Proof of principle of the chemoenzymatic cascade synthesis of

4.2.3 Proof of principle of the chemoenzymatic cascade synthesis of

well performed in the presence of biological matrices as has been previously described in other chemoenzymatic cascades.^16,145 The gradual increase of menthol formation could be correlated to the loss of isopulegol (Figure 58, chapter 3.2.3). Finally, 31 % menthol was detected in the final reaction sample after 44 h.

The following product isolation was also accompanied by a significant loss of product as after extraction, flash column chromatography and final solvent removal at the evaporator, 7 % isolated yield were obtained (6.3 mg). It is very likely that during solvent removal, also a lot of product evaporated because of the volatile nature of this terpene. The distinctly observable minty smell during this purification step underpins this assumption. Nevertheless, the solvent removal was continued until transparent crystals were formed. A trade-off of product purity and yield should be achieved and therefore, NMR analysis revealed that the final product did not contain remaining solvent. However, residual amounts of the isopulegol educt were detectable.

Nevertheless, the main side product, 3,7-dimethyloctanol, could be separated successfully from the desired menthol and no traces of this side product were detectable in the final menthol sample. This side product results as a final hydrogenation product of the previous byproducts of the bienzymatic cascade, hence, nerol, geraniol and citronellol (Figure 66). After flash column-chromatography, 15.5 mg of 3,7-dimethyloctanol were isolated in a separate fraction (16.3 % yield). Due to the R-selective ERED reduction, mainly the R-enantiomer of this side product will have been formed. However, it is interesting to note that the opposite S-enantiomer of 3,7-dimethyloctanol might be interesting as aroma chemical ingredient itself because it is described to possess a ‘[…] floral odour like clean rose […]’ scent.¹⁹⁶ Using wild-type NCR ERED in a whole cell approach with following chemical hydrogenation, this aroma chemical should be synthesised quantitatively with absolute enantiopurity.

Figure 66: Reaction scheme explaining obtained products in the herein described chemoenzymatic cascade reaction. The desired reaction path involved citral reduction by an ERED biocatalyst, followed by citronellal cyclization by an SHC biocatalyst and final reduction of isopulegol to menthol by hydrogen. However, especially after longer reaction times, remaining residual ADH activity resulted in the formation of small amounts of nerol and geraniol and larger amounts of citronellol because of the incomplete cyclization of citronellal to isopulegol. These alcohols were all fully reduced to 3,7-dimethyloctanol by hydrogen, which accumulated as only byproduct of the reaction.

Finally, this work successfully demonstrates that the initially envisioned chemoenzymatic cascade could be assembled successfully to synthesize (-)-menthol.

Potential inhibition effects in the bienzymatic cascade and necessary improvements in product purification were identified as potential bottlenecks of this approach. Hence, there still exists a large optimization potential to create a potentially efficient synthetic method. Although the herein described synthetic concept is due to its novelty still in an early developmental phase, it might be advantageous for a potential hypothetical application. Essentially, in particular the enzymatic reduction of citral and the following citronellal cyclization could be regarded as a ‘biocatalytic upgrade’ to the existent menthol production from citral. The advantages of these biocatalysts like the possible redundancy of a prior citral isomer separation might therefore initially be easier adapted in existent synthetic networks in contrast, for example, to a fermentative biotechnological procedure that is based on other resources than citral. Most recently, it was for example reported that a so far missing isomerase has been identified that should enable the transfer of the plant menthol biosynthesis in a by comparison better to handle E. coli strain.²⁹ Hence, such a fermentative menthol production based for example on a resource like glucose might be demonstrated in future. This would make a very different

in vivo approach in contrast to the herein described in vitro approach accessible. Both approaches have general advantages and disadvantages regarding for example operability, cost of catalyst and process workup that would need to be evaluated against each other in terms of economic and ecologic needs (chapter 1.6).¹⁴² In detail, this can be a complex endeavor, which with respect to current situations can also vary over time.

From a scientific point of view, it is, however, generally an asset to demonstrate the frontiers of our synthetic capabilities as broadly as possible.

Exploiting biological and mechanistic diversity to create new enzymes:

Next to the potential synthetic benefits of the chemoenzymatic synthesis that has been developed in this work, the chosen example serves to generally highlight how new enzymatic functions can be developed for a given synthetic challenge. While it is nowadays well known that enzymatic properties can be changed by genetic engineering, an important aspect is to find a suitable starting point for such an engineering.¹⁹⁷ This work demonstrates that novel enzymatic functions can be developed from two conceptually different starting points (Figure 67).

Figure 67: Conceptual strategy to find enzyme engineering targets by exploiting either biological or mechanistic diversity for application in synthetic biochemistry to enable the assembly of novel artificial biosyntheses. In this work, such a strategy is showcased by engineered ERED and SHC enzymes that have been combined with a chemocatalyst to create a novel synthetic route to (-)-menthol.

The figure has been adapted according to a previously published article.¹⁹⁷ The SHC active site depiction was also reported previously.¹²⁸

These different concepts can be distinguished by the exploitation of biological diversity on the one hand, and mechanistic diversity on the other hand. In the case of the altered enantioselectivity of NCR ERED, it was shown that the biologically present valuable reductase function could be diversified to adopt a novel selectivity. In contrast, the herein applied engineered AacSHC does not resemble its natural cyclization activity of the triterpene squalene, but rather exploits the inherent mechanistic diversity of its underlying Brønsted acid chemistry in a different type of chemical reaction.^117,129 Interestingly, although these starting points are very different, the methods for engineering the ERED and SHC enzyme towards their novel functions were quite similar. Both strategies identified potential hot-spot positions in the active sites of the respective enzymes and introduced new combinations that enabled the novel function.

Finally, this work represents a valuable showcase that such conceptually different derived novel enzymatic functions can not only be combined on the multienzymatic level, but also with ‘classical’ chemical reaction steps in a chemoenzymatic cascade reaction. This strategy should motivate to regard enzyme catalysis as a complementary opportunity in retrosynthetic analysis.