Expanding enzymatic substrate scope to transfer trifluoromethylcarbenes to

2. Diverse engineered heme proteins enable stereodivergent cyclopropanation of

2.4 Expanding enzymatic substrate scope to transfer trifluoromethylcarbenes to

2.4 Expanding enzymatic substrate scope to transfer trifluoromethyl-

(ApePgb “LQ”) and ApePgb Y60G F73W (ApePgb “GW”) which were active against both styrenyl and electron-deficient alkenes (Figure 2-13). Notably, the ApePgb LQ variant formed the cis diastereomer with electron-deficient alkenes in moderate to good stereoselectivity; these cis-trifluoromethyl-substituted cyclopropanes are difficult to access with reported synthetic methods. Cyclopropanation of unactivated alkenes was also tested, but ApePgb LQ and GW only show trace activity for the previous model unactivated alkenes, 1-octene and 4-phenyl-1-butene.

Figure 2-13. Substrate scope of ApePgb LQ (red) and GW (blue) variants against activated and electron-deficient alkenes. GC-MS data reported were collected by Dr. Xiongyi Huang and Lucas Schaus.

Dr. Huang transferred the mutations from ApePgb to the protoglobin from the mesophilic methanogen Methanosarcina acetivorans (MacPgb), as MacPgb has been structurally characterized previously,⁴⁸ as he intended to perform molecular dynamics simulations that required an accurate crystal structure. When that initial transfer of mutations to MacPgb yielded variants with comparable activity and selectivity to the ApePgb variants, I became interested in how general the effect on carbene transfer activity and selectivity is when transferring these mutations to other proteins in the protoglobin fold class. Transferring mutations from one scaffold to another has been quite successful for promiscuous native⁷³ as

well as new-to-nature⁷⁴ reactions – though there is certainly a positive bias in the literature toward successful transfers of mutations. I hypothesized that the carbene-transfer activity observed in ApePgb and MacPgb protein variants would be seen in protoglobin homologs and that the homologous proteins would provide additional starting points for directed evolution of carbene transferases.

To build a set of homologous proteins, I used Protein-BLAST to search by protein sequence identity to assemble a set of representative proteins from the protoglobin fold class, which has been identified in archaea and bacteria. Of this list, proteins from thermophilic organisms were selected; proteins from thermophilic origins are (usually) more stable than their mesophilic orthologs, and this stability helps counteract the destabilizing nature of most mutations.⁷⁵ The amino-acid residues mutated for DTFE cyclopropanation (W59, Y60, and F73) were conserved in all homologous protoglobins found, as were most first-shell active- site residues which would be prime targets for site-saturation mutagenesis. I will refer to these homologous protoglobins in general as XxxPgb. I ordered nine additional protoglobin sequences as linear DNA fragments (gBlocks, IDT) codon-optimized for E. coli and containing the LQ mutations (Table 2-5), and the oligonucleotides required to generate the GW variants using XxxPgb LQ genes as templatesThese XxxPgbs have between 51% and 83% pairwise amino-acid sequence identity (Figure 2-14).

Table 2-5. List of protoglobin proteins selected for the transfer of mutation experiment. The table lists the protoglobin names, the originating organism, and Uniprot ID or NCBI accession number.

ApePgb, MacPgb, and PfePgb were ordered and cloned prior to this gene acquisition.

Protein Organism UniProt ID

AauPgb Acetothermus autotrophicum H5SUA1 CthPgb Crenotalea thermophila A0A1I7NC60 ParPgb Pyrobaculum arsenaticum A4WIC7 PmePgb Pyrinomonas methylaliphatogenes A0A0B6WXB4

TamPgb Thermus amyloliquefaciens WP_038057460.1 (NCBI)

TarPgb Thermus arciformis A0A1G7GW55

TdaPgb Thermanaerothrix daxensis A0A0P6XZU8 ThuPgb Thermoflexus hugenholtzii A0A212QV80

TpePgb Thermorudis peleae A0A1E5BNX2

ApePgb Aeropyrum pernix Q9YFF4

MacPgb Methanosarcina acetivorans Q8TLY9 PfePgb Pyrobaculum ferrireducens G7VHJ7

Figure 2-14. Protein identity matrix showing the amino-acid sequence identity correlations for the various XxxPgbs. The lowest and highest sequence identities are CthPgb – ApePgb (51.8%) and PfePgb – ParPgb (82.7%).

Together with a Master’s student mentee, Lucas Schaus, I subcloned these new genes into the pET22b protein expression vector. The resulting XxxPgb LQ variant plasmids were used as PCR templates to generate the XxxPgb GW variants. E. coli competent cells were then transformed with the plasmids encoding both sets of protein variants to produce the proteins of interest. The biocatalysts were tested for their activity with DTFE against an array of activated, unactivated, and electron-deficient alkenes. Every successfully cloned XxxPgb

protein variant displayed carbene-transfer activity for most of the alkenyl substrates tested.

While ApePgb variants showed only trace activity the vinyl Weinreb amide substrate N- methoxy-N-methylacrylamide, variants of TarPgb and TdaPgb had high activity and diastereoselectivity against this substrate (Table 2-6). Weinreb amides are useful substrates as the corresponding products can be easily derivatized to generate myriad ketone-substituted cyclopropane products. These TarPgb and TdaPgb variants are therefore excellent starting points for directed evolution for biocatalytic synthesis and derivatizations to synthesize diverse trifluoromethyl-cyclopropylketones. Expansion of this reaction scope to include 2- substituted N-methoxy-N-methylacrylamides would enable the synthesis of trifluoromethyl- substituted cyclopropylketones with stereogenic quaternary carbons (Figure 2-15).

Following his Master’s thesis work, Lucas joined the Arnold laboratory as a graduate student and has continued this work.

Table 2-6. Activity and diastereoselectivity of TarPgb and TdaPgb wild type and variants in the synthesis of N-methoxy-N-methyl-2-(trifluoromethyl)cyclopropane-1-carboxamide. Activities were determined via NMR, using fluorobenzene as an internal standard. NMR data were collected by Lucas Schaus.

Protoglobin Yield TTN dr

TarPgb WT 21.5 % 600 36.3 %

TarPgb GW 21.8 % 2000 96.0 %

TdaPgb WT 17.6 % 1200 38.4 %

TdaPgb LQ 20.5 % 1800 93.9 %

Figure 2-15. Synthesis of Weinreb amide-substituted cyclopropanes. Carbene transfer to substituted N-methoxy-N-methylacrylamide forms a cyclopropane with a stereogenic quaternary carbon center. This compound can be further derivatized with Weinreb ketone synthesis to generate myriad carbonyl-substituted cyclopropanes.