Benchtop NMR for Online Reaction Monitoring of the Biocatalytic Synthesis of Aromatic Amino Alcohols

Article abstract of DOI:10.1002/cctc.201901910

Online analytics provides insights into the progress of an ongoing reaction without the need for extensive sampling and offline analysis. In this study, we investigated benchtop NMR as an online reaction monitoring tool for complex enzyme cascade reactions. Online NMR was used to monitor a two-step cascade beginning with an aromatic aldehyde and leading to an aromatic amino alcohol as the final product, applying two different enzymes and a variety of co-substrates and intermediates. Benchtop NMR enabled the concentration of the reaction components to be detected in buffered systems in the single-digit mM range without using deuterated solvent. The concentrations determined via NMR were correlated with offline samples analyzed via uHPLC and displayed a good correlation between the two methods. In summary, benchtop NMR proved to be a sensitive, selective and reliable method for online reaction monitoring in (multi-step) biosynthesis. In future, online analytic systems such as the benchtop NMR devices described might not only enable direct monitoring of the reaction, but may also form the basis for self-regulation in biocatalytic reactions.

Full text of DOI:10.1002/cctc.201901910

Accepted Article
Title: Benchtop NMR for online reaction monitoring of the biocatalytic
synthesis of aromatic amino alcohols
Authors: Christiane Claaßen, Kevin Mack, and Dörte Rother
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Benchtop NMR for online reaction monitoring of the biocatalytic
synthesis of aromatic amino alcohols
[a]
[a, b]
and D. Rother*[a, b]
C. Claaßen , K. Mack
Abstract: Online analytics provides insights into the progress
of an ongoing reaction without the need for extensive sampling
and offline analysis. In this study, we investigated benchtop
NMR as an online reaction monitoring tool for complex enzyme
cascade reactions. Online NMR was used to monitor a two-
step cascade beginning with an aromatic aldehyde and leading
to an aromatic amino alcohol as the final product, applying two
investigated over the past decade.[4] The product range
accessible in enzymatic cascade reactions increases with
each additional reaction step.[5] However, not only the
accessible product range increases, but also the overall
complexity of the reaction design. Among other aspects,
major challenges in designing suitable enzyme cascades
include side reactions due to enzyme-substrate
[4h, 6]
different enzymes and
a
variety of co-substrates and
promiscuity,
and balancing the different reaction steps
intermediates. Benchtop NMR enabled the concentration of the
reaction components to be detected in buffered systems in the
single-digit mM range without using deuterated solvent. The
concentrations determined via NMR were correlated with
offline samples analyzed via uHPLC and displayed a good
correlation between the two methods. In summary, benchtop
NMR proved to be a sensitive, selective and reliable method
for online reaction monitoring in (multi-step) biosynthesis. In
future, online analytic systems such as the benchtop NMR
devices described might not only enable direct monitoring of
the reaction, but may also form the basis for self-regulation in
biocatalytic reactions.
to achieve optimal conversions and specific space-time
yields for the whole cascade. Detailed kinetic knowledge
about the reactions would help, for example, in selecting
the right time point at which a cascade step reaches its
peak, so that the next reaction step could be started when
using a sequential cascade mode (i.e. by adding the
enzyme for the next reaction step). Another point is optimal
balancing of the amounts of catalyst in modularly attached
reactors (i.e. plug-flow devices). Selecting suitable
amounts of catalyst would be greatly facilitated if sensitive
and selective online analytics were available for each
reaction step and the overall process performance.
Furthermore, if the performance of complex reactions or
multi-step synthesis could be suitably monitored, it would
be easier to establish optimal reaction parameters for the
whole system in a more time-efficient manner.
Introduction
Recent advances with benchtop NMR (nuclear magnetic
resonance) spectroscopy makes online reaction monitoring
Enzymatic reactions typically show high substrate
specificities and both regio- and stereoselectivities, making
them ideal alternatives to classical chemical synthesis
strategies. This especially holds true if products with one or
more chiral centers and high optical purity are desired.[
Ongoing research in the field of biocatalysis provides a
broad variety of well-characterized enzymes catalyzing
reactions that are extremely challenging with classical
chemical synthesis.[2] The variety of enzymes available
provides access to a steadily increasing number of different
products.[3]
[7]
possible not only for chemical syntheses , but also for
[8]
[9]
fermentation processes,
and biocatalytic reactions
where low analyte concentrations and the necessity of
using deuterated solvents has impeded the use of NMR in
the past. A detailed overview of the advantages and
disadvantages of benchtop NMR for online reaction
monitoring in general can be found in references [7d, 10]. With
respect to the monitoring of biocatalytic reactions, online
NMR offers advantages such as the possibility of analyzing
the reaction mixture without destroying or changing the
sample composition and without the need for extensive
sampling. Altogether, this prevents the loss of valuable
product due to sampling and analysis. In addition to the
above-mentioned advantages, NMR in general enables
reaction components to be determined which can be
extremely challenging to quantify with other analytical
techniques, but which frequently serve as reaction
1]
Within the field of biocatalysis, the combination of enzymes
into multi-step reaction cascades has been increasingly
[
a]
b]
Prof. Dr. D. Rother, Dr. C. Claaßen, K. Mack
Institute of Bio- and Geosciences – Biotechnology (IBG-1)
Forschungszentrum Jülich GmbH
52425 Jülich (Germany)
E-mail: do.rother@fz-juelich.de
Prof. Dr. D. Rother, K. Mack
Aachen Biology and Biotechnology (ABBt)
RWTH Aachen University
components
isopropylamine
in
biocatalytic
or acetaldehyde
synthesis,
e.g.
[
[
4a, 4h, 11]
[1a, 12]
. The distinct
advantages mentioned above, are expected to lead to an
increasing relevance of NMR for applications in
biotechnological systems as already indicated by the first
few publications in this field.[8a, 9]
52074 Aachen (Germany)
Supporting information for this article is given via a link at the end of
the document.
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In this contribution, we use the example of an chiral
aromatic amino alcohol to show that substrates and
products for the enzymatic cascade reaction leading to 3-
selective transaminase BmTA[14] (see Scheme 1). This
cascade is especially interesting as the active catalyst in
both steps has to be sensitively regulated to avoid
undesired by-product formation or else the steps have to
be separated in time or space[4h]. To run the overall
cascade efficiently, optimal concentrations of both
enzymes and all participating co-substrates are needed,
which are usually obtained by extensive process
optimization on a small scale. With these prerequisites, the
cascade is a good example for online analytics to directly
(
2-amino-1-hydroxypropyl) phenol, originally published by
Erdmann et al.[4h], are detectable in a buffered solution
even in a low milli-molar range using the benchtop NMR
device. We show the signals of various buffers and
alternative co-substrates. Since
a suitable distinction
between the NMR signals of the reaction components is
given, concentrations of the different compounds can be
calculated even in the complex reaction mixture. In this way, monitor process design and selectivity to the final product.
we propose benchtop NMR-based online analysis for the
biocatalytic cascade reaction to gain direct insights into
product formation during the cascade reaction without the
need for extensive sampling. Furthermore, direct
information can be gained by means of the proposed online
reaction monitoring, thus enabling a quicker reaction to
parameter changes or in the future external regulation of
the system.
Cascade design
To assess whether signal overlap in the NMR spectrum will
occur between the reaction components during online
measurements, firstly offline spectra of each pure
component in aqueous solution were measured with the
benchtop NMR device. Signal overlaps between the
reaction components would complicate data evaluation.
However, it should be noted, that signal overlaps do not
necessarily prevent the usage of NMR spectroscopy for
online analytics. Nevertheless, for this publication we
chose the simplest system to verify the general suitability
of NMR analytics for enzyme cascades in buffered systems.
Figure 1 shows the positioning of the signals occurring
from the substrates/products (A), two possible co-
substrates for the first reaction step (B), three possible
amine donors/resulting by-products in the second reaction
step (C), and five alternative buffers and water (D) in the
Results and Discussion
The cascade reaction monitored here by NMR-based
online analysis is the sequential two-step cascade reaction
to (1S,2S)-3-(2-amino-1-hydroxypropyl)phenol (3OH-AHP)
previously published in a similar mode by Erdmann et al.
for another stereoisomer, the (1R,2S)-stereoisomer[4h]. The
cascade consists of two reaction steps, where first 3-
6
0 MHz benchtop NMR applied in this study. The original
spectra of the components in water can be found in the
supporting information. The aldehyde-CH- and CH -signals
hydroxy-benzaldehyde (3OH-BA) is converted by
a
carboligase to 1-hydroxy-1-(3-hydroxyphenyl) propan-2-
one (3OH-PAC). The second reaction step, a reductive
amination, catalyzed by a transaminase with an amine
donor molecule as co-substrate, leads to the final amino
alcohol (3OH-AHP).
3
of the substrates/products were clearly separated in the
NMR spectra, making it easy to distinguish between the
different components. However, the CH-protons of 3OH-
PAC and 3OH-AHP were buried under the broad water
signal.
The signal width, especially of the water signal, was
strongly dependent on the system shim status (Figure 1-D,
top row): As indicated by the darker coloring, the water
In general, the access to four different stereoisomers of
3
OH-AHP is possible by the combination of enzymes with
different stereoselectivities.[
4h]
For this publication, the
(
1S,2S)-stereoisomer was chosen for online reaction
monitoring. The stereoisomer was obtained by combining
the S-selective carboligase ApPDC E469G[ with an S-
13]
Scheme 1. Reaction scheme of a two-step enzymatic cascade yielding the aromatic amino alcohol 3-(2-amino-1-hydroxypropyl)phenol in (1S,2S)-
conformation. R=CH ; R´=CH , COOH, or phenyl depending on the amine donor applied The proton groups monitored by NMR-based online
analytic in this publication are highlighted in dark green.
3
3
.
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Figure 1. Position of NMR-signals of the reaction components, co-substrates, by-products and buffers for the cascade reaction. Some signals for
the substrates/products and buffers, especially CH-protons, were (partly) buried under the water signal so that these signals are not shown in the
figure. Corresponding amine donors and their keto-products formed upon amino-group transfer in C are indicated by the arrows. Water signal is also
shown in the buffer spectra in D. (3OH-BA=3-hydroxy-benzaldehyde; 3OH-PAC=1-hydroxy-1-(3-hydroxyphenyl) propan-2-one; 3OH-AHP=3-(2-
amino-1-hydroxypropyl)phenol).
signal was relatively narrow, even without any water
suppression, if the system had a very good shim status
height: 6.3 Hz; for the standard shimming sample, 10%
O in D O). If the shim status was not optimal (signal width
at 50% height: 0.69 Hz; signal width at 0.55% height:
H
2
2
(
signal width at 50% height: 0.28 Hz; signal width at 0.55%
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1
D
5.63 Hz; for the standard shimming sample, 10% H
O), a broader water signal was observed, as indicated by
the light blue coloring.
2
O in
separation of the signals seems to be more likely here.
Pyruvate is not only a good reaction partner for 3OH-BA
after decarboxylation through the carboligase enzyme, but
furthermore the acid derivatives have typically been shown
to be less toxic for the enzyme compared to the free
2
With respect to the investigated buffer systems, Kpi buffer
did not contribute additional signals to the spectra besides
the water signal, and the signal of Tris-buffer was almost in
the same ppm range as the water signal, while for the other
buffers (TEA, HEPES and MOPS) signals in addition to the
water signal occurred between 1.55 ppm and the water
signal. If a product signal in this ppm range is to be
monitored, the use of these buffers is not recommended,
as buffers are typically applied at higher concentration than
the substrates. This will most likely lead to the substrate
signal being completely buried under the buffer (“blind
region”). For our purposes, Kpi buffer was chosen as it is a
suitable buffer for the two-step cascade to the amino
alcohol[4h] and moreover has a lack of additional signals
except for the water signal thus preventing signal overlap
with reaction components during reaction monitoring
[
15]
aldehyde.
Therefore, pyruvate was chosen as the
partner for carboligation and for further analysis.
For the second reaction step, possible signal overlaps
cannot be precluded for isopropylamine (IPA), alanine, and
their ketone by-products with 3OH-PAC and 3OH-AHP. For
our purposes, α-MBA and acetophenone were the best
monitorable amine donors/acceptors and were not only
selected because of this advantage, but also due to the fact
that α-MBA pushed the reaction equilibrium to the product
side without the need of further adjustments.[16]
The signals used for quantification of substrate,
intermediate and product were the aldehyde CH of 3OH-
3
BA (@ 9.82 ppm), the CH -group of 3OH-PAC (@
2.07 ppm) and the CH -group of 3OH-AHP (@ 1.08 ppm).
3
The situation becomes more complicated with respect to
the carboligation partner for the first reaction step (Figure
Here, a suitable distinction is expected between the
reaction components, while the aromatic protons of the
components overlap largely in the NMR spectra. Whether
or not a signal overlap will ultimately occur, is largely
dependent on the concentration of the components and the
current operational status, especially line width, of the NMR
spectrometer. This parameter closely correlates with the
shim status of the system, where a manual correction to
obtain optimal operational status proved to be difficult
during the experiments.
1-B) and a number of possible amine donors (Figure 1-C).
For the first reaction step, an overlap was found between
the aldehyde CH of 3OH-BA (@ 9.82 ppm) and
acetaldehyde (AA) (@ 9.65 ppm), as well as an overlap
between the CH
CH -group of AA (@ 2.20 ppm), which is to be expected
due to the line width in the 60 MHz spectra. The signal of
pyruvate (@ 2.33 ppm) was in the same range as the CH
group of 3OH-PAC (@ 2.07 ppm). However, sufficient
3
-group of 3OH-PAC (@ 2.07 ppm) and
3
3
-
Figure 2. A: NMR setup (top) and measuring capillary adjusted in the sample holder (bottom); B: scheme of the NMR setup.
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NMR measuring parameters
relaxation times of the components would go beyond the scope
of this publication; interested readers are referred to the
publications with reference number [22] for further information.
The system which was used for online reaction monitoring is
depicted in Figure 2. It consists of the Spinsolve60ULTRA 60 MHz
NMR spectrometer with a glass measuring capillary, a
peristaltic pump and a computer with the control software. The
measuring capillary has a widened part serving as the
measuring cell, with the diameter of the measuring cell
corresponding to the diameter of a standard NMR tube (5 mm;
a scheme of the measuring cell with dimensions can be found
in the supporting information). The reaction vessel was linked
to the peristaltic pump and the measuring capillary with
standard HPLC tubing (Ø 3 mm).
1
Taking into account the longest T -time of the aldehyde CH-
proton of 3OH-BA and the required factor 7, the time between
acquisitions of spectra has to be at least 28.35 s. For all further
runs, the repetition time between the scans was therefore set
to 30 s.
Before starting online reaction monitoring, a number of
parameters that are important to ensure correct quantification
were determined with offline NMR samples. These data are
shown in the following subsections. Subsequently, we give the
results of the online reaction monitoring for the selected
cascade and correlate the data collected from the online NMR
measurements with offline samples analyzed via uHPLC.
Relaxation time
To ensure correct quantification of NMR signals, the
1
longitudinal relaxation time (T ) is one of the most important
values.[ Relaxation describes the process of dynamic return
17]
of the spins to the initial equilibrium state after a pulse
experiment and T
should be noted that individual T
1
is the time that this process takes.[18] It
-times are not substance
Figure 3. Relaxation times of protons that are intended to be used for
calculating concentrations during the reaction. (n=3)
1
constants, but dependent on experimental conditions i.e.
solvent, magnetic field strength, temperature etc.
For correct quantification, the time between two pulse
Limit of quantification
The limit of quantification (LOQ) was determined using a
concentration series of substrate (3OH-BA), intermediate
[
8a, 17, 19]
experiments (spectrum scans) should be at least 5-
to
20]
7
-times[ the value of the relaxation time to ensure that all
spins (99.9% in the case of 7xT
state before starting the next pulse sequence. If the time
between the scans is less than 5- to 7-times T , the signal
1
[20]) have returned to the initial
(
2
(
3OH-PAC) and final product (3OH-AHP) in the range of
.5 mM-25 mM, and determining the signal-to-noise ratio
SNR) depending on the number of scans used for spectra
1
intensity will successively decrease and is then no longer
directly correlated with the concentration of the component.
acquisition. The LOQ was defined as the concentration value
where the SNR was ten, which is an appropriate value
1
The highest T -time of a signal intended to be used for
[17, 23]
according to literature
, and was calculated from linear
calculating concentrations therefore determines the waiting
time between two scans.
T -times were measured with an inversion recovery pulse
1
sequence. For this pulse sequence, a 180° pulse is applied to
the sample, switching the magnetization antiparallel to the
magnetic field; then spectra are measured with a standard 90°
pulse after different evolution times (max. 15 sec in this
publication). If the applied time between the two pulses is less
regression. It should be noted that the SNR is additionally
dependent on the shim status of the spectrometer. A very good
shim status results in generally higher SNRs and smaller line
widths leading to lower LOQs, while a non-optimal shim status
results in lower SNRs and broader signals leading to higher
LOQs. The data shown below were acquired with an average
shim status for the standard shimming sample (signal width at
5
1
0% height: 0.41 Hz; signal width at 0.55% height: 11.6 Hz;
0% H O in D O). For a very good shim status, even lower
1
than the ln(2)*T -time, negative integral values are measured.
2
2
T
1
-times were calculated by fitting the curve according to the
LOQs are possible.
formula given in the experimental section.
As shown in Figure 4, the LOQs decreased with increasing
number of scans, due to the reduction of the noise in the
spectra resulting from repeated measurement. While LOQs
were between 3.7 mM (3OH-PAC) and 9.5 mM (3OH-AHP) for
only one scan, repetitions up to 16 spectra led to LOQs
between 0.8 mM (3OH-PAC) and 2.8 mM (3OH-AHP). LOQs
for 3OH-BA were between these values with 8.4 mM for one
scan and 2.7 mM for 16 scans. The difference in LOQs
Figure 3 shows the relaxation times of the protons used for
quantification in online reaction monitoring. It is obvious that
there were major differences between the relaxation time of the
3
OH-AHP CH
with 2.37 s and the 3OH-BA aldehyde CH-proton with 4.05 s.
The differences in the T -times were expected from the
3 3
-group with 0.71 s, the 3OH-PAC CH -group
1
molecular structure of the components and are in accordance
with literature.[21] A detailed discussion of the differences in the
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between the investigated components can be explained by the
correction factors accounting for the specific intensity decrease
number of protons and multiplicity of the signals. For 3OH-PAC, can be used as an alternative.[24]
3
the signal to be monitored is a CH -group without neighboring
protons meaning that the signal occurs as a singlet. For 3OH-
BA, the signal is a singlet as well, but only has one aldehyde
proton. Therefore, the intensity of the signal was lower
compared to the 3OH-PAC at the same concentration. For
3
3OH-AHP, the signal to be followed is a CH -group just like for
3
OH-PAC, but this time the CH -group has a neighboring
3
proton, meaning that the signal occurs as a duplet, again with
lower intensity compared to the singlet, which has three
protons in 3OH-PAC.
Figure 5. Left axis: Signal integral depending on the pumping speed in % of
the integral without pumping. (100% = signal integral without pumping) Right
axis: Pumping volume per minute depending on the pumping speed. (n=3)
Figure 5 depicts the percentage change of the signal intensity
at different pumping speeds based on the signal intensity
without pumping for the signals to be quantified during reaction
monitoring. A decrease in signal intensity of the aldehyde CH-
proton of 3OH-BA was determined for pumping speeds higher
-1
3
than 10 rpm (corresponding to 1.5 mL min ) while for the CH -
Figure 4. Left axis: Limit of quantification for educt and products of the
enzymatic cascade depending on the number of scans. Right axis:
Measuring time for one spectrum depending on the number of scans. (n=3)
groups of 3OH-PAC and 3OH-AHP no pronounced signal
decreases were noted up to 20 rpm (corresponding to
-1
3
mL min ). That spins with higher relaxation time are more
prone to signal decrease with increasing pumping speed is well
known from literature [24]. Consequently, to preclude any effects
of signal decrease on 3OH-BA due to pumping, a pumping
speed lower than 1.5 mL per minute, i.e. 3.5 rpm
corresponding to 0.51 mL min , was chosen for online reaction
monitoring in continuous flow.
The data clearly shows that low detection limits can be reached
with the benchtop NMR, but always at the expense of
measuring time and therefore time resolution. This means that
the optimal measuring parameters are always a tradeoff
between measuring time and detection limit. For very slow
reactions, 8 min of measuring time might be applicable, while
for very fast reactions even 2 min of measuring time might be
too long for the collection of appropriate kinetic data. For this
publication, 4 scans corresponding to a measuring time of
-1
Online reaction monitoring
For online reaction monitoring, 3-(trimethylsilyl)-2,2,3,3-
tetradeuteropropionic acid (TMSP-d
reaction mixture to serve as an internal standard for calculating
the concentrations. TMSP-d was chosen as internal standard,
4
) was added to the
2 min and resulting quantification limits of 4.5 mM for 3OH-BA,
.2 mM for 3OH-PAC and 5.5 mM for 3OH-AHP (all at average
2
4
shim status) were chosen for online reaction monitoring.
as it has a good water solubility, showed no visible signal
overlap with the compounds to be followed via reaction
monitoring, and additionally showed no interference with the
Influence of pumping speed on signal intensity
Earlier studies showed that above a certain pumping speed the
premagnetization of the sample in the NMR magnet is no
longer sufficient thus leading to a decrease in signal intensity
and an increase in line width.[ As this decrease in signal
intensity leads to the signal no longer being directly correlated
with the concentration, pumping speeds slower than those
leading to decreases in signal intensity have to be chosen for
online reaction monitoring. If it is not possible to stick to rather
low pumping speeds e.g. due to experimental limitations,
1
enzymes and the overall cascade reaction. The detected T -
time was with 2.85 s sufficiently low to allow correct
quantification (compare previous chapter). Data obtained from
online NMR measurements were correlated with data obtained
from samples taken during the reaction analyzed with uHPLC.
Figure 6 shows the data obtained during reaction monitoring
of the carboligation reaction. Depending on the reaction time
(Figure 6-A; time increases from bottom to top), the NMR
spectra show rapid changes: The aldehyde proton of 3OH-BA
7a]
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Figure 6. Online reaction monitoring for the carboligation reaction. A: Examples of NMR spectra recorded during the reaction; from bottom to top
measurements were taken after 3.4 min, 8.4 min, 18.3 min, 28.2 min, 38.3 min, 48.4 min, 58.3 min, and 68.4 min; B: Concentrations for reaction compounds
determined by NMR measurement and product additionally by uHPLC analysis (orange dots). Data were obtained with very good shim status of the system.
(HPLC n=2; NMR n=3)
at 9.82 ppm was already invisible in the fourth spectrum
corresponding to a reaction time of 28 min), while the signal of
occurs already in the pure substance, see supporting
information), and its relatively fast degradation in water.
(
3
3
OH-PAC increased successively with the decrease of the
OH-BA. Other changes in the spectra were visible in the
For the first part of the cascade, the carboligation reaction, it
can be seen, that all the concentrations depicted in Figure 6-B
reached a plateau corresponding to complete conversion of the
aldehyde after approx. 40 min of reaction time. In total, a final
concentration of 60.4 mM ± 1.6 mM was measured for the
aromatic protons region (6.5 ppm-7.7 ppm), but since an
overlap between the signals of 3OH-BA and 3OH-PAC was
evident there, a more detailed evaluation was not undertaken.
Additionally, the appearance of the CH-proton of 3OH-PAC at
intermediate 3OH-PAC via NMR. With uHPLC
a final
5
.27 ppm, partly superimposed with the water signal, was
concentration of 56.3 mM ± 0.2 mM for 3OH-PAC was
determined, which was slightly lower compared to the NMR
data, but nevertheless indicated the same reaction progression.
In summary, the deviation between the concentration values of
the two methods was 7%, which is a good correlation,
especially as uHPLC and NMR are two completely different
analytical methods, which were moreover calibrated differently.
uHPLC measurements furthermore confirmed complete
conversion of the 3OH-BA.
Figure 7 shows the data obtained for reaction monitoring of the
second part of the cascade, the reductive amination. The NMR-
spectra shown over time in Figure 7-A (time increasing from
bottom to top) indicate gradual changes in the spectra: The
detected.
To have a closer look at the reaction, concentrations (Figure
6
-B) were calculated based on the integrals of the CH-aldehyde
proton of 3OH-BA, the CH -group of 3OH-PAC and the CH
group of pyruvate (T -value of 4.53 s). The NMR-based initial
3
3
-
1
concentration for 3OH-BA was corrected by the emerging
amount of 3OH-PAC since the reaction was very fast and the
first measurement was taken after approx. 3 min as the solution
first had to be pumped into the measuring capillary. The
corrected initial concentration of 57.2 mM 3OH-BA showed
good correlation with the applied concentration of 3OH-BA
(
60 mM), while the initial concentration value determined for
pyruvate (124 mM ± 5 mM) was lower than the expected
amount (140 mM). One factor explaining this deviation could
have been the T -time of the CH -group of pyruvate, which
1 3
3
CH -group signal of 3OH-PAC at 2.05 ppm decreased until it
was almost completely disappeared in the last spectrum
(corresponding to a reaction time of 24 h). In the same manner
3
would require a repetition time of 31.92 s to be fully quantitative. the CH - groups signal of 3OH-AHP (1.08 ppm) increased. In
This deviation can however only account for an approximately
.55% (= 0.77 mM) lower detected concentration of pyruvate
addition, a decrease in the amine donor signal at 1.60 ppm was
visible, as the amino group was transferred to the final product
0
compared to the expected concentration and was therefore
ruled out as main factor. The reason for the deviation between
detected and expected pyruvate concentration is most likely a
combination of the purity of pyruvate (an additional signal
and the amine donor formed acetophenone (CH
at 2.61 ppm).
3
-groups signal
The concentrations (Figure 7-B) were calculated based on the
integrals of the CH -groups of 3OH-PAC, 3OH-AHP and α-
3
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Figure 7. Online reaction monitoring for the reductive amination reaction. A: Examples of NMR spectra recorded during the reaction; from bottom to top
measurements were taken after 3.4 min, 2 h, 4 h, 6 h, 10 h, 14 h, 20 h, 24 h; B: Concentrations for reaction compounds determined by NMR measurement
and product additionally by uHPLC analysis (orange triangles). Data were obtained with average shim status of the system. (n=3)
MBA (T
1
-value of 1.11 s). Again, the initially measured
the reaction when the much more bulky amine donor α-MBA is
arranged in the active site together with the substrate. Comparing
carboligation reaction and reductive amination, the amination
proceeded much more slowly as a quantitative conversion of
60 mM substrate was already achieved in the carboligation after
approx. 40 min of reaction time, while reductive amination of
20 mM of substrate took almost 24 h. It is additionally intriguing,
that double the mass of purified transaminase was applied for the
reaction compared to the carboligase. These data are in general
agreement with data previously published for similar cascade
reactions[4f-h] so that the online reaction monitoring data confirm
these findings. Furthermore, NMR and uHPLC analysis showed
identical trends with respect to reaction progression and almost
similar reaction times leading to full conversion. In summary,
concentrations (after 3 min of reaction time due to filling of the
measuring capillary) of 18.5 mM for 3OH-PAC and 39.1 mM for
α-MBA showed a good correlation with the amounts applied for
3
3
OH-PAC (20 mM) and α-MBA (40 mM). The concentration of
OH-PAC decreased successively while the concentration of the
final product 3OH-AHP increased, yielding a final concentration of
19.4 mM ± 1.2 mM 3OH-AHP corresponding to a conversion of
97.1% ± 6.2%. For 3OH-PAC, a residual amount of 0.4 mM ±
0.2 mM corresponding to 1.9% ± 1.0% was estimated, while for
α-MBA a residual amount of 22.8 mM ± 0.4 mM corresponding to
7% of the applied amount was determined, showing that the
5
mass balances were closed for all three components. The
concentrations of 3OH-AHP determined via uHPLC showed the
same trend compared to the NMR measurements, but the
concentrations determined via uHPLC tended to be higher
compared to the values measured with NMR. The measured final
concentration via uHPLC was 21.8 mM ± 3.0 mM, and thus 12%
higher compared to the NMR measurements.
benchtop
NMR
is
very
suitable
for
detecting
substrates/intermediates/by-product sensitively and for obtaining
direct information on their concentrations while running the
reaction.
During the reaction, approx. half of the amount of α-MBA was
consumed, corresponding to an equimolar ratio to the product.
Interestingly, pretrials also showed that when less than 40 mM of
α-MBA was applied, still only approx. half of the amount of α-MBA
was converted, indicating that the BmTA most likely can only
convert one of the α-MBA enantiomers, as the racemic mixture
was applied for the reaction. The kinetic resolution between the
two α-MBA enantiomers has been previously described in the
literature for a number of different ω-transaminases.[25]
Compared to data for the reductive amination of 3OH-PAC with
BmTA and the much smaller isopropylamine as amine donor from
our lab (data not shown), reductive amination with α-MBA
progressed more slowly. Most likely, a steric effect slows down
Conclusions
In this contribution, we showed that substrates and products for
the enzymatic cascade reaction leading to the aromatic amino
alcohol 3-(2-amino-1-hydroxypropyl)phenol (3OH-AHP) are
detectable in a low milli-molar range (approx. 2 mM-5 mM with
2
min measuring time) in buffered aqueous solution using online
benchtop NMR. Measurements were performed in standard
buffer solution, without the need to apply deuterated solvent or
components. Since a suitable distinction between the NMR
signals of the reaction components was given, the concentrations
of substrates/intermediates/products were calculated directly
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ChemCatChem
10.1002/cctc.201901910
FULL PAPER
from the NMR spectra with the help of an internal standard. In the
authors’ view, the distinction between the NMR signals is one of
the most crucial points permitting an easy reaction monitoring, as
data evaluation might be much more challenging (or even
impossible) for reactions where different signals overlap to a large
extent, e.g. due to similar chemical shifts or broader line widths.
For the two-step enzymatic reaction leading to 3OH-AHP online
reaction monitoring provided direct information on the
concentration of the reaction components during the reaction
without the need to take samples. The reaction monitoring was
shown to be very sensitive with regard to concentration changes
and at the same time very reproducible. Additionally, a high
correlation between concentrations determined via online NMR
and offline uHPLC samples was shown for the reaction products.
In future, online analytic systems such as the benchtop-NMR
device described may also be used for purposes other than direct
monitoring of the reaction in (multi-step) biosynthesis. Exemplarily,
this technique is believed to be a basis for self-regulation, as it is
not only sensitive and selective, but also sufficiently fast. This
could be especially helpful for reactions where high product
concentrations are so far hard to achieve due to substrate
insolubility or substrate toxicity.[26] In these cases, feeding
strategies could be set up for implementing feedback-control.
Also other types of interaction, such as subsequent catalyst
addition, would be adjustable. Furthermore, NMR could enable
the quantification of substrates that are extremely hard to analyze
with other techniques, e.g. acetaldehyde, to enable better process
control for these reactions. Altogether, the possibility of directly
controlling the reactions means that optimal regulation, for
example, regarding specific space-time yields, may be possible in
the near future.
detector) as well as a ZORBAX RRHD Eclipse Plus C18, 2.1x100 mm,
1.8 µm particle size and a precolumn. Details of sample preparation for
uHPLC measurement and uHPLC method can be found in the supporting
information. The uHPLC data were recorded and analyzed using Agilent
ChemStation software.
Offline NMR measurements
Recording of NMR spectra
NMR spectra of the components were recorded with the 60 MHz Spinsolve
NMR spectrometer at concentrations of 25 mM in the case of reaction
components as reference and 100 mM in the case of buffers in water.
Spectra were recorded with 16 scans per spectrum, an acquisition time of
3.2 s, repetition time of 30 s (time between the pulse experiments) and a
pulse angle of 90°.
Determination of relaxation time
Relaxation times were determined for the reaction components at a
concentration of 25 mM in water with 4 scans, an acquisition time of 6.4 s,
repetition time of 30 s, pulse angle of 90°, max. inversion time of 15 s and
1 steps. The signal integral was fitted to y=B+F*exp(-x*G) with G-1 being
2
the relaxation time according to the Mestrenova software package.
Determination of limit of quantification
The limit of quantification (LOQ) was determined by recording spectra of
the reaction components in water with different concentrations (2.5 mM,
5
mM, 7.5 mM, 10 mM and 25 mM), and determining the signal-to-noise
ratio (SNR) in the spectra for the signal to be quantified. Measuring
parameters were an acquisition time of 3.2 s, repetition time of 30 s, pulse
angle of 90° and different numbers of scan (1, 2, 4, 8 and 16). Linear
regressions of the signal-to-noise ratio for the different numbers of scans
were calculated based on SNR and concentration of the sample. With the
linear regression, the concentration at a signal–to-noise ratio of 10 was
determined (according to the commonly accepted ICH Harmonized
Tripartite Guidance Q2(R1)[23]).
Investigation of influence of pumping speed on signal intensity
To investigate the influence of pumping speed on signal intensity, solutions
of the reaction compounds with a concentration of 25 mM each were
pumped through the measuring capillary at different speeds with a
peristaltic pump. NMR spectra were recorded after at least 5 min of
continuous pumping (without stopping the pump during NMR signal
detection) with 4 scans, an acquisition time of 3.2 s, a repetition time of
Experimental Section
Materials
3-Hydroxy-benzaldehyde (3OH-BA), alpha-methylbenzylamine (α-MBA),
isopropylamine (IPA), acetaldehyde (AA), pyridoxalphosphate (PLP),
triethanolamine (TEA), and hydrochloric acid (HCl) were purchased from
30 s and a pulse angle of 90°. The pumping volume per minute resulting
Sigma-Aldrich
dihydrogenphosphate (KH
(Germany).
PO
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic
HEPES), 3-morpholinopropane-1-sulfonic acid (MOPS), sodium
Acetone,
), di-potassium hydrogenphosphate
acid
pyruvate,
potassium
from the rotating speed of the peristaltic pump was determined
gravimetrically.
2
4
2 4
(K HPO ),
(
Online reaction monitoring
hydroxide (NaOH), 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt
TMSP), trifluoroacetic acid (TFA), diethanolamine (DEA) were purchased
from Carl Roth (Germany). Other reagents were purchased from the
following sources (given in parentheses): 3-(2-amino-1-
hydroxypropyl)phenol (3OH-AHP; TRC, Canada); acetophenone
Reaction conditions of the enzymatic transformation
Carboligation: The carboligation reaction was performed with purified
enzyme (ApPDC E469G) at a protein concentration of 1.25 mg/mL in
(
1
00 mM Kpi buffer at pH 6.5 supplemented with 2.5 mM MgCl
ThDP, 0.2 mM PLP and 5 mM TMSP. Substrate concentrations were
0 mM 3OH-BA and 140 mM pyruvate. Reactions were carried out for 2 h
2
, 0.1 mM
(
(
AlfaAesar, Germany); alanine (Fluka, Germany); thiamine pyrophosphate
ThDP; AppliChem, Germany); 2-amino-2-(hydroxymethyl)propane-1,3-
6
at 30 °C.
diol (Tris; AlfaAesar, Germany); magnesium chloride hexahydrate
MgCl *6H O; Fluka, Germany).
(
2
2
Reductive amination: Reductive amination was performed with purified
enzyme (BmTA) at a protein concentration of 2.5 mg/mL in 100 mM Kpi
A Magritek Spinsolve 60 MHz NMR spectrometer was used as the NMR
system. NMR data were recorded using the Spinsolve 1.15.0 Software and
analyzed using Mestrenova (Version 12.4.0-22023). The NMR system was
equipped with a LongerPump® BT100-2J (DG-2). As the uHPLC system
an Agilent 1290 Infinity II was employed (consisting of solvent rack,
temperature-controlled sample rack, temperature-controlled multisampler,
temperature-controlled column oven, binary high-speed pump, diode array
2
buffer at pH 7.5 supplemented with 2.5 mM MgCl , 0.1 mM ThDP, 0.2 mM
PLP and 5 mM TMSP. 20 mM of the substrate (S)-3OH-PAC was applied
and 40 mM of the co-substrate α-MBA was added. Reactions were carried
out at 30 °C and allowed to proceed for 24 h.
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ChemCatChem
10.1002/cctc.201901910
FULL PAPER
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