Intramolecular C(sp3)H amination of arylsulfonyl azides with engineered and artificial myoglobin-based catalysts

Abstract

The direct conversion of aliphatic C—H bonds into C—N bonds provides an attractive approach to the introduction of nitrogen-containing functionalities in organic molecules. Following our recent discovery that cytochrome P450 enzymes can catalyze the cyclization of arylsulfonyl azide compounds via an intramolecular C(sp3)—H amination reaction, we have explored here the C—H amination reactivity of other hemoproteins. Various heme-containing proteins, and in particular myoglobin and horseradish peroxidase, were found to be capable of catalyzing this transformation. Based on this finding, a series of engineered and artificial myoglobin variants containing active site mutations and non-native Mn- and Co-protoporphyrin IX cofactors, respectively, were preparedWmvestigate the effect of these structural changes on the catalytic activity and selectivity of to these catalysts. Our studies showed that metallo-substituted myoglobins constitute viable C—H amination catalysts, revealing a distinctive reactivity trend as compared to synthetic metalloporphyrin counterparts. On the other hand, amino acid substitutions at the level of the heme pocket were found to be beneficial toward improving the stereo- and enantioselectivity of these Mb-catalyzed reactions. Mechanistic studies involving kinetic isotope effect experiments indicate that C—H bond cleavage is implicated in the rate-limiting step of myoglobin-catalyzed amination of arylsulfonyl azides. Altogether, these studies indicate that myoglobin constitutes a promising scaffold for the design and development of C— H amination catalysts.

Gram‐Scale Synthesis of Chiral Cyclopropane‐Containing Drugs and Drug Precursors with Engineered Myoglobin Catalysts Featuring Complementary Stereoselectivity

“…The biocatalytic systems developed here have enabled the stereoselective synthesis of multiple cyclopropane-containing drugs at the preparative scale, offering superior performance over currently available methods for asymmetric cyclopropanation (i.e., (+)-and (−)-16, 20, and 24) or granting a more concise route to their preparation (28). Along with the growing number of abiotic reactions accessible using engineered myoglobins, [4,[9][10]19] these results support the promise of these metalloproteins for the asymmetric synthesis of chiral drugs and synthons at a practical scale. …”

“…Previously, we found that mutations at the five amino acid positions defining the distal pocket in Mb (i.e., Leu29, Phe43, His64, Val68, and Ile107; Figure S1) significantly affected the activity and selectivity of this hemoprotein in carbene [4, 9] and nitrene [10] transfer reactions. In particular, mutation of the distal histidine residue (H64V) was determined to have a general activity enhancing effect in these reactions, possibly due to an increased accessibility of the heme pocket to the reactants.…”

“…We further demonstrate that these Mb-catalyzed reactions can be carried out and scaled up using whole cell systems. Using these stereocomplementary biocatalysts in combination with whole-cell transformations, it was possible to realize the asymmetric synthesis of the cyclopropane core of four different drugs, featuring both a trans-(1S,2S) and a trans-(1R,2R) configuration, at the multigram scale.Previously, we found that mutations at the five amino acid positions defining the distal pocket in Mb (i.e., Leu29, Phe43, His64, Val68, and Ile107; Figure S1) significantly affected the activity and selectivity of this hemoprotein in carbene [4, 9] and nitrene [10] transfer reactions. In particular, mutation of the distal histidine residue (H64V) was determined to have a general activity enhancing effect in these reactions, possibly due to an increased accessibility of the heme pocket to the reactants.…”

Advances in Engineered Hemoproteins that Promote Biocatalysis

“…Although less efficient than its manganese substituted myoglobin counterpart, substitution with Co(II) exhibits enhanced catalytic activity in comparison to its iron counterpart showing 64 TTN (total turnover numbers) also representing a case where the protein matrix provides significant activity enhancement since free Co(II)PPIX shows a TTN of five [41].…”

Genetic Optimization of Metalloenzymes: Enhancing Enzymes for Non‐Natural Reactions

“…[35] Mutation of the conserved distal histidine to valine (H64V) provides am odest increase in the catalyst activity. Thee nantioselectivity of the insertion can be modulated through modification of the active site.Ingeneral, myoglobin is less effective for reactions involving nitrenoid species than carbenoid species.T oaddress this issue,Fasan and co-workers explored exchanging the heme cofactor by Mn and Co hemin species,w hich are known to be more effective for amination under organic conditions.U nfortunately,t hey were less effective that Fe hemin.…”