Exploring the enzyme-catalyzed synthesis of isotope labeled cyclopropanes.

Cyclopropanes are commonly employed structural moieties in drug design since their incorporation is often associated with increased target affinity, improved metabolic stability, and increased rigidity to access bioactive conformations. Robust chemical cyclopropanation procedures have been developed which proceed with high yield and broad substrate scope, and have been applied to labeled substrates. Recently, engineered enzymes have been shown to perform cyclopropanations with remarkable diastereoselectivity and enantioselectivity, but this biocatalytic approach has not been applied to labeled substrates to date. In this study, the use of enzyme catalysis for the synthesis of labeled cyclopropanes was investigated. Two readily available enzymes, a modified CYP450 enzyme and a modified Aeropyrum pernix protoglobin, were investigated for the cyclopropanation of a variety of substituted styrenes. For this biocatalytic transformation, the enzymes required the use of ethyl diazoacetate. Due to the highly energetic nature of this molecule, alternatives were investigated. The final optimized cyclopropanation was successfully demonstrated using n-hexyl diazoacetate, resulting in moderate to high enantiomeric excess. The optimized procedure was used to generate labeled cyclopropanes from 13 C-glycine, forming all four labeled stereoisomers of phosphodiesterase type-IV inhibitor, MK0952. These reactions provide a convenient and effective biocatalytic route to stereoselective 13 C-labeled cyclopropanes and serve as a proof-of-concept for generating stereoselective labeled cyclopropanes.

Unexpected carbonate salt formation during isolation of an enantiopure intermediate by supercritical fluid chromatography.

The enantiomers of a chiral building block to be used in pre-clinical manufacturing were separated using supercritical fluid chromatography (SFC). Despite an extensive evaluation of different columns and solvent combinations followed by a careful optimization of the chromatographic method, the preparative separation suffered from low throughput and high solvent consumption. Consequently, additional improvements were necessary. By utilizing stacked injections, the chromatographic run time was almost halved, and the high solvent consumption was reduced by recycling of the two mobile phase components, carbon dioxide and methanol. The carbon dioxide was reprocessed by the SFC instrument, whereas methanol was evaporated and recycled from the fractions collected. Hence, the originally inefficient separation method was turned into a more sustainable one, and the desired enantiopure intermediate was delivered to be used in the following synthesis of the selected candidate drug. Unfortunately, when the intermediate was used in the subsequent amide coupling, a surprisingly poor yield was obtained. This was caused by an unexpected formation of a stable carbonate salt of the intermediate under the chromatographic conditions used. By removal of the carbonate prior to the amide coupling reaction, the manufacturing campaign could be saved, and the candidate drug was successfully delivered in time.