Biocatalytic production of tetrahydroisoquinolines

Article abstract of DOI:10.1016/j.tetlet.2011.12.089

The promiscuity of the enzyme norcoclaurine synthase is described. This biocatalyst yielded a diverse array of substituted tetrahydroisoquinolines by cyclizing dopamine with various acetaldehydes in a Pictet-Spengler reaction. This enzymatic reaction may provide a biocatalytic route to a range of tetrahydroisoquinoline alkaloids.

Full text of DOI:10.1016/j.tetlet.2011.12.089

Tetrahedron Letters 53 (2012) 1071–1074
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Biocatalytic production of tetrahydroisoquinolines
Bettina M. Ruff a,b, S. Bräse b, Sarah E. O’Connor c,d,
⇑
a Massachusetts Institute of Technology, Department of Chemistry, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
b Karlsruhe Institute of Technology, Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76137 Karlsruhe, Germany
c The John Innes Centre, Department of Biological Chemistry, Norwich NR4 7UH, UK
d The University of East Anglia, Norwich NR4 7TJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The promiscuity of the enzyme norcoclaurine synthase is described. This biocatalyst yielded a diverse
array of substituted tetrahydroisoquinolines by cyclizing dopamine with various acetaldehydes in a Pic-
tet–Spengler reaction. This enzymatic reaction may provide a biocatalytic route to a range of tetrahydro-
isoquinoline alkaloids.
Received 28 September 2011
Revised 6 December 2011
Accepted 19 December 2011
Available online 29 December 2011
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Biocatalysis
Pictet–Spengler reaction
Norcoclaurine synthase
Tetrahydroisoquinoline alkaloids
O
The tetrahydroisoquinoline moiety is found in many natural
products and synthetic pharmaceuticals. In particular, this chiral
N-heterocyclic scaffold is an integral part of all tetrahydroisoquin-
oline alkaloid natural products. The benzylisoquinoline biosyn-
thetic pathways in plants lead to a large group of alkaloids
including the well-known analgesics morphine and codeine, which
are isolated from the opium poppy. Additional examples of indus-
trially important tetrahydroisoquinolines include noscapine (1,
Fig. 1), which has been used as an antitussive since the 19th cen-
tury, and the anticancer properties of this compound were recently
reported.1 The (S)-configured antimuscarinic drug Michellamine B
(2),2 isolated from Ancistrocladus plants, is one of the many tetra-
hydroisoquinolines with anti-human immunodeficiency virus
(HIV) activity.3 The synthetic compound Solifenacin (3) has a uri-
nary antispasmodic effect.4
NCH3
O
OH
OH
H
H
O
H3CO
HN
O
H3CO
OCH3
OH OCH3
Noscapine (1)
CH3O
N
H
O
HO
N
O
NH
OH
Michellamine B (2)
Solifenacin (3)
Norcoclaurine synthase (NCS)—one of the three known Pictet–
Spenglerases5—catalyzes the C–C bond forming reaction between
4-hydroxyphenylacetaldehyde (4-HPAA, 4) and dopamine (5) to af-
ford (S)-norcoclaurine (6) (Table 1), a compound with antiallergic6
and b-adrenergic7 properties, and the biosynthetic precursor to all
known naturally occurring tetrahydroisoquinoline alkaloids.
Uncatalyzed Pictet–Spengler reactions with phenylethylamines
and aldehydes yield racemic mixtures of tetrahydroisoquinolines.8
Several catalysts have been developed for Pictet–Spengler reac-
tions utilizing substituted tryptamines to yield tetrahydro
-b-carbolines.9
Figure 1. Examples of naturally occurring and synthetic pharmaceutically active
tetrahydroisoquinoline alkaloids.
For the production of tetrahydroisoquinolines, the use of asym-
metric catalysts for the enantioselective hydrogenation of dihydro-
isoquinoline derivatives10 can be numbered among the few
stereoselective chemical methods reported to produce this class of
compounds.
The Pictet–Spenglerase enzymes could, in principle, be used to
produce enzymatically tetrahydroisoquinolines in an enantioselec-
tive fashion. The stereoselectivity of the NCS catalyzed reaction to
yield (S)-configured products has already been shown in various
⇑
Corresponding author.
E-mail address: sarah.o’connor@jic.ac.uk (S.E. O’Connor).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.12.089

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B. M. Ruff et al. / Tetrahedron Letters 53 (2012) 1071–1074
Table 1
Percent conversion of aldehydes 4, 7–25 with TfNCS after 3 h reaction time. Reaction conditions were as described in the text (dopamine (5) (1 mM), aldehyde 4, 7–25 (1 mM), TfNCS
(300 M), TRIS buffer (100 mM, pH 7)). All enzymatic reactions were performed alongside a control using boiled enzyme to ensure that no reaction occurs in the absence of enzyme
l
HO
NH2
HO
HO
HO
NH
H
dopamine (5)
TfNCS
R
+
O
R =
OH
R
H
(S)-norcoclaurine (6)
R =
OH
4-HPAA (4)
% Conv. after 3 h
R@
Product
R@
Product
% Conv. after 3 h
6
60
35
51
OH
16
17
4
26
27
65
66
36
37
68
61
S
F
7
8
F
18
28
29
71
66
38
39
71
42
F
19
9
O
O
10
11
20
30
31
32
58
57
69
40
52
—
21
Not detected
Not detected
12
22
H
—
23
OMe
OMe
13
CH3
24
33
34
69
65
Not detected
Not detected
—
—
OMe
14
OCF
3
15
25
examples in the literature.5c,d,11,12 However, a key limitation of bio-
catalytic approaches is the strict substrate specificity of many en-
zymes. An enzyme must have broad substrate specificity to be
practical for wide synthetic application. Here we describe the sub-

B. M. Ruff et al. / Tetrahedron Letters 53 (2012) 1071–1074
1073
strate scope and limitations of the Pictet–Spenglerase NCS, a study
that provides a foundation for developing a general biocatalytic
strategy for the synthesis of tetrahydroisoquinolines.
H
R1
R2
O
HO
NH
H
Affinity-tagged NCS from Thalictrum flavum (TfNCS) was overex-
pressed in Escherichia coli and purified using nickel resin in good
yields as previously reported.12 This readily produced heterologous
enzyme was used to probe systematically the enzymatic require-
ments for the aldehyde substrate using, in addition to the natural
substrate 4-HPAA (4),13 19 aldehyde analogs (7–24). These alde-
hydes were synthesized either by oxidation of the corresponding
alcohols with Dess–Martin periodinane (7–15, 17, 19, 20) or by
reduction of the corresponding Weinreb amide with LAH (18), or
were commercially available (16, 21–24).14 For the enzymatic as-
say, we chose to use one of the reported conditions under which
NCS had been assayed in previous studies. Specifically we used
TRIS-buffered aqueous media at neutral pH with substrate concen-
trations at 1 mM. While substantial background reaction can often
be observed for the Pictet–Spengler reaction, under these condi-
tions no tetrahydroisoquinoline product was detected after 3 h
reaction time with boiled enzyme by HPLC using UV detection at
228 nm. We further controlled for the non-enzymatic background
reaction by including a control using boiled (inactive) enzyme with
each reaction; in each case, no tetrahydroisoquinoline product was
detected by HPLC. Therefore, we could be certain that all tetrahy-
droisoquinoline products observed were generated enzymatically.
NCS
4-HPAA (4)
+
R1
R2
NH2
OH
( )-norcoclaurine (6)
2
S
R
1 = R2 = OH
1
R1 = H, R2 = F (41)
R1 = H, R2 = NH2 (42)
1 = OMe, R2 = H (43)
1 = R2 = H (44)
1 = OH, R2 = H (46)
1 = OH, R2 = OMe (47)
NH2
R
R
R
R
N
H
tryptamine (45)
Scheme 1. Phenethylamines 41–47 assayed with TfNCS and with co-substrate
4-HPAA (4).
average consumption of 65% dopamine (5) after 3 h. A slight disfa-
vor (42–52%) for the aliphatic compounds 20 and 21 as well as the
unsubstituted phenylacetaldehyde 16 could be observed (Table 1).
In summary, we found that TfNCS was able to catalyze the reaction
between dopamine and acetaldehydes containing more than 3 car-
bon atoms and that are unsubstituted at the a-position. Aromatic
Briefly, TfNCS (300 lM) was added to a solution of TRIS buffer
acetaldehydes appear to be slightly superior substrates compared
to bulky aliphatic compounds. Overall, however, all the aldehydes
appeared to be converted in qualitatively similar rates. The alde-
hyde substrate flexibility provides a general method for the enzy-
matic preparation of 1-(ortho-, meta- and para-substituted benzyl)-
1,2,3,4-tetrahydroisoquinolines.
In contrast to the relaxed substrate specificity observed for the
aldehyde substrate, NCS showed a strict requirement for the amine
substrate, dopamine (5). We failed to detect any product for enzy-
matic reactions utilizing the native aldehyde substrate 4-HPAA (4)
and the commercially available phenethylamines 41–44 (Scheme
1). Tryptamine (45), the natural substrate of strictosidine synthase,
the Pictet–Spenglerase that catalyzes the formation of tetrahydro-
b-carbolines,15 was also not turned over. Overall, this substrate
specificity is consistent with earlier mechanistic studies of NCS.
Luk et al.12 proposed the formation of a quinoid at the C-2 position
of the aromatic ring, which requires that the phenethylamine sub-
strate contains a hydroxy group at the C-2 position. Therefore,
while 46 and 47 (Scheme 1) could be turned over by NCS,12 sub-
strates 41–45 could not.
(100 mM, pH 7.0) containing dopamine (1 mM) and aldehyde
(1 mM). The enzymatic reaction was allowed to proceed at room
temperature for 1 h, after which it was quenched with MeOH,
and then analyzed by electrospray LC–MS. Authentic standards of
each norcoclaurine analogs 26–40 were synthesized on a milligram
scale, fully characterized by NMR and HRMS analysis and run along
with the enzymatic assay (Supplementary data).
TfNCS proved to have exceptionally broad aldehyde substrate
specificity, turning over aldehydes 4, 7–21 (Table 1). The structures
of substrates 4, 7–21 vary widely, and include phenylacetaldehy-
des substituted with various electron-withdrawing or donating
groups, heteroaromatic moieties, aromatic bicyclics, aliphatic (het-
ero) cycles and aliphatic open-chained compounds. Only the prod-
ucts of the smallest, acetaldehyde (23) and propionaldehyde (24)
and the a-substituted aldehydes, benzaldehyde (22) and 25 could
not be detected.
Reaction rates of aldehydes 4, 7–21 were evaluated by measur-
ing the amount of dopamine consumed over time by HPLC using
0.5, 1, 2 and 3 h time points (Supplementary data). These assays
show that 4, 7–21 are turned over at a comparable rate with an
Figure 2. (A) The norcoclaurine active site (2VQ5) surrounding the aldehyde substrate 4. The co-substrate 5 is in blue. Lys122 and Glu110 are important for catalysis.16 All
conserved residues that surround 5 are in green;⁄residues that vary among annotated NCS enzymes are in gray. (B) Alignment of annotated NCS enzymes. Residues labeled
⁄
with are shown in panel A and are conserved; are in panel A but are not conserved among the NCS homologs.

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B. M. Ruff et al. / Tetrahedron Letters 53 (2012) 1071–1074
The crystal structure of TfNCS, which was recently published in
References and notes
2009,16 revealed that this enzyme harbors a relatively shallow ac-
tive site (Fig. 2), which is consistent with the range of aldehydes
that can be turned over by NCS. With the exception of the residues
that directly interact with the aldehyde functional group, the bind-
ing pocket for substrate 4 appears to consist largely of hydrophobic
interactions that could presumably accommodate substrate ana-
logs 7–21. The aromatic 4-HPAA (4) and dopamine (5) appear to
stack together in the enzyme active site (Fig. 2A), but given the
turnover of aliphatic aldehyde substrates, this stacking interaction
must not be essential. We note that Ile143 appears to be close to
the alpha carbon of 4, and this residue may be responsible for pre-
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venting turnover of a-substituted aldehydes. We hypothesize that
the strict amine substrate specificity is not governed by the con-
fines of the enzyme active site, but instead by the reactivity
requirements of the substrate.
In summary, we have showed that the enzyme norcoclaurine
synthase can be used as a biocatalyst to yield a variety of substi-
tuted tetrahydroisoquinolines. We further note that the potential
of the enzyme in a multi-gram scale enantioselective preparation
of (S)-norcoclaurine itself was recently described.11 The easy over-
expression in E. coli and purification on large scale, as well as the
stability of the protein, suggests potential applications in synthetic
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Acknowledgments
13. Synthesized by Parikh–Doehring oxidation as described previously: Hirose, T.;
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Omura, S. Heterocycles 2000, 53, 777–784.
14. Aldehyde 25 was synthesized as described in: Hoffmann, S.; Nicoletti, M.; List,
B. J. Am. Chem Soc. 2006, 128, 13074–13075.
We thank the Karlsruhe House of Young Scientists (KHYS) for
financial support. We thank Anne Rüger, KIT, for the synthesis of
aldehyde 25.
15. Bernhardt, P.; Usera, A. R.; O’Connor, S. E. Tetrahedron Lett. 2010, 51, 4400–
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Supplementary data
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Pasquo, A.; Belluci, L.; Boffi, A. J. Biol. Chem. 2009, 284, 897–904.
Supplementary data (experimental procedures and spectral
characterization) associated with this article can be found, in the
online version, at doi:10.1016/j.tetlet.2011.12.089.