3. Diversity-oriented enzymatic synthesis of cyclopropane building blocks
3.4 Reaction engineering for preparative-scale production of cyclopropylboronates
3.4 Reaction engineering for preparative-scale production of
Figure 3-10. Nonlinear response behavior of the cyclopropylboronate compounds.
3.4.2 Lyophilized biocatalyst formulation
To enable access to these engineered biocatalysts for chemists without cell culture experience, I tested the formulation of the enzymes as lyophilized whole cells. Proteins have long been lyophilized to improve their shelf life and storage,31 and many commercially available enzymes sold today are formulated as lyophilized powders. Rather than preparing biocatalysts as fresh cells each time a reaction is required, the biocatalyst is produced in a large-scale fermentation, which eliminates batch-to-batch variability in specific expression and activity levels and allows for robust process optimization. Lyophilizing biocatalysts as whole Escherichia coli cells rather than cell lysate or purified protein decreases cost by removing additional lysis and purification steps, removes the requirement to exogenously add expensive redox cofactors, and in some cases provides enhanced stability to the biocatalyst relative to purified lyophilisate.32,33 I therefore prepared large-scale cultures (1 L) of RmaNOD THRAW and RmaNOD WAIHNM and lyophilized the whole-cell pellets. The resultant lyophilisate was ground into a fine powder with a mortar and pestle to facilitate weighing and adding catalyst to reaction vessels. The lyophilized cell powder could be dissolved into aqueous buffer and remained active for cyclopropylboronate formation.
Lyophilized biocatalysts allow for reactions being performed in the presence of minimal amounts of water. While water is viewed as a green solvent, starting the reaction in it simply adds a second waste stream if the downstream processing requires extracting the compounds into organic solvent. Biocatalysts have been used extensively in organic solvent systems.
Lipase reactions are a great example, as their reaction equilibrium is dependent on the ratio of water and substrate. Performing reactions directly in organic solvent is also beneficial in cases where the substrate is poorly soluble or unstable in water. Rother and others have shown multiple examples of the efficacy of biocatalytic reactions run in organic solvent and 1-5% water using lyophilized whole cells, either directly in solution32 or within “teabag”
membranes for easy catalyst removal.34 Analytical-scale experiments with lyophilized RmaNOD THRAW and RmaNOD WAIHNM cells in methyl tert-butyl ether with 1-5%
aqueous buffer added showed that the biocatalysts were still active under micro-aqueous conditions, though the drastically different reaction conditions prevent product extraction and comparison to the product standard calibration curves generated from aqueous samples.
Further work on micro-aqueous preparative-scale reaction optimization could enable an alternative biocatalytic synthesis and product isolation workflow; this could be particularly promising for cyclopropylboronate formation, as performing the reaction under low water content has the potential to decrease pinacolboronate hydrolysis.
3.4.3 EDA addition optimization
In scale-up reactions, whether with fresh or lyophilized whole cells, a pad of cell debris would form at the surface where the EDA was being added dropwise. Switching to a long needle that inserted into the solution instead of dropping EDA on top of it prevented the formation of this cell debris pad. I noted the distinct formation of gas bubbles at the point the needle tip inserted into solution, which indicated that the EDA was rapidly consumed at the point of addition, evolving nitrogen at that point to form a bubble. I hypothesized that the local concentration of EDA at the needle tip (and thus the concentration of the EDA solution added via slow addition) was a crucial parameter. Previously, the concentration of EDA in the syringe was 800 mM, the same as we used in our screening assay. After testing the solubility limit of EDA in water:ethanol mixtures, I diluted the EDA solution being added
via slow addition to 163 mM (increasing the water content of the EDA solution) while maintaining the same final reaction volume. The resultant RmaNOD THRAW preparative- scale reaction had decreased EDA dimer formation and was used in the trifluoroboration product isolation described below to yield the trifluoroborated enzymatic product in 52%
isolated yield.
3.4.4. Efficient product isolation
Downstream processing is a crucial consideration for any product synthesized in large scale;
an efficient process to separate and purify the product from the catalyst and byproducts needs to be developed. At laboratory scale, this is typically performed with some combination of acid-base extractions, solvent extractions, and silica column chromatography.
In the case of cyclopropylboronate synthesis, the final reaction mixture contains whole-cell biocatalysts, cyclopropylboronate product, the EDA dimer byproducts diethyl maleate and diethyl fumarate, and unreacted starting material. Due to the product’s partial hydrolysis to the cyclopropylboronic acid, silica column chromatography resulted in poor isolated yields.
We examined alternative methods to efficiently isolate the cyclopropylboronate product. One option was distillation; the vinylboronate starting material, EDA dimers, and cyclopropylboronate product have different boiling points. Using rotary evaporation to remove starting material and a Vigreux distillation column to remove EDA dimers, 3.7 g of cyclopropylboronate were purified from a crude reaction extract in a column free approach (36% isolated yield, 41% isolated yield based on recovered starting material, 95:5 dr, 99%
ee). Further iterative optimization of the reaction and downstream processing will improve the yields, while decreased starting material and byproducts in the final reaction mixture will make downstream processing more efficient.
A second option explored was the trifluoroboration of the cyclopropylboronate to the trifluoroborate-substituted cyclopropane. Previously, the trifluoroboration was carried out on purified cyclopropylboronate, but by performing the trifluoroboration on a crude reaction mixture, both the cyclopropylboronate and the free boronic acid can be converted to the trifluoroborate salt. This enables an alternative purification procedure for our cyclopropane
product. First, organic solvent is used to extract the cyclopropane, starting material, and EDA dimer byproducts from the aqueous reaction. Concentrating this mixture in vacuo removes the organic solvent and the volatile starting material. This remaining crude mixture, consisting of primarily cyclopropylboronate, cyclopropylboronic acid, and EDA dimer byproducts, is used as the starting material for a well-established trifluoroboration reaction.30 The reaction can then be lyophilized to remove the more volatile organic compounds, and the cyclopropane product can be separated from the fluoride salt with an acetone extraction.
This technique minimizes loss of cyclopropane product from pinacol deprotection and provides a column-free and scalable approach to the trifluoroborate salt. Using this approach, lyophilized whole cells harboring RmaNOD THRAW were used in a 1-mmol scale reaction to produce the cyclopropylboronate; the cyclopropylboronate was purified as the trifluoroborate salt with a 52% isolated yield across both steps. The trifluoroboration and distillation approaches can both be performed at large scale with further optimization.
3.4.5 Assessment of carbene precursor scope
The ability to generate diverse cyclopropanes from the cyclopropylboronate is limited in part by the carbene precursor used; we were therefore interested in testing diazo compounds in addition to EDA. RmaNOD THRAW and RmaNOD WAIHNM were examined for their ability to form and transfer five additional diazo esters and ketones to vinyl Bpin to produce alternate cyclopropylboronates. In each case, however, there was no formation of the cyclopropylboronate detected. To expand the cyclopropylboronate repertoire, it appears that a substrate-walk engineering approach is needed, where one first screens for carbene-transfer activity on styrene and other model substrates. Once the enzyme has been evolved to accept alternative diazo compounds as carbene precursors, these variants can be assayed for their ability to accept vinylboronate substrates. A similar approach was required for organoborane synthesis, with a separate engineered lineage for each diazo compound.35