Synthesis of Nω-Phospho-l-arginine by Biocatalytic Phosphorylation of l-Arginine

Article abstract of DOI:10.1002/cctc.201601080

The N_ω-phospho-l-arginine energy-buffering system is mainly present in invertebrates for regulating energy requirements when it is highly needed, such as in the flight muscles of an insect or when energy supply fluctuates, as in the medically important protozoa Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. The lack of availability of this important metabolite was due to a tedious chemical procedure, by which N_ω-phospho-l-arginine was prepared in over 5 reaction steps in a low yield. Therefore, we aimed at improving the synthetic methodology for the preparation of this important metabolite. As site- and enantioselective kinases have been very useful catalysts for biocatalytic phosphorylations in straightforward syntheses of phosphorylated metabolites, a stable and selective arginine kinase has been selected for the selective phosphorylation of l-arginine. The Arg gene has been cloned and expressed in E. coli and a highly active arginine kinase has been prepared. A simple synthesis of N_ω-phospho-l-arginine has been developed by arginine kinase catalyzed phosphorylation of l-arginine combined with the recycling of the phosphorylating agent ATP by using the phosphoenolpyruvate/pyruvate kinase system. After standard work-up, the desired product N_ω-phospho-l-arginine was obtained in gram quantities and in one step.

Full text of DOI:10.1002/cctc.201601080

Accepted Article
Title: Synthesis of Nω-Phospho-L-arginine by Biocatalytic
Phosphorylation of L-Arginine
Authors: Bernhard Schoenenberger, Agata Wszolek, Thomas
Milesi, Markus Obkircher, Henrike Brundiek, and Roland
Wohlgemuth
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: ChemCatChem 10.1002/cctc.201601080
Link to VoR: http://dx.doi.org/10.1002/cctc.201601080
A Journal of
www.chemcatchem.org

10.1002/cctc.201601080
ChemCatChem
FULL PAPER
Synthesis of Nω-Phospho-L-arginine by Biocatalytic
Phosphorylation of L-Arginine
Bernhard Schoenenberger [a], Agata Wszolek[b], Thomas Milesi[a], Henrike Brundiek[b] , Markus
Obkircher[a] and Roland Wohlgemuth*[a]
Abstract: The Nω-Phospho-L-arginine energy-buffering system is
mainly present in invertebrates for regulating energy requirements
when it is highly needed, as in the flight muscle of an insect or when
energy supply fluctuates, as in the medically important protozoa
Trypanosoma brucei, Trypanosoma cruzi and Leishmania major.
The lacking availability of this important metabolite was due to a
tedious chemical procedure, by which Nω-phospho-L-arginine was
prepared up to now over 5 reaction steps in a low yield. Therefore,
we aimed at improving the synthetic methodology for the preparation
of this important metabolite. As site- and enantioselective kinases
have been very useful catalysts for biocatalytic phosphorylations in
straightforward syntheses of phosphorylated metabolites, a stable
and selective arginine kinase has been selected for the selective
phosphorylation of L-arginine. The arg gene has been cloned and
expressed in E. coli and a highly active arginine kinase has been
prepared. A simple synthesis of Nω-phospho-L-arginine has been
developed by arginine kinase-catalyzed phosphorylation of L-
arginine combined with the recycling of the phosphorylating agent
ATP using the phosphoenolpyruvate/pyruvate kinase system. After
standard workup the desired product Nω-Phospho-L-arginine has
been obtained in gram quantities and in one step.
compounds.[3] Therefore, the metabolic pathway of phosphoar-
ginine is an attractive therapeutic target for parasitic diseases.[4]
Since the high-energy phosphoarginine is relatively small and a
highly diffusible molecule that provides fast energy supply when
energy consumption becomes critical, it stabilizes the cellular
ATP/ADP ratio and functions as temporal and spatial energy
buffer in the cell. It is through this buffering reaction that insect
cells can support bursts of nerve or muscle activity that would
otherwise drain ATP to levels that would not sustain other
essential functions.[5]
The phosphoarginine occurs as a free phosphoamino
acid and is associated with a specific arginine kinase (ArgK).
This cytoplasmic metabolite kinase catalyses the reversible and
ATP-dependent phosphorylation of guanidine acceptor
compounds, which act as phosphagen in muscle and nerve cells
of invertebrates. Under standard conditions, the reaction:
ATP + L-arginine ⇄ phospho-L-arginine + ADP operates close to
equilibrium. However, at times of high metabolic activity, when
ATP is low, the equilibrium shifts so as to yield net synthesis of
ATP. Hence, phosphoarginine acts as an ATP buffer in cells that
contain ArgK.[5c] Typically, ArgK are found as monomers targe-
ted to the cytoplasm, but true dimeric and contiguous dimeric
ArgKs as well as mitochondrial ArgK activities have been
observed, too.[3c] The N-phosphorylation of phosphagens like
phosphocreatine in vertebrates and phosphoarginine in
invertebrates has been studied well in physiology since 1927[6]
and has become also a paradigm for bimolecular enzymatic
reactions since the rise of molecular biology 30 years ago.[5b, 7]
Detailed solution structure NMR and high-resolution crystallo-
graphic analyses of arginine kinase in substrate-free, transition-
state and substrate-bound forms have provided mechanistic
details and linked substrate-associated motions to intrinsic
flexibility.[8]
Introduction
Nω-Phospho-L-arginine was first isolated from the muscle of
fresh-water crabs by Meyerhof and Lohmann in 1928[1] and has
been shown to play a key role in enzymatic phosphorylations in
the invertebrate muscle analogous to the role of phospho-
creatine in the vertebrate muscle.[2] It has been found in
biosynthetic pathways of parasites totally different from those
pathways found in mammalian host tissues. In a wide variety of
invertebrates and certain parasitic protozoa like Trypanosoma
brucei, Trypanosoma cruzi and Leishmania major, which cause
some of the most debilitating diseases of humankind like
leishmaniasis, African sleeping sickness, and Chagas disease,
phospho-arginine is the main reserve of high energy phosphate
Despite the fact that physiology, enzymology and
molecular biology of phosphagen synthesis has been well
understood, the availability of phosphoarginine has still been
lacking. This is partly due to the inherent instability of energy-
rich phosphoarginine, which makes chemical multistep synthesis
tedious. The instability of phosphoarginine results from the fact
that in phosphoarginine the phosphate is attached to a nitrogen
atom (N-phosphorylation) forming an acid-labile phosphor-
amidate bond, unlike in case of O-phosphorylation where the
phosphate group is attached to a hydroxyl group, yielding a
bond of lower acid lability. This is due to the protonation of the
bridging nitrogen, which induces considerable lengthening and
thereby weakening of the N-P bond [9]. Hence, the high energy
N-P bond in phosphoarginine is extremely acid labile, instable in
[a]
[b]
Dr.B.Schoenenberger, T.Milesi, Dr.M.Obkircher, Dr.R.Wohlgemuth*
Sigma-Aldrich, Member of Merck Group, Industriestrasse 25,
CH-9470 Buchs, Switzerland, E-mail: roland.wohlgemuth@sial.com
Dr.H.Brundiek, A.Wszolek
Enzymicals, Walther-Rathenau-Strasse 49a, 17489 Greifswald,
Germany
This article is protected by copyright. All rights reserved.

10.1002/cctc.201601080
ChemCatChem
FULL PAPER
hot alkali and sensitive to heat, rendering its isolation from
biological sources difficult, too [10]. Isolation of phosphoarginine,
e.g. from crayfish tail muscle or fresh water crab muscle (as a
barium salt) was low yielding, difficult to reproduce and involved
a lengthy and complex extraction procedure with average yields
of phosphoarginine less than 1% w/w.[11] Hence, several
multistep chemical synthesis routes have been employed for the
preparation of phosphoarginine.[12-15]
challenging product purification, different enzymatic ATP rege-
neration systems have been developed to overcome these prob-
lems.[17] For ATP regeneration from ADP the four systems acetyl
phosphate/acetate kinase,[18] phosphoenol pyruvate (PEP)/
pyruvate kinase (PK),[19] creatinphosphate/creatinkinase[20] or
polyphosphate/polyphosphate kinase[21] have been most often
applied. The PEP/PK-system for ATP regeneration from ADP
has typical total turnover numbers of 100 and the advantage of
the stable high-energy donor PEP, which together with PK is
commercially available at large scale and has been successfully
used in numerous preparative biocatalytic phospho-rylations.[22]
This is why we have chosen the PEP/PK-system for ATP
regeneration.
A few entirely chemical syntheses involving a number of reaction
steps have been published, but each route has its own bottle-
necks and challenges. Cramer and coworkers[12-13] described the
N-phosphorylation of Nα-Z-L-arginine-benzyl ester, the prepara-
tion of which requires several synthetic steps. Bis(p-nitrobenzyl)
phosphorochloridate was used for phosphorylation. Although
crystalline this reagent was not stable in our hands and had to
be prepared freshly each time before use. Hydrogenolytic depro-
tection provided crystalline N-phospho-L-arginine. Another
access to this energy rich phosphate described by the same
authors was by direct phosphoamidinylation of ornithine by (O-
Methyl-isoureido)-phosphonate, which is not available commer-
cially. Due to uncomplete turnover, the work up procedure
described is rather lengthy. Direct phosphorylation of arginine by
another commercially unavailable reagent, potassium phospho-
rous amidate, was applied for the peparation of NMR samples of
N-phosphoarginine.[14] Information on yield and any reaction
details were not given and the reaction mixture was not worked
up. Yet another synthesis starts from L-arginine and phospho-
rous oxychloride.[15] Due to the many side products formed this
procedure required a tedious workup and gave a very moderate
yield.
Results and Discussion
The objective of this study was to develop a simple and straight-
forward biocatalytic procedure for the phosphorylation of L-
arginine to N-phospho-L-arginine in gram-scale. Hence, we
chose
a
recombinant overexpression strategy for ArgK
employing the argK gene from Limulus polyphemus (Uniprot
P51541, KARG_LIMPO). This ArgK is one of the best characte-
rized ones and also different crystal structures are available for
this enzyme and its mutants in a substrate free form (pdb 3M10)
in transition state (pdb 1M15) or bound to different substrate
analogues (like pdb 4GW2, 4GWZ, 4GVY). The isolation of
ArgK-LP from biological sources like horseshoe crab sperm or
muscle would have been both inefficient and uneconomic as
[7b]
compared to the recombinant expression of this enzyme
.
Phosphorylating enzymes are abundant in nature and
kinases have been successfully utilized for selective biocatalytic
phosphorylations in straightforward syntheses of phosphorylated
metabolites.[16] Therefore, we aimed at implementing an impro-
ved and very efficient synthesis of Nω-Phospho-L-arginine in one
reaction step by a simple biocatalytic phosphorylation of L-argi-
Thus, ArgK-LP was overexpressed in E. coli BL21 (DE3) as a
soluble protein in high yield, i.e. around 30% of the protein was
localized in the soluble protein fraction of E. coli. The size of the
cytoplasmatic expressed monomer was approximately 40 kDa,
as determined by SDS-PAGE analysis (see supporting
information), which is similar to the sperm ArgK as determined
by Strong and Ellington.[23]
nine with
a recombinant ArgK. We describe the design,
expression, structural and functional characterization of the ArgK
from Limulus polyphemus and the biocatalytic synthesis of the
important metabolite, N-phospho-L-arginine, in gram scale.
Since the recombinant protein carried an N-terminal his-
tag, ArgK was successfully purified by IMAC purification (see
supporting information). The obtained yield of the purified ArgK
was 24 mg/L of E. coli culture. The specific activity was about 80
U/mg of purified protein and 834 U/ml in the crude extract.
In a previous study by Strong et al.,[23] in which no codon-
optimized gene was expressed, the concentration of soluble
ArgK-LP was much lower (~3 mg/L of culture) and most of the
recombinant protein aggregated as insoluble and inactive
protein.[15] In addition to that, the specific activity of the
recombinant ArgK-LP in our study was also ~28 times higher
than reported by Strong and Ellington.[23] The activity of arginine
kinase was determined by the pyruvate kinase/LDH assay
according to Oliveira et al.[24] using L-arginine as substrate.
Since storage stability could be a cause for loss of activity
when working in large scale, we studied the activity of arginine
kinase over one month in different formulations and storage
temperatures (4°C and -20°C). The storage temperature and the
addition of 30% glycerol had apparently a positive influence on
the enzyme activity. Without any precautions a maximum loss of
Figure 1. Arginine kinase-catalyzed phosphorylation of L-Arginine
As stoichiometric cofactor addition in ATP-dependent enzymatic
phosphorylations can lead to various complications in synthetic
applications such as inhibition by cofactor byproducts and
This article is protected by copyright. All rights reserved.

10.1002/cctc.201601080
ChemCatChem
FULL PAPER
40% was observed, when ArgK-LP was stored at 4°C. In case
the crude extract of ArgK was freeze-dried, a maximum loss of
50% was determined. When glycerol was added and Arg-LP
was stored at -20°C, only 13 % loss of activity could be detected
(Figure 2).
Figure 2. Storage stability investigation of recombinant arginine
kinase in different formulation
Arginine kinase activity measured in different enzyme formulations are shown
as relative activities compared to the activity of the freshly prepared crude
enzyme (white bar). From left to right: Activity of soluble enzyme stored over
one month at 4°C (grey bar), soluble enzyme stored over one month at 4°C
with additional of 30% glycerol (dotted bar), soluble enzyme stored over one
month at -20°C with additional of 30% glycerol (dashed bar), freeze-dried
arginine kinase (black bar).The activity results ± standard deviations are the
means of three independent experiments, each set measured in triplicate.
The development of biocatalytic phosphorylations is greatly
facilitated by direct 31P-NMR-analysis of product formation in the
reaction mixture.[25] The simultaneous consumption of the
phosphoenol pyruvate and the formation of the correct product is
thereby a great advantage and suitable reaction conditions can
be easily developed. The time course of a biocatalytic Nω-
phosphorylation of L-arginine was therefore followed by 31P-
NMR and is shown in figure 3. The reaction was carried out at
pH 8.0 and with ATP recycling using the PEP pyruvate kinase-
system with a slight sub-equimolar amount of PEP. A slight
excess of L-arginine compared to PEP was chosen to avoid
incomplete transformation of the latter which in our hands turned
out to be difficult to separate from the arginine-phosphate. In
Figure 3. A) Reaction kinetics of biocatalytic phosphorylation of L-arginine;
31P-NMR of reaction mixture from t=0 (lane 1) to t=5h (lane 7); signal at 0.80
ppm: PEP; signal at -3.65 ppm: Nω-Phospho-L-arginine B) Time course of
Nω-Phospho-L-arginine product formation and PEP consumption during
biocatalytic phosphorylation of L-arginine
Conclusions
The objective of this study was to develop a simple and straight-
forward biocatalytic procedure for the phosphorylation of L-
arginine to N-phospho-L-arginine in gram-scale. Tedious multi-
step chemical procedures of N-phospho-L-arginine described
with 5 reactions steps have been replaced by a straightforward
and highly efficient one-step biocatalytic phosphorylation of L-
arginine using a recombinant arginine kinase. A highly active
arginine kinase has been prepared by cloning and expressing
the gene for ArgK from Limulus polyphemus in E. coli. 31P-NMR
enabled the rapid development of the arginine kinase-catalyzed
phosphorylation of L-arginine combined with the recycling of the
phosphorylating agent ATP using the phosphoenolpyruvate/
pyruvate kinase system. The biocatalytic procedure and the sub-
sequent workup to the pure product Nω-Phospho-L-arginine has
been successfully demonstrated and opens up new opportu-
nities for the selective biocatalytic N-phosphorylation of interes-
ting small-molecular weight compounds and metabolites.[26]
1
contrast to H-NMR-spectra the 31P-NMR spectra are very clear
and the signal increase with increasing time (from bottom lane 1
to the top lane 7 in figure 3A) at -3.65 ppm demonstrates the
formation of the desired product Nω-phospho-L-arginine, while
the decreasing signal at 0.80 ppm shows the consumption of
PEP, in line with ATP turnover. The time required for complete
conversion can be optimized by selecting the total activity of the
arginine kinase utilized for the reaction, shown in figure 3B for a
phosphorylation completed within 24 hours. The method turned
out to be suitable for the synthesis of preparative quantities of
Nω-Phospho-L-arginine, whereby the use of crude extract of
ArgK and purified ArgK gave similar results.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201601080
ChemCatChem
FULL PAPER
Determination of arginine kinase activity
Experimental Section
Arginine kinase activity was measured spectrophotometrically at λ=340
nm based on ADP release by coupled assay with pyruvate kinase and
LDH, according to Strong and Ellington,[23] Oliveira et al.[24] ArgK was
assayed at room temperature by recording at λ= 340 nm the oxidation of
NADH (휀 =6.22 x103 M-1 cm-1 ). The assay mixture contained 100 mM
Tris–HCl buffer pH 7.5, 1 mM phosphoenol pyruvate, 5 mM MgCl2, 50
mM KCl, 0.15 mM NADH, 4 units of pyruvate kinase, 8 units of lactate
dehydrogenase, 2.5 mM ATP and 10 mM L-arginine in a total volume of
1ml. The reaction was started by the addition of recombinant protein.
One unit of enzyme activity is defined as the amount of enzyme
catalyzing the conversion of 1 µmol of substrate per minute at pH 7.5 and
25 ° C.
Unless otherwise stated, all chemicals were of analytical grade and were
purchased from Sigma-Aldrich.
NMR spectroscopy
NMR-spectra were measured in D2O at room temperature on a Bruker
Avance III 600 MHz spectrometer equipped with a BBO probe head with
z-gradient using 600.2 MHz for 1H-NMR and 150.9 MHz for 13C-NMR.
Gene synthesis and subcloning of ArgK
The arginine kinase gene ArgK was derived from Limulus polyphemus
(UniProtKB: P51541) as described by Strong and Ellington [15]. The
synthetic gene was codon optimized for E. coli and equipped with an N-
terminal 6x-his-tag and a TEV protease cleavage site for optional
removal of the affinity tag. The gene was synthesized and subcloned into
pET24a(+) via NdeI and NotI by Genscript (USA), giving vector pET24-
His(TV)-Karg-LimPo(op).
Determination of storage stability
The storage stability was investigated by determination of the catalytic
activity of arginine kinase for three different formulations and different
temperature. The aliquot in TrisHCl-buffer: 50 mM, pH 7.5 was
transferred into test tubes. One aliquot of the enzyme was stored as a
liquid at 4°C and -20°C for one month, to the other 30% of glycerol was
added as cryoprotectant to protect enzyme from freezing damage ant the
enzyme was stored as well as a liquid at 4°C and -20°C, another one
was freeze-dried (CHRIST beta 1-8 freeze dryer, Martin Christ Freeze
Dryers, Osterode am Harz, Germany) where the liquid was frozen at -
80°C. Dehydration step lasted for 20h at temperature of -53°C until dried
powder formed. The resulting lyophilized powders were rehydrated to its
original volume at room temperature with 50mM TrisHCl-buffer pH 7.5.
Then the samples were subjected to the subsequent activity tests. The
residual activity of the enzyme was measured on the same day.
Recombinant expression of ArgK in E. coli
E. coli strain BL21 (DE3) was transformed with the derived plasmid using
standard procedures.[27] Selected E. coli transformants were used for
expression tests, in which different media, induction times and
temperatures were optimized. The selected conditions were later used
for the overexpression of ArgK in E.coli BL21 (DE3). Firsty, an E.coli
overnight culture was prepared in 5 ml Luria Bertani (LB) medium.[28]
supplemented with 50 μg ml−1 kanamycin and incubated at 30 °C and
180 rpm. Next a 50 mL pre-culture was prepared at 37 °C and 150 rpm in
250 mL Erlenmayer flask with baffles. Therefore, 50 ml of Terrific Broth
[28]
(TB) medium supplemented with kanamycin (50 μg mL−1
)
was
Lab-scale preparation of Nω-Phospho-L-arginine
inoculated with 500 μL of the corresponding overnight culture. When the
pre-culture reached the mid-log phase, the main culture was started.
Hence, 400ml of fresh TB medium in a 2 L Erlenmeyer flask was
inoculated with 20 ml of the E. coli pre-culture. After the culture reached
an OD600 of 1.3 (optical density), protein expression was induced with
1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The main culture was
shaken at 110 rpm for 20 h at 25 °C. Cells were harvested by
centrifugation at 5000rpm for 30 min at 4°C and washed once with an
ice-cold 50 mM sodium phosphate buffer at pH 7.5. The E. coli cell
pellets containing the overexpressed enzyme (rec. ArgK) were frozen at -
20°C.
To 85ml of a 67mM solution of L-arginine in water, adjusted to pH 8.0
with diluted acetic acid, magnesiumchloride (20mM), ATP (0.05 equ.)
and PEP (0.95 equ.) were added and the pH again adjusted to 8.0. Then
200U pyruvate kinase and 200μl arginine kinase solution (see above)
were added and the reaction mixture again adjusted to pH 8 by addition
of a small amount of 0.1M acetic acid while stirring. The reaction
progress was monitored by 31P-NMR (fig.4), consumption of PEP was
complete after ~5h. After 2 days the reaction mixture was concentrated,
dried and worked up by column NP chromatography on silica gel
(methanol/water=1:1) to yield after conversion to the lithium salt 410mg
(28%) Nω-phospho-L-arginine.
Analytical Data: Single spot on TLC (silicagel, H2O/n-PrOH/NH4OH =
11:6:3); 1H-NMR: (D2O, 400MHz, δ) 3.45 (dd, 1H, J1≈J2≈6); 3.17
(t, 2H, J=6.8); 1.82 (m, 2H); 1.60 (m, 2H); 31P-NMR: (D2O, 162MHz,
δ, CPD) 3.26
Cell disruption
For cell disruption, frozen cells were adjusted to an OD600 of 40 in
potasium phosphate buffer (TrisHCl-buffer: 50 mM, pH 7.5) and mixed
with glass beads (Ø 0,25-05µm) at an approximate ratio of 1:1. Then, the
cells were disintegrated mechanically by a cell disruptor (Retsch Mixer
Mill MM 200: two cycles for 5 min at 600 rpm). The samples were cooled
on ice and insoluble cell debris was removed by centrifugation (5000
rpm, 30 min, 4°C). The supernatant, which contained the soluble protein
fraction, was immediately used for activity determination.
Gram scale peparation of Nω-Phospho-L-arginine
Based on the NMR experiments (Figures 4A and 4B) a lab scale
procedure for the gram scale preparation of Nω-Phospho-L-arginine was
performed. The amount of required kinase was determined in order to
convert 1g of L-Arginine within aprox. 6 hours. After complete consump-
tion of PEP as indicated by 1H- and 31P-NMR the reaction mixture was
purified by chromatography on silica gel as described in the lab-scale
preparation above. As considerable loss of the sensitive N-phosphate
occurred, there is room for further yield improvements by optimizing the
work up and isolation procedure.
IMAC-purification and protein content determination of rec.
ArgK
In order to purify the recombinant ArgK by IMAC (immobilized metal ion
chromatography), the soluble protein fraction was incubated with Talon®
resin on an orbital shaker (30 min, 4° C). Then, a standard gravity flow
protocol was applied to purify the target protein. Therefore, the resin was
washed at 4°C with 10 column volumes (CV) of TrisHCl-buffer (50 mM,
pH 7.5) containing 10 mM imidazole in order to get rid of unspecifically
bound protein. The target protein ArgK was eluted with 1.5 CV of elution
buffer (50mM TrisHCl-buffer, pH 7.5, 150 mM imidazole). Samples were
desalted on desalting columns in order to remove imidazole from the
purified protein samples (Zeba Spin columns, Pierce). The purified
protein was immediately used for activity determination.
The protein content of the purified protein was determined according to
the BCA method according to the manufacturer's protocol (Pierce Protein
Research Products, Thermo Scientific) using bovine serum albumin
(BSA) as a standard. The analysis of ArgK overexpressionn and the
purity was performed using 12, 5% SDS-polyacrylamide gels. Gels were
stained with Coomassie Blue. Gels were analyzed using software of
GEL ANALYZER (Version 2010a).
Acknowledgements
We thank Dr.Christine Hellriegel for the NMR kinetic data
aquisition and analysis. We also would like to thank the German
Federal Ministry of Education and Research (BMBF) for the
support of project P28 under the cluster of Biocatalysis2021.
Keywords: Nω-Phospho-L-arginine • Arginine kinase •
Phosphagen • Biocatalysis • Enzymatic Phosphorylation
This article is protected by copyright. All rights reserved.

10.1002/cctc.201601080
ChemCatChem
FULL PAPER
[1] O.Meyerhof, K. Lohmann, Biochem. Z. 1928, 196, 22-49 and 49-72.
[2] a) K. Lohmann, Biochem. Z. 1934, 271, 264-277; b) K.Lohmann,
Biochem. Z. 1935, 282, 109-119; c) K.Lohmann, Biochem. Z. 1936,
286, 28-44; d) S. Ochoa, Biochem J. 1938, 32, 237-242; e) J. K. Parnas,
Annu. Rev. Biochem. 1932, 1, 431-457.
[24] J. S. Oliveira, C. A. Pinto, L. A. Basso, D. S. Santos, Protein
Expression Purif. 2001, 22, 430-435.
[25] a) R. Matsumi, C. Hellriegel, B. Schoenenberger, T. Milesi, J. van der
Oost, R. Wohlgemuth, RSC Adv. 2014, 4, 12989-12994; b) D. Gauss,
B. Schoenenberger, R. Wohlgemuth, Carbohydr. Res. 2014, 389, 18-24.
[26] R. Wohlgemuth, Biotechnol. J. 2009, 4, 1253-1265.
[27] D.Hanahan, J. Mol. Biol. 1983 , 166, 557-580.
[28] J.Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning, Vol. 2,
New York: Cold Spring Harbor Laboratory Press, 1989.
[3] a) M. Parsons, E. A. Worthey, P. N. Ward, J. C. Mottram, BMC
Genomics 2005, 6:127, 1-19; b) C. A. Pereira, G. D. Alonso, M. C.
Paveto, A. Iribarren, M. L. Cabanas, H. N. Torres, M. M. Flawia, J.
Biol. Chem. 2000, 275, 1495-1501; c) K. Uda, N. Fujimoto, Y.
Akiyama, K. Mizuta, K. Tanaka, W. R. Ellington, T. Suzuki, Comp.
Biochem. Physiol., Part D: Genomics Proteomics 2006, 1, 209-218.
[4] C. A. Pereira, Infectious Disorders-Drug Targets 2014, 14(1), 30-36.
[5] a) A. Schneider, R. Wiesner, M. Grieshaber, Insect Biochem. 1989, 19,
471-480; b) G. Zhou, T. Somasundaram, E. Blanc, G. Parthasarathy, W.
R. Ellington, M. S. Chapman, Proc. Natl. Acad. Sci. U. S. A. 1998, 95,
8449-8454; c) R. Kucharski, R. Maleszka, Gene 1998, 211, 343-349.
[6] P. Eggleton, G. P. Eggleton, J. Physiol. 1928, 65, 15-24.
[7] a) J. Bolte, G. M. Whitesides, Bioorg. Chem. 1984, 12, 170-175; b) S. J.
Strong, W. R. Ellington, J. Exp. Zool. 1993, 267, 563-571; c) W. E.
Teague, G. P. Dobson, J. Biol. Chem. 1999, 274, 22459-22463; d) G.
Zhou, W. R. Ellington, M. S. Chapman, Biophys. J. 2000, 78, 1541-
1550; e) G. Zhou, G. Parthasarathy, T. Somasundaram, A. Ables, L.
Roy, S. J. Strong, W. R. Ellington, M. S. Chapman, Protein Sci. 1997,
6, 444-449.
[8] a) M. S. Yousef, F. Fabiola, J. L. Gattis, T. Somasundaram, M. S.
Chapman, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58(12),
2009-2017; b) X. Niu, L. Bruschweiler-Li, O. Davulcu, J. J. Skalicky,
R. Brüschweiler, M. S. Chapman, J. Mol. Biol. 2011, 405, 479-496.
[9] a) J. Fuhrmann, V. Subramanian, P. R. Thompson, Angew. Chem. Int.
Ed. 2015, 127(49), 14928-14931; b) E. A. Ruben, M. S. Chapman, J. D.
Evanseck, J. Phys. Chem. A 2007, 111, 10804-10814.
[10]H. Ouyang, C. Fu, S. Fu, Z. Ji, Y. Sun, P. Deng, Y. Zhao, Org. Biomol.
Chem. 2016, 14, 1925-1929.
[11]F. Marcus, J. Morrison, Biochem. J. 1964, 92, 429-435.
[12]F. Cramer, E. Scheiffele, A. Vollmar, Chem. Ber 1962, 95, 1670-1682.
[13]F. Cramer, A. Vollmar, E. Scheiffele, Angew. Chem. 1960, 72(6), 211-
211.
[14]T. Ruman, K. Długopolska, A. Jurkiewicz, D. Rut, T. Frączyk, J.
Cieśla, A. Leś, Z. Szewczuk, W. Rode, Bioorg. Chem. 2010, 38, 74-80.
[15]T. Nguyen Van, T. Nguyen Van, J. Roche, Bull. Soc. Chim. Biol. 1962,
44, 285-290.
[16] D. Gauss, B. Schoenenberger, G.S. Molla, B.M. Kinfu, J. Chow, A.
Liese, W. Streit, Biocatalytic Phosphorylation of Metabolites, in:
Applied Biocatalysis – From Fundamental Science to Industrial
Applications, Eds. A.Liese, L.Hilterhaus, U. Kettling, G.Antranikian,
Wiley-VCH, Weinheim, Germany, 2016.
[17] a) D. C. Crans, R. J. Kazlauskas, B. L. Hirschbein, C. H. Wong, O.
Abril, G. M. Whitesides, Methods Enzymol. 1987, 136, 263-280; b) J.
N. Andexer, M. Richter, ChemBioChem, 2015 , 16(3), 380-386.
[18] a) J. M. Lewis, S. L. Haynie, G. M. Whitesides, J. Org. Chem. 1979,
44, 864-865; b) C.-H. Wong, A. Pollak, S. D. McCurry, M. M. Sue, J.
R. Knowles, G. M. Whitesides, Methods Enzymol. 1982, 89, 108-121;
c) D. C. Crans, G. M. Whitesides, J. Org. Chem. 1983, 26, 3130-3132;
d) D. M. Kim, J. R. Swartz, Biotechnol. Bioeng. 2001, 74, 309-316.
[19] B. L. Hirschbein, F. P. Mazenod, G. M. Whitesides, J. Org. Chem.
1982, 47, 3765-3766; b)
[20] J. Zhang, B. Wu, Y. Zhang, P. Kowal, P.G. Wang, Org. Lett. 2003, 5,
2583-2586.
[21] a) K. Murata, T. Uchida, J. Kato, I. Chibata, Agric. Biol. Chem. 1988,
52, 1471–1477; b) S. M. Resnick, A. J. B. Zehnder, Appl. Environ.
Microbiol.2000, 66(5), 2045-2051; c) A. Kameda, T. Shiba, Y.
Kawazoe, Y. Satoh, Y. Ihara, M. Munekata, K. Ishige, T. Noguchi, J.
Biosci. Bioeng. 2001, 91(6), 557-563.
[22] a) A. Gross, O. Abril, J. M. Lewis, S. Geresh, G. M. Whitesides, J. Am.
Chem. Soc.1983, 105, 7428-7435; b) M. D. Bednarski, D. C. Crans, R.
DiCosimo, E. S. Simon, P. D. Stein, G. M. Whitesides, M. J. Schneider,
Tetrahedron Lett. 1988, 29, 427-430;
[23]S. J. Strong, W. R. Ellington, Comp. Biochem. Physiol., Part B:
Biochem. Mol. Biol. 1996, 113, 809-816.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201601080
ChemCatChem
FULL PAPER
Entry for the Table of Contents
FULL PAPER
A straightforward one-step synthesis
of Nω-phospho-L-arginine has been
developed by arginine kinase-cataly-
zed phosphorylation of L-arginine
using the phosphorylating agent ATP,
which was recycled with the phospho-
enolpyruvate/pyruvate kinase regene-
ration system for the selective
phosphorylation of L-arginine. A highly
active and stable arginine kinase has
been prepared by cloning and ex-
pressing the argK gene in E.coli .
Bernhard Schoenenberger, Agata
Wszolek, Thomas Milesi, Henrike
Brundiek, Markus Obkircher and
Roland Wohlgemuth*
Page No. – Page No.
Synthesis of N-Phospho-L-arginine
by Biocatalytic Phosphorylation of L-
Arginine
This article is protected by copyright. All rights reserved.