Simple synthesis of 1,3-cyclopentanedione derived probes for labeling sulfenic acid proteins

Article abstract of DOI:10.1039/c1cc12127h

Facile, two-step synthesis and kinetic characterization of new chemical probes for selective labeling of sulfenic acid (-SOH) in proteins are presented. The synthesis route relies on the simple and highly efficient Michael addition of thiol containing tags or linkers to 4-cyclopentene-1,3-dione, the unsaturated derivative of 1,3-cyclopentanedione.

Full text of DOI:10.1039/c1cc12127h

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COMMUNICATION
Simple synthesis of 1,3-cyclopentanedione derived probes for labeling
sulfenic acid proteinsw
Jiang Qian,a Chananat Klomsiri,b Marcus W. Wright,c S. Bruce King,c Allen W. Tsang,a
Leslie B. Pooleb and Cristina M. Furdui*a
Received 13th April 2011, Accepted 22nd June 2011
DOI: 10.1039/c1cc12127h
Facile, two-step synthesis and kinetic characterization of new
chemical probes for selective labeling of sulfenic acid (–SOH) in
proteins are presented. The synthesis route relies on the simple
and highly efficient Michael addition of thiol containing tags or
linkers to 4-cyclopentene-1,3-dione, the unsaturated derivative
of 1,3-cyclopentanedione.
biotinylated dimedone analogues5,10,12 for affinity purification,
azide ‘‘tailed’’ dimedone for ‘‘click’’ chemistry or Staudinger
ligation,13 and fluorophore-linked dimedone analogues for
visualization.5,14 While proven to be useful in labeling –SOH
proteins, these reagents are all centered on the classic reactive
group of dimedone, which imparts similar reactivity to its
derivatives. One particular disadvantage of the dimedone
moiety is the derivatization process which requires long
chemical synthetic steps with an overall low yield.5,10,13 Alter-
native reagents that may allow for a more facile derivatization
have not been reported. In this study we investigated whether
other 1,3-diketones, like 4-cyclopentene-1,3-dione, could be
used for synthesis of protein –SOH chemical probes. The
envisioned advantage of the 4-cyclopentene-1,3-dione is that
it could be directly conjugated to thiol-containing tags or linkers
through the highly efficient Michael addition.15 Protection of
the enol isomer, which is routinely employed in dimedone
derivatization strategies, would not be required. Furthermore,
fluorophores or enrichment tags (e.g. biotin) could be attached
in two easy steps and with high yield as it will be shown below.
To test whether 1,3-cyclopentanedione derivatives exhibit
similar reactivity and selectivity towards –SOH as dimedone,
we first synthesized 4-(ethylthio)cyclopentane-1,3-dione compound
(1, Scheme 1, RQSCH2CH3) following the procedure
described in Supplementary Material.w The C165S mutant of
AhpC was used as the prototype protein in these studies.5,9
AhpC is a cysteine-based peroxidase from bacteria known to
form –SOH at the reactive C46, by H2O2 oxidation. Mutation
of C165 to serine stabilizes the –SOH species at C46 and
allows for quantitative analysis of covalent adduct formation
Cysteine oxidative modification to sulfenic acid (–SOH), by
various types of reactive oxygen species (ROS) (e.g. H2O2), has
emerged as an important post-translational modification in
proteins and was shown to play a pivotal role in regulating
protein functions under both physiological and oxidative
stress conditions.1–2 During the modification process redox
sensitive cysteines are first oxidized to –SOH as an initially
formed product. Depending on the microenvironment where
the cysteine is located, this metastable intermediate is further
transformed into more stable products like disulfides and
sulfenamides—the sometimes cyclic condensation product of
sulfenic acid. These are readily reversed to yield the reduced
thiol form via the action of cellular reductants like thioredoxin,
glutaredoxin and glutathione.3 Hyperoxidation to sulfinic or
sulfonic acid may occur if sulfenic acids encounter high
oxidant concentrations, a process which is typically irreversible
in the cell. However, an exception to this is now recognized. A
specialized repair protein, sulfiredoxin, can rescue at least a
subset of cysteine-based peroxidases (peroxiredoxins) in which
hyperoxidation to sulfinic acid occurs.4 Given the significance
of cysteine oxidation in proteins, various analytical methods and
probes have been developed to identify the –SOH proteins.5-10
The main strategy of detecting –SOH modified proteins
relies on the reactivity of dimedone (5,5-dimethyl-1,3-cyclo-
hexanedione) with –SOH (Scheme 1), a reaction first recognized
by Allison et al. in 1974.11 Several dimedone-based derivatives
have recently been synthesized to label –SOH proteins, featuring
a Department of Internal Medicine, Section on Molecular Medicine,
Wake Forest School of Medicine, Medical Center Blvd,
Winston-Salem, NC 27157, USA. E-mail: cfurdui@wfubmc.edu;
Fax: +1 336 716-1214; Tel: +1 336 716-2697
b Department of Biochemistry, Wake Forest School of Medicine,
Medical Center Blvd, Winston-Salem, NC 27157, USA
c Department of Chemistry, Wake Forest University, Winston-Salem,
NC 27109, USA
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc12127h
Scheme 1 Reaction of sulfenic acid modified protein with dimedone,
1,3-cyclopentanedione and its derivatives (1 and 2).
c
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5 Æ 0.4 mM. Formation of a covalent adduct was confirmed
by nanoLC-MS/MS. A peptide containing the C46 modified
by 1 (Fig. S2Aw) was identified, thus confirming the covalent
bond formation between the sulfur at C46 in C165S AhpC and 1.
To address the selectivity of the probe with regard to other
cysteine oxidation states and amino acid side chains of proteins,
the compound 1 was incubated with the reduced form (–SH)
of C165S AhpC protein, the C165S AhpC–SO2H and the
disulfide bond (–S–S–) linked dimer of wt AhpC, respectively,
at pH 5.5 and pH 7.5 conditions. Results showed that 1
did not react with –SH, –SO2H and –S–S– forms of AhpC
(Fig. S3w), and is therefore selective for the –SOH oxidation
state. Since 1 was synthesized by introducing a thioether group
through Michael addition which is potentially reversible, we
tested the stability of the AhpC-1 adduct to other thiols. Data
in Fig. S4Aw show that treatment of AhpC-1 adduct with
50 mM DTT for 1 h did not alter the probe as evidenced by
lack of a mass shift, consistent with previous reports.16,17 The
thioether group in AhpC-1 was also resistant to oxidation with
5 and 10 mM H2O2 for 1 h (Fig. S5Bw). Oxidation of thioether
to sulfoxide and sulfone was observed, however, at higher
concentrations of H2O2 (50 and 100 mM, 1 h) (Fig. S5B) and
17 h incubation (Fig. S5D).w These oxidized species were
resistant to treatment with other thiols as well (Fig. S4B).w
Previous labeling studies using dimedone and dimedone
derivatives did not show a pH dependence for Escherichia coli
f RMsr.9 To determine whether this occurs with 1 as well, the
reaction of 1 with C165S AhpC–SOH was monitored using
buffers at pH 6.5, 5.5, 4.5 and 3.5. A strong increase of
reaction rate was observed at lower pH (Fig. 3), indicating
that the enol form instead of the enolate may act as the primary
species reacting with the –SOH as the reaction proceeds through
acid catalysis. Therefore, labeling and kinetics experiments were
performed at slightly acidic pH (pH 5.5) where protein loss was
limited while the reactivity of 1,3-cyclopentanedione derivatives 1
and 2 with –SOH proteins was increased relative to higher pH. It
should be noted that the stock solution of 1 was made in 0.5 M
Bis-Tris and DMSO (v : v = 1 : 1) to pre-buffer the acidity of
1 to around 6.5 before adding it to the reaction buffer, and final
pH values of 5.5 were confirmed. Final DMSO concentration
during labeling was 0.5%.
Fig. 1 Representative ESI-TOF MS spectra of C165S AhpC–SOH
reaction with 1,3-cyclopentanedione (left), dimedone (middle), and
1 (right) (AhpC: 50 mM, labeling reagent: 5 mM; buffer: 50 mM bis-tris-
citric acid pH 5.5).
with chemical probes. Labeling of C165S AhpC–SOH by 1
was monitored as a function of reaction time, and compared
with dimedone and 1,3-cyclopentanedione using electrospray
ionization time-of-flight mass spectrometry (ESI-TOF MS).
The three major peaks observed at 20 598, 20 616, and
20 632 a.m.u. correspond to sulfenamide (likely formed in
gas phase from sulfenic acid after loss of one molecule of
H2O), –SOH, and –SO2H C165S AhpC species, respectively.
The individual AhpC product adduct peaks at a.m.u. 20696 (*),
20 738 (#) and 20 756 (+) increased over time and correspond
to 1,3-cyclopentanedione, dimedone and 1 adducts, respectively
(Fig. 1). The adduct formation was then plotted against the
reaction time, as shown in Fig. 2A and Fig. S1, which in addi-
tion includes the data for AhpC sulfenamide, –SOH and
–SO2H species. Data were modeled using KinTek Explorer
with the following pseudo-first-order rates of adduct forma-
tion: 0.0018 minÀ1 (1,3-cyclopentanedione), 0.0024 minÀ1
(dimedone), and 0.0036 minÀ1 (1), indicating higher reactivity
of 1 towards –SOH compared with dimedone or 1,3-cyclo-
pentanedione (shown also in Fig. S1w).
Further studies were aimed at determining the dependence
of the reaction rate on the concentration of 1 (Fig. 2B).
Data were fit to a hyperbolic equation to obtain a K0.5 of
We then synthesized a biotin analogue of 1 (2, Scheme 2) as
described in the Supplementary Material.w First, cysteinamine
was reacted with 4-cyclopentene-1,3-dione to yield an inter-
mediate (Int, Scheme 2) that was then conjugated with biotin–
NHS to produce the biotin-tagged product, 2. The biotin
Fig. 2 (A) Plot of adduct formation of 1,3-cyclopentanedione (open
circles), dimedone (closed circles), and 1 (inverted triangle) with C165S
AhpC-SOH (50 mM). The adduct concentration was calculated based
on adduct abundance among the total ion abundances of the four
prominent species in MS spectrum as indicated in Fig. 1. (B).
Concentration dependence of AhpC–SOH labeling by 1. AhpC–SOH
(50 mM) was incubated with different concentration of 1 (0, 0.5, 1, 2, 5,
10 mM) at pH 5.5 for 85 min and at rt.
Fig. 3 MS spectra of C165S AhpC–SOH labeling with 1 at 40 min
reaction time and pH 3.5-6.5. Increased adduct formation (a.m.u. 20 756)
was observed as pH was decreased from 6.5 to 3.5.
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To identify some of the proteins that showed increased labeling
with 2 in the presence of H2O2, the labeled lysate was incubated
with streptavidin–agarose and probed for PLCg1, a signaling
protein recently identified as redox sensitive using a biotin
derivative of dimedone (DCP-Bio1).2 These results show that
diverse oxidation patterns exist for individual proteins depending
on the sensitivity to oxidation of specific cysteine sites and
reactivity of cysteine sulfenic acids with 2 or other thiols.
In summary, we present a new and facile route for the
synthesis of chemical probes to label SOH modified proteins.
The comparative kinetic studies show that 1 has improved
reactivity compared to dimedone. The reactivity of 1 with
–SOH was enhanced under acidic conditions. Compound 2,
the biotinylated derivative of 1, was easily synthesized in two
steps and shown to label C165S AhpC–SOH and –SOH
containing proteins in cell lysates. In this regard, we envision
that labeling using this biotinylated derivative is well suited
to monitor the –SOH formation in cells under oxidative
conditions.
Scheme 2 Synthesis of a biotin-tagged 1,3-cyclopentanedione, 2.
analogue 2 can therefore be constructed in two simple reactions
with an overall yield of 59–68%.
Its labeling of sulfenic acid proteins was also confirmed by
reaction with C165S AhpC–SOH, which was nearly all converted
to the adduct species within 4 h and at pH 5.5 (Fig. 4A). MS/MS
analysis of the labeled protein digests confirmed that C46 was
modified by 2 (Fig. S2Bw). To further demonstrate the appli-
cation of 2 in labeling –SOH containing proteins and profiling
the –SOH proteome, we incubated lysates obtained from NIH
3T3 cells with increased concentrations of H2O2 in the presence
of 2. The selectivity of 2 for –SOH was examined again by
prereducing the lysates with TCEP, a disulfide reductant,
which was also shown to reduce SOH to SH.18 Pre-reduced
C165S AhpC was added to lysates as a positive control. Lysates
were resolved by SDS-PAGE, transferred to nitrocellulose
membrane and probed for labeling with 2 using streptavidin–
HRP (Fig. 4B). The results showed that treatment with TCEP
almost completely prevented labeling of cellular proteins with
2 (Fig. 4B, lane 2). The slight labeling in the TCEP control
may have been due to the incomplete reduction of some highly
abundant –SOH proteins, which were heavily labeled in the
non-TCEP samples. Biotin signal intensity increased with
H2O2 concentration up to 100 mM and leveled off at higher
H2O2 concentrations (500 mM). Interestingly, equal labeling of
C165S AhpC was observed at all H2O2 concentrations
(Fig. 4B, lanes 3, 4, 5, and 6). This is likely because of the rapid
disulfide bond formation between C165S AhpC and other free
thiols (proteins or low molecular weight thiols like GSH).
This research was supported by the NIH R01 CA136810
(CMF), and R33 CA126659/CA126659Z (LBP).
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Fig. 4 Reaction products of pure protein and cell lysates with 2.
(A) ESI-TOF MS spectrum showing C165S AhpC-2 covalent adduct
formation. (B) Western-blot analysis of proteins from NIH 3T3 cell
lysates labeled with 2. The blot was probed with streptavidin-HRP,
and antibodies for AhpC and b-actin. Samples after enrichment in
proteins labeled with 2 were probed for PLCg1 (see Supporting
Materialw).
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 9203–9205 9205