Synthesis and evaluation of 2-ethynyl-adenosine-5′-triphosphate as a chemical reporter for protein AMPylation

Article abstract of DOI:10.1039/c5ob01081k

Protein AMPylation is a posttranslational modification (PTM) defined as the transfer of an adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl side-chain of a protein substrate. One recently reported AMPylator enzyme, Vibrio outer protein S (VopS), plays a role in pathogenesis by AMPylation of Rho GTPases, which disrupts crucial signaling pathways, leading to eventual cell death. Given the resurgent interest in this modification, there is a critical need for chemical tools that better facilitate the study of AMPylation and the enzymes responsible for this modification. Herein we report the synthesis of 2-ethynyl-adenosine-5′-triphosphate (2eATP) and its utilization as a non-radioactive chemical reporter for protein AMPylation.

Full text of DOI:10.1039/c5ob01081k

Organic &
Biomolecular Chemistry
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Synthesis and evaluation of 2-ethynyl-adenosine-
5’-triphosphate as a chemical reporter for protein
AMPylation†
Cite this: DOI: 10.1039/c5ob01081k
Christa Creech, Mukul Kanaujia and Corey P. Causey*
Protein AMPylation is a posttranslational modification (PTM) defined as the transfer of an adenosine
monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl side-chain of a protein substrate.
One recently reported AMPylator enzyme, Vibrio outer protein S (VopS), plays a role in pathogenesis by
AMPylation of Rho GTPases, which disrupts crucial signaling pathways, leading to eventual cell death.
Given the resurgent interest in this modification, there is a critical need for chemical tools that better
facilitate the study of AMPylation and the enzymes responsible for this modification. Herein we report the
synthesis of 2-ethynyl-adenosine-5’-triphosphate (2eATP) and its utilization as a non-radioactive chemi-
cal reporter for protein AMPylation.
Received 28th May 2015,
Accepted 8th July 2015
DOI: 10.1039/c5ob01081k
www.rsc.org/obc
Introduction
Posttranslational modifications (PTMs) are an evolutionary
solution to expand the relatively limited number of amino
acids used to synthesize all proteins found in nature. These
modifications, which transform functional groups on amino
acid side chains, can alter the function of
a protein
altogether.1 For example, lysine acetylation converts the side-
chain amino group into an amide functionality thereby
affecting hydrogen bonding and ion pairing potential, features
that can be important for conformational changes and
protein–protein interactions. Additionally, phosphorylation of
hydroxyl side chains is known to have a significant impact on
cell signaling pathways. Many PTMs are readily reversed by
complementary enzymes and can therefore serve as cellular
switches in vivo. For instance, the phosphoester bonds formed
by kinases during phosphorylation can be cleaved by phospha-
tases to reveal the original hydroxyl side chains.
Another lesser studied PTM, which is characterized by the
transfer of an AMP group from ATP to hydroxyl side-chains of
substrate proteins, is known as AMPylation (Fig. 1A). Although
first reported more than 40 years ago, this modification went
largely unstudied until recently.2 The resurgent interest in this
PTM is due to reports of adenyltranserases that are injected
into human cells by bacterial pathogens.3,4 Further investi-
gations revealed that these enzymes actively participate in
Fig. 1 Probing AMPylation. (A) AMPylator enzymes transfer AMP from
ATP to hydroxyl side chains. (B) N6-propargyl adenosine-5’-triphosphate
(N6pATP) and (C) N6-(6-amino)hexyl-adenosine-5’-triphosphate-5-car-
boxyl-fluorescein (Fl-ATP) have been previously utilized as chemical
reporters for protein AMPylation.
Department of Chemistry, University of North Florida, 1 UNF Drive, Jacksonville,
FL 32224, USA. E-mail: corey.causey@unf.edu
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob01081k
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pathogenesis.5,6 Among the AMPylator enzymes reported to
play roles in pathogenesis is Vibrio outer protein S (VopS) of
Vibrio parahaemolyticus. Once released into host cells, this
enzyme AMPylates a threonine residue in the Switch I region
of Rho GTPases, such as Cdc42.5 The addition of the AMP
group causes disruptions in crucial interactions with down-
stream proteins, leading to cell rounding and eventual cell
death. In addition to the discovery of AMPylator enzymes, a de-
AMPylator enzyme has also recently been identified,
suggesting that this PTM is dynamic and likely has significant
in vivo roles.7,8
Given the emerging interest in understanding the roles of
AMPylation, there is a need for tools that facilitate the study of
this PTM. Radioisotopically labeled ATP can be used to visual-
ize AMPylated protein, however this methodology requires
specialize training and equipment. Furthermore, isotopic
labeling does not aid in the selective isolation of modified sub-
strates, thus identification still required robust proteomic ana-
lysis.3,5,9,10 Such analysis of complex mixtures can be quite
laborious and difficult, especially when the relative abundance
of the analyte protein is low. For these reasons, the develop-
ment of non-radioactive chemical tools that enable the study
of AMPylation is quite advantageous.
In 2010, Hang and co-workers synthesized and utilized an
ATP-alkyne analog as a clickable chemical reporter for protein
AMPylation (Fig. 1B).11 Like ATP, this analog is enzymatically
transferred to substrate proteins, and the alkyne moiety
enables protein visualization through Cu(I)-catalyzed azide–
alkyne cycloaddition (CuAAC) reactions with azide-conjugated
reporter tags. In 2012, Thompson and co-workers reported the
use of a fluorescein-ATP conjugate (Fl-ATP) as a novel chemical
tool to identify AMPylated substrates (Fig. 1C).12 Substrate
specificity studies detailed in the same report also suggest
that, in addition to modifications at position 6, modifications
at position 2 of the adenine ring may also be tolerated by the
enzyme. Given the interest in studying this enzyme family, we
set out to synthesize an ATP analog, substituted with an alkyne
moiety at position 2, and evaluate its utility as a clickable
chemical probe to study protein AMPylation (Fig. 2). We
hypothesized that the structural differences (i.e., substitution
at position 2 rather than position 6) will yield an alternative
tool that may be effective to study not only known AMPylator
enzymes, but also as-of-yet unknown AMPylators with different
tolerances for structural modifications to the ATP substrate.
Herein we report the synthesis and in vitro evaluation of
Fig. 2 Detection of modified protein substrates. Once 2eATP is trans-
ferred to the substrate protein, detection of the modified protein can be
facilitated by the addition of reporter tags via CuAAC reactions.
radioactive reagents, use of these reporter tags can also enable
isolation methods such as immuno-precipitation. An investi-
gation into the substrate specificity of VopS indicated that
modifications to the adenine ring at positions 2 and 6 would
be tolerated by the enzyme.12 Since the probes utilized pre-
viously were modified at position 6, we chose to explore the
alternative position by installing an ethynyl group at position
2. Like the previously reported probe, the presence of the
alkyne will enable the addition of azide-conjugated reporter
tags via CuAAC reactions (Fig. 2). The addition of the alkyne to
this position also confers alternative steric constraints, and
therefore may prove useful in the study of subsequently discov-
ered AMPylators.
Synthesis of 2-ethynyl-adenosine-5′-triphosphate (2eATP)
The synthesis of 2-ethynyl-adenosine-5′-triphosphate (2eATP)
began from guanosine and proceeded in
7 total steps
(Scheme 1). The three hydroxyl groups were first acetylated, fol-
lowed by conversion to chloro-purine 2. A Sandmeyer reaction
was employed to replace the exocyclic amino group with an
iodo group. Treatment of 3 with ethanolic ammonia yielded
2-iodoadensine via substitution of the chloride and concurrent
removal of the three acetyl protecting groups. A Sonogashira
reaction between 4 and TMS-protected acetylene, followed by
deprotection of the silane, yielded 2-ethynyl-adenosine (6).
Treatment of 6 with phosphoryl chloride, followed by bis(tri-n-
butylaminium)pyrophosphate installed the triphosphate to
yield 2eATP (7) as the final product.
2-ethynyl-adenosine-5′-triphosphate (2eATP) as
a chemical
reporter for protein AMPylation.
Results and discussion
Fluorescent labeling of Cdc42
Design of chemical reporter
A major impetus for developing chemical probes for protein
Traditional methods, such as radioisotopic labeling and mass AMPylation is facile visualization of modified protein sub-
spectrometry analysis of complex mixtures have certain limit- strates. Although radioisotopically labeled ATP can be used,
ations that can be overcome by fluorescein or biotin labeling those experiments require radioactive reagents and equip-
of modified substrates. In addition to mitigating the need for ment. The use of 2eATP can mitigate these requirements when
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Scheme 1 Synthesis of 2-ethynyl-ATP. Reagents and conditions: (a) Ac2O, Et3N, DMAP, DMF, rt, 1 h; (b) POCl3, dimethylaniline, Et4NCl, ACN, reflux,
10 min; (c) CH2I2, I2, CuI, isopentyl nitrite, THF, reflux, 45 min; (d) NH3, EtOH, 0 °C → rt, 48 h; (e) PdCl2(PPh3)2, Et3N, CuI, TMS-acetylene, ACN, rt, 20
min; (f) NH3, MeOH, 0 °C → rt, 2.25 h; (g) POCl3, bis(tri-n-butyl)ammonium pyrophosphate, (MeO)3PO, 0 °C → rt.
used with a fluorescent reporter such as a fluorescein–azide.
To test 2eATP, both as a substrate and a fluorescent reporter
for protein AMPylation, a known AMPylator enzyme (VopS)
and decreasing amount of its cognate protein substrate
(Cdc42Q61L) were incubated with 2eATP at 30 °C for 1 h. Sub-
sequent to the transfer reaction, the mixture was treated with
fluorescein–azide under CuAAC conditions. The reaction mix-
tures were separated by SDS-PAGE and visualized fluorescently
using a Typhoon 9410 imaging system. The results of this
experiment show that 2eATP is effectively transferred to
Cdc42Q61L, as evidenced by the fluorescent bands; the limit
of detection is approximately 5 pmol of modified protein
(Fig. 3A). In addition to evaluating the transfer of 2eATP to pur-
ified protein substrate, the selectivity of this transfer was also
explored by conducting an analogous experiment in the pres-
ence of cell lysates from E. coli (Fig. 3B). Although there is a
slight decrease in the sensitivity for detection, which is likely
due to probe degradation by other ATP degrading enzymes, the
transfer of 2eATP to Cdc42Q61L is remarkably selective. This
latter finding is consistent with the fact that VopS shows very
little promiscuous activity. Note that at higher concentrations
a second lower band, which is likely a proteolyzed fragment, Fig. 3 Limit of detection and chemical selectivity. (A) Decreasing
amounts of Cdc42 were treated with 2eATP in the presence of VopS fol-
lowed by a CuAAC reaction with fluorescein–azide; fluorescence (top),
becomes visible.
coomassie (bottom). (B) Selectivity of labeling: decreasing amounts of
Cdc42 were added to E. coli cell lysates; fluorescence (top), coomassie.
Biotin labeling of Cdc42Q61L
(bottom).
The use of click chemistry has found great utility for develop-
ment of tools to study proteins. The ethynyl moiety of 2eATP
can facilitate not only the detection of modified protein by
fluorescent imaging, but also through the addition of a biotin reaction, biotin–azide was added via a CuAAC reaction, and
reporter tag. To demonstrate the ability to detect protein the samples were separated by SDS-PAGE. Western blot analy-
AMPylation using a biotin–azide conjugate, experiments analo- sis of these samples enabled visualization of the labeled
gous to those conducted with fluorescein–azide were under- protein to amounts as low as 10 pmol (Fig. 4A). In order to
taken. Decreasing amounts of Cdc42Q61L were incubated with demonstrate the selectivity of biotin labeling, an analogous
VopS and 2eATP at 30 °C for 1 h. Subsequent to the transfer experiment was conducted in the presence of cell lysate from
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1
from VWR. H, 13C, and 31P NMR spectra were collected on a
Varian 500 MHz spectrometer and chemical shifts are reported
(in ppm) relative to solvent peaks. Thin layer chromatography
was conducted on pre-coated silica gel plates and column
chromatography separations were conducted on silica gel
(230–450 mesh). RP-HPLC separations were conducted on a
Varian Prostar system with
250 mm).
a microsorb column (21 ×
2′,3′,5′-Triacetylguanosine (1)
To a mixture of guanosine (5 g, 17.7 mmol) and 4-diamino-
methylpyridine (0.16 g, 1.3 mmol) in acetonitrile were added
triethylamine (9.7 mL, 69.6 mmol) and acetic anhydride
(6 mL, 63.5 mmol). After stirring for 45 min, methanol (3 mL,
74 mmol) was added and the mixture was stirred for an
additional 10 min at rt. The reaction mixture was filtered, and
the filtrate was concentrated under reduced pressure. Isopropyl
alcohol (200 mL) was added to the resulting residue, and the
mixture was refluxed for 10 min. After cooling to rt, the
product was collected by filtration as a white powder (5.3 g,
12.9 mmol, 73%). 1H NMR (500 MHz, 10% DMSO-d6 in
CDCl3): δ 7.55 (s, 1H), 5.82 (d, J = 5 Hz, 1H), 5.77 (t, J = 5.5 Hz,
1H), 5.75 (br s, 2H), 5.58 (t, J = 5 Hz, 1H), 4.32 (dd, J = 11.5, 3.5
Hz, 1H), 4.26 (m, 1H), 4.22 (dd, J = 11.5, 5 Hz, 1H), 2.00 (s,
3H), 1.98 (s, 3H), 1.97 (s, 3H); 13C NMR (125 MHz, 10% DMSO-
d6 in CDCl3): δ 170.44, 169.48, 169.23, 157.59, 153.44, 150.65,
135.68, 118.00, 86.06, 79.51, 72.58, 70.31, 62.89, 20.57, 20.38,
Fig. 4 Limit of detection and chemical selectivity. (A) Decreasing
amounts of Cdc42 were treated with 2eATP in the presence of VopS
followed by a CuAAC reaction with biotin–azide; western blot (top),
coomassie (bottom). (B) Selectivity of labeling: decreasing amounts of
Cdc42 were added to E. coli cell lysates; western blot (top), coomassie.
(bottom).
+
20.27; HRMS (C16H20N5O8 ): calculated 410.1312, observed
410.1323.
E. coli. Fig. 4B shows that a similar detection limit was
achieved and that labeling was remarkably selective.
2-Amino-6-chloro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine (2)
Tetraethylammonium chloride (4.3 g, 26.2 mmol), N,N-di-
methylaniline (1.66 mL, 13.1 mmol) and freshly distilled phos-
phoryl chloride (7.35 mL, 76.6 mmol) were added to 2′,3′,5′-
triacetylguanosine (1) (5.3 g, 13 mmol) in dry acetonitrile
(27 mL). After heating the mixture under reflux for 10 min,
and the volatiles were immediately removed by vacuum distil-
lation. The remaining residue was dissolved in chloroform
(55 mL) and treated with cold water (36 mL) for 15 min at 0 °C
for 15 min. The layers were separated, and the aqueous layer
was further extracted with chloroform (3 × 20 mL). The organ-
ics were combined, washed with cold water (6 × 20 mL), 5%
sodium bicarbonate (6 × 40 mL), and dried over sodium
sulfate. The solution was filtered and added to isopropanol
(27 mL) and the resulting mixture was evaporated under
reduced pressure to a volume of ∼18 mL. The resulting solu-
tion was stored at 4 °C overnight to crystalize. The product was
Conclusions
In conclusion, we have successfully synthesized 2eATP and
demonstrated that this ATP analog is effectively transferred by
VopS to the cognate protein substrate, Cdc42Q61L. The alkyne
moiety enables the post-transfer addition of reporter tags to
the modified protein. These reporter tags, fluorescein–azide
and biotin–azide, enable the visual detection of modified
protein to quantities as low as 5 pmol. Given the growing inter-
ests in this PTM and the limited information available about
its in vivo roles, 2eATP should serve as a valuable tool to
further explore the roles of protein AMPylation.
Experimental
All chemicals and solvents were of reagent grade and were pur- isolated by filtration as a slightly yellow crystal (3.4 g,
1
ified and dried by standard methods prior to use. Bis(tri-n- 7.9 mmol, 61%). H NMR (500 MHz, DMSO-d6): δ 8.37 (s, 1H),
butylammonium) pyrophosphate was prepared as previously 7.08 (br, 2H), 6.11 (d, J = 6 Hz, 1H), 5.88 (t, J = 6 Hz, 1H), 5.54
described.13 Biotin–azide was purchased from Sigma-Aldrich (dd, J = 4.5, 6 Hz, 1H), 4.40 (dd, J = 3.5, 11 Hz, 1H), 4.38 (m,
and fluorescein–azide was synthesize from 1,4-diaminobutane 1H), 2.12 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H); 13C NMR (125 MHz,
and FITC (isomer I) in a manor analogous to the previous DMSO-d6): δ 170.14, 169.48, 169.33, 159.93, 153.71, 149.93,
report.14 PGEX-GST-TEV-VopSΔ30 and pRoEX-HTa Cdc42Q61L 141.30, 123.50, 84.87, 79.72, 71.91, 70.27, 62.98, 20.55, 20.42,
were both expressed and purified as previously described.12 20.23; HRMS (C16H19ClN5O7+): calculated 428.0973, observed
Streptavidin-HRP antibody (cat# EMOR-03L) was purchased 428.0981.
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6-Chloro-2-iodo-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-9H-
purine (3)
102.59, 91.22, 89.98, 87.01, 73.93, 71.41, 62.41, −0.62; HRMS
(C15H22N5O4Si+): calculated 364.1441, observed 364.1445.
Iodine (1.93 g, 7.6 mmol), diiodomethane (6.1 mL, 76 mmol),
copper iodide (1.61 g, 8.46 mmol), and isopentyl nitrite 2-Ethynyladenosine (6)
(3.0 mL, 22.5 mmol) were added to 2-amino-6-chloro-9-(2,3,5-
2-[2-(Trimethylsilyl)ethynyl]-adenosine (5) (71 mg, 0.2 mmol)
tri-O-acetyl-β-D-ribofuranosyl)purine (2) (3.25 g, 7.6 mmol) in
dry THF (35 mL). The mixture was stirred under reflux for
45 min, cooled to rt, filtered and concentrated. The product
(3.1 g, 5.7 mmol, 75%) was isolated by chromatography on
was dissolved in MeOH (5 mL) and cooled to 0 °C. Ammonia
was bubbled through the solution for 45 min. The reaction
was allowed to warm to rt and stir for an additional 1.5 h. The
solution was purged with air to remove excess ammonia and
the sample was concentrated under reduced pressure to yield
the title compound as a light brown solid (52 mg, 0.18 mmol,
90%). 1H NMR (500 MHz, DMSO-d6): δ 8.42 (s, 1H), 7.51 (s,
2H), 5.85 (d, J = 6 Hz, 1H), 5.46 (br s, 1H), 5.18 (br s, 2H), 4.54
(t, J = 5.5 Hz, 1H), 4.42 (t, J = 4.5 Hz, 1H), 4.00 (s, 1H), 3.95 (m,
1H), 3.66 (dd, J = 4, 12 Hz, 1H), 3.54 (d, J = 12 Hz, 1H); 13C
NMR (125 MHz, 10% DMSO-d6): δ 156.18, 149.48, 144.99,
141.23, 119.42, 88.03, 86.24, 83.42, 75.77, 74.05, 70.90, 61.89;
1
silica (99 : 1 DCM/methanol). H NMR (500 MHz, 10% DMSO-
d6 in CDCl3): δ 8.82 (s, 1H), 6.30 (d, J = 4.5 Hz, 1H), 5.88 (t, J =
5.5 Hz, 1H), 5.63 (t, J = 5.5 Hz, 1H), 4.41 (m, 2H), 4.28 (dd, J =
5.5, 11.5 Hz, 1H), 2.11 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H); 13C
NMR (125 MHz, DMSO-d6): δ 170.43, 169.75, 169.65, 152.36,
149.35, 146.29, 131.93, 118.57, 86.69, 80.05, 72.77, 70.00,
62.80, 20.83, 20.60, 20.49; HRMS (C16H17ClIN4O7+): calculated
538.9831, observed 538.9841.
+
HRMS (C12H14N5O4 ): calculated 292.1046, observed 292.1051.
2-Iodoadenosine (4)
2-Ethynyl-adenosine-5′-triphosphate (2eATP) (7)
6-Chloro-2-iodo-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-9H-purine
(3) (0.77 g, 1.42 mmol) was added to dry ethanol (80 mL) and
cooled to 0 °C. The reaction flask was sealed with a rubber
septum and ammonia was bubbled through the solution until
the starting material completely dissolved (∼3 h). The reaction
was allowed to warm to rt and stirred for an additional
48 h. Reaction progress was monitored by TLC (9 : 1 DCM/
methanol). The solution was purged with air to remove excess
ammonia and the solvent was removed under reduced
pressure. Water (5 mL) was added to the residue, and the
mixture was freeze dried. Water (5 mL) was added to the dried
product and the product was collect by centrifugation as a
white powder (0.46 g, 1.17 mmol, 83%). 1H NMR (500 MHz.
DMSO-d6): δ 8.30 (s, 1H), 7.75 (br s, 2H), 5.80 (d, J = 6. Hz, 1H),
5.48 (br s, 1H), 5.23 (br s, 1H), 5.06 (br s, 1H), 4.52 (t, J = 5.5
Hz, 1H), 4.12 (t, J = 4 Hz, 1H), 3.94 (q, J = 4 Hz, 1H), 3.65 (dd, J
= 4.5, 12.5 Hz, 1H), 3.54 (dd, J = 4, 12 Hz, 1H); 13C NMR
(125 MHz, DMSO-d6): δ 155.98, 149.80, 139.49, 120.94, 119.06,
2-Ethynyladenosine (6) (43.7 mg, 0.15 mmol) was dissolved in
dry trimethylphosphate (0.5 mL) and the solution was stirred
at 45 °C for 1.5 h. The solution was cooled to 0 °C and freshly
distilled phosphoryl chloride (29 mg, 0.19 mmol) was added
dropwise. Stirring was continued at 0 °C for 1 h, then a solu-
tion of bis(tri-n-butylammonium) pyrophosphate (1 M in DMF)
(0.75 mL, 0.75 mmol) was added dropwise and stirring was
continued at 0 °C. After 20 min, the reaction was allowed to
warm to rt and stir for an additional 5 min. Triethyl-
ammonium bicarbonate buffer (1 M, pH 8.0, AcOH) (2 mL)
was added and stirring was continued for 45 min at rt. The
reaction mixture was diluted with water (20 mL) and freeze-
dried. The residue was dissolved in water, and purified by
RP-HPLC eluting with a gradient of 100 mM TEAA (pH 7) and
1
acetonitrile (0–25%). H NMR (500 MHz, D2O): δ 8.49 (s, 1H),
5.95 (d, J = 6.5 Hz, 1H), 4.70 (m, 1H), 4.53 (m, 1H), 4.33 (m,
1H), 4.18 (m, 2H), 3.48 (s, 1H); 31P NMR (202 MHz, D2O): δ
−10.88 (d, J = 19.8 Hz), −11.45 (d, J = 19.8 Hz), −23.30 (t, J =
19.8 Hz); HRMS (C12H15N5O13P3−): calculated 529.9879,
observed 529.9914.
+
87.18, 85.85, 73.60, 70.56, 61.47; HRMS (C10H13IN5O4 ): calcu-
lated 394.0012, observed 394.0017.
2-[2-(Trimethylsilyl)ethynyl]-adenosine (5)
Limit of detection by fluorescence
2-Iodoadenosine (4) (0.1 g, 0.25 mmol), copper iodide (3.8 mg,
0.02 mmol), PdCl2(PPh3)2 (8.4 mg, 0.012 mmol) were added to Varying concentrations of Cdc42Q61L (0, 0.1, 0.25, 0.5, 1.0,
triethylamine (2 mL) and acetonitrile (2 mL). The reaction 2.5, 5, and 10 μM) were added to assay buffer (20 mM HEPES
mixture was stirred in the dark until all starting material was pH 8.0, 100 mM NaCl, 1 mM DTT, and 5 mM MgCl2), mixed
consumed (∼20 min). (TLC 15% EtOH in chloroform). The with 10 μM 2eATP, and 1 μM VopS to initiate the reaction. This
volatiles were removed under reduced pressure and the reaction mixture was incubated at 30 °C for 1 h. The CuAAC
product was purified on silica (eluting with chloroform, reaction was initiated by adding 10 μM fluorescein–azide,
chloroform/ether, ether, 5% MeOH in ether, 10% MeOH in 1 mM TCEP, 100 μM ligand (TBTA in 1 : 4 DMSO : butanol),
ether) and isolated as an off-white solid (77 mg, 0.21 mmol, and 2 mM CuSO4 to the reaction mixture. The reactions were
85%). 1H NMR (500 MHz, DMSO-d6): δ 8.41 (s, 1H), 5.83 (d, J = incubated in the dark for 1 h and quenched with SDS-loading
7 Hz, 1H), 4.43 (m, 1H), 4.15 (m, 1H), 3.92 (m, 1H), 3.65 (m, dye. The mixtures were heated at 95 °C for 10 min and separ-
1H), 3.57 (m, 1H), 0.21 (s, 9H); 13C NMR (125 MHz, 10% ated by SDS-PAGE. The gel fluorescence was measured using a
DMSO-d6 in CDCl3): δ 155.36, 148.50, 145.06, 141.17, 119.73, Typhoon 9410 imaging system.
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Limit of detection in a complex proteome by fluorescence
Acknowledgements
Purified VopS (1 μM) was incubated with E. coli lysate (25 μg
total protein) in assay buffer (50 mM HEPES pH 8.0, 100 mM
NaCl, 1 mM DTT, and 5 mM MgCl2) along with 10 μM 2eATP
and varying concentrations of Cdc42Q61L (0, 0.1, 0.25, 0.5, 1,
2.5, 5, and 10 μM) for 1 h at 30 °C. The CuAAC reaction was
initiated as described above. The samples were quenched with
SDS-loading dye, heated at 95 °C, and separated by SDS-PAGE.
Fluorescence was measured using a Typhoon 9410 imaging
system.
We thank Prof. Bryan Knuckley (University of North Florida)
for thoughtful and insightful discussions and Prof. Paul
R. Thompson (University of Massachusetts Medical School) for
the plasmids encoding VopS and Cdc42Q61L. We also thank
the University of North Florida for financial support.
Notes and references
Limit of detection by western blot
2eATP (10 μM) and VopS (1 μM) were added to assay buffer
(20 mM HEPES pH 8.0, 100 mM NaCl, 1 mM DTT, and 5 mM
MgCl2) containing various concentrations of Cdc42Q61L (0,
0.1, 0.25, 0.5, 1.0, 2.5, 5, and 10 μM). The reaction was incu-
bated at 30 °C for 1 h. Subsequently, the copper catalyzed click
reaction was initiated by adding 10 μM biotin–azide, 1 mM
TCEP, 100 μM ligand (TBTA in 1 : 4 DMSO : butanol), and
2 mM CuSO4 to the reaction mixture. The reaction was incu-
bated for 1 h before adding SDS-loading dye to quench the
reaction. The mixtures were heated at 95 °C for 10 min and
separated by SDS-PAGE in preparation for the western blot.
The samples were transferred from the SDS-PAGE to a nitro-
cellulose membrane in transfer buffer (25 mM Tris, 192 mM
glycine, 20% methanol, pH 8.3) at 80 V for 60 min. The mem-
brane was washed with TBS and TBST before incubating in
blocking buffer (5% BSA in TBS) overnight at 4 °C. The mem-
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Streptavidin-HRP antibody (1 : 7500) in blocking buffer for 1 h
at rt. The HRP signal was detected by applying a 1-step Ultra
TMB solution (Pierce #37574).
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Chem. Biol., 2011, 6, 466–476.
Org. Biomol. Chem.
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