Halohydrin dehalogenase-catalysed synthesis of enantiopure fluorinated building blocks: bottlenecks found and explained by applying a reaction engineering approach

Abstract

Optically pure fluorinated organic azides represent synthetically valuable building blocks in a range of industrial applications. Since direct fluorination of molecules is challenging from both economic and environmental perspective, the...

HHDH-Catalyzed Synthesis of Enantioenriched Fluorinated β-Hydroxy Nitrile─Process Advances through a Reaction Engineering Approach

“…On the other hand, in the presence of higher rac -1 concentrations, the deactivation was more severe, whereby the highest deactivation rate was recorded at the highest epoxide concentration (Figure B). This data trend insinuates that epoxide amount could have an important role in enzyme operational stability as well. ,, Such behavior could be due to esterification of protein carboxyl groups or nonspecific chemical reactions between epoxide rings and protein functional groups of a nucleophilic character, such as amino-, hydroxyl-, or thiol- groups, leading to random alterations on the protein surface. , …”

“…For example, epoxides act as inhibitors in both biocatalytic reactions. Moreover, inhibitions by the opposite enantiomers of the substrate, the formed biocatalytic product, the byproduct diol, and the cosolvent DMSO are present as well, 35 whereby the latter was also confirmed in other investigated HHDH-catalyzed reactions. 34 In our first studies regarding HHDH-catalyzed transformations of fluorinated styrene oxide derivatives, we have found that cyanolysis reactions, despite representing a high potential for the synthesis of optically pure valuable compounds, proceed with reduced efficiency on a higher concentration scale.…”

“…ISM-4 activity at certain time points was measured using the assay described previously. , Briefly, the enzymatic transformation of para -nitro-2-bromo-1-phenylethanol (PNSHH) to para -nitro styrene oxide (PNSO) (Scheme S1, SI) was monitored using the initial reaction rate method, whereby the reaction mixture was sampled 5 times within 10% conversion of the substrate. Reactions were performed in Eppendorf tubes on a ThermoMixer C (Eppendorf, Germany) under common conditions: V r = 500 μL, c PNSHH = 5 mM, γ ISM‑4 = 0.1 mg/mL, 100 mM Tris–SO 4 buffer pH 7.5 at 25 °C, 5% v/v DMSO, and 1000 rpm.…”

“…As already implied by enzyme incubation studies, the enzyme operational stability study revealed that the estimated k d values increase with higher initial concentrations of rac-1 in the reactor (Figure S7, Table S2); moreover, a regular hyperbolic trend of dependence was observed (Figure 3H). 35,43,44 The empirical equation describing the dependence of k d on rac-1 concentration is given in Table 1, eq 13, and the respective parameters (a, b) are listed in Table S1 (SI). The trend of enzyme deactivation with increasing epoxide concentration might be attributed to the nonspecific intramolecular interactions between epoxide rings and nucleophilic functional groups on the enzyme surface, as discussed earlier (see Section 3.2).…”

“…This data trend insinuates that epoxide amount could have an important role in enzyme operational stability as well. 35,43,44 Such behavior could be due to esterification of protein carboxyl groups or nonspecific chemical reactions between epoxide rings and protein functional groups of a nucleophilic character, such as amino-, hydroxyl-, or thiol-groups, leading to random alterations on the protein surface. 45,46…”