Bioorganic & Medicinal Chemistry Letters 19 (2009) 356–359
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Facile synthesis and biological evaluation of a cell-permeable probe
to detect redox-regulated proteins
Young Ho Seo a, Kate S. Carroll a,b,*
a
b
Life Sciences Institute, University of Michigan, 210 Washtenaw Ave. #4401, Ann Arbor, MI 48109-2216, USA
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-2216, USA
a r t i c l e
i n f o
Article history:
Received 15 October 2008
Revised 19 November 2008
Accepted 20 November 2008
Available online 24 November 2008
Keywords:
Redox regulation
Cysteine oxidation
Sulfenic acids
Tyrosine phosphatase
Chemical probes
Staudinger ligation
Click chemistry
a b s t r a c t
We have developed an improved synthesis for the cell-permeable, sulfenic acid probe DAz-1. Using
DAz-1, we detect sulfenic acid modifications in the cell-cycle regulatory phosphatase Cdc25A. In addition,
we show that DAz-1 has superior potency in cells compared to a biotinylated derivative. Collectively,
these findings set the stage for the development of activity-based inhibitors of Cdc25 cell-cycle phosphatases, which are sensitive to the redox state of the active-site cysteine and demonstrate the advantage of
bioorthogonal conjugation methods to detect protein sulfenic acids in cells.
Ó 2008 Elsevier Ltd. All rights reserved.
The reversible oxidation of cysteine is a widespread mechanism
in the regulation of protein function.1 In mammalian cells, activation of cell surface receptors can trigger enzymatic production of
reactive oxygen species (ROS). These oxidants function as second
messengers and modify signaling proteins through cysteine oxidation.2,3 For example, oxidation of a critical active-site cysteine
inhibits protein tyrosine phosphatases (PTPs) and results in increased phosphorylation levels.4 Redox-based signal transduction
plays an important role in many normal biological events, including cell proliferation, differentiation, migration and programed cell
death.3 In addition, abnormal ROS production correlates with cancer and aging-associated degenerative diseases.2,3 Because of the
central role that cysteine oxidation plays in cell signaling and human pathology, there has been considerable interest in developing
small-molecule probes to detect these post-translational modifications directly in cells.
Sulfenic acid (RSOH) is the simplest cysteine oxoform and is
formed by reaction of a thiolate anion (RS ) with cellular oxidants
such as hydrogen peroxide (H2O2). Sulfenic acids can oxidize further to sulfinic (RSO2H) and sulfonic (RSO3H) acid, condense with
a neighboring thiolate to form a disulfide, or be stabilized by the
protein microenvironment. The electrophilic sulfur atom in sulfenic acid reacts with carbon nucleophiles such as alkenes and eno-
* Corresponding author. Tel.: +1 734 615 2739.
E-mail address: katesc@umich.edu (K.S. Carroll).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.073
lates, including 5,5-dimethyl-1,3-cyclohexadione (1; dimedone,
Figure 1). Under aqueous conditions, dimedone reacts selectively
with cysteine sulfenic acids and not with other protein functional
groups.5–7 This chemoselective reaction has been exploited to
detect sulfenic acid modifications via mass analysis; fluorescentdimedone conjugates have also been reported.3 However, these
reagents are not suitable for proteomic studies since they lack an
affinity handle for isolating tagged proteins. To extend the utility
of the dimedone scaffold as a probe for protein sulfenic acid modifications we recently developed DAz-1 (2; Figure 1).7 DAz-1 covalently modifies sulfenic acid-modified proteins directly in cells and
Figure 1. Structures of small-molecule probes for sulfenic acids.
Y. H. Seo, K. S. Carroll / Bioorg. Med. Chem. Lett. 19 (2009) 356–359
has an azide chemical handle, which can be functionalized with a
wide variety of phosphine or alkyne-based reporter tags for
detection and isolation.8 In this report, we describe an improved
synthesis of DAz-1 and investigate sulfenic acid formation in the
cell-cycle regulatory phosphatase Cdc25A. In addition, we compare
the activity of DAz-1 to a biotinylated derivative in vitro and in
HeLa cells.
In a previous report, we pursued the synthesis of DAz-1 via
route A (Scheme 1, a ? b ? c ? f).7 Ni-catalyzed hydrogenation
of 3,5-dihydroxybenzoic acid (7) gave diketone 6 in 85% yield.9 Initial attempts to couple the carboxylic acid functional group on 6
with 3-azidopropylamine (8)10 using standard peptide coupling reagents such as PyBop/TEA, DIC/4-DMAP or TBTU/DIEA were not
successful and produced nucleophilic substitution products at the
carbonyl carbon of compound 6. To prevent these undesired reactions we protected the ketone 6 as the methyl ether. PTSA catalyzed protection was achieved in MeOH at room temperature to
give compound 5 in 97% yield. The amide coupling reaction of carboxylic acid 5 with amine 8 was carried out with TBTU and DIEA in
DMF to provide amide 3 in 99% yield. The synthesis of DAz-1 was
completed through deprotection of methyl vinyl ether with 2 N
HCl in THF. The overall yield of DAz-1 through route A was 64%.
In small-scale reactions amide coupling with TBTU, an HOBtbased aminium salt, afforded DAz-1 in excellent yield. However,
when the reaction was scaled up compound DAz-1 and HOBt were
not completely resolved by silica gel chromatography. Although reverse phase HPLC could be used to purify DAz-1 the procedure is
time consuming and reduced the overall yield to 20%. For this reason, we explored an alternate synthetic route B (Scheme 1,
a ? b ? d ? e ? f). Pentafluorophenyl trifluoroacetate (TFAPfp)
can activate carboxylic acids as Pfp esters in good yields and volatile side-products TFA and pentafluorophenol are easily removed
during workup.11 Therefore, we used TFAPfp as the activating reagent for the carboxylic acid functional group on 5. Esterification
of carboxylic acid 5 was achieved with TFAPfp and DIEA in DMF
in quantitative yield. Pfp ester 4 was then coupled with 3-azidopropylamine (8) to furnish compound 3 in high purity. For the final
step of the synthesis, we used cerium (IV) ammonium nitrate
(CAN) to deprotect 3. Non-acidic deprotection using catalytic
amount of CAN provided DAz-1 in 96%. Route B did not require
HPLC purification and furnished DAz-1 over five steps in 52% yield.
Having established an efficient synthesis for DAz-1, we next
investigated sulfenic acid formation in the cell-cycle regulatory
phosphatase Cdc25A (Scheme 2). In humans, three different
Cdc25 family members exist with Cdc25A required for the G1/S
357
transition and Cdc25B/Cdc25C involved in the G2/M.12 Like all tyrosine phosphatases, members of the Cdc family contain an activesite cysteine involved in formation of a phosphocysteine intermediate. The pKa of this active-site residue is significantly perturbed
from the typical 8.5 to 6.13 Cdc25 phosphatases also possess a
second conserved cysteine located 5 Å from the active-site residue. This additional cysteine is not required for activity, but can
form an intramolecular disulfide with the catalytic cysteine under
mild oxidizing conditions.14 The proposed function of this disulfide
is to prevent overoxidation of the catalytic cysteine to irreversible
sulfinic and sulfonic acid forms.
Biochemical studies indicate that the active-site cysteine in
Cdc25B and Cdc25C is sensitive to oxidants and that sulfenic acid
formation inhibits phosphatase activity.15 However, it is not
known whether Cdc25A—the only essential Cdc25 isoform—is susceptible to oxidation. Therefore, we treated the recombinant soluble catalytic domain of Cdc25A C384S16 with DAz-1 and
conjugated it to biotin reporter tags 9 or 10 using the Staudinger
ligation or click chemistry, respectively (Scheme 2). Western blot
analysis shows DAz-1-dependent labeling of Cdc25A C384S
(Fig. 2). Pre-treatment of the phosphatase with the reducing agent
dithiothreitol (DTT) significantly reduced labeling, as expected
(Fig. S1a). Trapping the sulfenic acid modification also blocked formation of disulfide-linked Cdc25A homodimers (Fig. S1b). Taken
together, these data indicate that the active-site cysteine in Cdc25A
can oxidize to a sulfenic acid, which can be trapped by DAz-1. Since
Cdc25A expression is elevated in a wide variety of cancers,12 smallmolecules inhibitors, which target the phosphatase active-site and
are sensitive to the redox state of the catalytic cysteine, may inhibit
proliferation of transformed cells that are associated with high levels of ROS.
In recent work, Charles and colleagues reported a biotinylated
dimedone derivative to monitor protein sulfenic acid formation
in peroxide-treated rat ventricular myocytes.17 In their studies,
Charles et al. observed protein labeling only when primary cells
were treated with hydrogen peroxide.17 One interpretation of
these data is that the basal level of cellular sulfenic acids in primary myocytes is below the threshold of detection. Alternatively,
since oxidants stimulate programed cell death and necrosis in cultured cells,18,19 it is possible that treatment compromised membrane integrity and allowed the dimedone-biotin derivative to
enter the dying cell. Consistent with the latter proposal, several recent reports demonstrate that direct conjugation of a reporter tag
such as biotin or a fluorophore to an inhibitor reduces potency
and prevents passive diffusion across cell membranes.20,21 For
Scheme 1. Synthesis of DAz-1 (1). Reagents and conditions: (a) T1 Raney nickel, NaOH, 750 psi, 70 °C, 85%; (b) PTSA, MeOH, rt, 10 min, 97%; (c) 3-azidopropylamine (8), TBTU,
DIEA, DMF, rt, 15 min, 99%; (d) TFAPfp, TEA, DMF, rt, 2 h, 100%; (e) 3-azidopropylamine (8), DIEA, DMF, rt, 3 min, 89%; (f) 2 N HCl, THF, rt, 1 h, 78% or CAN, H2O-MeCN, reflux,
1 h, 96%.
358
Y. H. Seo, K. S. Carroll / Bioorg. Med. Chem. Lett. 19 (2009) 356–359
Scheme 2. Strategy to detect sulfenic acid-modified Cdc25A with biotinylated reporter tags and the Staudinger ligation (top) or click chemistry (bottom) methods.
Figure 2. DAz-1 detects sulfenic acid modifications in the cell-cycle regulatory
phosphatase Cdc25A. Cdc25A C384S was reacted with 10 mM DAz-1, separated
from excess probe and conjugated to 9 or 10 using standard procedures.8 Samples
were analyzed by Western blot using HRP-conjugated streptavidin (top). Equivalent
protein loading for the Staudinger and click reactions was verified by SDS–PAGE
(bottom).
these reasons, we hypothesized that direct conjugation of the biotin reporter tag could decrease cellular activity.
To test this hypothesis, we synthesized DAz-1-biotin 11 (Fig. 1
and Scheme S1) and conducted a side-by-side comparison of sulfenic acid labeling in vitro and in cells. To generate DAz-1-biotin, Pfp
ester 5 was coupled to biotin-PEO3-amine (13). Subsequently,
methyl vinyl ether 12 was deprotected with 1 N HCl in THF. The
DAz-1-biotin 11 probe differs from the reagent reported by Charles
et al. only in the absence of a methyl substitutent at the C-5 position of the 1,3-cyclohexadione scaffold. In biochemical experiments, DAz-1 and DAz-1-biotin covalently tagged oxidized
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), as demonstrated by Western blot detection (Fig. S2a). In competition experiments, 250 lM DAz-1 was sufficient to block 90% of protein
labeling by 1 mM DAz-1-biotin (Fig. S2). Differences in the extent
and pattern of protein labeling by DAz-1-biotin and DAz-1 were
also observed in HeLa cell lysate (Fig. S2c). In particular, more individual sulfenic acid-modified proteins were visualized in DAz-1treated samples. Since the specificity of DAz-1 and dimedone
derivatives has been rigorously established in prior work5–7 the
simplest explanation for the observed differences is that the large,
hydrophobic reporter tag on DAz-1-biotin interfered with protein
binding. When tested in intact HeLa cells, DAz-1 was significantly
more potent than DAz-1-biotin as shown by immunofluorescence
microscopy (Fig. 3a) and Western blot (Fig. 3b). Using specific anti-
Figure 3. Immunofluorescence microscopy and Western blot showing sulfenic
acid-modified proteins labeled by DAz-1 in HeLa cells. (a) HeLa cells were incubated
in media containing 0.1 mM DAz-1 (top) or DAz-1-biotin (bottom). After fixation,
DAz-1-modified proteins were conjugated to p-biotin. Biotinylated proteins from
DAz-1 or DAz-1-biotin treated cells were detected by streptavidin-Alexa Fluor 555
(red). Nuclei were counterstained with DAPI (blue). (b) Protein was isolated from
cells incubated in media containing 10 mM DAz-1 or DAz-1-biotin. Protein lysate
from DAz-1 treated cells was conjugated to p-biotin. Biotinylated proteins from
DAz-1 or DAz-1-biotin treated cells were resolved by SDS–PAGE and detected by
Western blot analysis using HRP-conjugated streptavidin (top). Equal protein
loading was verified by reprobing the blot with an antibody against GAPDH
(bottom).
bodies we confirmed the identities of several DAz-1 labeled proteins as GAPDH, actin and peroxiredoxin (Fig. 3b), which are well
known to contain sulfenic acid modifications.1,3 Furthermore, no
significant differences in cell viability were observed among DMSO,
DAz-1-biotin and DAz-1-treated cells (Fig. S3). These data demonstrate the advantage of DAz-1 and bioorthogonal conjugation
methods for detecting protein sulfenic acids directly in living cells.
In summary, we have reported an improved synthesis for the
sulfenic acid probe DAz-1. This probe was successfully applied to
detect sulfenic acid modifications in the cell-cycle regulatory phosphatase Cdc25A. These findings set the stage for development of
activity-based tyrosine phosphatase inhibitors that are sensitive
to the redox state of the active-site cysteine. Furthermore, we show
that DAz-1 and bioorthogonal conjugation with reporter tags is the
method of choice for detecting protein sulfenic acids under physi-
Y. H. Seo, K. S. Carroll / Bioorg. Med. Chem. Lett. 19 (2009) 356–359
ological conditions in cells. Bi-functional probes such as DAz-1
should facilitate determination of the specific roles played by sulfenic acid modifications in redox-based cell signaling and in regulatory mechanisms that involve oxidation of cysteine residues.
Acknowledgments
We thank Prof. Mark Saper for the generous gift of Cdc25A
C384S and Prof. Howard Hang for alkyne-biotin reporter 10. We
also thank the Life Sciences Institute and the Leukemia & Lymphoma Society Special Fellows Award #3100-07 and the American
Heart Association Scientist Development Grant #0835419N to
K.S.C. for support of this work.
Supplementary data
Synthetic procedures, analysis data, and protocols for biochemical studies. Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2008.11.073.
References and notes
1. Reddie, K. G.; Carroll, K. S. Curr. Opin. Chem. Biol. 2008. doi:10.1016/
j.cbpa.2008.07.028. and references therein.
2. Miller, E. W.; Chang, C. J. Curr. Opin. Chem. Biol. 2007, 11, 620. and references
therein.
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3. Poole, L. B.; Nelson, K. J. Curr. Opin. Chem. Biol. 2008, 12, 18. and references
therein.
4. Tonks, N. K. Nat. Rev. 2006, 7, 833. and references therein.
5. Benitez, L. V.; Allison, W. S. J. Biol. Chem. 1974, 249, 6234.
6. Poole, L. B.; Zeng, B. B.; Knaggs, S. A.; Yakubu, M.; King, S. B. Bioconjug. Chem.
2005, 16, 1624.
7. Reddie, K. G.; Seo, Y. H.; Muse, W. B.; Leonard, S. E.; Carroll, K. S. Mol. Biosyst.
2008, 4, 521.
8. Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R. ACS Chem. Biol.
2006, 1, 644.
9. Van Tamelen, E. E.; Hildahl, G. T. J. Am. Chem. Soc. 1956, 78, 4405.
10. Hatzakis, S. N.; Engelkamp, H.; Velonia, K.; Hofkens, J.; Christianen, P. C. M.;
Svendsen, A.; Patkar, S. A.; Vind, J.; Maan, J. C.; Rowan, A. E.; Nolte, R. J. M. Chem.
Commun. (Camb) 2006, 2012.
11. Green, M.; Berman, J. Tet. Lett. 1990, 31, 5851.
12. Boutros, R.; Dozier, C.; Ducommun, B. Curr. Opin. Cell Biol. 2006, 18, 185. and
references therein.
13. Rudolph, J. Antioxid. Redox Signal. 2005, 7, 761.
14. Rudolph, J. Biochemistry 2007, 46, 3595.
15. Sohn, J.; Rudolph, J. Biochemistry 2003, 42, 10060.
16. The soluble catalytic domain of Cdc25A consists of residues 336–523; the
back-door cysteine, which is not required for phosphatase activity, has been
changed to serine (Cdc25A C384S).
17. Charles, R. L.; Schroder, E.; May, G.; Free, P.; Gaffney, P. R.; Wait, R.; Begum, S.;
Heads, R. J.; Eaton, P. Mol. Cell. Proteomics 2007, 6, 1473.
18. Kwon, S. H.; Pimentel, D. R.; Remondino, A.; Sawyer, D. B.; Colucci, W. S. J. Mol.
Cell. Cardiol. 2003, 35, 615.
19. Troyano, A.; Sancho, P.; Fernandez, C.; de Blas, E.; Bernardi, P.; Aller, P. Cell
Death Differ. 2003, 10, 889.
20. Cohen, M. S.; Hadjivassiliou, H.; Taunton, J. Nat. Chem. Biol. 2007, 3, 156.
21. Hang, H. C.; Loureiro, J.; Spooner, E.; van der Velden, A. W.; Kim, Y. M.;
Pollington, A. M.; Maehr, R.; Starnbach, M. N.; Ploegh, H. L. ACS Chem. Biol.
2006, 1, 713.
Supporting Information
Facile Synthesis and Biological Evaluation of a CellPermeable Probe to Detect Redox-Regulated Proteins
Young Ho Seo,a and Kate S. Carroll,a,b,*
a
b
Life Sciences Institute Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109-2216
General Experimental
Unless otherwise noted, all reactions were performed under an argon atmosphere in ovendried glassware.
All purchased materials were used without further purification.
Methylene
chloride and triethyl amine were distilled over calcium hydride prior to use.
Thin layer
chromatography (TLC) was carried out using Analtech Uniplate silica gel plates. TLC plates
were visualized using a combination of UV, p-anisaldehyde, ceric ammonium molybdate,
ninhydrin, and potassium permanganate staining.
Flash chromatography was performed
following the method of Still et al.1, using Sorbent Technologies Incorporated silica gel (32-63
µM, 60 Å pore size).
NMR spectra were obtained on a Varian Inova 400 (400 MHz for 1H; 100 MHz for 13C), or a
Varian Mercury 300 (300 MHz for 1H; 75 MHz for 13C NMR) spectrometer.
1
H and 13C NMR
chemical shifts are reported in parts per million (ppm) relative to TMS, with the residual solvent
peak used as an internal reference.
High resolution mass spectra (HRMS) were obtained on a
VG-70-250-s spectrometer manufactured by Micromass Corp. (Manchester UK) at the
University of Michigan Mass Spectrometry Laboratory. Reverse-phase HPLC purifications
were performed on a Beckman Coulter System Gold 126P equipped with System Gold 166P
detector (λ= 220) using a C18 (21.2×150 mm) Beckman Coulter Ultraprep with a gradient of
1
0.1% TFA in H2O and CH3CN as the mobile phase.
Experimental Procedures and Spectroscopic Data
Preparation of compound 4.
To an oven-dried round bottom flask purged with Ar was
added compound 3 (0.101g, 0.594 mmol) and 2 mL of DMF. To the solution was added
TEA (0.072g, 0.713 mmol) followed by TFAPfp (0.200g, 0.713 mmol) at rt under Ar.
The reaction mixture was stirred for 10 min, concentrated in vacuo at rt and purified by
silica gel chromatography, eluting with 9:1 hexanes:ethyl acetate to ethyl acetate. Rf: 0.19
(7:3 hexanes:ethyl acetate). 1H NMR (CDCl3, 300 MHz): δ 5.47 (s, 1H), 3.75 (s, 3H),
3.53-3.43 (m, 1H), 2.89-2.84 (m, 2H), 2.80-2.73 (m, 2H).
13
C NMR (CDCl3, 100 MHz):
δ 195.93, 176.27, 169.11, 102.48, 56.44, 38.35, 38.26, 30.87. ESI-HRMS calcd. For
C14H9F5NaO4 (M+Na+) 359.0319, found 359.0317.
Preparation of compound 3.
To a solution of compound 4 (0.0240 g, 0.0714 mmol) in
1 mL of DMF was added 3-azidopropylamine (0.0086 g, 0.0857 mmol) followed by
diisopropylethylamine (0.0148 mL, 0.0857 mmol) at rt under Ar. The reaction mixture
was stirred at rt for 0.5 h. The solution was concentrated in vacuo and purified by silica
gel chromatography to provide the product (0.0160 g, 0.0635 mmol) in 89% yield.
Spectroscopic data of the resulting product is identical to compound 3 synthesized via
route A. Rf: 0.38 (ethyl acetate). 1H NMR (CDCl3, 300 MHz): δ 6.13 (s, 1H), 5.37 (s,
1H), 3.71 (s, 3H), 3.36 (q, J = 6.3 Hz, 4H), 2.89-2.79 (m, 2H), 2.62-2.45 (m, 2H),
1.86-1.75 (m, 2H).
13
C NMR (CDCl3, 75 MHz): δ 197.27, 177.30, 172.20, 101.80, 56.03,
49.43, 40.72, 39.83, 37.43, 31.43, 28.60. ESI-HRMS calcd. For C11H16N4NaO3 (M+Na+)
275.1120, found 275.1122.
2
Preparation of compound 2 (DAz-1).
To a solution of compound 3 (0.033g, 0.131 mmol) in
1mL H2O2 and 1 mL MeCN was added CAN (7.0 mg, 0.0131 mmol) at rt. The solution was
heated to reflux for 1 h.
The resulting mixture was cooled to rt and subjected to silica gel
chromatography, eluting with ethyl acetate to provide the title compound (0.030 g, 0.126 mmol)
in 96% yield as a white solid.
Spectroscopic data of the resulting product is identical to DAz-1
synthesized by acidic deprotection in route A. Rf: 0.14 (3:1 ethyl acetate:methanol). 1H NMR
(DMSO-d6, 300 MHz): δ 7.99 (t, J = 5.1 Hz, 1H), 5.19 (s, 1H), 3.34 (t, J = 6.6 Hz, 2H), 3.11 (q,
J = 6.3 Hz, 2H), 2.91-2.80 (m, 1H), 2.43 (dd, J = 16.8 Hz, 11.1 Hz, 2H), 2.29 (dd, J = 16.8 Hz,
4.5 Hz, 2H), 1.69-1.60 (m, 2H).
13
C NMR (D2O, 100 MHz): δ191.11, 175.07, 48.55, 39.52,
36.64, 34.02, 27.44. ESI-HRMS calcd. For C10H14N4NaO3 (M+Na+) 261.0964, found 261.0974.
O
HN NH
S
O
H
N
O
3
O
13
O OH
5
OMe
HN NH
H
N
O
O
12
3
H
N
O
O
1N HCl
THF-H2O
4h, rt, 51%
O
O
HN NH
S
+
NH 2
TFAPfp
DIEA, DMF
3h, rt, 67%
O
S
OMe
H
N
O
O
11
Scheme S1. Synthesis of the DAz-1-biotin derivative.
3
3
H
N
O
O
Preparation of compound 12.
To a solution of compound 5 (0.20 g, 1.18 mmol) in DMF (5 mL)
was added DIEA (0.34 mL, 1.96 mmol) followed by TFAPfp under argon at rt.
After being
stirred for 30 min at rt, biotin-PEO3-amine (13, 0.438 g, 0.98 mmol) and DIEA (0.34 mL, 1.96
mmol) were added to the reaction mixture.
The reaction was stirred for 3 h at rt, concentrated
in vacuo and purified by silica gel chromatography, eluting with 1:1 ethyl acetate:methanol to
provide the title compound (0.40 g, 0.67 mmol) in 67 % yield. Rf: 0.45 (1:1 ethyl acetate:
methanol). 1H NMR (CD3OD, 400 MHz): δ 5.38 (s, 1H), 4.48 (dd, J = 4.8 Hz, 7.8 Hz, 1H), 4.29
(dd, J = 4.8 Hz, 7.2 Hz, 1H), 3.74 (s, 3H), 3.62-3.45 (m, 13H), 3.29-3.12 (m, 7H), 3.00-2.86 (m,
2H), 2.73-2.64 (m, 2H), 2.54-2.38 (m, 3H), 2.16 (t, J = 7.2, 2H), 1.87 (s, 1H), 1.78-1.51 (m, 8H),
1.40 (q, J = 7.5 Hz, 2H). ESI-LRMS calcd. For C28H47N4O8S (M+H+) 599.3, found 599.2.
Preparation of compound 11. To a solution of compound 12 (0.145 g, 0.284 mmol) in THF (2
mL) was added 1N HCl (2 mL) at rt.
After being stirred for 4 h at rt, the reaction was
concentrated in vacuo and purified by C18 reverse-phase HPLC (10 µm, 21.2×150 mm,
Beckman Coulter) with a gradient of 0% B to 50% B in 50 min (buffer A: 0.1% TFA in H2O;
buffer B: 0.1% TFA in CH3CN) at a flow rate of 10 mL/min to provide the title compound
(0.085 g, 0.145 mmol) in 51% yield.
Rt = 26.5 min.
1
H NMR (CD3OD, 300 MHz): δ 4.47 (dd,
J = 5.1 Hz, 7.8 Hz, 1H), 4.28 (dd, J = 4.5 Hz, 8.1 Hz, 1H), 3.71-3.47 (m, 13H), 3.32-3.15 (m,
7H), 3.00-2.87 (m, 2H), 2.73-2.38 (m, 5H), 2.18 (t, J = 7.2, 2H), 1.76-1.54 (m, 8H), 1.45 (q, J =
7.5 Hz, 2H). ESI-LRMS calcd. For C27H45N4O8S (M+H+) 585.3, found 585.2.
Stock solutions
Unless otherwise stated DAz-1, DAz-1-biotin and dimedone stocks were made up to a final
concentration of 200 mM in a 50:50 mixture of DMSO and 0.1 M Bis-Tris HCl pH 7.5.
4
In
experiments detailed below, “DMSO control” samples were treated with 50:50 mixture of
DMSO and 0.1 M Bis-Tris HCl pH 7.5.
The volume of buffered DMSO or probe stock solution
added to reactions were identical in all cases.
Phosphine and alkyne-biotin stocks were made
up to a final concentration of 5 mM in DMSO. All reagents were added to directly to purified
proteins, lysates and cells.
DAz-1 Labeling of Cdc25A C384S
Cdc25A C384S is a truncated protein consisting of residues 336-523 and was generously gifted
by Prof. Mark Saper (University of Michigan). Cdc25A C384S (20 µM in 250 mM Hepes, 200
mM NaCl pH 7.5) was reacted with DAz-1 (10 mM) for 10 min at rt.
Excess DAz-1 was
removed from the labeled proteins using an Amicon Ultra-4 10 KDa MWCO centrifugal filter
unit (Millipore).
Phosphine-biotin Labeling of Cdc25A C384S
Phosphine-biotin (p-biotin) was synthesized as previously described.2
Staudinger ligation
labeling was performed by incubation of azide-labeled Cdc25A C384S with phosphine-biotin
(250 µM) for 2 h at 37 oC.
Alkyne-biotin Labeling of Cdc25A C384S
Alkyne-biotin was synthesized as previously described3 and was a generous gift of Prof. Howard
Hang (Rockefeller University).
Copper-mediated [3+2] cycloaddition (click chemistry) was
performed by incubation of azide-labeled Cdc25A C384S with alkyne-biotin (100 µM), TCEP (1
mM), tris-triazolyl ligand (100 µM final concentration from 1.7 mM stock in 1:4 DMSO:tertbutanol) and CuSO4 (1 mM).
Samples were vortexed and allowed to react for 2 h at rt.
5
Immunoblotting
Biotin labeled proteins were separated by SDS-PAGE using Criterion XT 4-12% Bis-Tris gels
(Bio-Rad) in XT MES running buffer, transferred to a polyvinylidene fluoride membrane was
blocked with 5 % nonfat dried milk in TBST overnight at 4 oC or 1 h at rt. The membrane was
washed in TBST (2×10 min) and then incubated with 1:5,000-1:100,000 streptavidin-HRP (GE
Healthcare) in TBST for 1 h at rt, washed (1×5 min and 1×10 min) with TBST and developed
with chemiluminescence (Amersham ECL plus Western Blotting Detection Reagents).
To
assess equivalent loading, proteins were separated by SDS-PAGE and detected with Coomassie
Blue.
Detecting Sulfenic Acids in HeLa Cells with DAz-1 and DAz-1-biotin
HeLa cells were cultured in DMEM media supplemented with 10% fetal bovine serum (FBS),
1% penicillin-streptomycin-L-glutamate (PSG) and 1× non-essential amino acids (NEAA).
Cells were grown to 80~90% confluency, trypsinized, pelleted (200 g at rt), resuspended in
DMEM media containing sulfenic acid probe (10 mM) or DMSO control, 0.5 % FBS and 1×
NEAA and incubated for 2 h at 37 oC. After treatment, cells were washed 3 times with PBS,
scraped into ice-cold lysis buffer (1.0% NP-40, 50 mM Tris-HCl, 150 mM NaCl, and 2×
Complete Mini Protease Inhibitor Cocktail, pH 8.0) and incubated on ice for 30 min with
frequent mixing.
The supernatant was collected by centrifugation (20,000 g for 20 min) at 4 oC.
This procedure was repeated a second time to generate a clarified cell lysate.
concentration of the lysate was determined by Bradford assay (Bio-Rad).
The protein
Cell lysates were
incubated with p-biotin (200 µM in 50 µL lysis buffer) and DTT (5 mM) for 2 h at 37 oC; small
6
molecules were removed by three acetone precipitations.
Pellets were suspended in SDS-
protein loading buffer containing 10% 2-βME and immunoblotted as described above.
Detecting Sulfenic Acids in HeLa Cell Lysate with DAz-1 and DAz-1-biotin
HeLa cell lysate was prepared as described above.
Cell lysates were incubated with sulfenic
acid probe (1 mM) or DMSO control for 1 h at 37 oC and treated with alkyne-biotin (500 µM),
TCEP (1 mM), tris-triazolyl ligand (100 µM, from 1.7 mM stock in 1:4 DMSO:tert-butanol) and
CuSO4 (1 mM).
Samples were vortexed and allowed to react for 1 h at rt in the dark.
Reactions were diluted with methanol (400 µL), chloroform (150 µL) and H2O2 (300 µL) and
centrifuged at 4 oC for 1 min at 20,000 g. The top layer was carefully decanted, then methanol
(400 µL) was added, centrifuged at 4 oC for 1 min at 20,000 g and the supernatant was decanted.
This procedure was repeated once more as described.
Protein pellets were dissolved in 4% SDS
lysis buffer and mixed with SDS-protein loading buffer containing 10% 2-βME.
For
comparison of protein labeling 30 µg total protein for DMSO and DAz-1-treated samples and 3
µg total protein for DAz-1-biotin-treated samples were resolved by SDS-PAGE and
immunoblotted as described above.
Labeling Sulfenic Acid-Modified GAPDH with DAz-1 and DAz-1-biotin
GAPDH (2.89 mg/mL in 90 µL PBS; Sigma) was pre-reduced with 10 mM DTT.
incubation at rt for 15 min, DTT was removed using a P-30 spin column (Bio-Rad).
After
Reduced
GAPDH (11.6 µM in 30 µL PBS) was treated with H2O2 (43 µM) for 10 min at rt and incubated
with DAz-1 (0, 50, 100, 250, 500 or 1000 µM) or DAz-1-biotin (0, 50, 100, 250, 500 or 1000
µM) for 2 h at 37 oC.
Reactions were quenched by adding DTT (3.3 mM).
After removing
small molecules by P-30 spin column, samples were incubated with p-biotin (300 µM) for 2 h at
7
37 oC and immunoblotted as described above.
Labeling GAPDH with DAz-1-biotin Challenged by DAz-1
GAPDH (10 µM in 30 µL PBS; Sigma) was oxidized with H2O2 (50 µM) for 10 min at rt and
then treated with DAz-1 (0, 50, 100, 250, 500 or 1000 µM). After 2 h incubation at 37 oC,
DAz-1-biotin (1 mM) was added to each reaction, incubated for an additional 2 h at 37 oC and
immunoblotted as described above.
Fluorescence Microscopy
HeLa cells (~2.0 × 105) were seeded on collagen coated coverslips in 24-well plate containing
DMEM media (500 µL) supplemented with 10% FBS, 1% PSG and 1×NEAA 16 h before
analysis.
Cells were incubated with sulfenic acid probe (100 µM) or DMSO control for 15
min at 37 oC in 300 µL of DMEM media (2% FBS, 1% PSG and 1× NEAA).
Next, cells were
washed with PBS (500 µL) twice for 5 min, incubated in the absence of probe for 1 h at 37 oC
in DMEM media (2% FBS, 1% PSG and 1× NEAA), and fixed for 10 min with acetonemethanol (1:1) that had been pre-cooled to -20 oC. After 10 min fixation, cells were washed
with PBS (500 µL) for 5 min 3 times, and then incubated with p-biotin (100 µM in 300 µL) for
1 h at 37 oC. After treatment, cells were washed twice with DMEM media (2% FBS, 1% PSG
and 1× NEAA) and twice with PBS for 10 min, blocked with 5% FBS in PBS (300 µL) for 30
min at rt and washed 3 times with PBS for 10 min. Cells were stained with streptavidin-Alexa
Fluor 555 in PBS with 5% FBS and 0.025% Tween 20 for 30 min in the dark, washed with 5%
FBS in PBS twice for 5 min and stained with 0.1 µg/mL DAPI for 1 min.
Coverslips were
rinsed with PBS, mounted with Fluoromount G (Southern Biotech) and analyzed using a
Olympus BX-51 microscope.
8
Dimedone Labeling of Cdc25A C384S
Cdc25A C384S (20 µM in 250 mM Hepes, 200 mM NaCl pH 7.5) was incubated with H2O2 (0,
10, 20, or 40 µM) for 2 min at rt and treated with catalase (0.1 unit).
After treatment, oxidized
Cdc25A C384S was reacted with dimedone (0, 2, 10 or 50 mM) for 30 min at rt.
Samples were
treated with 10% SDS, resolved by nonreducing SDS-PAGE and analyzed by Coomassie Blue
staining.
DAz-1 Labeling of Cdc25A C384S in the Presence or Absence of DTT
Cdc25A C384S (20 µM in 250 mM Hepes, 200 mM NaCl pH 7.5) was incubated with DTT (0 or
5 mM) for 30 min at 0 oC and treated with DAz-1 (10 mM) for 10 min at rt.
Small molecules
were removed using an Amicon Ultra-4 10 KDa MWCO centrifugal filter unit and DAz-1 tagged
proteins were conjugated with p-biotin (250 µM) for 2 h at 37 oC and immunoblotted as
described above.
Viability Assay of HeLa Cells Treated with DMSO, DAz-1 or DAz-1-biotin
HeLa cells were cultured as described above.
Cells were grown to 80~90% confluency,
trypsinized, pelleted (200 g at rt) and resuspended in DMEM media containing 0.5 % FBS and
1× NEAA.
Cells (100 µL) were then mixed with trypan blue solution (100 µL) and dead cells
were quantified by counting with a hemocytometer.
Cells were further incubated in DMEM
media containing sulfenic acid probe (10 mM) or DMSO control, 0.5 % FBS and 1× NEAA for
2h at 37 oC.
After treatment, cell viability was quantified as described above.
9
Figure S1.
In vitro labeling of Cdc25A C384S. (a) Labeling of Cdc25A C384S with DAz-1.
Cdc25A C384S (20 µM) incubated in the presence (+) or absence (−) of DTT (5 mM) was first
reacted with DAz-1 (10 mM) for 10 min at rt and then incubated with p-biotin (250 µM) for 2 h
at 37 oC.
Samples were analyzed by HRP-streptavidin western blot. Total protein content was
determined using Coomassie Blue (Right). (b) Labeling of Cdc25A C384S with dimedone.
Cdc25A C384S (20 µM) was incubated with H2O2 for 2 min at rt and quenched with catalase
(0.1 unit).
Oxidized protein was then reacted with dimedone for 30 min at rt. Samples were
resolved on nonreducing SDS-PAGE and analyzed by Coomassie Blue staining.
10
Figure S2.
biotin.
Comparative labeling of sulfenic acid-modified proteins using DAz-1 and DAz-1-
(a) Labeling of sulfenic acid-modified GAPDH with DAz-1 and DAz-1-biotin.
GAPDH (11.6 µM) was treated with H2O2 (43 µM) for 10 min and then incubated with DAz-1 (0,
50, 100, 250, 500 or 1000 µM) or DAz-1-biotin (0, 50, 100, 250, 500 or 1000 µM) for 2 h at 37
o
C.
DAz-1 tagged GAPDH was conjugated with p-biotin (300 µM) for 2 h at 37 oC and
analyzed by HRP-streptavidin western blot. (b) Labeling of sulfenic acid-modified GAPDH
11
with DAz-1-biotin challenged by DAz-1.
GAPDH (10 µM) was incubated with H2O2 (50 µM)
for 10 min at rt and incubated with DAz-1 (0, 50, 100, 250, 500 or 1000 µM) for 2 h at 37 oC.
Each sample was then treated with DAz-1-biotin (1 mM) for 2 h for 37 oC.
(c) Labeling of
sulfenic acid-modified proteins in HeLa cell lysate with DAz-1 and DAz-1-biotin.
Cell lysates
were incubated with 1 mM probe (DMSO, DAz-1 or DAz-1-biotin) for 1 h at 37 oC and
conjugated with alkyne-biotin (500 µM) for 1 h at rt.
For comparison of protein labeling 30 µg
protein was loaded for DMSO or DAz-1-treated samples and 3 µg protein for DAz-1-biotintreated samples.
12
Figure S3.
Analysis of cell viability in DMSO, DAz-1 and DAz-1-biotin treated HeLa cells.
Cells were incubated with sulfenic acid probe (10 mM) or DMSO control for 2 h at 37 oC.
After incubation, cell viability was quantified by trypan blue exclusion and compared to cell
viability prior to treatment.
13
References
1. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.
2. Vocadlo, D. J.; Hang, H. C.; Kim, E. J.; Hanover, J. A.; Bertozzi, C. R. Proc. Nat. Acad. Sci.
U.S.A. 2003, 100, 9116.
3. Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004, 126, 15046.
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