Synthesis and use of a phosphonate amidine to generate an anti-phosphoarginine-specific antibody

Article abstract of DOI:10.1002/anie.201506737

Protein arginine phosphorylation is a post-translational modification (PTM) that is important for bacterial growth and virulence. Despite its biological relevance, the intrinsic acid lability of phosphoarginine (pArg) has impaired studies of this novel PTM. Herein, we report for the first time the development of phosphonate amidines and sulfonate amidines as isosteres of pArg and then use these mimics as haptens to develop the first high-affinity sequence independent anti-pArg specific antibody. Employing this anti-pArg antibody, we further showed that arginine phosphorylation is induced in Bacillus subtilis during oxidative stress. Overall, we expect this antibody to see widespread use in analyzing the biological significance of arginine phosphorylation. Additionally, the chemistry reported here will facilitate the generation of pArg mimetics as highly potent inhibitors of the enzymes that catalyze arginine phosphorylation/dephosphorylation. Arginine phosphorylation: Synthesis of stable phosphoarginine analogues enabled the generation of high-affinity sequence-independent anti-phosphoarginine antibodies. These antibodies can be used to directly detect and isolate arginine-phosphorylated proteins.

Full text of DOI:10.1002/anie.201506737

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201506737
German Edition: DOI: 10.1002/ange.201506737
Protein Modification
Synthesis and Use of a Phosphonate Amidine to Generate an Anti-
Phosphoarginine-Specific Antibody
Jakob Fuhrmann,* Venkataraman Subramanian, and Paul R. Thompson*
Abstract: Protein arginine phosphorylation is a post-transla-
tional modification (PTM) that is important for bacterial
growth and virulence. Despite its biological relevance, the
intrinsic acid lability of phosphoarginine (pArg) has impaired
studies of this novel PTM. Herein, we report for the first time
the development of phosphonate amidines and sulfonate
amidines as isosteres of pArg and then use these mimics as
haptens to develop the first high-affinity sequence independent
anti-pArg specific antibody. Employing this anti-pArg anti-
body, we further showed that arginine phosphorylation is
induced in Bacillus subtilis during oxidative stress. Overall, we
expect this antibody to see widespread use in analyzing the
biological significance of arginine phosphorylation. Addition-
ally, the chemistry reported here will facilitate the generation of
pArg mimetics as highly potent inhibitors of the enzymes that
catalyze arginine phosphorylation/dephosphorylation.
Protein arginine phosphorylation was originally found to
[
7]
occur on histone proteins. More recently, the first protein
[
8]
arginine kinase, McsB, was identified in bacteria, sparking
the subsequent identification of more than 100 phosphoargi-
nine (pArg) modification sites in the Gram-positive model
organism Bacillus subtilis using a ywlE arginine phosphatase
[9]
mutant strain. Given that this modification is even more
abundant than O-phosphorylation in B. subtilis, arginine
phosphorylation likely represents a ubiquitous regulatory
mechanism that plays a key role in cell physiology and
[9b]
survival under specific conditions. Moreover, the protein
arginine kinase McsB was shown to be important for the
virulence of Gram-positive pathogens such as Staphylococcus
[10]
aureus,
thus highlighting the antibacterial potential of
targeting this protein modification.
Current methods to detect cellular protein arginine
phosphorylation rely on cumbersome mass spectrometric
analyses that are carried out under acidic conditions leading
P
rotein phosphorylation regulates numerous biological
[9,11]
processes and intracellular signaling pathways in both
to a time dependent loss of signal.
Additionally, these
[
1]
eukaryotic and prokaryotic organisms. In eukaryotes, the
phosphorylation of serine, threonine and tyrosine residues,
that is, O-phosphorylation, represents a mechanism for the
control of cell signaling cascades. Apart from these phos-
phate monoesters, it is well established that histidine,
techniques are technically challenging and are not readily
available to all researchers in the field. Furthermore, they do
not allow for the rapid and facile detection of the changes in
arginine phosphorylation that occur in response to cell
signaling events. Given the limitations of current methods,
we sought to develop a facile method to visualize and enrich
for arginine phosphorylated proteins. Towards that goal, we
report the design and synthesis of non-cleavable phosphonate
and sulfonate amidines as isosteres of pArg and demonstrate
their use as haptens to generate the first high-affinity
sequence independent anti-pArg specific antibody.
[2]
[
3]
arginine, and lysine are N-phosphorylated. In contrast to
O-phosphorylation, the NÀP phosphoramidate bond is acid
[4]
labile and thus is readily hydrolyzed under acidic conditions.
This acid lability is due to the protonation of the bridging
nitrogen, which induces considerable lengthening and thereby
[
3c,5]
weakening of the NÀP bond.
Despite the challenging
nature of the NÀP bond, chemical biologists have recently
Previous attempts to generate anti-pArg antibodies using
classical immunizations of mice and rabbits failed; most likely
because the acid labile NÀP bond is cleaved during endo-
begun to focus on the development of suitable tools for
[3d,6]
analyzing protein N-phosphorylation.
For example, Muir
[12]
and colleagues recently reported the development of the first
pan-phospho-histidine (pHis) specific antibody using a stable
somal/lysosomal processing of the immunogen. An in vitro
phage display approach was used to generate an antibody
[6c]
[12]
triazole-based pHis analog as the hapten.
targeting a phosphoarginine containing peptide. While this
antibody is useful for in vitro studies with recombinant
[
12]
proteins, its low affinity for pArg (see below) has precluded
its use in cellular studies. To circumvent these drawbacks and
obtain high-affinity phosphoarginine-specific antibodies, we
set out to synthesize a non-cleavable, acid stable pArg
mimetic, hypothesizing that such a hapten would allow for
the generation of a sequence independent high-affinity anti-
pArg antibody. With this goal in mind, we further hypothe-
sized that amidines derivatized with either a sulfonate or
a phosphonate group might act as stable analogues of pArg
[
*] Dr. J. Fuhrmann
Department of Chemistry, The Scripps Research Institute
30 Scripps Way, Jupiter, FL 33458 (USA)
1
E-mail: jfuhrman@scripps.edu
Dr. V. Subramanian, Prof. Dr. P. R. Thompson
Department of Biochemistry and Molecular Pharmacology
UMass Medical School
3
64 Plantation Street, Worcester, MA 01605 (USA)
E-mail: paul.thompson@umassmed.edu
Dr. V. Subramanian, Prof. Dr. P. R. Thompson
Chemical Biology Interface Program, UMass Medical School
(
Figure 1)—the acid labile NÀP bond is replaced with a stable,
non-hydrolyzable CÀP bond. Under physiological conditions,
phosphoarginine exists in a zwitterionic form, containing a net
charge of À1, with the positive charge distributed over the
3
64 Plantation Street, Worcester, MA 01605 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201506737.
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Scheme 2. Synthesis of SO -amidine (6), 2-((6-aminohexyl)amino)-2-
3
iminoethane-1-sulfonic acid.
procedures and compound characterizations are provided in
the supporting material (Figure S7–S12). Since the phospho-
ryl group in phosphoarginine exists as the doubly negative
Figure 1. Structural representation of the electrostatic map of the side
chain of arginine, phosphoarginine and its stable analogs SO -amidine
3
as well as PO -amidine. The formal net charge of the individual
3
charged form (pK = 4.5) under conditions of physiological
a2
compounds is denoted by Q.
[3c]
pH, we evaluated the protonation state of the phosphonate
group in PO -amidine. Typically, the second ionization in
3
[13]
alkylphosphonates occurs at pH ꢀ 8. However, based on
P NMR pH titration experiments (Figure S13), the pK of
31
three nitrogens in the planar guanidinium moiety, whereas the
two negative charges are localized to the oxygen anions of the
phosphoryl group (Figure 1). Comparison of the calculated
electrostatic charge potentials highlights the electronic and
geometric similarities between phosphoarginine and its
anticipated analogues.
a2
PO -amidine was determined to be 6.0, indicating that the
second oxygen in the phosphono group ionizes under
physiological pH conditions. Interestingly, the pKa2 of PO3-
3
amidine is similar to aminomethylphosphonate (pK = 5.9)
a2
and thus highlights the strong pK lowering effect of the
a
Phosphonate amidine hexylamine (PO -amidine, 3), and
closely connected amidine moiety.
3
sulfonate amidine hexylamine (SO -amidine, 6), were pre-
pared as described in Scheme 1 and 2, respectively. In the case
The haptens thus generated were conjugated to KLH
carrier protein via their free amines, which are present distal
from the SO -amidine and PO -amidine groups, and then
3
of PO -amidine, Fmoc protected 1,6-diaminohexane (1) was
3
3
3
reacted with freshly prepared ethyl 2-(diethoxyphosphoryl)-
acetimidate to generate compound 2 that was deprotected
over two steps, using TMSBr to deprotect the ethyl esters and
piperidine to remove the Fmoc group, yielding the final
individually injected into rabbits to raise polyclonal anti-
bodies targeting these moieties (Figure 2a). After immuniza-
tion, the resulting sera were tested for phosphoarginine
recognition in Western blot analysis (Figure 2b and Fig-
ure S14a). The serum, obtained from rabbits immunized with
product PO -amidine (3) (Scheme 1 and Figure S1–S6 in the
3
PO -amidine, showed strong recognition of phosphoarginine
3
(Figure 2b, Figure S15), thereby validating this functional
group as a potent mimetic of phosphoarginine. By contrast,
immunization of rabbits with SO -amidine-KLH conjugates
3
only elicited SO -amidine specific antibodies that did not
3
show any significant cross reactivity to phosphoarginine
(Figure S14b).
To obtain a highly purified anti-pArg antibody, the crude
rabbit anti-serum from PO -amidine immunized rabbits was
3
affinity purified over an agarose column containing immobi-
lized arginine phosphorylated lysozyme (Figure S16 and S17).
The resulting antibody directly recognizes pArg with high
affinity and the limit of detection is approximately 10 fmol of
autophosphorylated McsB (Figure 2c). Moreover, direct
comparison with the previously described phage display
generated antibody revealed a more than 10-fold higher
sensitivity for the anti-pArg antibody developed using the
hapten approach (Figure S18). To evaluate the selectivity of
this anti-pArg antibody, we performed dot blot assays using
distinct phosphoproteins comprising pTyr, pThr, pSer, pHis,
pLys and pArg (Figure 2d). Notably, the antibody only
recognized arginine phosphorylated protein and not the
other phosphoproteins. Further highlighting the specificity
Scheme 1. Synthesis of PO -amidine (3), (2-((6-aminohexyl)amino)-2-
iminoethyl)phosphonic acid.
3
Supporting Information). The synthesis of the SO -amidine
3
derivative involves the reaction of Fmoc-1,6-diaminohexane
(
1) with chloroacetimidate to obtain the Cl-acetamidine
derivative 4 (Scheme 2). The sulfonate group was installed
by the displacement of the chloride with sodium sulfite to
synthesize 5. After Fmoc deprotection, the resulting SO3-
amidine (6) was obtained in good yield. The detailed synthetic
1
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Figure 3. Detection of arginine phosphorylated proteins using the anti-
pArg antibody in cellular extracts. a) Western blot analysis of arginine
phosphorylation in non-stressed and heat shocked (HS) cell lysates
derived from B. subtilis DywlE strain incubated with recombinant
McsB. For the loading control, equal McsB loading was verified by
western blot analysis using anti-McsB antibody. b) Strategy to detect
endogenous phosphoarginine in phosphoarginine containing proteins
using immunoprecipitation. The results below show the presence or
absence of immunoprecipitated, arginine phosphorylated ClpC and
GroEL obtained from distinct cellular samples. Abbreviations: DywlE,
B. subtilis DywlE strain; wt, B. subtilis wild-type strain.
Figure 2. Generation of phosphoarginine-specific antibodies using
a non-cleavable hapten approach. a) Strategy to develop anti-pArg
antibodies. b) Western blot of autophosphorylated McsB treated plus/
minus YwlE using anti-pArg antibody. The Ponceau stain was included
as the loading control. c) The sensitivity of the anti-pArg antibody was
determined by dot blot analysis using serial dilutions of autophos-
phorylated McsB. d) The specificity of the anti-pArg antibody was
monitored by dot blot analysis towards pTyr, pSer, pThr, pHis, pLys
and pArg modified BSA. The Coomassie stain was included as the
loading control.
For these experiments, we grew a B. subtilis DywlE strain that
[
9]
is known to harbor arginine phosphorylated proteins, and
used the anti-pArg antibody to immunoprecipitate pArg
containing proteins. As a proof of concept, we screened for
the chaperone proteins ClpC and GroEL, which are known
endogenous substrates of McsB mediated arginine phosphor-
of this antibody, pretreatment of the arginine phosphorylated
protein with the protein arginine phosphatase YwlE com-
pletely abolished the signal. Western blot analyses further
showed that this anti-pArg antibody selectively recognizes
arginine phosphorylated McsB and lysozyme, but not their
dephosphorylated counterparts, thereby confirming the gen-
eral utility and the context independent nature of this pan-
phosphoarginine targeted antibody (Figure S19).
With this data in hand, we next tested the recognition of
arginine phosphorylated proteins in cell lysates incubated
with the protein arginine kinase McsB. Interestingly, McsB
was barely active in non-stressed cell lysates, but shows strong
phosphorylation activity in heat shocked lysates, as evidenced
by the emergence of numerous arginine phosphorylated
protein bands (Figure 3a). Surprisingly, McsB activity is also
severely diminished in a sample containing a mixture of non-
stressed and stressed lysates (Figure 3a, lane 3), indicating the
presence for a potential inhibitor of McsB in non-stressed cell
lysates.
[
9]
ylation. As can be seen in Figure 3b, the anti-pArg antibody
efficiently immunoprecipitates ClpC and GroEL. Impor-
tantly, both signals were lost upon pre-incubation of the
lysates with recombinant arginine phosphatase YwlE. These
results further confirm and highlight the specificity of this
anti-pArg antibody assay (Figure 3b). Moreover, we were
curious to determine whether we could detect arginine
phosphorylated ClpC or GroEL in wild-type cells. However,
using the same assay setup we were unable to observe any
signal for arginine phosphorylation of these chaperones. Since
we had noticed that recombinant McsB activity is compro-
mised in non-stressed lysates, we next tested whether the
phosphorylation of these proteins was induced upon the
addition of the cellular stressor diamide, which was previously
shown to increase the level of arginine phosphorylation in a B.
[9b]
subtilis DywlE strain. For these studies, wild-type B. subtilis
cells were treated with diamide for 30 min, and then cells were
lysed and the phosphorylation status of ClpC and GroEL
monitored by immunoprecipitation and Western blotting.
Notably, both proteins were detected using cell lysates
derived from stressed wild-type cells, while there was no
To further demonstrate the utility of our anti-pArg
antibody, we next tested whether it could be used to enrich
for proteins that are endogenously arginine phosphorylated.
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signal in non-stressed cells (Figure 3b). We also performed
the same experiment using the previously described phage
display generated anti-pArg antibody. Notably, this low
affinity antibody was not able to enrich these arginine
phosphorylated proteins (Figure S20) highlighting the
requirement for a high-affinity antibody. Overall, these data
indicate that arginine phosphorylation is a highly regulated
PTM that is induced upon cellular stress in B. subtilis wild-
type cells.
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Keywords: antibodies · haptens · N-phosphorylation ·
phosphoarginine · phosphonate amidine
Received: July 21, 2015
How to cite: Angew. Chem. Int. Ed. 2015, 54, 14715–14718
Angew. Chem. 2015, 127, 14928–14931
Revised: September 8, 2015
Published online: October 12, 2015
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