Elucidation of the regio- and chemoselectivity of enzymatic allylic oxidations with Pleurotus sapidus - Conversion of selected spirocyclic terpenoids and computational analysis

Article abstract of DOI:10.3762/bjoc.9.262

Allylic oxidations of olefins to enones allow the efficient synthesis of value-added products from simple olefinic precursors like terpenes or terpenoids. Biocatalytic variants have a large potential for industrial applications, particularly in the pharmaceutical and food industry. Herein we report efficient biocatalytic allylic oxidations of spirocyclic terpenoids by a lyophilisate of the edible fungus Pleurotus sapidus. This ''mushroom catalysis'' is operationally simple and allows the conversion of various unsaturated spirocyclic terpenoids. A number of new spirocyclic enones have thus been obtained with good regio- and chemoselectivity and chiral separation protocols for enantiomeric mixtures have been developed. The oxidations follow a radical mechanism and the regioselectivity of the reaction is mainly determined by bond-dissociation energies of the available allylic CH-bonds and steric accessibility of the oxidation site.

Full text of DOI:10.3762/bjoc.9.262

Elucidation of the regio- and chemoselectivity of
enzymatic allylic oxidations with Pleurotus sapidus –
conversion of selected spirocyclic terpenoids and
computational analysis
Verena Weidmann1, Mathias Schaffrath2, Holger Zorn3, Julia Rehbein*4
and Wolfgang Maison*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 2233–2241.
1Department of Chemistry, University of Hamburg, Bundesstr. 45,
20146 Hamburg, Germany, 2LGCR Chemistry, Sanofi-Aventis
Deutschland GmbH, 65926 Frankfurt am Main, Germany,
3Justus-Liebig-University Giessen, Institute of Food Chemistry and
Food Biotechnology, Heinrich-Buff-Ring 58, 35392 Gießen and
4Department of Chemistry, University of Hamburg,
Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
doi:10.3762/bjoc.9.262
Received: 08 August 2013
Accepted: 10 October 2013
Published: 29 October 2013
This article is part of the Thematic Series "Natural products in synthesis
and biosynthesis".
Email:
Julia Rehbein* - rehbein@chemie.uni-hamburg.de;
Wolfgang Maison* - maison@chemie.uni-hamburg.de
Guest Editor: J. S. Dickschat
© 2013 Weidmann et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
allylic oxidation; CH-activation; chiral separation; enones; flavors;
natural products; terpenes
Abstract
Allylic oxidations of olefins to enones allow the efficient synthesis of value-added products from simple olefinic precursors like
terpenes or terpenoids. Biocatalytic variants have a large potential for industrial applications, particularly in the pharmaceutical and
food industry. Herein we report efficient biocatalytic allylic oxidations of spirocyclic terpenoids by a lyophilisate of the edible
fungus Pleurotus sapidus. This ‘’mushroom catalysis’’ is operationally simple and allows the conversion of various unsaturated
spirocyclic terpenoids. A number of new spirocyclic enones have thus been obtained with good regio- and chemoselectivity and
chiral separation protocols for enantiomeric mixtures have been developed. The oxidations follow a radical mechanism and the
regioselectivity of the reaction is mainly determined by bond-dissociation energies of the available allylic CH-bonds and steric
accessibility of the oxidation site.
Introduction
Selective oxidations of CH-bonds are attractive synthetic trans- convert relatively cheap precursors into value-added products
formations with a broad spectrum of applications in academia [1,2]. Among these transformations, allylic oxidations are of
and a high impact on the industrial chemical value chain as they high interest because the olefinic starting materials are readily
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Beilstein J. Org. Chem. 2013, 9, 2233–2241.
available as cheap bulk chemicals and many interesting deriva- pathways cannot be ruled out for other substrates. Reviewing
tives such as terpenes are available from renewable sources the available examples for PSA-mediated conversions in the
[3-5]. In addition, the resulting allyl alcohols [6-10] or α,β- literature, the regio- and chemoselectivity of these oxidations
unsaturated carbonyl compounds are attractive synthetic targets seems to be determined by the radical mechanism of the reac-
of high economic and scientific interest [11-17]. Allylic oxida- tions and would thus follow well-established rules for other
tions of olefins to enones have classically been performed with radical-type allylic oxidations [33-36].
strong oxidants such as chromium or other metal-based reagents
[18,19]. In addition, metal-free and biocatalytic methods have A notable example of an allylic oxidation with PSA is the
been reported [5]. Several of these biocatalytic protocols have conversion of theaspirane (1), a spirocyclic flavor compound of
been applied to the synthesis of fine chemicals [20-22], drugs tea, vanilla and different fruits [37] to the corresponding thea-
[23], and food ingredients [24-26]. A particularly interesting spirone (2) (Scheme 1) [32].
biocatalytic system for allylic oxidation is the edible fungus
Pleurotus sapidus (PSA), which is able to oxidize selected The reaction is quite clean and gives the enone 2 in good yield
terpenes and fatty acids [27-31]. We have recently shown that along with minor amounts of the corresponding allyl alcohol 3
the lyophilisate of PSA is able to affect allylic and benzylic and the epoxide 4a. This successful conversion of theaspirane
oxidations in a broad range of olefinic substrates including (1) encouraged us to investigate the oxidation of other spiro-
simple cyclohexene derivatives and several functionalized cyclic terpenoids. Many oxidized spiroethers are valuable flavor
terpenoids (Figure 1) [32].
compounds or have other interesting biological properties such
as phytotoxic activity. A few selected examples are depicted in
Biocatalytic allylic oxidations with PSA may be performed with Figure 2.
the lyophilisate from submerged cultures. Cyclic alkenes and
particularly cyclohexene derivatives are the preferred substrates In this paper, we report biocatalytic allylic oxidations of spiro-
of PSA. A PSA-derived dioxygenase has been shown to be re- cyclic model compounds and of the natural product vitispirane
sponsible for the allylic oxidation of valencene to nootkatone, with the lyophilisate of PSA. A rationalization of the observed
and the same enzyme oxidizes unsaturated fatty acids [29,31]. It selectivity is provided by means of computational determin-
is thus likely that this dioxygenase is the major oxidant in other ation of bond-dissociation enthalpies and correlation with struc-
allylic oxidations with PSA-lyophilisate, too. However, since tural and electronic features. In addition, a short synthesis of
the lyophilisate is a mixture of enzymes, alternative oxidation vitispirane is presented.
Figure 1: Selected biocatalytic allylic and benzylic oxidations with the lyophilisate of Pleurotus sapidus (PSA).
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Beilstein J. Org. Chem. 2013, 9, 2233–2241.
Scheme 1: Biocatalytic allylic oxidation of theaspirane (1) with lyophilisates of PSA. Only one enantiomer of racemic compounds is shown.
ing model compounds for the allylic oxidation with PSA
because they contain allylic positions with different stereoelec-
tronic properties.
All four spiroethers 7, 8, 11 and 12 were submitted to identical
conditions and were treated with PSA lyophilisate in Tris-buffer
at room temperature (Scheme 3). The reactions were monitored
by GC. The oxidation of allyl ether 7 may follow two alter-
native pathways either to the α,β-unsaturated lactone 14 or to
the enone 15 (allylic oxidations at exocyclic positions are gen-
erally less favorable if radical mechanisms are operating) [35].
Due to sterical hindrance at position 5 and the strong activation
Figure 2: Selected bioactive terpenoids based on spiroether back-
bones [38,39].
Results and Discussion
Allylic spiroethers, such as theaspirane (1) (Scheme 1) are suit- for hydrogen abstraction in position 2, spiroether 7 was selec-
able substrates for allylic oxidations with PSA. Due to their tively converted to the spirolactone 14. The alternative oxi-
interesting properties as flavor compounds, we focused our dation product 15 was not found by GC–MS. The regioiso-
attention to the oxidation of terpenoid spiroethers. As model meric spiroether 8 offers only one plausible path for allylic oxi-
compounds, unsaturated spiroethers 7, 8, 11 and 12 were dation in position 3. However, this spiroether was not converted
synthesized by the intramolecular silyl-modified Sakurai reac- at all and the starting material 8 was recovered almost quantita-
tion of precursors 5 and 10 and alkene 6 (Scheme 2). Two pairs tively from the reaction mixture. This finding may reflect the
of regioisomeric endocyclic derivatives 7, 8 and 11, 12 were increased sterical hindrance of the allylic position in 8
obtained as the major products of cyclization in a 1:1 mixture. compared to the site of oxidation in regioisomer 7 and a less
The third regioisomer 9 and 13 respectively, with an exocyclic stable radical intermediate.
double bond was observed in minor amounts only. Separation
of the regioisomeric spiroethers by column chromatography Similar results were obtained for oxidation of spiroethers 11
proved to be difficult, and in both cases only the two major and 12 which contain two separated double bonds. The allyl
isomers with endocyclic double bonds were isolated in pure ether 11 allows three plausible oxidation products 17–19.
form. The regioisomeric spiroethers 7, 8, 11 and 12 are interest- However, due to our experiences with oxidations of 7, 8 and
Scheme 2: Intramolecular silyl modified Sakurai reaction to spiroethers 7–9 and 11–13.
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Beilstein J. Org. Chem. 2013, 9, 2233–2241.
Scheme 3: Biocatalytic allylic oxidation of spiroethers 7, 8, 11 and 12 with the lyophilisate of PSA. Conversion was measured by GC and yields are
given after isolation of the oxidation products and purification by chromatography.
theaspirane (1), position 2 should be activated most strongly for tures 11A and 11B that contain key structural features of 11 and
hydrogen abstraction and should thus be privileged for oxi- 12 (Table 1). According to the data summarized in Table 1 and
dation. As a consequence, the α,β-unsaturated lactone 17 is Table 2 B3LYP/6-31+G** gives reasonable results and is a
indeed the major product along with some unidentified more suitable method to predict at least relative BDH-values of
complex oxidation products. Again, oxidation in 5-position of C–H bonds of the spiro compounds at hand. In absolute terms,
11 to the enone 18 was not observed. In addition, the allylic B3LYP underestimates the BDH systematically by
hydrogens in 9-position are obviously also of low reactivity and 2.9–4.6 kcal/mol.
the corresponding oxidation product 19 was also not detectable.
In agreement with the oxidation of 8, substrate 12 was not The experimental observation as summarized in Scheme 3, e.g.,
oxidized by PSA at all and the starting material was reisolated the selective oxidation in 2-position of an allyl ether subunit
almost quantitatively.
may be rationalized by the particularly low BDH298 for the
corresponding C–H bond compared to the other allylic C–H
Computational analysis (for full computational details see bonds (Table 2). Carbon-centered radicals adjacent to an
Supporting Information File 1) of relative radical stabilities and oxygen atom are commonly known to be stabilized as they
bond-dissociation enthalpies (BDH298) based on DFT (B3LYP/ benefit from inductive effects as well as from orbital interac-
6-31+G** and B3LYP/6-31G* + PCM) and composite method tions of the p-type lone pair of the oxygen atom with the half-
(CBS-QB3) calculations enable a quantification and visual filled p-orbital of the mainly sp2 hybridized radical [40]. The
rationalization of the observed experimental results. Since PCM picture of the SOMO and the mapped out spin density of 11-R3
and gas phase results do not differ significantly neither in terms illustrate the latter effect (Figure 3). In addition, the methyl
of geometry nor energy, we will restrict the discussion to gas group in 3-position of the allylic system in 11-R3 as well as in
phase values only unless otherwise stated (for details of the the model system 11B-R3 helps to stabilize the radical further,
PCM results see Supporting Information File 1). The CBS-QB3 mainly by hyperconjugation. In total, both substituent effects
method has been used to obtain accurate energies and to eval- result in a rather low C–H bond-dissociation enthalpy of
uate the DFT-energies in terms of relative and absolute values. 79.5 kcal/mol at CBS-QB3 level of theory (74.9 kcal/mol
For this comparison CBS-QB3 has been applied to model struc- B3LYP/6-31+G**, Table 2).
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Beilstein J. Org. Chem. 2013, 9, 2233–2241.
Table 1: Model substrates 11A and 11B that allowed a determination of accurate BDH298 values with the CBS-QB3 method.
CBS-QB3
entry
compd
BDH298 [kcal/mol]
ΔG [kcal/mol]
1
2
3
11A-R1
11B-R2
11B-R3
83.5
86.9
79.5
75.0
77.4
70.8
Table 2: Optimized geometries and BDH298 values of radicals derived from 11 and 12 for gas phase and PCM calculations.
B3LYP/6-31+G**
B3LYP/6-31G* + PCM
ΔGa
entry
compd
ΔHa
ΔGa
BDH298
ΔHa
BDH298
[kcal/mol]
[kcal/mol]
[kcal/mol]
[kcal/mol]
[kcal/mol]
[kcal/mol]
1
2
3
4
5
6
7
11
1.2
−1.5
0.5
1.3
−0.2
0.7
—
1.0
−1.9
0.3
1.2
−0.3
0.7
0.0
0.0
2.0
0.0
—
11-R1
11-R2
11-R3
12
80.9
82.9
74.9
—
81.6
83.7
74.6
—
−7.5
0.0
−6.4
0.0
−7.8
0.0
12-R1
12-R2
−3.3
0.0
−2.1
0.0
80.3
83.6
3.4
80.1
83.4
0.0
aReferring to 12 for closed-shell species and 12-R2 for radicals.
Compared to the other potential allylic radicals like 11-R1 and ences are less pronounced. Equally, the reasons for the small
11-R2, 11-R3 is up to 8 kcal/mol more stable. Within the subset variations in BDH298 values are more subtle and might be
of allylic radicals that differ only by their alkyl substitution referred to additional steric strain imposed by a change in
pattern (11-R1, 11-R2, 12-R1 and 12-R2) the energetic differ- hybridization of the carbon atom from sp3 to sp2 upon radical
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Figure 3: Bond-dissociation enthalpies for three allylic C–H bonds in 11. Double stabilization of the radical in 11-R3 by the adjacent oxygen and
hyperconjugation lead to a rather low BDH of 74.9 kcal/mol (B3LYP/6-31+G**).
formation and hyperconjugation effects (Table 2). Correlating alternative protocol, we used Yamamoto´s conditions [47] for
the experimental observations with the computational results, the isomerization of epoxides (TMP, n-BuLi, Et2AlCl) and
one can deduce that a BHD298 of around 80 kcal/mol (CBS- obtained allyl alcohol 22 in excellent 93% yield. The conver-
QB3) seems to be a threshold that might be used as a guideline sion of allyl alcohol 22 to vitispirane (23) has been reported
to decide whether an allylic oxidation of spiro compounds with POCl3 in pyridine. Again, this known protocol did not
related to 11 and 12 with PSA takes places or not.
work satisfactory for us resulting in only minor amounts of the
desired vitispirane (23). As a consequence, we decided to use an
As a further test on the substrate scope of the reaction, we were alternative method via acetylation and Pd-catalyzed elimination.
interested in the biocatalytic oxidation of vitispirane as a This conversion gave vitispirane (23) in good yield [48].
terpenoid with a conjugated double bond and a sterically Overall, this improved protocol gave racemic vitispirane (23) in
hindered allylic position (Figure 2). Vitispirane is a flavor com- three steps from theaspirane (trans-1) in excellent 72% overall
pound of vanilla and quince fruit and was identified in grape yield. It should be noted that the gas chromatographic sep-
juice and wine [41,42]. Various syntheses of vitispirane have aration of racemic vitispirane (23) has been reported by
been reported in the literature [43-45]. To get sufficient quan- Schreier [45]. In addition, we have been able to separate
tity of the oxidation precursor, we focused on a strategy racemic vitispirane by HPLC on a chiral stationary phase (see
reported by Ohloff starting from commercially available thea- Supporting Information File 1).
spirane (1) [46]. As described by Ohloff and coworkers, the
separation of vitispirane diastereoisomers is quite difficult. Vitispirane (23) is a challenging substrate for allylic oxidation
Therefore we decided to start with diastereomerically pure thea- with PSA. As outlined above, the oxidation with PSA is quite
spirane (trans-1) that was obtained by chromatographic sep- sensitive to steric and electronic factors and the allylic position
aration of the commercial diastereomeric mixture of cis- and 9 in vitispirane (23) is sterically hindered (Scheme 5).
trans-theaspirane (1). As depicted in Scheme 4, trans-theaspi-
rane (trans-1) was converted to the corresponding epoxides 4a Treatment of vitispirane (23) with PSA was indeed not a clean
and 4b following the literature protocol using m-chloroper- conversion giving a number of different oxidation products.
benzoic acid. The major epoxide 4a was obtained in good yield From this mixture, however, three main products 24, 26a and
and a diastereoselectivity of 11:1.
26b were identified unambiguously. So far, we have never
detected similar oxidation products with PSA and rationalize
Isomerisation of epoxide 4a to allyl alcohol 22 was reported their formation by an initial epoxidation of the endocyclic
with aluminium triisopropoxide at 140 °C [46]. However, in our double bond to give the allyl epoxide 25, which might then be
hands this procedure gave only complex reaction mixtures hydrolyzed to two diastereomeric alcohols 26a and 26b. The
containing minor amounts of the target allyl alcohol 22. In an latter reaction is known for similar allyl epoxides under slightly
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Beilstein J. Org. Chem. 2013, 9, 2233–2241.
Scheme 4: Improved 3-step synthesis of vitispirane (23) from theaspirane (1). Only one enantiomer of racemic compounds is shown.
Scheme 5: Oxidation of vitispirane (23) with PSA gave enone 24 and two diastereomeric allyl alcohols 26a and 26b. A putative intermediate is
epoxide 25, which upon hydrolysis would give allyl alcohols 26a and 26b. Oxidation of the latter might provide enone 24. Only one enantiomer of
racemic compounds is shown.
acidic conditions [49]. However, participation of hydrolases It should be noted, that all three compounds 24, 26a and 26b are
from the PSA lyophilisate is also possible. A fraction of new derivatives of vitispirane 24 with potentially interesting
the resulting alcohols 26 would then finally be oxidized to properties as flavors. The relative stereochemistry of 26a and
give enone 24. Support for an epoxide intermediate comes 26b was evaluated after HPLC separation of the two diastereo-
from oxidations of substrates containing similarly hindered isomers by 2D-NOESY NMR.
allylic CH-bonds. For β-ionone, for example, we have
previously observed epoxidation to be the major oxidation The compounds 24, 26a and 26b were obtained as racemic
pathway.
mixtures. However, we found HPLC protocols for the sep-
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Beilstein J. Org. Chem. 2013, 9, 2233–2241.
aration of these terpenoids with commercial chiral stationary fungal research”. M. Schaffrath would like to thank Peter
phases (see Supporting Information File 1). This allows the Hamley and Martin Will from Sanofi for their support.
isolation of large quantities of the enantiomerically pure deriva-
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