An artificial cytochrome P450 that hydroxylates unactivated carbons with regio- and stereoselectivity and useful catalytic turnovers

A catalyst has been synthesized comprising a manganese porphyrin carrying four beta-cyclodextrin groups. It catalyzes the hydroxylation of substrates of appropriate size carrying tert-butylphenyl groups that can hydrophobically bind into the cyclodextrin cavities. In one example as many as 650 catalytic turnovers are seen before the catalyst is oxidatively destroyed, and with a rate comparable to that of typical cytochrome P450 enzymes. In another example, a steroid derivative is regio- and stereoselectively hydroxylated at a single unactivated carbon atom, but more slowly and with fewer turnovers. The carbon attacked is not the most chemically reactive, and the selectivity is determined by the geometry of the catalyst-substrate complex. Nonbinding substrates are not reactive under the conditions used, and substrates with more flexible binding geometries give more than a single product.

The Decisive Step in Betaxanthin Biosynthesis Is a Spontaneous Reaction1

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris “Golden Beet”) showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris “Altamo”) plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.