Theoretical Study on the Mechanism of Spirocyclization in Spiroviolene Biosynthesis

Abstract

Spiroviolene is a spirocyclic triquinane diterpene produced by Streptomyces violens. Recently, a biosynthetic pathway that includes secondary carbocation intermediates and a complicated concerted skeletal rearrangement was proposed for spiroviolene, based upon careful labeling experiments. On the basis of density functional theory (DFT) calculations, we propose a revised pathway for spiroviolene biosynthesis, involving a multistep carbocation cascade that bypasses the formation of unstable secondary carbocations by breaking the adjacent C–C bond to form a more stable tertiary carbocation (IM3) and by Wagner–Meerwein 1,2-methyl rearrangement (IM7).

Introduction

Terpene/terpenoids are biosynthesized via tandem reactions catalyzed by terpene cyclase.^1–3) Such carbocation cascades contain multiple short-lived/reactive intermediates, which makes mechanistic investigation very challenging. Recently, however, computational chemistry has enabled us to unveil in detail terpene/terpenoid biosynthetic pathways involving various types of rearrangement reactions.⁴⁾

Spiroviolene, featuring a 5/5/5/5 tetracyclic system with a spirocyclic skeleton, was isolated from Streptomyces violens by Dickschat and colleagues in 2017.⁵⁾ They characterized the terpene cyclase SvS that catalyzes spiroviolene formation, carried out ¹³C-labeling experiments, and proposed the reaction mechanism shown in Fig. 1A. In 2020, the stereochemistry of spiroviolene was revised by Snyder and colleagues on the basis of their total synthesis.⁶⁾ Then in 2021, Xu and Dickschat performed a mechanistic investigation of spiroviolene biosynthesis by means of stereoselective labeling experiments, and revised the biosynthetic pathway⁷⁾ (Fig. 1B). Interestingly, the originally proposed pathway contains a 5/5/7/4 tetracyclic intermediate (cyclobutylcarbinyl 3° cation, 2) as a precursor of the methyl shift, whereas the revised pathway involves the formation of a 5/5/5/6 tetracyclic intermediate (2° cation, 7)^8–10) followed by a non-classical carbocation transition state (TS_7–8) for the methyl shift. We have recently established a powerful combination of quantum-chemical calculations with the artificial force induced reaction (AFIR) method¹¹⁾ to unveil complicated biosynthetic pathways/mechanisms, such as those leading to trichobrasilenol,¹²⁾ quiannulatene,¹³⁾ and cyclooctatin.^14,15) In this study, we investigated the spiroviolene biosynthetic pathway by means of density functional theory (DFT) calculations, focusing on (i) whether or not the putative secondary (2°) cations are viable intermediates, and (ii) the mechanism of the methyl shift (particularly the feasibility of the non-classical cation transition state). Although our calculations amend and refine the earlier mechanistic proposals, our proposed pathway remains fully consistent with all the experimental findings.

Fig. 1. (A) Originally Proposed Biosynthetic Pathway of Spiroviolene⁵⁾; (B) Revised Proposed Biosynthetic Pathway of Spiroviolene⁷⁾

(Color figure can be accessed in the online version.)

Results and Discussion

Our DFT calculations indicate that the full reaction pathway for the conversion of GGPP (SM) into IM8, and the energy diagram (relative energies with respect to IM1) are as shown in Figs. 2 and 3, respectively. These calculations indicate that the spiroviolene biosynthetic pathway contains 16 steps, including conformational changes. The carbocation reaction cascade begins with dissociation of the pyrophosphate, which yields an allylic carbocation (IM1) that is partially stabilized by a cation–π interaction with distal C10–C11 and C14–C15 double bonds. Then, double annulation takes place to form a 5/11 bicyclic intermediate (IM2a). Next, ring expansion (the C13–C14 σ bond shift towards the C15 carbocation center) followed by C10–C11 bond cleavage reaction affords a 3° carbocation at C11 and the C10=C14 double bond (IM3a). Notably, the 6,11-bicyclic 2° carbocation structure (X) is not a minimum on the potential energy surface (PES). IM3 has three equilibrium structures within an energy range of about 4 kcal mol⁻¹, and interconversion (conformational change) between homoallyl carbocations IM3a and IM3c proceeds via cyclopropyl carbinyl cation IM3b. Similar equilibria between cyclopropylcarbinyl and homoallyl cations are often seen in terpene/terpenoid biosynthesis.^14–16) Subsequently, cation-mediated annulation with C11–C14 bond formation proceeds to give the so-called humulyl cation intermediate IM4 with a very low activation energy (2.6 kcal mol^–1).¹⁷⁾ IM4 is an extraordinarily stable 2° cation due to transannular cation-π interactions with two (C2–C3 and C6–C7) double bonds, and it undergoes double annulation to give a cyclobutylcarbinyl 3° cation intermediate (IM5a). The 5/5/7/4 tetracyclic structure of IM5 is essentially the same as that of 2 in the initially proposed biosynthetic pathway, except for the stereochemistry of the C18 methyl group (Fig. 1). The highly distorted cyclobutane C–C bonds in the cyclobutylcarbinyl cation effectively stabilize the adjacent carbocation. Thus, the C3–C6 bond in IM5a is elongated to 1.81 Å by hyperconjugation to stabilize the C7 carbocation. After the conformational changes from IM5a to IM5c, C3–C6 σ bond cleavage together with H2 migration proceeds with an activation barrier of 9.9 kcal mol⁻¹ to form the 5/5/9 tricyclic system (IM6a) with the generation of a 3° carbocation at the C2 position. Then, the unique methyl shift assisted by the C2 carbocation take place to form a non-classical TS (TS_6c–7a) with an activation energy of 18.1 kcal mol⁻¹, affording the 5/5/5/6 tetracyclic intermediate IM7a. The details of this drastic skeletal rearrangement will be discussed later (vide infra). After conformational changes, C2–C3 σ bond migration to the C7 carbocation (ring contraction) takes place with a small activation energy (5.0 kcal mol⁻¹) to complete a 5/5/5/5 spirocyclic skeleton (IM8), which undergoes deprotonation, either enzymatically or non-enzymatically, to give spiroviolene.

Fig. 2. Results of DFT Evaluation of the Whole Biosynthetic Pathway of Spiroviolene

Potential energies (kcal mol⁻¹, Gibbs free energies calculated at the mPW1PW91/6–31+G(d, p) level based on M06-2X/6–31+G(d, p) geometries) relative to IM1 are shown in purple. IM stands for intermediate. TS stands for transition state. (Color figure can be accessed in the online version.)

Fig. 3. Energy Diagram of the Proposed Spiroviolene Biosynthetic Pathway

Potential energies (kcal mol⁻¹, Gibbs free energies calculated at the mPW1PW91/6–31+G(d, p) level based on M06-2X/6–31+G(d, p) geometries) relative to IM1 are shown in parentheses. (Color figure can be accessed in the online version.)

The energy profile suggests that this carbocation-mediated tandem reaction pathway is thermodynamically and kinetically favorable: (1) no energy barrier is larger than 20 kcal mol⁻¹, and (2) the overall exothermicity is about 50 kcal mol⁻¹. Overall, the computed biosynthetic pathway of spiroviolene is similar in many respects to that of mangicol, which we have very recently uncovered in detail by means of theoretical/computational study.¹⁸⁾ The most important difference is the unique skeletal rearrangement process (IM6c→TS_6c–7a), which is the rate-determining step in spiroviolene biosynthesis, with the activation energy of 18.1 kcal mol⁻¹. The rearrangement involves two chemical events in one step, i.e., (i) skeletal rearrangement from the 5/5/9 tricyclic to the 5/5/5/6 tetracyclic system, and (ii) 1,2-shift of the C19 methyl group. In general, methyl shift is a less common process, not only in biosynthesis but also in vitro, compared to 1°/2°/3°-alkyl or hydrogen shift.¹⁹⁾ Nevertheless, the activation energy is sufficiently low for the reaction to proceed smoothly at ambient temperature. Thus, we carefully explored this unique skeletal rearrangement process along the intrinsic reaction coordinate (IRC) (Fig. 4). Close examination revealed shoulder peaks (shoulder_6c–7a_1 and shoulder_6c–7a_2, respectively) located before and after TS_6c–7a. As judged from the elongated C2–C6 (2.48 Å) and C2–C7 (2.56 Å) bond distances, we consider that shoulder_6c–7a_1 is close to the putative cation–π interaction structure in which the 3° carbocation at C2 is stabilized by the C6–C7 π bond. Moving towards TS_6c–7a, as C2–C7 σ bond formation proceeds, a transient 2° carbocation is formed at the C6 position. Then, the methyl group at the C19 position migrates very smoothly to the C6 cation center (Wagner–Meerwein rearrangement) via shoulder_6c–7a_2 to form a tertiary carbocation intermediate (IM7a). As seen in Fig. 4, this unique skeletal rearrangement is actually concerted but involves two asynchronous events, i.e., 1) the cation–π interaction-induced C2–C7 σ bond formation and 2) the Wagner–Meerwein-type 1,2-methyl rearrangement. Note that in the early stage of this skeletal rearrangement, the C19 methyl group (the C7–C19 σ bond) is almost orthogonal (i.e., it does not contribute any stabilization) to the three-membered non-classical (C2–C6–C7) carbocation ring, but as the C2–C7 bond formation proceeds, the methyl group plays a crucial role in stabilizing the developing carbocation at the C6 position through the three-membered non-classical (C6–C19–C7) structure.

Fig. 4. Representative Example of the Evolution of Key Bond Lengths in the Conversion of IM6c to IM7a

Potential energies (hartree, calculated at the M06-2X/6–31+G(d, p) level) are shown in parentheses. (Color figure can be accessed in the online version.)

Conclusion

We have scrutinized in detail the biosynthetic pathway of spiroviolene by means of DFT calculation. Our computational study has uncovered a complex 16-steps carbocation cascade leading to formation of the spiroviolene skeleton. This cascade bypasses the formation of unstable secondary carbocations by breaking the adjacent C–C bond to form a more stable tertiary carbocation (IM3) and by Wagner–Meerwein 1,2-methyl rearrangement (IM7). We further propose a new, energetically viable pathway for 5/5/5/5 spirotetracycle formation in spiroviolene biosynthesis. We believe these findings are helpful to update the picture of spiroviolene biosynthesis, and also offer interesting insights into the stability and reactivity of various carbocations putatively involved in terpene biosynthesis. We are continuing to investigate the biosynthetic reaction pathways/mechanisms of terpenes/terpenoids and other natural products.

Experimental

All calculations were carried out using the Gaussian 16 package.²⁰⁾ Structure optimizations were done with the M06-2X²¹⁾ density functional theory method and the 6–31+G(d, p) basis set without any symmetry restrictions. Vibrational frequency calculations at the same level of theory with optimization were performed to verify that each local minimum has no imaginary frequency and that each TS has only a single imaginary frequency. IRC calculations^22–25) for all TSs were performed with GRRM11^26–29) based on Gaussian 16. Single-point energies were calculated at the mPW1PW91/6–31+G(d, p) level³⁰⁾ based on the optimized structure by the M06-2X method. The utility of relative Gibbs free energy energies (G_rel) based on single-point energy and frequency calculation at the mPW1PW91 level has been previously validated for a wide variety of terpene-forming reactions.³¹⁾

Acknowledgments

This work was supported by JSPS KAKENHI (S) (No. 17H06173 (M.U.)), MEXT Leading Initiative for Excellent Young Researchers (No. JPMXS0320200422 (H.S.)), JSPS Grant-in-Aid for Scientific Research on Innovative Areas (No. 19H04643 (M.U.)), and JST CREST (No. JPMJCR19R2 (M.U.)).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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