Cat-ELCCA: A robust method to monitor the fatty acid acyltransferase activity of ghrelin O-acyltransferase (GOAT)

Article abstract of DOI:10.1002/anie.201003387

Assays armed with catalytic signal amplification have arisen as superior systems for ultrasensitive detection of analytes. A conceptually new enzyme assay called cat-ELCCA (catalytic assay using enzyme-linked click-chemistry) is described, in which an enzyme-linked azide is utilized to arm the assay with catalytic fluorescence signal amplification. By using this assay technology, the first high-throughput screen for recently disclosed ghrelin O-acyltransferase (GOAT) was developed. Copyright

Full text of DOI:10.1002/anie.201003387

Communications
DOI: 10.1002/anie.201003387
Enzyme Assays
cat-ELCCA: A Robust Method To Monitor the
Fatty Acid Acyltransferase Activity of Ghrelin
O-Acyltransferase (GOAT)**
Amanda L. Garner and Kim D. Janda*
Angewandte
Chemie
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Assays armed with catalytic signal amplification have arisen
as superior systems for ultrasensitive detection of analytes.[1,2]
In the biocatalysis field, while fluorogenic and colorimetric
enzyme assays designed to examine cleavage reactions have
taken advantage of this strategy,[3] many important enzyme
classes, including acyltransferases, have mechanistically been
excluded from such analytical techniques. As a result, the
visualization of activity for these enzymes (i.e. addition of a
post-translational modification) typically relies on autoradio-
graphic methods, which are often not high-throughput and are
hazardous, precluding library-based screening against these
targets.[4] For example, in the acyltransferase field, [3H]-
labeled fatty acids have traditionally been utilized to detect
protein fatty acid acylation using liquid scintillation counting,
although exceptions have been reported.[5] Despite these
efforts, high-throughput, fluorescence-based assays still
remain highly sought after in this field.
Figure 1. Comparison of A) cat-ELISA and B) catalytic assay using
enzyme-linked click-chemistry assay (cat-ELCCA). E=enzyme. S=sub-
strate.
As an alternative approach, several groups have recently
reported the use of click-chemistry-based approaches utiliz-
ing azido- and alkynyl-tagged fatty acids for the nonradioac-
tive detection of protein fatty-acylation in living cells.[6] In
specific, the use of CuI-catalyzed Huisgen [3+2] cycloaddition
chemistry[7] has proved to be a robust detection method for
such efforts. Interestingly, while click chemistry has found
widespread success in activity-labeling approaches such as
those described above, bioconjugation efforts[8] and enzyme
inhibitor discovery,[9] it has yet to be utilized in the develop-
ment of high-throughput screening assays, despite its biolog-
ical and chemical compatibility and near-quantitative
yields.[7c] Herein, we describe our efforts toward the develop-
ment of a click-chemistry-based, high-throughput screen to
monitor acyltransferase activity.
Taking a similar approach, we proposed to immobilize an
acyltransferase-recognized, biotinylated peptide fragment to
a streptavidin microtiter plate. This immobilized peptide
would then be incubated with an alkynyl-tagged fatty acid in
the presence of the enzyme (Figure 1B). Following this
enzyme-catalyzed acyl transfer, the tagged fatty acid ester
would then be converted to the enzyme-linked 1,2,3-triazole
using an azido-tagged enzyme by CuI-catalyzed Huisgen
[3+2] cycloaddition chemistry.[12] Subsequent addition of a
fluorogenic substrate of the linked enzyme would provide
catalytic signal amplification. To keep with the terminology of
cat-ELISA, we have named this assay technology catalytic
assay using enzyme-linked click-chemistry assay (cat-
ELCCA).
As a model enzyme to demonstrate this technology, we
chose to examine the recently disclosed acyltransferase,
ghrelin-O-acyltransferase (GOAT).[13] This membrane-
bound O-acyltransferase (MBOAT)[14] family member cata-
lyzes the n-octanoylation of ghrelin (Figure 2), a 28-amino
acid growth-hormone-releasing peptide implicated in feeding,
weight gain, and the regulation of energy homeostasis, and
identified as a leading anti-obesity drug target.[15] Importantly,
the functions of this peptide hormone are solely dependent on
its n-octanoylation at Ser-3 by GOAT.[16] Of further signifi-
cance, ghrelin is the only known peptide to contain an n-
Inspired by enzyme immunoassays,[10] in particular cat-
ELISA (catalytic assay using ELISA),[11] we sought to design
an acyltransferase assay that takes advantage of the catalytic
signal amplification obtained with enzyme-linked detection
antibodies. In cat-ELISA (Figure 1A), a solid-supported
substrate is first treated with a catalytic antibody (or
enzyme) to obtain a product, which is subsequently bound
with a product-specific antibody. This antibody is then
detected with an enzyme-linked secondary antibody, the
enzyme of which catalyzes the turnover of a fluorescent or
colorimetric substrate in solution to provide signal amplifica-
tion necessary for sensitive quantitation.
[*] Dr. A. L. Garner, Prof. K. D. Janda
Departments of Chemistry and Immunology
The Skaggs Institute for Chemical Biology and
The Worm Institute for Research and Medicine
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: (+1)858-784-2595
E-mail: kdjanda@scripps.edu
[**] This work was supported by the Skaggs Institute for Chemical
Biology (K.D.J.) and by the NIH (postdoctoral fellowship F32-
DK083179 to A.L.G.). We are grateful to Damian Ekiert for
assistance with baculovirus expression, Prof. M. G. Finn for helpful
discussions, and Vu Hong for donation of THPTA.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201003387.
Figure 2. GOAT-catalyzed n-octanoyl transfer to ghrelin.
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octanoyl post-translational modification,[16] thus, this peptide
known HPLC traces of n-octanoyl–ghrelin(1–28) and the
unacylated peptide, the activity of GOAT in this crude extract
was confirmed (data not shown). Additionally, no octanoyl
transfer was observed in uninfected Sf9 cell membranes (data
not shown).
is most likely the only substrate for GOAT and inhibitors of
this enzyme should be highly specific. As our group has
previously examined a vaccine-based approach for sequester-
ing[17] and/or catalyzing the hydrolysis[18] of ghrelin using
monoclonal antibodies as anti-obesity strategies, we were also
interested in establishing a small-molecule-based program
targeting GOAT since currently no validated chemical probes
for this enzyme have been reported.[19a]
Next, we turned our attention toward the preparation of
suitable ghrelin and azido-HRP substrates. For an immobi-
lized ghrelin substrate, we chose a ghrelin(1–5) pentapeptide
containing a C-terminal biotin, which was synthesized using
solid-phase peptide synthesis (see the Supporting Informa-
tion). Ghrelin(1–5) was chosen as this has been shown to be
the minimum recognition sequence for both GOAT activi-
ty[19a] and ghrelinꢀs receptor, growth hormone secretagogue
receptor 1a (GHS-R1a),[20] and is synthetically tractable for
high-throughput efforts. As anticipated, this ghrelin substrate
was found to be recognized by GOAT (HPLC data not
shown). To prepare the azido-tagged HRP, we used an
aldehyde-functionalized peroxidase as an amine-reactive
HRP, and this activated HRP was treated with 11-azido-
3,6,9-trioxaundecan-1-amine to obtain azido-tagged HRP
(see the Supporting Information).
With the assay components in hand, we set out to test our
design hypothesis. The biotinylated ghrelin(1–5)pentapeptide
was first immobilized to the wells of a streptavidin-coated
microtiter plate (96-well, black), which was to be followed
with the GOAT-catalyzed acyl transfer reaction. Here, the
solid-supported ghrelin substrate was first incubated with
membrane-bound GOAT ( ꢀ 50 mg) in HEPES buffer (50 mm
pH 7.0) for 5 min at 378C. Palmitoyl-CoA (50 mm) and n-
octynoyl-CoA (1 mm) were then added to the mixture to
initiate the reaction. Palmitoyl-CoA was included as a
competing fatty acid thioester to reduce any background
hydrolysis of n-octynoyl-CoA due to esterases in the crude
membrane extract.[19a] The click reaction was then performed
with azido-tagged HRP (Figure 3A). It is important to note
here that in model studies all attempts to use the commer-
cially available tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]a-
mine (TBTA)[21] ligand at low concentrations of substrate
failed; however, use of the more hydrophilic ligand, tris[(3-
hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine (THPTA)
(Figure 3A)[22] recently disclosed by the Finn group, resulted
in consistently successful coupling regardless of substrate
To date, only two in vitro assay systems have been
reported for screening GOAT activity.[19] Similar to other
acyltransferase assays, both rely on autoradiographic methods
and neither is amenable to high-throughput screening. Since
we envisioned establishing a library-based screening effort
against GOAT and the current screening assays impede such
efforts, we wanted to use our cat-ELCCA approach to design
a robust assay for GOAT. Our assay design is shown in
Figure 3. In brief, a biotinylated ghrelin peptide immobilized
on a streptavidin-modified microtiter plate would be incu-
bated with GOATand alkynyl-tagged n-octynoyl-CoA for the
acyl transfer reaction; the wells would then be subjected to
click-chemistry conditions. For the azide-labeled enzyme, we
chose horseradish peroxidase (HRP), as activated HRP
derivatives for bioconjugation and fluorescent/colorimetric
substrates for this enzyme are commercially available.
Following the click reaction, the HRP-triazole product
would be detected using amplex red as a fluorogenic substrate
in the presence of hydrogen peroxide to provide catalytic
fluorescence signal amplification of the octynoyl ester–HRP
triazole product concentration.
To test the applicability of cat-ELCCA to the GOAT
problem, we first isolated the GOAT enzyme. As previously
described,[19a] Sf9 insect cells were infected with a recombi-
nant baculovirus encoding mouse GOAT for protein over-
expression. Since GOAT is a transmembrane protein and
efforts to purify this enzyme to homogeneity have previously
failed,[19a] the crude GOAT-containing membrane fraction
was isolated by ultracentrifugation techniques, and the
resultant membrane fraction was used as such for all
subsequent assays. The acyltransferase activity of the isolated
membrane-bound GOAT was confirmed with a control
reaction using ghrelin(1–28) and n-octanoyl-CoA. Based on
Figure 3. cat-ELCCA for screening GOAT activity. A) Assay design. B) Proof-of-concept data. ++=Sample treated with both GOAT-containing
membranes and n-octynoyl-CoA. RFU=normalized relative fluorescence units at 585 nm.
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concentration (data not shown). After the click reaction, the
wells were treated with amplex red and hydrogen peroxide,
and the resulting resorufin fluorescence was measured (lex =
560 nm, lem = 585 nm). As shown in Figure 3B, our assay
design was successful, and under these conditions a 7.5-fold
fluorescence enhancement (raw fluorescence data) was
observed over wells not treated with GOAT or n-octynoyl-
CoA. It is important to note here that this is the first time that
GOAT has been shown to accept an alkynyl-CoA substrate.
Given these successful results, we next characterized the
GOAT-catalyzed acyl transfer reaction using our cat-ELCCA
system. We first examined the time dependence of n-octanoyl
transfer to the immobilized ghrelin(1–5)pentapeptide. As
Figure 4A shows, the formation of n-octynoyl-ghrelin(1–
5)pentapeptide was linear with time for ca. 2 min. The assay
was also linear with respect to the amount of GOAT-
containing membranes (up to ca. 25 mg) added to the wells
(Figure 4B). It is important to note here that beyond the
range shown in Figure 4B, a slight decrease in product
formation was observed, which is likely due to an increased
concentration of competing esterases in the crude membrane
preparation. As Figure 4C and D show, the assay also
exhibited saturation kinetics with respect to the concentra-
tions of n-octynoyl-CoA and immobilized ghrelin(1–5)penta-
peptide.
v5.0a) showed that the data fit Michaelis–Menten kinetics
(see the Supporting Information). The apparent Km values
were 67.5 nm and 99.8 nm for n-octynoyl-CoA and the
immobilized ghrelin(1–5)pentapeptide, respectively. In addi-
tion, to assess the suitability of cat-ELCCA for high-
throughput screening, we also determined the signal-to-
noise ratio (S/N), signal-to-background ratio (S/B) and Z’
factor, a statistical parameter for scoring assay perfor-
mance.[23] The S/N ratio was 24, the S/B ratio was 3.5 and
the Z’ factor was 0.63 (see the Supporting Information),
indicating the potential of our assay for high-throughput
screening. Most important of these statistical indicators is the
Z’ factor, which is reflective of both dynamic range and data
variation associated with the signal measurement, and Z’
values between 0.5 and 1 are typically regarded as excellent
assays.
In summary, we have described a conceptually new
enzyme assay based on cat-ELISA, catalytic assay using
enzyme-linked click-chemistry assay, or cat-ELCCA. By
utilizing an enzyme-linked click-chemistry substrate, we are
able to arm the assay with catalytic signal amplification
necessary for ultrasensitive detection. Using this assay
technology, we developed a screening system for the acyl-
transferase of ghrelin, ghrelin O-acyltransferase (GOAT),
that is efficient, sensitive, and inexpensive, and importantly,
obviates the need for radioisotopes. As GOAT has been
implicated as a potential anti-obesity target and much more is
still to be learned about this recently disclosed MBOAT
family member, we envision that cat-ELCCA will be useful
for discovering antagonists and chemical probes for this
enzyme, a goal that we are currently pursuing. Finally, as the
immobilized peptide and alkynyl
Because we envision that this assay will be used to screen
for antagonists against this potential anti-obesity target, it was
also important to determine the apparent Km values for
GOAT. Initial velocities were measured for varying concen-
trations of n-octynoyl-CoA and immobilized ghrelin(1–
5)pentapeptide. Nonlinear regression (GraphPad Prism
substrates are modular, cat-
ELCCA should find additional
uses with other fatty acid acyl-
transferases, such as myristoyl-
transferases and palmitoyltrans-
ferases, other biologically rele-
vant acyltransferases (e.g. histone
acetyltransferases, N-acetyltrans-
ferases), and the discovery of
previously unidentified proteins
with acyltransferase activity.
Experimental Section
Membrane-bound GOAT (ca. 50 mg)
and HEPES buffer (50 mm, pH 7.0)
were added to wells of a black, 96-
well Reacti-Bind Streptavidin High-
Binding Capacity microtiter plate
containing immobilized ghrelin(1–
5)pentapeptide, and the plate was
incubated at 378C for 5 min. Palmi-
toyl-CoA (50 mm) and n-octynoyl-
CoA (1.0 mm) were then added to
Figure 4. Characterization of GOAT acyltransferase activity using cat-ELCCA. A) Time dependence.
B) Dependence on the amount of GOAT membranes. C) Dependence on n-octynoyl-CoA concentration.
D) Dependence on immobilized ghrelin(1–5)pentapeptide concentration. RFU=normalized relative
fluorescence units at 585 nm.
the mixture to initiate the enzyme-
catalyzed reaction (total volume
100 mL), and the plate was incubated
at 378C for another 5 min. The
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GOAT reaction mixture was then removed, and the wells were
[7] a) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless,
Angew. Chem. 2002, 114, 2708 – 2711; Angew. Chem. Int. Ed.
2002, 41, 2596 – 2599; b) C. W. Tornøe, C. Christensen, M.
Meldal, J. Org. Chem. 2002, 67, 3057 – 3064; c) M. Meldal,
C. W. Tornoe, Chem. Rev. 2008, 108, 2952 – 3015.
[8] Q. Wang, T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless,
M. G. Finn, J. Am. Chem. Soc. 2003, 125, 3192 – 3193.
[9] a) A. Krasinski, Z. Radic, R. Manetsch, J. Raushel, P. Taylor,
K. B. Sharpless, H. C. Kolb, J. Am. Chem. Soc. 2005, 127, 6686 –
6692; b) V. P. Mocharla, B. Colasson, L. V. Lee, S. Rꢂper, K. B.
Sharpless, C.-H. Wong, H. C. Kolb, Angew. Chem. 2005, 117,
118 – 122; Angew. Chem. Int. Ed. 2005, 44, 116 – 120; c) R.
Srinivasan, J. Li, S. Ling Ng, K. A. Kalesh, S. Q. Yao, Nat. Protoc.
2007, 2, 2655 – 2664.
washed with TBST (3 ꢁ 200 mL) followed by PBS (pH 7.4) (3 ꢁ
200 mL). Fresh PBS (pH 7.4), HRP-N3 (1.0 mm), CuSO4 (100 mm),
THPTA (500 mm), and sodium ascorbate (5 mm) were then added
(total volume 100 mL), and the plate was covered and allowed to
shake at 258C for 2 h. The click reaction mixture was then removed,
and the wells were washed again with TBST (3 ꢁ 200 mL) and PBS
(pH 7.4) (3 ꢁ 200 mL). For detection, 100 mL of amplex red/H2O2
solution (QuantaRed Enhanced Chemifluorescent HRP Substrate
kit) was added to each well, and the fluorescence (lex = 560 nm, lem
=
585 nm) was measured over time (ca. 10 min) on a SpectraMax M2e
Microplate Reader (Molecular Devices). A standard curve was
generated using known concentrations of HRP-N3 in the reaction
buffer.
[10] a) G. B. Wisdom, Clin. Chem. 1976, 22, 1243 – 1255; b) J. P.
Gosling, Clin. Chem. 1990, 36, 1406 – 1427.
Received: June 4, 2010
[11] D. S. Tawfik, B. S. Green, R. Chap, M. Sela, Z. Eshhar, Proc.
Natl. Acad. Sci. USA 1993, 90, 373 – 377.
Published online: September 15, 2010
[12] Immobilized substrates have previously been utilized in click
chemistry, see M. C. Bryan, F. Fazio, H.-K. Lee, C.-Y. Huang, A.
Chang, M. D. Best, D. A. Calarese, O. Blixt, J. C. Paulson, D.
Burton, I. A. Wilson, C.-H. Wong, J. Am. Chem. Soc. 2004, 126,
8640 – 8641.
[13] a) J. Yang, M. S. Brown, G. Liang, N. V. Grishin, J. L. Goldstein,
Cell 2008, 132, 387 – 396; b) J. A. Gutierrez, P. J. Solenberg, D. R.
Perkins, J. A. Willency, M. D. Knierman, Z. Jin, D. R. Witcher, S.
Luo, J. E. Onyia, J. E. Hale, Proc. Natl. Acad. Sci. USA 2008, 105,
6320 – 6325.
[14] K. Hofmann, Trends Biochem. Sci. 2000, 25, 111 – 112.
[15] a) M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, K.
Kangawa, Nature 1999, 402, 656 – 660; b) M. H. Tschꢂp, D. L.
Smiley, M. L. Heiman, Nature 2000, 407, 908 – 913; c) T. R.
Castaꢅeda, J. Tong, R. Datta, M. Culler, M. H. Tschꢂp, Front.
Neuroendocrinol. 2010, 31, 44 – 60.
[16] M. Kojima, K. Kangawa, Physiol. Rev. 2005, 85, 495 – 522.
[17] E. P. Zorrilla, S. Iwasaki, J. A. Moss, J. Chang, J. Otsuji, K. Inoue,
M. M. Meijler, K. D. Janda, Proc. Natl. Acad. Sci. USA 2006, 103,
13226 – 13231.
[18] A. V. Mayorov, N. Amara, J. Y. Chang, J. A. Moss, M. S. Hixon,
D. I. Ruiz, M. M. Meijler, E. P. Zorrilla, K. D. Janda, Proc. Natl.
Acad. Sci. USA 2008, 105, 17487 – 17492.
[19] An initial report of product-based inhibitors has been described
in Ref. [19a]; a) J. Yang, T.-J. Zhao, J. L. Goldstein, M. S. Brown,
Proc. Natl. Acad. Sci. USA 2008, 105, 10750 – 10755; b) H.
Ohgusu, K. Shirouzu, Y. Nakamura, Y. Nakashima, T. Ida, T.
Sato, M. Kojima, Biochem. Biophys. Res. Commun. 2009, 386,
153 – 158.
[20] a) M. A. Bednarek, S. D. Feighner, S.-S. Pong, K. K. McKee,
D. L. Hreniuk, M. V. Silva, V. A. Warren, A. D. Howard,
L. H. Y. Van der Ploeg, J. V. Heck, J. Med. Chem. 2000, 43,
4370 – 4376; b) M. Matsumoto, H. Hosoda, Y. Kitajima, N.
Morozumi, Y. Minamitake, S. Tanaka, H. Matsuo, M. Kojima, Y.
Hayashi, K. Kangawa, Biochem. Biophys. Res. Commun. 2001,
287, 142 – 146.
Keywords: alkynes · azides · enzymes · peptides
.
[1] W. J. Blaedel, R. C. Boguslaski, Anal. Chem. 1978, 50, 1026 –
1032.
[2] a) L. Zhu, V. M. Lynch, E. V. Anslyn, Tetrahedron 2004, 60,
7267 – 7275; b) Q. Wu, E. V. Anslyn, J. Am. Chem. Soc. 2004,
126, 14682 – 14683; c) Q. Wu, E. V. Anslyn, J. Mater. Chem. 2005,
15, 2815 – 2819; d) R. J. T. Houk, E. V. Anslyn, New J. Chem.
2007, 31, 729 – 735; e) N. C. Gianneschi, S. T. Nguyen, C. A.
Mirkin, J. Am. Chem. Soc. 2005, 127, 1644 – 1645; f) M. S.
Masar III, N. C. Gianneschi, C. G. Oliveri, C. L. Stern, S. T.
Nguyen, C. A. Mirkin, J. Am. Chem. Soc. 2007, 129, 10149 –
10158; g) N. Graf, M. Gꢂritz, R. Krꢃmer, Angew. Chem. 2006,
118, 4117 – 4119; . Chem. Int. Ed. 2006, 45, 4013 – 4015; h) N.
Graf, R. Krꢃmer, Chem. Commun. 2006, 4375 – 4376; i) F. Song,
A. L. Garner, K. Koide, J. Am. Chem. Soc. 2007, 129, 12354 –
12355; j) A. L. Garner, K. Koide, J. Am. Chem. Soc. 2008, 130,
16472 – 16473; k) A. L. Garner, F. Song, K. Koide, J. Am. Chem.
Soc. 2009, 131, 5163 – 5171.
[3] a) B. Rotman, Proc. Natl. Acad. Sci. USA 1961, 47, 1981 – 1991;
b) J.-L. Reymond, V. S. Fluxꢄ, N. Maillard, Chem. Commun.
2009, 34 – 46.
[4] M. D. Resh, Methods 2006, 40, 191 – 197.
[5] a) A. S. Varner, M. L. De Vos, S. P. Creaser, B. R. Peterson, C. D.
Smith, Anal. Biochem. 2002, 308, 160 – 167; b) A. S. Varner, C. E.
Ducker, Z. Xia, Y. Zhuang, M. L. De Vos, C. D. Smith, Biochem.
J. 2003, 373, 91 – 99; c) A. T. Lada, M. Davis, C. Kent, J.
Chapman, H. Tomoda, S. Omura, L. L. Rudel, J. Lipid Res. 2004,
45, 378 – 386; d) C. E. Ducker, L. K. Griffel, R. A. Smith, S. N.
Keller, Y. Zhuang, Z. Xia, J. D. Diller, C. D. Smith, Mol. Cancer
Ther. 2006, 5, 1647 – 1659.
[6] a) H. C. Hang, E.-J. Geutjes, G. Grotenberg, A. M. Pollington,
M. J. Bijlmakers, H. L. Ploegh, J. Am. Chem. Soc. 2007, 129,
2744 – 2745; b) A. Raghavan, G. Charron, J. Flexner, H. C. Hang,
Bioorg. Med. Chem. Lett. 2008, 18, 5982 – 5986; c) B. R. Martin,
B. F. Cravatt, Nat. Methods 2009, 6, 135 – 138; d) G. Charron,
M. M. Zhang, J. S. Yount, J. Wilson, A. Raghaven, E. Shamir,
H. C. Hang, J. Am. Chem. Soc. 2009, 131, 4967 – 4975; e) R. N.
Hannoush, N. Arenas-Ramirez, ACS Chem. Biol. 2009, 4, 581 –
587; f) M. M. Zhang, L. K. Tsou, G. Charron, A. Raghaven,
H. C. Hang, Proc. Natl. Acad. Sci. USA 2010, 107, 8627 – 8632.
[21] T. R. Chan, R. Hilgraf, K. B. Sharpless, V. V. Fokin, Org. Lett.
2004, 6, 2853 – 2855.
[22] V. Hong, S. I. Presolski, C. Ma, M. G. Finn, Angew. Chem. 2009,
121, 10063 – 10067; Angew. Chem. Int. Ed. 2009, 48, 9879 – 9883.
[23] J.-H. Zhang, T. D. Y. Chung, K. R. Oldenburg, J. Biomol.
Screening 1999, 4, 67 – 73.
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