Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes

Article abstract of DOI:10.1002/adsc.201900154

The selective and direct reduction of carboxylic acids into the corresponding aldehydes by chemical methods is still a challenging task in synthesis. Several reductive and oxidative chemical methods are known to produce aldehydes, but most of them require

Full text of DOI:10.1002/adsc.201900154

Title: Substrate-Independent High-Throughput Assay for the
Quantification of Aldehydes
Authors: Anna Ressmann, Daniel Schwendenwein, Simon
Leonhartsbgerger, Marko Mihovilovic, Uwe Bornscheuer,
Margit Winkler, and Florian Rudroff
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201900154
Link to VoR: http://dx.doi.org/10.1002/adsc.201900154

10.1002/adsc.201900154
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Substrate-Independent High-Throughput Assay for the
Quantification of Aldehydes
Anna K. Ressmann,a Daniel Schwendenwein,b Simon Leonhartsberger,a Marko D. Mihovilovic,a
Uwe T. Bornscheuer,c Margit Winkler,b* Florian Rudroff a*
a Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/OC-163, 1060 Vienna, Austria
b acib GmbH, Petersgasse 14, 8010 Graz, Austria
c
Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-
Str. 4, 17487 Greifswald, Germany
Correspondence should be addressed to: M.W. (margit.winkler@acib.at) and F.R.
(florian.rudroff@tuwien.ac.at)
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://pubs.acs.org######.
Abstract: The selective and direct reduction of carboxylic We report a simple and fast HTA that allows the substrate-
acids into the corresponding aldehydes by chemical methods independent and chemoselective quantification of
is still a challenging task in synthesis. Several reductive and aldehydes (irrespective of their chemical structure) and is
oxidative chemical methods are known to produce sensitive to the nM range. The HTA was validated by NMR
aldehydes, but most of them require expensive reagents, and GC analyses and in microbial cells by reexamination of
special reaction conditions, are two-step procedures and the substrate scope of CAR from Nocardia iowensis
often lack chemoselectivity. Nature provides an elegant tool, (CARNi). The results were fully consistent with reported
so called carboxylic acid reductases (CARs) for the direct data.
reduction of carboxylic acids to aldehydes. Discovery as
well as engineering of novel CAR enzymes necessitates a
robust, product selective high-throughput assay (HTA).
Keywords:
aldehyde
quantification,
2-amino-
benzamidoxime, dihydroquinazoline, high-throughput
screening, cell compatible assay
essential for the synthesis of complex molecules for
fine chemicals and pharmaceutical ingredients, they
also play an important role in food and flavor
Introduction
ingredients.[3]
Chemically,
aldehydes
are
Synthetic chemists have been intrigued by the
structural complexity of natural compounds and
always endeavored synthesizing them from readily
available building blocks. To fulfill unprecedented
needs, novel methodologies were developed,
especially in the fields of catalysis (metal-, photo-,
organo-, and biocatalysis)[1] to synthesize the most
challenging molecules in as few steps as possible with
satisfying yield and high optical purity.[2] Many
syntheses rely on the availability of the highly
reactive aldehyde functional group, which can be used
for (asymmetric)-aldol-, and Mannich-type reactions
as well as Grignard additions, Wittig reactions,
oxidations and reductions. Aldehydes are not only
predominantly synthesized by oxidation of primary
alcohols, by ozonolysis or the reduction of activated
carboxylic acid derivatives (e.g. acid halides, esters).
Nature provides us with numerous enzymes that yield
aldehydes as the products. Alcohol oxidases and
alcohol dehydrogenases, for example, convert
alcohols to aldehydes.[4] Carboxylic acid reductases
(CARs) and aldehyde oxidoreductases (AORs) reduce
carboxylates to aldehydes.[5] In order to screen for
new and engineer known enzymes towards
operational fitness for industrial applications, a fast,
simple, structure independent, qualitative, and
quantitative assay is required, enabling efficient
1
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900154
Advanced Synthesis & Catalysis
identification of desired variants or novel enzymes in
a reliable fashion by the first law of directed
evolution: "you get what you screen for".[6] The
design and accuracy of the assay is crucial for success
and consequently, a robust high-throughput assay
(HTA) is absolutely required to enable the generation
of enzyme toolboxes with desired substrate scope,
stereoselectivity, stability under process conditions
etc.
should allow for screening of large mutant libraries of
aldehyde producing enzymes irrespective of the
chemical structure of the substrate.
The assay described herein is based on UV and
fluorescence, can be operated from the nM to mM
concentration range, requires minimal sample
preparation (pH-adjustment
&
filtration or
centrifugation) and it can be quantified.
Current analytical methods to quantify aldehydes rely
on time-consuming GC and HPLC methods rather
than on fast photometric assays. Commercial kits for
the colorimetric detection of small aldehydes in µmol
ranges in proprietary buffer solutions and serum
samples are available. However, those kits are costly
and have not been established for whole cell
systems.[7] So far, no general application of these kits
on structurally different aldehydes could be shown.
In the past, a number of HTAs have been developed
to determine activity for various enzymes: esterases
and lipases via fluorescence,[8] an in vivo selection
method for esterases based on cell viability,[9]
a
method based on ‘fluorescence-activated droplet
sorting’ for retro-aldolases,[10] colorimetric assays for
the determination of racemase activity on agar
plates,[11] for terpene synthases,[12] and for
transaminases.[13] The latter ones rely on the indirect
detection of side products formed during the actual
target reaction, the addition of an auxiliary enzyme
and a dye. Recently, Schnepel et al. published a
fluorescence-based quantitative method for the
determination of tryptophan halogenase activity in
crude E. coli lysate after derivatization of their
Scheme 1. High-throughput assay (HTA) for structurally diverse
aldehyde detection and application of whole cell biocatalysts.
Results and Discussion
The prerequisites for a suitable HTA are the fast
reaction of the target aldehyde and the probe
precursor in aqueous media at room temperature as
well as the formation of a stable, UV active (high
absorption coefficient) or fluorescent molecular
probe, which is selective towards the aldehyde
functionality but relaxed with respect to the overall
chemical structure of the aldehyde. In a recent paper,
Kitov et al. investigated different nucleophilic
derivatization reagents to probe aldehyde containing
proteins and to study protein functionalization.[17]
Within their work, they investigated the rapid
cyclization-reaction of 2-amino-benzamidoxime
(ABAO) derivatives (e.g. 1α–β) and a highly
activated model aldehyde (2a) in acidic aqueous
sodium acetate buffer, forming dihydroquinazoline
products (e.g. 3α–β) (Scheme 2). The latter species
displayed a high absorption coefficient, showed
fluorescence properties and could be detected down to
100 µM concentration. The study prompted us to
investigate this reaction in more detail and to adapt it
towards a general methodology for the detection of a
large variety of chemically diverse aldehydes derived
halogenated products via
a
Suzuki-Miyaura
coupling.[14] Yan et al. developed an elegant method
where product formation was directly analyzed on
agar plates by using a qualitative multicomponent
analytical imaging method.[15] Lee et al. developed a
growth dependent selection system for novel
threonine aldolases based on the consumption of toxic
acetaldehyde. Clones with high threonine aldolase
activity consume acetaldehyde faster and exhibit a
higher growth rate than others.[16] Most of the
presented assays either require a specific “screening
substrate”, include several operative steps, run at
harsh conditions or they are incompatible with living
cells.
The aim of this study was the development of a
reliable high-throughput assay for aldehyde detection
in the presence of resting cells (Scheme 1); the assay
2
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900154
Advanced Synthesis & Catalysis
from enzyme mediated whole-cell transformations.
sodium acetate, pH 4.5) at room temperature.[17]
Reaction progress was monitored via UV absorbance
at 405 nm (1α, Supporting information Fig. S4) and
NMR measurements (1α-, Supporting information,
Fig. S5).
In a first step, we investigated the reaction of six
ABAO derivatives 1α-β displaying different
electronic properties with phenylacetaldehyde (2b) as
aliphatic model aldehyde in aqueous buffer (100 mM
Scheme 2. Aldehydes, ABAO-derivatives and chemical probes (dihydroquinazolines) investigated in this study. Top:
Formation of dihydroquinazoline products (3) as reported by Kitov et al.[17] These compounds were used as a basis for the
development of the HTA. Bottom: A set of 23 different aldehydes (2) was investigated with different ABAO derivatives (1)
(only selected ABAO derivatives are shown, for a comprehensive list see Supporting information Fig. S2). Different
structures and substitutions were chosen to underline the general applicability and structural independence of the assay.
demonstrated by converting more than 20 aldehydes
in NaOAc-buffer (Scheme 2, e.g. aliphatic aldehydes
Quantitative product formation to 2-was observed
for all ABAO derivatives (1-) after 5 min. In the
following, we decided to focus on unsubstituted
ABAO (1α) and 5-methoxy ABAO (1β), mainly due
to their overall high reactivity,[17] and their promising
UV as well as fluorescence levels (Supporting
information, Table S1 & S2). Unsubstituted ABAO
(1α) was chosen for UV measurements based on its
low price of starting material, easy and high yielding
synthesis, and good solubility in buffer (Supporting
information, Table S1, entry 1). The 5-methoxy-
dihydroquinazoline derivative (3β) was outstanding
with respect to fluorescence sensitivity (Supporting
information, Table S1, entry 2).
(2b–d),
benzaldehyde
derivatives
(2e–p),
naphthaldehyde (2q), thiophene carboxaldehyde (2r),
unsaturated (2s–v) and saturated cinnamic aldehydes
(2w–x). The corresponding dihydroquinazoline
products were obtained in quantitative yields (3–b,
3αc–x, 3βi) within 3-20 min (Supporting information,
Table S4) reaction time. The reaction kinetics were
determined via UV measurements and the emerging
cyclization products 3 were confirmed by NMR
analysis (Supporting information, Chapter 4).
The assay can be used for quantification within the
low µM range applying UV detection and semi-
quantified down to nM concentrations by using
fluorescence measurements. The latter were impeded
by the noise of the fluorescence signal and the strong
After optimization of the reaction conditions (ABAO
equivalents, organic solvents, Supporting information,
Chapter 4.4) the versatility of the assay was
3
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900154
Advanced Synthesis & Catalysis
influence of the co-solvent. Furthermore, the
quantification of the assay does not rely on tedious
synthesis of reference compounds: we performed
reactions with a constant amount of ABAO and
varying concentrations of 2, and since all reactions
proceeded to completion, the system can be calibrated
“in situ” (Supporting information, Fig. S13). Next, the
chemoselectivity of 1α was verified in the presence of
different functional groups (alcohol, ketone,
carboxylic acid). When monitoring the reaction at
405 nm, no product formation was observed with
biotransformations.
Application of the ABAO assay
Chemoselectivity and the freedom to apply any
aldehyde of choice prompted us to perform a time-
resolved substrate screening of the well-known
[19]
CARNi
to validate the HTA. The expression of
CARNi was performed in an engineered E. coli strain
with Reduced Aldehyde Reduction[20] to minimize
over-reduction of aldehydes to the corresponding
alcohols
caused
by
endogenous
alcohol
phenylacetic
acid (4b),
2-phenylethanol (5b),
dehydrogenases and aldo-keto-reductases present in
the host.[20-21] Initially, reaction conditions for CARNi
mediated whole-cell reduction to phenylacetaldehyde
(2b) were optimized. We investigated different
concentrations of phenylacetic acid (4b) and
identified a substrate concentration of 2.5 mM of
carboxylic acid 4b as ideal (Supporting information,
Fig. S19). Different carboxylic acids (4) including
aliphatic-, aromatic-, and olefinic acids were
investigated and their aldehyde yields are shown in
Figure 1. Benzoic acid derivatives were generally
well reduced by CAR, but with increasing steric bulk
in 2-position the yields decreased significantly (2f–i),
which was in accordance to reported results.[22]
phenylacetone (6b) or acetophenone (6e) (Supporting
information, Fig. S14). However, phenylacetaldehyde
(2b) showed quantitative product formation in the
presence of 4–6b, which confirmed the
chemoselectivity of this assay for aldehydes.
Next, a protocol for aldehyde detection in the
presence of resting cells was developed. First, we
investigated the importance of the pH value for the
formation of the dihydroquinazoline product 3αb.
Resting cell transformations are typically performed
at neutral pH, whereas the assay requires acidic
conditions (pH 4.5).[17] In case of phenylacetaldehyde
(2b), performing the reaction in resting cell media at
pH 7 decreased the dihydroquinazoline formation rate
tremendously and only 20% of the desired product
3αb was detected after 20 min, whereas quantitative
product formation was observed in less than 5 min at
pH 4.5. Thus, we introduced a dilution step to adjust
the pH from 7.0 to 4.5 ensuring full conversion of
aldehyde 2b and restoring the fast reaction kinetics
observed at pH 4.5 (Supporting information, Fig.
S15). Additionally, we observed that the presence of
glucose in the medium resulted in a background
reaction caused by the reducing aldehyde group
(Supporting information, Fig. S16). This reaction was
very slow,[17] mainly due to the very low
concentration of the free aldehyde form of glucose in
water (<0.01%).[18] Nevertheless, reaction parameters
such as dilution and ABAO (1) amount were
optimized, which resulted in a significant suppression
of the background signal. The accuracy of the HTA
was confirmed by comparison of reaction yields
measured by GC and the ABAO assay. Aldehyde
yields determined by both analytical methods were
highly consistent, emphasizing that the assay can be
used as quantitative analytical tool in the presence of
resting cells (Supporting information, Fig. S18).
Finally, sample preparation for the HTA
(centrifugation, dilution, pH adjustment & on-plate
calibration) was adapted in view of whole-cell
4
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900154
Advanced Synthesis & Catalysis
cells, carboxylic acids, alcohols and ketones.
Furthermore, this selectivity and the very fast reaction
rates
enable
future
in-depth
mechanistic
investigations for the reactivity of aldehydes in
organic synthesis. In summary, we present a valuable
and convenient tool for the detection of aldehydes in
enzymatic as well as chemical reactions based on UV
absorption and fluorescence. With this assay in hands
first random mutagenesis studies of aldehyde
producing enzymes become possible.[23]
Experimental section
For detailed experimental procedures and NMR spectra of
novel compounds see Supporting information.
General Procedure for the preparation of 2-
aminobenzamide oximes (1)
2-Aminobenzonitrile derivatives (4.00-14.00 mmol) were
dissolved in ethanol (0.54 mmol/mL) and hydroxylamine
hydrochloride (1.1 eq.) was added. NaHCO3 (1.2 eq.) in
water (1 mL/125 mg hydroxylamine hydrochloride) was
added and the solution was stirred for 15 min at room
temperature. The solution was refluxed overnight. The
mixture was concentrated, diluted with water (2 mL/mmol
2-aminobenzonitrile) and neutralized with 1 N HCl. The
aqueous solution was extracted with EtOAc, then the
combined organic layers were dried over Na2SO4 and
concentrated. The obtained solid was washed with
DCM/PE to obtain 2-amino-benzamide oxime derivatives
1.
Figure 1. Time dependent substrate screening of structural
diverse carboxylic acids (4) in the presence of CARNi
expressing whole cells (2.5 mM 4, resting cell OD590 = 10,
25 °C). Aldehyde (2) yields were detected with the
developed ABAO assay using 1α as the probe. Reaction
yields were reported up to 5 h; prolonged reaction times led
to significantly decreased yields due to aldehyde volatility
or side reactions of the whole-cell host.
General procedure for plate reader assays: To 190 µL of
a 5.3 mM solution of different ABAO derivatives 1
dissolved in NaOAc buffer (100 mM, pH = 4.5, Supporting
information chapter 4.1) in a 96-well plate, 10 µL of
phenylacetaldehyde (2b) in acetonitrile were added to
obtain final concentrations of 1 mM aldehyde 2b and 5 mM
unsubstituted ABAO 1. After 10 minutes incubation at
room temperature, the absorbance was measured at
405 nm.
Conclusion
The lack of general high-throughput analytics for the
enzymatic synthesis of aldehydes necessitates the
development of a fast and simple assay. The
presented HTA is sensitive (up to nM range), robust,
inexpensive, compatible to aqueous systems as well
as microbial cells and can be performed with standard
laboratory equipment. By comparison of the yields
obtained via GC and this ABAO assay, sensitivity and
accuracy were validated. The assay can be conducted
in cuvettes or 96 well format and requires, for very
unreactive aldehydes about 0.5 h for completion,
including 10 min incubation time at room
temperature. The ABAO assay is suitable for various
aliphatic and aromatic aldehydes. Its selectivity
General procedure for product validation by NMR:
Phenylacetaldehyde (2b, 350 µL of a 2 mM solution in
deuterated NMR buffer) and ABAO derivatives (1, 350
µL of a 10 mM solution in deuterated buffer, Supporting
information, chapter 4.1) were mixed in a NMR tube to
obtain final concentrations of 1 mM aldehyde 2b and 5 mM
ABAO 1 and these were immediately measured.
General procedure for the preparation of resting cells:
A single colony of the desired E. coli RARE transformant
was incubated in 4 mL LB-0.8G pre-culture medium
supplemented with ampicillin (100 µg/mL) at 37 °C and
200 rpm for 14 h. The main culture medium LB-5052
supplemented with ampicillin (100 µg/mL) was inoculated
towards
aldehydes
allows
the
quantitative
determination of enzyme activity in the presence of
5
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900154
Advanced Synthesis & Catalysis
de Souza, L. S. M. Miranda, U. T. Bornscheuer, Chem.
Eur. J. 2017, 23, 12040-12063; d) M. Hönig, P.
Sondermann, N. J. Turner, E. M. Carreira, Angew.
Chem. Int. Ed. 2017, 56, 8942-8973.
A. L. Carroll, S. H. Desai, S. Atsumi, Y.-S. Jin, J.-H.
Seo, Curr. Opin. Biotechnol. 2016, 37, 8-15.
W. Kroutil, H. Mang, K. Edegger, K. Faber, Adv Synth
Catal 2004, 346, 125-142.
K. Napora-Wijata, G. A. Strohmeier, M. Winkler,
Biotechnol J. 2014, 9, 822-843.
C. Schmidt-Dannert, F. H. Arnold, Trends Biotechnol.
1999, 17, 135-136.
with 0.2% (ν/ν) of the pre-culture and incubated at 37°C
and 150 rpm for 4 h. Then the temperature was lowered to
25 °C and the main culture shaken at 150 rpm for 20 h.[24]
For resting cell preparation, cells were harvested by
centrifugation (6,000 x g at 4°C, 15 min). The pellet was
re-suspended in 1/10 volume of the main culture in resting
cell medium (M9 medium without nitrogen) to a cell
density of OD590 = 10. Resting cells were stored at 4 °C up
to one day (Supporting information chapter 5.3).
[3]
[4]
[5]
[6]
[7]
Biotransformations in screw-cap vials: A screw-cap vial
(8 mL) was loaded with resting cells (1,975 µL,
OD590 = 10) and carboxylic acid (4, 25 µL, 200 mM)
dissolved in acetonitrile or 1 M KOH was added, to obtain
a final acid concentration of 2.5 mM (optimized conditions,
Supporting information chapter 5.3). The vials were shaken
at 200 rpm at 25 °C in a Multitron shaker and samples were
taken immediately, after 1, 3, 5, and 24 h. Reactions were
performed in triplicates. Samples were at 405 nm analyzed
using the ABAO reaction in cuvettes and/or by GC as
described below.
a)
http://www.sigmaaldrich.com/catalog/product/sigma/ma
k141?lang=de&region=AT,
2017;
b)
http://www.abcam.com/aldehyde-quantification-assay-
kit-colorimetric-ab112113.html, 2017.
M. Konarzycka-Bessler, U. T. Bornscheuer, Angew.
Chem. Int. Ed. 2003, 42, 1418-1420.
E. Fernandez-Alvaro, R. Snajdrova, H. Jochens, T.
Davids, D. Böttcher, U. T. Bornscheuer, Angew. Chem.
Int. Ed. 2011, 50, 8584-8587.
R. Obexer, M. Pott, C. Zeymer, A. D. Griffiths, D.
Hilvert, Prot. Engin. Des. Selec. 2016, 29, 355-365.
S. C. Willies, J. L. White, N. J. Turner, Tetrahedron
2012, 68, 7564-7567.
R. Lauchli, K. S. Rabe, K. Z. Kalbarczyk, A. Tata, T.
Heel, R. Z. Kitto, F. H. Arnold, Angew. Chem. Int. Ed.
2013, 52, 5571-5574.
M. S. Weiss, I. V. Pavlidis, C. Vickers, M. Hohne, U. T.
Bornscheuer, Anal. Chem. 2014, 86, 11847-11853.
C. Schnepel, H. Minges, M. Frese, N. Sewald, Angew.
Chem. Int. Ed. 2016, 55, 14159-14163.
C. Y. Yan, F. Parmeggiani, E. A. Jones, E. Claude, S.
A. Hussain, N. J. Turner, S. L. Flitsch, P. E. Barran, J.
Am. Chem. Soc. 2017, 139, 1408-1411.
[8]
[9]
[10]
[11]
[12]
GC sample preparation: The reaction mixture (100 µL)
was treated with 20 µl of 2 N HCl and extracted into
200 µL of ethyl acetate containing 1 mM methyl benzoate
as internal standard in an Eppendorf vessel. The organic
layer was removed after centrifugation for 10 s at
5,000 x g. The extraction was repeated once. The combined
organic layers were dried over anhydrous Na2SO4 and
analyzed via GC. Conversions were calculated by linear
interpolation of calibration with authentic standards
(Supporting information chapter 5.3).
[13]
[14]
[15]
[16]
a) S. J. Lee, H. Y. Kang, Y. Lee, J Mol Catal B-Enzym
2003, 26, 265-272; b) K. Fesko, Appl Microbiol Biot
2016, 100, 2579-2590.
Acknowledgements
[17]
[18]
[19]
P. I. Kitov, D. F. Vinals, S. Ng, K. F. Tjhung, R. Derda,
J. Am. Chem. Soc. 2014, 136, 8149-8152.
K. N. Drew, J. Zajicek, G. Bondo, B. Bose, A. S.
Serianni, Carb. Res. 1998, 307, 199-209.
A. He, T. Li, L. Daniels, I. Fotheringham, J. P. N.
Rosazza, App. Environ. Mircobiol. 2004, 70, 1874-
1881.
A. M. Kunjapur, Y. Tarasova, K. L. J. Prather, J. Am.
Chem. Soc. 2014, 136, 11644-11654.
G. M. Rodriguez, S. Atsumi, Metabol. Engin. 2014, 25,
227-237.
W. Finnigan, A. Thomas, H. Cromar, B. Gough, R.
Snajdrova, J. P. Adams, J. A. Littlechild, N. J. Harmer,
ChemCatChem 2017, 9, 1005-1017.
Financial support by FWF (P28477-B21) and TU Wien ABC-Top
Anschubfinanzierung are gratefully acknowledged. This work has
been supported by the Austrian BMWD, BMVIT, SFG,
Standortagentur Tirol, Government of Lower Austria and
Business Agency Vienna through the Austrian FFG-COMET-
Funding Program. We would like to thank K.L.J. Prather for
providing the RARE strain.
[20]
[21]
[22]
References
[1]
a) J. Twilton, C. Le, P. Zhang, M. H. Shaw, R. W.
Evans, D. W. C. MacMillan, Nat. Rev. Chem. 2017, 1,
0052; b) D.-F. Chen, Z.-Y. Han, X.-L. Zhou, L.-Z.
Gong, Acc. Chem. Res. 2014, 47, 2365-2377; c) U. T.
Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz,
J. C. Moore, K. Robins, Nature 2012, 485, 185-194; d)
F. Rudroff, D. M. Mihovilovic, H. Gröger, R.
Snajdrova, H. Iding, T. U. Bornscheuer, Nat. Catal.
2018, 12-22.
[23]
[24]
D. Schwendenwein, A. K. Ressmann, M. Doerr, M.
Höhne, U. T. Bornscheuer, M. D. Mihovilovic, F.
Rudroff,
M.
Winkler,
2019,
361,
DOI:
10.1002/adsc.201900155.
a) F. W. Studier, Protein expression and purification
2005, 41, 207-234; b) F. W. Studier, in Structural
Genomics: General Applications (Ed.: Y. W. Chen),
Humana Press, Totowa, NJ, 2014, pp. 17-32.
[2]
a) E. J. Corey, Angew. Chem. Int. Ed. 1991, 30, 455-
465; b) S. Warren, P. Wyatt, Editors, Workbook for
Organic Synthesis: the Disconnection Approach, 2nd
Edition, John Wiley & Sons, Inc., 2010; c) R. O. M. A.
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900154
Advanced Synthesis & Catalysis
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900154
Advanced Synthesis & Catalysis
FULL PAPER
Substrate-Independent High-Hhroughput Assay for
the Quantification of Aldehydes
Adv. Synth. Catal. Year, Volume, Page – Page
Anna K. Ressmann, Daniel Schwendenwein,
Simon Leonhartsberger, Marko D. Mihovilovic,
Uwe T. Bornscheuer, Margit Winkler, Florian
Rudroff
8
This article is protected by copyright. All rights reserved.