Evaluation of sulfatase-directed quinone methide traps for proteomics

Article abstract of DOI:10.1016/j.bmc.2011.04.044

Sulfatases hydrolytically cleave sulfate esters through a unique catalytic aldehyde, which is introduced by a posttranslational oxidation. To profile active sulfatases in health and disease, activity-based proteomic tools are needed. Herein, quinone methi

Full text of DOI:10.1016/j.bmc.2011.04.044

Bioorganic & Medicinal Chemistry 20 (2012) 622–627
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of sulfatase-directed quinone methide traps for proteomics
Janina Lenger a,d, Marius Schröder a, Eva C. Ennemann b, Benjamin Müller c, Chi-Huey Wong d, Thomas Noll c,
a,
Thomas Dierks b, Sarah R. Hanson d, , Norbert Sewald
⇑
⇑
a Bioorganic and Organic Chemistry, Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany
b Biochemistry I, Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany
c Cell Culture Technology, Faculty of Technology, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany
d Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, 92037 CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Sulfatases hydrolytically cleave sulfate esters through a unique catalytic aldehyde, which is introduced by
a posttranslational oxidation. To profile active sulfatases in health and disease, activity-based proteomic
tools are needed. Herein, quinone methide (QM) traps directed against sulfatases are evaluated as
Received 31 January 2011
Revised 12 April 2011
Accepted 20 April 2011
Available online 24 April 2011
activity-based proteomic probes (ABPPs). Starting from
a p-fluoromethylphenyl sulfate scaffold,
enzymatically generated QM-traps can inactivate bacterial aryl sulfatases from Pseudomonas aeruginosa
and Klebsiella pneumoniae, and human steroid sulfatase. However, multiple enzyme-generated QMs form,
diffuse, and non-specifically label purified enzyme. In complex proteomes, QM labeling is sulfatase-
dependent but also non-specific. Thus, fluoromethylphenyl sulfates are poor ABPPs for sulfatases.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Quinone methide
Enzyme inhibitor
Suicide inhibitor
Sulfatase probe
Activity-based proteomics
1. Introduction
enzymes.2–4 Because sulfatases require activation, protein abun-
dance does not necessarily reflect biological activity. Multiple
The goal of activity-based proteomics (ABP) is to isolate and
characterize specific enzyme classes within the context of the pro-
teome. ABP targets active enzyme classes by using activity-based
proteomic probes (ABPPs), which are most commonly equipped
with broadly active mechanism-based inhibitors that can elicit
turnover-dependent capture. In doing so, important information
on catalytically active enzyme members is provided by ABPPs.
Powerful ABPPs are available for several types of enzymes, includ-
ing cysteine proteases and serine hydrolases; however, many other
enzyme classes await the development of ABP tools.1
The biochemical characterization of sulfatases would benefit
from ABP tools. Type I sulfatases (EC 3.1.5.6) are a large class of en-
zymes found in prokaryotes and eukaryotes that cleave sulfate es-
ters by a unique hydrolytic mechanism dependent on the catalytic
aldehyde, formylglycine (FGly).2 FGly is installed post-translation-
ally in eukaryotic sulfatases from encoded cysteine residues by
formylglycine generating enzyme (FGE, also known as SUMF1);
while, in prokaryotes FGly can originate from either cysteine or
serine residues by SUMF1 (activates Cys-type sulfatases), AtsB
(activates Ser-type sulfatases), and potentially other activating
sulfatase deficiency (MSD) is a fatal human genetic disorder char-
acterized by normal sulfatase protein production but inadequate
FGly activation by FGE.2 To date, eight different human disorders
are known to be caused by the deficiency of single sulfatase genes.5
The human aryl sulfatases (ARS) D–K have been identified, but
have unknown substrates and biological functions.2,3 Furthermore,
sulfatases are increasingly found in pathological settings such as
bacterial infection, cancer, and inflammation.2,6 ABP tools can help
to better define the roles of active sulfatases in these processes.
We have been interested in developing ABP tools for the selec-
tive detection and isolation of sulfatases within complex mixtures
using quinone methide (QM) traps (Fig. 1).7 QM-traps are consid-
ered promising general ABPs for hydrolytic enzymes.8,9 The
QM-trap concept involves in situ generation of a reactive QM inter-
mediate that is dependent on enzymatic turnover of a suicide
inhibitor. Typically, QMs are the product of hydrolytically liberated
fluoromethylphenolates, which then spontaneously fragment by
fluoride elimination. Importantly, the enzymatically-generated
QM features an electrophilic carbon atom poised to covalently
capture a nucleophile via Michael-addition. In an ideal ABPP, the
QM will capture a properly disposed active site residue conserved
in the targeted enzyme class, resulting in turnover-dependent
inactivation and specific protein labeling. Although active site cap-
ture is likely to result in a single specific enzyme-labeling event,
the requirements for specific labeling of a target enzyme class in
⇑
Corresponding authors. Tel.: +1 858 784 2440; fax: +1 858 784 2409 (S.R.H.);
tel.: +49 521 106 2051; fax: +49 521 106 8094 (N.S.).
E-mail addresses: shanson@scripps.edu (S.R. Hanson), norbert.sewald@
uni-bielefeld.de (N. Sewald).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.044

J. Lenger et al. / Bioorg. Med. Chem. 20 (2012) 622–627
623
F
a
b
Nuc
F
F
1. Nuc-
2. H+
O
S
O
F
sulfatase
sulfatase
HO
O
HO
O
1
5a
5b
1. Nuc-
2. H+
O
S
O
HO
Nuc
O
HO
O
F
F
F
F
2
F
Nuc
R
R
sulfatase
1. Nuc-
2. H+
O
S
O
R
O
HO
O
HO
O
3, 4
5c,d
HO
H
3, 5c R =
4, 5d R =
N
O
O
O
NH2
S
O
O
H
N
O
N
H
N
OH
O
H
O
Figure 1. Capture schemes for sulfatase-directed QM-traps starting from masked fluoromethylphenyl sulfates, including: (a) difluoromethylphenyl sulfates (DFPSs 1 and 2)
and (b) monofluoromethylphenyl sulfates (MFPS probes 3 and 4 see Supplementary data, Scheme S1 for synthesis). Specific sulfatase capture requires sulfatase-catalyzed
sulfate cleavage and fluoride elimination to release a reactive QM (5a–d), which can then covalently trap nucleophiles for specific labeling of sulfatases.
ABPP are still met if non-catalytic nucleophiles are specifically cap-
tured instead. Herein, we evaluate the utility of sulfatase-directed
QM-traps as ABPPs for sulfatases. As shown in Figure 1, our QM-
traps are based on fluoromethylphenyl sulfate substrates. By con-
taining the masked fluoromethyl within the minimal phenyl sul-
fate motif, these traps are designed to have broad-ranged
reactivity against sulfatases, which are known to be generally tol-
erant of small aryl sulfate substrates.2
specifically captured outside of the active site is suitable in the
context of a proteomics approach, provided it occurs before the
QM diffuses into a generally reactive space. As such, we next
turned to creating sulfatase-directed ABPP 4 to monitor sulfatase
labeling. This probe also included a more reactive QM-trap.
We postulated that a more reactive sulfatase-directed QM-trap
would be afforded starting from a monofluoromethylphenyl sul-
fate (MFPS) scaffold (Fig. 1b). This scaffold offers several potential
improvements to QM reactivity, specificity, and labeling over the
masked difluoro analogs 1 and 2. First, the monofluoro analogs
cannot undergo side reactions that can complicate QM formation
and inactivation kinetics.8f,11 Second, QMs generated from mono-
fluoromethyl phenolates form and react faster than those gener-
ated from difluoromethyl phenolates.8f,12 Thus, MFPS containing
structures like 3 and 4 should accelerate sulfatase capture, thereby
increasing the likelihood of specificity, perhaps even labeling with-
in the active site. Importantly, the MFPS probes still contain the
key phenyl sulfate motif that should make them broadly accessible
to the sulfatase enzyme class.2 For a complete sulfatase-directed
ABPP, the MFPS trapping scaffold was combined with a reporter
group for detection in a proteomic mixture (e.g., fluorescein in 4;
see Supplementary data, Scheme S1 for synthetic details).
To begin ABPP characterization, the broad-ranged biochemical
activity of model MFPS probe 3 was tested against a panel of sulfat-
ases. Four disparate sulfatase members were included: recombi-
nantly expressed and purified Cys- and Ser-type aryl sulfatases
from bacteria (PARS7 and KARS13), as well as human aryl sulfatase
G (ARSG14) and steroid sulfatase (STS15). Each has distinct features:
the bacterial sulfatases are active against a broad range of aryl sul-
fate substrates under basic (pH of 8.9 for PARS) and neutral (pH 7.5
for KARS) conditions; STS is a membrane-bound sulfatase of the
endoplasmic reticulum with neutral pH optimum, a high specific-
ity for steroid sulfates, and a noted involvement in hormone-
dependent cancers; and, ARSG is a newly discovered lysosomal
enzyme, with acidic pH optimum and unknown biological sub-
2. Results and discussion
A good ABPP must be able to specifically label a targeted en-
zyme class within a complex proteome. We designed sulfatase-di-
rected QM probes 1–4 and evaluated their ability to inhibit and
label sulfatases to develop ABPPs for this class of enzymes. Inhibi-
tion studies will indicate, first, if the QM probes can bind in the ac-
tive site as competitive substrates, and second, if they are
irreversible inhibitors (also known as mechanism-based or suicide
inhibitors), which inactivate active site residues in a turnover-
dependent manner. Standard inhibition by a competitive substrate
will be concentration-dependent, whereas irreversible inhibition
will shut down catalysis resulting in time- and concentration-
dependent inhibition.10 Early work with QM-traps based on diflu-
oromethylphenyl sulfates (DFPS 1 and 2, Fig. 1a) indicated that
these compounds are inhibitors, but not of the irreversible type,
of aryl sulfatases as demonstrated with an aryl sulfatase from Pseu-
domonas aeruginosa (PARS)7 and from Klebsiella pneumoniae (KARS;
see Supplementary data, Fig. S1). Although 1 and 2 have substrate-
like properties with PARS and KARS, their utility as ABPPs is ques-
tionable, since the enzymatically-formed QM-traps 5a and 5b do
not inactivate sulfatases even over long periods of time. However,
it is possible that the QM adducts resulting from 1 and 2 form with
nucleophiles situated outside of the active site. For ABPPs, specific
labeling of the target enzyme is paramount to active site
inactivation. Thus, the scenario of a turnover-dependent QM being

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J. Lenger et al. / Bioorg. Med. Chem. 20 (2012) 622–627
strate and function.2–4,6i,14 Sulfatase activity was determined by
standard assay methods (see Section 4). Briefly, KARS, PARS, and
ARSG activities were monitored using a para-nitrocatechol sulfate
(pNCS) colorimetric assay, and STS activity was determined using
a radioactive dehydroepiandrosterone-3-sulfate (3DHEAS) assay.16
To test MFPS inhibition, enzymes were pre-incubated with model
ABPP 3, before being diluted into appropriate assay buffer to mea-
sure activity; final activities are reported as a percentage of the
uninhibited control.
The dose-dependent inhibitory activity of MFPS probe 3 against
the sulfatase panel at representative time points is shown in Fig-
ure 2. The inhibition potential was variable, with the strongest
inactivation in shortest time periods being observed for the bacte-
rial enzymes (i.e., roughly estimated 50% inactivation of KARS and
PARS after a 30-min pre-incubation with 0.8 and 5 mM 3, respec-
tively), intermediate inactivation of STS (i.e., around 50% after a
60 min pre-incubation at 15 mM 3) and very little inactivation of
ARSG. It is surprising that ARSG remained unaffected at all tested
pre-incubation concentrations of 3, even after overnight incuba-
tion (see Supplementary data, Fig. S2), because the enzyme is
known to accept aryl sulfate substrates, albeit with a preference
for ortho-hydroxylated aryl sulfates.14 Possibly, the acidic pH con-
ditions needed for ARSG activity affect QM-trap formation (e.g.,
pH-dependent thiol additions to QM have been observed17). In
addition to dose-dependence, KARS, PARS and STS also exhibited
time-dependent inhibition, as exemplified for KARS in Figure 3a
(also see Supplementary data, Fig. S2). Time-dependent inactiva-
tion indicates that the sulfatase active site can be shut down by
the MFPS probes, an improvement over the original DFPS 1 and 2
probes that did not show any time-dependent inhibition (see Sup-
plementary data, Fig. S1).
To investigate if the time-dependent inactivation of sulfatases
by MFPS trap 3 (as clearly shown for KARS in Fig 3a) is caused by
specific labeling, the stoichiometry of QM activation and QM-ad-
duct formation was next gauged by 19F NMR and matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-ToF MS). As shown in Figure 3b, real-time formation of
the QM-trap 5c is visible in 19F NMR experiments: in the NMR-
spectrum, probe 3 has a chemical shift of d = ꢀ180 ppm; while
the free fluoride signal at d = ꢀ120 ppm emerges with QM forma-
tion, that is, as sulfate is cleaved and fluoride is eliminated.8a,b
Using low activity MFPS probe concentrations with KARS
Figure 3. Evaluation of monofluoro-QM-trap 3. (a) Time- and concentration-
dependent inhibition of KARS by 3. (b) Real-time generation of QM intermediate 5c
as monitored by 19F NMR via the generation of free fluoride (d = ꢀ120 ppm) after
(140 lM 3 and 70 nM KARS), slow QM activation kinetics were
monitored over 2 weeks. Fluoride release ceased after 12 h due to
complete enzyme inactivation (notably, under the same condi-
tions, untreated enzyme remained active and probe 3 did not
decompose). At complete inactivation, the integral ratio of 19F
NMR signals between 3-bound fluorine and liberated fluoride indi-
KARS-catalyzed sulfate cleavage of 3 (d = ꢀ180 ppm). Conditions: 140
lM 3, 70 nM
KARS; spectral recording times as indicated; integrals were determined to add up to
one. (c and d) Formation of multiple QM adducts, as shown by MALDI-TOF MS-
spectral overlay of sulfatases (c) KARS and (d) PARS with spectra for untreated
enzyme shown in black and for enzyme treated with 3 in gray. Conditions: labeling
with 5 mM 3 (KARS for 12 h, PARS for 1 h). Untreated enzyme (black line) shows
expecteþd masses: KARS with and without N-terminal signal peptide,
½M þ HꢁKARS = 65 and 63 kDa, corresponding to inactive and active enzyme respec-
tively; PARS, ½M þ HꢁþPARS = 61 kDa. Shifted and broadened distributions in molecular
weight after treatment with 3 demonstrates multiple QM adducts labeling (each
QM adduct adds 280 Da). The active KARS peak disappears with QM probe
treatment (shift is 2 kDa), which corresponds to up to seven QM labels (although
the inactive KARS complicates the discernment of a true quantitative average). PARS
shows an average of five QM adducts (shift 1.5 kDa). See Supplementary data for
protein sequence details.
cates that approximately 20% of probe 3 was converted to QM.
Thus, nearly 400 molecules of probe must be turned over on aver-
age by each enzyme molecule before it is inactivated, when consid-
ering the 2000-fold molar excess of probe 3 over KARS. Although
the 400:1 MFPS 3 to KARS inactivation ratio is surprisingly better
than those previously reported with other hydrolases,8a,b such a
high ratio is consistent with non-specific labeling. Indeed, after
treatment with 3, KARS multiply labeled with up to 7 QM-adducts
Figure 2. Dose-dependent inhibition of KARS, PARS, STS, and ARSG after pre-
incubation with and without
3 (time is indicated below enzyme name. Bars
represent the percentage of activity relative to control; with SD, n = 3. (White:
control, no inhibitor; gray: 1 mM 3; black: 10 mM 3).

J. Lenger et al. / Bioorg. Med. Chem. 20 (2012) 622–627
625
is apparent by MALDI-ToF MS (Fig. 3c). PARS shows similar QM
activation by 19F NMR (Supplementary data, Fig. S3) and an aver-
age of five QM-enzyme adducts by MALDI-ToF MS after incubation
with 3 (Fig. 3d). Thus, the data in Figure 3 suggest that inactivation
of purified sulfatases by MFPS probes is consistent with non-spe-
cific protein labeling.
As a final test for ABPP utility, the MFPS probe 4 was applied to
complex proteomic mixtures. Probe activation and selectivity was
assessed using three model proteomes containing, (i) an active sul-
fatase, (ii) an inactive sulfatase, or (iii) no sulfatase. These proteo-
most of the proteins in the sample population. Non-specific label-
ing of enzymes from a wide range of protein classes, including ki-
nases, dehydrogenases, transaldolases, and a methyl transferase,
was confirmed by trypsination and liquid chromatography tandem
mass spectrometry (LC–MS/MS) analysis of fluorescent gel spots
(see Supplementary data, Fig. S4 and Table S1). Adding small-mol-
ecule nucleophiles (e.g., ethanolamine) to trap diffused activated
QM species resulted in decreased labeling of off-target proteins
and KARS, suggesting that even the labeling of the QM-generating
enzyme is non-specific (Supplementary data, Fig. S5). Similar re-
sults were obtained when labeling other sulfatase-expressing com-
plex proteomes (e.g., expressing ARSG or STS, resp., see
Supplementary data, Fig. S6 and S7). Thus, in complex proteomes
QMs that are generated in a sulfatase-dependent manner do not
lead to specific sulfatase labeling.
Our studies with QM-traps based on general masked monofluo-
romethylphenyl sulfates are consistent with sulfatase-generated
QMs that label sulfatases non-specifically. The combined behavior
of slow inactivation and multiple labeling events suggest that the
QM can indiscriminately react with sulfatase protein nucleophiles
within and outside of the active site, presumably because it dif-
fuses after turn-over-dependent activation. QM diffusion is also
supported by off-target labeling in proteomes. Notably, selected
protein identifications by LC–MS/MS revealed that these hits con-
tain cysteine residues, for which QMs have been described to have
increased affinity (see Supplementary data for sequences).8h
Although KARS does not contain any Cys residues, it is possible that
sulfatases with exposed Cys residues may exhibit a decrease of
non-specific QM-labeling. However, STS, which has surface ex-
posed Cys residues still shows active site inactivation and non-spe-
cific labeling in proteomes (see Supplementary data, Fig. S7) which
suggests that the labeling will remain non-specific overall.
mic samples consisted of crude lysates from Escherichia coli DH5
a
cells that expressed (i) recombinant KARS together with its post-
translational activating enzyme AtsB,13 (ii) recombinant KARS
alone, which will remain inactive without AtsB, and (iii) no sulfa-
tase (null vector control). Notably, E. coli does not express endoge-
nous sulfatases and shows no endogenous KARS activation
activity.13 Proteomes were labeled with 1 mM of the fluorescent
probe 4 for 60 min, separated by 2D-PAGE, and visualized by fluo-
rescence scanning and then Coomassie staining (Fig. 4).
As shown in Figure 4, fluorescent labeling by 4 is turnover-
dependent. The development of fluorescent spots is significant
only in the presence of active KARS (Fig. 4a) and not in control pro-
teomes (inactive KARS in Fig. 4b; and without sulfatases in Fig. 4c).
However, most of the Coomassie stained proteins in the active
KARS proteome also show up in the fluorescence scan of the gel
(Figure 4a, left versus right panel), indicating that the probe labels
During the course of our studies, efforts toward developing STS-
directed QM-traps were put forth, which reinforce our findings.8e,f
Recombinantly over-expressed STS can be labeled with a biotinyl-
ated MFPS probe similar to 4, although non-specific labeling was
apparent in crude mammalian lysates.8e Apparently, STS can also
evade QM-based inactivation by a masked difluorophenyl sulfate
derivative of estrone sulfate (as with compounds 1 and 2 against
PARS and KARS), whereas the corresponding monofluoromethyl
analog will irreversibly inactivate STS (as with 3 against PARS,
KARS, and STS).8f Although the estrone sulfate-masked QMs were
not applied to proteomic endeavors, their corresponding STS-gen-
erated QMs showed diffusive behavior and high QM to STS inacti-
vation ratios.8f It is interesting that the highly substrate-like
scaffold of these estrone-based QM-traps did not improve the spec-
ificity of STS labeling. Specific capture of protein tyrosine phospha-
tases (PTPs) was recently achieved by making short
phosphotyrosine peptide substrate analogs armed with a masked
phosphotyrosine QM.18 Although, flanking amino acid character
has also been used to hone the specificity of protease-dependent
quinole imine capture,9 the PTP example is the first to show spe-
cific capture of a quinone methide in the context of a complex pro-
teome.18 Indeed, previous efforts using simple fluoromethylphenyl
phosphates against PTPs resulted in a large degree of non-specific
protein labeling,18 reflecting the non-specific nature of many other
hydrolytically generated QMs from masked fluoromethyl pheno-
lates,8 including our own against sulfatases.
3. Conclusion
Figure 4. Labeling of model proteomic lysates using fluorescent MFPS probe 4 and
2D-PAGE. Model proteomes comprised of E. coli lysates containing: (a) active KARS,
(b) inactive KARS, (c) no sulfatase were incubated for 60 min with 1 mM probe 4.
Panels show 2D-gel images by fluorescence scanning (left) and Coomassie staining
(right). Fluorescent labeling only occurs with active KARS; however, it is non-
specific. Black frames indicate KARS, as verified by LC–MS/MS analysis (see
Supplementary data).
Sulfatases are key players in disease6 making ABPPs important
for providing information about their catalytic integrity in the
underlying pathobiochemical processes. As such, we examined
sulfatase-directed QM-traps based on masked fluoromethylphenyl

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J. Lenger et al. / Bioorg. Med. Chem. 20 (2012) 622–627
sulfates as ABPPs for sulfatases. New sulfatase-directed probes
based on MFPSs (3 and 4) were synthesized and found to have
superior irreversible inactivation properties against aryl sulfatases
in comparison to previously designed DFPSs (1 and 2).7 MFPS
probes were active against aryl sulfatases operating at neutral
and basic pH, including bacterial PARS and KARS and human STS;
however, they were not active against the human lysosomal en-
zyme ARSG. Despite good inactivation and reasonable reactivity to-
ward the enzyme class, further biochemical characterization of the
MFPS probes with purified enzyme demonstrated that sulfatase
inactivation occurs only after multiple QM-traps are enzymatically
generated. This leads to multiple enzyme labeling events, presum-
ably through non-specific QM capture by sulfatase nucleophiles,
both inside and outside of the active site. The activity of MFPS
probe 4 in complex proteomes confirmed that the sulfatase-direc-
ted QM-traps required enzymatic activation, but lead to off-target
labeling of many different enzyme classes. Unfortunately, such
behavior does not meet the stringent requirements for a sulfa-
tase-directed ABPP, which must be able to decisively report on
the sulfatase activity by turnover-dependent labeling in the con-
text of the greater proteome.1,9
NaOH. For ARSG the assay was conducted as follows: ARSG
(20 nM, in 0.5 M NaOAc, pH 5.6) was incubated with 3 for 3 h
and for 18 h at 37 °C. Sulfatase activity assay was then initiated
by adding pNCS to a final concentration of 8 mM in 0.5 M NaOAc,
pH 5.6, with a total volume of 150
to react for 60 min at 37 °C. The reaction was stopped by addition
of 150 L 1 M NaOH. See Fig. 2, Fig 3a, and Supplementary data,
lL, and the enzyme was allowed
l
Figure S2 for time- and concentration-dependent inhibition studies
with all sulfatases.
4.2.2. Steroidsulfatase (STS) activity assay
STS lysate (5
lL) was pre-incubated with inhibitor for 0, 1, 2 and
4 h at 37 °C with different concentrations of 3 in 50 mM Tris,
pH 7.4 (final volume of 47.5 lL). STS activity was determined using
3H-dehydroepiandrosterone-3-sulfate (3DHEAS) as substrate.15,16
The reaction was started by adding DHEAS to a final concentration
of 5
Bernhard Schmidt, Institut für Biochemie II, Universität Göttingen)
in a final volume of 50 L. After 10 min of incubation at 37 °C the
reaction was stopped by addition of 25
l
M containing 25,000 cpm 3H-DHEAS (kindly supplied by
l
l
L 1 M NaOH. Radioactivity
of the product 3H-DHEA was detected by liquid scintillation count-
ing as previously described.15 All measurements were done in trip-
licate and repeated twice.
4. Experimental
4.2.3. 19F NMR real-time experiments of sulfate cleavage of
probe 3
4.1. Sulfatase protein expression
All experiments were performed in standard glass NMR tubes at
Literature procedures were followed for the preparation of:
purified recombinant KARS (1.4 mg/mL in 50 mM Tris, pH 7.4,
250 mM NaCl, TBS)13; enriched KARS (0.7 mg/mL in TBS)19; puri-
fied recombinant PARS (3 mg/mL in 100 mM Tris, pH 7.5 with
37 °C on a Bruker Avance 600. KARS (70 nM) was measured with
compound 3 (140
lM) in D2O containing 10 mM Tris, pH 7.5 over
14 d. PARS (70 M) and 3 (5 mM) in D2O containing 100 mM Tris,
l
pH 8.5 were measured for 12 h.
20% glycerol)7; and purified ARSG (17
l
g/mL) from HT1080
stable Tet-on
ARSG-His cells.14 STS was expressed using
a
4.2.4. MALDI-ToF MS analysis of probe 3-labeled KARS and PARS
MALDI-ToF MS measurements were performed on an ultrafleX-
treme mass spectrometer (Bruker Daltonik). The spectra were ac-
quired in the linear mode. 5000 single spectra were summarized
with a 1 kHz smartbeam-II laser for each sample. Spectral process-
ing (smoothing and baseline subtraction) was done in flexAnalysis
HT1080 cell line.15 Cells were cultured in 10% fetal calf serum con-
taining Dulbecco’s modified Eagle’s medium (Gibco) at 37 °C with
5% CO2. The cells were transiently transfected in 6-wells with
4 lg of the bidirectional, tet-responsive pBI-STS+FGE-HA expres-
sion plasmid15 coding for STS and FGE using Lipofectamine LTX
(Invitrogen) as recommended. After transfection for 5 h, protein
expression was induced for 24 h by replacing the normal medium
(Bruker Daltonik). Purified KARS and PARS (20
with 5 mM 3 for 1 and 12 h, respectively, at room temperature.
For MALDI-ToF MS analysis: 2 L of the labeling mixture was
mixed with 2 L of 2% TFA and 2 L of matrix solution (7.6 mg
2,5-dihydroxyacetophenone dissolved in 375 L EtOH and 125
of a solution containing 18 mg/mL aqueous diammonium hydro-
gen citrate solution). The protein–matrix mixture (0.5 L) was
lL) were labeled
with medium containing 2
Cells were harvested by trypsination and cell pellets were frozen
L TBS, pH 8.0, con-
lg/mL doxycycline (Fisher Scientific).
l
l
l
at ꢀ20 °C. STS Pellets were resuspended in 150
l
l
lL
taining proteinase inhibitor cocktail 1:100 (Sigma), and lysed by
3 ꢂ 10 s sonication at 4 °C. Total protein concentration was
5.2 mg/mL. All protein concentrations were determined using Coo-
massie Plus Bradford reagent (Pierce).
l
spotted onto a ground steel target for MALDI-ToF analysis.
4.2.5. Proteome labeling with 4
4.2. Inhibition assays with QM-inhibitor 3
Model proteome (50
for 60 min in 10 L buffer (50 mM Tris, pH 7.5, 250 mM NaCl),
at room temperature. The sample was diluted with 330 L DeS-
lg) was incubated with 1 mM probe 4
l
4.2.1. Nitrocatechol sulfate (pNCS) activity assay
l
Turnover of pNCS by sulfatases to p-nitrocatechol (pNC) is mea-
sured by absorbance at 515 nm after stopping the enzyme reac-
tions with 0.33 M NaOH. Absorption measurements were made
in flat transparent 96-well plates (Greiner) using an infinite
M200 microplate reader (TECAN). All measurements were per-
formed in triplicate and repeated twice.
treak Rehydration Solution (GE Healthcare) containing 0.5% IPG
Buffer, pH 3–10 (GE Healthcare) and submitted to isoelectric
focusing on Immobiline DryStrip gels pH 3–10 NL, 18 cm (GE
Healthcare). An Ettan IPGphor (GE Healthcare) with the following
program was used for focusing: (1) step, 10 V for 1 h; (2) step,
30 V for 12 h; (3) step 500 V for 1 h; (4) gradient, 1000 V for
8 h; (5) gradient, 8000 V for 3 h; (6) step, 8000 V for 2 h. Second
dimension separation was achieved with a 10% polyacrylamide
gel, visualization of the fluorescence signal was done using a
Fujifilm LAS 3000 with an excitation of kex = 460 nm and detec-
tion using a FL-Y515 filter.
KARS (1
M, in 50 mM Tris, pH 8.5) were incubated for various times
with different concentrations of inhibitor 3 in 10 L assay volume
at room temperature. After pre-incubation, the sulfatase activity
assay was initiated by adding pNCS to a final volume of 310
lM, in 50 mM Tris, pH 7.5, 250 mM NaCl) or PARS
(1
l
l
lL
with a final assay composition of 35 nM sulfatase, 8 mM pNCS
and 8 mM Tris (at pH 7.5 or pH 8.5, for KARS and PARS, respec-
tively). The enzyme reaction was allowed to proceed for 10 min
4.3. Synthesis of sulfatase-directed QM probes
at room temperature before quenching by addition of 600 lL 1 M
See Supplementary data online.

J. Lenger et al. / Bioorg. Med. Chem. 20 (2012) 622–627
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Acknowledgments
We thank Dr. Marc-André Frese for kindly providing purified
ARSG and Thomas Kramer for synthesis of probe 4. J.L. acknowl-
edges graduate support from the Studienstiftung des Deutschen
Volkes. This work was supported by the Deutsche Forschungs-
gemeinschaft and the Fonds der Chemischen Industrie. C.-H.W.
and S.R.H. acknowledge support from NIH Grant AI072155.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.04.044.
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