Bypassing stereoselectivity in the early steps of alkaloid biosynthesis

Article abstract of DOI:10.1039/b916027m

Total synthesis of glycosylated seco-iridoid stereoisomers allows the identification and bypassing of the stereoselectivity of early steps in monoterpene indole alkaloid biosynthesis.

Full text of DOI:10.1039/b916027m

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Bypassing stereoselectivity in the early steps of alkaloid biosynthesis†
Peter Bernhardt,‡ Nancy Yerkes‡ and Sarah E. O’Connor*
Received 4th August 2009, Accepted 6th August 2009
First published as an Advance Article on the web 13th August 2009
DOI: 10.1039/b916027m
Total synthesis of glycosylated seco-iridoid stereoisomers
allows the identification and bypassing of the stereoselectivity
of early steps in monoterpene indole alkaloid biosynthesis.
Different heteroyohimbine alkaloid stereoisomers have differ-
ent pharmacological activities. The heteroyohimbine alkaloid
ajmalicine (raubasine) 5a acts as a smooth muscle relaxant and
10–12
as an a1 anti-adrenergic,
an a2 anti-adrenergic.
while tetrahydroalstonine 5b acts as
12
Stereochemistry often determines how a natural product interacts
with biological systems. For example, different stereosisomers
The stereogenic centers of strictosidine 3a, the central inter-
mediate for all monoterpene indole alkaloids, are either derived
from the densely functionalized secologanin dihydropyran (C-15,
C-20, and C-21) or the prochiral aldehyde carbon that is converted
to the C-3 stereogenic center in strictosidine (Scheme 1). We
recently reported that strictosidine glucosidase promotes not only
deglucosylation of strictosidine 3a, but also deglucosylation of
1
can have different pharmacological profiles. The biosynthetic
enzymes that form natural products typically allow the pro-
duction of only one stereoisomer, so fermentation of products
with alternate stereochemistry must therefore bypass the stereo-
chemical restrictions of biosynthetic pathway enzymes. Using
synthetic seco-iridoid and strictosidine starting materials we
show that the heteroyohimbine branch of monoterpene indole
alkaloid (MIA) biosynthesis in the medicinal plant Catharanthus
13
vincoside 3b (the 3-(R) diastereomer of 3a). We report here that
strictosidine glucosidase has an 80-fold higher specificity constant
2,3
(
k
cat/K
M
) for 3a compared to 3b (Table 1). The kinetics show that
the change in kcat/K is due to a change in turnover number (kcat
while the Michaelis constant (K ) remains the same for the two
diastereomers (Table 1). In the crystal structure of SGD from the
roseus has a surprisingly broad tolerance for stereochemical
perturbations in vitro.
M
)
Strictosidine synthase catalyzes the first step in monoterpene
indole alkaloid biosynthesis: an asymmetric Pictet–Spengler reac-
tion between tryptamine 1 and secologanin 2 to yield strictosidine
M
14
closely related Rauvolfia serpentina homolog (PDB: 2FJ6), the
C-3 carbon atom of strictosidine 3a is located near the surface
of the protein. We speculate that the orientation of the glucose
moiety, which is in the interior of the glucosidase, remains
unchanged during turnover of vincoside 3b, while the binding
pocket for the more distal regions of the substrate, including the
C-3 carbon, can accommodate 3b.
4,5
3
a (Scheme 1). In the second step of this alkaloid pathway, 3a is
6,7
deglucosylated by strictosidine-b-D-glucosidase. This results in
the formation of a hemiacetal that rearranges into a mixture of
products that is channeled into one of several pathway branches.
The enzymes responsible for these branching reactions are un-
known at the genetic and biochemical levels. However, entry into
the heteroyohimbine class of alkaloids (5a–c) is likely controlled
We asked whether strictosidine glucosidase can catalyze the
conversion of alternate dihydropyran stereoisomers of 3a, derived
from the C-2 and C-4 positions of secologanin 2. This has not been
tested previously, as alternate stereoisomers of 2 are not readily
available. To access alternate configurations, we applied and
expanded the previously reported synthesis of acetal-protected
8,9
by one or several NADPH-dependent reductases (Scheme 1).
15,16
9
,10-nor-secologanin aglycones. The key step is Tietze’s tan-
dem Knoevenagel–hetero-Diels–Alder (KHDA) reaction, which
assembles the dihydropyran from 7, 8, and an electron rich
dienophile (e.g. vinyl ether). We adopted this strategy to, for the
first time, obtain O-glucosylated 9,10-nor-strictosidine 12a and
its stereoisomers 12 (Scheme 2). The KHDA substrate, 2,3,4,6-
tetrabenzyl vinyl-glucose 6, was synthesized by Ir-catalyzed vinyl
17
transfer from vinyl acetate to the corresponding alcohol. After
the KHDA reaction of 6, 7, and 8, in the presence of potassium flu-
oride, the cycloadduct was subjected to methanolysis to generate
methyl ester 9. Pd-catalyzed debenzylation of 9 afforded acetal-
protected secologanin 10 as a mixture of inseparable stereoisomers.
After acetal hydrolysis, 9,10-nor-secologanin 11 was carried into
either an enzymatic or a non-enzymatic Pictet–Spengler reaction
to generate the corresponding tetrahydro-b-carboline products
Scheme 1 Heteroyohimbine biosynthesis. STS (strictosidine synthase)
and SGD (strictosidine glucosidase) catalyze reactions that lead to a
hemiacetal, which rearranges into a mixture of isomers; cathenamine 4
is shown. Reduction leads to heteroyohimbines 5a–c.
MIT Department of Chemistry, 77 Massachusetts Avenue, 18-592,
Cambridge, MA 02139, USA. E-mail: soc@mit.edu; Fax: +1 617 324 0505;
Tel: +1 617 324 0180
12a and 12 (Scheme 2).
Isomers 12 were synthesized in acidic buffer from 1 and 11
†
Electronic supplementary information (ESI) available: Chemical synthe-
sis, supplementary figures, methods. See DOI: 10.1039/b916027m
(Scheme 2), and purified by preparative HPLC. Each separable
1
‡
These authors contributed equally to this work
peak was characterized by H NMR (ESI†). Analysis of the
4
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Table 1 Steady-state kinetic constants
Strictosidine
glucosidase
kinetics
a
cat [s-1]
[M-1 s-1]
b
max [U mg-1]b
-1
-1 b
k
K
M
[mM]
k
cat/K
M
Reductase kinetics
V
K
M
[mM]
M
Vmax/K [U M mg ]
2
2
3
3
a
b
15 ± 0.8
0.15 ± 0.03 1.0 ¥ 10 ± 0.2 ¥ 10
Deglycosylated 3a
Deglycosylated 3b
0.027 ± 0.001
0.012 ± 0.0003
0.10 ± 0.02 0.26 ± 0.04
-2
-2
0.19 ± 0.01 0.15 ± 0.04 1.3 ± 0.3
0.66 ± 0.04 1.9 ¥ 10 ± 0.1 ¥ 10
a
◦
b
Activity quantified by monitoring the disappearance of starting material at pH 6.0, 30 C. 1 U = 1 mmol product formed per minute at pH 7.0,
◦
3
0 C. Apparent kinetic constants obtained from partially purified enzyme.
Scheme 2 Synthesis of 12 and 12a, 13a, and 13. a) KF, toluene; b) DBU,
CH OH, 38% over 2 steps; c) Pd/C, CH OH, 77%; d) PPTS (2 eq.,
.2 M), acetone/H O (2:1), ~10%; e) 1, C. roseus STS, NaP (0.05 M,
3
3
0
2
i
pH 7.0), 89%; f) 1, maleic acid buffer (0.01 M, pH 2.0); g) C. roseus
SGD, citrate-phosphate buffer (0.15 M, pH 6.0); h) B. stearothermophilus
a-glucosidase, almond b-glucosidase, strictosidine glucosidase, citrate
phosphate buffer (0.15 M, pH 6.0); (i) reductase activity from C. roseus
Fig. 1 A Remaining starting material after deglucosylation of 12
m/z 505.1 ± 0.5) by C. roseus strictosidine glucosidase (ii), almond
b-glucosidase (b, iii), and B. stearothermophilus a-glucosidase (a, iv)
monitored by UPLC-MS, (i) no-enzyme control; B. Reductase assay.
(
i
cell suspension culture, NADPH, NaP (0.05 M, pH 7.0).
mixture by UPLC-MS resolved six peaks, each with a mass
(
i) reduction of 12 yields two separable stereoisomers 13; (ii) reduction
of deglucosylated 12a yields a single product 13a; (iii) reduction of deglu-
cosylated d -strictosidine 3a (m/z 357); (iv) reduction of deglucosylated
+
consistent with 18,19-nor-strictosidine 12 (m/z = 505 [M + H] )
(
Fig. 1A (i): pk 1–6). In contrast, the strictosidine synthase-
4
catalyzed reaction between 1 and 11 resulted in the appearance
of a single product, 12a, which was isolated and characterized
by NMR (ESI†). The doublet of a doublet at 5.8 ppm suggested
d4-vincoside 3b (m/z 357); (v) ajmalicine 5a standard (m/z 353).
Strictosidine glucosidase could efficiently deglucosylate
tetrahydro-b-carboline 12a, the isomer with the natural 2,4-trans
configuration. When strictosidine glucosidase was incubated with
all isomers of 12, only two of the six separable peaks decreased
in area (Fig. 1A (ii): pk 1 and pk 3). One peak (pk 3) co-
eluted with 12a (not shown), suggesting that the compound with
natural trans stereochemistry is turned over. Pk 1 was isolated
by preparative HPLC and subjected to NMR spectroscopy. The
15,16
a dihydropyran 2,4-trans configuration (Scheme 1),
and the
doublet at 4.8 ppm with a J-coupling constant of 7.8 Hz suggested
18
a b-anomeric configuration for the glycoside linkage. Compound
2a therefore contains the same relative stereochemistry found
1
in natural strictosidine. Compound 12a co-eluted with the third
peak observed in the LC chromatogram of 12, indicating the
presence and location of the natural stereoisomer among the
mixture of the chemical reaction products. Therefore, strictosidine
synthase, when challenged with an array of stereoisomers, displays
a stringent preference for the diastereomer displaying the 2,4-trans
configuration and b-glycoside bond found in the natural substrate.
As an alternative to enzymatic synthesis, chemical synthesis of
1
H NMR spectrum showed the presence of two stereoisomers,
both with H15/H21-cis configuration, as evidenced by the triplets
15,16
at 5.9 and 5.7 ppm, assigned to H-21 (ESI†).
The anomeric
configuration of the glucose moiety is likely b as indicated by the
18
J-coupling constant of 8.0 Hz. We conclude that strictosidine
glucosidase accommodates stereochemical perturbation in the
secologanin dihydropyran moiety, but the anomeric configuration
must be b for turnover to occur.
12 from 1 and 11 allowed us to bypass the stringent selectivity
of strictosidine synthase and assess stereochemical restrictions
of subsequent biosynthetic steps. Analysis of the reaction of
unnatural strictosidine stereoisomers with downstream enzymes
therefore utilized chemically synthesized 12.
To fully explore the stereochemical promiscuity of the subse-
quent reductase catalyzed step, complete deglucosylation of 12 is
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required. However, strictosidine glucosidase only deglycosylated
a few of the stereoisomers of 12. Since it has been previously
shown that strictosidine 3a can be deglycosylated by bacterial
this reductase(s) will have broad applications in chemoenzymatic
synthesis after efforts to clone the enzyme are successful. Synthetic
installation of the vinyl group on 11 will allow access to an even
broader range of alkaloid structures.
19
glycosidases, we examined whether two commercially available
glucosyl hydrolases, Bacillus stearothermophilus a-glucosidase and
almond b-glucosidase, display different deglucosylation patterns
compared to strictosidine glucosidase. Almond b-glucosidase was
considerably more permissive than strictosidine glucosidase, con-
suming four out of the six peaks in the chromatogram (Fig. 1A (iii):
pk 1, 3, 5, and 6), B. stearothermophilus a-glucosidase facilitated
the consumption of two peaks that were not converted by either
b-glucosidase (Fig. 1A (iv): pk 2 and 4). Strictosidine glucosidase
therefore appears to be more specific for its substrate, 12a, while
the two commercially available glucosidases likely have active sites
that allow a greater diversity of substrates to be accepted. By using
glucosidases from different metabolic pathways, it is possible to
bypass the native biosynthetic pathway to fully deglucosylate 12.
In the heteroyohimbine biosynthetic pathway, one or more
reductases catalyze the NADPH-dependent reduction of degly-
cosylated strictosidine 3a to form monoterpene indole alkaloids
such as 5a–c (Scheme 1). A cell-free extract from C. roseus cell
suspension culture was used to reconstitute the reductase activity.
Control experiments revealed that 3a, strictosidine glucosidase,
NADPH, and the reductase activity were each a necessary
component for formation of a reduced product that eluted near
an authentic standard of ajmalicine 5a (Fig. 1B). We determined
the steady-state kinetics for reduction of the natural substrate,
deglucosylated strictosidine 3a, and the unnatural substrate,
deglucosylated vincoside 3b (Table 1). The enzyme showed a
The first step of the pathway, catalyzed by strictosidine synthase,
may have evolved stringent substrate specificity to ensure the
integrity of the first committed intermediate strictosidine 3a.
Strictosidine glucosidase also shows strict stereocontrol, but
accepts at least one unnatural dihydropyran stereoisomer with
relative cis stereochemistry. Recruitment of glucosidases from
other metabolic pathways highlights the potential to bypass stereo-
chemical restrictions and to diversify alkaloid biosynthesis. Assay
of 3a, 3b, 12a, and 12 with heteroyohimbine reductase activity
suggests that C. roseus harbors at least one enzyme that converts
these stereoisomers to reduced alkaloids in vitro. Regardless of
whether the observed activity is functional in vivo, this enzyme can
be used in heterologous expression systems to yield novel variants
of the heteroyohimbine framework. This approach requires the
gene encoding this enzyme, and efforts to identify the NADPH
dependent reductases of C. roseus are ongoing.
Acknowledgements
We gratefully acknowledge funding from GM074820.
Notes and references
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1
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This journal is © The Royal Society of Chemistry 2009