Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Aug 22;140(33):10464-10472.
doi: 10.1021/jacs.8b04742. Epub 2018 Aug 13.

Overriding Traditional Electronic Effects in Biocatalytic Baeyer-Villiger Reactions by Directed Evolution

Guangyue Li  1   2   3 , Marc Garcia-Borràs  4 , Maximilian J L J Fürst  5 , Adriana Ilie  2   3 , Marco W Fraaije  5 , K N Houk  4 , Manfred T Reetz  2   3
Affiliations

Overriding Traditional Electronic Effects in Biocatalytic Baeyer-Villiger Reactions by Directed Evolution

Guangyue Li et al. J Am Chem Soc. .

Abstract

Controlling the regioselectivity of Baeyer-Villiger (BV) reactions remains an ongoing issue in organic chemistry, be it by synthetic catalysts or enzymes of the type Baeyer-Villiger monooxygenases (BVMOs). Herein, we address the challenging problem of switching normal to abnormal BVMO regioselectivity by directed evolution using three linear ketones as substrates, which are not structurally biased toward abnormal reactivity. Upon applying iterative saturation mutagenesis at sites lining the binding pocket of the thermostable BVMO from Thermocrispum municipale DSM 44069 (TmCHMO) and using 4-phenyl-2-butanone as substrate, the regioselectivity was reversed from 99:1 (wild-type enzyme in favor of the normal product undergoing 2-phenylethyl migration) to 2:98 in favor of methyl migration when applying the best mutant. This also stands in stark contrast to the respective reaction using the synthetic reagent m-CPBA, which provides solely the normal product. Reversal of regioselectivity was also achieved in the BV reaction of two other linear ketones. Kinetic parameters and melting temperatures revealed that most of the evolved mutants retained catalytic activity, as well as thermostability. In order to shed light on the origin of switched regioselectivity in reactions of 4-phenyl-2-butanone and phenylacetone, extensive QM/MM and MD simulations were performed. It was found that the mutations introduced by directed evolution induce crucial changes in the conformation of the respective Criegee intermediates and transition states in the binding pocket of the enzyme. In mutants that destabilize the normally preferred migration transition state, a reversal of regioselectivity is observed. This conformational control of regioselectivity overrides electronic control, which normally causes preferential migration of the group that is best able to stabilize positive charge. The results can be expected to aid future protein engineering of BVMOs.

PubMed Disclaimer

Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
TmCHMO structure model showing docked 4-phenyl-butan-2-one (1) (in cyan) based on the crystal structure of wild-type TmCHMO (PDB code 5M10), and the 11 CAST amino acids lining 1 displayed in purple. Green: NADP+; Yellow: FAD.
Figure 2.
Figure 2.
Iterative saturation mutagenesis (ISM) exploration of TmCHMO as catalyst in the reactions with focus on reversal of regioselectivity in favor of the abnormal product using substrate 1 (a), 4 (b), or 7 (c).
Figure 3.
Figure 3.
Analysis of 1-R-Criegee intermediate conformations though 500 ns MD simulations when bound in the (a) WT enzyme; and (b) LGY3-D-E1 variant. Black symbols denote selected snapshots used for further analysis in Figure 4.
Figure 4.
Figure 4.
Active site arrangement in selected snapshots obtained from 500 ns MD trajectories of the 1-R-Criegee intermediate bound into the (a) WT enzyme (snapshot at 400 ns in gray); and (b) LGY3-D-E1 variant (snapshots at 100 ns in purple, and 300 ns in orange). The active sites are shown from the same perspective. Substrate 1 in the 1-R-Criegee intermediate is shown in blue. (c) Superimposition of the WT (snapshot at 400 ns in gray) and LGY3-D-E1 (snapshot at 300 ns in orange) 1-R-Criegee intermediate bound active sites. QM/MM optimized transition state geometries (only atoms included in the QM-region are shown) for selected snapshots obtained from (d) WT 1-TS-normal phenylethyl migration (snapshot at 400 ns); (e) LGY3-D-E1 1-TS-normal phenylethyl migration (snapshot at 100 ns); and (f) LGY3-D-E1 1-TS-abnormal methyl migration (snapshot at 300 ns). Energies are given in kcal·mol−1, distances in Å, and O(peroxy1)−O(peroxy2)−C(carbonyl)−C(migrating) dihedral angles in degrees. (g) Superimposition of the 1-R-Criegee intermediate QM/MM optimized 1-TS-normal structures (only Criegee intermediate atoms are shown) in the WT (snapshot at 400 ns in gray) and LGY3-D-E1 (snapshot at 100 ns in purple).
Scheme 1.
Scheme 1.
Baeyer−Villiger Reaction of Unsymmetrical Acyclic Ketones with Potential Formation of Two Different Esters
Scheme 2.
Scheme 2.
Baeyer−Villiger Oxidation of Model Ketones 1, 4, and 7 Catalyzed by TmCHMO Used in This Study
See this image and copyright information in PMC

References

    1. Baeyer A; Villiger V Ber. Dtsch. Chem. Ges 1899, 32, 3625–3633.
    1. Reviews of Baeyer−Villiger reactions: (a) Krow GR Org. React 1993, 43, 251–256.
    2. (b) Renz M; Meunier B Eur. J. Org. Chem 1999, 4, 737–750.
    3. (c) Ten Brink G-J; Arends IWCE; Sheldon RA Chem. Rev 2004, 104, 4105–4124. - PubMed
    4. (d) Mihovilovic MD; Rudroff F; Grötzl B Curr. Org. Chem 2004, 8, 1057–1069.
    5. (e) Jiménez-Sanchidriań C; Ruiz JR Tetrahedron 2008, 64, 2011–2026.
    6. (f) Uyanik M; Ishihara K ACS Catal 2013, 3, 513–520.
    1. Asymmetric BV reactions using synthetic transition metal catalysts: (a) Bolm C; Schlingloff G; Weickhardt K Angew. Chem., Int. Ed. Engl 1994, 33, 1848–1849.
    2. (b) Ito K; Ishii A; Kuroda T; Katsuki T Synlett 2003, 643–646.
    3. (c) Strukul G Angew. Chem., Int. Ed 1998, 37, 1198–1209. - PubMed
    4. (d) Frison J-C; Palazzi C; Bolm C Tetrahedron 2006, 62, 6700–6706.
    5. (e) Malkov AV; Friscourt F; Bell M; Swarbrick ME; Kocovsky P J. Org. Chem 2008, 73, 3996–4003. - PubMed
    6. (f) Zhou L; Liu X; Ji J; Zhang Y; Hu X; Lin L; Feng XJ Am. Chem. Soc 2012, 134, 17023–17026. - PubMed
    7. (g) Zhou L; Liu X; Ji J; Zhang Y; Wu W; Liu Y; Lin L; Feng X Org. Lett 2014, 16, 3938–3941. - PubMed
    8. (h) Poudel PP; Arimitsu K; Yamamoto K Chem. Commun 2016, 52, 4163–4166. - PubMed
    9. (i) Russo A; De Fusco C; Lattanzi A ChemCatChem 2012, 4, 901–916.
    1. Asymmetric BV reactions using chiral organocatalysts: (a) Murahashi S; Ono S; Imada Y Angew. Chem., Int. Ed 2002, 41, 2366–2370. - PubMed
    2. (b) Imada Y; Iida H; Murahashi SI; Naota T Angew. Chem., Int. Ed 2005, 44, 1704–1706. - PubMed
    3. (c) Wang B; Shen YM; Shi YJ Org. Chem 2006, 71, 9519–9521. - PubMed
    4. (d) Peris G; Miller SJ Org. Lett 2008, 10, 3049–3052. - PMC - PubMed
    5. (e) Xu S; Wang Z; Zhang X; Ding K Angew. Chem., Int. Ed 2008, 47, 2840–2843. - PubMed
    6. (f) Abascal NC; Miller SJ Org. Lett 2016, 18, 4646–4649. - PMC - PubMed
    1. (a) Yadav VK Steric and Stereoelectronic Effects in Organic Chemistry; Springer, 2016.
    2. (b) Alabugin IV Stereoelectronic Effects: A Bridge between Structure and Reactivity; John Wiley & Sons, 2016.
    3. (c) Deslongchamps P Stereoelectronic Effects in Organic Chemistry; Pergamon Press: New York, 1983.

Publication types