Organelle-targetable fluorescent probes for imaging hydrogen peroxide in living cells via SNAP-Tag protein labeling

Article abstract of DOI:10.1021/ja100117u

Hydrogen peroxide (H₂O₂) Is a potent small-molecule oxidant that can exert a diverse array of physiological and/or pathological effects within living systems depending on the timing and location of Its production, accumulation, traff

Full text of DOI:10.1021/ja100117u

Published on Web 03/04/2010
Organelle-Targetable Fluorescent Probes for Imaging
Hydrogen Peroxide in Living Cells via SNAP-Tag Protein
Labeling
Duangkhae Srikun,† Aaron E. Albers,† Christine I. Nam,†,‡ Anthony T. Iavarone,§
and Christopher J. Chang*,†,‡
Department of Chemistry and the Howard Hughes Medical Institute, UniVersity of California,
Berkeley, California 94720, and QB3/Chemistry Mass Spectrometry Facility,
Berkeley, California 94720
Received January 6, 2010; E-mail: chrischang@berkeley.edu
Abstract: Hydrogen peroxide (H2O2) is a potent small-molecule oxidant that can exert a diverse array of
physiological and/or pathological effects within living systems depending on the timing and location of its
production, accumulation, trafficking, and consumption. To help study the chemistry and biology of this
reactive oxygen species (ROS) in its native cellular context, we now present a new method for monitoring
local, subcellular changes in H2O2 levels by fluorescence imaging. Specifically, we have exploited the
versatility of the SNAP-tag technology for site-specific protein labeling with small molecules on the surface
or interior of living cells with the use of boronate-capped dyes to selectively visualize H2O2. The resulting
SNAP-Peroxy-Green (SNAP-PG) probes consist of appropriately derivatized boronates bioconjugated to
SNAP-tag fusion proteins. Spectroscopic measurements of the SNAP-PG constructs confirm their ability
to detect H2O2 with specificity over other biologically relevant ROS. Moreover, these hybrid small-molecule/
protein reporters can be used in live mammalian cells expressing SNAP-tag fusion proteins directed to the
plasma membrane, nucleus, mitochondria, and endoplasmic reticulum. Imaging experiments using scanning
confocal microscopy establish organelle-specific localization of the SNAP-tag probes and their fluorescence
turn-on in response to changes in local H2O2 levels. This work provides a general molecular imaging platform
for assaying H2O2 chemistry in living cells with subcellular resolution.
to neurodegeneration.16-18 However, organisms that live in
aerobic environments can also harness and incorporate ROS
Introduction
Reactive oxygen species (ROS) derived from the metabolism
of oxygen by living organisms are classically known as
indicators for oxidative stress and potential contributors to
aging1-6 and diseases ranging from cancer7-12 to diabetes13-15
chemistry into their normal physiology by means of regulated
production, release, and compartmentalization of these oxygen
metabolites.19-26 A prime example of ROS chemistry used for
beneficial purposes is the oxidative burst within phagosomes
of macrophages and neutrophils that serves as a defense against
invading pathogens.27-29 In this context, a major ROS produced
in living systems is hydrogen peroxide (H2O2), which is a newly
recognized messenger in cellular signal transduction but also a
† Department of Chemistry, University of California, Berkeley.
‡ Howard Hughes Medical Institute, University of California, Berkeley.
§ QB3/Chemistry Mass Spectrometry Facility.
(1) Harman, D. J. Gerontol. 1956, 11, 298–300.
(2) Finkel, T.; Holbrook, N. J. 2000, 408, 239–247.
(3) Stadtman, E. R. Free Radical Res. 2006, 40, 1250–1258.
(4) Droge, W.; Schipper, H. M. Aging Cell 2007, 6, 361–370.
(5) Muller, F. L.; Lustgarten, M. S.; Jang, Y.; Richardson, A.; Van
Remmen, H. Free Radical Biol. Med. 2007, 43, 477–503.
(6) Sharpless, N. E.; Depinho, R. A. Nat. ReV. Mol. Cell Biol. 2007, 8,
703–713.
(15) Pop-Busui, R.; Sima, A.; Stevens, M. Diabetes-Metab. Res. ReV. 2006,
22, 257–273.
(16) Barnham, K. J.; Masters, C. L.; Bush, A. I. Nat. ReV. Drug DiscoV.
2004, 3, 205–214.
(17) Lin, M. T.; Beal, M. F. Nature 2006, 443, 787–795.
(18) DiMauro, S.; Schon, E. A. Annu. ReV. Neurosci. 2008, 31, 91–123.
(19) Rhee, S. G. Science 2006, 312, 1882–1883.
(7) Brandon, M.; Baldi, P.; Wallace, D. C. Oncogene 2006, 25, 4647–
4662.
(20) Stone, J. R.; Yang, S. Antioxid. Redox Signal. 2006, 8, 243–270.
(21) D’AutrEaux, B.; Toledano, M. B. Nat. ReV. Mol. Cell Biol. 2007, 8,
813–824.
(8) Finkel, T.; Serrano, M.; Blasco, M. A. Nature 2007, 448, 767–774.
(9) Fruehauf, J. P.; Meyskens, F. L. Clin. Cancer Res. 2007, 13, 789–
794.
(22) Giorgio, M.; Trinei, M.; Migliaccio, E.; Pelicci, P. G. Nat. ReV. Mol.
Cell Biol. 2007, 8, 722–728.
(10) Ishikawa, K.; Takenaga, K.; Akimoto, M.; Koshikawa, N.; Yamaguchi,
A.; Imanishi, H.; Nakada, K.; Honma, Y.; Hayashi, J. Science 2008,
320, 661–664.
(23) Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T. D.; Mazur, M.;
Telser, J. Int. J. Biochem. Cell Biol. 2007, 39, 44–84.
(24) Miller, E. W.; Chang, C. J. Curr. Opin. Chem. Biol. 2007, 11, 620–
625.
(11) Goetz, M. E.; Luch, A. Cancer Lett. 2008, 266, 73–83.
(12) Rossi, D. J.; Jamieson, C. H. M.; Weissman, I. L. Cell 2008, 132,
681–696.
(25) Veal, E. A.; Day, A. M.; Morgan, B. A. Mol. Cell 2007, 26, 1–14.
(26) Winterbourn, C. C. Nat. Chem. Biol. 2008, 4, 278–286.
(27) Segal, A. W.; Abo, A. Trends Biochem. Sci. 1993, 18, 43–47.
(28) Lambeth, J. D. Nat. ReV. Immunol. 2004, 4, 181–189.
(29) Bedard, K.; Krause, K. H. Physiol. ReV. 2007, 87, 245–313.
(13) Houstis, N.; Rosen, E. D.; Lander, E. S. Nature 2006, 440, 944–948.
(14) Jay, D.; Hitomi, H.; Griendling, K. K. Free Radical Biol. Med. 2006,
40, 183–192.
9
10.1021/ja100117u  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 4455–4465 4455

A R T I C L E S
Srikun et al.
participant in oxidative stress and damage. Whereas high
concentrations of H2O2 can trigger apoptotic or necrotic cell
death, controlled bursts of H2O2 produced in response to
stimulation by various growth factors, cytokines, and neu-
rotransmitters can act as secondary messengers to signal cell
growth, proliferation, and differentiation.19-26,30-39
(HaloTag),53-55 formation of covalent adduct between cutinase
and p-nitrophenyl phosphonate (pNPP) derivatives,56 labeling
of AGT (O6-alkyguanine-DNA alkyltransferase) fusion proteins
with SNAP/CLIP substrates,57-59 and covalent labeling by
enzymes involved in post-translation modifications such as
phosphopantetheine transferase,60,61 transglutaminase,62,63 biotin
ligase BirA,64,65 lipoic acid ligase,66 sortaseA,67,68 protein
farnesyltransferase,69 and formylglycine generating enzyme.70,71
In particular, we were attracted to the SNAP tag technology,
which within a short time after its invention by Johnsson and
co-workers, has found broad utility in a variety of biological
applications. Use of the SNAP tag allows precise targeting of
bioactive reagents or probes to specific subcellular locales; recent
examples include photosensitizers for chomophore-assisted laser
inactivation (CALI) of fusion proteins72 and fluorescent sensors
for intracellular metal ions including zinc(II)73 and calcium(II).74
Simultaneous labeling of two different targets can also be
achieved using tandem SNAP and CLIP tags.59 The SNAP tag
has been used for protein tracking with spatial and temporal
resolution, where a fusion protein at a given time point is initially
marked by a fluorescent or nonfluorescent SNAP tag substrate
label, and subsequent newly synthesized proteins can be
differentiated by a second label.59,75 Other applications of the
SNAP tag technology include its use in combination with
fluorescent proteins as FRET-based reporters for studying
Because variations in the spatial and temporal production of
H2O2 lead to disparate downstream biological effects, new
methods that allow detection of this ROS in living systems with
subcellular resolution can help decipher its complex cellular
chemistry. To this end, an elegant example of a fluorescent
protein sensor based on a circularly permuted YFP with an
OxyR insert has been reported for cellular H2O2 detection.40
We envisioned developing an alternative chemical biology
approach that combines the versatility of small-molecule report-
ers with the targetability of genetically encodable protein
scaffolds for subcellular localization. Currently, selective protein
labeling in living cells with synthetic compounds is feasible in
either a covalent or noncovalent manner.41-45 Noncovalent
labeling methods utilizing the specific binding of ligands to short
peptide or protein domains include the binding of biarsenic
compounds to tetracysteine haiprins (FlAsH, ReAsH),46 the
binding of a metal complex to oligo-His47,48 or oligo-Asp
motifs,49 the recognition of trimethoprim by dihydrofolate
reductase,50,51 and a high affinity binding interaction of protein
FKBP12(F36 V) with its ligand FK-SLF.52 Examples of
covalent self-labeling mediated by fusion enzymes include
attachment of alkyl chlorides to dehalogenase enzymes
(53) Zhang, Y.; So, M. K.; Loening, A. M.; Yao, H. Q.; Gambhir, S. S.;
Rao, J. H. Angew. Chem., Int. Ed. 2006, 45, 4936–4940.
(54) Los, G. V.; et al. ACS Chem. Biol. 2008, 3, 373–382.
(55) Watkins, R. W.; Lavis, L. D.; Kung, V. M.; Los, G. V.; Raines, R. T.
Org. Biomol. Chem. 2009, 7, 3969–3975.
(30) Sundaresan, M.; Yu, Z. X.; Ferrans, V. J.; Irani, K.; Finkel, T. Science
1995, 270, 296–299.
(56) Bonasio, R.; Carman, C. V.; Kim, E.; Sage, P. T.; Love, K. R.;
Mempel, T. R.; Springer, T. A.; von Andrian, U. H. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 14753–14758.
(31) Bae, Y. S.; Kang, S. W.; Seo, M. S.; Baines, I. C.; Tekle, E.; Chock,
P. B.; Rhee, S. G. J. Biol. Chem. 1997, 272, 217–221.
(32) Avshalumov, M. V.; Chen, B. T.; Marshall, S. P.; Pena, D. M.; Rice,
M. E. J. Neurosci. 2003, 23, 2744–2750.
(57) Juillerat, A.; Gronemeyer, T.; Keppler, A.; Gendreizig, S.; Pick, H.;
Vogel, H.; Johnsson, K. Chem. Biol. 2003, 10, 313–317.
(58) Keppler, A.; Kindermann, M.; Gendreizig, S.; Pick, H.; Vogel, H.;
Johnsson, K. Methods 2004, 32, 437–444.
(33) Tonks, N. K. Cell 2005, 121, 667–670.
(34) Poole, L. B.; Nelson, K. J. Curr. Opin. Chem. Biol. 2008, 12, 18–24.
(35) Bao, L.; Avshalumov, M. V.; Patel, J. C.; Lee, C. R.; Miller, E. W.;
Chang, C. J.; Rice, M. E. J. Neurosci. 2009, 29, 9002–9010.
(36) Eligini, S.; Arenaz, I.; Barbieri, S. S.; Faleri, M. L.; Crisci, M.; Tremoli,
E.; Colli, S. Free Radical Biol. Med. 2009, 46, 1428–1436.
(37) Kim, J. S.; Huang, T. Y.; Bokoch, G. M. Mol. Biol. Cell 2009, 20,
2650–2660.
(59) Gautier, A.; Juillerat, A.; Heinis, C.; Correa, I. R.; Kindermann, M.;
Beaufils, F.; Johnsson, K. Chem. Biol. 2008, 15, 128–136.
(60) Yin, J.; Straight, P. D.; McLoughlin, S. M.; Zhou, Z.; Lin, A. J.; Golan,
D. E.; Kelleher, N. L.; Kolter, R.; Walsh, C. T. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 15815–15820.
(61) Zhou, Z.; Cironi, P.; Lin, A. J.; Xu, Y. Q.; Hrvatin, S.; Golan, D. E.;
Silver, P. A.; Walsh, C. T.; Yin, J. ACS Chem. Biol. 2007, 2, 337–
346.
(38) Leonard, S. E.; Reddie, K. G.; Carroll, K. S. ACS Chem. Biol. 2009,
4, 783–799.
(39) Niethammer, P.; Grabher, C.; Look, A. T.; Mitchison, T. J. Nature
2009, 459, 996–999.
(62) Lorand, L.; Graham, R. M. Nat. ReV. Mol. Cell Biol. 2003, 4, 140–
156.
(40) Belousov, V. V.; Fradkov, A. F.; Lukyanov, K. A.; Staroverov, D. B.;
Shakhbazov, K. S.; Terskikh, A. V.; Lukyanov, S. Nat. Methods 2006,
3, 281–286.
(63) Lin, C. W.; Ting, A. Y. J. Am. Chem. Soc. 2006, 128, 4542–4543.
(64) Chen, I.; Howarth, M.; Lin, W. Y.; Ting, A. Y. Nat. Methods 2005,
2, 99–104.
(41) Chen, I.; Ting, A. Y. Curr. Opin. Biotechnol. 2005, 16, 35–40.
(42) Gronemeyer, T.; Godin, G.; Johnsson, K. Curr. Opin. Biotechnol. 2005,
16, 453–458.
(65) Howarth, M.; Takao, K.; Hayashi, Y.; Ting, A. Y. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 7583–7588.
(66) Fernandez-Suarez, M.; Baruah, H.; Martinez-Hernandez, L.; Xie, K. T.;
Baskin, J. M.; Bertozzi, C. R.; Ting, A. Y. Nat. Biotechnol. 2007, 25,
1483–1487.
(43) Miller, L. W.; Cornish, V. W. Curr. Opin. Chem. Biol. 2005, 9, 56–
61.
(44) Chattopadhaya, S.; Abu Bakar, F. B.; Yao, S. Q. Curr. Med. Chem.
2009, 16, 4527–4543.
(67) Popp, M. W.; Antos, J. M.; Grotenbreg, G. M.; Spooner, E.; Ploegh,
H. L. Nat. Chem. Biol. 2007, 3, 707–708.
(45) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974–
6998.
(68) Tanaka, T.; Yamamoto, T.; Tsukiji, S.; Nagamune, T. ChemBioChem
2008, 9, 802–807.
(46) Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269–
272.
(69) Duckworth, B. P.; Zhang, Z. Y.; Hosokawa, A.; Distefano, M. D.
ChemBioChem 2007, 8, 98–105.
(47) Guignet, E. G.; Hovius, R.; Vogel, H. Nat. Biotechnol. 2004, 22, 440–
444.
(70) Carrico, I. S.; Carlson, B. L.; Bertozzi, C. R. Nat. Chem. Biol. 2007,
3, 321–322.
(48) Hauser, C. T.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
3693–3697.
(71) Wu, P.; Shui, W. Q.; Carlson, B. L.; Hu, N.; Rabuka, D.; Lee, J.;
Bertozzi, C. R. Proc. Nat. Acad. Sci. U. S. A. 2009, 106, 3000–3005.
(72) Keppler, A.; Ellenberg, J. ACS Chem. Biol. 2009, 4, 127–138.
(73) Tomat, E.; Nolan, E. M.; Jaworski, J.; Lippard, S. J. J. Am. Chem.
Soc. 2008, 130, 15776–15777.
(49) Ojida, A.; Honda, K.; Shinmi, D.; Kiyonaka, S.; Mori, Y.; Hamachi,
I. J. Am. Chem. Soc. 2006, 128, 10452–10459.
(50) Calloway, N. T.; Choob, M.; Sanz, A.; Sheetz, M. P.; Miller, L. W.;
Cornish, V. W. ChemBioChem 2007, 8, 767–774.
(51) Gallagher, S. S.; Sable, J. E.; Sheetz, M. P.; Cornish, V. W. ACS Chem.
Biol. 2009, 4, 547–556.
(74) Bannwarth, M.; Correa, I. R.; Sztretye, M.; Pouvreau, S.; Fellay, C.;
Aebischer, A.; Royer, L.; Rios, E.; Johnsson, K. ACS Chem. Biol.
2009, 4, 179–190.
(52) Marks, K. M.; Braun, P. D.; Nolan, G. P. Proc. Natl. Acad. Sci. U. S. A.
2004, 101, 9982–9987.
(75) Farr, G. A.; Hull, M.; Mellman, I.; Caplan, M. J. J. Cell. Biol. 2009,
186, 269–282.
9
4456 J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010

Imaging Hydrogen Peroxide in Living Cells
A R T I C L E S
Figure 1. Design strategy for organelle-specific hydrogen peroxide reporters using the SNAP tag methodology.
Scheme 1. Synthesis of SNAP-Tag Substrates
protein-protein interactions76 and the development of semi-
synthetic fluorescent sensors or photoactivable switches.77,78
In this report, we present the design, synthesis, properties,
and cellular applications of a new class of organelle-targetable
fluorescent probes for H2O2 based on SNAP-AGT bioconjuga-
tion chemistry. We have prepared and characterized a pair of
boronate-caged, Peroxy Green-type fluorescent peroxide indica-
tors bearing the SNAP substrates, where one is a membrane-
permeable version for intracellular use and another is a
membrane-impermeable version for cell surface labeling, and
coupled them to AGT proteins expressed in various cellular
compartments. The resulting SNAP-Peroxy Green (SNAP-PG)
hybrid small-molecule/protein conjugates are capable of detect-
ing H2O2 over a variety of ROS and can be targeted to various
subcellular locales, including the plasma membrane, nucleus,
mitochondrial inner membrane, and endoplasmic reticulum, to
sense changes in H2O2 levels within or on the surface of living
cells. This method provides a general platform for studying the
chemistry of H2O2 by molecular imaging in living biological
specimens with subcellular resolution.
of AGT with proteins containing a signaling sequence allows
expression of AGT in defined subcellular compartments. These
localized AGT scaffolds can then be tagged by covalent labeling
at Cys145 with a boronate PG fluorescent probe bearing an
appended SNAP substrate. The resulting SNAP-PG conjugates
are hybrid small-molecule/protein reporters that give a fluores-
cent turn-on response upon H2O2-mediated boronate cleavage.
To this end, we previously reported the synthesis and
application of Peroxy Green 1 (PG1),87 a cell-permeable and
selective H2O2 probe that can be activated by a single boronate
deprotection. Along these lines, we sought to create PG1 analogs
for organelle-specific labeling by replacing its 4-methoxy moiety
with benzylguanine or benzyl-2-chloro-6-aminopyrimidine SNAP
tag substrates. Scheme 1 depicts the synthesis of SNAP tag
derivatives for coupling to a PG1 scaffold. Benzylguanine 4
was synthesized according to literature protocols.58 In a similar
manner, benzyl-2-chloro-6-aminopyrimide 3 was synthesized
by nucleophilic substitution of 2,6-dichloropyrimidin-4-amine
with trifluoro-N-(4-hydroxymethyl-benzyl)-acetamide 1. The
trifluoroacetamide protecting group was subsequently removed
with 33% methylamine in ethanol.
Results and Discussion
Design and Synthesis of SNAP-Peroxy-Green Bioconjugates
as Organelle-Targetable Fluorescent Reporters for Cellular Hy-
drogen Peroxide. Figure 1 depicts our overall strategy for creating
subcellular-targetable fluorescent H2O2 probes by combining the
SNAP-tag methodology for site-specific labeling79,80 with the
chemoselective H2O2-mediated deprotection of boronate esters
to phenols for reaction-based detection of H2O2.81-86 Fusion
(76) Maurel, D.; Comps-Agrar, L.; Brock, C.; Rives, M. L.; Bourrier, E.;
Ayoub, M. A.; Bazin, H.; Tinel, N.; Durroux, T.; Prezeau, L.; Trinquet,
E.; Pin, J. P. Nat. Methods 2008, 5, 561–567.
Scheme 2 details the synthesis of SNAP-Peroxy Green-1
(SPG1) and SNAP-Peroxy Green-2 (SPG2) probes by coupling
the aforementioned SNAP tag substrates with a carboxylate-
derivatized PG1; the latter is prepared in five steps from the
fluorescein-like dye Tokyo Green (TG).88 Treatment of TG 5
with boron tribromide at -78 °C in dry CH2Cl2 furnishes the
phenolic TG 6. O-alkylation of 6 in anhydrous DMF with excess
(77) Mao, S.; Benninger, R. K. P.; Yan, Y. L.; Petchprayoon, C.; Jackson,
D.; Easley, C. J.; Piston, D. W.; Marriott, G. Biophys. J. 2008, 94,
4515–4524.
(78) Brun, M. A.; Tan, K. T.; Nakata, E.; Hinner, M. J.; Johnsson, K. J. Am.
Chem. Soc. 2009, 131, 5873–5884.
(79) Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.;
Johnsson, K. Nat. Biotechnol. 2003, 21, 86–89.
(80) Keppler, A.; Pick, H.; Arrivoli, C.; Vogel, H.; Johnsson, K. Proc.
Nat. Acad. Sci. U. S. A. 2004, 101, 9955–9959.
(81) Chang, M. C. Y.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem.
Soc. 2004, 126, 15392–15393.
(85) Srikun, D.; Miller, E. W.; Domaille, D. W.; Chang, C. J. J. Am. Chem.
Soc. 2008, 130, 4596–4597.
(82) Miller, E. W.; Albers, A. E.; Pralle, A.; Isacoff, E. Y.; Chang, C. J.
J. Am. Chem. Soc. 2005, 127, 16652–16659.
(86) Dickinson, B. C.; Chang, C. J. J. Am. Chem. Soc. 2008, 130, 11561–
11562.
(83) Albers, A. E.; Okreglak, V. S.; Chang, C. J. J. Am. Chem. Soc. 2006,
128, 9640–9641.
(87) Miller, E. W.; Tulyathan, O.; Isacoff, E. Y.; Chang, C. J. Nat. Chem.
Biol. 2007, 3, 263–267.
(84) Albers, A. E.; Dickinson, B. C.; Miller, E. W.; Chang, C. J. Bioorg.
Med. Chem. Lett. 2008, 18, 5948–5950.
(88) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano, T.
J. Am. Chem. Soc. 2005, 127, 4888–4894.
9
J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010 4457

A R T I C L E S
Srikun et al.
Scheme 2. Synthesis of SPG1 and SPG2
Scheme 3. Synthesis of STG1 and STG2
cesium carbonate and a slow addition of tert-butylbromoacetate
gives tert-butyl ester TG 7. Reaction of 7 with bis(trifluo-
romethane)sulfonamide affords triflate 8. Palladium-mediated
borylation of triflate 8 with bis(pinacolato)diboron, cyclohexyl
JohnPhos, and diisopropylethylamine in anhydrous 1,4-dioxane
provides boronic ester 9. Subsequent treatment with trifluoro-
acetic acid in anhydrous CH2Cl2 delivers carboxylate PG 10.
Amide bond formation between the carboxyl PG 10 and the
SNAP substrate 4 or 3 mediated by N,N,N′,N’-tetramethyl-O-
(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU)
affords SPG1 (11) or SPG2 (12), respectively.
coupling of 13 with either 4 or 3 provides SNAP-TG-1 (STG1,
14) and SNAP-TG-2 (STG2, 15), respectively.
Spectroscopic Properties, In Vitro Protein AGT Labeling,
and Peroxide Responses of SNAP-PG and SNAP-TG Fluoro-
phores. Table 1 summarizes the spectroscopic properties of the
SNAP-PG and SNAP-TG fluorophores in aqueous buffer at
neutral pH (20 mM HEPES, pH 7). SPG1 and SPG2 both show
visible absorption bands centered at 465 nm (SPG1: ε ) 10 200
M-1 cm-1, SPG2: ε ) 9800 M-1 cm-1) and weak fluorescence
with an emission maximum at 515 nm. In contrast, the STG1
and STG2 fluorophores exhibit a prominent absorption at 495
nm (STG1: ε ) 36 000 M-1 cm-1, STG2: ε ) 37 000 M-1
cm-1) and fluorescent emission maximum at 513 nm. The
quantum yield of STG1 (Φ ) 0.12) is much lower than that of
STG2 (Φ ) 0.87), presumably due to the greater quenching
Finally, we also synthesized TG analogs bearing SNAP tags
3 or 4 as control compounds for cell labeling and imaging as
shown in Scheme 3. Treatment of tert-butyl ester TG 7 with
TFA furnishes carboxyl TG 13. The HATU-mediated amide
9
4458 J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010

Imaging Hydrogen Peroxide in Living Cells
A R T I C L E S
Table 1. Spectroscopic Properties of SNAP Dyes and Their AGT
Bioconjugates
compound
λexc (nm)
(M-1 cm-1
)
λem (nm)
Φ
STG1
STG2
AGT-TG
SPG1
SPG2
495
495
500
465
465
465
36000
37000
32000
10200
9800
513
513
515
515
515
515
0.12
0.87
0.57
0.10
0.09
0.07
AGT-PG
11500
effect of guanidine moiety; a similar effect has been observed
in other SNAP fluorophores.73 We thus proceeded to test the
H2O2 reactivity of these free dyes with the SPG2 analog.
Reaction of SPG2 with 1 mM H2O2 triggers a ca. 32-fold
fluorescence turn-on for the dye (Figure S11) confirming the
peroxide response of these SNAP tag reporters. Kinetics
measurements of the SPG2 boronate deprotection performed
under pseudo-first-order conditions (1 µM dye, 1 mM H2O2)
Figure 2. Fluorescence responses of 1 µM AGT-PG, a conjugate formed
from the reaction of AGT with SPG2, to 100, 250, 500, and 1000 µM H2O2
in 20 mM HEPES, pH 7 at 25 °C. The plot shows emission responses at 0,
10, 20, 30, 40, 50, 60, and 120 min after H2O2 addition. The reactions are
not complete at these early time points. The collected emission was
integrated between 500-650 nm (λexc ) 488 nm).
give an observed rate constant of kobs ) 1.0(1) × 10-3 s-1
.
We next proceeded to test the ability of the SPG and STG
dyes to label purified AGT protein samples and to evaluate the
spectroscopic properties and peroxide responses of the isolated
fluorophore-protein conjugates. For these in vitro protein
labeling assays, hexahistidine-tagged AGT (His-AGT) was
overexpressed in E. coli and purified with a standard Ni-NTA
affinity column. After incubation of His-tagged AGT with SPG1,
SPG2, STG1, or STG2 dyes in 20 mM HEPES at pH 7 for 30
min at 37 °C, we confirmed the formation of the desired
covalently labeled products, AGT-PG and AGT-TG, by both
LC-MS and 488 nm excitation in-gel fluorescence measurements
(Supporting Information, Figures S1 to S7). Specifically, AGT-
TG and AGT-PG show mass increases of 478 and 506 Da,
respectively, from His-tagged AGT; these values are consistent
with the calculated mass of the protein tagged with TG or the
boronic acid form of PG, respectively. We purified the AGT-
PG and AGT-TG bioconjugates for further spectroscopic
evaluation using MWCO spin columns and size-exclusive gel
chromatography to remove the free organic fluorophores. This
dual purification method is essential to obtain clean dye-protein
conjugates without interference from small-molecule impurities.
In addition, the labeling proceeds well under oxidizing condi-
tions, as we observe covalent adduct formation in the presence
up to 1 mM H2O2 (Figure S2). AGT-TG possesses a dominant
absorption maximum in the visible region centered at 500 nm,
which is slightly red-shifted relative the free TG dye. The AGT-
TG complex shows prominent absorption at 280 (standard
protein absorption) and 500 nm (TG dye), consistent with
labeling of the AGT protein with the small-molecule TG payload
(Figure S8). Furthermore, formation of AGT-TG conjugate is
also reflected in the observed fluorescence quantum yield, as
binding of STG1 to AGT ejects guanidine from the dye and
eliminates its quenching effect, resulting in a 6-fold fluorescence
increase (Figure S9). The absorption and emission spectra of
AGT-PG are similar to SPG1 and SPG2, with an additional
280 nm absorption due to the AGT protein.
Figure 3. Absorption spectra of 1 µM AGT-PG in 20 mM HEPES, pH 7
upon addition of 1 mM H2O2. Spectra were acquired at 1 h intervals for
8 h. An isosbestic point was observed at 460 nm.
Figure 4. Fluorescence responses of 1 µM AGT-PG to various concentra-
tions of added H2O2. Spectra were acquired in 20 mM HEPES, pH 7 at 25
°C after incubation of the probe with H2O2 for 30 min. Reactions are not
complete at this early time point. The collected emission was integrated
between 500-650 nm (λexc ) 488 nm). Error bars represent standard
deviation.
Importantly, the SPG dyes are able to retain their fluorescence
response to H2O2 upon protein labeling, as purified AGT-PG
conjugates exhibit a marked turn-on emission response to added
H2O2 (Figure 2). The H2O2-induced fluorescence increases occur
with concomitant growth of the characteristic TG absorption at
500 nm, consistent with boronate cleavage of PG to produce
the phenol TG product (Figure 3). Finally, we observe a linear
correlation between various H2O2 concentrations added and
observed fluorescence emission responses (Figure 4).
Fluorescence Detection of H2O2 in Various Compartments
of Living Cells with SNAP-AGT PG Reporters. With spectro-
scopic data establishing the formation of AGT-PG conjugates
and their fluorescence turn-on response to H2O2, we next sought
to apply SPG1 and SPG2 for optical detection of H2O2 in living
biological samples. Specifically, we focused on directing these
9
J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010 4459

A R T I C L E S
Srikun et al.
probes to a broad range of subcellular compartments that are
either capable of locally generating H2O2 in response to various
stimuli or to sites where their function and regulation are
sensitive to H2O2 flux. In this study, we targeted SPG reporters
to a broad spectrum of locations. Included are the plasma
membrane, mitochondria, endoplasmic reticulum, and nucleus.
To this end, plasmids for expression of the SNAP-tag in live
mammalian cells were obtained from Covalys and then subjected
to further modification as necessary. The parent SNAP-tag
without a signaling sequence (pSNAP), encoding a genetically
optimized AGT protein, gives a uniform intracellular expression
of SNAP-tag in the cytoplasm and the nucleus. Localization of
the SNAP-tag to nucleus is achieved by fusion of SNAP-tag to
the C-terminus of the histone H2B protein (SNAP-H2B). Fusion
of the SNAP-tag to the C-terminus of cytochrome c oxidase
subunit 8 (Cox8A) enables its localization to the mitochondrial
inner membrane (SNAP-Cox8A). For plasma membrane target-
ing, the SNAP-tag is expressed as a fusion to the N-terminus
of neurokinin-1 receptor (NK1R) and the C-terminus of 5HT3A
serotonin receptor signaling sequence, resulting in a SNAP-tag
exposed to the extracellular space (SNAP-NK1R). Addition of
KDEL, a signaling peptide for retention of the protein in
endoplasmic reticulum (ER), to the C-terminus of SNAP results
in an ER localized SNAP-tag (SNAP-KDEL).
We first tested the site-specific labeling of various SNAP
fusion proteins with control STG1 and STG2 dyes in live
HEK293T cells. To our surprise, we found that STG1 and STG2
exhibit marked differences in cell permeability that can be
exploited to obtain more selective conjugation to extracellular
and intracellular spaces. Specifically, whereas both STG1 and
STG2 gave excellent membrane labeling with SNAP-NK1R,
intracellular tagging of pSNAP and SNAP-H2B was observed
only with STG2 (Figure 5). The same trend applies to SPG1
and SPG2, where only the latter is membrane permeable. Our
findings are consistent with previous work that reports that
benzylguanine can hamper the cell permeability of its conjugated
partners in a fluorophore-dependent manner.58,89 STG2 shows
desirable properties for intracellular site-specific labeling ap-
plications, as the dye is cell permeable and the unbound probe
can be readily washed out. This latter property is crucial to
minimizing background fluorescence from nonspecific or off-
target labeling. Indeed, in HEK293T cells transiently expressing
SNAP-KDEL and SNAP-Cox8A to target the endoplasmic
reticulum and mitochondria, respectively, we were able to
successfully tag these organelles with STG2. The subcellular
localization of this dye to these compartments was confirmed
by fluorescence overlay of green emissive STG2 with red
emission from mCherry-KDEL and mCherry-Cox8A constructs
(Figure 6).
Recognizing these SNAP-tag-dependent differences in mem-
brane permeability, we then proceeded to evaluate the boronate
SPG1 and SPG2 reporters for site-specific subcellular H2O2
detection, employing SPG1 for extracellular plasma membrane
targeting and SPG2 for intracellular labeling. We utilized real-
time imaging of the same cells before and after oxidative
stimulation or in untreated samples. This method allows us to
perform an important control for varying levels of protein
expression as well as a control to show that photodynamic
oxidation is not significant with these systems. For extracellular
measurements, HEK293T cells transiently expressing SNAP-
Figure 5. Targeted localization of STG1 and STG2 in living HEK293T
cells by conjugation to SNAP-AGT fusion proteins. Cells were incubated
with 5 µM STG1 or STG2 for 30 min, and washed with fresh DMEM +
10% FBS for 30 min (2 × 1 mL) before image acquisition. Rows (A) and
(B) show HEK 293T cells transiently expressing SNAP-NK1R. Rows (C)
and (D) display HEK 293T cells transiently expressing pSNAP for
nonspecific intracellular tagging, and row (E) presents HEK 293T cells
transiently expressing SNAP-H2B. For each series: (1) emission from
labeling with STG1 or STG2, (2) nuclear staining with Hoechst 33342,
and (3) brightfield image. Scale bar ) 20 µm.
NK1R display weak but detectable green fluorescent emission
localized to the plasma membrane after SPG1 conjugation. The
relatively poor membrane permeability of this probe allows for
rapid and selective labeling of cell surfaces with washing.
Treatment of SPG1-labeled cells with 100 µM H2O2 triggers a
marked increase in fluorescence from the plasma membrane
region as shown by time lapse image acquisitions (Figure 7a).
Importantly, negligible emission comes from nontransfected
cells in the same image frame even after treatment with H2O2,
establishing that specific SPG1 binding to the SNAP-NK1R
fusion protein scaffold is required for imaging changes in H2O2
levels localized to the plasma membrane. As a further control,
we do not observe fluorescence turn-on responses for SPG1-
loaded cells in the absence of H2O2, and a combination of
nuclear staining and brightfield transmission measurements
confirm that the cells are viable throughout the imaging
experiments.
To label intracellular compartments, we subjected cells to 5
µM SPG2 for 30 min followed by a 30 min incubation in fresh
complete medium (DMEM + 10% FBS) to wash out any
unbound probe. HEK293T cells transiently expressing pSNAP
display weak intracellular fluorescent emission after labeling
with SPG2. Addition of 100 µM H2O2 to the cells results in a
patent increase in overall intracellular fluorescence from this
(89) Keppler, A.; Arrivoli, C.; Sironi, L.; Ellenberg, J. Biotechniques 2006,
41, 167–175.
9
4460 J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010

Imaging Hydrogen Peroxide in Living Cells
A R T I C L E S
Figure 6. Targeted labeling of endoplasmic reticulum and mitochondria organelles with STG2. Row (A) shows HEK 293T cells expressing SNAP-KDEL
in the ER lumen, and row (B) depicts HEK 293T cells expressing SNAP-Cox8A for mitochondrial tagging. For each series: (1) emission from labeling with
STG2, (2) emission from (a) mCherry-KDEL or (b) mCherry-Cox8A, (3) overlay of STG2 and mCherrry, (4) nuclear staining with Hoechst 33342, and (5)
brightfield image. Scale bar ) 20 µm.
Figure 7. Fluorescence detection of H2O2 on the surface of or within living HEK 293T cells. Row (A) shows cells transiently expressing SNAP-NK1R
tagged with SPG1, row (B) displays cells transiently expressing pSNAP tagged with SPG2, and row (C) presents cells transiently expressing SNAP-H2B
tagged with SPG2. H2O2 was added from 50 mM solution in Mili-Q water. Time-lapse image acquisition was assisted by a motorized stage equipped with
incubator and humidifier maintaining 37 °C and 5% CO2 atmosphere. For each series: (1-4) pseudocolor mode of fluorescent emission at 0, 10, 20, and 30
min after addition of 100 µM H2O2, (5, 6) fluorescent emission before (5) and after (6) treatment of cells with 100 µM H2O2 for 30 min (λexc ) 488 nm, λem
) 500-550 nm), (7) nuclear staining with Hoechst 33342, (8) brightfield image. Scale bar ) 20 µm.
Figure 8. Fluorescence detection of H2O2 in living HEK 293T cells transiently expressing SNAP-KDEL and mCherry-KDEL. Panels (A) and (B) show
fluorescent emission from cells labeled with SPG2 before (A) and after (B) treatment with 100 µM H2O2 for 30 min (λexc ) 488 nm, λem ) 500-550 nm),
panel (C) presents emission from mCherry-KDEL, panel (D) displays the overlay of (B) and (C), panel (E) shows nuclear staining with Hoechst 33342, and
panels (F-I) display pseudocolor fluorescent emission at 0, 10, 20, and 30 min after addition of 100 µM H2O2. Panel (J) presents the brightfield image of
cells in (I). Scale bar ) 20 µm.
protein/small-molecule reporter (Figure 7b). Likewise, SPG2-
loaded HEK293T cells transiently expressing SNAP-H2B show
nuclear-localized fluorescence that becomes more intense after
stimulation with H2O2 (Figure 7c). Moreover, SPG2 can be
precisely immobilized onto the mitochondrial inner membrane
and ER regions of HEK293T cells transiently expressing SNAP-
Cox8A (Figure 8) and SNAP-KDEL (Figure 9), respectively.
In both cases, the turn-on green fluorescence emission from the
SNAP-PG probes after H2O2 treatment display good colocal-
ization with the red mCherry emission of the corresponding
SNAP protein target, and the staining pattern can persist for
many hours. Because the mitochondrial and ER structures are
9
J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010 4461

A R T I C L E S
Srikun et al.
Figure 9. Fluorescence detection of H2O2 in living HEK 293T cells transiently expressing SNAP-Cox8A and mCherry-Cox8A. Panels (A) and (B) show
fluorescent emission from cells labeled with SPG2 before (A) and after (B) treatment with 100 µM H2O2 for 30 min (λexc ) 488 nm, λem ) 500-550 nm),
panel (C) presents emission from mCherry-Cox8A, panel (D) displays the overlay of (B) and (C), panel (E) shows nuclear staining with Hoechst 33342, and
panels (F-I) display pseudocolor fluorescent emission at 0, 10, 20, and 30 min after addition of 100 µM H2O2. Panel (J) presents the brightfield image of
cells in (I). Scale bar ) 20 µm.
presented here combines the precision of site-specific labeling
using genetically encodable tags with the chemical versatility
of small-molecule probes. Ongoing efforts are focused on
expanding the expanding color palette of targetable H2O2 probes,
optimizing their sensitivities and dynamic turn-on responses to
H2O2, creating targetable reporters with ratiometric readouts,
and developing additional labeling methodologies for multi-
channel imaging. We anticipate that simultaneous measurements
of H2O2 and other ROS fluxes in multiple locales using hybrid
small-molecule/protein conjugates will contribute to a better
understanding of oxidation biology in states of health, aging,
and disease, with particular interest in elucidating cellular redox
regulation and signaling in multiple cellular compartments.
Figure 10. Relative fluorescence emission from time-lapse confocal images
of SPG1 or SPG2 labeled HEK 293T cells in response to added 100 µM
Experimental Section
Materials and Methods. All reactions were carried out under a
dry nitrogen atmosphere. Silica gel 60 (230-400 mesh, Silicycle)
was used for column chromatography. Palladium(II) acetate
H2O2 at 0, 10, 20, 30 min. Error bars represent standard error of the mean
of five experiments.
[Pd(OAc)2],
dichloro[1,1′-bis(diphenylphosphino)ferrocene]-
relatively small compared to the overall volume of the cell,
fluorescence emission from these organelles could be easily
overwhelmed by noise from any nonspecific staining in cyto-
plasm. These experiments clearly demonstrate that nonspecific
binding is not an issue with the SPG2 probe, as unbound dye
can be effectively removed from cells with a simple washing
step. Taken together, these data show that SPG1 and SPG2 can
be used to label and sense changes in H2O2 levels in specific
subcellular compartments via selective bioconjugation with a
variety of localizable SNAP-tag fusion constructs (Figure 10).
palladium(II) [Pd(dppf)Cl2], and 2-(dicyclohexylphosphino)biphenyl
[cyclohexyl JohnPhos] were purchased from Strem Chemicals
(Newburyport, MA). Bis(pinacolato)diboron was purchased from
Boron Molecular (Research Triangle Park, NC). 2,2,2-Trifluoro-
N-(4hydroxylmethyl-benzyl)-acetamide (1) and O6-(4-Amino-meth-
yl-benzyl)guanine (4) were synthesized following literature proce-
dures.58 All other chemicals were purchased from Sigma-Aldrich
(St. Louis, MO) and were used as received. 1H NMR and 13C NMR
spectra were collected in CDCl3, DMSO-d6, or CD3OD (Cambridge
Isotope Laboratories, Cambridge, MA) at 25 °C using a Bruker
AVQ-400 spectrometer at the College of Chemistry NMR Facility
at the University of California, Berkeley. All chemical shifts are
reported in the standard δ notation of parts per million. Low-
resolution mass spectral analyses were carried out using GC-MS
(Agilent Technology 5975C, inert MSD with triple axis detector)
or LC-MS (Agilent Technology 6130, Quadrupole LC/MS). High-
resolution mass spectral analyses (ESI-MS, FAB-MS) were carried
out at the College of Chemistry Mass Spectrometry Facility at the
University of California, Berkeley.
Restiction endonucleases and T4 DNA ligases were purchased
from New England Biolabs (Ipswich, MA). Hoechst 33342,
monoclonal ANTI-FLAG M2, antibody and Alexa Fluor 488 Goat
antimouse lgG antibody were obtained from Invitrogen (Carlsbad,
CA).pCEMS-CLIP10m-NK1R,pSEMS-SNAP26m-ADBR,pCEMS1-
H2B-CLIP10m, and pSEMS1-Cox8A-SNAP26m plasmids were
purchased from Covalys Biosciences (Witterswil, Switzerland).
pmCherry and DsRed Golgi were obtained from Clontech Labo-
ratories (Mountain View, CA). pET28a(+) was obtained from
Concluding Remarks
In summary, we have described the synthesis, spectroscopic
properties, H2O2 responses, and live-cell imaging applications
of a new class of organelle-targeted reporters for cellular H2O2.
SPG1 and SPG2 combine boronate-caged Tokyo Green deriva-
tives for H2O2 detection with SNAP tags for site-specific protein
labeling at extracellular and intracellular locations, respectively.
When SPG1 and SPG2 are exposed to AGT or an AGT fusion
protein, these dyes form covalent AGT-PG adducts that respond
to H2O2 by a turn-on fluorescence increase upon H2O2-mediated
boronate deprotection. These hybrid small-molecule/protein
reporters can be used to label a variety of subcellular locations,
including the plasma membrane, nucleus, mitochondria, and
endoplasmic reticulum, and detect changes in local H2O2 fluxes
in living cells by confocal microscopy. The methodology
9
4462 J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010

Imaging Hydrogen Peroxide in Living Cells
A R T I C L E S
Novagen (EMD Chemicals, Gibbstown, NJ). Primers for PCR
amplification were synthesized by Intergrated DNA Technologies
(San Diego, CA).
dissolved in anhydrous DMF (3 mL) and added dropwise to the
stirring basic solution over 5 min. The mixture was stirred at room
temperature overnight, diluted in toluene (200 mL), washed with
H2O (3 × 100 mL), and dried over MgSO4. Purification by flash
column chromatography (silica gel, EtOAc:hexanes 1:4 to 1:2
gradient) gave 8 as a puffy, orange crystalline solid (930 mg, 67%
N-(4-((2-Amino-6-chloropyrimidin-4-yloxy)methyl)benzyl)-2,2,2-
trifluoroacetamide (2). Compound 1 (1.0 g, 4.3 mmol) in anhydrous
DMF (7 mL) was added to a dry 50 mL Schlenk flask under a
nitrogen atmosphere. NaH (60% in mineral oil, 515 mg, 12.8 mmol)
was then added slowly to the reaction flask. After stirring at room
temperature for 15 min, the solution appeared blue-green in color.
A solution of 4,6-dichloropyrimidin-4-amine (700 mg, 4.3 mmol)
in anhydrous DMF (5 mL) was added dropwise to the reaction flask
over a 5 min period, giving a brown-colored solution. The reaction
was then heated to 90 °C for 6 h. The reaction was cooled to room
temperature, quenched with slow addition of cold water (20 mL),
extracted with EtOAc (3 × 30 mL), and dried over Na2SO4.
Purification by column chromatography (silica gel, EtOAc:hexanes
1
yield). H NMR (CDCl3, 400 MHz): δ 7.39 (1H, d, J ) 2.4 Hz),
7.18 (1H, d, J ) 8.8 Hz), 7.09 (2H, d, J ) 8.8, 2.4 Hz), 7.01 (1H,
d, J ) 10.0 Hz), 6.95 (1H, d, J ) 2.4 Hz), 6.91 (1H, dd, J ) 10,
2.4 Hz), 6.60 (1H, dd, J ) 10.0 2.0 Hz), 6.44 (1H, d, J ) 2.0 Hz),
4.61 (2H, s), 2.05 (3H, s), 1.54 (9H, s). LC-MS: calculated for
[MH+] 565, found 565.
tert-Butyl-2-(3-methyl-4-(3-oxo-6-(4,4,5,5-tetramethyl-1,3,2-diox-
aborolan-2-yl)-3H-xanthen-9-yl)phenoxy)acetate (9). Compound 8
(641 mg, 1.14 mmol), Pd(OAc)2 (84 mg, 0.12 mmol), cyclohexyl
JohnPhos (201 mg, 0.57 mmol), and bis(pinacolato)diboron (446
mg, 1.76 mmol) were added to a dried 25 mL scintillation vial in
a glovebox. Anhydrous 1,4-dioxane (10 mL) was added and the
solution stirred at room temperature for 5 min. Anhydrous DIPEA
(742 mg, 1.00 mL, 5.74 mmol) was added dropwise by syringe
and the reaction mixture was stirred at room temperature overnight.
The reaction vial was removed from the glovebox, poured into
saturated aqueous NH4Cl (30 mL), extracted with EtOAc (3 × 30
mL), and dried over MgSO4. Purification by flash column chro-
matography (silica gel, EtOAc:hexanes 1:4) delivered 9 as an orange
crystalline solid (615 mg, 99% yield). 1H NMR (CDCl3, 400 MHz):
δ 7.90 (1H, s), 7.57 (1H, d, J ) 8.0 Hz), 7.08 (1H, J ) 8.4 Hz),
7.07 (1H, d, J ) 8.0 Hz), 7.01 (1H, d, J ) 9.6 Hz), 6.94 (1H, d,
J ) 2.4 Hz), 6.89 (1H, dd, J ) 8.4, 2.4 Hz), 6.59 (1H, dd, J ) 9.6,
2.0 Hz) 6.43 (1H, d, J ) 2.0 Hz), 4.61 (2H, s), 2.03 (3H, s), 1.54
(9H, s), 1.37 (12H, s). LC-MS: calculated for [MH+] 543, found
543.
2-(3-Methyl-4-(3-oxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)-3H-xanthen-9-yl)phenoxy)acetic acid (10). tert-Butyl PG 9
(542 mg, 1.0 mmol) was dissolved in CH2Cl2 (3 mL) and added
dropwise by Pasteur pipet to a stirring mixture of CH2Cl2:TFA (1.0:
1.0, 25 mL). The solution was allowed to stir for 4 h and the solvent
removed under reduced pressure. Purification by flash column
chromatography (silica gel, MeOH:CH2Cl2 1:10) gave 10 as an
orange film (419 mg, 86% yield). 1H NMR (CDCl3, 400 MHz): δ
8.23 (1H, s), 7.82 (1H, d, J ) 8.0 Hz), 7.41 (1H, d, J ) 8.0 Hz),
7.40 (1H, d, J ) 8.8 Hz) 7.15 (3H, m), 7.04 (1H, s), 7.00 (1H, d,
J ) 8.8 Hz), 4.83 (2H, s), 2.01 (3H, s), 1.39 (12H, s). LC-MS:
calculated for [MH+] 487, found 487.
N-(4-((2-Amino-9H-purin-6-yloxy)methyl)benzyl)-2-(3-methyl-
4-(3-oxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-xanthen-
9-yl)phenoxy)acetamide, SPG1 (11). Compound 10 (99 mg, 0.20
mmol) was added to a dried 25 mL Schlenk flask. Anhydrous DMF
(3 mL) was added by syringe and the solution was stirred at room
temperature for 5 min. HATU (89 mg, 0.23 mmol), 6-(4-
(aminomethyl)benzyloxy)-9H-purin-2-amine 4 (63 mg, 0.23 mmol),
and DIPEA (266 mg, 0.359 mL, 2.06 mmol) were added, and the
reaction stirred at room temperature overnight. The reaction was
poured into an ice/H2O slurry (30 mL) and stirred to precipitate an
orange solid. The solid was collected by vacuum filtration and dried
in vacuo. Purification by flash column chromatography (silica gel,
MeOH:CH2Cl2 1:10) provided SPG1 11 as an orange film (17.1
mg, 11% yield). 1H NMR (DMSO-d6, 400 MHz): δ 12.41 (1H, s),
8.75 (1H, s), 7.78 (1H, s), 7.71 (1H, s), 7.55 (1H, d, J ) 7.6 Hz),
7.45 (2H, d, J ) 7.2 Hz) 7.31 (2H, d, J ) 7.6 Hz), 7.24 (1H, d, J
) 8.4 Hz), 7.11 (1H, s), 7.04 (2H, d, J ) 8.4 Hz), 6.94 (1H, d, J
) 9.6 Hz), 6.50 (1H, d, J ) 9.6 Hz), 6.29 (2H, s), 6.25 (1H, s),
5.44 (2H, s), 4.67 (2H, s), 4.39 (2H, d, J ) 6.4 Hz), 1.98 (3H, s),
1.31 (12H, s). HRFAB-MS: calculated for [MH+] 739.3042, found
739.3046.
1
2:1) gave 2 as a clear viscous oil (300 mg, 19% yield). H NMR
(DMSO-d6, 400 MHz): δ 9.99 (1H, br-s), 7.39 (2H, d, J ) 8.4
Hz), 7.26 (2H, d, J ) 8.4 Hz), 7.09 (2H, s), 6.10 (1H, s), 5.26 (2H,
s), 4.36 (2H, s). GC-MS: calculated for [M+] 360, found 360.
4(4-(Aminomethyl)benzyloxy)-6-chloropyrimidin-2-amine (3).
Methylamine (33% in EtOH, 2.5 mL) was added to the solution of
2 (250 mg, 0.69 mmol) in MeOH (5 mL). The mixture was stirred
at room temperature for 24 h. The solvent was removed in vacuo,
1
providing product 3 as a white powder (175 mg, 95% yield) H
NMR (DMSO-d6, 400 MHz): δ 7.33 (2H, d, J ) 8.0 Hz), 7.31
(2H, d, J ) 8.0 Hz), 7.10 (2H, br-s), 6.10 (1H, s), 5.25 (2H, s),
3.68 (2H, s) LC-MS: calculated for [M+] 265.1, found 265.1.
6-Hydroxy-9-(4-hydroxy-2-methylphenyl)-3H-xanthen-3-one (6).
Tokyo Green 5 (6.3 g, 18.9 mmol) was added to a dried 500 mL
round-bottom flask. Anhydrous CH2Cl2 (300 mL) was added by
cannula and the resulting insoluble mixture stirred at room
temperature for 5 min. The flask was cooled to -78 °C in a CO2(s)/
acetone bath and boron tribromide (14.2 g, 5.4 mL, 56.7 mmol)
was added dropwise over 15 min. The reaction was allowed to warm
to room temperature overnight and added slowly to 500 mL of
vigorously stirred ice/H2O slurry, causing precipitation of a reddish
orange solid. The mixture was stirred for an additional 24 h and
the solid collected by vacuum filtration. Purification by flash column
chromatography (silica gel, MeOH:CH2Cl2 1:10) afforded 4 as a
brick red solid (5.3 g, 87% yield). 1H NMR (DMSO-d6, 400 MHz):
δ 10.00 (1H, s), 7.35 (2H, d, J ) 9.2 Hz), 7.08 (2H, d, J ) 8.0
Hz), 7.07 (1H, s), 7.03 (2H, dd, J ) 9.2, 2.4), 6.87 (1H, d, J ) 2.4
Hz), 6.83 (2H, dd, J ) 8.0, 2.4), 1.90 (3H, s). LC-MS: calculated
for [MH+] 319, found 319.
tert-Butyl 2-(4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-3-methylphe-
noxy)acetate (7). Phenolic TG 6 (3.13 g, 10.0 mmol) was added to
a dried 500 mL Schlenk flask. Anhydrous DMF (300 mL) and
cesium carbonate (22.2 g, 68.1 mmol) were added, and the mixture
stirred at room temperature for 60 min. tert-Butylbromoacetate (1.95
g, 1.46 mL, 10.0 mmol) was added by micropipettor and the
resulting reaction mixture stirred at room temperature overnight.
The reaction was filtered and solvent concentrated under reduced
pressure, giving a dark brown mass. H2O (100 mL) was added and
the solution neutralized with 1 M HCl, giving a red orange
precipitate. The solid was collected by vacuum filtration, washed
with H2O (100 mL), and dried in air. Purification by flash column
chromatography (silica gel, MeOH:CH2Cl2 1:20) furnished 5 as a
1
brick red solid (1.54 g, 36% yield). H NMR (CDCl3, 400 MHz):
δ 7.10 (2H, d, J ) 9.2 Hz), 7.09 (1H, d, J ) 8.4 Hz), 6.93 (1H, d,
J ) 2.4 Hz), 6.89 (1H, s), 6.88 (2H, s), 6.83 (2H, dd, J ) 9.2, 2.4
Hz), 4.61 (2H, s), 2.03 (3H, s), 1.54 (9H, s). LC-MS: calculated
for [MH+] 433, found 433.
tert-Butyl-2-(3-methyl-4-(3-oxo-6-(trifluoromethylsulfonyloxy)-
3H-xanthen-9-yl)phenoxy)acetate (8). tert-Butyl TG 7 (1.06 g, 2.44
mmol) was added to a dried 25 mL Schlenk flask. Anhydrous DMF
(5 mL) and DIPEA (1.57 g, 2.1 mL, 12.2 mmol) were added, and
the resulting mixture stirred at room temperature for 60 min.
N-Phenyl bis-trifluoromethane sulfonimide (1.37 g, 3.84 mmol) was
N-(4-((2-Amino-6-chloropyrimidin-4-yloxy)methyl)benzyl)-2-(3-
methyl-4-(3-oxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-
xanthen-9-yl)phenoxy)acetamide, SPG2 (12). Compound 10 (25
mg, 0.051 mmol) dissolved in anhydrous DMF (2 mL) was added
9
J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010 4463

A R T I C L E S
Srikun et al.
to a dried 5 mL round-bottom flask and the solution was stirred at
room temperature for 5 min. HATU (25 mg, 0.066 mmol),
compound 3 (18 mg, 0.068 mmol), and DIPEA (25 uL) were added,
and the reaction was stirred at room temperature overnight. DMF
was removed under vacuo and the orange oil was redissolved in
10% MeOH in CH2Cl2. Purification by flash column chromatog-
raphy (silica gel, MeOH:CH2Cl2 1:10) afforded SPG2 12 as an
orange film (20 mg, 0.027 mmol, 53% yield). 1H NMR (20% MeOD
in CDCl3) δ 7.92 (1H, s), 7.55 (1H, d, J ) 6.4 Hz), 7.33 (2H, d,
J ) 6.4 Hz), 7.28 (2H, d, J ) 6.4 Hz), 7.07 (1H, d, J ) 6.8 Hz),
7.02 (1H, d, J ) 6.4 Hz), 6.90-7.00 (3H, m), 6.58 (1H, d, J ) 7.6
Hz), 6.43 (1H, s), 6.06 (1H, s), 5.26 (2H, s), 4.61 (2H, s), 4.53
(2H, d, J ) 3.6 Hz), 1.98 (3H, s), 1.32 (12H, s). ESI-MS: calculated
for [MH+] 733.25, found 733.26.
2-(4-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-3-methylphenoxy)ace-
tic acid (13). Compound 7 (200 mg, 0.46 mmol) was stirred in a
mixture of CH2Cl2:TFA (1:2, 20 mL) for 1 h. Removal of the
solvent under vacuum gave a viscous orange oil. EtOAc (10 mL)
was added, and the mixture was sonicated for 30 min. Slow addition
of the EtOAc mixture into a beaker of vigorously stirring hexane
(30 mL) gave carboxyl TG 13 as orange solid precipitate (120 mg,
0.32 mmol, 69% yield). 1H NMR (CD3OD, 400 MHz) δ 7.32 (2H,
d, J ) 8.6 Hz), 7.19 (1H, d, J ) 8.4 Hz), 7.08 (1H, d, J ) 2.0 Hz),
7.03 (1H, dd, J ) 8.4, 2.0 Hz), 6.96 (2H, s), 6.92 (2H, d, J ) 8.6
Hz), 4.80 (2H, s), 1.97 (3H, s). LC-MS: calculated for [MH+] 377.1,
found 377.0.
N-(4-((2-Amino-9H-purin-6-yloxy)methyl)benzyl)-2-(4-(6-hydroxy-
3-oxo-3H-xanthen-9-yl)-3-methylphenoxy)acetamide, SNAP-TG1
(14). Compound 13 (235 mg, 0.67 mmol) was added to a dried 25
mL Schlenk flask. Anhydrous DMF (5 mL) was added by syringe
and the solution was stirred at room temperature for 5 min. HATU
(256 mg, 0.67 mmol), 6-(4-(aminomethyl)benzyloxy)-9H-purin-2-
amine 4 (182 mg, 0.67 mmol), and DIPEA (0.87 mL) were added,
and the reaction stirred at room temperature overnight. The reaction
was poured into an ice/H2O slurry (30 mL) and stirred to precipitate
an orange solid. The solid was collected by vacuum filtration and
dried in Vacuo. Purification by flash column chromatography (silica
gel, MeOH:CH2Cl2 1:10) delivered STG1 14 as an orange solid
(197 mg, 0.31 mmol, 46% yield). 1H NMR (DMSO-d6, 400 MHz)
δ 12.42 (1H, br-s), 8.74 (1H, s), 7.82 (1H, s), 7.45 (2H, d, J ) 7.2
Hz), 7.29 (2H, d, J ) 7.2 Hz), 7.20 (1H, d, J ) 8.4 Hz), 7.09 (1H,
s), 7.02 (1H, d, J ) 8.0 Hz), 6.87 (2H, d, J ) 8.8 Hz), 6.50-6.80
(4H, m), 6.29 (2H, br s), 5.44 (2H, s), 4.66 (2H, s), 4.38 (2H, d, J
) 5.2 Hz), 3.16 (1H, s), 1.98 (3H, s). ESI-MS: calculated for [MH+]
629.21, found 629.21.
optical density (OD600nm) of 0.8 was reached. Expression of His-
AGT was induced by addition of 0.5 mM isopropyl-ꢀ-D-thioga-
lactopyranoside (IPTG). The culture was grown at 16 °C for 12 h
and was harvested by centrifugation at 8000 rpm for 10 min at 4
°C. Cell lysates were prepared by resuspending the pellet in buffer
(PBS with 0.5 mM phenylmethylsulfonyl fluoride) followed by
FRENCH press (Thermo Scientific). Insoluble protein and cell
debris were removed by centrifugation at 10000 rpm for 30 min.
For purification of His-AGT, Ni-NTA (Qiagen) was used according
to the supplier’s protocol. The fractions with His-AGT were
combined and concentrated using an Amicon Ultra-15 Ultracel-
10K centrifugal unit (Millipore). The concentrated His-AGT
solution was then desalted using G25 Sephadex (Sigma Aldrich)
with the following elution buffer: 150 mM NaCl, 25 mM Tris-Cl,
1 mM DTT pH 7.5. The purified His-AGT protein was concentrated
using an Amicon Ultra-4 Ultracel-10K and stored at -20 °C. The
protein concentration was determined using the BCA assay, yielding
8.6 mg of purified His-AGT (1.5 mM in 250 µL elution buffer).
In Vitro SNAP Labeling and Analysis by ESI-MS and
In-Gel Fluorescence Scanning. Purified His-AGT (0.5 uM) was
incubated in reaction buffer (PBS pH 7.4, 1 mM DTT) at 37 °C
with 0.5 µM of either SPG1, SPG2, STG1, or STG2 for 30 min at
37 °C. Binding of the fluorescent dyes to His-AGT was analyzed
by SDS-PAGE, followed by in-gel fluorescence scanning (488 nm
Argon excitation, 520 BP 40 emission filter, Typhoon 9410 imaging
system, GE Healthcare) and Coomassie protein staining. For mass
spectrometric analysis and spectroscopic measurements, His-AGT
(1 µM) was incubated in 20 mM HEPES buffer pH 7 (1 mL) with
5 µM of either SPG1, SPG2, STG1, or STG2 for 30 min at 37 °C.
The reaction was concentrated to 50 µM with an Amicon 4 10K
column (Milipore) and subjected to size-exclusive gel filtration with
Bio-Rad Micro Bio-Spin 6 chromatography column to remove the
excess dye. The deconvoluted MS data were collected by Dr.
Anthony T. Iavarone at UC Berkeley QB3 Mass Spectrometry
Facility.
Spectroscopic Materials and Methods. Millipore water was
used to prepare all aqueous solutions. All spectroscopic measure-
ments were performed in 20 mM HEPES buffer, pH 7.0, 25 °C.
Absorption spectra were recorded using a Varian Cary 50 spec-
trophotometer (Walnut Creek, CA). Fluorescence spectra were
recorded using a Photon Technology International Quanta Master
4 L-format scanning spectrofluorometer (Lawrenceville, NJ) equipped
with an LPS-220B 75-W xenon lamp and power supply, A-1010B
lamp housing with integrated igniter, switchable 814 photon-
counting/analog photomultiplier detection unit, and MD5020 motor
driver. Samples for absorption and fluorescence measurements were
contained in 1-cm × 1-cm quartz cuvettes (1.4 mL volume, Starna,
Atascadero, CA). Fluorescein in 0.1 M NaOH (Φ ) 0.85)90 was
used as standard for quantum yield measurements.
Cell Culture and Labeling Procedures. HEK 293T cells were
cultured in DMEM (Invitrogen) supplemented with 10% fetal
bovine serum (FBS, Hyclone) and glutamine (2 mM). One day
before transfection, cells were passaged and plated on 4-wells Lab-
Tek borosilicate chambered coverglass (Nunc). Transient expression
of SNAP fusion protein or mCherry was performed by following
the standard protocol of Lipofectamine 2000. SNAP-tag labeling
was achieved by incubating cells in DPBS containing 5 µM dye
and 1 µM Hoechst 33342 for 30 min at 37 °C. For membrane
labeling with cell-impermeable SPG1 or STG1, cells were washed
with fresh DPBS (2 × 1 mL) before image acquisition. Labeling
of intracellular targets with STG2 or SPG2 required incubation of
cells in fresh DMEM with 10% FBS (2 × 1 mL) for 30 min after
dye loading to remove any unbound fluorophore. All confocal
fluorescence images were acquired in DPBS media.
N-(3-((2-Amino-6-chloropyrimidin-4-yloxy)methyl)benzyl)-2-(4-
(6-hydroxy-3-oxo-3H-xanthen-9-yl)-3-methylphenoxy)acetamide,
STG2 (15). Compound 13 (30 mg, 0.079 mmol) in anhydrous DMF
(2 mL) and HATU (30 mg, 0.078 mmol) were added to a dried 5
mL round-bottom flask and the solution was stirred at room
temperature for 5 min. Compound 3 (21 mg, 0.079 mmol), and
DIPEA (100 µL) were added, and the reaction was stirred at room
temperature overnight. DMF was removed in Vacuo, and the orange
oil was redissolved in 10% MeOH in CH2Cl2. Purification by flash
column chromatography (silica gel, MeOH:CH2Cl2 1:10) afforded
1
STG2 15 as an orange film (45 mg, 0.072 mmol, 91% yield). H
NMR (20% CD3OD in CDCl3) δ 7.35 (1H, t, J ) 6.0 Hz), 7.25
(2H, d, J ) 8.2), 7.21 (2H, J ) 8.2 Hz), 6.98 (1H, d, J ) 8.0),
6.91 (2H, d, J ) 8.8 Hz), 6.85 (1H, s), 6.82 (1H, d, J ) 8.0), 6.41
(2H, d, J ) 2.0 Hz), 6.60 (2H, dd, J ) 8.8, 2.0 Hz), 5.97 (1H, s),
5.16 (2H, s), 4.52 (2H, s), 4.44 (2H, d, J ) 6.0 Hz), 1.97 (3H, s)
ESI-MS: calculated for [MH+] 623.16, found 623.17.
Expression and Purification of His-AGT for SNAP
Labeling. The sequence encoding SNAP was PCR amplifed from
pSEMS-SNAP-ADBR (Covalys) and subcloned into PET28a(+)
(Novagen) using NheI/XhoI restriction sites. The resulting plasmid,
after verified by DNA sequencing, was transformed into E. coli
strain BL-21(DE3). A bacterial culture was grown at 37 °C in TB
medium (50 mL) with 50 µg/mL Kanamycin for 6-8 h until an
Fluorescence Imaging Experiments. Confocal fluorescence
imaging studies were performed with a Zeiss LSM510 NLO
Axiovert 200 Laser scanning microscope and a 40× oil-immersion
(90) Parker, C. A.; Rees, W. T. Analyst 1960, 85, 587–600.
9
4464 J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010

Imaging Hydrogen Peroxide in Living Cells
A R T I C L E S
objective lens. The motorized stage on the microscope was equipped
with an incubator, maintaining the sample at 37 °C in a 5% CO2
humidified atmosphere. Excitation of the SNAP tag probe at 488
nm was carried out with an argon laser and emission was collected
using a 500-550 nm filter. Excitation of mCherry was carried out
with a helium-neon 543 nm laser and emission was collected using
a LP560 filter. Excitation of Hoechst 33342 was carried out using
a MaiTai two-photon laser at 770 nm pulses (mode-locked
Ti:sapphire laser, Tsunami Spectra Physics) and emission was
collected through 385-425 nm filter. Image analysis was performed
in ImageJ (National Institute of Health).
a scholarship from the Ministry of Science, Thailand. A.E.A. thanks
the NIH Chemical Biology Graduate Program (T32 GM066698)
for support, as well as the ACS Organic Division for an Emmanuil
Troyansky Graduate Fellowship and UC Berkeley for a Chancellor’s
Opportunity Fellowship. We thank Holly Aaron (UCB Molecular
Imaging Center), Ann Fischer (UCB Tissue Culture Facility), and
Prof. Michelle Chang (UCB Department of Chemistry) for expert
technical assistance and helpful discussions.
Supporting Information Available: Mass spectrometry and
in-gel fluorescence analysis of intact AGT as well as AGT-PG
and AGT-TG adducts, details of construction of plasmids, and
complete ref 54. This material is available free of charge via
the Internet at http://pubs.acs.org.
Acknowledgment. We thank the Packard and Sloan Founda-
tions, the Hellman Faculty Fund (UC Berkeley), Amgen, Astra
Zeneca, and the National Institute of General Medical Sciences
(NIH GM 79465) for funding this work. C.J.C. is an Investigator
with the Howard Hughes Medical Institute. D.S. was supported by
JA100117U
9
J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010 4465