Asymmetric Total Synthesis and Biosynthetic Implications of Perovskones, Hydrangenone, and Hydrangenone B.

Perovskones and hydrangenones are a family of structurally complex triterpenoids that were mainly isolated from the genus Salvia medicinal plants. These isoprenoids exhibit a broad range of biological activities, such as antitumor and antiplasmodial activities. Here, we report the collective total synthesis of perovskone, perovskones C, D, F, hydrangenone, and hydrangenone B. The key strategies in this work include the following: (1) an asymmetric photoenolization/Diels-Alder reaction was developed to construct a tricyclic ring bearing three contiguous quaternary centers, which was used to build the core icetexane skeleton; (2) a bioinspired Diels-Alder reaction of perovskatone D with trans-α-ocimene was applied to stereospecifically generate perovskones; (3) late-stage oxidations and ring forming steps were developed to synthesize perovskones and hydrangenones. Our synthetic work suggests that (1) perovskatone D may serve as the precursor of the biosynthesis of perovskones and (2) the formation of hydrangenone and hydrangenone B, containing a five-membered D ring, may involve an oxidative ring cleavage and ring regeneration process.

Dehydration-Induced Carotenoid Cleavage Dioxygenase 1 Reveals a Novel Route for ß-Ionone Formation during Tea (Camellia sinensis) Withering.

ß-Ionone is a carotenoid-derived flavor and fragrance compound with a complex fruity and woody scent, known for its violet aroma. Due to the low odor threshold, ß-ionone dramatically affects the aroma and quality of tea. Previous studies have shown that ß-ionone increases during tea withering; however, its formation and regulation during the withering process are far from being understood. As dehydration is the most important stress during the withering of the tea leaves, we isolated a dehydration-induced gene belonging to the subfamily of carotenoid cleavage dioxygenases called carotenoid cleavage dioxygenase 1a (CsCCD1a) from Camellia sinensis and expressed it in Escherichia coli. The recombinant protein could generate volatile ß-ionone and pseudoionone from carotenoids. CsCCD1a was induced by dehydration stress, and its expression was related to the ß-ionone accumulation during tea withering. Overall, this study elucidated that CsCCD1a catalyzes the formation of ß-ionone in C. sinensis and enhanced the understanding of the ß-ionone formation under multiple stresses during the processing of tea.

Carotenoid Cleavage Dioxygenase 4 Catalyzes the Formation of Carotenoid-Derived Volatile ß-Ionone during Tea (Camellia sinensis) Withering.

The carotenoid-derived volatile ß-ionone plays an important role in the formation of green and black tea flavors due to its low odor threshold, but its formation and the gene(s) involved in its biosynthesis during the tea withering process is(are) still unknown. In this study, we found that the content of ß-ionone increased during the tea withering process catalyzed by an unknown enzyme(s). Correlation analysis of expression patterns of Camellia sinensis carotenoid cleavage dioxygenase genes (CsCCDs) and the ß-ionone content during the withering period revealed CsCCD4 as the most promising candidate. The full-length CsCCD4 gene was amplified from C. sinensis, and the biochemical function of the recombinant CsCCD4 protein was studied after coexpression in Escherichia coli strains engineered to accumulate ß-carotene. The recombinant protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' double bonds. Volatile ß-ionone was detected as the main product by gas and liquid chromatography-mass spectrometry. The accumulation of ß-ionone was consistent with the expression levels of CsCCD4 in different tissues and during the withering process. The CsCCD4 expression was induced by low temperature and mechanical damage stress but not by dehydration stress. The results demonstrate that CsCCD4 catalyzes the production of ß-ionone in the tea plant and provide insight into its formation mechanism during the withering process.

In Vitro Regio- and Stereoselective Oxidation of ß-Ionone by Human Liver Microsomes.

The metabolism of the norisoprenoid ß-ionone was investigated in vitro using human liver microsomes and 11 different recombinant cytochrome P450 enzymes expressed in Trichoplusia ni cells. ß-Ionone was found to be oxidized via 4S-hydroxylation by CYP2B6 in human liver microsomes. CYP1A2 also regioselectively catalyzed the hydroxylation of ß-ionone to yield 4-hydroxylation; this conversion was not stereoselective. Further kinetic analysis revealed that CYP2B6 exhibited the highest activity for ß-ionone 4-hydroxylation. Kinetic analysis showed that Km and Vmax for oxidation of ß-ionone by CYP1A2 and CYP2B6 was 107.9 ± 36.0 µM and 3200.3 ± 323.0 nmol/min/nmol P450 and 5.6 ± 1.2 µM and 572.8 ± 29.8 nmol/min/nmol P450, respectively. The reaction rates observed using human liver microsomes and recombinant CYP2B6 were very high compared with those of other CYP2B6 substrates reported thus far. These results suggest that ß-ionone, a norisoprenoid present in nature, is one of the effective substrates for CYP2B enzymes in human liver microsomes. To the best of our knowledge, this is the first time that 4-hydroxy ß-ionone has been described as a human metabolite of ß-ionone.

Applications of biocatalysis in fragrance chemistry: the enantiomers of alpha-, beta-, and gamma-irones.

The history of iris extracts, and of the isolation and enzyme-mediated synthesis of their odoriferous principle, the "irones", will be used to describe the improvement brought about by chemistry and biocatalysis in the development of natural fragrances. In particular, this tutorial review will discuss how the progress in the field of enzyme chemistry allowed the optimisation of accelerated procedures for the preparation of natural irone extracts, and the synthesis of all the ten isomers of irone, starting from commercial irone alpha.

A carotenoid cleavage dioxygenase from Vitis vinifera L.: functional characterization and expression during grape berry development in relation to C13-norisoprenoid accumulation.

A potential Carotenoid Cleavage Dioxygenase (CCD) gene was identified among a Vitis vinifera L. EST collection and a full-length cDNA (VvCCD1) was isolated. Recombinant expression of VvCCD1 confirmed that the gene encoded a functional CCD. Experimental evidence was obtained that VvCCD1 cleaves zeaxanthin symmetrically yielding 3-hydroxy-beta-ionone, a C(13)-norisoprenoidic compound, and a C(14)-dialdehyde. Expression of the gene was studied by real-time PCR at different developmental stages of grape berries from Muscat of Alexandria and Shiraz cultivars. A significant induction of the gene expression approaching véraison was observed in both cultivars. In parallel, the C(13)-norisoprenoid level increased from véraison to maturity in both cultivars.

Production of tobacco aroma from lutein. Specific role of the microorganisms involved in the process.

A mixed culture formed by Bacillus sp. and Geotrichum sp. produced tobacco aroma compounds from the carotenoid lutein through the formation of the intermediate beta-ionone. Both microorganisms can grow independently in a medium supplemented with lutein, but only Geotrichum produces beta-ionone. This intermediate was incorporated by the bacilli, converted to aroma and this product excreted to the culture medium. Bacillus sp. did not utilize beta-ionone for growth but modified it. We conclude that, in the bioconversion of lutein to products with tobacco aroma, Geotrichum sp. is involved in carotenoid oxidation to produce beta-ionone and Bacillus sp. is responsible for the norisoprenoid reduction to produce 7,8-dihydro-beta-ionone and 7,8-dihydro-beta-ionol.