Enzymatic Synthesis of Trimethyl-ε-caprolactone: Process Intensification and Demonstration on a 100 L Scale

Abstract

Optimization and scale up of the Baeyer-Villiger oxidation of 3,3,5-trimethyl-cyclohexanone to trimethyl-ε-caprolactones (CHL) was studied in order to demonstrate this technology on 100 L pilot plant scale. The reaction was catalyzed by a cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) that utilizes the costly redox cofactor NADPH, which was regenerated by a glucose dehydrogenase (GDH). As a first stage, different cyclohexanone monooxygenase formulations were tested: cell free extract, whole cells, fermentation broth and sonicated fermentation broth. Using broth resulted in the highest yield (63%) and required the least biocatalyst preparation effort. Two commercial glucose dehydrogenases (GDH-105 and GDH-01) were evaluated resulting in similar performances. Substrate dosing rate and biocatalyst loadings were optimized. At 30 mL scale, the best conditions were found when 30 mM h-1 dosing rate, 10% (v/v) cyclohexanone monooxygenase broth and 0.05% (v/v) of glucose dehydrogenase (GDH-01) liquid enzyme formulation were applied. These same conditions (with oxygen instead of air) were applied at 1 liter scale with 92% conversion, achieving a specific activity of 13.3 U g-1 cell wet weight (cww), a space time yield of 3.4 g CHL L-1 h-1 and a biocatalyst yield of 0.83 g CHL g-1 cww. A final 100 liter demonstration was performed in a pilot plant facility. After 9 hours, the reaction reached 85% conversion, 12.8 U g-1 cww, a space time yield of 2.7 g L-1 h-1 and a biocatalyst yield of 0.60 g CHL g-1 cww. The extraction of product resulted in 2.58 kg isolated final product. The overall isolated CHL yield was 76% (distal lactone 47% and proximal lactone 53%).

Baeyer–Villiger Monooxygenases: Tunable Oxidative Biocatalysts

“…On a 100 L scale, the 2.58 kg obtained material contained 84.5% product, and the final yield was 76%. 331 Although the biocatalyst yield (gram product per gram enzyme/ cells) seems much lower compared with the same reaction with immobilized enzyme, 330 it is actually difficult to compare these values, since a lot of costs go into the preparation and immobilization of the enzymes, in contrast to the fermentation broth used in this study. 331 This demonstration is a great example of how to reach large scale production with a BVMO reaction by addressing important challenges, in particular the oxygen limitation through careful supply of pure oxygen.…”

“…Despite the success, it would be interesting to see whether the low levels of cofactor in the cells affected this particular reaction, as was described by Milker et al 179 The low number of preparative-scale applications (Table 5) can be explained by considering the obstacles that arise from upscaling. Oxygen limitation and substrate inhibition have been successfully addressed in recent demonstrations, 316,331 although one could argue that oxygen supply remains suboptimal with respect to using whole cells. 180 On the other hand, obstacles such as enzyme stability and product inhibition are not easy to solve in any general way, since these will be case specific due to the use of different BVMOs and different products.…”

“…From the examples (Table 5), we can conclude that scaling up a BVMO reaction typically demands (1) resources, such as time to engineer the biocatalyst 218,271 and the process, and (2) facilities, such as proper reactors with equipment to control oxygen levels. 316,331 Successful industrial implementation of BVMO-processes will rely on companies that can bring together expertise in enzyme and process development.…”

Biocatalytic and Chemo-Enzymatic Approaches for the Synthesis of Heterocycles

“…Very recently, Schürmann and Guillén reported an enzymatic synthesis of trimethyl--caprolactones 68 and 69 exploiting cyclohexanone monooxygenase (CHMO) from Thermocrispum municipale (Scheme 16). 31 A glucose dehydrogenase (GDH) was used to regenerate the cofactor NADPH. As a result, the CHMO-catalyzed Baeyer-Villiger oxidation of 3,3,5-trimethylcyclohexanone to trimethyl-caprolactone occurred smoothly and could be performed on a 100 L scale, although a mixture of two regioisomers was obtained as the products.…”

Enzymatic Preparation of the Chiral (S)-Sulfoxide Drug Esomeprazole at Pilot-Scale Levels

“…17,19−21 Despite achieving laboratory-scale bioconversion, scale-up of the biocatalytic oxidation process for esomeprazole production remains challenging. Several other successful scaleup reports on biocatalytic oxidation processes, such as the 200 L scale bio-oxidation of bicyclo[3.2.0]hept-2-en-6-one, 22 100 L scale synthesis of trimethyl-ε-caprolactone, and 100 L scale synthesis of the chiral sulfoxide intermediate of the anticancer drug AZD6738, 23,24 inspired us to further explore the limiting issues for bio-oxidation of omeprazole sulfide.…”