Ochratoxins synthesis

This chemistry can be very powerful, since the amide product itself offers further possibilities for functionalization by lithiation. The synthesis of the natural product ochra-toxin A illustrates this point. Ochratoxins A 66 and B 67 (Scheme 34) are metabolites of Aspergillus ochraceus and Penicillium viridicatum whose presence in agricultural products... 

Total synthesis has benefited from key DoM reactions. The sequence 48 —> 49 —> 50 —> 51 (Scheme 12) en route to the natural product ochratoxin A (52) takes multiple advantage of anion chemistry (53) ortho-metalation of the powerful OCONEtj group (step 1), anionic Fries rearrangement (step 2), in-between DoM and chain-extension by Li-Mg transmetalation (step 3) [12]. 

Scheme 13. ArOCONEt2 DoM / ortho-Fries strategy towards the total synthesis of ochratoxin A.

Bavaresco, L., Vezzulli, S., Battilani, P., Giorni, P., Pietri, A. and Bertuzzi, T. (2003) Effect of ochratoxin A-producing Aspergilli on stilbenic phytoalexin synthesis in grapes, J. Agric. Food Chem., 51, 6151-6157. 

Ochratoxin A 57 is a highly toxic metabolite isolated from Aspergillus ochraceus Wilh The structure assigned to it on the basis of spectral data, was confirmed by an anambiguous synthesis, which is shown below >. 

Two mechanisms of action have been recognized for ochratoxin A inhibition of protein synthesis and enhancement of oxygen reactive radical production with lipid peroxidation. The first effect can be counteracted by the presence of amino acids in the diet (the sweetener aspartame may be one source), the second by the presence in the diet of antioxidants, and coffee itself contains such compounds. 

As summarized previously by this Committee, there was no evidence of DNA repair as a result of possible DNA damage in bacteria, whereas DNA single-strand breaks were consistently induced in cultured mammalian cells and were also observed in vivo in spleen, liver and kidney cells of mice after intraperitoneal injection of ochratoxin A. DNA repair, manifested as unscheduled DNA synthesis, was observed in most studies with primary cultures of rat and mouse hepatocytes, porcine epithelial cells from bladder and human urothelial cells (Annex 1, reference 153). 

Induction of unscheduled DNA synthesis in primary human urothelial cells by the mycotoxin ochratoxin A. Toxicol. Sci. 53, 271-277. 

Stetina, R. Votava, M. (1986) Induction of DNA single-strand breaks and DNA synthesis inhibition by patulin, ochratoxin A, citrinin, and aflatoxin B, in cell lines CHO and AWRF. Folia Biol. 32, 128-144. 

All ochratoxins and their derivatives have been obtained by total syntheses. The first so-produced were ochratoxins A (323) and B (324) by Steyn et al. in 1967 (263), two years after they were first reported as a class of mycotoxins. This group was able to S3mthesize racemic ochratoxin a (326) in six steps. However, for the stereoselective synthesis of ochratoxins A (323) and B (324), (/ )-ochratoxin a (326) obtained through hydrolysis of the natural product was used as the starting material. 

It took another 35 years until the first (and still the only known) enantioselective total synthesis of (/ )-ochratoxin a (326), and therefore of ochratoxins A and B, was published by Gill et al. in 2002 (264, 265). Scheme 6.1 shows six steps of the nine-step synthesis, which was achieved with 10% overall yield. The first three steps of the procedure are not shown and comprise the preparation of 327 from (/ )-2-methyloxirane according to ref. (266). Ketene dimethyl acetal and acetylenic ester 327 react in an intermolecular cycloaddition to give 328. This diene undergoes a Diels-Alder reaction with methyl propiolate to yield 329. Lactonization ( 330), demethylation ( 331), chlorination ( 332), and methyl ester cleavage finally furnished enantiomerically pure ochratoxin a (326) (267). 

Scheme 6.1 Total synthesis of (/f)-ochratoxin a (326) as well as ochratoxins A-C (323-325). Reagents and conditions a) ketene dimethyl acetal, sealed tube, 165°C, 23 h b) methyl propiolate, sealed tube, 145 C, 22 h, 69% over two steps c) p-TsOH, CH2CI2, rt, 72 h, 82% d) BCI3, CH2CI2, 0°C, 10 min, 92% e) SO2CI2, CH2CI2, rt, 48 h f) MeOH, LiOH H20, reflux, 5 h, 68% over two steps g) L-phenylalanine t-butyl ester, EEDQ, THF, rt, 15 h h) EtOH...

In 1985, Snieckus et al. reported another method for the preparation of racemic ochratoxin a (326) and its dechloro analog 331 in only four steps (269). They used the readily accessible 0-aryl carbamates 333a and 333b (270) as starting materials for their synthesis (Scheme 6.2). The 0-carbamate benzamides 334a and 334b were obtained by metalation of the carbamates followed by quenching with diethylcarbamoyl chloride. The next step consisted of a 1,3-carbamoyl... 

In 2009, Gabriele et al. published a new and expedient synthesis of racemic ochratoxin ot (326) (277). After coupling with protected L-phenylalanine, they were able to separate the resulting diastereoisomers by preparative TLC. Thus, they obtained enantiomerically pure (/ )-ochratoxin A (323) and its (35)-diastereomer 343a in six steps and 9% and 6% overall yields, from commercially available starting materials (Scheme 6.3). Furthermore, they were able to synthesize (is-ochratoxin A (342b) and its (35)-diastereomer 343b. The former can be used as an internal standard in a stable isotope dilution assay that is an important quantification tool for micro components in food such as the ochratoxins (272). 

Scheme 6.3 Total synthesis of ochratoxin A, d5-ochratoxin A and their (3S)-diastereomers. Reagents and ermditions a) NaH, bnt-2-ynal, 10°C, 6 h, 48% b) LDA, MeCHO, —78°C, 15 min thai 0°C, 15 min 70% c) SO2CI2, CH2O2, rt, 24 h then LiOH, MeOH, reflux, 5 h, 69% d) Ha04, r-BuOAc, rt, 12 h, 91% (341a), 90% (341b) e) rac-326, EDC HC1, HOBt, GHCI3, rt, 20 h then TFA, CH2O2, rt, 5 h then preparative TLC, 46% (323), 34% (343a), 47% (342b), 36% (343b)...

Scheme 6.4 Total synthesis of all ochratoxin A stereoisomers. Reagents and conditions a) DIPEA, HATU, DMF, rt, 2 h, 88% then NaOH, rt, 12 h, 62%...

Steyn PS, Holzapfel CW (1967) The Synthesis of Ochratoxins A and B, Metabolites of Aspergillus ochraceus Wilh. Tetrahedron 23 4449... 

Donner CD, Gill M (2002) Pigments of Fungi. LXEX. Total Synthesis of (/f)-Ochratoxin a and the Formal Total Synthesis of Ochratoxin A. Aust J Chem 55 213... 

Roberts JC, Woollven P (1970) Studies in Mycological Chemistry. Part XXIV. Synthesis of Ochratoxin A, a Metabolite of Aspergillus ochraceus Wilh. J Chem Soc C 278... 

Sibi MP, Chattopadhyay S, Dankwardt JW, Snieckus V (1985) Combinational 0-Aryl Carbamate and Benzamide Directed Ortho Metalation Reactions. Synthesis of Ochratoxin A and Ochratoxin B. J Am Chem Soc 107 6312... 

Gabriele B, Attya M, Fazio A, Di Donna L, Plastina P, Sindona G (2009) A New and Expedient Total Synthesis of Ochratoxin A and ds-Ochratoxin A. Synthesis 1815... 

Cramer B, Harrer H, Nakamura K, Uemura D, Humpf H-U (2010) Total Synthesis and Cytotoxicity Evaluation of All Ochratoxin A Stereoisomers. Bioorg Med Chem 18 343... 

Giraudi, G., Anfossi, L., Baggiani, C., Giovannoli, C., and Tozzi, C. 2007. Solid-phase extraction of ochratoxin A from wine based on a binding hexapeptide prepared by combinatorial synthesis. J. Chromatogr. A 1175 174-178. 

Heller, K., and R. Roschenthaler Inhibition of Protein Synthesis in Streptococcus faecalis by Ochratoxin A. Canad. J. Microbiol. 24, 466 (1978). 

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