Biocatalytic Activity of Fresh Passiflora Spp. Leaves in EnantioselectiveOxido-Reduction

The biocatalytic activity of five species of Passiflora leaves (i.e. P. amethyst, P. incarnata, P. quadrangularis, P. edulis, P. cerulea) was tested in the reduction of the ketone “cocktail” (i.e. 5-hexen-2-one 1, acetophenone 2, cisbicyclo[ 3.2.0]hept-2-en-6-one 3 and 2-methylcyclohexanone 4) and in the oxidation of the corresponding alcohols “cocktail” (i.e. 5-hexen-2-ol 5, 1-phenylethanol 6, endo-bicyclo[3.2.0]hept-2-en-6-ol 7, exo-bicyclo[3.2.0] hept-2-en- 6-ol 8, trans-2-methylcyclohexanol 9 and cis-2-methylcyclohexanol 10). P. amethyst and P. incarnata show the best activity in the reduction, while P. quadrangularis affords low yield in reduction but gives appreciable results in oxidation towards a cocktail of model substrates. This simple screening permits to test the potential of parts of fresh plants that can be used as biocatalysts in more ecologically and economically promising transformations.


Introduction
Asymmetric synthesis remains a challenge for synthetic chemists as the demand for enantiomerically pure compounds continues to increase. Several of these compounds are potential chiral building blocks of pharmaceutically important molecules, agrochemicals, flavours and asymmetric chiral ligands [1]. Moreover the enantiomers of a number of chiral drugs may exhibit great differences with regard to pharmacodynamics, pharmacokinetics and toxicological properties [2].
In this field the biotransformations, defined as chemical reactions catalysed by isolated enzymes, microorganisms or cell cultures [3], are a "green opportunity" to obtain enantiomerically pure building blocks. The use of purified enzymes [4], whole microorganisms [5] or plant cell cultures [6], mainly at industrial scale, is limited by cost and by the difficulty to handle them, due to their great sensitivity to changes in pH or temperature, requirements of additional cofactors, long and tedious procedures [7]. In order to overcome these limitations, the possible use of vegetable whole organs, as leaves, without any preliminary preparation or transformation, has been considered [8].
Since the biochemical potential of plant cell cultures to produce specific secondary metabolites such as drugs, flavours, pigments and agrochemicals is of considerable interest in connection with their biotechnological utilisation [9], plants have the potential to integrate the greening of organic chemistry with the use of cheap and effective reagents.
Moreover, this methodology offers numerous advantages both in terms of time-saving, since fastidious steps of preparation, extraction, purification and multiplication of the biocatalyst are not necessary thus promoting the preservation of a maximum catalytic activity of the enzymes [10], and in terms of environment-saving, since the reactions are performed in aqueous media, at room temperature, and generate only biodegradable waste [11].
In a green context, several reports have so far described the possibility of using parts of fresh plants as biocatalysts, i.e. the reduction of aromatic aldehydes using of fresh leaves of banana and maize plants [12], the stereoselective reduction of prochiral ketones by wild tissues of South American endemic plants [13] and the bioreduction of acenaphthenequinones by using peach and carrot [14].
Passiflora commonly known as passion fruit plant belongs to the family Passifloraceae, which comprises nearly 500 species. This genus, native to Tropical America and Brazil, and widely distributed all over the world [15], is principally popular for their fruits, but, traditionally, in American and European countries leaves are largely used for tea infusion [16]. Machado and co-workers have reported the use of fruit peel of Passiflora edulis for the reduction of aromatic aldehydes and ketones, obtaining very good yields and moderate enantioselectivity [17].
The final research objective was to find new biocatalysts readily accessible from both wild and commercially available natural resources, as a sustainable alternative to traditional chemical methods.

Results and Discussion
The biotransformation procedure was very simple owing the easy availability of plants, the use of water without carbon source and the simple work up, because no emulsion has formed.
Also P. incarnata (1 g) oxidized with good yields 5-hexen-2-ol 5 and the mixture of bicycloheptenols 7 and 8 to give the corresponding ketones 1 (50%) and 3 (41%) with only a good kinetic resolution of the R-alcohol 5 (50%, ee 47%). Similar or worst results were obtained with 5 g of the same leaves.

Conclusions
The use of a cocktail of ketones and alcohols allows preliminary information of oxido-reduction potential of Passiflora leaves using a simple and fast methodology that permits to test the potential of parts of fresh plants that can be used as biocatalysts in more ecologically and economically promising transformations. P. amethyst and P. incarnata appear to have a higher inclination towards the reduction with respect to other species, while towards the oxidation there is a greater regularity of behaviour (low yields and enantiomeric excesses) even if P. amethyst, P. incarnata and P. quadrangularis show a higher activity towards the substrate cocktail.