## 3 Results
### Biotransformation of (1\(R\), 4\(R\))-(+)-Dihydrocarvone (**2**)  
As mentioned before, (+)-dihydrocarvone is not available as a pure diastereoisomer, but in the form of two _cis_ and _trans_ isomers. Using the Aldrich catalog description, it is known that the ratio between _trans_ and _cis_ isomers is 70:30, which can additionally be confirmed by the following chromatogram made from a commercially available mixture of (+)-dihydrocarvone (Figure 3).  
Figure 2: Biotransformation of 4\(R\))-(–)-carvone (**1**) via a comminuted potato.  
The diastereomers were separated by column chromatography using a ratio of hexane:acetone (20:1) as an eluent. After purification, the specific rotation of (1\(R\), 4\(R\))-(+)-dihydrocarvone (**2**) was measured and compared to the literature (\([\alpha]^{20}_{D}\) = +14.6 CHCl\({}_{3}\), c = 1.0; lit [22]; \([\alpha]^{20}_{D}\) = 14.2, CHCl\({}_{3}\), c = 0.8).  
The obtained pure (1\(R\), 4\(R\))-(+)-dihydrocarvone (**2**) was biotransformed using previously selected plant biocatalysts. The composition of the reaction mixture was determined by comparing the chromatograms of the standards and the reaction mixtures.  
The results obtained during biotransformation are summarized in Table 3. This evidence supports the conclusion that the substrate was transformed by each of the catalysts used. In contrast to the biotransformation of (4\(R\))-(-)-carvone (**1**) described above, the enzymatic system of potato turned out to be a poor biocatalyst in the reduction of (1\(R\), 4\(R\))-(+)-dihydrocarvone (**2**). The largest loss of substrate was observed using the carrot enzymatic system as a bioreagent.  
As _Daucus carota_ emerged as the best biocatalyst, a preparative biotransformation was carried out, which would allow us to attain a greater amount of product for spectroscopic investigation. The chromatograms (Figure 4) demonstrate that (1\(R\), 4\(R\))-(+)-dihydrocarvone (**2**) was converted to alcohol. After the separation of the reaction mixture into its components by column chromatography, however, it appeared that two diastereomeric alcohols, (**4**) and (**5**), were the reaction products. These alcohols were visible in the GC analysis as one signal and, despite numerous attempts, we were not able to separate them.  
Figure 3: Chromatogram of the (+)-dihydrocarvone mixture of two diastereoisomers: _trans_ (1\(R\), 4\(R\))-(+)-dihydrocarvone (**2**) with R\({}_{\rm f}\) = 7.114 and _cis_ (1\(S\), 4\(R\))-(+)-dihydrocarvone (**3**) with R\({}_{\rm f}\) = 7.363.  
\begin{table}
\begin{tabular}{c c c} \hline
**Biocatalyst** & **Substrate (\%)** & **Product (\%)** \\ \hline _Daucus carota_ L. (carrot) & 27.9 & 72.1 \\ _Apium graveolens_ L. var. _rapaceum_ (celerac) & 53.0 & 47.0 \\ _Petroselinum sativum_ Hoffm: (parsley) & 58.6 & 41.4 \\ _Solanum tuberosum_ L. (potato) & 74.7 & 25.3 \\ _Beta vulgaris_ L. (beetroot) & 94.1 & 5.9 \\ _Malus pumila_ L.(apple) “Golden” & 87.4 & 12.3 \\ _Malus pumila_ L.(apple) “Kortland” & 93.8 & 6.2 \\ _Pyrus communis_ L.(pear) “Konferenjá” & 61.2 & 38.8 \\ \hline \end{tabular}
\end{table}
Table 3: Results of the (1\(R\), 4\(R\))-(+)-dihydrocarvone (**2**) biotransformation.