Enzymatic and Chemoenzymatic Three-Step Cascades for the Synthesis of Stereochemically Complementary Trisubstituted Tetrahydroisoquinolines

Article abstract of DOI:10.1002/anie.201705855

Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4-trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in a step-efficient and selective manner without intermediate purification. The cascade employs inexpensive substrates (3-hydroxybenzaldehyde and pyruvate), and involves a carboligation step, a subsequent transamination, and finally a Pictet–Spengler reaction with a carbonyl cosubstrate. Appropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)-metaraminol. Subsequent cyclization catalyzed either enzymatically by a norcoclaurine synthase or chemically by phosphate resulted in opposite stereoselectivities in the products at the C1 position, thus providing access to both orientations of the THIQ C1 substituent. This highlights the importance of selecting from both chemo- and biocatalysts for optimal results.

Full text of DOI:10.1002/anie.201705855

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Accepted Article
Title: Enzymatic and chemoenzymatic 3-step cascades for the
synthesis of stereochemically complementary trisubstituted
tetrahydroisoquinolines
Authors: Vanessa Erdmann, Benjamin R. Lichmann, Jianxiong Zhao,
Robert C. Simon, Wolfgang Kroutil, John M. Ward, Helen C.
Hailes, and Dörte Rother
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201705855
Angew. Chem. 10.1002/ange.201705855
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10.1002/anie.201705855
Angewandte Chemie International Edition
COMMUNICATION
Enzymatic and chemoenzymatic 3-step cascades for the
synthesis of stereochemically complementary trisubstituted
tetrahydroisoquinolines
Vanessa Erdmann, Benjamin R. Lichman, Jianxiong Zhao, Robert C. Simon, Wolfgang Kroutil, John M.
Ward, Helen C. Hailes, and Dörte Rother*
The tetrahydroisoquinoline (THIQ) moiety is a ‘privileged
scaffold’[1] and, as such, it is found in numerous bioactive natural
products (e.g. noscapine[2], dioncophyllines[3]) and synthetic
pharmaceutical drugs (e.g. solifenacin[4]). THIQ containing
compounds demonstrate a wide range of bioactivities, including
anti-tumour[2], anti-parasitic[3] and anti-cholinergic[4] properties.
Natural derived THIQs may be obtained by extraction or
fermentation[5], but in many cases only small quantities are
available, typically as one component of a mixture. Chemical
syntheses are a frequent source of these compounds, but
complex multi-step asymmetric routes that give high
stereoselectivities can be challenging including at scale and are
often dependent on the use of toxic or environmentally harmful
chemicals.[6] Therefore novel synthetic routes towards THIQs are
of significant interest.
Modular biocatalytic cascades[23] for the stereoselective
production of pharmaceutical compounds are exemplified by the
work conducted on phenylpropanolamines.[24–26] All four possible
isomers of nor(pseudo)ephedrine are available from simple
starting materials, in 1-pot 2-enzyme cascades by selection of
the appropriate biocatalysts.[26] Phenylpropanolamines with an
additional 3-hydroxy group on the aromatic ring were considered
to be suitable substrates for cascades including PSRs catalysed
by NCS or phosphate: indeed, a previous screen of Coptis
japonica NCS (CjNCS2) had suggested that (1R,2S)-2-amino-1-
(3-hydroxyphenyl)propan-1-ol (also referred to as metaraminol)
was accepted as a substrate in low yield.[13] With this in mind,
two 3-step cascades, using either 3 enzymes or 2 enzymes and
one chemo-step, were designed for the stereoselective
formation of 1,3,4-trisubstituted-THIQs (Scheme 1).
In vitro biocatalysis provides a viable method of producing
complex THIQs in high stereoselectivities and under mild
conditions.[7–9] Previous studies have shown norcoclaurine
Here, we present the first examples of 3-step
(chemo)enzymatic cascades for the stereoselective formation of
1,3,4-trisubstituted-THIQs from cheap starting materials. The
synthase
(NCS),
the
Pictet-Spenglerase
from
plant
cascades
involved
first
a
carboligation
using
3-
benzylisoquinoline alkaloid biosynthesis[10,11], to be a versatile
biocatalyst. NCS can convert 3-hydroxyphenethylamines (e.g.
dopamine) and a carbonyl co-substrate into a (1S)-THIQ in high
hydroxybenzaldehyde 1 (Scheme 1) and pyruvate to form the
acyloin 2; subsequent transamination revealed a 2-amino-1-(3-
hydroxyphenyl)propan-1-ol 3, which could then, together with a
carbonyl co-substrate, undergo a PSR. Most importantly, NCS
and phosphate catalysis of the PSR-step resulted in opposite
stereoselectivities, thus enabling access to both orientations of
the THIQ 1-substituent, highlighting the importance of selecting
from both chemo- and biocatalysts.
stereoselectivities.[12–15] The enzyme has demonstrated
remarkably wide carbonyl co-substrate tolerance, accepting a
variety of aldehydes and ketones.[13–20]
NCS has also been successfully employed in chemo-
enzymatic cascades.[12,15,16,21] As an alternative to NCS,
inorganic phosphate can be used to catalyse the Pictet-Spengler
reaction (PSR), forming racemic THIQs under aqueous
conditions.[22] This can be successfully combined with enzyme
steps.[21]
a
The first requirement for chemoenzymatic cascades towards
complex trisubstituted THIQs was the development of a 2-step
cascade for the production of the amino alcohol (1R,2S)-3
(Scheme 1). The initial step in the cascade was the carboligation
of 3-hydroxybenzaldehyde (1) and acetaldehyde (after
decarboxylation from pyruvate) to form (R)-1-hydroxy-1-(3-
hydroxyphenyl)propan-2-one ((R)-2). This was catalysed by the
ThDP-dependent acetohydroxy acid synthase I from Escherichia
coli (EcAHAS-I)[27], which was also capable of decarboxylating
pyruvate to acetaldehyde prior to carboligation in the same
active site. After a 1 hour incubation, the reaction was complete
with excellent conversions (conversion 95%, yield 95 ± 2%) and
excellent enantiomeric excess (ee 99%). To form (1R,2S)-3, the
hydroxy ketone intermediate (R)-2 was immediately subjected to
transamination by the transaminase from Chromobacterium
violaceum (Cv2025)[28], using isopropylamine as amine donor.
[*]
M.Sc. V. Erdmann, Jun.-Prof. Dr. D. Rother
IBG-1: Biotechnology, Forschungszentrum Jülich GmbH
D-52425 Jülich, Germany
E-Mail: do.rother@fz-juelich.de
Dr. B.R. Lichman
John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
M.Sc. J. Zhao, Prof. Dr. H. C. Hailes
Department of Chemistry, University College London, London,
WC1H 0AJ, UK
Prof. Dr. J. M. Ward
Department of Biochemical Engineering, University College London,
London, WC1E 6BT, UK
Dr. R. C. Simon
Dr. R. C. Simon
Roche-Diagnostics GmbH, DOZCBE, 82377 Penzberg, Germany
Prof. Dr. W. Kroutil
Department of Chemistry, University of Graz, 8010 Graz, Austria
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10.1002/anie.201705855
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Scheme 1. Enzymatic (a) and chemoenzymatic (b) 3-step cascades to isoquinoline derivatives (reaction conditions step 1: 100 mM HEPES, pH 7.5, 5 mM MgCl2,
0.1 mM ThDP, 0.05 mM FAD, 10 mM 3HBA (1), 20 mM sodium pyruvate, 2,5 % DMSO (v/v), 0.5 mg/mL acetohydroxy acid synthase I from Escherichia coli
(EcAHAS-I), 30 °C, 750 rpm, step 2: 0.2 mM PLP, 100 mM IPA, 3.1 % DMSO (v/v), 3 mg/mL transaminase from Chromobacterium violaceum (Cv2025), 30 °C,
750 rpm, step 3a: 9.5 mM PAA (4), 2,5 % DMSO (v/v), 0.5 mg/mL norcoclaurine synthase variant from Thalictrum flavum (ΔTfNCS-A79I), 37 °C, 750 rpm, step
3b: 10 mM 2-BrBA (6), 200 mM potassium phosphate buffer, pH 7, 50 °C, 750 rpm) (more detailed information on the set-up is given in SI-2).
This step was completed in 8 hours and provided excellent
conversions (conversion 93 ± 1%, yield 91 ± 3%) and excellent
isomeric ratios (97 ± 1%, ir definition SI-5). The enzymes
described for this 2-step cascade were valuable catalysts for
cascade reactions with unsubstituted aromatic aldehydes and
hydroxy ketones.[25,29] Here, they also proved to be successful
catalysts for a substituted benzaldehyde, yielding biocatalytic
access to (1R,2S)-3 (metaraminol), a α1 adrenergic receptor
agonist that acts as vasoconstrictor.[30]
The formed 3 was then used for a subsequent PSR. A vital
feature of 3 was the presence of the meta-hydroxy group, which
is mechanistically required for both the mild phosphate catalysed
or enzyme catalysed PSR.[22,31] The carbonyl co-substrate
benzaldehyde (1), pyruvate (2) and isopropylamine in two
enzyme catalysed steps. Subsequently, TfNCS-A79I and 4 were
added. Reaction conversions and stereoselectivities for this last
step were excellent (conversion 99 ± 1%, dr >99%). Overall the
cascade conversion from 1 to the (1S,3S,4R)-5 was achieved in
88% yield (calculated from substrate depletion, SI-8, Fig. 39)
with excellent optical purities demonstrated at each step
(Scheme 1a, also with HPLC and GC-TOF-MS data only one
product is identified, SI-3, Fig. 8d/12). The final isomeric ratio (ir)
value was >97%. This formation of a complex trisubstituted
THIQ, with the highly selective installation of three chiral centres,
high reaction yields, and no purification of intermediates is a
clear demonstration of the power of biocatalytic cascades for the
production of complex chemicals. What is crucial to note in this
case is that NCS is a particularly promiscuous enzyme with
respect to the carbonyl co-substrate, with no observed loss in
stereoselectivity[13–15], and therefore, this cascade can be easily
adapted to produce a wide variety of (1S)-THIQs.
The PSR with meta-hydroxy substituted phenylethylamines
and aldehydes can also be very effectively catalysed by
inorganic phosphate.[22] In the initial screening with (1R,2S)-3
and 4, phosphate catalysis was observed to give rise to three
products (SI-3.4), where one of the minor products was
(1S,3S,4R)-5. In order to probe stereoselectivities of the
phosphate catalysed PSR, alternative aldehydes were examined.
A number of carbonyl co-substrates were screened and the
product selectivities were monitored (SI-7). The use of more
sterically challenging aldehydes increased reaction selectivities.
Notably, the aldehyde 2-bromobenzaldehyde (6) provided the
best stereoselectivity in the product (although priority naming
rules dictate the major product here to be an (1S)-THIQ, the 1-
stereocentre is the opposite arrangement to THIQs produced by
NCS). To isolate and characterise the product (1S,3S,4R)-1-(2-
bromophenyl)-1,2,3,4-tetrahydroisoquinoline-4,6-diol (7), this
phenylacetaldehyde (4) was selected as
a representative
aldehyde for the proof-of-principle reactions. To investigate
reaction conditions, NCS wildtype Coptis japonica and
Thalictrum flavum enzymes and available variants were
screened against 3 and 4 and the formation of products was
monitored by HPLC (for details see SI-6). The variant TfNCS-
A79I was found to produce the largest quantity of product. In a
recent study, this variant also demonstrated increased
conversion compared to wildtype for reactions with dopamine
and ketones.[18] Therefore, TfNCS-A79I was selected for a
preparative (50 µmol) biotransformation reaction for product
identification, using 4 and (1R,2S)-3 from a commercial source.
The THIQ product (1S,3S,4R)-1-benzyl-3-methyl-1,2,3,4-
tetrahydroisoquinoline-4,6-diol (5) was isolated in excellent yield
(92%, 5.HCl) and purity (de 99%). Analysis by both chiral HPLC
and two-dimensional NMR spectroscopy verified the presence of
(1S,3S,4R)-5 (SI-4.1).
With the isolated product 5 and analytics for stereoisomer
isolation in hand (SI-3), the entire 3-step 3-enzyme cascade was
demonstrated. As described above and shown in scheme 1,
(1R,2S)-3 was formed biocatalytically from 3-hydroxy
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reaction was scaled up (20 µmol), and the product was purified
by extraction and recrystallisation, to give 7.HCl in 54% isolated
yield (97% de). Analysis by both chiral HPLC and two-
dimensional NMR spectroscopy verified the compound as
(1S,3S,4R)-7 (ee before recrystallisation 97%, after
recrystallisation 99%) (SI-4.2).
The 3-step chemoenzymatic cascade was then performed
(Scheme 1b). The amino alcohol (1R,2S)-3 was formed in two
enzyme catalysed steps as described above, enzymes were
removed by ultrafiltration, and potassium phosphate and 6 were
added. This final step led to high conversions (85%, 24 h) and
excellent stereoselectivities (dr 99%, SI-3.5 Fig. 9c). The major
product of the cascade was the anticipated (1S,3S,4R)-1-(2-
bromophenyl)-3-methyl-1,2,3,4-tetrahydroisoquinoline-4,6-diol
(7). Minor side products were also formed; these are believed to
(Fig. 1). In the phosphate catalysed reaction, the favoured
transition state also has the methyl and hydroxyl substituents in
pseudo-equatorial positions. Bulkier aldehyde substituents
increase the stereoselectivity, presumably due to increased
steric clashes and raising the energy of the less favoured
transition state (in which the methyl and hydroxyl groups are
pseudo-axial). This is an example of a substrate-controlled
diastereoselectivity, in which the extant stereocentres determine
the final installed stereocentre. The enzymes EcAHAS-I and
Cv2025 thus establish the stereocentres in the amino alcohol (3),
which in turn determines the THIQ C-1 stereocentre. It is
pertinent to note that the C-1 stereocentre of the THIQs
available with the phosphate catalysed method here is opposite
to those available from NCS. Thus, here we demonstrate direct
PSR access to ‘unnatural’ THIQ stereoisomers without the
requirement of discovering or engineering a novel ‘epi-NCS’
biocatalyst.
Figure 1. Rationale for reaction selectivity.
The substrate-controlled outcome of the phosphate
catalysed reaction contrasts with the catalyst-controlled
stereoselectivity in the enzyme catalysed reactions. In contrast
to transaminases and imine reductases[32,33] that are known for
their changing stereopreference, dependent on substrate and
co-substrate choice, NCS can only stabilise one transition state,
due to the arrangement of active site side chains, giving rise to
only (1S)-THIQs (Fig. 1).[13,14,34] In this transition state the
aldehyde derived substituent is pseudo-equatorial and oriented
towards the active site entrance, whilst the methyl and hydroxyl
substituents are in pseudo-axial positions. In this scenario the
constraints of the biocatalysts active site architecture
overwhelms any conformational preferences of the intermediate.
Although the phosphate and enzyme catalysed reactions
provide access to the opposite selectivities at C-1 in the PSR
with (1R,2S)-3, the aldehyde co-substrates required for
maximum conversion and stereoselectivity differ. Aldehyde 4, an
excellent substrate for NCS, seems to be `insufficiently bulky` to
be the ortho-cyclised products 1-(2-bromophenyl)-3-methyl-
1,2,3,4-tetra-hydroisoquinoline-4,8-diol (amount <4%, see SI-3.5
and 4.3, and validated by GC-TOF-MS, HPLC and NMR by
correlation to the non-cascade KPi reaction products).[16] Overall,
conversion of the cascade, from 1 to (1S,3S,4R)-7, was 77%,
based on substrate depletion of each step. Also here the final
isomeric ratio (ir) value was excellent >97%. The enzymatic
synthesis of the opposite stereoisomer (1R,3S,4R)-7 was not
possible, as the NCS enzymes screened did not accept the
sterically demanding aldehyde 2-bromobenzaldehyde as
substrate.
The origin of the stereoselective PSR phosphate catalysed
reaction with (1R,2S)-3 is the phenethylamine substrate. In the
transition state of the phosphate PSR cyclisation step, the
molecule likely adopts a pseudo-chair conformation with the
aldehyde R-group occupying a pseudo-equatorial orientation
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COMMUNICATION
COMMUNICATION
Tetrahydroisoquinolines can
be produced by enzymatic and
chemoenzymatic 3-step
cascade reactions with a
combination of carboligases,
transaminases and
Vanessa Erdmann, Benjamin R.
Lichman, Jianxiong Zhao,
Robert C. Simon, Wolfgang
Kroutil, John M. Ward, Helen C.
Hailes, and Dörte Rother*
Page No. – Page No.
norcoclaurine synthases or,
alternatively, phosphate as
catalyst. With NCS and
phosphate, products with
opposite stereoselectivity are
gained.
Enzymatic and
chemoenzymatic 3-step
cascades for the synthesis of
stereochemically
complementary trisubstituted
tetrahydroisoquinolines
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