. Pedro, 11510 Puerto Real, Cádiz, Spain.

Biotransformation of 6-methylthiochroman-4-one (1) and 6-chlorothiochroman-4-one (2) was performed using Trichoderma viride in order to obtain new derivatives with antifungal properties against the phytopathogen Botrytis cinerea. Two thiochromanone derivatives are described for the first time. Antifungal activity of these compounds was tested against two different strains of Botrytis cinerea; 1 and 2 gave 100% inhibition of Bc2100 at 100-250 μg/mL, and 3 gave a maximal inhibition of 96% of BcUCA992 at 200 μg/mL. The detoxification mechanism of 1 and 2 by B. cinerea was also investigated.
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http://dx.doi.org/10.1021/acs.jnatprod.7b00298DOI Listing
April 2018

Phenotypic Effects and Inhibition of Botrydial Biosynthesis Induced by Different Plant-Based Elicitors in Botrytis cinerea.

Curr Microbiol 2018 Apr 17;75(4):431-440. Epub 2017 Nov 17.

Microbiology Laboratory, Andalusian Center for Grape and Grapevine Research, Faculty of Marine and Environmental Sciences, University of Cadiz, Polígono Rio San Pedro s/n, 11510, Puerto Real, Cádiz, Spain.

Botrytis cinerea is considered a model organism for the study of plant-pathogen interaction showing great genetic diversity and a high degree of morphological variability depending on environmental conditions. The use of new compounds and plant-based elicitors may trigger the expression of different B. cinerea genes, providing new sources of virulence factors. This work is focused on elucidating the phenotypic effect in B. cinerea of different carbon sources such as glucose, cellulose and tomato cell walls (TCW). Production of botrydial and dihydrobotrydial toxins was evaluated using thin-layer chromatography (TLC), proton nuclear magnetic resonance spectroscopy (H-NMR) and mass spectrometry (UPLC-HRESIMS). Expression of the toxin biosynthesis gene BcBOT2 was followed using RT-qPCR. Results show an inhibition of the toxin biosynthesis pathway when TCW are present as a sole carbon source, suggesting that the toxin is only produced when rich molecules, like glucose, are available for fungal metabolism. That suggests a connection between gene expression of virulence factors and environmental conditions, where the silent genes can be induced by different culture conditions.
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http://dx.doi.org/10.1007/s00284-017-1399-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842495PMC
April 2018

Mild Epoxidation of Allylic Alcohols Catalyzed by Titanium(III) Complexes: Selectivity and Mechanism.

ACS Omega 2017 Jul 3;2(7):3083-3090. Epub 2017 Jul 3.

Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas, Universidad de Cádiz, Campus Universitario Puerto Real s/n, 11510 Puerto Real, Cádiz, Spain.

A novel methodology for the epoxidation of a broad range of primary, secondary, and tertiary allylic alcohols is described using -butyl hydroperoxide as oxidant and Ti(III) species generated by reduction of Ti(IV) complexes, with manganese (0) in 1,4-dioxane under mild reaction conditions. The reaction proceeded with wide substrate scope and high chemo- and diastereoselectivity. A mechanistic pathway for the reaction is also discussed.
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http://dx.doi.org/10.1021/acsomega.7b00386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6641598PMC
July 2017

The formation of sesquiterpenoid presilphiperfolane and cameroonane metabolites in the Bcbot4 null mutant of Botrytis cinerea.

Org Biomol Chem 2017 Jun;15(25):5357-5363

Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain.

Botrytis cinerea is a polyphagous fungal parasite which causes serious damage to more than 200 plant species and consequent economic losses for commercial crops. This pathogen produces two families of phytotoxins, the botryanes and botcinins, which are involved in the infection mechanism. The B. cinerea genome has provided a complete picture of the genes involved in the biosynthesis of its secondary metabolites. The botrydial biosynthetic gene cluster has been identified. This cluster consists of seven genes, where the genes BcBOT1, BcBOT3 and BcBOT4 encode three mono-oxygenases. A study of the Bcbot4Δ null mutant revealed that this mono-oxygenase was involved in the hydroxylation at C-4 of the probotryane skeleton (C-11 of the presilphiperfolane skeleton). A detailed study of the Bcbot4Δ null mutant has been undertaken in order to study the metabolic fate of the presilphiperfolan-8-ol intermediate biosynthesized by this organism and in particular by this strain. As a result three new presilphiperfolanes and three new cameroonanes have been identified. The results suggest that the absence of the oxygen function at C-11 of the presilphiperfolane skeleton permits rearrangement to a cameroonane whilst hydroxylation at C-11 precludes this rearrangement. It is possible that the interactions of the C-11 hydroxylated derivatives perturb the stereo-electronic requirements for the migration of the C-11:C-7 sigma bond to C-8.
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http://dx.doi.org/10.1039/c7ob01088eDOI Listing
June 2017

Lathyrane Diterpenes from the Latex of Euphorbia laurifolia.

Nat Prod Commun 2017 May;12(5):671-673

Two new macrocyclic diterpenes, 2-epi-latazienone (4) and 15β-acetoxy-7β-nicotinoyloxy-3β,8α-di-(2-methylpropanoyloxy)-4αH,9αH,l1αH-lathyra- 5E,12E-dien-14-one (5), and three known lathyrane-type diterpenes (1-3) were isolated from Euphorbia laurifolia latex. Their structures were determined on the basis of a detailed analysis of their 1D and 2D NMR spectroscopic and mass spectral data.
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May 2017

Efficient -Acylation of Alcohols and Phenol Using CpTiCl as a Reaction Promoter.

European J Org Chem 2016 Jul 4;2016(21):3584-3591. Epub 2016 Jul 4.

Departamento de Química Orgánica University of Cádiz Facultad de Ciencias Polígono Río San Pedro s/n 11510 Puerto Real Cádiz Spain.

A method has been developed for the conversion of primary, secondary, and tertiary alcohols, and phenol, into the corresponding esters at room temperature. The method uses a titanium(III) species generated from a substoichiometric amount of titanocene dichloride together with manganese(0) as a reductant, as well as methylene diiodide. It involves a transesterification from an ethyl ester, or a reaction with an acyl chloride. A radical mechanism is proposed for these transformations.
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http://dx.doi.org/10.1002/ejoc.201600496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095770PMC
July 2016

Genetic and Molecular Basis of Botrydial Biosynthesis: Connecting Cytochrome P450-Encoding Genes to Biosynthetic Intermediates.

ACS Chem Biol 2016 10 1;11(10):2838-2846. Epub 2016 Sep 1.

Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz , Puerto Real, 11510 Cádiz, Spain.

Over two hundred species of plants can be infected by the phytopathogenic fungus Botrytis cinerea under a range of different environmental conditions. In response to these, the fungus produces unique terpenoid and polyketide metabolites. Parts of the plants may be killed by the phytotoxin botrydial, enabling the fungus to feed on the dead cells. In this paper, we describe the genetic and molecular basis of botrydial biosynthesis and the function of the five genes of the genome of B. cinerea that together constitute the botrydial biosynthetic gene cluster. Genes BcBOT3 and BcBOT4, encoding two cytochrome P450 monooxygenases, were inactivated by homologous recombination and were shown to catalyze regio- and stereospecific hydroxylations at the carbons C-10 and C-4, respectively, of the presilphiperfolan-8β-ol skeleton. The null mutants, bcbot3Δ and bcbot4Δ, accumulated key intermediates in the botrydial biosynthesis enabling the complete genetic and molecular basis of the botrydial biosynthetic pathway to be established. Furthermore, the bcbot4Δ mutant overproduced a significant number of polyketides, which included, in addition to known botcinins, botrylactones and cinbotolide A, two new botrylactones and two new cinbotolides, cinbotolides B and C.
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http://dx.doi.org/10.1021/acschembio.6b00581DOI Listing
October 2016

Trichothecenes and aspinolides produced by Trichoderma arundinaceum regulate expression of Botrytis cinerea genes involved in virulence and growth.

Environ Microbiol 2016 11 15;18(11):3991-4004. Epub 2016 Jul 15.

Area of Microbiology, Universitary School of Agricultural Engineers. University of León, Ponferrada, Spain.

Trichoderma arundinaceum (Ta37) and Botrytis cinerea (B05.10) produce the sesquiterpenoids harzianum A (HA) and botrydial (BOT), respectively. TaΔTri5, an HA non-producer mutant, produces high levels of the polyketide compounds aspinolides (Asp) B and C. We analyzed the role of HA and Asp in the B. cinerea-T. arundinaceum interaction, including changes in BOT production as well as transcriptomic changes of BcBOT genes involved in BOT biosynthesis, and also of genes associated with virulence and ergosterol biosynthesis. We found that exogenously added HA up-regulated the expression of the BcBOT and all the virulence genes analyzed when B. cinerea was grown alone. However, a decrease in the amount of BOT and a down-regulation of BcBOT gene expression was observed in the interaction zone of B05.10-Ta37 dual cultures, compared to TaΔTri5. Thus, the confrontation with T. arundinaceum results in an up-regulation of most of the B. cinerea genes involved in virulence yet the presence of T. arundinaceum secondary metabolites, HA and AspC, act separately and together to down-regulate the B. cinerea genes analyzed. The present work emphasizes the existence of a chemical cross-regulation between B. cinerea and T. arundinaceum and contributes to understanding how a biocontrol fungus and its prey interact with each other.
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http://dx.doi.org/10.1111/1462-2920.13410DOI Listing
November 2016

Antifungal and Cytotoxic Assessment of Lapachol Derivatives Produced by Fungal Biotransformation.

Nat Prod Commun 2016 Jan;11(1):95-8

In the screening for biological active compounds, the biotransformation processes catalyzed by filamentous fungi are useful because they can provide information about the possible appearance of toxic metabolites after oral administration and also generate new leads. In this paper, biotransformation of lapachol (1) by three fungal strains, Mucor circinelloides NRRL3631, Botrytis cinerea UCA992 and Botrytis cinerea 2100, has been investigated for the first time. Lapachol (1) was biotransformed into avicequinone-A (2) by M circinelloides, 3'-hydroxylapachol (3) by B. cinerea, and into dehydro-α-lapachone (4) by both fungi. All these compounds were evaluated for their cytotoxic activities. The metabolite 2 displayed non-selective cytotoxicity against tumor and normal cell lines, 3 did not show cytotoxicity against the same cells, while 4 showed higher cytotoxicity against cancer cell lines than lapachol (1). The transformation of 1 into harmless and reactive metabolites evidences the importance of the evaluation of drug metabolism in the drug discovery process. Antifungal potential of lapachol (1) and its metabolites 2 and 4 against B. cinerea has also been evaluated. Dehydro-α-lapachone (4) has been shown to be less toxic to fungal growth than lapachol (1), which indicates a detoxification mechanism of the phytopathogen.
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January 2016

Chemically Induced Cryptic Sesquiterpenoids and Expression of Sesquiterpene Cyclases in Botrytis cinerea Revealed New Sporogenic (+)-4-Epieremophil-9-en-11-ols.

ACS Chem Biol 2016 05 8;11(5):1391-400. Epub 2016 Mar 8.

Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz , Campus Universitario Río San Pedro s/n, Torre sur, 4° planta, 11510, Puerto Real, Cádiz, Spain.

The sequencing of the genomes of the B05.10 and T4 strains of the fungus Botrytis cinerea revealed an abundance of novel biosynthetic gene clusters, the majority of which were unexpected on the basis of the previous analyses of the fermentation of these and closely related species. By systematic alteration of easy accessible cultivation parameters, using chemical induction with copper sulfate, we have found a cryptic sesquiterpenoid family with new structures related to eremophil-9-ene, which had the basic structure of the sesquiterpene (+)-5-epiaristolochene ((+)-4-epieremophil-9-ene). An expression study of the sesquiterpene cyclase genes present in the Botrytis cinerea genome, under culture conditions, is reported. In general, a 3 day delay and a higher BcSTC genes expression were observed when copper (5 ppm) was fed to the fermentation broth. In addition, to the observed effect on the BcBOT2 (BcSTC1) gene, involved in the biosynthesis of the botrydial toxin, a higher expression level for BcSTC3 and BcSTC4 was observed with respect to the control in the strain B05.10. Interestingly, under copper conditions, the BcSTC4 gene was the most expressed gene in the Botrytis cinerea UCA992 strain. In vitro evaluation of the biological role of these metabolites indicates that they contributed to the conidial development in B. cinerea and appear to be involved in self-regulation of the production of asexual spores. Furthermore, they promoted the formation of complex appressoria or infection cushions.
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http://dx.doi.org/10.1021/acschembio.5b00931DOI Listing
May 2016

Botrydial and botcinins produced by Botrytis cinerea regulate the expression of Trichoderma arundinaceum genes involved in trichothecene biosynthesis.

Mol Plant Pathol 2016 09 8;17(7):1017-31. Epub 2016 Feb 8.

Area of Microbiology, University School of Agricultural Engineers, University of León, Campus de Ponferrada, 24400 Ponferrada, Spain.

Trichoderma arundinaceum IBT 40837 (Ta37) and Botrytis cinerea produce the sesquiterpenes harzianum A (HA) and botrydial (BOT), respectively, and also the polyketides aspinolides and botcinins (Botcs), respectively. We analysed the role of BOT and Botcs in the Ta37-B. cinerea interaction, including the transcriptomic changes in the genes involved in HA (tri) and ergosterol biosynthesis, as well as changes in the level of HA and squalene-ergosterol. We found that, when confronted with B. cinerea, the tri biosynthetic genes were up-regulated in all dual cultures analysed, but at higher levels when Ta37 was confronted with the BOT non-producer mutant bcbot2Δ. The production of HA was also higher in the interaction area with this mutant. In Ta37-bcbot2Δ confrontation experiments, the expression of the hmgR gene, encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the first enzyme of the terpene biosynthetic pathway, was also up-regulated, resulting in an increase in squalene production compared with the confrontation with B. cinerea B05.10. Botcs had an up-regulatory effect on the tri biosynthetic genes, with BotcA having a stronger effect than BotcB. The results indicate that the interaction between Ta37 and B. cinerea exerts a stimulatory effect on the expression of the tri biosynthetic genes, which, in the interaction zone, can be attenuated by BOT produced by B. cinerea B05.10. The present work provides evidence for a metabolic dialogue between T. arundinaceum and B. cinerea that is mediated by sesquiterpenes and polyketides, and that affects the outcome of the interaction of these fungi with each other and their environment.
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http://dx.doi.org/10.1111/mpp.12343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638445PMC
September 2016

Biological activity of natural sesquiterpenoids containing a gem-dimethylcyclopropane unit.

Nat Prod Rep 2015 Aug;32(8):1236-48

Department of Organic Chemistry, Faculty of Science, University of Cádiz, Polígono Río San Pedro s/n, 11510, Puerto Real, Cádiz, Spain.

The biological activities of aristolane, aromadendrane, ent-1,10-secoaromadendrane, 2,3-secoaromadendrane, ent-5,10-cycloaromadendrane, bicyclogermacrene, lepidozane, and maaliane terpenoids which contain the gem-dimethylcyclopropyl unit are described. Particular attention is given to their anti-viral, anti-microbial and cytotoxic activities. In the main text there are 119 references covering the literature from 1963-2014. The ESI contains tables listing 332 of these terpenoids, their occurrence and biological activity together with the related references.
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http://dx.doi.org/10.1039/c5np00024fDOI Listing
August 2015

nor-Mevaldic acid surrogates as selective antifungal agent leads against Botrytis cinerea. Enantioselective preparation of 4-hydroxy-6-(1-phenylethoxy)tetrahydro-2H-pyran-2-one.

Bioorg Med Chem 2015 Jul 23;23(13):3379-87. Epub 2015 Apr 23.

Departamento de Química Orgánica, Facultad de Ciencias, Campus Universitario Puerto Real, Universidad de Cádiz, Puerto Real, Cádiz 11510, Spain. Electronic address:

Solvent-free desymmetrisation of meso-dialdehyde 1 with chiral 1-phenylethan-1-ol, led to preparation of 4-silyloxy-6-alkyloxytetrahydro-2H-pyran-2-one (+)-3a with a 96:4 dr Deprotected lactone (+)-19a and the related racemic lactones 16a-18a present a lactone moiety resembling the natural substrate of HMG-CoA reductase and their antifungal properties have been evaluated against the phytopathogenic fungi Botrytis cinerea and Colletotrichum gloeosporioides. These compounds were selectively active against B. cinerea, while inactive against C. gloeosporioides.
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http://dx.doi.org/10.1016/j.bmc.2015.04.048DOI Listing
July 2015

The asymmetric total synthesis of cinbotolide: a revision of the original structure.

J Org Chem 2014 Dec 14;79(23):11349-58. Epub 2014 Nov 14.

Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas, Campus Universitario Rio San Pedro s/n, Universidad de Cádiz , 11510 Puerto Real, Cádiz, Spain.

The structure 3,4-dihydroxy-2,4,6,8-tetramethyldec-8-enolide (1) was assigned to a metabolite of Botrytis cinerea, but the spectra of several synthetic analogues had significant differences from that of 1. Examination of the constituents of a B. cinerea mutant that overproduces polyketides gave sufficient quantities of 1, now named cinbotolide, for chemical transformations. These led to a revised γ-butyrolactone structure for the metabolite. This structure has been confirmed by an asymmetric total synthesis, which also established its absolute configuration.
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http://dx.doi.org/10.1021/jo501471mDOI Listing
December 2014

Novel aspinolide production by Trichoderma arundinaceum with a potential role in Botrytis cinerea antagonistic activity and plant defence priming.

Environ Microbiol 2015 Apr 25;17(4):1103-18. Epub 2014 Jun 25.

Area of Microbiology, Universitary School of Agricultural Engineers, University of León, Campus de Ponferrada, Avda. Astorga s/n, Ponferrada, 24400, Spain.

Harzianum A (HA), a trichothecene produced by Trichoderma arundinaceum, has recently been described to have antagonistic activity against fungal plant pathogens and to induce plant defence genes. In the present work, we have shown that a tri5 gene-disrupted mutant that lacks HA production overproduces two polyketides, aspinolides B and C, which were not detected in the wild-type strain. Furthermore, four new aspinolides (D-G) were characterized. These compounds confirm that a terpene-polyketide cross-pathway exists in T. arundinaceum, and they may be responsible for the antifungal activity and the plant sensitization effect observed with the tri5-disrupted mutant. In addition, the molecular changes involving virulence factors in the phytopathogenic fungus Botrytis cinerea 98 (Bc98) during interaction with T. arundinaceum were investigated. The expression of genes involved in the production of botrydial by Bc98 was relatively repressed by HA, whereas other virulence genes of this pathogen were induced by the presence of T. arundinaceum, for example atrB and pg1 which encode for an ABC transporter and endopolygalacturonase 1 respectively. In addition, the interaction with Bc98 significantly repressed the production of HA by T. arundinaceum, indicating that a bidirectional transcriptional regulation is established between these two antagonistic fungi.
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http://dx.doi.org/10.1111/1462-2920.12514DOI Listing
April 2015

Biologically active diterpenes containing a gem-dimethylcyclopropane subunit: an intriguing source of PKC modulators.

Nat Prod Rep 2014 Jul 21;31(7):940-52. Epub 2014 May 21.

Department of Organic Chemistry, Faculty of Science, University of Cádiz, Polígono Río San Pedro s/n, 11510, Puerto Real (Cádiz), Spain.

Covering: 1973 to 2013. The biological activities of tigliane, lathyrane, ingenane, casbane, jatropholane and premyrsinane diterpenoids which contain the gem-dimethylcyclopropyl unit are described. Particular attention is given to their anti-viral, anti-microbial and cytotoxic activities. In the main text there are 132 references. The electronic supplementary information contains tables listing 424 of these diterpenoids, their occurrence and biological activity together with the references.
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http://dx.doi.org/10.1039/c4np00008kDOI Listing
July 2014

Further mulinane and azorellane diterpenoids isolated from Mulinum crassifolium and Azorella compacta.

Molecules 2014 Mar 28;19(4):3898-908. Epub 2014 Mar 28.

Laboratorio de Productos Naturales, Departamento de Química, Universidad de Antofagasta, Av. Coloso S-N, Antofagasta 1240000, Chile.

The new mulinane diterpenoids 1 and 2 were isolated from the EtOAc extract of Mulinum crassifolium, while the rearranged mulinane 5, which was isolated for the first time from a natural source, was isolated from Azorella compacta. Compounds 1-2 were prepared by semi-synthesis thorough acetylation of the diterpene 17-acetoxymulinic acid (3). A mechanism of reaction was proposed, while the structures of the new compounds were elucidated on the basis of comprehensive spectroscopic analysis and computational methods.
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http://dx.doi.org/10.3390/molecules19043898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6271538PMC
March 2014

Multiple anticancer effects of damsin and coronopilin isolated from Ambrosia arborescens on cell cultures.

Anticancer Res 2013 Sep;33(9):3799-805

Biomedical Center B11, Gastroenterology and Nutrition Lab, Institution of Clinical Sciences, Lund University, S-221 84 Lund, Sweden.

Terpenoids in plants are important sources for drug discovery. In this study, we extracted damsin and coronopilin, two sesquiterpene lactones, from Ambrosia arborescens and examined their anticancer effects on cell cultures. Damsin and coronopilin inhibited cell proliferation, DNA biosynthesis and formation of cytoplasmic DNA histone complexes in Caco-2 cells, with damsin being more potent than coronopilin. Further studies using the luciferase reporter system showed that damsin and coronopilin also inhibited expressions of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription-3 (STAT3), indicating that these sesquiterpenes can interfere with NF-κB and STAT3 pathways. Finally, we examined the effects of two synthetic dibrominated derivatives of damsin, 11α,13-dibromodamsin and 11β,13-dibromodamsin. While bromination appeared to weaken the antiproliferative effects of damsin, the β epimer had strong inhibitory effects on STAT3 activation. In conclusion, the sesquiterpene lactones damsin and coronopilin have inhibitory effects on cell proliferation, DNA biosynthesis and NF-κB and STAT3 pathways, thus being potentially important for discovery of drugs against cancer.
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September 2013

Phytotoxic activity and metabolism of Botrytis cinerea and structure-activity relationships of isocaryolane derivatives.

J Nat Prod 2013 Jun 3;76(6):1016-24. Epub 2013 Jun 3.

Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627, 31270-901, Belo Horizonte, MG, Brazil.

Research has been conducted on the biotransformation of (8S,9R)-isocaryolan-9-ol (4a) and (1S,2S,5R,8S)-8-methylene-1,4,4-trimethyltricyclo[6.2.1.0(2,5)]undecan-12-ol (5a) by the fungal phytopathogen Botrytis cinerea. The biotransformation of compound 4a yielded compounds 6-9, while the biotransformation of compound 5a yielded compounds 10-13. The activity of compounds 4a and 5a against B. cinerea has been evaluated. (8R,9R)-Isocaryolane-8,9-diol (6), a major metabolite of compound 4a, shows activity compared to its parent compound 4a, which is inactive. The effect of isocaryolanes 3, 4a, and 5a, together with their biotransformation products 6-8, 10, and 14-17, on the germination and radicle and shoot growth of Lactuca sativa (lettuce) has also been determined. Compounds 7-13 are described for the first time.
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http://dx.doi.org/10.1021/np3009013DOI Listing
June 2013