A para-nitrophenol phosphonate probe labels distinct serine hydrolases of Arabidopsis

Article abstract of DOI:10.1016/j.bmc.2011.06.041

Activity-based protein profiling represents a powerful methodology to probe the activity state of enzymes under various physiological conditions. Here we present the development of a para-nitrophenol phosphonate activity-based probe with structural simila

Full text of DOI:10.1016/j.bmc.2011.06.041

Bioorganic & Medicinal Chemistry 20 (2012) 601–606
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A para-nitrophenol phosphonate probe labels distinct serine hydrolases
of Arabidopsis
Sabrina Nickel a, Farnusch Kaschani b, Tom Colby c, Renier A. L. van der Hoorn b, Markus Kaiser a,
⇑
a Zentrum für Medizinische Biotechnologie, Fakultät Biologie, Universität Duisburg-Essen, Universitätsstr. 2, D-45117 Essen, Germany
b The Plant Chemetics Laboratory, Chemical Genomics Centre of the Max Planck Society, Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, D-50829 Cologne,
Germany
c Mass Spectrometry Group, Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, D-50829 Cologne, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 15 July 2011
Activity-based protein profiling represents a powerful methodology to probe the activity state of
enzymes under various physiological conditions. Here we present the development of a para-nitrophenol
phosphonate activity-based probe with structural similarities to the potent agrochemical paraoxon. We
demonstrate that this probes labels distinct serine hydrolases with the carboxylesterase CXE12 as the
predominant target in Arabidopsis thaliana. The designed probe features a distinct labeling pattern and
therefore represents a promising chemical tool to investigate physiological roles of selected serine hydro-
lases such as CXE12 in plant biology.
Keywords:
Activity-based protein profiling
Phosphonate probe
Paraoxon
Carboxylesterase
Nitrophenol
Ó 2011 Elsevier Ltd. All rights reserved.
Arabidopsis thaliana
1. Introduction
In the last years, we have worked intensively to establish ABPP
in plant biology research,3 for example, by generating and estab-
Activity-based protein profiling (ABPP) is a powerful chemical
biology methodology to probe the activity state and thus biolog-
ical function of enzymes.1 To this end, small molecules known as
activity-based probes (ABPs) are applied to cell lysates, intact
cells or even living organisms, where they react in an irreversible,
covalent manner with their target enzymes. Labeled proteins are
then detected and identified by a combination of protein gels and
mass spectrometry. ABPs are composed of a ‘warhead’, that is, an
irreversible, reactive enzyme inhibitor moiety, a tag such as biotin
or the fluorophore rhodamine for purification and/or visualiza-
tion, and a linker joining the two. While the ABPP methodology
is well-established, a limitation of its scope is the number and
labeling properties of currently available ABPs. To date, ABPs for
several enzyme classes such as proteases, phosphatases or glyco-
sidases have already been reported.1,2 These ABPs however often
differ in their in-class target selectivities: while some act as
broadband probes, labeling a wide range of enzymes, others have
rather narrow and thus highly-selective labeling properties.
Broadband probes can be used to compare enzyme activity pat-
terns of different biological samples; selective probes, in contrast,
are powerful chemical tools for studying the function of a specific
enzyme and are crucial for advanced applications such as activ-
ity-imaging.
lishing probes and protocols that have been used to elucidate the
biological role of the pathogen-induced small molecule protea-
some inhibitor Syringolin A.4 However, further advancement of
ABPP in plant biology is hampered by the limited availability of
ABPs (both broadband and selective) with characterized labeling
properties for plant proteomes.
Carboxylesterases (CXEs) are serine hydrolases present in all
kingdoms of life with roles in diverse biological processes.5 In
plants, CXEs have been reported to participate in development,
xenobiotic detoxification, secondary metabolism and defense.6
CXEs have been labeled in ABPP studies with fluorophosphonate
(FP)-based ABPs such as FP-Rh (1, Fig. 1),7 but these are broadband
probes labeling a multitude of other serine hydrolases in addition
to several CXEs.8 For ABPP-based functional studies of CXEs, more
selective ABPs which only label predominantly distinct members
within this serine hydrolase class therefore would be highly desir-
able. In 2007, the Baker group reported a co-complex structure of
the Actinidia eriantha carboxylesterase CXE1 and the p-nitrophenyl
phosphate agrochemical paraoxon (2, Fig. 1).9 The structure re-
vealed—as expected for a phosphate-based inhibitor—a covalent
binding mode. It furthermore indicated that careful modifications
of paraoxon could allow the generation of a CXE-targeting ABP.
Here, we report the synthesis of a para-nitrophenol phosphonate-
based ABP as a structural mimic of paraoxon and the subsequent
identification of its corresponding targets in Arabidopsis leaf
extracts.
⇑
Corresponding author. Tel.: +49 201 183 2955; fax: +49 201 183 3672.
E-mail address: markus.kaiser@uni-due.de (M. Kaiser).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.041

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S. Nickel et al. / Bioorg. Med. Chem. 20 (2012) 601–606
Figure 1. Chemical structures of the broadband serine hydrolase probe FP-Rh (1), of the agrochemical paraoxon (2), and of the designed trifunctional para-nitrophenol
phosphonate TriNP (3).
azide functionality required for the click reaction) resulted in the
2. Results and discussion
generation of the desired target molecule TriNP (3).
2.1. Design and chemical synthesis of a paraoxon-like para-
nitrophenol phosphonate ABP
2.2. Profiling and target identification with TriNP (3)
We first investigated the general target selectivity of our TriNP
probe in Arabidopsis leaf extracts (Fig. 3A). To this end, Arabidopsis
leaf extracts were preincubated with either 100 lM paraoxon or
DMSO as a negative control for 30 min. Subsequently, a standard
labeling experiment was performed with either the broadband ser-
ine hydrolase probe FP-Rh or with TriNP. As previously reported,7
labeling with FP-Rh on leaf extracts resulted in a plethora of sig-
nals. Preincubation with paraoxon resulted in decreased FP-Rh sig-
nals predominantly in the 30 and 40 kDa region. Labeling with
The X-ray structure of paraoxon (2, Fig. 1) bound to CXE1 re-
vealed a diethyl phosphate moiety linked to the active site serine.
Paraoxon’s para-nitrophenol group, however, was absent, confirm-
ing its role as the leaving group during the covalent modification of
the active site serine. Importantly, one of the ethoxy groups ex-
tends into the lipid binding cleft, indicating that replacement of
this group with a suitably long linker, followed by a rhodamine
visualization and biotin purification tag would not hamper binding
efficiency. To limit unspecific binding and to ensure water solubil-
ity, a PEG linker was chosen. To incorporate the rhodamine and
biotin tags, a Huisgen’s [3+2] click chemistry approach was envis-
aged, making a modular synthesis of the desired ABP possible.
These considerations led to the trifunctional para-nitrophenol
phosphonate probe TriNP (Figs. 1 and 3). Although para-nitrophe-
nol phosphonate-based probes have been reported,10 a biotin- and
rhodamine-containing ABP has (to the best of our knowledge) not
yet been generated and tested on plant proteomes.
2 lM TriNP, however, gave one major band at around 40 kDa,
which was paraoxon-sensitive. These experiments revealed a sig-
nificantly enhanced selectivity of TriNP when compared to FP-Rh.
Importantly, all TriNP-derived signals were competable by prein-
cubation with paraoxon, demonstrating that the attachment of
the linker and of the biotin/rhodamine-tags did not lead to unspe-
cific labelling.
We next identified TriNP-labeled proteins in Arabidopsis leaf
extracts using affinity purification on avidin beads. After labeling
For the generation of the paraoxon-like phosphonate warhead,
we followed in the first steps the previously reported FP-Rh synthe-
sis strategy of Cravatt (Fig. 2).8,11 To this end, commercially available
tetraethylene glycol (4) was protected on one of its terminal hydro-
xyl groups as a tert-butyl dimethyl silyl ether, followed by conver-
sion of the remaining hydroxyl group into an iodide residue. The
resulting intermediate 6 was converted into the phosphonate 7
using standard Arbuzov conditions, followed by TBAF-mediated
cleavage of the tert-butyl dimetyhl silyl protecting group. With the
hydoxyl phosphonate 8 in hand, the reactive intermediate 9 was
formed by reaction with N,N0-dihydroxysuccinimide carbonate.
The subsequent addition of propargylamine led to urethane 10,
which upon treatment with oxalylchloride in diethyl ether resulted
in chloro-phosphonate derivative 11.10 Treatment of this intermedi-
ate with para-nitrophenol and triethylamine in toluene finished the
synthesis of the reactive para-nitrophenol phosphonate warhead of
the ABP. Subsequent [3+2] Huisgen ‘click’ reaction of intermediate
12 with the trifunctional tag 1312 (which contains a fluorescent rho-
damine moiety, a biotin residue for affinity purification and the
of the extracts with 5 lM of TriNP, affinity capture, and separation
on SDS protein gels, fluorescent bands were excised from the gel
and in gel-digested with trypsin.
NanoLC-ESI-MS/MS-analysis identified CXE12 as the predomi-
nant target. Several other serine hydrolases [tripeptidyl-peptidase
2 (TPP2), prolyloligopeptidase-like (POPL), serine carboxylpepti-
dase 48 (SCPL48) and carboxylesterase 7 (CXE7)] were identified
as well, consistent with a phosphonate-based ABP (Fig. 3B, Supple-
mentary Fig. 1 and Table 1).7,8 Interestingly, no signals could be
visualized in the 30 kDa region, even though preincubation with
paraoxon indicated a 30 kDa putative paraoxon target (Fig. 3).
The most likely target of FP in this region is the methylesterase
MES2.7,13 Crystallographic studies on
a MES2 homolog from
Nicotiana tabacum however indicate that the active site pocket of
this enzyme has strict size limitations.6a In order to investigate if
the observed target selectivity from TriNP results from the bulky
rhodamine and biotin tags, we performed an additional 2-step
labeling strategy, using alkyne 12 as an ABP and click chemistry

S. Nickel et al. / Bioorg. Med. Chem. 20 (2012) 601–606
603
Figure 2. Chemical synthesis of the ABP TriNP (3). (a) TBDMS–Cl, imidazole, DCM, rt, 12 h, 43%; (b) I2, PPh3, imidazole, toluene, rt, 1.5 h, 77%; (c) P(OEt)3, 150 °C, 1 h, 91%; (d)
TBAF, THF, 0 °C to rt, 12 h, 73%; (e) N,N0-disuccinimidyl carbonate, NEt3, DMF, rt, 12 h, 49%; (f) propargylamine, NaHCO3, MeOH, rt, 12 h, 80%; (g) oxalyl chloride, DCM, rt, 12 h,
99%; (h) para-nitrophenol, NEt3, toluene, rt, 3 h, 40%; (i) 13, TCEP, TBTA, CuSO4, H2O, rt, 12 h, 28%.
Figure 3. TriNP is more specific than FP-Rh and predominantly labels the serine hydrolase CXE12. (A) An extract from Arabidopsis thaliana leaves was preincubated with
100
Proteins were separated on protein gels, and fluorescent signals were detected by fluorescence scanning. (B) Affinity purification of TriNP targets and subsequent
identification by nanoLC-ESI-MS/MS. Labeled proteins were affinity purified on avidin beads after activity-dependent labeling with 5 M TriNP. The purified proteins were
lM paraoxon or DMSO for 30 min, followed by the addition of either 2 lM FP-Rh or 2 lM TriNP for 1 h. The reaction was stopped by addition of 4ꢀ gel loading buffer.
l
then separated by SDS–PAGE and visualized with a fluorescent scanner. Fluorescent gel regions were excised and subjected to in-gel digestion with trypsin. The obtained
peptides were analyzed by nanoLC-ESI-MS/MS.
with 13 after labeling to visualize labeled proteins (Supplementary
Fig. 2). The 2-step labeling profile is highly similar to the one from
TriNP. Only TPP2 was less efficiently labelled, most probably as a
result of the overall lower labeling efficiency resulting from the
additional click chemistry step. We therefore propose that the ob-
served enhanced target selectivity of TriNP versus FP-Rh is a conse-

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S. Nickel et al. / Bioorg. Med. Chem. 20 (2012) 601–606
4. Experimental
4.1. General
All reagents were purchased with highest purity available
from Sigma–Aldrich, Acros, Fisher Scientific, Merck, or Fluka,
and were used without further purification. Anhydrous, as well
as p.a. grade solvents, were purchased from commercial suppliers.
All reactions were carried out under an argon atmosphere. Flash
silica gel chromatography was performed with silica gel from Ac-
ros (particle size 35–70
lm) and Li-Chroprep RP 18 from Merck
(particle size 40–63 m). Thin layer chromatography (TLC) was
l
carried out on Merck aluminium precoated silica gel plates
(20 ꢀ 20 cm, 60F254 and 60 RP-18 F254S) using UV irradiation at
254 and 366 nm or the following developing reagents (Reagent
A: 20 g of phosphomolybdic acid hydrate in 80 mL ethanol, Re-
agent B: 1.5 g KMnO4, 10 g K2CO3 and 1.25 mL of 10% aq NaOH
in 200 mL water). Eluents and Rf values are given in the respec-
tive experiment. LC–MS analyses were performed on a HPLC sys-
tem from Agilent (1200 series) with an Eclipse XDB-C18, 5 lm
column from Agilent (peak detection at 210 nm) and a Thermo
Finnigan LCQ Advantage Max ESI-Spectrometer. A linear gradient
of solvent B (0.1% formic acid in acetonitrile) in solvent A (0.1%
formic acid in water) was used at 1 mL/min flow rate. Nuclear
magnetic resonance (NMR) spectra were recorded on a Varian
Mercury 400 system (400 MHz for 1H-, 100 MHz for 13C-, and
Figure 4. Specific labeling of agroinfiltrated CXE12. Extracts from agroinfiltrated
leaves overexpressing CXE12 with silencing suppressor p19 or p19 alone were pre-
incubated for 30 min with or without 100 lM paraoxon and labeled with 2 lM FP-
Rh or TriNP for 1 h. After separation on a protein gel, fluorescent labeled proteins
were visualized with a fluorescence scanner.
162 MHz for 31P-NMR),
a Bruker Avance DRX 500 system
(500 MHz for 1H- and 126 MHz for 13C-NMR) or a Varian Unity
Inova 600 system (600 MHz for 1H- and 151 MHz for 13C-NMR).
1H NMR spectra are reported in the following manner: chemical
shifts (d) in ppm calculated with reference to the residual signals
of undeuterated solvent, multiplicity (s, singlet; d, doublet; t, trip-
let; dt, doublet of triplet; m, multiplet), coupling constants (J) in
Hertz (Hz), and number of protons (H).
quence of either the change of the leaving group on the phospho-
nate moiety and/or the substitution of the alkyl to a PEG linker.
To confirm the labeling of CXE12 by the paraoxon-derived
probe, we performed labeling experiments with CXE12 transiently
overexpressed in Nicotiana benthamiana. The CXE12 cDNA was
cloned into a binary vector which was used to transform Agrobac-
terium tumefaciens. Cultures of the A. tumefaciens strain carrying
CXE12 were then mixed with strains carrying the RNA silencing
inhibitor p19 and co-infiltrated into N. benthamiana leaves.7,14 As
a negative control for this experiment, we also infiltrated leaves
with only the silencing suppressor p19. At 5 days post-infiltration,
leaves were harvested and used for labeling studies. Preincubation
of leaf extracts from CXE12 overexpressing leaves as well as prein-
cubation of extracts of leaves expressing only the p19 suppressor
with either paraoxon or DMSO, confirmed that CXE12 is a target
of TriNP (Fig. 4). The molecular basis of the double CXE12 signal
is unknown at this stage.
4.2. Synthesis
4.2.1. Synthesis of 2,2,3,3-tetramethyl-4,7,10-13-tetraoxa-3-
silapentadecan-15-ol (5)
To a solution of tetraethylene glycol (4) (10 g, 51.5 mmol) in
DCM (80 mL) was added imidazole (7 g, 103 mmol, 2 equiv) and
TBDMS–Cl (7.8 g, 51.5 mmol, 1 equiv), and the resulting solution
was stirred over night at room temperature. The reaction was
quenched by addition of saturated aq NaHCO3, and extracted twice
with ethyl acetate. The combined organic phases were washed
with brine and the organic phase was dried over sodium sulfate.
After purification on silica gel column (cyclohexane/ethyl acetate
1:2), 6.7 g (22.1 mmol, 43%) of the desired product 5 was isolated.
1H NMR (CDCl3) d 3.75 (t, 2H, J = 5.4 Hz), 3.71 (m, 2H), 3.65 (d, 8H,
J = 4.3 Hz), 3.59 (m, 2H), 3.54 (t, 2H, J = 5.4 Hz), 0.88 (s, 9H), 0.05
(s, 6H); 13C NMR (CDCl3) d 72.6, 70.8, 70.5, 62.8, 61.9, 26.1, 18.5,
ꢁ5.2; TLC (cylohexane/ethyl acetate 1:2) Rf = 0.25; ESI-MS: tR
4.21 min, m/z 308.87 [M+H]+, 309.21 calcd for C14H33O5Si+.
3. Conclusions
Our studies have resulted in the development of a paraoxon-
like para-nitrophenol phosphonate ABP that labels predominantly
CXE12 in Arabidopsis. CXE12 has specifically been implicated in
bioactivation of herbicides, for example, by hydrolysis of the pro-
herbicide methyl-2,4-dichlorphenoxyacetate into the phytotoxic
agent 2,4-dichlorophenoxy acetic acid.6c Besides its role in bioher-
bicide activation, no other biological roles of CXE12 are known. The
ABP TriNP (3) could therefore represent a promising chemical tool
to further investigate the function of CXE12 due to its altered,
highly specific labeling profile. In this context it should be noted
that a probe for labeling CXE12 has recently been reported which,
however, requires cautious use due to potential problems with
labeling intensity and off-target labelling.15
4.2.2. Synthesis of 15-Iodo-2,2,3,3-tetramethyl-4,7,10,13-
tetraoxa-3-silapentadecane (6)
To a solution of 5 (6.0 g, 19.7 mmol) in toluene (150 mL) was
added triphenylphosphine (25.8 g, 98.5 mmol, 5 equiv), iodine
(20.0 g, 78.8 mmol, 4.5 equiv) and imidazole (7.0 g, 102.4 mmol,
5.2 equiv). The heterogenic solution was stirred for 1.5 h at room
temperature and was then filtered and washed with ethyl acetate.
A saturated aq sodium thiosulfate solution was added, the organic
phase was washed with water and brine, and the organic phase
was dried over sodium sulfate. After silica gel chromatography
(cyclohexane/ethyl acetate 5:1), 6.3 g (15.1 mmol, 77%) of the de-
sired product 6 was isolated. 1H NMR (CDCl3): d 3.78–3.73
Interestingly, a previous study on para-nitrophenol phospho-
nate-based inhibitors demonstrated that this type of warhead also
inhibits cutinases.10 Plant pathogens often employ cutinases dur-
ing infection.16 Consequently, TriNP (3) might also find important
applications in plant-pathogen studies.

S. Nickel et al. / Bioorg. Med. Chem. 20 (2012) 601–606
605
(m, 4H), 3.65 (m, 8H), 3.55 (t, 2H, J = 5.4 Hz), 3.25 (t, 2H, J = 7.0 Hz),
0.88 (s, 9H), 0.05 (s, 6H); 13C NMR (CDCl3): d 72.8, 72.1, 70.9, 70.8,
70.4, 26.1, 18.5, 3.1, ꢁ5.1; TLC (cyclohexane/ethyl acetate 5:1):
Rf = 0.35; ESI-MS: tR 4.31 min, m/z 419.20 [M+H]+, 419.11 calcd
for C14H33IO4Si+.
69.6, 65.2, 64.4, 61.9, 30.9, 27.8, 16.6; TLC (CHCl3/methanol
19:1): Rf = 0.30; LC–MS (ESI): tR 7.19 min, m/z 396.00 [M+H]+,
396.18 calcd for C16H31NO8P+.
4.2.7. Synthesis of 2-(2-(2-(2-(chloro)ethoxy)phosphory)ethoxy)
ethoxy)ethoxy)ethyl prop-2-yn-1-yl carbamate (11)
4.2.3. Synthesis of diethyl(2,2,3,3-tetramethyl-4,7,10,13-
tetraoxa-3-silapentadecan-15-yl-phosphonate (7)
To a solution of 10 (80 mg, 0.2 mmol) in DCM (1 mL) was added
dropwise oxalyl chloride (0.34 ml, 4.0 mmol, 20 equiv) and the
resulting mixture was stirred over night at room temperature.
The solvent was removed under a stream of argon and dried under
high vacuum. The remaining residue 11 (76.4 mg, 0.19 mmol, 99%)
was used in the next step without any further purification. 1H NMR
(CDCl3): d4.51–4.45 (m, 3H), 4.42–4.18 (m, 3H), 3.89–3.75 (m, 4H),
3.69–3.61 (m, 8H), 2.53 (dt, 2H, J = 18.1, 7.4 Hz), 2.32 (t, 1H,
J = 2.4 Hz), 1.39 (t, 3H, J = 7.1 Hz); 13C NMR (CDCl3): d 152.1, 76.4,
73.0, 70.8, 70.7, 70.6, 68.4, 68.1, 64.6, 35.4, 33.2, 16.1; 31P NMR
(CDCl3): d 40.6.
Compound
6 (5.5 g, 13.1 mmol) was dissolved in P(OEt)3
(12.4 mL, 71 mmol, 5.4 equiv) and heated up to 150 °C and stirred
for 1 h at this temperature. The excess of P(OEt)3 was subsequently
removed in high vacuum and the residue purified on silica gel col-
umn (DCM/methanol 50:1), yielding 5.1 g (12 mmol, 91%) of the
desired product 7. 1H NMR (CDCl3): d 4.14–4.03 (m, 4H), 3.73 (m,
4H), 3.65–3.58 (m, 8H), 3.54 (t, 2H, J = 5.4 Hz), 2.16–2.07 (m, 2H),
1.30 (t, 6H, J = 7.1 Hz), 0.88 (s, 9H), 0.05 (s, 6H); 13C NMR (CDCl3):
d 72.8, 70.9, 70.6, 70.3, 65.3, 62.8, 61.8, 27.8, 26.4, 26.1, 16.6, ꢁ5.1;
31P NMR (CDCl3): d 29.6; TLC (DCM/methanol 50:1): Rf = 0.20;
ESI-MS: tR 4.32 min, m/z 429.20 [M+H]+, 429.25 calcd for
4.2.8. Synthesis of 2-(2-(2-(2-(ethoxy(4-nitrophenoxy)
phosphoryl)ethoxy)ethoxy)ethoxy)ethyl prop-2-yn-1-yl
carbamate (12)
C
18H42O7PSi+.
4.2.4. Synthesis of diethyl(2-(2-(2-(2-hydroxyethoxy)ethoxy)
ethoxy)ethyl)phosphonate (8)
To a solution of 11 (76.4 mg, 0.19 mmol) in toluene (1 mL) was
added dropwise
a
solution of para-nitrophenol (55.6 mg,
A solution of 7 (5.1 g, 12.0 mmol) in THF (150 mL) was cooled to
0 °C and TBAF (13.1 mL of a 1 M solution in THF, 14.0 mmol,
1.25 equiv) was added slowly. The resulting mixture was stirred
over night and warmed to room temperature. The reaction was
quenched by addition of water and the resulting mixture was ex-
tracted three times with DCM. Drying of the organic phase over so-
dium sulfate and purification by silica gel chromatography (DCM/
methanol 19:1) led to 2.8 g (8.8 mmol, 73%) of the desired product
7. 1H NMR (CDCl3): d 4.13–4.03 (m, 4H), 3.75–3.57 (m, 14 H), 2.12
(dt, 2H, J = 18.8, 7.6 Hz), 1.30 (t, 6H, J = 7.1 Hz); 13C NMR (CDCl3): d
72.7, 70.7, 70.5, 70.2, 65.3, 61.8, 27.7, 26.4, 16.6; TLC (DCM/MeOH
19:1): Rf = 0.40; ESI-MS: tR 4.31 min, m/z 315.13 [M+H]+, 315.16
calcd for C12H28O7P+.
0.38 mmol, 2 equiv) and triethylamine (0.14 mL, 0.95 mmol,
5 equiv) in toluene (1 mL). The resulting mixture was stirred for
3 h at room temperature and then evaporated to dryness. The sub-
sequent purification by silica gel column chromatography (CHCl3/
methanol 50:1) yielded 38.7 mg (0.08 mmol, 40%) of the desired
product 12. 1H NMR (CDCl3): d 8.23 (d, 2H, J = 9.2 Hz), 7.39 (d,
2H, J = 9.2 Hz), 4.30–4.14 (m, 4H), 3.96 (d, 2H, J = 2.5 Hz), 3.87–
3.77 (m, 2H), 3.69–3.65 (m, 2H), 3.61 (d, 8H, J = 6.5 Hz), 2.33
(dt, 2H, J = 18.6, 7.1 Hz), 2.22 (t, 1H, J = 2.4 Hz), 1.32 (t, 3H,
J = 7.1 Hz); 13C NMR (CDCl3): d 155.7, 155.3, 144.7, 125.8, 121.3,
78.9, 71.6, 70.7, 70.5, 69.6, 64.8, 64.4, 63.4, 30.9, 28.2, 16.5;
31P NMR (CDCl3): d 28.0; TLC (CHCl3/methanol 50:1): Rf = 0.06;
LC–MS (ESI): tR 8.58 min, m/z 489.00 [M+H]+, 489.17 calcd for
C
20H30N2O10P+.
4.2.5. Synthesis of 2-(2-(2-(2-(diethoxyphosphoryl)ethoxy)
ethoxy)ethoxy)ethyl(2,5-dioxopyrrolidin-1-yl)carbonate (9)
Compound 8 (2.0 g, 6.4 mmol) was dissolved in DMF (18.7 mL,
0.34 M) and N,N0-disuccinimidyl carbonate (3.6 g, 14.1 mmol,
2.2 equiv) and NEt3 (2.2 mL, 16.0 mmol, 2.5 equiv) were added.
The resulting mixture was stirred over night at room temperature.
Water and DCM were added and the organic and water phase were
separated. The organic layer was washed three times with brine,
separated and the organic phase was dried over sodium sulfate.
After silica gel chromatography (DCM/methanol 50:1), 1.44 g
(3.2 mmol, 49%) of the desired product 9 was obtained. 1H NMR
(CDCl3): d 4.45–4.42 (m, 2H), 4.12–4.03 (m, 4H), 3.79–3.57 (m,
12H), 2.82 (s, 4H), 2.11 (dt, 2H, J = 18.7, 7.6 Hz), 1.29 (t, 6H,
J = 7.1 Hz); 13C NMR (CDCl3): d 168.7, 151.7, 71.0, 70.7, 70.3, 68.4,
65.2, 61.8, 27.7, 25.6, 16.6; TLC (DCM/methanol 50:1): Rf = 0.20;
ESI-MS: tR 4.21 min, m/z 456.20 [M+H]+, 455.16 calcd for
4.2.9. Synthesis of TriNP (3)
To a solution of 12 (0.4 mg, 0.9
water (0.3 mL) was added TCEP (3
l
l
mol) and 13 (1 mg, 0.9
L of a 50 mM solution in water,
L of a 100 mM solution in DMSO,
mol, 0.5 equiv) and CuSO4 (30 L of a 5 mM solution in water,
lmol, 0.25 equiv). The resulting mixture was stirred in the
lmol) in
0.15 lmol, 0.25 equiv), TBTA (3 l
0.3
l
l
0.15
dark over night at room temperature. The mixture was evaporated
to dryness and the crude residue was purified by C18 reversed
phase silica gel (water with 20% CH3CN–50% CH3CN) to yield
0.4 mg (0.26 mmol, 28%) of the desired product 3. TLC (CH3CN/
water 1:1): Rf = 0.45; LC–MS (ESI): tR 7.48 min, m/z 1567.60
[M+H]+, 1589.53 [M+Na]+, 784.80 [M+2H]2+, 1567.71 calcd for
C
75H104N14O19PS+.
4.3. Profiling and target identification
C
17H31NO11P+.
4.3.1. Sample preparation
4.2.6. Synthesis of 2-(2-(2-(2-(diethoxyphophoryl)ethoxy)
ethoxy) ethoxy)ethyl prop-2-yn-1-yl carbamate (10)
Arabidopsis thaliana leaf extracts were obtained by grinding 2 g
of frozen leaves of 4-week-old A. thaliana ecotype Col-0 in a mortar
at room temperature (22–24 °C) to a homogenous green paste. The
paste was mixed with 5–6 mL of distilled water and cleared by
centrifugation (5 min at 16,000ꢀg). The protein concentration
was determined by using the Reducing agent Compatible/Deter-
gent Compatible (RC/DC) Protein Assay (Bio-Rad) following the
manufacturer’s instructions.
To a solution of 9 (405.5 mg, 0.89 mmol) in methanol (5 mL)
were added propargylamine (73.5 mg, 1.3 mmol, 1.5 equiv) and so-
dium bicarbonate and the resulting mixture was stirred over night
at room temperature. The solvent was evaporated and purification
on silica gel column (CHCl3/methanol 19:1) yielded 281.1 mg
(0.71 mmol, 80%) of the desired product 10. 1H NMR (CDCl3): d
4.26–4.21 (m, 2H), 4.14–4.04 (m, 4H), 3.95 (s, 2H), 3.76–3.59 (m,
12H), 2.22 (t, 1H, J = 2.3 Hz), 2.12 (dt, 2H, J = 18.7, 7.5 Hz), 1.31 (t,
6H, J = 7.1 Hz); 13C NMR (CDCl3): d 162.7, 79.9, 71.5, 70.7, 70.3,
Full-length CXE12 (At3g48690) cDNA was amplified from an A.
thaliana cDNA library (kindly provided by Dr. Hans Sommer, Max
Planck Institute for Plant Breeding Research) using the primers

606
S. Nickel et al. / Bioorg. Med. Chem. 20 (2012) 601–606
r434 (forward, 50-GAT CCC ATG GAT TCC GAG ATC GCC GTC
GAC-30) and r435 (reverse, 50-GAT CCT GCA GCT AGT TCC CTC
CCT TAA TAA ACC C-30). The fragment was cloned into the cloning
vector pFK26 using the NcoI and PstI restriction sites, resulting in
pFK161.17 The 35S::CXE12::terminator cassette was excised from
pFK161 with XbaI and EcoRI and shuttled into pTP5.17 The result-
ing binary vector pBP8 was transformed into A. tumefaciens strain
GV3101 pMP90.18 Transient overexpression of CXE12 was
achieved by co-infiltrating cultures of Agrobacterium strains carry-
ing pBP8 together with cultures carrying silencing inhibitor p19 in
fully expanded leaves of 4-week-old N. benthamiana.14 Leaves were
harvested after 3 days, and the proteins were extracted as de-
scribed above.
The resulting MS2 spectra matches were assembled and filtered
in Proteome Discoverer. Spectra matches were retained when their
confidence score was ‘medium’ or ‘high’. Protein identification re-
quired the matching of at least two peptides per protein not shared
with any other unrelated protein in the database (matches to iso-
forms coming from the same gene locus were allowed).
Acknowledgements
The authors would like to thank Bikram Pandey for providing
pBP8 and Sarah Tully and Benjamin F. Cravatt for a kind donation
of FP-Rh. Support of this work by the Deutsche Forschungsge-
meinschaft (KA 2894/1-1 to M.K., HO 3983/3-2 and HO3983/4-1
to R.H.) is greatly acknowledged.
4.3.2. Sample preparation for target identification
The labeling reactions and the affinity purification of labeled
target proteins was carried out as previously described.7 Affinity
purified, labeled target proteins were separated by 1D SDS–PAGE
and visualized on a Typhoon 8600 (GE Healthcare, Pittsburgh,
USA) fluorescence scanner. Fluorescent areas were cut out with a
disposable steel blade and washed several times with water. The
gel slices were then subjected to the in-gel-digestion procedure
and obtained peptides used for the subsequent nanoLC-ESI-MS/
MS analysis.7
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.041.
References and notes
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4.4. Mass spectrometry
Experiments were performed on a Thermo LTQ Velos mass
spectrometer coupled to a Proxeon EASY-nLC. The LTQ Velos mass
spectrometer was operated using Xcalibur software (version 2.1).
The mass spectrometer was set in the positive ion mode, survey
scanning between m/z of 400 and 1600, with an ionization poten-
tial of 3.61 kV. Peptides were separated on a single reverse phase
C18 column (inner diameter 75
lm, packed with 12 cm ReproSil-
Pur C18-AQ (3 m)) using an acetonitrile gradient (5–40% over
l
90 min), at a flowrate of 300 nL/min. Peptides were fragmented
by collision-induced decay (CID) in a data-directed fashion, frag-
menting the 20 most intense multiply-charged precursors in each
MS scan. CID collision energy was set to 35% for the generation of
MS2 spectra. Following fragmentation, precursors were excluded
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consisting of 500 members maximum.
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sequences from the TAIR10_pep_20101028.fasta database (version
October 2010, www.arabidopsis.org). In order to increase the
statistical significance of peptide matches, the database was
supplemented with Pseudomonas aeruginosa sequences from
the Pseudomonas_aeruginosa_PAO1.txt (version November 2009,
www.pseudomonas.com) and sequences for chicken avidin,
human keratins and porcine trypsin (the latter sequences were ob-
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41012 protein sequence entries. SEQUEST searches allowed for oxi-
dation of methionine residues (16 Da), static modification of cys-
teine residues (57 Da; due to alkylation), tryptic peptides with
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one missed cleavage allowed, and
a mass tolerance set to
0.25 Da for precursor mass and 0.8 Da for product ion masses.
The ‘Decoy Database Search’ was turned on.