Simplified Synthesis and Stability Assessment of Aflatoxin B1-Lysine and Aflatoxin G1-Lysine.

Aflatoxins B1 (AFB1) and G1 (AFG1) are carcinogenic mycotoxins that contaminate crops such as maize and groundnuts worldwide. The broadly accepted method to assess chronic human aflatoxin exposure is by quantifying the amount of aflatoxin adducted to human serum albumin. This has been reported using ELISA, HPLC, or LC-MS/MS to measure the amount of AFB1-lysine released after proteolysis of serum albumin. LC-MS/MS is the most accurate method but requires both isotopically labelled and unlabelled AFB1-lysine standards, which are not commercially available. In this work, we report a simplified synthetic route to produce unlabelled, deuterated and 13C6 15N2 labelled aflatoxin B1-lysine and for the first-time aflatoxin G1-lysine. Additionally, we report on the stability of these compounds during storage. This simplified synthetic approach will make the production of these important standards more feasible for laboratories performing aflatoxin exposure studies.

Organocatalytic enantioselective direct alkylation of phloroglucinol derivatives: asymmetric total synthesis of (+)-aflatoxin B2.

The organocatalytic enantioselective Friedel-Crafts alkylation of phloroglucinol derivatives with enals is reported, providing general access to the benzylic chiral centers shown in a variety of phloroglucinol natural products. The synthetic utility is demonstrated by the very concise asymmetric total synthesis of aflatoxins B2.

Effect of dietary acids on the formation of aflatoxin B2a as a means to detoxify aflatoxin B1.

Aflatoxin B1 (AFB1) is a class 1 carcinogen and a common food contaminant worldwide with widely uncontrolled human exposure. The ability of organic acids to transform AFB1 into a known detoxified form, aflatoxin B2a (AFB2a), was investigated using high performance liquid chromatography-electrospray ionisation-time of flight mass spectrometry (HPLC/ESI/TOF/MS). The identity of the transformation product was confirmed by accurate mass measurement, chromatographic separation from other aflatoxins, H(1)-nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. Of the weak acids tested, citric acid was found to be the most effective for AFB2a formation. At room temperature, 1 M citric acid was able to convert > 97% of AFB1 to AFB2a over 96 h of treatment. Up to 98% transformation was achieved by boiling AFB1 in the presence of citric acid for 20 min. AFB1 hydration after ingestion was explored by spiking AFB1 into simulated gastric fluid containing citric acid. Under these conditions, > 71% of AFB1 was hydrated to AFB2a and did not show any reversion to the parent compound after being transferred to a neutral solution. These results provide a basis for a practical and effective method for detoxification of AFB1 in contaminated foods.

What makes Aspergillus fumigatus a successful pathogen? Genes and molecules involved in invasive aspergillosis

Dos secciones incluyen los genes y moléculas relacionadas con la absorción de nutrientes, la señalización y las regulaciones metabólicas implicadas en la virulencia, incluyendo enzimas, como las serin-proteasas (alp/asp f 13, alp2 y asp f 18), metaloproteasas (mep/asp f 5, mepB y mep20), aspártico-proteasas (pep/asp f 10, pep2 y ctsD), dipeptidilpeptidasas (dppIV y dppV) y fosfolipasas (plb1-3 y fosfolipasa C); sideróforos y la adquisición de hierro (sidA-G sreA, ftrA, fetC, mirB-C y amcA); adquisición de zinc (zrfA-H, zafA, y pacC); biosíntesis de aminoácidos, absorción de nitrógeno, y regulación por Cross-pathway Control (areA, rhbA, mcsA, lysF, cpcA/gcn4p y cpcC/gcn2p); vías de biosíntesis generales (pyrG, hcsA, y pabaA) y biosíntesis de trehalosa (tpsA y tpsB); otras vías de regulación, como MAP quinasas (sakA/hogA, mpkA-C, ste7, pbs2, mkk2, steC/ste11, bck1, ssk2 y sho1), proteínas G (gpaA, sfaD y cpgA), AMPc-PKA (acyA, gpaB, pkaC1 y pkaR), histidin-quinasas (fos1 y tcsB), señalización de Ca2+(calA/cnaA, crzA, gprC y gprD), familia Ras (rasA, rasB y rhbA), y otros (ace2, medA, y srbA). Por último, también se comentan los efectos de los alérgenos de A. fumigatus (Asp f 1 a Asp f 34) en la AI. Los datos obtenidos generan un complejo rompecabezas, cuyas piezas serían factores de virulencia o diferentes actividades del hongo, que se deben reunir para obtener una visión conjunta de la virulencia de A. fumigatus. Los estudios de expresión mediante microarrays de ADN podrían ser útiles para entender esta compleja virulencia, y para detectar dianas para desarrollar métodos rápidos de diagnóstico y nuevos agentes antifúngicos. Aspergillus fumigatus es un patógeno oportunista que causa el 90% de las aspergilosis invasoras (AI) con un 50–95% de mortalidad. Se ha postulado la existencia de factores de virulencia característicos, pero en A. fumigatus existe una gran variabilidad de factores de virulencia «no clásicos». Todos los estudios han demostrado que la virulencia de este hongo es multifactorial, asociada a su estructura, su capacidad de crecimiento y adaptación a condiciones de estrés, sus mecanismos de evasión del sistema inmune y su capacidad de causar daños en un huésped. En esta revisión se pretende dar una visión general de los genes y moléculas que intervienen en el desarrollo de la AI. La sección de termotolerancia incluye cinco genes relacionados con la capacidad de que el hongo crezca a más de 30°C (thtA, cgrA, afpmt1, kre2/afmnt1 y hsp1/asp f 12). En las siguientes secciones se discuten las moléculas y los genes relacionados con la interacción con el huésped y con la respuesta inmune. Estas secciones incluyen el β-glucano, el α-glucano, la quitina, el galactomanano, galactomanoproteinas (afmp1/asp f 17 y afmp2), hidrofobinas (rodA/hyp1 y rodB), la DHN-melanina, sus respectivas enzimas sintasas (fks1, rho1-4, ags1-3, chsA-G, och1-4, mnn9, van1, anp1, glfA, pksP/alb1, arp1, arp2, abr1, abr2 y ayg1) y enzimas modificantes (gel1-7, bgt1, eng1, ecm33, afpigA, afpmt1-2, afpmt4, kre2/afmnt1, afmnt2-3, afcwh41 y pmi), varias enzimas relacionadas con la protección del estrés oxidativo como catalasas (catA, cat1/catB, cat2/katG, catC y catE), superóxido dismutasas (sod1-2, sod3/asp f 6 y sod4), oxigenasas de ácidos grasos (ppoA-C), glutatión transferasas (gstA-E) y otros (afyap1, skn7 y pes1), y los transportadores de moléculas (mdr1-4, atrF, abcA-E y msfA-E)...(AU)

Two sections cover genes and molecules related with nutrient uptake, signaling and metabolic regulations involved in virulence, including enzymes, such as serine proteases (alp/asp f 13, alp2, and asp f 18), metalloproteases (mep/asp f 5, mepB, and mep20), aspartic proteases (pep/asp f 10, pep2, and ctsD), dipeptidylpeptidases (dppIV and dppV), and phospholipases (plb1–3 and phospholipase C); siderophores and iron acquisition (sidA–G, sreA, ftrA, fetC, mirB–C, and amcA); zinc acquisition (zrfA–H, zafA, and pacC); amino acid biosynthesis, nitrogen uptake, and cross-pathways control (areA, rhbA, mcsA, lysF, cpcA/gcn4p, and cpcC/gcn2p); general biosynthetic pathway (pyrG, hcsA, and pabaA), trehalose biosynthesis (tpsA and tpsB), and other regulation pathways such as those of the MAP kinases (sakA/hogA, mpkA–C, ste7, pbs2, mkk2, steC/ste11, bck1, ssk2, and sho1), G-proteins (gpaA, sfaD, and cpgA), cAMP-PKA signaling (acyA, gpaB, pkaC1, and pkaR), His kinases (fos1 and tcsB), Ca2+ signaling (calA/cnaA, crzA, gprC and gprD), and Ras family (rasA, rasB, and rhbA), and others (ace2, medA, and srbA). Finally, we also comment on the effect of A. fumigatus allergens (Asp f 1–Asp f 34) on IA. The data gathered generate a complex puzzle, the pieces representing virulence factors or the different activities of the fungus, and these need to be arranged to obtain a comprehensive vision of the virulence of A. fumigatus. The most recent gene expression studies using DNA-microarrays may be help us to understand this complex virulence, and to detect targets to develop rapid diagnostic methods and new antifungal agents. Aspergillus fumigatus is an opportunistic pathogen that causes 90% of invasive aspergillosis (IA) due to Aspergillus genus, with a 50–95% mortality rate. It has been postulated that certain virulence factors are characteristic of A. fumigatus, but the “non-classical” virulence factors seem to be highly variable. Overall, published studies have demonstrated that the virulence of this fungus is multifactorial, associated with its structure, its capacity for growth and adaptation to stress conditions, its mechanisms for evading the immune system and its ability to cause damage to the host. In this review we intend to give a general overview of the genes and molecules involved in the development of IA. The thermotolerance section focuses on five genes related with the capacity of the fungus to grow at temperatures above 30°C (thtA, cgrA, afpmt1, kre2/afmnt1, and hsp1/asp f 12)... (AU)

Short, enantioselective total synthesis of aflatoxin B2 using an asymmetric [3+2]-cycloaddition step.

A highly enantioselective [3+2]-cycloaddition reaction of 2,3-dihydrofuran with 1,4-benzoquinones using a chiral oxazaborolidinium triflimidate as catalyst has been developed which allows rapid access to a variety of chiral phenolic tricycles (enantiomeric excesses ranging from 91 to 98%). The utility of this new methodology is demonstrated by a short synthesis of the important pentacyclic natural product, aflatoxin B2. This exploratory study indicates that an even broader application of these catalysts to enantioselective cycloadditions may be possible.

Palladium catalyzed kinetic and dynamic kinetic asymmetric transformations of gamma-acyloxybutenolides. Enantioselective total synthesis of (+)-Aflatoxin B1 and B2a.

The reaction of gamma-tert-butoxycarbonyloxy-2-butenolide with phenol nucleophiles in the presence of a Pd(0) complex with chiral ligands may be performed under conditions that favor either a kinetic resolution or a kinetic asymmetric transformation (KAT) or dynamic kinetic asymmetric transformation (DYKAT). Performing the reaction at high concentration (0.5 M) in the presence of a carbonate base favors the former, i.e., KAT; whereas, running the reaction at 0.1M in the presence of tetra-n-butylammonium chloride favors the DYKAT process. Syntheses of aflatoxin B(1) and B(2a) employs the DYKAT to introduce the stereochemistry. Starting with Pechmann condensation of the monomethyl ether of phloroglucinol, the requisite phenol nucleophile is constructed in two steps. The DYKAT proceeds with > 95% ee. A reductive Heck cyclization followed by a lanthanide catalyzed intramolecular acylation completes the synthesis of the pentacyclic nucleus in 3 steps. Reduction of the lactone provides aflatoxin B(2a) and its dehydration product B(1). This synthetic strategy creates an asymmetric synthesis of the former in only 7 steps and the latter in 9 steps. Thus, the ultimate synthetic sequence involves 3 + 5 --> 39 --> 40 --> 42 --> 43 --> 46 --> 47 --> 48 (aflatoxin B(2a)) --> 49 (aflatoxin B(1)).

Quinone approaches toward the synthesis of aflatoxin B(2).

[reaction: see text] Quinones bearing electron-withdrawing groups can serve as useful precursors to furobenzofuran ring systems through their reaction with 2,3-dihydrofuran. Formal racemic and stereoselective syntheses of the fungal metabolite aflatoxin B(2) are described that utilize this approach to construct the tricyclic ABC-ring core of the molecule.

Preparation of protein conjugatable aflatoxin oxime derivative from significantly reduced starting quantities.

A modification of an existing technique for the preparation of aflatoxin B1-(0-carboxymethyl) oxime is presented which allows for a significantly reduced starting amount of aflatoxin. About 80% of the aflatoxin was converted to its oxime. The new technique will be particularly valuable when protein conjugatable oximes of more expensive aflatoxin metabolites, such as aflatoxin M1, are required for immunoassay production.

Preparation and characterization of aflatox-n B1-1-(O-carboxymethyl) oxime.

A method is described for the preparation and purification of aflatoxin B1-1(O-carboxymethyl) oxime from aflatoxin B1. The overall yield was about 73-83%. The new aflatoxin B1 derivative was characterized by mass, ultraviolet, infrared, and nuclear magnetic resonance spectral analyses, and was nontoxic to 8-day-old chicken embryos when tested at a concentration of 3.48 microgram/egg.