Direct monitoring of biocatalytic deacetylation of amino acid substrates by1H NMR reveals fine details of substrate specificity

Article abstract of DOI:10.1039/d1ob00122a

Amino acids are key synthetic building blocks that can be prepared in an enantiopure form by biocatalytic methods. We show that thel-selective ornithine deacetylase ArgE catalyses hydrolysis of a wide-range ofN-acyl-amino acid substrates. This activity was revealed by¹H NMR spectroscopy that monitored the appearance of the well resolved signal of the acetate product. Furthermore, the assay was used to probe the subtle structural selectivity of the biocatalyst using a substrate that could adopt different rotameric conformations.

Full text of DOI:10.1039/d1ob00122a

Organic &
Biomolecular Chemistry
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Direct monitoring of biocatalytic deacetylation
of amino acid substrates by 1H NMR reveals fine
details of substrate specificity†
Cite this: Org. Biomol. Chem., 2021,
19, 4904
Silvia De Cesare,a Catherine A. McKenna,a Nicholas Mulholland,b Lorna Murray,a
a
Juraj Bellaa and Dominic J. Campopiano
*
Amino acids are key synthetic building blocks that can be prepared in an enantiopure form by biocatalytic
methods. We show that the L-selective ornithine deacetylase ArgE catalyses hydrolysis of a wide-range of
N-acyl-amino acid substrates. This activity was revealed by 1H NMR spectroscopy that monitored the appear-
ance of the well resolved signal of the acetate product. Furthermore, the assay was used to probe the subtle
structural selectivity of the biocatalyst using a substrate that could adopt different rotameric conformations.
Received 22nd January 2021,
Accepted 30th April 2021
DOI: 10.1039/d1ob00122a
rsc.li/obc
reverse” to catalyse the formation of amides from amino
Introduction
sugars, amino acids and various carboxylic acids.11,12
Reaction monitoring remains a pillar of biocatalysis. It has
A classic approach to calculate the conversions of these bio-
been employed in the study of the activity, selectivity and sub- transformations is via chiral HPLC analysis.13,14 However, this
strate scope of wild type enzymes and it is a fundamental plat- approach can only be employed when UV-active substrates (e.g.
form upon which directed evolution can be applied to engin- aromatic, heterocyclic) are used. Alternative HPLC assays
eer novel, more efficient biocatalysts.1–4 Therefore, the develop- monitor the kinetics of the hydrolytic reaction by observing
ment of robust and reproducible high-throughput (HTP) the disappearance of the N-acylated starting material.15–17
assays is necessary to achieve fast and reliable measurement However, given the weak absorbance of the amide bond in the
of enzymatic activity, kinetic parameters and reaction UV region (214 nm, ε = 103 M−1 cm−1),15 the sensitivity of this
conversions.5
Hydrolases form a well-characterised class of stereoselective orimetric and fluorometric assays use two main strategies to
analysis is quite poor. To overcome this limitation, various col-
biocatalysts, usually employed to develop kinetic resolution
(KR), dynamic kinetic resolution (DKR) or de-symmetrisation
processes for the isolation of small, enantiopure molecules.6
Aminoacylases or acylases (EC 3.5.1.14) are important
members of this family of enzymes; they specifically catalyse
the hydrolysis of the amide bonds of canonical N-acetylated
amino acids (N-Ac-AAs) or derivatives, releasing the free amino
acid along with acetate into solution (Scheme 1). Given the
high stereoselectivity of these class of enzymes, they are
usually employed in the resolution of optically active products
from racemic starting materials7 and to synthesise a range of
amides including the penicillin-derived antibiotics.8 They can
be readily combined with N-acetyl amino acid racemases
(NAAARs or NSARs) to achieve a DKR that can convert racemic
N-acetylated amino acids into enantiopure products.9,10
Moreover, some acylases have also been shown to act “in
monitor biocatalysed deacetylation reactions.
The first strategy consists of exploiting a HTP continuous
enzymatic assay.5 For example, three enzymes, an acetate
kinase (AK), a pyruvate kinase (PK) and a D-lactate dehydrogen-
ase (LDH) are coupled together in the presence of ATP and
phosphoenolpyruvate (PEP) to measure the free acetate formed
by linking it directly to the oxidation of NADH to NAD+
(Scheme S1A†).18 Similarly, the formation of L-glutamate
(L-Glu) can be easily monitored using an NAD+-dependant
L-Glu dehydrogenase (GDH) that catalyses the oxidative deami-
nation of L-Glu to α-ketoglutarate.19 Sánchez Carrón et al.,20
recently reported a HTP colorimetric assay that detects free
aSchool of Chemistry, University of Edinburgh, David Brewster Road,
King’s Buildings, Edinburgh, EH9 3FJ, UK. E-mail: Dominic.Campopiano@ed.ac.uk
bSyngenta, Jealott’s Hill, Warfield, Bracknell RG42 6EY, UK
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1ob00122a
Scheme 1 Synthesis of enantiopure D- or L-amino acids via hydrolysis
of N-acetylated substrates using
a stereoselective D- or L-acylase
biocatalyst.
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amino acids by coupling their production with an FAD-depen-
dant L-amino acid oxidase (L-AAO) and a horseradish peroxi-
dase (HRP) (Scheme S1B†). The resulting H2O2 can be linked
Results and discussion
1H NMR assay proof of concept
to the oxidation of o-dianisidine at 436 nm (ε = 5700 M−1 Our target biocatalyst for proof-of-concept studies is the
cm−1) or a similar colorimetric reporter. Although these HTP Escherichia coli N-acetyl ornithine (N-Ac-Orn) deacetylase (ArgE,
continuous assays are useful when measuring biocatalyst Uniprot code: P23908, Fig. 1A) which is a di-metal (e.g. Zn2+)
activity and kinetics, they are not suitable for quantitative ana- dependant L-selective acylase with a broad substrate scope.15,16
lysis as the formation of the product is monitored indirectly 1H NMR is ideal for monitoring deacetylations as the H NMR
1
via a cascade of reactions with a limited linear range.
spectrum of an N-acetylated amino acid has at least two very
A second popular strategy often employed for the analysis distinctive signals, which undergo a shift during hydrolysis
of free amino acids is the use of derivatization reagents to (Fig. 1B and C). Catalytic cleavage of the amide bond results in
produce a product with either a strong absorbance or fluo- the C-α-proton shifting from 4–5 ppm to 3–4 ppm, while the
rescence in the UV-Vis region. These derivatives are easily CH3 group singlet shifts from the amide substrate at
detected via HPLC or LC-MS analysis. Such reagents include: 2.1–1.9 ppm to the acetate product at 1.9–1.8 ppm. By compar-
ninhydrin,21 o-phthalaldehyde (OPA),22,23 Marfey’s reagent ing the ratios integrals of these signals, it is possible to calcu-
(MR)24 and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD).25 late the relative conversions of the biocatalysed reaction.33
Use of MR also allows for the determination of the enantio-
A biotransformation (Fig. 1A) was set up using the
meric excess (% ee) of the desired reaction product, since it natural substrate of the ArgE deacetylase, N-Ac-L-Orn (1a).
forms two easily separated diastereoisomers (Scheme S1C†). Recombinant E. coli ArgE was expressed, purified and charac-
Amino acids can also be derivatised using various agents, such terised as reported in ESI (see ESI, Fig. S1 and S2†). The sub-
as alcohols or chloroformates to obtain a volatile product that strate N-Ac-L-Orn, 1a (20 mM) was incubated with ArgE and
is readily analysed by GC.26–29 Unfortunately, all derivatization CoCl2 at 40 °C for 24 h in a final volume of 500 μL. CoCl2 was
reactions suffer from a lack of reproducibility, given the poss- added to the reaction mixture, since it has been reported that
ible formation of side-products in the assay mixture. Recently, Co(II) increases the activity of ArgE approximately 2-fold.16
various LC-MS protocols for the quantitation of free amino
acids and MR derivatives in solution have also been
developed.30–32
Alongside these methods, an ideal candidate for biocatalyst
1
reaction monitoring is H NMR spectroscopy. This technique
allows the easy calculation of reaction conversions using a rela-
tive quantitation method, or the determination of the absolute
concentration of starting material and product present in the
sample when an appropriate internal standard is employed in
a quantitative NMR (qNMR) experiment.33 Given the various
1
advantages of H NMR spectroscopy, such as the easy analysis
set-up, short measurement time, direct structural information,
high accuracy and its non-destructive nature, this technique
had already been applied for the quantitative analysis of active
ingredients (AI), natural products and complex biological
mixtures.33,34 Furthermore, 1H NMR spectroscopy has been
used to measure biocatalysed reactions; examples include
lipase-catalysed acylations35 and monophosphates ring
opening reactions,36 determination of the % ee of hydrolytic
KRs,37,38 and to characterise complex carbohydrate
derivatives.39,40 Here we report the development, optimization
and application of a novel 1H NMR assay to monitor amide
hydrolysis catalysed by
a
highly active deacetylase
(Scheme S1D†). This was achieved by direct observation (both
time dependent and end-point) of the sharp, well-resolved
singlet peak associated with the methyl group of the acetate
product. Furthermore, we show the wide utility and applica-
Fig. 1 1H NMR assay proof of concept. (A) Scheme for the ArgE-cata-
lysed deacetylation of N-Ac-L-Orn (1a) with 1H NMR spectra with anno-
tated signals. (B) N-Ac-L-Orn (1a) substrate standard. (C) L-Orn (2a)
product standard. (D) ArgE biotransformation reaction shows complete
conversion of 1a to 2a during 24 h at pH 8.0 (the signal at 3.68 ppm is
due to the TRIS buffer used during E. coli ArgE purification).
1
bility of this assay, by applying an optimised H NMR protocol
to determine the % conversion for a series of N-acetylated,
non-canonical amino acids (NCAAs). Finally, we provide
insight into the subtle biocatalyst substrate preference of
N-acyl amide rotamer conformations.
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Once L-ornithine (2a) formation was confirmed by LC-TOF MS strates with higher sensitivity compared to classic spectropho-
analysis, the biotransformation was quenched by addition of tometric analysis.
HClconc to precipitate the enzyme. 100 μL of D2O was mixed in
Reaction monitoring
an NMR tube with 400 μL of the crude reaction mixture and
the 1H NMR spectrum of the sample was recorded on a Bruker
AVA500 (500 MHz) NMR. To achieve a higher signal to noise
ratio (S/N), a solvent signal suppression experiment was
employed. For reference, an NMR spectrum of a standard
samples of substrate 1a and product 2a were also recorded. By
comparing the spectra of standards 1a, 2a (Fig. 1B and C) with
the reaction mixture (Fig. 1D) it was possible to conclude that
the reaction went to completion, following 24 h incubation at
40 °C. This was confirmed by the disappearance of the signals
from the C-α proton (multiplet at 4.15 ppm) and the CH3 from
the amide (2.00 ppm) (Fig. 1D). This was also accompanied by
the clear CH3 signal observed for the acetate product at
1.88 ppm (Fig. 1D). It is worth noting that depending on the
nature of the substrate side chain, the C-α proton signal of
N-acetylated amino acids (4–5 ppm region) could fall close to
or overlap with the residual peak of water (4.79 ppm). This
superimposition can alter the intensity of this signal during
the solvent suppression experiment, rendering peak inte-
gration inaccurate. However, the acetate singlets of the amide
Thus far, we have simply employed 1H NMR spectroscopy to
calculate the reaction conversion in an end-point type assay,
after prolonged incubation (24 h). However, NMR has been
successfully employed to run kinetic experiments to directly
monitor various reactions over time.41–43 Similarly, we
employed this technique to follow the progress of the ArgE-cat-
alysed deacetylation reaction. To run these real-time, reaction
monitoring experiments N-Ac-L-Val (Fig. 2A) was selected as
the substrate since this N-acetylated amino acid has two
signals that fall at low field in the 1H NMR spectrum. These
can be employed to follow the biotransformation progress. The
1H NMR spectrum was recorded for both the N-acyl-L-Val sub-
strate and L-Val reaction product to use as reference (Fig. S3†).
We observed both the N-acyl singlet (N-Ac at 2.16 ppm) and
the signals generated from the two –CH3 groups of the isopro-
pyl side-chain (iPr, βR at 0.98 pm) for N-Ac-L-Val.
Two identical reactions were set up in NMR tubes and incu-
bated at 25 °C and 40 °C respectively. A 1H NMR spectrum was
recorded on a Bruker AVA 400 (400 MHz) NMR every 10 min for
2.5 h (Fig. 2B); the first time-point measurement was taken
15 min after the addition of ArgE to the reaction mixture. This
lag phase was necessary to set up the experiment and to give
enough time for the NMR probe to reach the desired tempera-
substrate fall in
a low-field region of the spectrum
(2.1–1.7 ppm), far away from the solvent residual peak, thus, it
can be integrated with high accuracy to give a good estimate of
the reaction conversion. Furthermore, this signal is generated
by three equivalent protons, which should deliver a better S/N
ratio and higher assay sensitivity; indeed, sodium acetate is a
common internal standard used in qNMR experiments for the
determination of the concentrations of the species present in
the sample.32
Encouraged by the success of our first analysis, we set up a
screen of biotransformations following the same reaction pro-
tocol, to prove the wide applicability of this assay in the study
of enzymatic deacetylations. A panel of nineteen N-acetylated
L-proteinogenic amino acids (with the exception of N-acetyl
serine, available only as a racemate) was tested as ArgE sub-
strates (Scheme S2†). All reaction conversions were calculated
by comparing the intensity of the methyl signal of the
N-bound amide substrate and free acetate product (apart from
the N-Ac-Gln substrate where we used the signals from the C-
α-proton, see ESI†). Amino acid product formation was also
1
confirmed by detection via LC-MS analysis to back up the H
NMR data. The screening results, reported in Table S1,† are in
broad agreement with the data reported in literature which
used the less sensitive UV-Vis amide bond assay (λmax
=
214 nm) to determine the kinetic parameters of ArgE.14 We
found that ArgE displayed a preference for aliphatic and posi-
tively charged substrates, whereas acidic (glutamic and aspar-
Fig. 2 Direct time-dependant monitoring of the biocatalytic N-Ac-L-
tic acid) and aromatic (phenylalanine (<2.0% conversion), tyro- Val deacetylation by 1H NMR spectroscopy. (A) Reaction scheme of the
sine and tryptophan) N-acylated amino acids are not hydro-
lysed by this L-acylase. In addition, our assay was able to ident-
ArgE-catalysed N-Ac-L-Val hydrolysis. The iPr groups of the substrate
(βR) and product (βP) are labelled. (B) Superimposition of 1H NMR
spectra of the N-Ac-L-Val hydrolysis at 40 °C, pH 8.0 during incubation
from 15 (dark red) to 125 min (dark blue). The signals for the substrate
ify some novel substrates, such as N-Ac-L-valine, arginine, histi-
1
dine and proline. This data shows how H NMR spectroscopy
(βR at 0.98 ppm and N-Ac at 2.16 ppm) reduce and new signals for the
can be exploited to obtain reliable assays for these amide sub- L-Val product (βP 1.12 ppm and Ac, 2.02 ppm) appear.
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ture. In this initial NMR spectrum, effectively 15 min into the range of NCAAs side-chain functionalities. The best substrates
reaction, we observed the appearance of a new signals due to were N-Ac-Orn (1a), N-Ac-ω-nitro-Arg (1e), N-Ac-β-chloro-Ala (1i)
the formation of the acetate (Ac, 2.02 ppm) and the L-Val and also included N-Ac-cyclopropyl-Gly (1f). The substrate pre-
product (βP at 1.12 pm). Integration of these revealed the reac- ference defined by the 1H NMR assay can now be compared
tion progress to be 34%, for the reaction incubated at 25 °C. with that measured using the coupled L-AAO method
We then monitored these signals over time (every 10 min) for (Scheme S1B, Table S2†). This revealed a similar substrate
3 h. By superimposing the recorded spectra, it was possible to profile however the 1H NMR assay has the advantage that it
observe the progress of the reaction, with the characteristic reports directly on ArgE specificity and removes the impact of
signals of the N-Ac-L-Val decreasing over time and reached the substrate preference of the L-AAO coupling enzyme.20
completion at 125 min (Fig. 2B, coloured dark red at 15 min
Conformational analysis of substrate specificity
moving to dark blue). At the same time, we observed an
increase in the relative abundance of the L-Val product signals Finally, we wanted to further explore the selectivity of ArgE
(normalised to the acetate CH3 signal at 2.02 ppm, Fig. 2B). By using a substrate that displayed the ability to adopt different
measuring the integrals of the substrate and product signals conformations in solution. We selected trans N-acetyl 2-amino-
for every 1H NMR spectrum recorded, we were able to calculate 5-methylhex-3-enoic acid (1j) (Fig. 3A, see ESI†) and observed a
the percentage conversion of the reaction for each time point series of signals in the double bond region of the 1H NMR
at both 25 and 40 °C (Fig. S4A†). We found that 40 °C was the spectrum of the starting material (Fig. 3B). N-Acyl amides are
best incubation temperature for ArgE activity, with full conver- known to form two different conformations (rotamers), due to
sion (>99%) reached after 2 h incubation, compared with a the restricted rotation around the amide bond. The ratio of
conversion of 81% for the reaction incubated at room tempera- minor : major rotamers is dictated by the thermodynamic
ture. We also confirmed that the conversion of the substrate stability of each conformation.44,45 We assigned the two
follows a linear initial rate after ArgE addition and determined doublet of doublets at 5.81 and 5.68 ppm (J = 15.6, 6.5 Hz) as
similar specific activities for the reaction monitored by both Hd and Hb of the minor and major rotamers respectively, with
the 1H NMR and the coupled L-AAO assays (Fig. S4A and S4B†). a ratio of 0.7 : 1 of Hd to Hb (Fig. 3B). These assignments were
confirmed by 2D NMR spectroscopy (see ESI† for NMR charac-
terization). In addition, the multiplet at 5.48–5.41 pm is
caused by the overlapping signals of Ha and Hc of the major
N-Acetylated NCAAs library screening
1
To further emphasise the broad applicability of this H NMR
assay we prepared and screened a small library of N-acetylated,
non-canonical L-amino acids (NCAAs). The substrates used in
the screen were prepared synthetically from the free amino
acid via acetylation with an excess of acetic anhydride (see
ESI†). The screen of the N-Ac-NCAAs substrates was carried out
using the standard protocol (see ESI†) and the final conver-
sions, following incubation with ArgE for 24 h, are reported in
Table 1.
No activity was detected towards N-Ac-tert Leu (1g) and
N-Ac-tert Bu-Ala (1h), probably caused by the large steric bulk
of the tert-butyl group preventing it being a good ArgE sub-
strate. In contrast, good-to-excellent activity was observed with
the eight other substrates tested which displayed a broad
Table 1 Conversion percentages determined from the integral of the
acetate peak in the 1H NMR spectra (Fig. 1). The m/z values are from
positive ion LC-TOF MS analysis for the ArgE-catalysed production of
the corresponding product L-NCAAs (2a–j) (N.D. product not detected)
m/z [M + H]+
Fig. 3 Determination of the conformational selectivity of E. coli ArgE.
Substrate
R (side-chain)
Conv (%)
(A) Reaction scheme for the hydrolysis of the substrate trans-2-acetoa-
mido-5-methylhex-3-enoic acid 1j, present in solution as a mixture of
rotamers indicated by the rotation arrow. (B) The double bond region
(5–6 ppm) of the 1H NMR spectrum of the two trans protons are anno-
tated as follows: Ha and Hb for the major rotamer, Hc and Hd for the
minor rotamer. (C) The same chemical shift region as (B) after incubation
of 1j with ArgE (0.25 mg mL−1) and CoCl2 (100 μM) at 40 °C for 24 h. The
1H NMR integrals for the major rotamer do not change whereas the inte-
grals for the minor rotamer decrease. The appearance of new signals
that correspond to the product 2j are annotated as He and Hf.
1a
1b
1c
1d
1e
1f
1g
1h
1i
–CH2CH2CH2NH2
–CH2CH2SOCH3
–CH2CH2SO2CH3
–CH2SCH3
–CH2CH2CH2NHCNHNHNO2
–Cyclopropyl
–C(CH3)3
–CH2C(CH3)3
–CH2Cl
–CHCHCH(CH3)2
>99.0
94.3
76.4
50.7
>99.0
60.9
N.D.
N.D.
>99.0
28.3
133
166
182
136
220
116
N.D.
N.D.
124
144
1j
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and minor rotamers respectively (Fig. 3B). Upon incubation
Conflicts of interest
with ArgE we observed the formation of a new signal at
1
5.90 ppm, Hf, which we assign to the vinyl H of 2j, as well as The authors declare no conflict on interests.
the reduction of the Hd signal at 5.81 ppm (Fig. 3C). The other
vinyl 1H of the product He, appears in the multiplet at
5.48–5.41 ppm (Fig. 3C). Our analysis suggests that ArgE
hydrolyses only one of the rotamers (the minor conformation).
Acknowledgements
However, at present we cannot define the absolute confor- We thank the Engineering and Physical Sciences Research
mation of the preferred rotamer (attempts to further define Council (EPSRC), Syngenta and CRITICAT Centre for Doctoral
these using variable temperature NMR did not resolve the con- Training for financial support [Ph.D. studentships to S. D. C.
formations). This conformational selectivity (atropisomerism) and C. A. McK.; Grant Code: EP/L016419/1]. The NMR facility
has been observed during the design of N-acyl amide inhibi- is supported by EPSRC grant EP/R030065/1. We also thank
tors and it is now incorporated into the design of Prof. Guy Lloyd-Jones and Dr Andrew Hall (University of
pharmaceuticals.46,47 Such detailed substrate analysis is not Edinburgh) for their help and useful comments, as well as
possible with the HPLC, MS and HTP colorimetric methods.
Dr Dimitrios Papasotiriou and Dr Peter Howe (Syngenta,
Bracknell) for their help in the initial optimization of the H
1
NMR and LC-MS experimental protocols. We appreciate the
insight provided by Dr Manuela Tosin (University of Warwick)
and Dr Amanda Jarvis (University of Edinburgh).
Conclusions
In conclusion, by employing 1H NMR spectroscopy we were
able to develop a quick and efficient assay to monitor biocata-
lytic deacetylation of various amides. For proof of concept we
have used E. coli N-Ac-L-ornithine deacetylase (ArgE) and a
wide range of canonical N-Ac-L-AAs. The comparison of the
integrals of the clear signals for the amide starting material
and the free acetate product allows for a fast and accurate
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