Figure 3 - Biocatalytic cascade reaction including UPOs to retain an API Note: Adapted from Heckmann et al.
Notably, the UPO with the highest overoxidation activity (UPO-O) is not the same as the one with the highest hydroxylation activity (UPO-K for R, UPO-C for S). Depending on the desired oxyfunctionalisation and substrate, a different UPO may turn out to be the most suitable biocatalyst, which illustrates the benefit of a panel screening.
However, Hara et al . discovered one KGO biocatalyst that could synthesise trans-3-L-hydroxyproline from L-proline with 90% conversion under the given reaction conditions (Figure 4).
revolutionise the chemical industry. 1 In this work, Aminoverse’s UPO kit was used to identify a suitable biocatalyst for a three-step biocatalytic reaction to synthesise the chiral compound and the API (1S,3R)-3-hydroxycycloh exanecarbonitrile.
KGO-catalysed reaction
This study shows that nature's toolbox contains biocatalysts of incredible value for industrial applications. Biocatalyst kits like this can thus open access to cutting-edge synthesis routes, new intermediates, and novel products by significantly accelerating the timeline towards identification of an optimal catalyst
Starting from cyclohexene-1-nitril, the first step is the overoxidisation of the non-activated C-H in cyclohexene-1-nitril towards the ketone 3-oxocyclohexane-1- carbonitrile. This overoxidation is achieved best by UPO-O, with over 80% conversion. The intermediate is then converted to the final API by an ene reductase and an alcohol dehydrogenase (Figure 3).
The launch of the world’s first KGO kit by Aminoverse provides a means of synthesising new molecules that were even inaccessible with UPOs. A study by Hara et al . showed how KGOs can impact the search for a range of APIs. 2
As industry often uses (2S,4R)- 4-hydroxypyrrolidine-2-carboxylic acid (L-hydroxyproline), its isomer, (2S,3S)-4-hydroxypyrrolidine- 2-carboxylic (trans-3-Lhydroxyproline ), represents an important building block for further synthesis. By using various biocatalysts, it was possible to produce the three other isomers: (2S,4R)-4-hydroxypyrrolidine- 2-carboxylic acid, (2S,4S)-4- hydroxypyrrolidine-2-carboxylic acid and (2S,3R)-4-hydroxypyrrolidine-2- carboxylic.
Limits & potential of biocatalysis
While a steadily growing number of companies start and expand the use of biocatalysis, the worldwide adoption across all chemical industries is still facing obstacles. The main concerns for PIs and research managers are costs, IP concerns, short-term availability and development times.
The authors also investigated the stereoselectivity of the tested UPO, emphasising its diversity. In the first reaction using cyclohexene-1-nitrile as substrate, six R-selective and two S-selective UPOs were found to yield 30 –70% of the corresponding 3-hydroxycyclohex-1-ene-1- carbonitrile enantiomer under nonoptimised conditions.
Biocatalysts are typically produced in biological cells, requiring some isolation of the catalyst prior to its use in a chemical process. These two steps of production and isolation account for the largest cost factor and are typically offset by increasing production scale.
The lack of a highly selective enzyme made the biocatalytic production of trans-3-Lhydroxyproline inconceivable.
60 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981