Reaction Mechanism of a Nonheme Iron Enzyme Catalyzed Oxidative

Letter Cite This: Org. Lett. 2019, 21, 228−232

pubs.acs.org/OrgLett

Reaction Mechanism of a Nonheme Iron Enzyme Catalyzed Oxidative Cyclization via C−C Bond Formation Wei-chen Chang,*,† Zhi-Jie Yang,‡ Yueh-Hua Tu,‡ and Tun-Cheng Chien*,‡ †

Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States Department of Chemistry, National Taiwan Normal University, Taipei 11677, Taiwan

‡

Org. Lett. 2019.21:228-232. Downloaded from pubs.acs.org by TULANE UNIV on 01/09/19. For personal use only.

S Supporting Information *

ABSTRACT: A complementary study including design of mechanistic probes, biochemical assays, model analysis, and liquid chromatography coupled mass spectrometry was conducted to establish the reaction mechanism for a nonheme iron enzyme catalyzed (−)-podophyllotoxin formation. Our results indicate that the originally proposed hydroxylated intermediate is unlikely to be involved in this reaction. Instead, the formation of benzylic radical/ carbocation intermediate can be utilized to trigger the C−C bond formation to construct the C-ring of (−)-podophyllotoxin.

E

from the plant Podophyllum, which is followed by chemical elaboration to produce etoposide.15 Recently, the biosynthetic pathway of (−)-podophyllotoxin has been established using a transcriptome mining approach.4 In this pathway, 2-ODD, a member of the mononuclear nonheme iron and 2-oxoglutarate (Fe/2OG) enzyme family, is found to catalyze a novel oxidative cyclization (Figure 1). The Fe/2OG enzyme family is composed of a large number of members and catalyzes versatile reactions to regulate primary and secondary metabolite production.16,17 To the best of our knowledge, compared to other reaction types, such as hydroxylation, desaturation, halogenation, epoxidation, and endoperoxdation,9,16,17 oxidative cyclization via a C−C bond formation catalyzed by 2-ODD represents an unexplored reaction type in Fe/2OG enzyme catalysis. Elucidating the 2ODD catalyzed reaction pathway and investigating the corresponding mechanism will provide insight into understanding this unique transformation. In the majority of Fe/2OG enzyme catalyzed reactions, a consensus step involving hydrogen atom transfer (HAT) by the Fe(IV)-oxo species to form the substrate radical has been repeatedly utilized.17,18 The reaction is then followed by diverse but well-controlled reaction outcomes. For example, in hydroxylation, subsequent to the HAT step, the substrate radical reacts with the Fe(III)−OH intermediate to form the C−O bond and generates the Fe(II) to complete the catalysis. This step is also known as OH group rebound.19 Recently, discovery of Fe/2OG enzyme catalyzed endoperoxide and olefin installation suggests that an alternative pathway involving a radical or a cation species can be used to trigger CC or C−O bond formation.20,21 Therefore, in the 2-ODD catalyzed reaction, a few different pathways can be envisioned

nzyme catalyzed oxidative cyclization through stereo- and regio-specific C−H bond activation represents an effective way to construct molecule complexity in natural product biosynthesis as found in amaryllidaceae alkaloids, etoposide, matacycloprodigiosin, menaquinone, morphine, nogalamycin, premarineosin, streptide, and streptorubin.1−8 Proteins containing a cofactor or a transition metal center, such as mononuclear nonheme iron, cytochrome P-450, Rieske-type cluster, or [4Fe−4S] and S-adenosylmethionine (SAM), are known to catalyze this type of transformation.9−12 From a mechanistic point of view, despite the diverse substrate structures, a consensus pathway involving substrate radical generation followed by C−C bond formation has been suggested.9−12 Although a few possible pathways for the C− C bond formation have been proposed, their mechanisms remain to be further explored. Herein, we investigate the plausible mechanism of a nonheme iron enzyme, 2oxoglutarate/Fe(II)-dependent dioxygenase (2-ODD), which catalyzes the oxidative cyclization for the core structure in constructing (−)-podophyllotoxin, an important precursor in producing etoposide (Figure 1). Etoposide is listed on the World Health Organization list of essential medicine and is used in chemotherapy regimens for various malignancies.13,14 Currently, production of etoposide requires isolation of its alycone, (−)-podophyllotoxin (1),

Figure 1. An Fe/2OG enzyme, 2-ODD, catalyzes oxidative cyclization found in the etoposide biosynthetic pathway. © 2018 American Chemical Society

Received: November 16, 2018 Published: December 14, 2018 228

DOI: 10.1021/acs.orglett.8b03670 Org. Lett. 2019, 21, 228−232

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depicted. In these pathways, the reaction can proceed through radical induced cyclization followed by a second HAT (pathway III) or cation induced Friedel−Crafts-type cyclization accompanied by proton removal (pathway IV) to complete the reaction. In order to elucidate the abovementioned pathways, we designed mechanistic probes (6−9, Scheme 1) to examine the 2-ODD reaction in vitro and establish the plausible 2-ODD reaction mechanism. To study the 2-ODD catalyzed reaction, N-terminal hexahistidine tagged 2-ODD was heterologously expressed and purified (see Supporting Information for details). The synthesis of mechanistic probes, including yatein and deoxypodophyllotoxin analogs, started with β-piperonyl-γbutyrolactone (3-(3,4-methylenedioxybenzyl)butyrolactone, 11) as a common precursor. Lactone 11 was prepared from piperonal and dimethyl succinate by literature procedures.22−24 Subjecting 11 to the reaction with lithium diisopropylamide (LDA) at −78 °C followed by alkylation of the resulting butyrolactone enolate with benzyl bromide or benzyl-α,α-D2 bromide afforded the yatein analog 6 or its 7′,7′-D2 analog 7.23−27 The reaction of the corresponding enolate with benzaldehyde gave the diastereomeric mixture of 7′-hydroxyyatein analog 9.23,28,29 Treatment of 9 with trifluoroacetic acid promoted the intramolecular Friedel−Crafts-type ring closure to form the epimeric mixtures of deoxypodophyllotoxin analog 10 (Scheme 2A).23,28,30 To prepare 8, the reaction of 11 in

subsequent to the substrate radical formation to construct the designated C−C bond (Scheme 1). Scheme 1. (Top) Proposed Reaction Pathways to Account for 2-ODD Catalyzed Cyclization; (Bottom) Mechanistic Probes (6−9) Used in This Studya

Scheme 2. Preparation of 6−10 Used in This Studya

a

Tested compounds 6−8 are a pair of enantiomers, and 9 and 10 are mixtures of racemic diastereomers.

sodium deuteroxide solution (NaOD in D2O) furnished α,αdideuteration with concomitant saponification. Upon relactonization in acidic condition, 12 was obtained in a good yield with 90% D-incorporation. Subsequent α-benzylation provided 8 with 85% D-retention (Scheme 2B). It is worth mentioning that all the compounds were prepared as racemic forms for subsequent studies. Having 2-ODD, substrate analogs, and the proposed product in hand, we first incubated 2-ODD with Fe(II), 2OG, substrate analog 6, and O2, with the molar ratio of 2-ODD/Fe(II)/6/ 2OG being 1.2:1:5:10 and with a final concentration of 250 μM of 6. Compared to the product standard (10), liquid chromatography coupled mass spectrometry (LC-MS) analysis of the 2-ODD reaction yielded a peak with the same retention time and an identical m/z value to that of 10. Additionally, a peak with the retention time and m/z value that matched the hydroxylated standard (9) was also detected (Figure 2a). In the absence of 2OG or O2 (data not shown), neither substrate consumption nor product formation could be observed. These results demonstrate that 2-ODD can catalyze O2 and 2OGdependent cyclization using 6 as the substrate. Furthermore,

a In pathway I, a quinone methide (3) serves as the key intermediate. An alternative desaturated intermediate can be utilized in pathway II. In pathways III and IV, a benzylic radical (2) or carbocation (5) is used to trigger C−C bond formation, respectively.

In pathway I, subsequent to substrate radical (2) formation and OH rebound at the benzylic position, C−C bond formation is triggered via the quinone methide intermediate (3), which is formed by the para-methoxy group facilitated hydroxyl group removal. Aromatization via deprotonation completes the reaction to produce 1. 4 Alternatively, dehydration can also occur through a pathway involving C9proton removal of intermediate (4). The reaction then proceeds analogously to form the C−C bond (pathway II). In this pathway, the acidity of C9-proton is increased by the adjacent carbonyl group and does not involve a quinone methide intermediate. A third and fourth pathway involving a radical (2) or a carbocation (5) as the key species can also be 229

DOI: 10.1021/acs.orglett.8b03670 Org. Lett. 2019, 21, 228−232

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ranges from 20 to 100.31−36 In some cases, the decrease of HAT efficiency when the deuterium analog is used results in reduction of substrate consumption and product formation. Examples include CarC, clavaminate synthase, CytC3, IsnB, and TauD.31−33,35,36 Alternatively, in other cases, due to deuterium incorporation at the designated HAT site, the Fe(IV)O species now attacks an alternative C−H bond and prompts a non-native reaction outcome. Thus, the substrate consumption will not be substantially decreased and alternate reaction product(s) can be detected. In NapI, SyrB2, and VioC studies, when deuterated analogs are used, the reactions are redirected to non-native hydroxylation.20,37 Compared with the results obtained using 6 as the substrate, we observe a similar level of substrate consumption (∼2/3 of substrate consumption of 6, Figure S20), no cyclized product formation, and alternative hydroxylation when 7 is used indicating that the HAT site is likely at the benzylic (7′) position during the regular reaction. Next, to evaluate the involvement of C9−H during the enzyme reaction, 8 was tested with 2-ODD. If C9proton removal occurs during the reaction, we anticipate to observe, at least partially, deuterium washout during the reaction when analog 8 is used. As shown in Figure 2c, LC-MS analysis reveals exclusive deuterium retention in both cyclized and hydroxylated products (m/z 334.1 → 332.1 and 350.1) and the ratio of the cyclized product to the hydroxylated species is not perturbed. This observation suggests that C9−H is unlikely to participate in 2-ODD reaction and disfavors pathway II. Even though formation of 10 using 6 disfavors the pathway involving the quinone methide intermediate, due to lack of 2ODD structural information, one cannot completely rule out the possibility of OH group removal with the aid of potential protein−substrate interaction. To examine the possible role of the hydroxylated product 9 during 2-ODD reaction, 9 was incubated with 2-ODD under both anaerobic and aerobic conditions with a ratio of 9 to 2-ODD of ∼10 to 1. Under these conditions, no detectable cyclized product 10 could be observed (Figure 2d). Although we cannot exclude the possibility that 9 cannot enter the active site of 2-ODD due to a conformational change during the reaction, our observation implies that 9 is likely the off-pathway product and not involved in cyclization. To gain insight into the possible intermediate and distinguish radical or cation-induced cyclization during 2-ODD catalysis, nonenzymatic model studies were carried out using 9 (see Supporting Information for experimental details). As summarized in Scheme 2A, when 9 was treated with trifluoroacetic acid, 10 was produced as epimeric mixtures with a ratio of ∼1/1. This reaction most likely proceeded through trifluoroacetic acid promoted intramolecular Friedel−Crafts ring closure via a carbocation intermediate. Although the normal 2-ODD reaction may be different from the model study where epimeric mixtures of 10 were obtained, this observation provides a correlation between carbocation intermediate and cyclization and suggests a possibility of utilizing a carbocation as the key intermediate to trigger cyclization. In summary, combining mechanistic probe design, model studies, in vitro biochemical assays, and LC-MS analysis of 2ODD catalyzed reactions, we explored the plausible reaction pathways of 2-ODD catalyzed oxidative cyclization. On the basis of these results, the 2-ODD catalyzed reaction initiates from the benzylic hydrogen atom abstraction of the substrate. The reaction is likely followed by benzylic carbocation

Figure 2. LC-MS chromatograms of 2-ODD catalyzed reaction using 6−8 as substrates. (a) Production of 9 and 10 at the expense of 6, O2, and 2OG; (b) alternative hydroxylated product formation when 7 was tested; (c) production of 2H-9 and 2H-10 when using 8 as the substrate; (d) no detectable 10 formation under aerobic or anaerobic conditions when 9 was used. All analytes are detected as their corresponding sodium ion adduct form using selected ion monitoring.

due to the lack of a para-methoxy group, which is proposed to trigger elimination of the hydroxyl group (most likely at its protonated form), 6 is less likely to form the corresponding quinone methide intermediate as proposed in pathway I. Thus, production of cyclized product (10) using 6 where the paramethoxy group is replaced by a proton implies that cyclization is unlikely to proceed via pathway I. Next, to identify the HAT site, analog 7 was tested. Different from the results obtained using 6, although 7 was consumed at a similar level (see Figure S20), no obvious cyclized product (m/z 335.1 → 333.1, 332.1, or 331.1 (−2H, −D−H, −2D)) could be observed. Instead, a peak with the m/z value corresponding to hydroxylation with retention of two deuterons (m/z 335.1 → 351.1) was detected (Figure 2b). The obvious retention time difference of this product to 9 along with both deuterons being retained on the tentative hydroxylated product suggest that the reaction most likely occurs at a carbon other than the deuterated position (C7′) when 7 was used. We reason that observation of alternative hydroxylation and retardation of cyclization is probably driven by the intrinsic hydrogen/deuterium (H/D) isotope effect related to the HAT step. In the majority of characterized Fe/ 2OG enzymes, when the designated HAT hydrogen atom is replaced by deuterium, the measured H/D isotope effect 230

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Catalyzes Formation of the Para-Para’ C-C Phenol Couple in the Amaryllidaceae Alkaloids. Front. Plant Sci. 2016, 7, 225. (4) Lau, W.; Sattely, E. S. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone. Science 2015, 349, 1224−8. (5) Salem, S. M.; Kancharla, P.; Florova, G.; Gupta, S.; Lu, W.; Reynolds, K. A. Elucidation of final steps of the marineosins biosynthetic pathway through identification and characterization of the corresponding gene cluster. J. Am. Chem. Soc. 2014, 136, 4565− 74. (6) Schramma, K. R.; Bushin, L. B.; Seyedsayamdost, M. R. Structure and biosynthesis of a macrocyclic peptide containing an unprecedented lysine-to-tryptophan crosslink. Nat. Chem. 2015, 7, 431−7. (7) Siitonen, V.; Selvaraj, B.; Niiranen, L.; Lindqvist, Y.; Schneider, G.; Metsa-Ketela, M. Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 5251−6. (8) Sydor, P. K.; Barry, S. M.; Odulate, O. M.; Barona-Gomez, F.; Haynes, S. W.; Corre, C.; Song, L.; Challis, G. L. Regio- and stereodivergent antibiotic oxidative carbocyclizations catalysed by Rieske oxygenase-like enzymes. Nat. Chem. 2011, 3, 388−92. (9) Gao, S. S.; Naowarojna, N.; Cheng, R.; Liu, X.; Liu, P. Recent examples of alpha-ketoglutarate-dependent mononuclear non-haem iron enzymes in natural product biosyntheses. Nat. Prod. Rep. 2018, 35, 792−837. (10) Perry, C.; de Los Santos, E. L. C.; Alkhalaf, L. M.; Challis, G. L. Rieske non-heme iron-dependent oxygenases catalyse diverse reactions in natural product biosynthesis. Nat. Prod. Rep. 2018, 35, 622−32. (11) Tang, M. C.; Zou, Y.; Watanabe, K.; Walsh, C. T.; Tang, Y. Oxidative Cyclization in Natural Product Biosynthesis. Chem. Rev. 2017, 117, 5226−333. (12) Yokoyama, K.; Lilla, E. A. C-C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products. Nat. Prod. Rep. 2018, 35, 660−94. (13) Gordaliza, M.; Garcia, P. A.; Miguel del Corral, J. M.; Castro, M. A.; Gomez-Zurita, M. A. Podophyllotoxin: distribution, sources, applications and new cytotoxic derivatives. Toxicon 2004, 44, 441−59. (14) Stahelin, H. F.; von Wartburg, A. The chemical and biological route from podophyllotoxin glucoside to etoposide: ninth Cain memorial Award lecture. Cancer Res. 1991, 51, 5−15. (15) Kamal, A.; Ali Hussaini, S. M.; Rahim, A.; Riyaz, S. Podophyllotoxin derivatives: a patent review (2012 - 2014). Expert Opin. Ther. Pat. 2015, 25, 1025−34. (16) Islam, M. S.; Leissing, T. M.; Chowdhury, R.; Hopkinson, R. J.; Schofield, C. J. 2-Oxoglutarate-Dependent Oxygenases. Annu. Rev. Biochem. 2018, 87, 585−620. (17) Martinez, S.; Hausinger, R. P. Catalytic Mechanisms of Fe(II)and 2-Oxoglutarate-dependent Oxygenases. J. Biol. Chem. 2015, 290, 20702−11. (18) Bollinger, J. M., Jr.; Chang, W.-c.; Matthews, M. L.; Martinie, R. J.; Boal, A. K.; Krebs, C. Mechanisms of 2-Oxoglutarate-Dependent Oxygenases: The Hydroxylation Paradigm and Beyond. In 2Oxoglutarate-Dependent Oxygenases; Hausinger, R. P., Schofield, C. J., Eds.; Royal Society of Chemistry: London, 2015; pp 95−122. (19) Groves, J. T. Key Elements of the Chemistry of Cytochrome-P450 - the Oxygen Rebound Mechanism. J. Chem. Educ. 1985, 62, 928−31. (20) Dunham, N. P.; Chang, W.-c.; Mitchell, A. J.; Martinie, R. J.; Zhang, B.; Bergman, J. A.; Rajakovich, L. J.; Wang, B.; Silakov, A.; Krebs, C.; Boal, A. K.; Bollinger, J. M., Jr. Two Distinct Mechanisms for C-C Desaturation by Iron(II)- and 2-(Oxo)glutarate-Dependent Oxygenases: Importance of alpha-Heteroatom Assistance. J. Am. Chem. Soc. 2018, 140, 7116−26. (21) Yan, W.; Song, H.; Song, F.; Guo, Y.; Wu, C. H.; Her, A. S.; Pu, Y.; Wang, S.; Naowarojna, N.; Weitz, A.; Hendrich, M. P.; Costello, C. E.; Zhang, L.; Liu, P.; Zhang, Y. J. Endoperoxide formation by an

formation which is used to trigger C−C bond installation. The aromatization via deprotonation and reformation of Fe(II) species completes the reaction. It is also worth noting that instead of a stepwise mechanism as shown in pathway iv, Scheme 1, the reaction may proceed through a concerted mechanism. Formation of the carbocation species requires ejection of a single electron from the substrate radical to the Fe(III)−OH species. Experiments to investigate the timing and the controlling factors that direct the reaction outcome from canonical hydroxylation into C−C bond installation are in progress. Alternatively, it is also possible that the use of the analog has triggered an alternative pathway, unlike the native pathway, leading to formation of the cyclized product. In addition, our approach using regioselective deuteron incorporated analogs coupled with LC-MS analysis provides a simple and straightforward method to investigate reaction mechanisms that can be applied to other Fe/2OG enzyme catalyzed reactions.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b03670.

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Experimental procedures, supplementary scheme and figures, and NMR and MS spectra (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (W.-c.C.). *E-mail: [email protected] (T.-C.C.). ORCID

Wei-chen Chang: 0000-0002-2341-9846 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by North Carolina State University, National Taiwan Normal University, and research grant MOST 107-2113-M-003-002 from Ministry of Science and Technology, Taiwan. We thank Dr. Bo Zhang at Penn State University, Prof. Yisong Guo at Carnegie Mellon University, and Prof. Christian Melander at University of Notre Dame for helpful discussion.

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REFERENCES

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