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 13C‐glycine, forming all four labeled stereoisomers of phosphodiesterase type‐IV inhibitor, MK0952. These reactions provide a convenient and effective biocatalytic route to stereoselective 13C‐labeled cyclopropanes and serve as a proof‐of‐concept for generating stereoselective labeled cyclopropanes.


| INTRODUCTION
Cyclopropanes are often metabolically stable motifs in drugs and drug candidates and make an intelligent location for labeling with stable isotopes or radionuclides in drug development studies. Cyclopropanes are featured in many natural compounds, including pheromones and terpenes, 1 and the introduction of this subunit into pharmacologically active synthetic compounds dates back to the 1960s. 2, 3 Talele et al 2 have reviewed the frequent appearance of cyclopropyl rings in drug molecules and stated that the cyclopropane unit can be used as a less lipophilic surrogate for hydrophobic ring systems and can be used to increase affinity and rigidity, and to improve metabolic stability. This ring also offers controlled stereochemistry, with alternate stereoisomers often having very different biological activities. 2,4 Many synthetic methods have been developed for the synthesis of cyclopropanes, including the Simmons-Smith reaction and the transition-metal-catalyzed decomposition of diazo compounds. 1 Despite the synthetic utility of these methods, they can be difficult to use in the synthesis of isotopically labeled compounds due to the lack of desirable labeled starting materials.
Biocatalysis is an important tool in organic synthesis, often exhibiting exquisite chemo-, regio-, and stereoselective control, [5][6][7] combined with low environmental impact and improved safety parameters. Arnold and co-workers 4,8 engineered cytochrome P450 enzymes (CYP450) to form cyclopropanes with high stereo-and enantioselectivities from olefins. Similarly, Hartwig and co-workers 9 described a modified CYP450 containing iridium rather than iron in the heme. This modified enzyme is suitable for a broad range of alkenes forming cyclopropanes with high stereoselectivity. In addition to CYP450-enzymes, another iron-based heme-dependent enzyme, myoglobin, has also been engineered by Fasan and co-workers 10 to transform alkenes into cyclopropanes. The reaction exhibits broad substrate scope and has been applied to the synthesis of Tasimelteon, a TRPV1 inhibitor, and ticagrelor ( Figure 1) on a gram scale. 11,12 In contrast to many synthetic procedures, 13 enzymatic cyclopropanations are performed in aqueous media. By replacing organic solvent with water and stoichiometric metal for catalytic iron, the environmental impact of the reaction should be improved. In addition, and perhaps more importantly, the reactions afford cyclopropanes in a high diastereoselectivity and F I G U R E 1 Selection of pharmaceutically important cyclopropanes S C H E M E 1 Cyclopropanation methodologies enantioselectivity, limiting the formation of any by-products. However, the enzyme-mediated cyclopropanations reported to date use ethyl diazoacetate (EDA (1)) as the methylene source, which exhibits high impact sensitivity (1730 J/g). 14,15 Inspired by the works of Arnold, Fasan, and Hartwig, we explored the synthesis of various substituted cyclopropanes with readily available enzymes (Scheme 1) using EDA (1) as co-substrate as a first objective. For this feasibility study, enzymes previously engineered for 1-octene were chosen. As described before, enzymes engineered for a target molecule provide the ability to produce cyclopropanes with both high diastereoselectivity (cis or trans) and enantioselectivity. Our second objective was to develop a diazoacetate substrate with a higher carbon content to mitigate the impact sensitivity. With this alternative diazoacetate and optimized conditions, 13 C-labeled MK0952 was successfully synthesized.

| RESULTS AND DISCUSSION
We initiated our optimization study with the enzyme P411-UA and enzyme ApePgb AGW, provided by T A B L E 1 Scope of cyclopropanation using EDA (1) Professor Frances Arnold, using EDA (1) and styrene (2). 4 Various parameters were investigated, including concentration, EDA (1) equivalents, and reaction time.
The summarized results of this optimization study can be found in the experimental section (Table E1, in the  Experimental). By adjusting the parameters, a shift of the diastereomers to either cis or trans could be achieved. When using 2 equiv EDA (1), 15 mM styrene (2), and 2.8 mg/mL enzyme, a conversion of 100% is obtained with a d.r. of 7:3 (cis:trans) after shaking 22 h (Table E1, entry 9). Given that any optimization requires a target molecule and that this work represents a feasibility study rather than a study tailored to a specific target molecule, the conditions found for styrene (2) were applied to investigate the substrate scope.
The cis-cyclopropanes obtained from enzyme P411-UA also displayed a higher e.e. in comparison to the trans-cyclopropanes. One electron-rich and several electron-deficient styrenes provided modest to excellent conversions. In general, a higher conversion resulted in higher selectivity towards cis. The e.e. of the ciscyclopropanes was moderate to excellent, while that of the trans-cyclopropanes was poor. The results using enzyme ApePgb AGW are more difficult to categorize, but the e.e. were good to excellent for both cis-and trans-cyclopropanes. In addition, ortho substitution and heterocycles were not tolerated for either enzyme.
A robust substrate scope was demonstrated by this study, although these readily available enzymes were evolved using 1-octene as a substrate. It is likely that by using a styrene as a substrate and further engineering of the enzyme, higher specificity for the desired product could be achieved. Nevertheless, the e.e. and d.r. obtained are useful and may be further manipulated Overall, moderate conversions were obtained. This can be explained by the limited conversion for most cyclopropanations, the small scale of the reactions, and the dimerization of EDA (1) forming maleate and fumarate. Additionally, the isolation of phenylcyclopropane 3 was challenging due to volatility. Unfortunately, only a small mass peak was observed for 2-vinylnaphthalene 9, along with all ortho-substituted styrenes (10)(11)(12), suggesting that these reactions need further optimization or may represent a limitation of this enzyme. Finally, based on gas chromatography-mass spectrometry (GC-MS) analysis, heterocyclic ring systems did not provide any product formation (13-15).

| Finding an alternative for EDA (1)
EDA (1) plays a central role in many cyclopropanation reactions; however, the industrial use of EDA (1) requires special precautions due to safety concerns associated with its instability and high reactivity. 16 To reduce safety concerns, diazoacetates with longer alkyl chains were synthesized and investigated. It was expected that extending the alkyl side chain would reduce the inherent energetics of the compound while having a minimal effect upon the enzymatic conversion. The differential scanning calorimetry (DSC) results show that while onset temperature was raised significantly by lengthening the alkyl side chain, the J/g was not significantly affected (Table 2).
Initially, benzyl diazoacetate (16) was investigated as the enzymatic substrate; however, no product formation in the enzyme-catalyzed cyclopropanation was observed. We hypothesized that the benzyl chain may not fit in the pocket in the enzyme where the reaction takes place; therefore, longer alkyl chain were used instead. Product formation was observed when using n-hexyldiazoacetate (18). The following scope was performed using 18 as neither 19 nor 20 significantly increased the onset temperature, although they reduced the energetic content of the molecule.

| Screening of substituted styrenes with n-hexyldiazoacetates
A small panel of substrates was investigated using nhexyl diazoacetate (18) ( selectivities obtained from these reactions are comparable with the results found in Table 1. The synthesis of isomers 24 and 25 was performed using labeled n-hexyl diazoacetate ( 13 C-18). This diazoacetate is synthesized in two steps, starting from commercially available labeled glycine 26 with a yield of 73% over two steps. C-13 labeling was used here as a model for C-14 labeling, avoiding unnecessary radiochemical handling and the generation of difficult-todispose radiochemical waste. The two labeled isomers, 24 and 25, were synthesized to compare the effect of halogens at two different positions, changing from meta to ortho. Both compounds gave similar results in terms of conversion and d.r., and 25 was isolated with good yield. p-Bromo cyclopropane 25 is a precursor for the selective phosphodiesterase type-IV inhibitor, MK0952, and is only three steps away from the final drug. 17 MK0952 was previously synthesized using an enantioselective Simmons-Smith cyclopropanation followed by a Suzuki coupling. Using labeled glycine 26 as the starting material, MK0952 was successfully synthesized with an overall yield of 27% by the enzymatic approach (Scheme 2). The metabolic profile reported by Gallant et al 17 indicates that the targeted cyclopropane ring is a metabolically stable position and labeling there would be desirable.

| CONCLUSION
In summary, the enzyme-catalyzed cyclopropanation with readily available enzymes was implemented successfully with various styrenes. This transformation was applied to a variety of substituted styrenes and afforded moderate to high levels of diastereoselectivity and enantioselectivity. The reaction could be tuned towards the desired diastereomer by adjusting the concentration of the reaction, the equivalents of EDA (1), and/or the amount of enzyme. Since the enzymes were designed for the transformation of 1-octene, further engineering of the enzymes towards substituted styrenes could result in considerably improved d.r. and e.e.
Moreover, a selection of diazoacetates was investigated, and n-hexyl diazoacetate (18) displayed a higher onset time by DSC, making this a safer alternative to EDA (1). Furthermore, n-hexyl diazoacetate (18) performed well in the biocatalytic system. The biocatalytic strategy was successfully applied to the asymmetric synthesis of labeled cyclopropanation using modified enzymes and 13 C-labeled diazoacetate to synthesize 13 C-MK0952 with good yield and moderate stereoselectivity.

| General information
All reagents were purchased from commercial suppliers and used without further purification unless mentioned otherwise. Anhydrous solvents were purchased from Sigma-Aldrich and stored under a nitrogen atmosphere. Racemates of the cyclopropane products were synthesized using a literature procedure. 4 For literature known compounds, the analytical data were in agreement with those reported previously. The corresponding literature reference is given for each molecule. Yields are based on styrene (as the limiting reagent) and refer to purified, isolated, homogeneous product and spectroscopically pure material (analyzed by NMR), unless stated otherwise.
For each substrate, a 500-or 1000-mM stock solution in ethanol was prepared and used for the enzymatic cyclopropanation. Ethyl diazoacetate in ethanol was prepared by co-evaporation of the commercially available EDA in CH 2 Cl 2 with ethanol (2x), without concentration to dryness. The concentration of 1 in EtOH was determined by NMR analysis.
All diazoacetates were stored in CH 2 Cl 2 ; however, for the DSC analysis, the solvent was switched to toluene, due to solvent interference during the analysis. DSC analyses were measured on a DSC3 + by Mettler Toledo using 10-20 mg of diazoacetates in toluene (10-15 wt%) using either of the following gradients: 1. Method A: 5 K/min, 30-300 C 2. Method B: 3 K/min, 30-350 C All reactions were carried out under a nitrogen atmosphere and were magnetically stirred or shaken. Electric heating plates and DrySyn were used for elevated temperatures, and a stated temperature corresponds to the external DrySyn temperature. Concentration was performed on a rotary evaporator with a heating bath at 40 C, unless stated otherwise.
Crude reaction mixtures were assayed by GC-MS and/or liquid chromatography-MS (LC-MS) or SFC for diastereomeric ratio and enantiomeric ratio, respectively. Diastereomeric ratio (d.r.) (%) is based on the area under the curve (AUC) for the cis-product and trans-product in GCMS analysis, and e.e. (%) is based on the AUC of the two enantiomers in chiral HPLC or chiral supercritical fluid chromatography (SFC) analysis. 18 Enantiomer 1 is the main enantiomer obtained by the enzyme P411-UAcatalyzed cyclopropanation. The four different enantiomers that could be obtained from the enzyme-catalyzed reaction are displayed in Scheme 3.
GC-MS (EI) analysis was performed on an Agilent 7890A GC system and Agilent 5975C inert MSD system equipped with an Agilent 19091S-433L (30 m Â 250 μ Â 0.25 μm) capillary column using a gradient: (SHALLOW) 40-150 C with a rate of 15 C/min followed by 150-300 C with a rate of 60 C/min and electron impact ionization at 70 eV or (STANDARD) gradient 40-250 C with a rate of 50 C/min, followed by 250-300 C with a rate of 13 C/min, and electron impact ionization at 70 eV.
Chiral HPLC: Waters Acquity ARC HPLC -equipped with PDA detector, sample Manager FTN-R, Quaternary solvent manager-R, and 30S column manager.
Thin layer chromatography was carried out using E. Merck silica glass plates (60F-254) with UV light (254 nm) and/or potassium permanganate as the visualization agent.

| General procedure B: Alkyl 2-diazoacetates
A biphasic mixture of alkyl 2-aminoacetate (1 equiv) in CH 2 Cl 2 and water (1:1, 0.33 M) at 0 C was stirred as sodium nitrite (1.5 equiv, 4.55 M in H 2 O) was added dropwise over a period of 5 min, and the reaction mixture was warmed to room temperature. Upon completion, sat. aq. NaHCO 3 was added, and the layers were separated. The aqueous layer was extracted with CH 2 Cl 2 (3x), and the combined organic layers were dried over MgSO 4 , filtered, and partly concentrated in vacuo. The crude yellow oil was purified by flash column chromatography on silica gel (10-15% Et 2 O in n-pentane) to give the alkyl 2-diazoacetate as a yellow solution. The product was stored as a solution in CH 2 Cl 2 .

| General procedure C: Enzymatic cyclopropanation
Enzyme P411-Ua/ApePgb AGW (150 mg) was added to a reaction tube which was sealed and flushed with N 2 for at least 30 min. Under N 2 flow were added M9-N buffer (28.5 mL), sodium dithionite (4.5 mL, 0.11 mmol, 500 mM stock solution, 0.2 equiv), styrene (1.05 mL, 0.53 mmol, 500 mM stock solution, 1 equiv), and EDA (1) (1.05 mmol, 1306 mM stock solution, 2.00 equiv) with 6% EtOH as co-solvent. The N 2 flow was removed, and the reaction tube was shaken at room temperature for 24 h. The reaction was acidified by the addition of HCl (3 M, 0.7 mL), after which the pH was adjusted to 3. EtOAc (10 mL) was added to the reaction mixture, which was filtered through a pad of Celite to remove the enzyme and to break the emulsion. The Celite pad was rinsed with additional water (2 Â 10 mL) and EtOAc (2 Â 50 mL). The layers were separated, and the aqueous layer wasextracted with EtOAc (2 Â 100 mL). The combined organic layers were dried over sodium sulfate and filtered. The organic solvents were partly removed in vacuo.