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Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation.

Srivastava P, Yang H, Ellis-Guardiola K, Lewis JC - Nat Commun (2015)

Bottom Line: The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water.This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction.Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

View Article: PubMed Central - PubMed

Affiliation: Coskata Inc. 4575 Weaver Parkway, Warrenville, IL 60555, USA.

ABSTRACT
Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkyne-substituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor-acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

No MeSH data available.


CD spectra for POP variants and ArMs.(a) Comparing different constructs (10 μM). (b) CD spectra of POP-ZA4-HFF acquired at 10 °C intervals from 50 to 100 °C (see also Supplementary Fig. 7).
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f4: CD spectra for POP variants and ArMs.(a) Comparing different constructs (10 μM). (b) CD spectra of POP-ZA4-HFF acquired at 10 °C intervals from 50 to 100 °C (see also Supplementary Fig. 7).

Mentions: The unique catalytic properties of POP-ZA4-HFF-1 result from the introduction of eight mutations and dirhodium cofactor 1 into the interior of the POP scaffold, far more mutations than described in most ArM efforts7. Despite these perturbations, essentially identical circular dichroism (CD) spectra were obtained for several POP variants and POP-ZA4-HFF-1, suggesting little difference in secondary structures of these proteins (Fig. 4a)35. Remarkably, the CD spectrum of POP-ZA4-HFF-1 remains unchanged up to 100 °C (Fig. 4b), indicating that the stability of POP itself is also not reduced to a relevant extent. This stability clearly highlights the utility of protein scaffolds from hyperthermophilic organisms that can form robust ArMs even when extensive mutagenesis is required to achieve high selectivity and will greatly facilitate further efforts to evolve ArMs derived from the POP scaffold36. Of the mutations introduced, L328H led to the largest improvements in both selectivity, conversion and activity (Table 1, entry 8). As previously noted, this mutation was introduced based on the improved selectivity of peptide-based dirhodium catalysts containing a histidine residue capable of coordinating to Rh33. It is important to note, however, that axial coordination of ligands to dirhodium complexes in peptide and small molecule catalysts typically leads to decreased activity33. Given the difference in the effects of histidine incorporation into peptide catalysts and POP-ZA4-HFF-1, several additional ArM variants were examined to clarify the role of H328 in POP-ZA4-HFF-1 (Table 1, entries 4–8). First, POP-ZA4-L328F-1 was prepared to examine the impact of a non-coordinating aromatic residue at position 328. The L328F variant possesses significantly lower selectivity than the L328H variant, suggesting that purely steric factors are not responsible for the improved selectivity of the latter. In addition, the L328M and L328C variants show that other residues capable of coordinating to Rh also improve ArM selectivity. The structural differences between histidine, methionine and cysteine suggest that their common metal-coordinating ability is responsible for the improved selectivity ArMs containing these residues, including POP-ZA4-HFF-1. Initial attempts to characterize histidine coordination to 1 in this ArM via NMR spectroscopy3738 and ultraviolet–vis spectroscopy333940 have been complicated by the high molecular weight of POP (ca. 70 kDa) and the weak absorbance associated with the diagnostic Rh–Rh π*–σ* transition41 in 1, respectively. Further spectroscopic and crystallographic analysis of this ArM is underway to rigorously characterize the nature of cofactor binding within its active site and thus provide a mechanistic rationale for its high selectivity and improved specificity.


Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation.

Srivastava P, Yang H, Ellis-Guardiola K, Lewis JC - Nat Commun (2015)

CD spectra for POP variants and ArMs.(a) Comparing different constructs (10 μM). (b) CD spectra of POP-ZA4-HFF acquired at 10 °C intervals from 50 to 100 °C (see also Supplementary Fig. 7).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4525152&req=5

f4: CD spectra for POP variants and ArMs.(a) Comparing different constructs (10 μM). (b) CD spectra of POP-ZA4-HFF acquired at 10 °C intervals from 50 to 100 °C (see also Supplementary Fig. 7).
Mentions: The unique catalytic properties of POP-ZA4-HFF-1 result from the introduction of eight mutations and dirhodium cofactor 1 into the interior of the POP scaffold, far more mutations than described in most ArM efforts7. Despite these perturbations, essentially identical circular dichroism (CD) spectra were obtained for several POP variants and POP-ZA4-HFF-1, suggesting little difference in secondary structures of these proteins (Fig. 4a)35. Remarkably, the CD spectrum of POP-ZA4-HFF-1 remains unchanged up to 100 °C (Fig. 4b), indicating that the stability of POP itself is also not reduced to a relevant extent. This stability clearly highlights the utility of protein scaffolds from hyperthermophilic organisms that can form robust ArMs even when extensive mutagenesis is required to achieve high selectivity and will greatly facilitate further efforts to evolve ArMs derived from the POP scaffold36. Of the mutations introduced, L328H led to the largest improvements in both selectivity, conversion and activity (Table 1, entry 8). As previously noted, this mutation was introduced based on the improved selectivity of peptide-based dirhodium catalysts containing a histidine residue capable of coordinating to Rh33. It is important to note, however, that axial coordination of ligands to dirhodium complexes in peptide and small molecule catalysts typically leads to decreased activity33. Given the difference in the effects of histidine incorporation into peptide catalysts and POP-ZA4-HFF-1, several additional ArM variants were examined to clarify the role of H328 in POP-ZA4-HFF-1 (Table 1, entries 4–8). First, POP-ZA4-L328F-1 was prepared to examine the impact of a non-coordinating aromatic residue at position 328. The L328F variant possesses significantly lower selectivity than the L328H variant, suggesting that purely steric factors are not responsible for the improved selectivity of the latter. In addition, the L328M and L328C variants show that other residues capable of coordinating to Rh also improve ArM selectivity. The structural differences between histidine, methionine and cysteine suggest that their common metal-coordinating ability is responsible for the improved selectivity ArMs containing these residues, including POP-ZA4-HFF-1. Initial attempts to characterize histidine coordination to 1 in this ArM via NMR spectroscopy3738 and ultraviolet–vis spectroscopy333940 have been complicated by the high molecular weight of POP (ca. 70 kDa) and the weak absorbance associated with the diagnostic Rh–Rh π*–σ* transition41 in 1, respectively. Further spectroscopic and crystallographic analysis of this ArM is underway to rigorously characterize the nature of cofactor binding within its active site and thus provide a mechanistic rationale for its high selectivity and improved specificity.

Bottom Line: The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water.This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction.Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

View Article: PubMed Central - PubMed

Affiliation: Coskata Inc. 4575 Weaver Parkway, Warrenville, IL 60555, USA.

ABSTRACT
Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkyne-substituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor-acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

No MeSH data available.