Two steps in one pot: Enzyme cascade for the synthesis of nor(pseudo)ephedrine from inexpensive starting materials

Article abstract of DOI:10.1002/anie.201300718

Two steps in one pot: An enzyme cascade consisting of a lyase and an (R)- or (S)-selective ω-transaminase (TA) provides (1R,2R)-norpseudoephedrine and (1R,2S)-norephedrine in only two steps. The intermediate is not isolated in this one-pot reaction and the products are obtained in high enantio- and diastereomeric purity. Moreover, the by-product from the second reaction can be recycled to serve as the substrate for the first reaction. Copyright

Full text of DOI:10.1002/anie.201300718

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Angewandte
Communications
DOI: 10.1002/anie.201300718
Biocatalysis
Two Steps in One Pot: Enzyme Cascade for the Synthesis of
Nor(pseudo)ephedrine from Inexpensive Starting Materials**
Torsten Sehl, Helen C. Hailes, John M. Ward, Rainer Wardenga, Eric von Lieres,
Heike Offermann, Robert Westphal, Martina Pohl, and Dçrte Rother*
A challenging task for chemical researchers in the next
decade is the development of cleaner and more environ-
mentally friendly reactions.[1] The traditional chemical syn-
theses of enantiomerically pure compounds often require
multistep protocols with protection–deprotection steps as
well as the isolation of potentially unstable intermediates,
lowering the yields and sustainability of the overall process.[2]
Phenylpropanolamines, members of the amphetamine
family of ephedra alkaloids, are compounds with multifunc-
tional applications but challenging syntheses routes. The
stereoisomers norpseudoephedrine (NPE) and norephedrine
(NE) are used as building blocks for the preparation of
ligands and chiral auxiliaries in organic syntheses[3] and also
have direct applications as pharmaceutically active mole-
cules.[4] Reported synthetic approaches to these compounds
have disadvantages such as relatively expensive reagents,
multistep preparative routes, and only moderate enantio- and
diastereoselectivity.[5] Recently, a novel highly stereoselective
method was described for the synthesis of all phenylpropanol-
amine isomers with ee and de values exceeding 99%.[6]
Norephedrine isomers were accessible in four steps (40%
yield) and norpseudoephedrine in seven steps (35% yield)
starting from 2-phenyl-2-trimethylsilyloxyacetonitrile.
vented[7] and thus the eco-efficiency increased.[8] Here we
present an enzymatic one-pot two-step reaction for the
synthesis of stereomerically pure (1R,2S)-NE and (1R,2R)-
NPE from benzaldehyde and pyruvate (Scheme 1). A number
of different ways to perform enzyme cascade reactions have
already been described (for more details see Chapter 1 in the
Scheme 1. One-pot two-step reaction for the synthesis of norpseudo-
ephedrine (NPE) and norephedrine (NE).
Supporting Information).[1b,2a,8a–c,9] Our one-pot two-step
reaction combines many advantages of known synthesis
strategies like high stereoselectivities, inexpensive starting
materials, high step economy (only two steps), and an
equilibrium shift without addition of further enzymes or
cosubstrates.
Synthetic enzyme cascades are valuable alternative routes
for the stereoselective production of fine chemicals. Since the
chemo- and stereoselectivities are typically high, the isolation
of by-products and reaction intermediates can be circum-
In the first step pyruvate is decarboxylated and subse-
quently ligated to benzaldehyde yielding (R)-phenylacetyl-
carbinol ((R)-PAC). The reaction is catalyzed by the thiamine
diphosphate(ThDP)-dependent acetohydroxyacid synthase I
(AHAS-I) from E. coli which performs the decarboxylation
of pyruvate and the subsequent carboligation without releas-
ing the hydroxyethyl-ThDP (see Scheme 2).[10] (R)-PAC is
obtained with high stereoselectivity (ee > 98%) and can be
converted directly to the desired (1R,2S)-NE and to (1R,2R)-
NPE in the second step of the cascade (reductive amination)
by selectively using (S)- and (R)-selective w-transaminases
(TAs), respectively. In our previous work a set of 18 different
(S)-selective wild-type (S)TAs had been screened for the
conversion of 2-hydroxy ketones.[11] For the reductive amina-
tion of (R)-PAC, the Cv-(S)TA from Chromobacterium
violaceum gave the most promising results. To gain access to
(1R,2R)-NPE, seven different (R)-selective (R)TAs from
Enzymicals AG (see Chapter 2 in the Supporting Informa-
tion) were tested.
[*] M. Sc. T. Sehl, Dr. E. von Lieres, H. Offermann,
Dipl.-Biotechnol. R. Westphal, Prof. Dr. M. Pohl, Dr. D. Rother
Institute of Bio- and Geosciences
IBG-1: Biotechnology, Forschungszentrum Jꢀlich GmbH
Leo-Brandt-Strasse 1, 52425 Jꢀlich, (Germany)
E-mail: do.rother@fz-juelich.de
Prof. H. C. Hailes
Department of Chemistry, University College London
20 Gordon Street, London WC1H OAJ (UK)
Prof. J. M. Ward
The Advanced Centre for Biochemical Engineering
Department of Biochemical Engineering, University College London
Torrington Place, London, WC1E 7JE (UK)
Dr. R. Wardenga
Enzymicals AG
The enzymatic reductive amination requires an amine
donor as a cosubstrate. Through the clever combination of
cosubstrates (here: alanine) and enzymes, the resulting by-
product (here: pyruvate) of the second reaction step can serve
as the substrate for the first step. This novel type of cascade
design is referred to as a “recycling cascade” (Scheme 2 and
Chapter 1 in the Supporting Information). We determined the
Walther-Rathenau-Strasse 49a, 17489 Greifswald (Germany)
[**] This work was supported by the CLIB Graduate Cluster Industrial
Biotechnology of the Heinrich-Heine-University Dꢀsseldorf and
financed by the DFG (German Research Foundation) in the frame of
Research Group FOR 1296.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201300718.
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Scheme 2. One-pot two-step reaction for the synthesis of nor(pseudo)ephedrine performed as a recycling cascade combining acetohydroxyacid
synthase I (AHAS-I) and a (S)- or (R)-selective w-transaminases ((R)TA, (S)TA).
thermodynamic equilibrium constant for the reductive ami-
nation of PAC with alanine as the amine donor to be 2.31 ꢀ
10À3 (Chapter 4 in the Supporting Information). Conse-
quently, when equimolar concentrations of alanine and PAC
are used and the by-product pyruvate is not removed,
a theoretical conversion of less than 5% is obtained (see
Chapter 4.4 in the Supporting Information). In our reaction
setup, pyruvate can be removed by two different carboliga-
tion reactions mediated by AHAS-I: 1) the carboligation with
benzaldehyde yielding PAC or 2) a carboligation with another
pyruvate molecule yielding acetolactate. The reversible
reaction giving acetolactate is kinetically favored, whereas
the reaction equilibrium lies on the side of PAC formation.[10]
Thus, acetolacetate is a suitable substrate for the carboliga-
tion of (R)-PAC by the cleavage reaction to pyruvate and
hydroxyethyl-ThDP.
Figure 1. Conversion curves for the reductive amination of benzalde-
hyde (10 mm) and PAC (10 mm) by A) w-TA Cv-(S)TA (1 mgmLÀ1
)
and by B) At-(R)TA (1 mgmLÀ1), respectively. The reaction was carried
out in 100 mm HEPES (pH 7.5 with 200 mm pyridoxal-5’-phosphate
(PLP), 50 mm flavine adenine dinucleotide (FAD), 100 mm ThDP, 5 mm
MgCl2) containing (S)- or (R)-a-methylbenzylamine (10 mm) as amine
donor.
A challenge in this one-pot two-step cascade is the fact
that the starting material benzaldehyde might serve as
a substrate for AHAS-I as well as for the w-transaminases.
As a consequence of the higher chemical reactivity of
aldehydes relative to ketones and steric constraints in the
active site of w-TAs, it was not possible to find an enzyme
among the 25 screened w-TAs for which the reductive
amination of PAC was kinetically favored over the reductive
amination of benzaldehyde. The most promising (S)-selective
transaminase Cv-(S)TA has a roughly 17-fold higher initial
rate in the reaction with benzaldehyde than with PAC
(Figure 1A). As a consequence, in a one-pot two-step cascade
reaction where AHAS-I and Cv-(S)TA were added simulta-
neously, 98% of the benzaldehyde was converted to benzyl-
amine (Figure 2A). However, in the case of the (R)-selective
w-TAs we could surprisingly identify enzymes for which the
simultaneous one-pot two-step reaction provided (1R,2R)-
NPE with conversions of up to 85% (Figure 2A). The initial
rate activities of the w-TA from Aspergillus terreus (At-
(R)TA) for reactions with PAC and benzaldehyde were on the
same order of magnitude (Figure 1B), but roughly ten times
lower than the initial rates for the (R)-PAC formation
catalyzed by AHAS-I. These differences suffice to reduce
the amount of formed by-product (benzylamine) to merely
10%.
In line with these experimental data, the NE/benzylamine
ratio is low when the one-pot two-step reaction is performed
as a simultaneous cascade including a recycling step. Here,
both enzymes were added simultaneously to a mixture of
20 mm benzaldehyde, 10 mm pyruvate, and 50 mm alanine.
Since no further pyruvate was added, product concentrations
higher than 10 mm (NE or NPE) are only possible by the
successful recycling of pyruvate that is generated by deam-
ination of alanine. Remarkably, with At-(R)TA about 14 mm
(1R,2R)-NPE and only 5.5 mm benzylamine were formed in
this simultaneous recycling mode, while in case of the Cv-
(S)TA the major product is benzylamine.
Although benzylamine can be separated from NPE and
NE by column chromatography (mobile phase EtOAc/
MeOH/NH3 = 85:10:5), it is more advantageous to reduce
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Figure 2. A) One-pot two-step simultaneous cascade and B) one-pot
two-step simultaneously recycling cascade with Cv-(S)TA and seven
(R)-selective TAs. For the one-pot two-step reaction 10 mm benzalde-
hyde, 10 mm pyruvate, and 50 mm d- or l-alanine were dissolved in
100 mm HEPES (pH 7.5 with 200 mm PLP, 50 mm FAD, 100 mm ThDP,
5 mm MgCl2) and the two enzymes (AHAS-I and w-TA) were added
simultaneously. The recycling cascade reactions (B) were performed
analogously with 20 mm benzaldehyde instead of 10 mm.
Figure 3. Synthesis of A) (1R,2S)-NE and B) (1R,2R)-NPE performed as
a sequential one-pot two-step reaction with an additional recycling
step (time-dependent reaction curve can be found in Chapter 6 in the
Supporting Information). Reaction conditions: 100 mm HEPES (pH 7.5
with 200 mm PLP, 50 mm FAD, 100 mm ThDP, 5 mm MgCl2), 258C,
100 rpm. One-pot two-step reaction: Carboligation (1 h): 10 mm ben-
zaldehyde, 10 mm pyruvate, 0.5 mgmLÀ1 AHAS; reductive amination
(12 h): +50 mm alanine, +1 mgmLÀ1 TA. Recycling step:step a:
+10 mm benzaldehyde, +0.5 mgmLÀ1 AHAS-I (A: 1.5 h, B: 5 h);
step b: +1 mgmLÀ1 TA (A: Cv-(S)TA, 12 h, B: At-(R)TA, 5 h).
the formation of byproducts by appropriate process engineer-
ing in order to increase process efficiency. There are two
general ways to perform a cascade reaction: one is the already
described simultaneous mode, the other one the so-called
sequential mode, where the catalysts are added consecu-
tively.[9] In our sequential synthetic enzyme cascade, the
limiting step is the reductive amination. In order to circum-
vent this bottleneck, we optimized the reaction parameters of
the reductive amination step regarding pH, temperature, the
concentrations of transaminase and AHAS-I, and the amine
donor/PAC ratio (see Chapter 5 in the Supporting Informa-
tion). For the enzyme combination Cv-(S)TA/AHAS-I con-
versions exceeding 80% could be achieved under optimized
cascade conditions (pH 7.5, 258C, 1 mgmLÀ1 Cv-(S)TA,
0.5 mgmLÀ1 AHAS-I, alanine/PAC = 5:1).
These optimized conditions were applied in the one-pot
two-step sequential mode. Here, the transaminase was added
after the benzaldehyde had been completely consumed in the
AHAS-I-catalyzed carboligation step (after 1 h 100% con-
version was achieved, Figure 3A). This increased the con-
version of (1R,2S)-NE from 2% (Figure 2A: one-pot two-
step simultaneous cascade) to 78% (7.8 mm, Figure 3A) with
the combination AHAS-I/Cv-(S)TA. Under these conditions
the undesired by-product benzylamine amounted to less than
0.5 mm (Figure 3A). Upon subsequent addition of further
10 mm of benzaldehyde and fresh AHAS-I, PAC was formed
in a second carboligation step. Since no further pyruvate was
added, this result demonstrates that the recycling of pyruvate,
generated by deamination of alanine, was successful in this
sequential enzyme recycling cascade mode. However, the
reaction resulted in the formation of only 4.7 mm PAC (47%
conversion), which is most likely due to the instability of
acetolactate. If the latter is chemically decarboxylated to
acetoin, it is no longer available for PAC formation. More-
over, acetoin (and probably also acetolactate) can act as
substrates for Cv-(S)TA as described previously.[11] Further,
neither the NE nor the benzylamine concentration increased
significantly, which suggests almost complete inactivation of
Cv-(S)TA (Figure 3A). Addition of fresh Cv-(S)TA started
the reaction again yielding 12.9 mm (1R,2S)-NE (de > 98%,
ee > 99%). This corresponds to roughly 65% of the possible
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reaction analysis, cascade optimizations, and equilibrium determina-
tion can be found in the Supporting Information.
product concentration (related to a total benzaldehyde
concentration of 20 mm, Figure 3A).
In the case of At-(R)TA the cascade reaction is even more
efficient (Figure 3B): (1R,2R)-NPE is accessible in the one-
pot two-step sequential cascade with conversions greater than
96% and very high stereomeric purity (de > 98%, ee > 99%).
After addition of another 10 mm benzaldehyde and fresh
AHAS-I, the At-(R)TAwas still active. Thus, without addition
of further transaminase, 16.6 mm (1R,2R)-NPE (83% con-
version) was obtained in 5 h by the complete one-pot two-step
recycling cascade. Further addition of At-(R)TA did not
considerably increase the final product concentration.
In summary, we have developed a strategy for the
synthesis of (1R,2S)-NE and (1R,2R)-NPE. Both compounds
are accessible in a biocatalytic one-pot two-step reaction in
high stereoisomeric purity (de > 98%, ee > 99%) from inex-
pensive starting materials without isolation of the intermedi-
ate product. Additionally, these cascade reactions can be
performed in a novel “recycling mode” in which the
coproduct of the second step is removed without addition of
further catalysts or cosubstrates and recycled as a substrate
for the first cascade step.
By combining reaction and process optimization, the
sequential cascade consisting of AHAS-I and Cv-(S)TA
provided (1R,2S)-NE with a conversion of 80% (8.0 mm).
For the production of (1R,2R)-NPE we could identify (R)-
selective w-TAs catalyzing the one-pot two-step cascade in
a simultaneous mode with conversions up to 85% (8.5 mm).
Moreover, in the sequential mode, formation of the side
product (benzylamine) is reduced and (1R,2R)-NPE was
obtained with a conversion exceeding 96% within 13 h. In the
recycling step (addition of another 10 mm benzaldehyde, but
no pyruvate) a total concentration of 16.6 mm (1R,2R)-NPE
(83% conversion) was observed after further 5 h reaction
time.
Received: January 27, 2013
Published online: May 9, 2013
Keywords: asymmetric synthesis · biocatalysis ·
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enzyme cascades · phenylpropanolamine · w-transaminase
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The recycling mode can be applied to any set of reactions
for which a clever combination of cosubstrates and copro-
ducts is possible, such that the coproducts of one reaction can
be reused as substrates for the other. This approach optimizes
the atom economy of the reaction by reducing the waste
production.
Experimental Section
l-alanine (Merck), d-alanine (Sigma Aldrich), and pyruvate (Sigma–
Aldrich) were of > 99% purity. Benzaldehyde (Sigma–Aldrich) was
freshly distilled before use. The preparation of the catalysts Cv-(S)TA
and AHAS-I is described in the supporting information. (R)-selective
TAs are commercially available from Enzymicals AG (Germany) as
lyophilized crude cell extracts. Descriptions of the reaction details,
[11] T. Sehl, R. C. Simon, H. C. Hailes, J. M. Ward, U. Schell, M. Pohl,
D. Rother, J. Biotechnol. 2012, 159, 188 – 194.
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