Biocatalytic asymmetric formation of tetrahydro-β-carbolines

Article abstract of DOI:10.1016/j.tetlet.2010.06.075

Strictosidine synthase triggers the formation of strictosidine from tryptamine and secologanin, thereby generating a carbon-carbon bond and a new stereogenic center. Strictosidine contains a tetrahydro-β-carboline moiety - an important N-heterocyclic framework found in a range of natural products and synthetic pharmaceuticals. Stereoselective methods to produce tetrahydro-β-carboline enantiomers are greatly valued. We report that strictosidine synthase from Ophiorrhiza pumila utilizes a range of simple achiral aldehydes and substituted tryptamines to form highly enantioenriched (ee >98%) tetrahydro-β-carbolines via a Pictet-Spengler reaction. This is the first example of aldehyde substrate promiscuity in the strictosidine synthase family of enzymes and represents a first step toward developing a general biocatalytic strategy to access chiral tetrahydro-β-carbolines.

Full text of DOI:10.1016/j.tetlet.2010.06.075

Tetrahedron Letters 51 (2010) 4400–4402
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Tetrahedron Letters
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Biocatalytic asymmetric formation of tetrahydro-b-carbolines
*
Peter Bernhardt, Aimee R. Usera, Sarah E. O’Connor
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Strictosidine synthase triggers the formation of strictosidine from tryptamine and secologanin, thereby
generating a carbon–carbon bond and a new stereogenic center. Strictosidine contains a tetrahydro-b-
carboline moiety—an important N-heterocyclic framework found in a range of natural products and syn-
thetic pharmaceuticals. Stereoselective methods to produce tetrahydro-b-carboline enantiomers are
greatly valued. We report that strictosidine synthase from Ophiorrhiza pumila utilizes a range of simple
achiral aldehydes and substituted tryptamines to form highly enantioenriched (ee >98%) tetrahydro-b-
carbolines via a Pictet–Spengler reaction. This is the first example of aldehyde substrate promiscuity in
the strictosidine synthase family of enzymes and represents a first step toward developing a general bio-
catalytic strategy to access chiral tetrahydro-b-carbolines.
Received 23 April 2010
Revised 4 June 2010
Accepted 14 June 2010
Available online 18 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
The Pictet–Spengler reaction between tryptamine and an alde-
hyde yields tetrahydro-b-carboline moiety, a heterocyclic frame-
work found in a wide range of natural products and synthetic
pharmaceuticals. Catalytic methodology has been developed to
stereoselectively generate the C–C bond formed in this reaction.1
Chiral tetrahydro-b-carbolines are also formed enzymatically in
natural product biosynthetic pathways (Fig. 1).2 Although biocatal-
ysis has been proven useful for a number of synthetic transforma-
tions,3 natural product biosynthetic enzymes often have narrow
substrate scopes that limit the use of these enzymes as biocata-
lysts. Herein, we describe a Pictet–Spenglerase with a broad
substrate range that asymmetrically generates tetrahydro-b-carbo-
lines from tryptamine and a range of aldehydes.
Strictosidine synthase (STS), a Pictet–Spenglerase utilized in
monoterpene and quinoline alkaloid biosynthesis, catalyzes the
conversion of tryptamine 1 and secologanin 2 to the tetrahydro-
b-carboline strictosidine 3 ( Fig. 1).2 Two strictosidine synthase
homologs from the medicinal plants Rauvolfia serpentina (RsSTS)4a
and Catharanthus roseus (CrSTS),4b which share 82% amino acid se-
quence identity overall and near-identical active sites, have been
studied in detail. Both these homologs can turnover a variety of
electron-deficient and electron-substituted tryptamine analogs.5,6
Site-directed mutagenesis of these enzymes has been successfully
used to broaden the scope of strictosidine synthase for larger trypt-
amine derivatives.7a,b However, only minor changes to the alde-
hyde substrate 2 are tolerated.5,7c Therefore, any tetrahydro-b-
carbolines generated by strictosidine synthase will have little
application outside the context of alkaloid biosynthetic pathways
that use secologanin 2.
The recently cloned strictosidine synthase from Ophiorrhiza
pumila (OpSTS)4c has a lower sequence identity to both CrSTS
(54%) and RsSTS (60%). This observation suggested that the active
site of OpSTS could have different structural features, perhaps a re-
sult of the evolutionary distance between the Apocynaceae plant
family from which CrSTS and RsSTS are derived, and the Rubiaceae
family from which OpSTS is derived. Sequence alignments showed
that OpSTS has a four-amino acid deletion that corresponds to
D
[Gln282His283Gly284Arg285] in CrSTS and D[Met276His277-
Gly278Arg279] in RsSTS. The crystal structure of RsSTS in complex
with secologanin 2 (PDB: 2FPC)6 indicated that these amino acids
are in the proximity of the secologanin binding site. Moreover,
His283 (His277 in RsSTS), which is missing in OpSTS, is within
hydrogen bonding distance to the glucose moiety of 2. This obser-
vation prompted us to assess whether OpSTS has a broader, and
therefore potentially more useful, aldehyde substrate scope com-
pared to CrSTS and RsSTS.
Pseudo steady-state kinetics with 1, secologanin 2, and OpSTS
(kcat = 1.1 sꢀ1, Km,2 = 21 M) or CrSTS (kcat = 5.1 sꢀ1, Km,2 = 64
l lM)
verified that the heterologously expressed enzyme is active (Sup-
plementary data). Aldehydes 4–14 (1 mM) and tryptamine
(1 mM) were incubated with OpSTS (0.2 mol % catalyst, 2
lM) or
CrSTS (1.0 mol % catalyst, 10 M) in aqueous buffer (pH 7.0). The
l
resulting tetrahydro-b-carboline products were identified by mass
spectrometry and by comparison with a conveniently prepared
racemic authentic standard (Supplementary data).8 CrSTS turned
over substrate 4 poorly, while no products formed with substrates
5–12 (Table 1). In contrast, OpSTS turned over aldehydes 4–10.
Although OpSTS displayed a preference for the natural substrate
2 by at least 1000-fold, this enzyme nevertheless catalyzed the
conversion of a relatively broad range of substrates, including
hydrogen bond accepting (4), aliphatic (5–6 and 8) and aromatic
(7) aldehydes with similar rates (kobs = 0.013–0.048 minꢀ1). The
* Corresponding author. Tel.: +1 617 324 0180.
E-mail address: soc@mit.edu (S.E. O’Connor).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.06.075

P. Bernhardt et al. / Tetrahedron Letters 51 (2010) 4400–4402
4401
CHO
NH
O-β-D-Glc
*
N
H
H
NH2
O-β-D-Glc
N
O
H
MeO2C
2
1
3
O
MeO2C
Figure 1. Pictet–Spengler reaction catalyzed by strictosidine synthase. The tetrahydro-b-carboline moiety is highlighted in red, the C–C bond formed is shown in bold, and
the stereogenic center (C3) is marked with an asterisk.
Table 1
full kinetic analysis was not performed because of the high esti-
mated Km values for the aldehydes (P10 mM) and substrate solu-
bility limitations.
Aldehyde scope for OpSTS and CrSTS
kobs (minꢀ1
)
a
Starting material R =
The enantiomeric excess (ee) of OpSTS was >98% as evidenced
by chiral HPLC (Supplementary data).9 The absolute configuration
was determined for the enzymatic product derived from 1 and 8.
The reaction was carried out on a milligram scale using OpSTS
immobilized to a solid support (Supplementary data),10 and com-
pared to an authentic 3(S)-standard obtained by an organocatalytic
method.1a This confirmed that the absolute configuration of this
enzymatic product is 3(S), which is the configuration observed
with the natural substrate, secologanin 2 (Supplementary data).
Notably, the stereoselectivity observed for the reaction between
1 and 2 with RsSTS, CrSTS, and OpSTS was preserved with achiral
aldehydes, indicating that stereoselectivity does not rely on the
chirality of substrate 2. The configuration at C3 is determined
during the cyclization of iminium 13 (derived from condensation
of 1 and 2) to yield 14, which is then deprotonated to form 3
(Fig. 2A).11a,b
To develop a model for stereoselectivity, computationally de-
rived structures of 1411a were docked into the crystal structure of
RsSTS (Supplementary data). The 14-2(R)3(R) isomer could only
be docked after considerable changes to the active site structure
and substrate orientation, suggesting that this intermediate is
not formed in the active site (Supplementary data). Out of the three
remaining isomers [14-2(R)3(S), 14-2(S)3(S), and 14-2(S)3(R)], only
14-2(R)3(S) was appropriately positioned for deprotonation at C-2
by the catalytic base Glu309 (Fig. 2B and Supplementary data). We
propose that although alternate diastereomers of 14 may be
OpSTS
0.048
CrSTS
4
0.0002
O
Not obsdb
Not obsdb
Not obsdb
5
6
7
0.037
0.032
0.022
Not obsdb
Not obsdb
8
9
0.013
<0.0001c
<0.0001c
Not obsdb
Not obsdb
Not obsdb
Not obsdb
Not obsdb
10
11
12
a
kobs measured by monitoring product formation with
1 at 280 nm using
reverse-phase HPLC with a PDA detector.
Not observed, detection limit ꢁ10ꢀ5 minꢀ1
.
b
Quantitation limit ꢁ10ꢀ4 minꢀ1. kobs of 4 was 230-fold improved for OpSTS
c
compared to CrSTS; kobs of 5–10 were at least 3700-fold improved, given the
detection limit of 10ꢀ5 minꢀ1
.
kobs values clearly indicated that OpSTS preferred aldehydes
unsubstituted at the alpha position (compare 4–8 with 10–12). A
Figure 2. (A) Reaction sequence leading to 3. (B) Computer-generated models of two diastereomers of 14 docked in RsSTS (RsSTS numbering).6 Arrows highlight H-2 that is
deprotonated to form 3.

4402
P. Bernhardt et al. / Tetrahedron Letters 51 (2010) 4400–4402
Stockigt, J. Chem. Biol. 2007, 14, 979–985; (c) Chen, S.; Galan, M. C.; Coltharp, C.;
O’Connor, S. E. Chem. Biol. 2006, 13, 1137–1141.
formed within the active site, they cannot be deprotonated. In-
stead, these intermediates would undergo the reverse reaction,
reforming iminium 13, and cyclization would be repeated until
the productive 14-2(R)3(S) isomer is formed. This facial deprotona-
tion model is consistent with the reversible nature of the Pictet–
Spengler reaction,11c but should be interpreted cautiously, since
it has not been experimentally validated.11d Nevertheless, this
model serves as a basis for understanding and engineering the ste-
reoselectivity of Pictet–Spenglerases.
8. Racemic authentic standards were synthesized from tryptamine hydrochloride
(0.5 M) and achiral aldehydes (0.5 M) in aqueous maleic acid buffer (10 mM,
pH 2.0, total volume 2 mL) at 60 °C. Either during the reaction or upon
standing, the tetrahydro-b-carboline product precipitated and could be filtered
and washed with water (3 ꢂ 2 mL) to afford analytically pure product. See
Supplementary data for NMR spectra and structures of products 15 through 21.
1-((Tetrahydro-2H-pyran-4-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
(product 15 from 1 and 4): 1H NMR (methanol-d4): d 7.48 (1H, dt, J = 0.9, 7.9),
7.36 (1H, dt, J = 0.9, 8.2), 7.16 (1H, td, J = 1.1, 8.2), 7.06 (1H, td, J = 0.9, 8.0), 4.86–
4.82 (1H, m), 4.03–3.96 (2H, m), 3.75 (1H, dt, J = 1.3, 4.1, 12.4), 3.55–3.44 (3H,
m), 3.12–3.07 (2H, m), 2.21–2.15 (1H, m), 1.95–1.83 (3H, m), 1.68 (1H, td,
J = 1.9, 13.1), 1.50–1.37 (2H, m); 13C NMR (methanol-d4): d 137.61, 129.58,
126.73, 122.80, 119.91, 118.34, 111.63, 106.54, 68.03, 67.87, 51.08, 42.39,
39.94, 33.93, 32.40, 31.09, 18.80; ESI-MS (C17H23N2O+) m/z calcd: 271.1810
[M+H]+, found: 271.1805 [M+H]+.
In an attempt to broaden the substrate scope of CrSTS, we mu-
tated His283 for Gly/Leu/Phe and deleted
D[282–285]. However,
these modifications did not confer the substrate scope of OpSTS
to CrSTS. His313 (His307 in RsSTS, His299 in OpSTS) is also within
hydrogen bonding distance to the glucose moiety of secologanin 2.
However, neither His313Leu nor the double mutant His283Leu/Hi-
s313Leu resulted in a broadened aldehyde substrate specificity.
The factors that control the substrate specificity of 2 are complex,
and mutations distant from the active site to impact substrate
specificity are currently being explored.
The Rubiaceae family of plants contains chiral tetrahydro-b-
carbolines different from those found in the Apocynaceae family.12
The enzymes that catalyze the formation of the precursor for these
natural products are unknown, but it is tempting to speculate that
they resemble OpSTS. Screening of Rubiaceae plants for new strict-
osidine synthase homologs and assay of the predicted substrates
will experimentally test this possibility, and potentially broaden
the base of biocatalysts available for use in this reaction. Although
the catalytic rates using OpSTS are relatively low with simple alde-
hyde substrates, high stereoselectivity is maintained. Protein engi-
neering may improve catalytic efficiency. This discovery allows the
asymmetric biocatalytic formation of a wide range of tetrahydro-b-
carbolines directly from tryptamine, and represents the first report
of a Pictet–Spenglerase with broad aldehyde specificity.
1-(Cyclohexylmethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (product 16
from 1 and 5): 1H NMR (methanol-d4): d 7.47–7.44 (1H, m), 7.36 (1H, td,
J = 0.8, 8.2), 7.14 (1H, ddd, J = 1.1, 7.1, 8.2), 7.04 (1H, ddd, J = 1.0, 7.1, 8.0), 4.81–
4.74 (1H, m), 3.71 (1H, ddd, J = 3.5, 5.5, 12.5), 3.41 (1H, ddd, J = 5.5, 9.9, 12.6),
3.09 (1H, dddd, J = 1.9, 5.5, 9.8, 15.4), 3.05–2.99 (1H, m), 2.12 (1H, ddd, J = 3.7,
10.2, 14.3), 2.08–2.00 (1H, m), 1.87–1.60 (6H, m), 1.49–1.34 (2H, m), 1.33–1.22
(1H, m), 1.19–1.02 (2H, m); 13C NMR (methanol-d4): d 137.59, 129.95, 126.75,
122.69, 119.84, 118.31, 111.64, 106.39, 51.57, 42.46, 40.53, 34.56, 33.59, 32.41,
26.74, 26.54, 26.16, 18.81; ESI-MS (C18H25N2þ) m/z calcd: 269.2012 [M+H]+,
found: 269.2014 [M+H]+.
1-Isobutyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (product 17 from 1 and 6):
1H NMR (10:1, (methanol-d4/DMSO-d6) d 7.50 (1H, td, J = 1.0, 7.8), 7.39 (1H, td,
J = 0.9, 8.2), 7.17 (1H, ddd, J = 1.2, 7.1, 8.2), 7.08 (1H, ddd, J = 1.0, 7.1, 8.0), 4.82–
4.79 (1H, m), 3.74 (1H, ddd, J = 3.9, 5.3, 12.6), 3.47 (1H, ddd, J = 5.9, 9.3, 12.6),
3.16–3.02 (2H, m), 2.08 (1H, ddd, J = 3.9, 10.2, 14.2), 1.99 (1H, dqd, J = 3.9, 6.5,
19.5), 1.85 (1H, ddd, J = 3.9, 10.3, 14.2), 1.15 (3H, d, J = 6.4), 1.10 (3H, d, J = 6.5);
13C NMR (DMSO-d6): d 137.22, 131.64, 126.98, 122.74, 120.07, 119.09, 112.46,
106.81, 51.51, 41.94, 24.67, 24.53, 22.51, 19.16; ESI-MS (C15H21N2þ) m/z calcd:
229.1699 [M+H]+, found: 229.1697 [M+H]+.
1-Benzyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (product 18 from 1 and 7):
1H NMR (methanol-d4): d 7.55–7.37 (m, 7H), 7.21 (1H, ddd, J = 1.1, 7.1, 8.2,),
7.11 (1H, dd, J = 1.0, 7.1, 8.0), 5.06–5.01 (1H, m), 3.79–3.73 (1H, m), 3.62 (1H,
ddd, J = 3.6, 5.3, 12.5), 3.36 (1H, ddd, J = 5.7, 9.6, 12.6), 3.21–3.01 (3H, m); 13C
NMR (methanol-d4): d 136.68, 135.82, 130.10, 129.87, 127.71, 126.26, 122.3þ6,
119.54, 118.59, 111.90, 106.72, 54.20, 41.90, 37.76, 18.45; ESI-MS (C18H19N2
)
m/z calcd: 263.1543 [M+H]+, found: 263.1540 [M+H]+.
1-Pentyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (product 19 from 1 and 8):
1H NMR (methanol-d4): d 7.48 (1H, dt, J = 0.9, 7.9), 7.36 (1H, dt, J = 0.9, 8.2), 7.15
(1H, td, J = 1.1, 8.2), 7.06 (1H, td, J = 0.9, 8.0), 4.69 (1H, dd, J = 4.1, 8.9) 3.73 (1H,
ddd, J = 3.8, 5.5, 12.5), 3.44 (1H, ddd, J = 5.6, 9.6, 12.6), 3.11 (1H, dddd, J = 1.9,
5.6, 9.5, 15.1), 3.05 (1H, dddd, J = 1.3, 3.8, 5.3, 16.3), 2.31–2.23 (1H, m), 1.97–
1.89 (1H, m), 1.64–1.54 (2H, m), 1.53–1.37 (4H, m), 0.97 (3H, t, J = 7.1); 13C
NMR (methanol-d4): d 138.36, 130.32, 127.46, 123.48, 120.59, 119.10, 112.39,
107.18, 55.17, 43.19, 33.38, 32.79, 25.92, 23.48, 19.54, 14.37; ESI-MS
(C16 23N þ) m/z calcd: 243.1856 [M+H]+, found: 243.1851 [M+H]+.
Acknowledgments
This work was funded by the NIH (GM074820); A.R.U. acknowl-
edges NIH for a postdoctoral fellowship. We thank Omar Ahmad
for assistance with chiral HPLC.
H
2
1-Ethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (product 20 from 1 and 9):
1H NMR (methanol-d4): d 7.47 (1H, td, J = 0.9, 7.9), 7.37 (1H, td, J = 0.9, 8.2), 7.15
(1H, ddd, J = 1.1, 7.1, 8.2), 7.06 (1H, ddd, J = 1.0, 7.1, 8.0), 4.63–4.57 (1H, m), 3.71
(1H, ddd, J = 3.7, 5.6, 12.5), 3.39 (1H, ddd, J = 5.5, 9.7, 12.6), 3.10 (1H, dddd,
J = 2.0, 5.6, 9.7, 15.3), 3.01 (1H, dddd, J = 1.4, 3.7, 5.3, 16.3), 2.33 (1H, dqd, J = 4.2,
7.6, 15.1), 2.05–1.93 (1H, m), 1.19 (3H, t, J = 7.5); 13C NMR (methanol-d4): d
138.30, 130.14, 127.42, 123.46, 120.57, 119.13, 112.38, 107.25, 56.19, 43.15,
26.33, 19.52, 9.93; ESI-MS (C13H17N2þ) m/z calcd: 201.1386 [M+H]+, found:
[M+H]+ 201.1385.
Supplementary data
Supplementary data (experimental methods, modeling data and
NMR spectra) associated with this article can be found, in the on-
line version, at doi:10.1016/j.tetlet.2010.06.075.
1-Cyclohexyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (product 21 from 1 and
10): 1H NMR (methanol-d4): d 7.45 (1H, d, J = 7.9), 7.38 (1H, d, J = 8.2), 7.14 (1H,
ddd, J = 1.1, 7.2, 8.2), 7.04 (1H, ddd, J = 1.0, 7.2, 8.0), 4.60–4.57 (1H, m), 3.71 (1H,
ddd, J = 3.2, 5.5, 12.4), 3.38 (1H, ddd, J = 5.4, 10.1, 12.5), 3.10 (1H, dddd, J = 2.0,
5.6, 10.1, 15.8), 3.04–2.95 (1H, m), 2.35–2.22 (1H, m), 1.98–1.68 (4H, m), 1.57–
1.10 (6H, m); 13C NMR (methanol-d4): d 137.64, 128.27, 126.71, 122.76, 119.84,
References and notes
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þ
18.70; ESI-MS (C17H23N2
)
m/z calcd: 255.1856 [M+H]+, found: 255.1858
[M+H]+.
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