Copper-free sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells

Article abstract of DOI:10.1021/ja2066913

Bioorthogonal reactions suitable for functionalization of genetically or metabolically encoded alkynes, for example, copper-catalyzed azide-alkyne cycloaddition reaction ("click chemistry"), have provided chemical tools to study biomolecular dynamics and function in living systems. Despite its prominence in organic synthesis, copper-free Sonogashira cross-coupling reaction suitable for biological applications has not been reported. In this work, we report the discovery of a robust aminopyrimidine-palladium(II) complex for copper-free Sonogashira cross-coupling that enables selective functionalization of a homopropargylglycine (HPG)-encoded ubiquitin protein in aqueous medium. A wide range of aromatic groups including fluorophores and fluorinated aromatic compounds can be readily introduced into the HPG-containing ubiquitin under mild conditions with good to excellent yields. The suitability of this reaction for functionalization of HPG-encoded ubiquitin in Escherichia coli was also demonstrated. The high efficiency of this new catalytic system should greatly enhance the utility of Sonogashira cross-coupling in bioorthogonal chemistry.

Full text of DOI:10.1021/ja2066913

COMMUNICATION
pubs.acs.org/JACS
Copper-Free Sonogashira Cross-Coupling for Functionalization of
Alkyne-Encoded Proteins in Aqueous Medium and in Bacterial Cells
Nan Li, Reyna K. V. Lim, Selvakumar Edwardraja, and Qing Lin*
Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260-3000, United States
S Supporting Information
b
Table 1. Ligand Screen and Reaction Condition
Optimizationa
ABSTRACT: Bioorthogonal reactions suitable for functio-
nalization of genetically or metabolically encoded alkynes,
for example, copper-catalyzed azideÀalkyne cycloaddition
reaction (“click chemistry”), have provided chemical tools
to study biomolecular dynamics and function in living
systems. Despite its prominence in organic synthesis, cop-
per-free Sonogashira cross-coupling reaction suitable for
biological applications has not been reported. In this work,
we report the discovery of a robust aminopyrimidineÀ
palladium(II) complex for copper-free Sonogashira cross-
coupling that enables selective functionalization of a homo-
propargylglycine (HPG)-encoded ubiquitin protein in aqu-
eous medium. A wide range of aromatic groups including
fluorophores and fluorinated aromatic compounds can be
readily introduced into the HPG-containing ubiquitin under
mild conditions with good to excellent yields. The suitability
of this reaction for functionalization of HPG-encoded
ubiquitin in Escherichia coli was also demonstrated. The high
efficiency of this new catalytic system should greatly
enhance the utility of Sonogashira cross-coupling in bio-
entry
1a: 2a
Pd ligand
ligand
time (min)
yield (%)b
orthogonal chemistry.
3
1
2
3
4
5
6
7
8
1.05:1.00
1.05:1.00
1.05:1.00
1.05:1.00
1.05:1.00
1.05:1.00
1.05:1.00
1.00:2.40
20%
20%
20%
20%
20%
20%
20%
30%
L1
L2
L3
L4
L5
L6
L7
L2
30
30
30
30
30
30
30
40
NR
59
<5
<5
29
<5
13
91
ecause of their small sizes and ease in the genetic/metabolic
B
incorporation, simple organic groups such as azide, aldehyde,
terminal alkyne, and terminal alkenes are attractive bioorthogo-
nal reporters for studying biomolecular dynamics and functions
in their native environment.1 One of the key driving forces for
this approach has been the emergence of a growing repertoire
of bioorthogonal reactions2 through which designed small-
molecule probes can be selectively conjugated to pretagged bio-
molecules. For tracking biological dynamics, bioorthogonal
functionalization needs to be rapid, highly selective, and high
yielding. Indeed, an aldehyde can be selectively functionalized
with the hydrazide or alkoxyamine-based probes via nucleophilic
addition;3 an azide can be functionalized with the alkyne probes
via copper-catalyzed click chemistry,4 strained-promoted cyclo-
addition,5 or Staudinger ligation;6 and terminal alkenes can be
functionalized via either photoclick chemistry7 or ruthenium-
catalyzed olefin metathesis.8 While many aqueous reactions
involving terminal alkynes have been reported,9 the viable option
for functionalization of terminal alkynes in biological systems
remains to be click chemistry.10
a Reactions were carried out under ambient conditions without the need
of insert gas protection. b Yields were derived by comparing the
integration area of 3a in their respective HPLC traces to that of a
standard curve. See Supporting Information for details.
aqueous medium containing 18% DMSO.11 However, a low yield
of 25% was achieved after 80-min incubation at 6 °C under N2
protection; in addition, copper(I) salt was used as a cocatalyst,
which further limits its potential in cellular applications because
of the known cytotoxicity associated with copper.12 Inspired
by the recent report of a robust Suzuki-Miyaura cross-coupling
with protein substrates in aqueous medium,13 we envisioned that
with a suitable palladium catalyst, efficient copper-free Sonoga-
shira cross-coupling with protein substrates could be realized.
In an effort to apply palladium-catalyzed reactions to modify
proteins in vitro, Yokoyama and co-workers previously reported a
Sonogashira cross-coupling reaction between N-(prop-2-yn-1-yl)-
biotinamide and an iodophenyl-containing Ras protein in an
Received: July 18, 2011
Published: September 08, 2011
r
2011 American Chemical Society
15316
dx.doi.org/10.1021/ja2066913 J. Am. Chem. Soc. 2011, 133, 15316–15319
|

Journal of the American Chemical Society
COMMUNICATION
Table 2. Aryl Iodides in Aqueous Copper-Free Sonogashira
Cross-Coupling Reactionsa
Table 3. Aryl Iodide Substrates for Cross-Coupling with
HPGÀUba
a Reactions were carried out on a 4 μmol scale at 37 °C with 1:2a ratio
of 1:2.4. b Conversions were calculated based on the disappearance of
aryl iodides in the reaction mixtures in the HPLC. See Supporting
Information for details.
Here, we report the discovery of a new water-soluble palladium
complex, and its utility in copper-free Sonogashira cross-coupling
reactions in clean and rapid formation of new carbonÀcarbon
bonds between a terminal alkyne-containing peptide/protein
and a wide range of aryl iodides in aqueous medium. Furthermore,
we demonstrate the suitability of this reaction for fluorescent
labeling of an alkyne-encoded protein in Escherichia coli cells.
Our initial study focused on the Sonogashira cross-coupling
between fluorescein iodide 1a and a homopropargylglycine
(HPG)-containing peptide 2a using a water-soluble 2-amino-
4,6-dihydroxypyrimidine (ADHP) as the palladium ligand be-
cause ADHP has been previously used successfully in the copper-
free Sonogashira reactions in organic solvents.14 However, no
cross-coupling product was detected after 30 min (Table 1, entry 1);
similar result was obtained when the reaction was extended to
3 h. We attributed this lack of reactivity to the hydration of
ADHP at either the two hydroxy groups or the amino group in
aqueous medium. To alter the hydration shell of ADHP and fine-
tune the electron density of the pyrimidine ring, a series of
pyrimidine-based ligands (L2ÀL7) were synthesized and their
catalytic activities were examined in the model reaction in
potassium phosphate buffer (Table 1). To our delight, we found
that ligands L2 and L5 with the dimethylamino substituent
gave the desired product 3a in 59% and 29% yield, respectively
(entries 2 and 5). Blocking the two hydroxyl groups led to trace
amount of product (entries 3 and 4). Replacement of the amino
group with bulkier morpholine or piperidine group resulted
in substantially lower yields (entries 6 and 7). Using the
palladiumÀligand L2 complex, we obtained the cross-coupled
product 3a in 91% yield when palladium complex usage was
a Reactions were carried out with 5À10 μM of HPGÀUb and 50 equiv
of aryl iodide and palladium complex at 37 °C under ambient conditions.
b Yields were determined based on LCÀMS analysis of the reaction
mixtures based on the equation: yield % = Iproduct/(IHPGÀUb + Iproduct).
When side products were detected, the yield was calculated using
the equation: yield % = Iproduct/(IHPGÀUb + Iproduct + Iside product), where
I
HPGÀUb, Iproduct, and Iside product represent the ion counts of the
remaining HPGÀUb, product, and side product, respectively. See Sup-
porting Information for details. c Hydrolysis of the lactone ring was
observed in mass spectrum.
increased to 30% along with an excess of 2a and a slightly longer
reaction time (entry 8).
With the optimal conditions in hand, we then investigated the
generality of this protocol for various aryl iodide (Table 2). Both
ortho- or para-substituted aryl iodides gave good conversions
(entries 1, 3, and 5). Sterically hindered 2,4,6-trimethylphenyl
15317
dx.doi.org/10.1021/ja2066913 |J. Am. Chem. Soc. 2011, 133, 15316–15319

Journal of the American Chemical Society
COMMUNICATION
Figure 2. Fluorescent labeling of HPGÀUb by fluorescein iodide 1a in
E. coli cells via copper-free Sonogashira cross-coupling. (a) Fluorescence
image of the cell pellets upon excitation at 365 nm. Pellet 1, cells
collected after treatment with 100 μM of fluorescein iodide 1a but not
palladium complex; Pellet 2, cells collected after treatment with 100 μM
1a, 1 mM palladium complex, 5 mM sodium ascorbate in Na2HPO4
buffer at 37 °C for 4 h; Pellet 3, cells collected after treatment with the
preactivated mixture of palladium complex (1 mM) and 1a (100 μM).
See Supporting Information for the treatment details. (b) In-gel
fluorescence (left panel) and Coomassie blue staining (right panel) of
SDSÀPAGE analysis of the proteins captured by Ni-NTA-agarose beads
from the lysates of the three cell pellets.
(SDSÀPAGE) followed by in-gel fluorescence analysis. A clear
green fluorescent band was detected only for HPGÀUb but not
for Ub (Figure S2). We then examined a range of aryl iodides
containing various functional groups including hydroxy (1i),
carboxylic acid (1h), fluorine (1j, 1k), nitro (1l), and thiophene
(1c) groups (Table 3). In general, Sonogashira cross-coupling
reactions with HPGÀUb gave the modified proteins in good
yields (55À93%) independent of the electronic structures of aryl
iodides. Sterically hindered substrates such as 1g and 1m gave
lower yields (entries 7 and 13), even with extended reaction times.
To verify the yield and selectivity of the reaction, HPGÀUb was
incubated with 50 equiv of phenyl iodide covalently linked to
monodisperse polyethylene glycol (1n, MW ≈ 5 kDa) in a
sodium phosphate buffer at 37 °C for 30 min, and the mixture
was then analyzed by SDSÀPAGE (Figure 1). A distinct higher
molecular weight band corresponding to UbÀmPEG with an
estimated 84% yield was observed, matching closely the yield for
analogous substrate, 1-iodo-4-methoxy benzene (1f; Table 3,
entry 6).18 Importantly, no PEGylated products were detected in
the absence of the palladium complex, and the reaction pro-
ceeded only with HPGÀUb and not with Ub (lane 1 in Figure 1),
indicating that PEGylation occurs selectively with HPG via the
Sonogashira cross-coupling.
Since many enzyme-mediated biochemical transformations
are facilitated by transition metals,19 selective palladium-cata-
lyzed transformations inside living cells may offer an organome-
tallic tool to manipulate biological processes. To assess whether
the copper-free Sonogashira cross-coupling reaction can be
employed to modify HPGÀUb in bacterial cells, we added
1 mM of palladium complex, 100 μM of fluorescein iodide 1a,
and 5 mM of sodium ascorbate in sodium phosphate buffer to
HPGÀUb-overexpressing M15A cells. After growing at 37 °C for
4 h, cells were collected and washed extensively with 0.9% NaCl
solution to remove the excess reagents. The palladium complex-
treated cells showed green fluorescence upon 365-nm excitation
while the untreated cells did not (compare cell pellets 2 and 3 to
1 in Figure 2a). Subsequently, cells were lysed and HPGÀUb
proteins in the cell lysates were captured with Ni-NTA-agarose
beads. The beads were re-suspended in a protein elution buffer
and heated to 95 °C for 5 min. After centrifugation, the super-
natants were analyzed by SDSÀPAGE. In the in-gel fluorescence
Figure 1. Selective PEGylation of HPGÀUb with an mPEG-linked
phenyl iodide 1n via copper-free Sonogashira cross-coupling. (a) Reaction
scheme. (b) Coomassie blue staining of SDSÀPAGE gel showing
selective formation of the PEGylated Ub (UbÀmPEG): lane 1, control
reaction with wild-type ubiquitin; lane 2, control reaction with HPGÀ
Ub in the presence of 50 equiv of 1n but absence of palladium complex;
lane 3, reaction with HPGÀUb in the presence of 50 equiv of 1n and
palladium complex.
iodide also participated in the reaction and gave a 72% conver-
sion (entry 6). Heterocyclic aryl iodide, 2-iodothiophene, under-
went successful reaction with 2a to afford the cross-coupled
product in 95% conversion (entry 2). A fluorogenic 4-iodo-7-
methoxy-coumarin was also a good substrate (entry 4). However,
electron-deficient aryl iodides such as 4-nitrophenyl iodide gave
poor conversions under these conditions.
The encouraging results with peptide substrate 2a prompted
us to examine whether these conditions can be employed to
modify a HPG-encoded protein. We selected ubiquitin as a protein
model because of its small size and robust fold.15 Since HPG has
been shown in the literature to be an efficient methionine
surrogate for metabolic incorporation,16 we appended a Met-
Gly-Gly sequence to the C-terminus of ubiquitin on a pQE-80L
construct, and expressed the HPG-containing ubiquitin in M15A, a
methionine auxotroph, in the presence of HPG. After Ni-NTA
affinity chromatography, the N-terminal His-tag, together with
the first Met or HPG in the sequence, was cleaved by treating
the protein with PreScission protease. The resulting ubiquitin
was purified by FPLC to afford either wild-type ubiquitin (Ub)
or HPG-encoded ubiquitin (HPGÀUb) with one HPG at its
C-terminus. The HPG occupancy was determined to be 92%
based on the LCÀMS analysis. To test Sonogashira reaction with
the HPG-containing proteins, we incubated HPGÀUb with 50
equiv of 1a and 50 equiv of palladium complex using a two-step
addition procedure.17 Gratifyingly, the reaction reached comple-
tion in 30 min (Table 3, entry 1) as monitored by LCÀMS.
Control experiments showed that the reaction did not proceed
in the absence of palladium complex. By taking advantage of
the intrinsic fluorescence of 1a, we subjected the reaction mixture
to sodium dodecyl sulfate polyacrylamide gel electrophoresis
15318
dx.doi.org/10.1021/ja2066913 |J. Am. Chem. Soc. 2011, 133, 15316–15319

Journal of the American Chemical Society
COMMUNICATION
analysis, clear fluorescent bands were observed for samples
derived from cell pellets 2 and 3, but not for the sample derived
from pellet 1 (left panel, Figure 2b), indicating selec-
tive cross-coupling of 1a with HPGÀUb inside E. coli cells.
The identities of the fluorescent HPGÀUb bands were con-
firmed by Coomassie blue staining of the same gel (right panel,
Figure 2b). The lower abundance of HPGÀUb protein seen in
sample 2 was probably due to the inhibition of protein expression by
thepalladiumcomplex and 1a when they were added separately.20
In conclusion, we have discovered a new palladium complex
for the aqueous copper-free Sonogashira cross-coupling reactions.
This palladium complex promoted the efficient cross-coupling of
a wide range of aryl iodides with the terminal alkyne-containing
peptide and protein substrates in good to excellent yields. The
capability of this cross-coupling reaction in functionalizing a
metabolically encoded alkyne-containing protein in E. coli was
also demonstrated, indicating a potential utility of this reaction in
monitoring and manipulating proteins in cellular systems. Efforts
to further optimize this palladium-catalyzed cross-coupling
reaction for cellular applications, for example, by immobilizing
the complex onto nanoparticles to improve cell penetration and
reduce complex loading,21 are currently underway.
E. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 9482. (e) Blackman, M. L.;
Royzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008, 130, 13518.
(6) (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007. (b)
Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000, 2, 1939.
(c) K€ohn, M.; Breinbauer, R. Angew. Chem., Int. Ed. 2004, 43, 3106.
(7) Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew.
Chem., Int. Ed. 2008, 47, 2832. (b) Song, W.; Wang, Y.; Qu, J.; Lin, Q.
J. Am. Chem. Soc. 2008, 130, 9654. (c) Wang, Y.; Song, W.; Hu, W. J.; Lin,
Q. Angew. Chem., Int. Ed. 2009, 48, 5330. (d) Song, W.; Wang, Y.; Yu, Z.;
Vera, C. I.; Qu, J.; Lin, Q. ACS Chem. Biol. 2010, 5, 875. (e) Wang, J.;
Zhang, W.; Song, W.; Wang, Y.; Yu, Z.; Li, J.; Wu, M.; Wang, L.; Zang, J.;
Lin, Q. J. Am. Chem. Soc. 2010, 132, 14812.(f) Lim, R. K.; Lin, Q., Acc.
Chem. Res. 2011, ASAP, DOI: 10.1021/ar200021p.
(8) (a) Lin, Y. A.; Chalker, J. M.; Floyd, N.; Bernardes, G. J. L.; Davis,
B. G. J. Am. Chem. Soc. 2008, 130, 9642. (b) Ai, H.-w.; Shen, W.; Brustad,
E.; Schultz, P. G. Angew. Chem., Int. Ed. 2010, 49, 935. (c) Lin, Y. A.;
Chalker, J. M.; Davis, B. G. J. Am. Chem. Soc. 2010, 132, 16805.
(9) (a) Herrerías, C. I.; Yao, X.; Li, Z.; Li, C. J. Chem. Rev. 2007,
107, 2546. (b) Doucet, H.; Hierso, J. ÀC. Angew. Chem., Int. Ed. 2007,
46, 834. (c) Firouzabadi, H.; Iranpoor, N.; Gholinejad, M. J. Mol. Catal.
A: Chem. 2010, 321, 110.
(10) For a recent example, see:Besanceney-Webler, C.; Jiang, H.;
Zheng, T.; Feng, L.; Soriano del Amo, D.; Wang, W.; Klivansky, L. M.;
Marlow, F. L.; Liu, Y.; Wu, P. Angew. Chem., Int. Ed. 2011, 50, 8051.
(11) Kodama, K.; Fukuzawa, S.; Nakayama, H.; Sakamoto, K.;
Kigawa, T.; Yabuki, T.; Matsuda, N.; Shirouzu, M.; Takio, K.; Yokoyama,
S.; Tachibana, K. ChemBioChem 2007, 8, 232.
’ ASSOCIATED CONTENT
(12) (a) Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem. Soc.
2003, 125, 4686. (b) Kennedy, D. C.; Lyn, R. K.; Pezacki, J. P. J. Am.
Chem. Soc. 2009, 131, 2444.
(13) (a) Chalker, J. M.; Wood, C. S. C.; Davis, B. G. J. Am. Chem. Soc.
2009, 131, 16346. (b) Spicer, C. D.; Davis, B. G. Chem. Commun. 2011,
47, 1698.
S
Supporting Information. Synthetic schemes for new
b
ligands, experimental procedures, and characterization of all com-
pounds. This material is available free of charge via the Internet at
http://pubs.acs.org.
(14) Li, J. H.; Zhang, X. D.; Xie, Y. X. Eur. J. Org. Chem. 2005, 4256.
(15) Vijay-Kumar, S.; Bugg, C. E.; Cook, W. J. J. Mol. Biol. 1987,
194, 531.
(16) van Hest, J. C. M.; Kiick, K. L.; Tirrell, D. A. J. Am. Chem. Soc.
2000, 122, 1282.
’ AUTHOR INFORMATION
Corresponding Author
qinglin@buffalo.edu
(17) (a) Our preliminary studies indicated that excess amount of the
palladium complex was necessary to achieve high conversions due to
nonspecific sequestration of the palladium complex by the proteins; see
Figure S1 in Supporting Information for details. Similar phenomena
were also observed in the palladium-catalyzed Suzuki-Miyaura cross-
coupling reactions with protein substrates; see ref 13. To further alleviate
this problem, we also pre-incubated the palladium complex with the aryl
iodide (“pre-activation”) to minimize the effect of non-specific binding.
(b) 2.4% DMSO was used in the reaction to help dissolve fluorescein
iodide. (c) We found that adding the palladium complex/1a mixture pre-
incubated for 1 h (“pre-activated”) into the HPGÀUb solution in two
portions instead of one improved the reaction yield.
’ ACKNOWLEDGMENT
We gratefully acknowledge the National Institutes of Health
(R01 GM 085092) for financial support.
’ REFERENCES
(1) (a) Dieterich, D. C.; Hodas, J. J.; Gouzer, G.; Shadrin, I. Y.; Ngo,
J. T.; Triller, A.; Tirrell, D. A.; Schuman, E. M. Nat. Neurosci. 2010, 13,
897. (b) Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R.
Science 2008, 320, 664.
(18) The greater than 5 kDa increase in apparent MW is typically
observed in electrophoresis of PEGylated proteins: see ref 7c as well as
Lim, R. K.; Lin, Q. Chem. Commun. 2010, 46, 7993. Deiters, A.; Cropp,
T. A.; Summerer, D.; Mukherji, M.; Schultz, P. G. Bioorg. Med. Chem.
Lett. 2004, 14, 5743.
(19) (a) Lippard, S. J.; Berg, J. M. Principles of Bioinorganic Chemistry;
University Science Books: Mill Valley, CA, 1994. (b) Waldron, K. J.;
Rutherford, J. C.; Ford, D.; Robinson, N. J. Nature 2009, 460, 823. (c)
Antos, J. M.; Francis, M. B. Curr. Opin. Chem. Biol. 2006, 10, 253.
(20) In our cytotoxicity studies based on colony formation assay and
growth curve measurement, the pre-activated palladium complex/1a
mixture and 1a itself did not show toxicity to E. coli cells while palladium
complex and palladium complex/1a mixture added separately showed
some level of cytotoxicity. See Figure S4 in Supporting Information for
details.
(2) (a) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009,
48, 6974. (b) Lim, R. K.; Lin, Q. Chem. Commun. 2010, 46, 1589.(c)
Chalker, J. M.; Bernardes, G. J. L.; Davis, B. G. Acc. Chem. Res. 2011,
ASAP, DOI: 10.1021/ar200056q.
(3) (a) Carrico, I. S.; Carlson, B. L.; Bertozzi, C. R. Nat. Chem. Biol.
2007, 3, 321. (b) Zeng, Y.; Ramya, T. N.; Dirksen, A.; Dawson, P. E.;
Paulson, J. C. Nat. Methods 2009, 6, 207.
(4) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596. (b) Tornøe, C. W.; Christensen,
C.; Meldal, M. J. Org. Chem. 2002, 67, 3057. (c) Wang, Q.; Chan, T. R.;
Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc.
2003, 125, 3192.
(5) (a) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc.
2004, 126, 15046. (b) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.;
Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi,
C. R. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793. (c) Ning, X.; Guo, J.;
Wolfert, M. A.; Boons, G.-J. Angew. Chem., Int. Ed. 2008, 47, 2253.
(d) Dieterich, D. C.; Link, A. J.; Graumann, J.; Tirrell, D. A.; Schuman,
(21) Yusop, R. M.; Unciti-Broceta, A.; Johansson, E. M. V.; Sꢀanchez-
Martín, R. M.; Bradley, M. Nat. Chem. 2011, 3, 239.
15319
dx.doi.org/10.1021/ja2066913 |J. Am. Chem. Soc. 2011, 133, 15316–15319