Confirmation of the stereochemistry of spiroviolene

We confirm the previously revised stereochemistry of spiroviolene by X-ray crystallographically characterizing a hydrazone derivative of 9-oxospiroviolane, which is synthesized by hydroboration/oxidation of spiroviolene followed by oxidation of the resultant hydroxy group. An unexpected thermal boron migration occurred during the hydroboration process of spiroviolene that resulted in the production of a mixture of 1α-hydroxyspiroviolane, 9α- and 9β-hydroxyspiroviolane after oxidation. The assertion of the cis-orientation of the 19- and 20-methyl groups provided further support for the revised cyclization mechanism of spiroviolene.


Introduction
Terpenes represent one of the most fascinating family of natural products due to their structural complexity and diversity, as well as their indispensable biological functions that would be potentially applied as fragrances, pharmaceuticals etc.Until now, more than 80,000 terpenoid structures have been reported, which are found in all domains of life [1][2][3].Despite their remarkable chemodiversity, the biosynthetic logic of terpenes is relatively neat [4].All terpenes are originated from two key C5 building blocks, namely isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are biosynthesized via either methylerythritol phosphate (MEP) pathway or mevalonic acid (MVA) pathway by using the primary metabolites.Different numbers of IPP and DMAPP are assembled by prenyltransferases to afford oligoprenyl pyrophosphates, such as farnesyl pyrophosphate (FPP, 3 × C5) and geranylgeranyl pyrophosphate (GGPP, 4 × C5), with varied C5 units.The linear oligoprenyl pyrophosphates are typically converted by terpene synthases in a chemo-and stereodefined process to form complex terpene skeletons, normally with multiple stereocenters.In this context, the 3D-defined cyclization products retain the rich information of the complex cyclization process.Thus, assignment of the stereochemistry of terpene skeleton with high confidence is crucial for proposing a reasonable cyclization mechanism [5].

Figure 1: Structures of spiroviolene and related natural products
Spiroviolene (1, Figure 1) was identified by Dickschat and co-workers as a nascent cyclization product of spiroviolene synthase (SvS) cloned from Streptomyces violens NRRL ISP-5597 [6].Its unique spiro-fused linear triquinane to cyclopentane skeleton, as well as its stereochemistry, was originally elucidated as 1′ as shown in Figure 1, on the basis of detailed analysis of NMR spectroscopy.Spiroviolene was also found to be produced by several bacterial strains harboring SvS homologs [6,7], as well as putative ancestors of SvS generated by ancestral sequence reconstruction [8,9].Related natural products with the same 5-5-5-5 tetracyclic ring system, including spirograterpene A (2) from Penicillium granulatum MCCC 3A00475 [10], and GJ1012A (3) from an engineered E. coli strain harboring FgGS (FgJ07623) cloned from Fusarium graminearum GJ1012 [11], have been reported almost at the same period.The discrepancy of the stereochemistry at C3 between 1′ and 2 was first noticed by Snyder and co-workers [12].The same stereochemistry at C3 was later confirmed by the conversion of a synthetic intermediate of 2 to spiroviolene.However, the reassignment of spiroviolene to 1 was mainly relied on DFT calculation of the transition state of the key hydroboration reaction and NOE correlation analysis of the resultant product, while direct evidences such as single-crystal X-ray diffraction results were not obtained.
However, no related natural products that would be derived from the intermediates of this pathway have been found so far.
A third cyclization mechanism (Scheme 3C) leading to the same spirocyclic skeleton of spiroviolene with an altered stereochemistry at C7 found in GJ1012A (3) could be proposed [11,17,18].A 3,6-or 3,7-cyclization of cation 4 through a conformation shown as 4-2 with β-oriented 20-methyl group, would generate either 13 or 15 cations.A direct dyotropic rearrangement, or two stepwise 1,2-alkyl migrations of 13, are possible pathways en route to cation 14.The presence of these intermediates 13-15 could be inferred by the identification of GJ1012B/D (16/17, Scheme 1D) [11], cattleyene (18) [ 19,20], and fusaterpenol (19, GJ1012E) [17].A similar 1,2-alky shift of 14, followed by deprotonation of the formed spirocyclic cation 20, afforded 3. Furthermore, a hypothetical product 7-epi-spiroviolene (1′′) can be generated if a 1,4-hydride shift of cation 20 followed by deprotonation of 21 occurred.In this case, hydride from C1, which is closer to the C3 cation in the 3D model, rather than that from C2, shifts from the α-face.Although previous isotope labeling experiments did not support this pathway for spiroviolene cyclization, it should be noted that a subtle alteration of stereochemical assignment of spiroviolene would have consequences for a different mechanistic proposal.We herein report the production of spiroviolene (1) in a heterologous host by taking advantage of an artificial isopentenol utilization pathway [21][22][23][24][25][26], and confirmation of its stereochemistry by X-ray crystallography using a hydrazone derivative of 1.Our work commenced with the production of spiroviolene with an engineered E. coli by using a recently developed isopentenol utilization pathway for the efficient supply of two C5 precursors for terpene biosynthesis (Figure 2) [21][22][23][24][25][26].In this artificial generated pathway, DMAPP and IPP could be easily generated from prenol and isoprenol respectively, by the effect of two kinases, such as hydroxyethylthiazole kinase from E. coli (EcTHIM) and isopentenyl phosphate kinase from Methanocaldococcus jannaschii (MjIPK).Thus, we have cloned genes coding EcTHIM, MjIPK, IDI, IspA and CrtE into the multiple cloning site-2 of pCDFDeut-1 for GGPP production.Also, we have cloned SvS-coding gene directly from Streptomyces violens CGMCC 4.1786 (= NRRL ISP-5597) into pET28a.The two plasmids were then cotransformed into E. coli for diterpene production.Spiroviolene could be produced by feeding prenol and isoprenol to the fermentation broth after the engineered E. coli being induced by IPTG, and fermented at 18 o C for 72 h.GC-MS analysis (Figure 2A) of the EtOAc-extract of fermentation broth gave a single peak, whose EI-MS spectrum (Figure 2B) matches that of spiroviolene.We then carried out large-scale fermentation using shake flasks, and isolated spiroviolene in 40 mg/L yield.The physicochemical data of the isolated material are consistent with those reported for spiroviolene [6].With sufficient amount of spiroviolene in hand, we next attempted to obtain a crystalline compound suitable for X-ray diffraction by introducing functional groups (e.g.hydroxy group, or ketone group) for further derivatization.Spiroviolene was not transformed when subjected to conditions for allylic oxidation (SeO2) even at elevated temperature [27], and the starting material was fully recovered.

Results and Discussion
We have also tried hydroboration-oxidation conditions for transforming the double bond in a congested environment of spiroviolene (Scheme 2).Low conversion was observed when a tetrahydrofuran (THF) solution of 1 was treated with BH3•THF at ambient temperature.The hydroboration reaction could be driven to synthetically useful yield when 1 was directly dissolved in 1M BH3•THF in THF, and heated at 60 o C for 3 days.After oxidative treatment of the resultant alkylborane products with NaOH/H2O2, we have obtained three derivatives (22)(23) with one hydroxy group in 37%, 30% and 21% isolated yield, respectively, as well as recovery of 10% of the starting material.After detailed analysis of their NMR spectra, we have found that besides the normal hydroboration-oxidation product 1α-hydroxy-spiroviolane ( 22), 9α-( 23) and 9β-hydroxy-spiroviolane (24) resulted from a formal boration at the homoallylic position C9 were also produced.The stereochemistry of the newly generated stereocenters were elucidated on the basis of NOESY spectra.Thus, the key NOE correlations of H-1/H-6, H-1/H3-19, and H-2/H3-20 of 22 allowed to assign the 1-OH to be α-oriented, while correlations of H-9/H-14 of 23, and H-9/H-3 of 24, supported the assignment of 9α-and 9β-oriented hydroxy groups, respectively.
The formation of all three products 22-24 can be explained as follows (Scheme 1A) [28][29][30][31].Due to the favorable formation of cis-5,5-fused B/C ring system, the borane reagent is preferred to approach the double bond of 1 from the α-face, to give either a  [31].
It is worth noting that significant discrepancies of the NMR data between 23 and 24 are easily to be noticed.To further advance the intermediate to crystalline hydrazone product (Scheme 2B), we have found that both 23 and 24 can be oxidized to the same 9-oxo-spiroviolane 25 in high yield, hence confirming the structural assignment of 23 and 24.By reacting with 2,4-dinitrohydrazine [32], ketone 25 was further converted to hydrazone derivative 26, which gave a brownish-yellow crystal suitable for X-ray diffraction [33].The crystal structure of 26 clearly showed that 19-and 20-methyl groups are cis-oriented in D-ring that is consistent with that of spriograterpene A. This structural data reaffirms the revised structure of spiroviolene, and further support the unified cyclization process of fungi-derived deoxyconidiogenol and bacteria-derived spiroviolene by sharing a common C6-cation intermediate 6 with cyclopiane skeleton (Scheme 1A).

Conclusion
The complex structures and congested NMR signals have made the elucidation of terpene structures a challenging task.The same stereochemistry of spiroviolene and spirograterpene A was recently confirmed based on their total synthesis by Snyder and co-workers, that have led to the structural revision of spiroviolene.Stereoselective isotope labelling experiments allowed more precise attribution of the NMR data, and further support the reassignment of the C3 stereochemistry.We have clearly confirmed this structural revision by obtaining a suitable crystal of hydrazone derivative of 9-oxospiroviolane, which is synthesized from hydroboration-oxidation of spiroviolene followed by converting the resultant hydroxy group to a ketone group, for single-crystal X-ray diffraction.The confirmation of the cis-oriented 19-and 20-methyl groups of spiroviolene would further support the unified cyclization process that bifurcate at the C6-cation intermediate 6 with a cyclopiane skeleton proposed for spiroviolene and deoxyconidiogenol.In our study, the production of spiroviolene was secured by feeding prenol and isoprenol to an engineered E. coli strain harboring an artificial isoprenol utilization pathway for the supply of DMAPP and IPP.In the hydroboration-oxidation reaction of spiroviolene, besides the normal 1-hydroxy-spiroviolane product, an unexpected thermal 1,2-boron migration of the unstable tertiary 2-organoborane occurred, resulted in the formation of both 9αand 9β-hydroxy-spiroviolane due to the epimerization during the oxidation step.

secondary 1 - 25 - 2 .
organoborane intermediate 25-1, or a tertiary 2-organoborane intermediate Oxidation of 25-1 could stereoselectively furnish the normal hydroboration-oxidation product 22.On the other hand, the unstable tertiary 2organoborane 25-2 would undergo a thermal isomerization to give 25-1 through the borane-olefin complex 26-1, or two consecutive 1,2-boron migrations to yield 9-organoborane intermediate 25-3 through borane-olefin complexes 26-2 and 26-3.The suprafacial nature of the boron migration allowed the boron to be α-oriented in intermediate 25-3, which would give both 23 and 24 after NaOH/H2O2 oxidation.The low diastereoselectivity in this case might be explained by the epimerization during the oxidation step, which has been reported previously