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SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis

Nature volume 549, pages 502–506 (2017)Cite this article

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Abstract

Pericyclic reactions—which proceed in a concerted fashion through a cyclic transition state—are among the most powerful synthetic transformations used to make multiple regioselective and stereoselective carbon–carbon bonds1. They have been widely applied to the synthesis of biologically active complex natural products containing contiguous stereogenic carbon centres2,3,4,5,6. Despite the prominence of pericyclic reactions in total synthesis, only three naturally existing enzymatic examples (the intramolecular Diels–Alder reaction7, and the Cope8 and the Claisen rearrangements9) have been characterized. Here we report a versatile S-adenosyl-l-methionine (SAM)-dependent enzyme, LepI, that can catalyse stereoselective dehydration followed by three pericyclic transformations: intramolecular Diels–Alder and hetero-Diels–Alder reactions via a single ambimodal transition state, and a retro-Claisen rearrangement. Together, these transformations lead to the formation of the dihydropyran core of the fungal natural product, leporin10. Combined in vitro enzymatic characterization and computational studies provide insight into how LepI regulates these bifurcating biosynthetic reaction pathways by using SAM as the cofactor. These pathways converge to the desired biosynthetic end product via the (SAM-dependent) retro-Claisen rearrangement catalysed by LepI. We expect that more pericyclic biosynthetic enzymatic transformations remain to be discovered in naturally occurring enzyme ‘toolboxes’11. The new role of the versatile cofactor SAM is likely to be found in other examples of enzyme catalysis.

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Figure 1: Enzyme-catalysed pericyclic reactions and the proposed naturally occurring inverse electron demand hetero-Diels-Alder (HDA) reactions.
Figure 2: HPLC analysis showing the reactions catalysed by LepI.
Figure 3: LepI-catalysed reactions are SAM-dependent.
Figure 4: Energetics and transition states of the ambimodal IMDA/HDA, and retro-Claisen pericyclic reactions leading to formation of 2 from 4.

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Acknowledgements

This work was supported by the NIH (1DP1GM106413 and 1R35GM118056), the NSF (CHE-1361104 to K.N.H.), and the JSPS Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (G2604 to K.W.).

Author information

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, California, USA

    Masao Ohashi, Yang Hai, Mengbin Chen, Man-cheng Tang, Michio Sato, K. N. Houk & Yi Tang

  2. Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, 422-8526, Japan

    Masao Ohashi, Michio Sato & Kenji Watanabe

  3. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, USA

    Fang Liu, Zhongyue Yang, K. N. Houk & Yi Tang

Authors
  1. Masao Ohashi
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Contributions

M.O., F.L., Y.H., K.N.H. and Y.T. developed the hypothesis and designed the study. M.O. performed all in vivo and in vitro experiments, as well as compound isolation and characterization. M.O., Y.H. and M.C. performed protein purification. M.S. and M.-C.T. performed compound characterization. F.L., Z.Y. and K.N.H. performed the computational experiments. All authors analysed and discussed the results. M.O., F.L., K.W., K.N.H. and Y.T. prepared the manuscript.

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Correspondence to Kenji Watanabe, K. N. Houk or Yi Tang.

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Extended data figures and tables

Extended Data Figure 1 LC–MS analysis of the in vitro reaction of 3 with LepF.

The extracted ion chromatograms (EIC) under positive ionization are shown. The mass/charge (m/z) ratio of alcohols 4 and 4′ is 354 under positive ionization. Because the enzymatic activity of LepF is low and 4 is very unstable, we were not able to obtain enough 4 to use it as the substrate for in vitro reaction of LepI. Thus, we obtained 4 by reducing ketone 3 with NaBH4. Since this reduction proceeds non-stereoselectively, 4 and diastereomer 4′ were formed. After the isolation of 4 and 4′ by HPLC, the fractions containing 4 and 4′ were not concentrated and were immediately used as the substrate. The stereochemistry of the secondary alcohol in 4 and 4′ was not determined.

Extended Data Figure 2 HPLC analysis of the chemical reduction of 3 with NaBH4.

The reaction mixture containing 1 mM 3 and 10 mM NaBH4 with EtOH (50 μl) was incubated at 0 °C for 1 min, then the reaction was quenched with water. After centrifugation, the supernatant was analysed by HPLC. The reduction of 3 gave the alcohol 4 and diastereomer 4′. The spontaneous dehydration of both alcohols resulted in the formation of HDA and IMDA products via the E/Z mixture of quinone methide 5. The isolated 4 and 4′ also readily dehydrated and converted to a mixture of the desired HDA (2) and the undesired HDA (9) and IMDA (6–8) products, showing the instability of these compounds. The structures (right) show the relative stereochemistry.

Extended Data Figure 3 Reaction analysis of 6–9 under heating.

6 (dissolved in 5% DMSO with H2O) was heated at 95 °C for 1 h. 7–9 (dissolved in 5% DMSO with H2O) were heated at 95 °C for 10 h. 6 was completely converted to 2 via [3,3]-sigmatropic retro-Claisen rearrangement. This reaction is irreversible under these conditions. It should be noted that the conversion of 6 to 2 via cycloreversion can be ruled out, since 6 was completely converted to 2 without any other IMDA/HDA side products. No reactions occurred in the case of 7. 8 and 9 can be interconverted via Claisen rearrangement. In this case, retro-Claisen rearrangement (8 to 9) is preferable to forward Claisen rearrangement (9 to 8). The structures show the relative stereochemistry.

Extended Data Figure 4 Analysis of the substrate specificity of LepI.

a, In vitro reactions of other IMDA products 7–9 with 30 μM LepI for 12 h. (i) 8 in buffer, (ii) 8 with LepI, (iii) 7 in buffer, (iv) 7 with LepI, (v) 9 in buffer, (vi) 9 with LepI. The experimental details are described in Methods. b, Elucidation of inhibitory activity of 7 on LepI-catalysed retro-Claisen rearrangement of 6 to 2. The experimental details are described in Methods. The IC50 value is mean ± standard deviation (s.d.) of three independent experiments. The structures show the relative stereochemistry.

Extended Data Figure 5 Time-course analysis of the LepI-catalysed retro-Claisen rearrangement of 6 to 2.

The experimental details are described in Methods. The data show one representative experiment from at least three independent replicates.

Extended Data Figure 6 HPLC analysis showing that purified LepI retains SAM.

SAM was detected in the supernatant of denatured (by acetonitrile) LepI. When LepI was denatured by heating the sample at 95 °C for 10 min, a single peak corresponding to 5′-deoxy-5′-(methylthio)adenosine (MTA), a major degradation product of SAM25, was detected from the supernatant of boiled LepI. Since SAM to MTA conversion is nearly quantitative and an MTA standard curve can be readily constructed, we found that about 90% of LepI still retains SAM after purification. Shown are HPLC profiles of (i) LepI denatured by acetonitrile, (ii) LepI heated at 95 °C for 10 min, (iii) the authentic reference of SAM, (iv) SAM heated at 95 °C for 10 min, and (v) the authentic reference of MTA. The experimental details are described in Methods.

Extended Data Figure 7 HPLC analysis of SAM-dependent LepI-catalysed reactions.

a, Analysis of in vitro reaction of 240 μM 4 with 300 nM LepI at 30 °C for 5 min in the presence and absence of cofactors. The concentrations of SAH, SAM and sinefungin used in this experiment are 250 μM, 100 μM and 100 μM, respectively. The data show one representative experiment from at least three independent replicates. b, Analysis of the in vitro reaction of 140 μM 6 with 300 nM LepI at 30 °C for 4 min in the presence and absence of cofactors. The concentrations of SAH, SAM and sinefungin used in this experiment are 250 μM, 100 μM and 100 μM, respectively. The data show one representative experiment from at least three independent replicates.

Extended Data Figure 8 SAH is a competitive inhibitor of LepI retro-Claisen rearrangement.

a, Dose-dependent inhibition of retro-Claisen rearrangement by SAH. b, Dose-dependent recovery of retro-Claisen rearrangement by SAM in the presence of 250 μM SAH. The experimental details are described in Methods. Error bars, s.d. of three independent experiments.

Extended Data Figure 9 Time-course analysis of the production of 2 divided by the sum of the production of 2 and 6.

The substrate used in this study is alcohol 4. a, LepI-catalysed reaction with or without SAH (250 μM). b, Non-enzymatic reaction. The initial production ratio (IMDA (6) versus HDA products (2)) between LepI-catalysed (about 1:1 periselectivity) and non-catalysed reactions (about 94:6 periselectivity) are clearly different. These data support the suggestion that LepI catalyses the competitive IMDA/HDA reactions by changing the product distribution resulting from IMDA versus HDA reactions.

Extended Data Figure 10 Calculated free energies and bond distances.

Data are shown for the ambimodal transition state (TS-1) and the transition state for the retro-Claisen rearrangement (TS-2), uncatalysed and with various catalysts, calculated with B3LYP-D3/6-311+G(d,p)//6-31G(d), CPCM water. Positions of the bonds are shown in the structures above.

Supplementary information

Supplementary Information

This file contains Supplementary Methods 1-4, Supplementary Tables 1-9, Supplementary Figures 1-32, Supplementary Data and additional references. (PDF 17133 kb)

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Ohashi, M., Liu, F., Hai, Y. et al. SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis. Nature 549, 502–506 (2017). https://doi.org/10.1038/nature23882

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