Tuning the regio- and stereoselectivity of C-H activation in n-octanes by cytochrome P450 BM-3 with fluorine substituents: Evidence for interactions between a C-F bond and aromatic π systems

Li Lan Wu, Chung Ling Yang, Feng Chun Lo, Chih Hsiang Chiang, Chun Wei Chang, Kok Yaoh Ng, Ho-Hsuan Chou, Huei Ying Hung, Sunney I. Chan, Steve S.F. Yu

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

We employed the water- soluble cytochrome P450 BM-3 to study the activity and regiospecificity of oxidation of fluorinated n-octanes. Three mutations, A74G, F87V, and L188Q, were introduced into P450 BM-3 to allow the system to undergo n-octane oxidation. In addition, the alanine at residue 328 was replaced with a phenylalanine to introduce an aromatic residue into the hydrophobic pocket to examine whether or not van der Waals interactions between a C-F substituent in the substrate and the polarizable π system of the phenylalanine may be used to steer the positioning of the substrate within the active-site pocket of the enzyme and control the regioselectivity and stereoselectivity of hydroxylation. Interestingly, not only was the regioselectivity controlled when the fluorine substituent was judiciously positioned in the substrate, but the electron input into the iron-heme group became tightly coupled to the formation of product, essentially without abortive side reactions. Remarkable enhancement of the coupling efficiency between electron input and product formation was observed for a range of fluorinated octanes in the enzyme even without the A328F mutation, presumably because of interactions of the C-F substituent with the π system of the porphyrin macrocycle within the active-site pocket. Evidently, tightening the protein domain containing the heme pocket tunes the distribution of accessible enzyme conformations and the associated protein dynamics that activate the iron porphyrin for substrate hydroxylation to allow the reactions mediated by the high-valent FeIV=O to become kinetically more commensurate with electron transfer from the flavin adenine dinucleotide (FAD)/flavin mononucleotide (FMN) reductase. These observations lend compelling evidence to support significant van der Waals interactions between the CF2 group and aromatic π systems within the heme pocket when the fluorinated octane substrate is bound. Activation of F-octanes: Cytochrome P450 BM-3-A74GF87VL188Q (see figure) and the A328F variant regionselectively converted fluorinated C8 alkanes to the corresponding secondary alcohols. The pattern of reactivity, especially the unprecedented regio- and stereoselectivity, observed for 4,4-difluorooctane suggested that specific interactions of the fluorinated substituent with aromatic π systems within the active site could tune the reactivity.

Original languageEnglish
Pages (from-to)4774-4787
Number of pages14
JournalChemistry - A European Journal
Volume17
Issue number17
DOIs
Publication statusPublished - 2011 Apr 18

Fingerprint

Stereoselectivity
Regioselectivity
Fluorine
Cytochrome P-450 Enzyme System
Tuning
Chemical activation
Octanes
Heme
Substrates
Hydroxylation
Enzymes
Porphyrins
Phenylalanine
Electrons
FMN Reductase
Iron
Proteins
Oxidation
Flavin-Adenine Dinucleotide
Alkanes

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Organic Chemistry

Cite this

Wu, Li Lan ; Yang, Chung Ling ; Lo, Feng Chun ; Chiang, Chih Hsiang ; Chang, Chun Wei ; Ng, Kok Yaoh ; Chou, Ho-Hsuan ; Hung, Huei Ying ; Chan, Sunney I. ; Yu, Steve S.F. / Tuning the regio- and stereoselectivity of C-H activation in n-octanes by cytochrome P450 BM-3 with fluorine substituents : Evidence for interactions between a C-F bond and aromatic π systems. In: Chemistry - A European Journal. 2011 ; Vol. 17, No. 17. pp. 4774-4787.
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abstract = "We employed the water- soluble cytochrome P450 BM-3 to study the activity and regiospecificity of oxidation of fluorinated n-octanes. Three mutations, A74G, F87V, and L188Q, were introduced into P450 BM-3 to allow the system to undergo n-octane oxidation. In addition, the alanine at residue 328 was replaced with a phenylalanine to introduce an aromatic residue into the hydrophobic pocket to examine whether or not van der Waals interactions between a C-F substituent in the substrate and the polarizable π system of the phenylalanine may be used to steer the positioning of the substrate within the active-site pocket of the enzyme and control the regioselectivity and stereoselectivity of hydroxylation. Interestingly, not only was the regioselectivity controlled when the fluorine substituent was judiciously positioned in the substrate, but the electron input into the iron-heme group became tightly coupled to the formation of product, essentially without abortive side reactions. Remarkable enhancement of the coupling efficiency between electron input and product formation was observed for a range of fluorinated octanes in the enzyme even without the A328F mutation, presumably because of interactions of the C-F substituent with the π system of the porphyrin macrocycle within the active-site pocket. Evidently, tightening the protein domain containing the heme pocket tunes the distribution of accessible enzyme conformations and the associated protein dynamics that activate the iron porphyrin for substrate hydroxylation to allow the reactions mediated by the high-valent FeIV=O to become kinetically more commensurate with electron transfer from the flavin adenine dinucleotide (FAD)/flavin mononucleotide (FMN) reductase. These observations lend compelling evidence to support significant van der Waals interactions between the CF2 group and aromatic π systems within the heme pocket when the fluorinated octane substrate is bound. Activation of F-octanes: Cytochrome P450 BM-3-A74GF87VL188Q (see figure) and the A328F variant regionselectively converted fluorinated C8 alkanes to the corresponding secondary alcohols. The pattern of reactivity, especially the unprecedented regio- and stereoselectivity, observed for 4,4-difluorooctane suggested that specific interactions of the fluorinated substituent with aromatic π systems within the active site could tune the reactivity.",
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Tuning the regio- and stereoselectivity of C-H activation in n-octanes by cytochrome P450 BM-3 with fluorine substituents : Evidence for interactions between a C-F bond and aromatic π systems. / Wu, Li Lan; Yang, Chung Ling; Lo, Feng Chun; Chiang, Chih Hsiang; Chang, Chun Wei; Ng, Kok Yaoh; Chou, Ho-Hsuan; Hung, Huei Ying; Chan, Sunney I.; Yu, Steve S.F.

In: Chemistry - A European Journal, Vol. 17, No. 17, 18.04.2011, p. 4774-4787.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Tuning the regio- and stereoselectivity of C-H activation in n-octanes by cytochrome P450 BM-3 with fluorine substituents

T2 - Evidence for interactions between a C-F bond and aromatic π systems

AU - Wu, Li Lan

AU - Yang, Chung Ling

AU - Lo, Feng Chun

AU - Chiang, Chih Hsiang

AU - Chang, Chun Wei

AU - Ng, Kok Yaoh

AU - Chou, Ho-Hsuan

AU - Hung, Huei Ying

AU - Chan, Sunney I.

AU - Yu, Steve S.F.

PY - 2011/4/18

Y1 - 2011/4/18

N2 - We employed the water- soluble cytochrome P450 BM-3 to study the activity and regiospecificity of oxidation of fluorinated n-octanes. Three mutations, A74G, F87V, and L188Q, were introduced into P450 BM-3 to allow the system to undergo n-octane oxidation. In addition, the alanine at residue 328 was replaced with a phenylalanine to introduce an aromatic residue into the hydrophobic pocket to examine whether or not van der Waals interactions between a C-F substituent in the substrate and the polarizable π system of the phenylalanine may be used to steer the positioning of the substrate within the active-site pocket of the enzyme and control the regioselectivity and stereoselectivity of hydroxylation. Interestingly, not only was the regioselectivity controlled when the fluorine substituent was judiciously positioned in the substrate, but the electron input into the iron-heme group became tightly coupled to the formation of product, essentially without abortive side reactions. Remarkable enhancement of the coupling efficiency between electron input and product formation was observed for a range of fluorinated octanes in the enzyme even without the A328F mutation, presumably because of interactions of the C-F substituent with the π system of the porphyrin macrocycle within the active-site pocket. Evidently, tightening the protein domain containing the heme pocket tunes the distribution of accessible enzyme conformations and the associated protein dynamics that activate the iron porphyrin for substrate hydroxylation to allow the reactions mediated by the high-valent FeIV=O to become kinetically more commensurate with electron transfer from the flavin adenine dinucleotide (FAD)/flavin mononucleotide (FMN) reductase. These observations lend compelling evidence to support significant van der Waals interactions between the CF2 group and aromatic π systems within the heme pocket when the fluorinated octane substrate is bound. Activation of F-octanes: Cytochrome P450 BM-3-A74GF87VL188Q (see figure) and the A328F variant regionselectively converted fluorinated C8 alkanes to the corresponding secondary alcohols. The pattern of reactivity, especially the unprecedented regio- and stereoselectivity, observed for 4,4-difluorooctane suggested that specific interactions of the fluorinated substituent with aromatic π systems within the active site could tune the reactivity.

AB - We employed the water- soluble cytochrome P450 BM-3 to study the activity and regiospecificity of oxidation of fluorinated n-octanes. Three mutations, A74G, F87V, and L188Q, were introduced into P450 BM-3 to allow the system to undergo n-octane oxidation. In addition, the alanine at residue 328 was replaced with a phenylalanine to introduce an aromatic residue into the hydrophobic pocket to examine whether or not van der Waals interactions between a C-F substituent in the substrate and the polarizable π system of the phenylalanine may be used to steer the positioning of the substrate within the active-site pocket of the enzyme and control the regioselectivity and stereoselectivity of hydroxylation. Interestingly, not only was the regioselectivity controlled when the fluorine substituent was judiciously positioned in the substrate, but the electron input into the iron-heme group became tightly coupled to the formation of product, essentially without abortive side reactions. Remarkable enhancement of the coupling efficiency between electron input and product formation was observed for a range of fluorinated octanes in the enzyme even without the A328F mutation, presumably because of interactions of the C-F substituent with the π system of the porphyrin macrocycle within the active-site pocket. Evidently, tightening the protein domain containing the heme pocket tunes the distribution of accessible enzyme conformations and the associated protein dynamics that activate the iron porphyrin for substrate hydroxylation to allow the reactions mediated by the high-valent FeIV=O to become kinetically more commensurate with electron transfer from the flavin adenine dinucleotide (FAD)/flavin mononucleotide (FMN) reductase. These observations lend compelling evidence to support significant van der Waals interactions between the CF2 group and aromatic π systems within the heme pocket when the fluorinated octane substrate is bound. Activation of F-octanes: Cytochrome P450 BM-3-A74GF87VL188Q (see figure) and the A328F variant regionselectively converted fluorinated C8 alkanes to the corresponding secondary alcohols. The pattern of reactivity, especially the unprecedented regio- and stereoselectivity, observed for 4,4-difluorooctane suggested that specific interactions of the fluorinated substituent with aromatic π systems within the active site could tune the reactivity.

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