Diversification of monoterpene indole alkaloid analogs through cross-coupling

Article abstract of DOI:10.1021/ol401179k

Catharanthus roseus monoterpene indole alkaloid analogs have been produced via a combination of biosynthetic and chemical strategies. Specifically, introduction of a chemical handle - a chlorine or a bromine - into the target molecule by mutasynthesis, followed by postbiosynthetic chemical derivatization using Pd-catalyzed Suzuki-Miyaura cross-coupling reactions robustly afforded aryl and heteroaryl analogs of these alkaloids.

Full text of DOI:10.1021/ol401179k

ORGANIC
LETTERS
2013
Vol. 15, No. 11
2850–2853
Diversification of Monoterpene Indole
Alkaloid Analogs through Cross-Coupling
Weerawat Runguphan‡, and Sarah E. O’Connor*,†,§
The John Innes Centre, Department of Biological Chemistry, Norwich, NR4 7UH, U.K.,
The University of East Anglia, School of Chemistry, Norwich NR4 7TJ, U.K., and
Massachusetts Institute of Technology, Department of Chemistry, 77 Massachusetts
Avenue, Cambridge, Massachusetts 02139, United States
Sarah.O’Connor@jic.ac.uk
Received April 26, 2013
ABSTRACT
Catharanthus roseus monoterpene indole alkaloid analogs have been produced via a combination of biosynthetic and chemical strategies.
Specifically, introduction of a chemical handle;a chlorine or a bromine;into the target molecule by mutasynthesis, followed by postbiosynthetic
chemical derivatization using Pd-catalyzed Suzuki-Miyaura cross-coupling reactions robustly afforded aryl and heteroaryl analogs of these alkaloids.
Modification of natural products can yield analogs, or
“unnatural products”, with improved or novel medicinal
properties.1 Traditional approaches to generate natural
product analogs include total and semisyntheses, precursor-
directed biosynthesis, mutasynthesis, and combinatorial
biosynthesis.2 More recently, these strategies have been
combined to yield an even broader array of compounds in
a cost-effective and rapid manner. For example, analogs of
the uridyl peptide antibiotic pacidamycin have been suc-
cessfully generated by the genetic manipulation of a bio-
synthetic pathway to yield unnatural halogenated analogs,
followed by postbiosynthetic chemical derivatization.3
Monoterpene indole alkaloid biosynthesis in the medi-
cinal plant Catharanthus roseus, which is only partially
characterized at the enzymatic level, produces a variety of
alkaloids with diverse biological activities and complex
molecular architecture. While numerous semi-2a and bio-
synthetic4 methods have been applied to generate analogs
of these alkaloids, new approaches to broaden the scope
of modifications that can be made to these biologically
important scaffolds would be advantageous. For example,
while halogenation in and of itself often has profound
effects on the bioactivity of natural products, the halides
also serve as a useful handle for further chemical deriva-
tization. Here we demonstrate that the introduction of
a chemical handle, a halide, into the indole moiety of the
monoterpene indole alkaloids by mutasynthesis or by
introducing prokaryotic halogenases into plant cell cul-
tures, followed by subsequent chemical derivatizations
using Pd-catalyzed SuzukiÀMiyaura cross-coupling reac-
tions, robustly afforded aryl and heteroaryl analogs of
monoterpene indole alkaloids.
† The John Innes Centre.
‡ Massachusetts Institute of Technology.
§ The University of East Anglia.
Current address: Joint BioEnergy Institute, 5885 Hollis Street, Emeryville,
CA 94608, USA.
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r
10.1021/ol401179k
Published on Web 05/28/2013
2013 American Chemical Society

Figure 1. Strategy for synthetic diversification of monoterpene indole alkaloids using Pd-catalyzed SuzukiÀMiyaura coupling reaction.
Alkaloid analogs with chlorine and bromine at the 7-position of the indole ring were obtained by culturing transgenic tryptophan
decarboxylase-suppressed hairy root cultures in the presence of 7-chlorotryptamine 1a or 7-bromotryptamine 1b (a). Alkaloid analogs
with chlorine at the 6-position of the indole ring were obtained by culturing hairy root cultures that express a mutant strictosidine
synthase enzyme (V214M) in the presence of 6-chlorotryptamine 1c (b). Alkaloid analogs with chlorine at the 7-position of the indole
ring could also be obtained from hairy root cultures that express prokaryotic halogenases, RebH and RebF (c).
Monoterpene indole alkaloids are derived from trypt-
amine 1. Endogenous tryptamine biosynthesis can be sup-
pressed by silencing the enzyme tryptophan decarboxylase
in C. roseus hairy root culture. This silenced culture can
then be subjected to precursor directed feeding, in which
halogenated tryptamine 1 precursors are cocultivated with
the root tissue. This strategy, termed mutasynthesis, has
allowed generation of chlorinated and brominated mono-
terpene indole analogs.5 The desired, halogenated alkaloid
profile is streamlined in these tissues since no natural,
nonhalogenated alkaloids derived from endogenous trypt-
amine 1 are present. We opted to use these tryptophan
decarboxylase-suppressed hairy root lines to obtain 7-ha-
logenated analogs for subsequent cross-coupling derivati-
zation. Alkaloid analogs with chlorine and bromine at the
7-position of the indole ring were obtained by culturing
transgenic tryptophan decarboxylase-suppressed hairy
root cultures in liquid Gamborg’s B5 media in the presence
of either 0.6 mM 7-chlorotryptamine 1a or 7-bromotrypt-
amine 1b (Figure 1a). After one week of culture, the plant
material was lysed and alkaloids were extracted into
methanol. Liquid chromatography mass spectral (LC-MS)
analysis of these extracts indicated accumulation of one
major product, 12-chloro-19,20-dihydroakuammicine 2a
or 12-bromo-19,20-dihydroakuammicine 2b, as previously
demonstrated.7
With halogenated analogs readily available as a compo-
nent of crude plant extracts, we then set out to explore
chemical functionalization reactions that would derivatize
halogenated analogs under mild reaction conditions. While
cross-coupling reactions of aryl iodides and bromides with
arylboronic acids are well precedented, reactions involving
aryl chlorides require the use of catalyst systems that employ
highly active and sterically demanding ligands, such as
Sphos.6 Therefore, we turned our attention to the Pd-
catalyzed SuzukiÀMiyaura cross-coupling reaction condi-
tions refined by Goss et al. to derivatize chlorinated pada-
mycin analogs.3 Since the nonhalogenated metabolites that
are present in crude extracts are unlikely to interfere with the
progression of the cross-coupling reactions, crude plant
extracts containing chlorinated or brominated alkaloids
were used directly in the cross-coupling reactions without
any purification.
(6) Barder, T. E.; Walker, S. D.; Martinelli, J. R.; Buchwald, S. L. J.
Am. Chem. Soc. 2005, 127, 4685.
(7) Runguphan, W.; Qu, X.; O’Connor, S. E. Nature 2010, 468, 461.
(5) Runguphan, W.; Maresh, J. J.; O’Connor, S. E. Proc. Natl. Acad.
Sci. U.S.A. 2009, 106, 13673.
Org. Lett., Vol. 15, No. 11, 2013
2851

used as substrates for the SuzukiÀMiyaura cross-coupling
reactions with three representative boronic acids, phenyl-
boronic acid 3d, 4-methoxyphenylboronic acid 3f, and
4-methylphenylboronic acid 3g. LC-MS analysis of crude
reaction mixtures demonstrated that the reactions went to
completion in <1 h at 90 °C (Figure 2b).
Table 1. SuzukiÀMiyaura Cross-Coupling Reactions of 12-
Chloro-19,20-dihydroakuammicine 2a with Aryl and Hetero-
aryl Boronic Acids 3dÀi
Figure 2. LC-MS traces showing the progression of SuzukiÀ
Miyaura cross-coupling reactions of TDC-suppressed hairy
root extracts containing 12-chloro-19,20-dihydroakuammicine
2a (m/z 359, bottom) and 12-bromo-19,20-dihydroakuammi-
cine 2b (m/z 403, top) with phenylboronic acid 3d to form 12-
phenyl-19,20-dihydroakuammicine 2d (m/z 401) (a). LC-MS
traces showing the progression of SuzukiÀMiyaura cross-cou-
pling reactions of RebH/F hairy root extracts containing 2a (m/z
359) with phenylboronic acid 3d (bottom), 4-methylphenylboro-
nic acid 3g (middle), and 4-methoxyphenylboronic acid 3f (top).
Expected products were 12-phenyl-19,20-dihydroakuammicine
2d (m/z 401), 12-(4-methylphenyl)-19,20-dihydroakuammicine
2g (m/z 415), and 12-(4-methoxyphenyl)-19,20-dihydroakuam-
micine 2f (m/z 431) (b).
a Compounds were purified using reversed-phase preparative HPLC.
We also wished to assess the potential for derivatization
at an additional position at the indole ring. However,
incorporation of tryptamine analogs with substituents at
the 6-position present a challenge, since these substrate
analogs are not accepted by an early biosynthetic enzyme,
strictosidine synthase. Therefore, alkaloids incorporating
6-halo-tryptamine analogs cannot be produced using the
tryprophan decarboxylase-silenced cultures described in
the preceding section. Instead, we used hairy root cultures
that express a mutant strictosidine synthase enzyme
(V214M) that has been designed to turnover these try-
ptamine analogs.8 These cultures were grown in liquid
Gamborg’s B5 media in the presence of 0.6 mM 6-chloro-
tryptamine 1c, and after one week of culture, the plant
We performed the SuzukiÀMiyaura cross-coupling re-
actions using six aryl and heteroaryl boronic acid sub-
strates 3dÀi (Table 1). Gratifyingly, small-scale coupling
reactions of crude methanolic extracts containing either
12-chloro-19,20-dihydroakuammicine 2a or 12-bromo-
19,20-dihydroakuammicine 2b from 100 mg of fresh hairy
roots with each boronic acid went to completion in <1 h at
90 °C, as monitored by LC-MS, to form products 2dÀi
(Figure 2a). Product identity on this small scale was
evidenced by high-resolution mass spectrometry, tandem
MS/MS, and UV absorption spectra (Supporting Infor-
mation (SI)).
Crude extracts from transgenic hairy roots that express
prokaryotic 7-halogenases (RebF and RebH)7 were also
(8) Runguphan, W.; O’Connor, S. E. Nat. Chem. Biol. 2009, 5, 151.
Org. Lett., Vol. 15, No. 11, 2013
2852

11-chlorotabersonine 6c were good substrates for the
SuzukiÀMiyaura cross-coupling reactions using the same
set of aryl and heteroaryl boronic acid substrates used to
derivatizethe 7-halogenatedalkaloids (Figure3). Ofthe six
boronic acids tested, only the electron-deficient 4-carboxy-
phenyl boronic acid did not yield the desired analogs.
High-resolution mass spectrometry, tandem MS/MS,
and UV absorption spectra were used to confirm the
identities of all alkaloid analogs (SI).
Encouraged by these results, we performed large-scale
cross-coupling reactions to more rigorously structurally
characterize several representative products. Crude ex-
tracts containing 12-chloro-19,20-dihydroakuammicine
2a from 16 g of hairy roots were used as the substrate.
Three representative boronic acids;phenyl, 4-fluorophe-
nyl, and furanyl boronic acids (1d, 1e, and1i, respectively);
were tested under these scaled-up conditions. Standard
workup of the reaction followed by reversed-phase HPLC
purification of the reaction mixture afforded milligram
quantities of these analogs. 1H, 13C, and 19F (when
applicable) NMR spectroscopy, in addition to high-reso-
lution mass spectrometry, tandem MS/MS, and UV ab-
sorption spectra, confirmed the structural identities of the
resulting products (SI).
In summary, we have applied a chemogenetic ap-
proach;installation of a chemical handle onto the alka-
loid framework via mutasynthesis or precursor directed
biosynthesis, followed by chemical derivatization;to pro-
duce analogs of C. roseus monoterpene indole alkaloids.
Using this strategy, we have successfully obtained milli-
gram quantities of three alkaloid analogs. The effective-
ness of this approach should facilitate rational modi-
fication of other classes of plant-derived natural products
via cross-coupling for biological activity screening and
drug development.
Figure 3. LC-MS traces showing the progression of SuzukiÀ
Miyaura cross-coupling reactions of 11-chlorotabersonine 6c
(m/z 371) with phenylboronic acid 3d (bottom), 4-methylphe-
nylboronic acid 3g (middle), and 4-methoxyphenylboronic
acid 3f (top). Expected products were 11-phenyltabersonine 6d
(m/z 413), 11-(4-methylphenyl)-tabersonine 6g (m/z 427), and
11-(4-methoxyphenyl)-tabersonine 6f (m/z 443). LC-MS traces
showing the progression of SuzukiÀMiyaura cross-coupling
reactions of 6c (m/z 371) with 4-fluorophenylboronic acid 3e
(bottom), 4-carboxyphenylboronic acid 3h (middle), and fura-
nylboronic acid 3i (top). Expected products were 11-(4-
fluorophenyl)-tabersonine 6e (m/z 431), 11-(4-carboxyphenyl)-
tabersonine 6h (m/z 457), and 11-furanyl-tabersonine 6i (m/z 403).
Acknowledgment. We gratefully acknowledge support
from NIH GM074820.
material was lysed and extracted (Figure 1b). LC-MS
analysis of these extracts indicated accumulation of three
major analogs: 11-chloro-19,20-dihydroakuammicine 4c,
11-chloroakuammicine 5c, and 11-chlorotabersonine 6c
(Figure S2, SI).
Supporting Information Available. Experimental pro-
cedures and compound characterization. This material is
available free of charge via the Internet at http://pubs.acs.
org.
Crude extracts containing 11-chloro-19,20-dihy-
droakuammicine 4c, 11-chloro-akuammicine 5c, and
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 11, 2013
2853