Exploiting the Synthetic Potential of Sesquiterpene Cyclases for Generating Unnatural Terpenoids

Article abstract of DOI:10.1002/anie.201805526

The substrate flexibility of eight purified sesquiterpene cyclases was evaluated using six new heteroatom-modified farnesyl pyrophosphates, and the formation of six new heteroatom-modified macrocyclic and tricyclic sesquiterpenoids is described. GC-O analysis revealed that tricyclic tetrahydrofuran exhibits an ethereal, peppery, and camphor-like olfactoric scent.

Full text of DOI:10.1002/anie.201805526

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Accepted Article
Title: Exploiting the synthetic potential of sesquiterpene cyclases for
generating unnatural terpenoids
Authors: Andreas Kirschning, Benjamin Schröder, Vanessa Harms,
Clara Oberhauser, Katja Seidel, Kimia Ekramzadeh, Sascha
Beutel, Jeroen Dickschat, Lukas Lauterbach, and Sven
Winkler
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201805526
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10.1002/anie.201805526
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Exploiting the synthetic potential of sesquiterpene cyclases for
generating unnatural terpenoids
Clara Oberhauser+,[a] Vanessa Harms+,[a] Katja Seidel,[a] Benjamin Schröder,[a] Kimia Ekramzadeh,[b]
Sascha Beutel,[b] Sven Winkler,[c] Lukas Lauterbach,[d] Jeroen S. Dickschat[d] and Andreas
Kirschning*[a]
Dedicated to Prof. Ernst Schaumann on the occasion of his 75th birthday.
Abstract: The substrate flexibility of eight purified sesquiterpene
cyclases was evaluated using six new heteroatom modified farnesyl
pyrophosphates and the formation of six new heteroatom-modified
macrocyclic and tricyclic sesquiterpenoids is described. GC-O
analysis revealed that tricyclic furan exhibits an ethereal, peppery
and camphor-like olfactoric scent.
derivatives and even new terpenoid backbones.
Oxyfunctionalized mono- and sesquiterpenes are widely applied
in the flavor and fragrance industries. However the products of
the TCs, so called terpene hydrocarbons, often do not show
beneficial olfactoric properties and are therefore commonly
separated from the oxidized derivatives.[6] In fact, the growing
flavor and fragrance industries constantly demand for new
terpenoids with olfactoric potential. Noteworthy, the concept of
transforming heteroatom-modified pyrophosphate precursors to
new terpenoids using recombinant TCs has not been pursued in
the arena of fragrances and flavors so far.
Terpenes are structurally the most diverse class of secondary
metabolites. These complex backbones are generated from a linear
precursor
pyrophosphate (FPP, 17) - by means of a cascade reaction
composed of -solvolytic steps and Wagner-Meerwein
- in the case of sesquiterpenes this is farnesyl
rearrangements. This unique cascade is catalyzed by terpene
cyclases (TCs) and is commonly finalized either by proton
abstraction or by trapping of the final carbocation with a nucleophile,
commonly water.[1] TCs have seen increased interest lately, because
several of these enzymes were expressed as functional proteins and
served to study the mechanisms of cyclization in detail.[2,[3]
In selected biosynthetic studies unnatural substrates, mainly
fluoro- and hydroxyl-modified derivatives of FPP, were
administered to sesquiterpene cyclases (STCs). E.g. 12-
hydroxy-FPP was transformed by the amorphadiene synthase
from Artemisia annua to yield the artemisinin precursor
dihydroartemisinic aldehyde in 34% yield.[4] With the natural
substrate 17 the yield could recently be raised to over 90%
under continuous flow conditions.[5] These few examples
demonstrate that the field of chemo-enzymatic use of TCs is an
emerging field of research with prospects to access new terpene
[a]
C. Oberhauser, V. Harms, Dr. K. Seidel, Dr. B. Schröder, Prof. Dr.
A. Kirschning
Institute of Organic Chemistry and Center of Biomolecular Drug
Research (BMWZ)
Leibniz Universität Hannover
Schneiderberg 1B, 30167 Hannover (Germany)
Fax: (+49) 511-762-3011
E-mail: andreas.kirschning@oci.uni-hannover.de
K. Ekramzadeh, Dr. S. Beutel
[b]
Institute of Technical Chemistry and Center of Biomolecular Drug
Research (BMWZ)
Leibniz Universität Hannover
Callinstr. 5, 30167 Hannover (Germany)
S. Winkler, Symrise AG, Mühlenfeldstrasse 1, 37603 Holzminden,
(Germany)
Prof. Dr. J. S. Dickschat, L. Lauterbach
Kekulé-Institute of Organic Chemistry and Biochemistry
University of Bonn
[c]
[d]
Figure 1. Reported main products 1-16 of sesquiterpene cyclases studied
here (from plants (light grey): Pts= patchoulol synthase, Tps32= viridiflorene
synthase, Hvs1= vetispiradiene synthase; from bacteria (grey): GcoA=
caryolan-1-ol synthase, TmS= T-muurolol synthase, PenA= pentalenene
synthase; from fungi (dark grey): Bot2= presilphiperfolan-8-β-ol synthase,
Cop4= cubebol synthase), FPP 17 and its derivatives 18-23 employed in the
present studies.
Gerhard-Domagk-Straße 1, 53121 Bonn (Germany)
These authors contributed equally.
+
Supporting information under http://www.angewandte.org or from
the authors.
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10.1002/anie.201805526
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In this report we study the substrate flexibility of eight
recombinant and purified STCs with respect to their ability to
transform oxygen-, nitrogen- and sulfur-containing unnatural
FPPs and additionally disclose preliminary results on the
olfactoric properties of selected terpenoids formed. For all STCs
chosen, the initial cyclization of FPP proceeds between the
positions 1 and 10 or 11, respectively (Figure 1).[7] Three of the
chosen TCs are plant-based, two are found in fungi and three
cyclases are of bacterial origin. Plant-derived STCs included the
patchoulol synthase (Pts) from Pogostemon cablin that provides
patchoulol (1), -bulnesene (2), -guaiene (3) and -
caryophyllene (4) as the main products.[8] Additionally,
viridiflorene synthase (Tps32, yielding 5) found in Solanum
lycopersicum[9] and vetispiradiene synthase (Hvs1, yielding 6),
originating from Hyoscamus muticus and a sequence homology
of 90% were included.[10] The bacterial caryolan-1-ol-synthase
(GcoA) from Streptomyces griseus catalyzes the transformation
of FPP (17) to (+)-caryolan-1-ol (7) and (+)-β-caryophyllene
(4),[11] and (+)-T-muurolol synthase (TmS) from Roseiflexus
castenholzii catalyzes the conversion of FPP (17) into (+)-T-
muurolol (16).[12] The pentalenene (8) synthase (PenA) is
obtained from Streptomyces exfoliatus UC5319.[13] In addition,
the two fungal STCs presilphiperfolan-8-β-ol synthase (Bot2)
from Botrytis cinerea being responsible for the formation of the
tricyclic sesquiterpene alcohol 9[14] and cubebol synthase (Cop4)
that originates from Coprinus cinereus and which yields several
cyclization products (10-15) in temperature and pH dependent
proportions were employed. Commonly, germacrene D (10) and
cubebol (11) are the main products.[15]
First enzymes were cloned and expressed in E. coli. In vitro
enzyme tests were used to determine enzyme activity and the
substrate tolerance. Semipreparative transformations were
optimized with respect to temperature, pH-value as well as
substrate and enzyme concentration to avoid denaturation and
effects of inhibition. Moreover, the solubility of enzyme and
substrate in semi-preparative scale were optimized (see SI).
We found that Tps32 yielded vetispiradiene (6) instead of
viridiflorene (5) from 17. This was confirmed NMR-
spectroscopically and by GC-coinjection of the FPP-cyclization
products obtained with Tps32 and Hvs1, respectively. In no case
variations of conditions (pH, temperature and Mg-concentration)
yielded traces of 5. The mechanistic formation of both terpenes
is alike and initiated by a 1→10 cyclization. The resulting
carbocation is stabilized by deprotonation either with formation
of a terminal alkene (for 6) or a cyclopropane (for 5). Then a
proton-induced cyclization that either provides a decahydro-
azulene (for 5) or alternatively a decahydronaphthalene (for 6)
backbone occurs. Clearly, subtle conformational changes in the
active site lead to substantial structural deviations.
An overview on the outcome of the biotransformations for
individual enzymes is shown in Scheme 1. The nitrogen
functionalized analogues 19 and 22 generally did not serve as
substrates for any TC (Pts, Tps32, Hvs1, GcoA, PenA, Cop4
and Bot2). In contrast, ethers 18 and 21 as well as thioethers 20
and 23, respectively, are well suited to create new heteroatom-
containing macrocycles 24, 26, 29-31 and in one case a tricyclic
terpenoid 25 (Scheme 1).
Scheme 1. New terpenoid products 24-31 from FPP-derivatives 18, 20, 21
and 23 (bold: main product; marked in grey: enzyme used for preparative
scale; italic: traces of product).
Also the rearranged tertiary alcohols 27 and 28 are formed from
the precursor pyrophosphates 20 and 21, respectively,
noteworthy only under enzyme-catalysis conditions.
The four new FPP derivatives 18, 20, 21 and 23 were prepared
from geraniol and after standard preparations of the different
building blocks these were merged by means of the Williamson
ether synthesis (see SI). The key step for preparing
corresponding new amines 19 and 22 was the nucleophilic
substitution of the metallated Boc-protected amines with the
appropriate bromides. The pyrophosphatyl group was introduced
by Appel chlorination of the terminal allylic alcohols followed by
Scheme 2. Mechanistic considerations on the formation of new terpenoids 24,
26 and 29-31.
The STCs Pts, Tps32, Hvs1, GcoA, Cop4 and Bot2 are all able
to generate macrocyclic ether 24, despite the fact that they
resemble 1→10 as well as 1→11 cyclases. The corresponding
substitution
with
tris(tetra-n-butylammonium)
hydrogen
pyrophosphate (see SI).[15]
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10.1002/anie.201805526
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either takes place much faster than conformational
reorganization of the humulyl cation 37 and that it is
synchronous with the generation of 37. These stereochemical
results contrast our findings for 25.
thioether 20 was cyclized by Tps32, PenA and Bot2 to the
corresponding macrocyclic thioether 26.
The isomeric ether 21 and thioether 23 yielded the
corresponding macrocycles 29 (catalyzed by TmS) and 30
(catalyzed by Pts, Tps32, PenA, Cop4, Bot2 and TmS). The
formation of products 24, 26, 29 and 30 is expectable as they
are generated after pyrophosphate activation, cyclization via
C10 (cationic intermediates 32-35) and formation of the 1,1-
disubstituted alkene after deprotonation. In the presence of
GcoA 23 was transformed into 1,3-diene 31, whose formation
can be rationalized if one assumes that a 1,2-hydride shift
occurs after formation of cation 35 and deprotonation on
intermediate allylic cation 36 occurs.
The
most
remarkable
product
is
the
tricyclic
cyclobuta[3,4]cyclohepta[1,2-c]furan derivative 25 obtained from
ether 18. Despite the structural differences between
presilphiperfolan-8--ol (9) and the derivative 25 a sensible
proposal of its formation is depicted in Scheme 3. The
biosynthesis of 9 from FPP (17)[14] is initiated by formation of the
humulyl cation 37. Next,
a cationic cascade leads to
cyclopentaindene 40 via cyclobutane 38, bicyclic bridged cation
39, followed by a third cyclization. Tantillo noted, that the 1,2-
alkyl shift and cation–alkene cyclization may proceed via a
transition state structure more closely resembled by
a
nonclassical carbocation. A hydride migration provides cation 41
which is finally trapped by water to provide the carbinol moiety in
9. In a similar manner ether 18 yields cationic cyclobutane
intermediate 43 that resembles cation 38 via 42 (Scheme 2,
case B). At this stage the two proposed biosynthetic sequences
diverge. Instead of a Wagner-Meerwein rearrangement the
cation in 43 is trapped by the remaining alkene and the resulting
cation 44 yields terpenoid furan 25 after deprotonation.
Intermediate 43 is the key intermediate of this route, as unlike
for FPP, the extra oxygen atom is able to exert a slightly
different substrate fold in the enzyme and furthermore
anchimerically can stabilize the cation by formation of an oxiran-
1-ium cation. The relative stereochemistry was elucidated by
determining selected nuclear Overhauser effects, and these are
summarized in Scheme 3. Clearly, the cyclobutane ring is fused
to the central seven-membered ring in an anti fashion while the
furan ring is attached in a syn manner to the cycloheptane which
can be rationalized from the shown conformations of 18 and 42
in Scheme 3B. The mechanistic considerations on the formation
of terpenoid 25 also provide information on the likely absolute
stereochemistry. The carbon atom labelled in grey is formed
during cyclobutane formation and remains unaltered for both
mechanistic pathways (leading to 9 and 25, respectively). The
relative as well as the absolute configurations of
Scheme 3. Mechanistic considerations on the
formation of presilphiperfolan-8-β-ol
9
(A)[14] and
terpenoid 25 (B) catalyzed by Bot2 and noe
correlations relevant for determining the relative
configuration.The stereogenic center that is identical in
9 and 25 is marked in grey and acts as a reference for
the absolute stereochemistry of 25;  and  refer to the nomenclature
used in steroids.
Clearly, the insertion of an extra oxygen atom into the linear
precursor leads to subtle conformational alterations when being
incorporated into the active site of Bot2, so that the cyclobutane
formation may be effected, and additionally the remaining
olefinic double bond can act as a nucleophile and induce a new
final ring closure.
A GC-O (gas chromatography-olfactometry) olfactory evaluation
was conducted for new farnesol derivatives (S7, S16, S20 and
S31, see SI) as well as for isolated products 24-31. This analysis
revealed, that tricyclic furan 25 shows an ethereal, peppery and
camphor scent, a sensory profile that is comparable with the
sesquiterpene rotundone 45 in terms of the peppery note.
Interestingly, both molecules contain a similar bicyclic ring
system that is related to hydroazulene. Rotundone is found in
many essential oils such as patchouli oil, agarwood or various
pepper oils. Moreover, it can be part of spicy wines.[22]
In conclusion, we report on the use of eight STCs from fungal,
bacterial and plant sources for the transformation of six
heteroatom functionalized FPP derivatives to create six
unnatural heteroatom-modified macrocyclic terpenoids. We
encountered that viridiflorene synthase (Tps32) clearly yielded
vetispiradiene instead of viridiflorene. Most remarkably, Bot2
furnished a tricyclic product from FPP derivative 18 in 36% yield.
The olfactoric analysis revealed an ethereal, peppery and
camphoric scent for tricyclic terpenoid 25. In this work we paved
presilphiperfolan-8--ol (9) were unequivocally determined
spectroscopically[17], by derivatization to silphiperfol-6-enes,[17] by
X-ray crystallographic analysis of the p-nitrobenzoate[18] and
recently by total synthesis.[19] Therefore, we assume that the C-
atom marked in grey in 25 is (R)-configured.[20]
Unlike other authors[21] Cane and coworkers proposed that the
stereochemistry of the caryophyllenyl cation 38 formation is cis.[14]
This assumption was based on the use of specifically deuterated
FPPs and on the analysis of the deuterated presilphiperfolan-8-β-ols
formed with respect to the presence and position of the label. It is
further argued that formation of the cyclobutyl carbinyl cation 38
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10.1002/anie.201805526
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COMMUNICATION
the way to create new terpenoid backbones to serve as starting
point for semisynthetic derivatizations such as oxidations,
creating potentially new terpenoids with new olfactoric properties.
[8]
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General experimental procedure for biotransformations with unnatural FPPs:
Biotransformations were carried out in a fed-batch mode. The initial conditions
such as concentrations of enzyme, substrate and magnesium, the pH-value,
temperature additives such as Tween, pyrophosphatase (from S. cerevisiae)
and a hydrophobic adsorber (Amberlite XAD-4) are listed in the SI. The
biotransformations were carried out in an orbital shaker while the
pyrosphosphate derivatives 18-23 were added either portionwise or
continuously through a syringe. When biotransformations took longer than 4
hours new portions of enzyme were added at intervals of 3-5 h. Under these
circumstances also 1 to 2 portions of pyrophosphatase were added. Then,
shaking was elongated for up to 10 h. The biotransformation was terminated
by addition of proteinase K (from Tritirachium album) and an aqueous solution
of CaCl2. After 3-5 h protein precipitation had markedly decreased. Workup
was carried out either by multiple extraction with pentane or by removal of the
hydrophobic adsorber. This was washed with water before the product was
eluted with pentane. The mixture was dried (MgSO4) and concentrated under
mildly reduced pressure (900 mbar) followed by a flow of nitrogen gas at -5°C.
Results of preparative biotransformations (isolated yields): a) Bot2,
pyrophosphate 17: 46% yield of 9; b) Bot2, pyrophosphate 18: 36% yield of
25; c) Cop4, pyrophosphate 17: 30% yield of 11; d) Cop4, pyrophosphate 20:
27% yield of 27; e) Tps32, pyrophosphate 17: 30% yield of 6; f) Tps32,
pyrophosphate 20: 20% yield of 26; g) Tps32, pyrophosphate 23: 18% yield of
30; h) GcoA, pyrophosphate 17: 16% yield of 7; i) GcoA, pyrophosphate 23:
17% yield of 31; j) PenA, pyrophosphate 17: 89% yield of 8.
[10] J. Chappell, J. Biol. Chem. 1995, 270, 7375–7381.
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Acknowledgements
We thank J. Fohrer (LUH) for support in NMR spectroscopic
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Keywords: biotransformations • farnesyl pyrophosphates •
scent • terpene cyclases • terpenoids
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G; for details see SI.
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In our hands, trichodiene synthase (Tri5), zizaene synthase (ZIZS) and
epi-isozizaene synthase (EIZS/Cyc1) with initial 1,6-cyclization did not
accept substrates 18-23.
[7]
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Entry for the Table of Contents
COMMUNICATION
Flexibility for new scents: Eight
sesquiterpene cyclases from
fungal, bacterial and plant
sources were tested to transform
6 heteroatom functionalized
farnesylpyrophosphate
derivatives. Bot2 yielded a novel
tricyclic product in 36 % yield. The
olfactoric analysis revealed an
ethereal, peppery and camphoric
scent.
C. Oberhauser, V. Harms, K.
Seidel, B. Schröder, K.
Ekramzadeh, S. Beutel, L.
Lauterbach, J. S. Dickschat, A.
Kirschning
Page No. – Page No.
Exploiting the synthetic
potential of sesquiterpene
cyclases for generating
unnatural terpenoids
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