Early and Late Steps of Quinine Biosynthesis

Article abstract of DOI:10.1021/acs.orglett.1c00206

The enzymatic basis for quinine 1 biosynthesis was investigated. Transcriptomic data from the producing plant led to the discovery of three enzymes involved in the early and late steps of the pathway. A medium-chain alcohol dehydrogenase (CpDCS) and an esterase (CpDCE) yielded the biosynthetic intermediate dihydrocorynantheal 2 from strictosidine aglycone 3. Additionally, the discovery of an O-methyltransferase specific for 6′-hydroxycinchoninone 4 suggested the final step order to be cinchoninone 16/17 hydroxylation, methylation, and keto-reduction.

Full text of DOI:10.1021/acs.orglett.1c00206

pubs.acs.org/OrgLett
Letter
Early and Late Steps of Quinine Biosynthesis
Francesco Trenti, Kotaro Yamamoto, Benke Hong, Christian Paetz, Yoko Nakamura,
and Sarah E. O’Connor*
Cite This: Org. Lett. 2021, 23, 1793−1797
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: The enzymatic basis for quinine 1 biosynthesis was investigated. Transcriptomic data from the producing plant led to
the discovery of three enzymes involved in the early and late steps of the pathway. A medium-chain alcohol dehydrogenase
(CpDCS) and an esterase (CpDCE) yielded the biosynthetic intermediate dihydrocorynantheal 2 from strictosidine aglycone 3.
Additionally, the discovery of an O-methyltransferase specific for 6′-hydroxycinchoninone 4 suggested the final step order to be
cinchoninone 16/17 hydroxylation, methylation, and keto-reduction.
compounds were identified by comparison with purchased
standards. RNA was prepared from the same organs and was
subjected to sequencing.
uinine 1 is an alkaloid produced by Cinchona trees of
Q the Rubiaceae family, historically used as an antima-
larial drug and as a flavor ingredient in beverages such as tonic
water.1−3 Although the total synthesis of quinine has been
achieved,4 the enzymes of this biosynthetic pathway remain
undiscovered. In the present work, we combined metabolomics
and transcriptomics data from different tissues of Cinchona
pubescens to identify genes involved in the biosynthesis of
quinine and related compounds. We report the discovery of
the enzymes required to form the early biosynthetic
intermediate dihydrocorynantheal 2 from strictosidine agly-
cone 3 via reduction and esterase-triggered decarboxylation.
Additionally, we identified an O-methyltransferase specific for
6′-hydroxycinchoninone 4, suggesting a preferred order for the
late steps of quinine biosynthesis in planta.
Methanolic extracts from roots, stems, and leaves in different
developmental stages of the quinine-producing plant C.
pubescens were subjected to untargeted metabolomic analysis.
Roots and stem presented a similar metabolomic profile,
showing the accumulation of quinine 1 as well as the
derivatives quinidine 5, cinchonine 6, and cinchonidine 7.
Notably, dihydroquinine 8, dihydroquinidine 9, dihydrocin-
chonine 10, and dihydrocinchonidine 11, derivatives in which
the terminal methylene group is reduced, were also observed in
roots and stem. Leaves contained 6, 7, 10, and 11 but not the
methoxy derivatives 1, 5, 8, and 9 (Figure 1, Figure S1). These
Figure 1. Metabolomic analysis of Cinchona alkaloids in plant roots,
stems, and leaves.
Received: January 19, 2021
Published: February 24, 2021
© 2021 The Authors. Published by
American Chemical Society
https://dx.doi.org/10.1021/acs.orglett.1c00206
Org. Lett. 2021, 23, 1793−1797
1793

Organic Letters
pubs.acs.org/OrgLett
Letter
To identify biosynthetic gene candidates from the RNA-seq
data set, we first developed a hypothesis for the enzymatic
transformations required for quinine biosynthesis based on
previously reported feeding studies in Cinchona spp. as a
starting point. Hay et al. conclusively demonstrated through
feeding of radiolabeled secologanin and tryptamine that 1 is a
derivative of strictosidine 12, which is the central intermediate
in the biosynthesis of all monoterpene indole alkaloids (MIAs)
including aspidosperma, iboga, and quinoline scaffolds.5,6
Battersby et al. used evidence from in planta tracer experiments
to propose the early biosynthetic intermediate corynantheal
13.7 Because nearly all MIA pathways begin with the
deglycosylation of strictosidine 12, we proposed that
strictosidine aglycone 3 is reduced to form corynantheine
aldehyde 14, which can be de-esterified to enable decarbox-
ylation to form 13 (Scheme 1). Battersby proposed that
which catalyzes hydrolysis of the methyl ester of an MIA,
triggering a spontaneous decarboxylation (Figure S2).13 We
reasoned that reduction catalyzed by a geissoschizine synthase
orthologue followed by hydrolysis and decarboxylation
catalyzed by a polyneuridine aldehyde esterase orthologue
would yield corynantheal 13 from strictosidine aglycone. We
further hypothesized that orthologues of tabersonine 16-
hydroxylase and 16-hydroxytabersonine O-methyltransferase
could be responsible for the late-stage methoxylation of the
quinoline scaffold, namely, the conversion of cinchonidine 7 to
quinine 1. We focused on transcripts that displayed high
expression levels in stem and roots, correlating with the
presence of 1 in those organs. Ultimately, 16 MDHs, 6
esterases, 20 P450s, and 7 OMTs were cloned and successfully
expressed in E. coli (MDHs, OMTs, esterases) or S. cerevisiae
(P450s).
All 16 MDH/geissoschizine synthase orthologue candidates
cloned from C. pubescens were tested in combination with C.
roseus strictosidine glucosidase (CrSGD) using strictosidine 12
as a substrate to generate strictosidine aglycone 3.14,15 A single
MDH completely consumed the strictosidine aglycone 3
starting material, with the major product 18 having a detected
mass of m/z 355.20 (Figure 2, Figure S3). Although the
Scheme 1. Key Intermediates on the Hypothetical Pathway
a
of Quinine Biosynthesis
a
Steps elucidated in this study are highlighted in red.
corynantheal 13 could be converted through a series of redox
reactions to cinchonaminal 15.7a The indole moiety of
cinchonaminal 15 could then rearrange to the quinoline
structure found in cinchoninone 16 and cinchonidone 17,
again presumably through redox transformations (Scheme
1).7b Note that for simplicity, we refer to this mixture of 16
and 17, which are epimers, as cinchoninone 16/17. Isaac et al.
observed the NADPH-dependent keto-reduction of the
desmethoxy derivatives 16/17 to the epimers cinchonine 6
and cinchonidine 7 using protein extracts from C. ledgeriana
suspension cultures (Scheme 1).8 Finally, the hydroxylation
and methylation of cinchonine 6 and cinchonidine 7 would
subsequently yield quinidine 5 and quinine 1, respectively.
Note that the order of reduction, hydroxylation, and O-
methylation to go from 16/17 to 1 is not known.
On the basis of this biosynthetic hypothesis, RNA-seq data
were mined for oxidases, reductases, esterases, and O-
methyltransferases. We hypothesized that quinine biosynthetic
genes would be evolutionarily related to other MIA
biosynthetic enzymes. Therefore, we limited our search
among these classes of enzymes to orthologues of MIA
biosynthetic genes from other plant species that have been
previously reported (Figure S2).9,10 Specifically, we searched
the C. pubescens RNA-seq data for orthologues of geissoschi-
zine synthase, a medium-chain alcohol dehydrogenase (MDH)
that reduces strictosidine aglycone in vinblastine biosynthesis
in Catharanthus roseus;11 orthologues of tabersonine-16-
hydroxylase, a cytochrome P450 (P450) that hydroxylates an
MIA in C. roseus;12a orthologues of 16-hydroxytabersonine O-
methyltransferase (OMT), a methyl transferase that methylates
an aromatic hydroxyl group of an MIA;12b and orthologues of
polyneuridine aldehyde esterase from Rauwolfia serpentina,
Figure 2. TIC traces of enzymatic assays featuring combinations of
CpDCS and CpDCE.
expected m/z of corynantheine aldehyde 14 is m/z 353.19, we
speculated that the product of this enzyme could be the over-
reduced product dihydrocorynantheine aldehyde 18, the
putative biosynthetic intermediate for the dihydro-quinine
compounds 8−11. Because metabolomic analysis showed that
members of the dihydro-series of quinine-related compounds
were highly abundant in these plant tissues, it was not
surprising to find a reductase capable of catalyzing this reaction
(Figure 1). Unfortunately, efforts to purify this compound
resulted only in decomposition. However, when this enzymatic
product was further incubated with one of the six
polyneuridine aldehyde esterase orthologues, a new broad
peak with m/z 297.20 was observed. This molecular weight is
1794
https://dx.doi.org/10.1021/acs.orglett.1c00206
Org. Lett. 2021, 23, 1793−1797

Organic Letters
pubs.acs.org/OrgLett
Letter
consistent with the structure of dihydrocorynantheal 2, the
product of methyl ester hydrolysis and the subsequent
decarboxylation of 18 (Figure 2). Approximately 1 mg of
this enzymatic product was purified and subjected to full NMR
characterization, which validated the predicted structure
(Figures S4−S26, Table S3). Therefore, we named the
MDH DihydroCorynantheine aldehyde Synthase (CpDCS)
and the esterase DihydroCorynantheine aldehyde Esterase
(CpDCE).
cinchonidine 7 failed to yield any new product. The
hydroxylation of the quinoline scaffold may not be catalyzed
by an orthologue of tabersonine 16-hydroxylase, and more
extensive candidate screening is required to identify the
enzyme for this step. However, an orthologue of 16-
hydroxytabersonine O-methyltransferase (CpOMT1) appeared
to methylate a semisynthetically generated standard of 6′-
hydroxycinchoninone 4 (Figure 3A, Figures S28−S31). The
We then considered the mechanism that the reductase
CpDCS uses to catalyze the formation of 18 (Scheme 2a).
a
Scheme 2. Formation of (Dihydro)corynantheal
a
(a) Proposed mechanism of CpCDS, leading to the dihydro-series
(route A) or quinine (route B). (b) Proposed action of CpDCE.
After SGD-catalyzed deglycosylation, the open aglycone 3
forms corynantheine iminium 19, which exists in equilibrium
with dehydrogeissoschizine 20. The reductase CpDCS might
act on the iminium of 19 to yield the quinine intermediate
corynantheine aldehyde 14 (Scheme 2a, route A). Alter-
natively, when the alkene is in conjugation with the iminium,
as is the case with dehydrogeissoschizine 20, the reductase can
catalyze a 1,4-reduction of 20 followed by a 1,2 reduction of
22, leading to the dihydro-series (Scheme 2a, route B). This
consideration opens the possibility that the cell environment of
Cinchona might regulate the flux toward quinine or the
dihydro-series by tuning the equilibrium of dehydrogeissoschi-
zine and corynantheine iminium 19, thereby enhancing the
variety of quinine-like scaffolds. Surprisingly, we never
observed evidence of formation of corynantheine aldehyde
14 (m/z 353.19) or corynantheal 13 (m/z 295.18) with any of
the 16 MDH candidates, even under limiting concentrations of
NADPH cofactor. Either an as-yet unidentified reductase is
responsible for the formation of corynantheine aldehyde 14 or
the conditions that were used for our in vitro assays favor the
formation of the dehydrogeissoschizine isomer 20 (Scheme 2);
however, because CpDCS catalyzes a 1,2-iminium reduction in
the formation of dihydrocorynantheine aldehyde 18, it seems
reasonable that this enzyme could also catalyze the 1,2-
reduction of 19 to yield 14 (Scheme 2a). De-esterification of
18 by CpDCE then leads to a carboxylic acid that could
spontaneously decarboxylate (Scheme 2b).
Figure 3. TIC traces of CpOMT1 assays using substrates 4 (A), 25
(B), and 24 (C) incubated with CpOMT1 for 12 h. Notice that the
ketone analogs 4 are in an inseparable tautomeric equilibrium, as
indicated by the two peaks in the chromatogram.
product was confirmed to be 6′-methoxycinchoninone 23 by
comparison with an authentic chemical standard. This
methyltransferase was also assayed with 6′-hydroxycinchoni-
dine 25 and 6′-hydroxycinchonine 24 standards, but only a
modest amount of 25 was converted to quinine 1 (Figure
3B,C). Quantification of substrate conversion with CpOMT1
in an endpoint assay with 100 μM substrate showed that only
17
1% of 6′-hydroxycinchonidine 25 was consumed,
whereas 80.4
consumed (Figures S32 and S33).
0.4% of 6′-hydroxycinchoninone 4 was
The higher relative activity of CpOMT1 for 6′-hydrox-
ycinchoninone 4 suggests that the hydroxylation and
methylation of the quinine scaffold on 16/17, followed by
NADPH-dependent keto-reduction, may be the preferred
biosynthetic route in planta (Scheme 3, route A). However,
we cannot exclude on the basis of the in vitro assays that the
biosynthesis of quinine 1 (and quinidine 5) might also go
through hydroxylation and O-methylation of chinchonidine 7
(and chinchonine 6), respectively (Scheme 3, route B).
In conclusion, we identified a medium-chain alcohol
dehydrogenase that reduces strictosidine aglycone 3 to yield
We next tested whether the orthologues of tabersonine 16-
hydroxylase and 16-hydroxytabersonine O-methyltransferase
were responsible for the late-stage methoxylation of the
quinoline scaffold. An assay of 20 tabersonine-16-hydroxylase
orthologues with cinchoninone 16/17, cinchonine 6, and
1795
https://dx.doi.org/10.1021/acs.orglett.1c00206
Org. Lett. 2021, 23, 1793−1797

Organic Letters
pubs.acs.org/OrgLett
Letter
a
Benke Hong − Department of Natural Product Biosynthesis,
Max Planck Institute for Chemical Ecology, 07745 Jena,
Germany
Scheme 3. Proposed Order of Final Steps
Christian Paetz − Department of Natural Product
Biosynthesis, Max Planck Institute for Chemical Ecology,
07745 Jena, Germany
Yoko Nakamura − Department of Natural Product
Biosynthesis, Max Planck Institute for Chemical Ecology,
07745 Jena, Germany
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c00206
a
In vitro enzyme assays suggest that the substrate specificity of
Author Contributions
CpOMT1 favors route A over route B.
The manuscript was written through the contributions of all
authors. F.T. and S.E.O. designed the experiments. F.T.
performed the metabolomics, cloning, protein expression, and
enzyme assays. K.Y. isolated the RNA. B.H. synthesized the
cinchona standards. C.P. and Y.N. performed the NMR
analysis. All authors have given approval to the final version of
the manuscript.
dihydrocorynantheine aldehyde 18. This chemical step also
occurs in the biosynthesis of mitragynine, and we could
identify an additional orthologue in the producer plant
Mitragyna speciosa. We named these genes CpDCS (GenBank
MW456554) and MsDCS (GenBank MW456555). Although
we only observed the over-reduced product 18 that leads to
the dihydro-series 8−11, we hypothesize that control of the
equilibrium of strictosidine aglycone 3 would yield corynan-
theine aldehyde 14. We also discovered an orthologue of
polyneuridine aldehyde esterase from Rauwolfia serpentina,
CpDCE (GenBank MW456556) that acts on 18, leading to
demethylation, decarboxylation, and the formation of dihy-
drocorynantheal 2. Finally, the discovery of CpOMT1
(GenBank MW456557) that acts preferentially on 6′-
hydroxycinchoninone 4 rather than on the keto-reduced 6′-
hydroxycinchonine 24 and 6′-hydroxycinchonidine 25 suggests
that hydroxylation and methylation of the quinine scaffold
occur on cinchoninone 16/17 followed by keto-reduction. The
discovery of the enzymes that catalyze these three steps sets
the stage for the elucidation of the remaining enzymes involved
in quinine biosynthesis.
Notes
The authors declare no competing financial interest.
Transcriptomic data is uploaded at Data Dryad (https://doi.
org/10.5061/dryad.z34tmpgcc).
ACKNOWLEDGMENTS
■
We gratefully acknowledge the European Research Council
(788301) and Horizon 2020 (MIAMI) for financial support of
this work. B.H. is grateful to the Alexander von Humboldt
Foundation for the postdoctoral fellowship. We thank
Mohamed Omar Kamileen (Max Planck Institute for Chemical
Ecology, MPI-CE) for the assistance with preparing the RNA,
Maritta Kunert (MPI-CE) and Delia Ayled Serna Guerrero
(MPI-CE) for the assistance with the mass spectrometry, and
Eva Rothe and the Greenhouse team (MPI-CE) for the
cultivation of plants. C. pubescens was obtained through the
Edinburgh Botanical Garden. The plant art in Figure 1 was
generated with BioRender.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.1c00206.
REFERENCES
■
(1) Achan, J.; Talisuna, A. O.; Erhart, A.; Yeka, A.; Tibenderana, J.
K.; Baliraine, F. N.; Rosenthal, P. J.; D’Alessandro, U. Quinine, an old
anti-malarial drug in a modern world: role in the treatment of malaria.
Malar. J. 2011, 10, 144.
(2) Eyal, S. The fever tree: from malaria to neurological diseases.
Toxins 2018, 10, 491.
Detailed biochemical, molecular biology, metabolomic
and synthetic procedures, enzyme assays, compound
characterization, NMR spectra, additional experimental
procedures, and spectroscopic data for all new
compounds (PDF)
(3) Yohalem, S. B. Quinine in tonic water. JAMA 1953, 153 (14),
1304.
AUTHOR INFORMATION
Corresponding Author
■
(4) (a) Woodward, R. B.; Doering, W. E. The total synthesis of
quinine. J. Am. Chem. Soc. 1944, 66, 849. (b) Stork, G.; Niu, D.;
Fujimoto, R. A.; Koft, E. R.; Balkovec, J. M.; Tata, J. R.; Dake, G. R.
The first stereoselective total synthesis of quinine. J. Am. Chem. Soc.
2001, 123, 3239−3242. (c) Smith, A. C.; Williams, R. M. Rabe rest in
peace: confirmation of the rabe-kindler conversion of D-quinotoxine
into quinine: experimental affirmation of the Woodward-Doering
formal total synthesis of quinine. Angew. Chem., Int. Ed. 2008, 47,
1736−1740. (d) O’Donovan, D. H.; Aillard, P.; Berger, M.; de la
Torre, A.; Petkova, D.; Knittl-Frank, C.; Geerdink, D.; Kaiser, M.;
Maulide, N. C-H activation enables a concise total synthesis of
quinine and analogues with enhanced antimalarial activity. Angew.
Chem., Int. Ed. 2018, 57, 10737−10741. (e) Lee, Y.; Chen, D. Y.-K. A
Local-Desymmetrization-Based Divergent Synthesis of Quinine and
Quinidine. Angew. Chem., Int. Ed. 2019, 58, 488−493.
Sarah E. O’Connor − Department of Natural Product
Biosynthesis, Max Planck Institute for Chemical Ecology,
07745 Jena, Germany; orcid.org/0000-0003-0356-6213;
Email: oconnor@ice.mpg.de
Authors
Francesco Trenti − Department of Natural Product
Biosynthesis, Max Planck Institute for Chemical Ecology,
07745 Jena, Germany
Kotaro Yamamoto − Department of Natural Product
Biosynthesis, Max Planck Institute for Chemical Ecology,
07745 Jena, Germany
1796
https://dx.doi.org/10.1021/acs.orglett.1c00206
Org. Lett. 2021, 23, 1793−1797

Organic Letters
pubs.acs.org/OrgLett
Letter
(5) Hay, C. A.; Anderson, L. A.; Phillipson, J. D.; Curless, D.;
Brown, R. T. In vitro cultures of Cinchona species. Part II: Time-
course studies on the uptake of radio-labelled alkaloid precursors and
alkaloids by C. ledgeriana root suspension cultures. Plant Cell, Tissue
Organ Cult. 1987, 9, 197−206.
(6) Guirimand, G.; Courdavault, V.; Lanoue, A.; Mahroug, S.;
Guihur, A.; Blanc, N.; Giglioli-Guivarc’h, N.; St-Pierre, B.; Burlat, V.
Strictosidine activation in Apocynaceae: towards a “nuclear time
bomb”? BMC Plant Biol. 2010, 10, 182.
(7) (a) Battersby, A. R.; Parry, R. J. Biosynthesis of the cinchona
alkaloids: middle stages of the pathway. J. Chem. Soc. D 1971, 30−31.
(b) Battersby, A. R.; Parry, R. J. Biosynthesis of the cinchona
alkaloids: late stage of the pathway. J. Chem. Soc. D 1971, 31−32.
(8) Isaac, J. E.; Robins, R. J.; Rhodes, M. J. C. Cinchoninone:
NADPH oxidoreductases I and II - novel enzymes in the biosynthesis
of quinoline alkaloids in Cinchona ledgeriana. Phytochemistry 1987,
26, 393−399.
(9) Caputi, L.; Franke, J.; Farrow, S. C.; Chung, K.; Payne, R. M. E.;
Nguyen, T.-D.; Dang, T.-T. T.; Soares Teto Carqueijeiro, I.;
Koudounas, K.; Dugé de Bernonville, T.; Ameyaw, B.; Jones, D.
M.; Vieira, I. J. C.; Courdavault, V.; O’Connor, S. E. Missing enzymes
in the biosynthesis of the anticancer drug vinblastine in Madagascar
periwinkle. Science 2018, 360, 1235−1239.
(10) Dang, T. T.; Franke, J.; Carqueijeiro, I. S. T.; Langley, C.;
Courdavault, V.; O’Connor, S. E. Sarpagan bridge enzyme has
substrate-controlled cyclization and aromatization modes. Nat. Chem.
Biol. 2018, 14, 760−763.
(11) Tatsis, E. C.; Carqueijeiro, I.; Dugé de Bernonville, T.; Franke,
J.; Dang, T. T.; Oudin, A.; Lanoue, A.; Lafontaine, F.; Stavrinides, A.
K.; Clastre, M.; Courdavault, V.; O’Connor, S. E. A three enzyme
system to generate the Strychnos alkaloid scaffold from a central
biosynthetic intermediate. Nat. Commun. 2017, 8, 316.
̈
(12) (a) Schroder, G.; Unterbusch, E.; Kaltenbach, M.; Schmidt, J.;
̈
Strack, D.; De Luca, V.; Schroder, J. Light-induced cytochrome P450-
dependent enzyme in indole alkaloid biosynthesis: tabersonine 16-
hydroxylase. FEBS Lett. 1999, 458, 97−102. (b) Levac, D.; Murata, J.;
Kim, W. S.; De Luca, V. Application of carborundum abrasion for
investigating the leaf epidermis: molecular cloning of Catharanthus
roseus 16-hydroxytabersonine-16-O-methyltransferase. Plant J. 2008,
53 (2), 225−236.
(13) Dogru, E.; Warzecha, H.; Seibel, F.; Haebel, S.; Lottspeich, F.;
̈
Stockigt, J. The gene encoding polyneuridine aldehyde esterase of
monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of
the alpha/beta hydrolase super family. Eur. J. Biochem. 2000, 267,
1397−1406.
(14) Maresh, J. J.; Giddings, L. A.; Friedrich, A.; Loris, E. A.;
̈
Panjikar, S.; Trout, B. L.; Stockigt, J.; Peters, B.; O’Connor, S. E.
Strictosidine synthase: mechanism of a Pictet-Spengler catalyzing
enzyme. J. Am. Chem. Soc. 2008, 130, 710−723.
(15) Luijendijk, T. J. C.; Stevens, L. H.; Verpoorte, R. Purification
and characterisation of strictosidine β-D-glucosidase from Cathar-
anthus roseus cell suspension cultures. Plant Physiol. Biochem. 1998, 36
(6), 419−425.
1797
https://dx.doi.org/10.1021/acs.orglett.1c00206
Org. Lett. 2021, 23, 1793−1797