Rapid cross-metathesis for reversible protein modifications via chemical access to se-allyl-selenocysteine in proteins

Article abstract of DOI:10.1021/ja403191g

Cross-metathesis (CM) has recently emerged as a viable strategy for protein modification. Here, efficient protein CM has been demonstrated through biomimetic chemical access to Se-allyl-selenocysteine (Seac), a metathesis-reactive amino acid substrate, via dehydroalanine. On-protein reaction kinetics reveal a rapid reaction with rate constants of Seac-mediated-CM comparable or superior to off-protein rates of many current bioconjugations. This use of Se-relayed Seac CM on proteins has now enabled reactions with substrates (allyl GlcNAc, N-allyl acetamide) that were previously not possible for the corresponding sulfur analogue. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (KAc, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. Moreover, Cope-type selenoxide elimination allowed this putative marker (and function) to be chemically expunged, regenerating an H3 that can be rewritten to complete a chemically enabled "write (CM)-erase (ox)-rewrite (CM)" cycle.

Full text of DOI:10.1021/ja403191g

Communication
pubs.acs.org/JACS
Rapid Cross-Metathesis for Reversible Protein Modifications via
Chemical Access to Se-Allyl-selenocysteine in Proteins
Yuya A. Lin, Omar Boutureira,† Lukas Lercher, Bhaskar Bhushan, Robert S. Paton,
and Benjamin G. Davis*
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
S
* Supporting Information
Scheme 1. Allyl Chalcogen-Assisted Cross-Metathesis
ABSTRACT: Cross-metathesis (CM) has recently
emerged as a viable strategy for protein modification.
Here, efficient protein CM has been demonstrated
through biomimetic chemical access to Se-allyl-selenocys-
teine (Seac), a metathesis-reactive amino acid substrate, via
dehydroalanine. On-protein reaction kinetics reveal a rapid
reaction with rate constants of Seac-mediated-CM
comparable or superior to off-protein rates of many
current bioconjugations. This use of Se-relayed Seac CM
on proteins has now enabled reactions with substrates
(allyl GlcNAc, N-allyl acetamide) that were previously not
possible for the corresponding sulfur analogue. This CM
strategy was applied to histone proteins to install a mimic
of acetylated lysine (KAc, an epigenetic marker). The
resulting synthetic H3 was successfully recognized by
antibody that binds natural H3-K9Ac. Moreover, Cope-
type selenoxide elimination allowed this putative marker
(and function) to be chemically expunged, regenerating an
tional incorporation of Seac has not yet been demonstrated,
H3 that can be rewritten to complete a chemically enabled
likely due to rapid oxidative cellular metabolism, also observed
for selenomethionine and S-methyl-selenocysteine.12−14 Despite
the precedence for incorporation of Sec into recombinant
proteins in Escherichia coli, this approach requires more complex
genetic manipulation of both DNA and RNA15,16 that limits
widespread utility. Moreover, our attempts to convert Sec to Seac
via a desulfurative 2,3-sigmatropic rearrangement failed in model
systems (see Supporting Information (SI) p S9). Notably, a
recent study has revealed a biosynthetic mechanism for
selenocysteine generation via conjugate C−Se bond formation
from a dehydroalanyl-tRNASec intermediate.17 Inspired by
nature, we therefore considered an alternative biomimetic
route to incorporate Seac into proteins via dehydroalanine
(Dha), which has recently emerged as a versatile chemical handle
for protein modifications.18−22
First, the generation of an appropriate conjugate, allyl
selenolate nucleophile, was explored on model Dha 3 (Scheme
2a). In situ generation of allyl selenolate via cleavage of methyl
benzoselenoate 2a failed (Table S1). Next, we considered
reductive access. Diallyl diselenide (2b) is unstable and exists in
mixture with diallyl selenide.23 Moreover, allyl selenolate has
previously only been reductively generated under aprotic
conditions.23 Nevertheless, treatment of 2b with a stoichiometric
amount of NaBH4 in degassed MeOH generated allyl selenolate;
subsequent addition of this solution to Dha 3 in water yielded
“write (CM)−erase (ox)−rewrite (CM)” cycle.
eveloping strategies for site-specific protein modifications
1−3 For over a
Dis an ongoing challenge in chemical biology.
decade, allylic alcohols have been identified as reactive in olefin
metathesis.4,5 However, only in recent years has a generalized
allylic heteroatom effect begun to emerge in cross-metathesis
(CM).5 A mechanism involving pre-coordination to the metal
center of catalyst 1 has been proposed to explain the observed
reactivity of privileged substrates, such as amino acid S-allyl-
cysteine (Sac, Scheme 1).6 This “heteroatom-relay” has enabled
CM to be applied as a bioconjugation technique in selective
protein modifications,7−11 and so greatly increased the bench-
mark for complexity in the substrate scope. Such putative
beneficial effects of allylic chalcogens suggest a likely increase in
the reactivity of higher chalcogens as Lewis bases that may be
more preferred, softer ligands for ruthenium complexes. Indeed,
simple allyl selenides are reactive substrates in aqueous CM.11
We show here that installation into peptides and proteins of the
simplest amino acid residue containing an allyl selenide moiety,
the unnatural amino acid Se-allyl-selenocysteine (Seac), enables
rapid and efficient CM.
Possible approaches to incorporating Seac into proteins were
considered that relied on installation of the Se heteroatom either
co- (either using Seac directly or from a precursor such as
selenocysteine (Sec)) or post-translationally. Direct co-transla-
Received: March 30, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja403191g | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Scheme 2. Biomimetic Synthesis of Se-Allyl-selenocysteine via
Conjugate Addition of Allyl Selenolate to Dehydroalanine
(Table S1)
Scheme 3. Enhanced CM of Seac-SBL 6a, cf. Sac-SBL 9a
53% of the desired Seac product 4 (Table S1). Allyl
selenocyanate (2c), a potentially more stable,24 alternative allyl
selenolate source, was investigated next. Pleasingly, due to
cleaner pre-reduction, the desired addition product 4 was
obtained with an improved yield of 86% after 1 h at room
temperature (Scheme 2a). Notably, although allyl selenolate is
sensitive to acidic conditions (allylselenol quickly decomposes to
2b in the absence of radical inhibitor23), under buffered basic
conditions the lifetime of allyl selenolate is apparently long
enough to successfully react with Dha in aqueous media.
Figure 1. Kinetic studies of CM between SBL-156Seac (6a) and allyl
alcohol. (a) LC-MS of reaction showing conversions over time. (b)
Changes in [product] as a function of time. Inset shows kinetics of CM
with SBL-156Sac (9a); note difference in time scale. Conditions:
[protein] = 0.01 mM, [cat.] = 1 mM, [allyl alcohol] = 52 mM, [MgCl2·
6H2O] = 26 mM in 3:7 tBuOH/NaPi (50 mM, pH 5) at room
temperature. (c) CM of 6a with various concentrations of allyl alcohol.
(d) Determination of the second-order CM rate constant for 6a.
With promising results observed on amino acid models, we
next explored installation of Seac via this biomimetic strategy in
two differing protein structural motifs/folds: three-layer α/β-
Rossman-fold protein subtilisin from Bacillus lentus (SBL) and
all-β-helix protein 275−276 from Nostoc punctiforme (termed
Npβ),25 respectively. Accordingly, Dha substrate proteins SBL-
156Dha (5) and Npβ-61Dha (7) were synthesized from
corresponding cysteine mutants following reported bis-alkylative
elimination procedures.21 Use of selenolate derived from 2a or
2b failed on proteins and led to only a mixture of products.
However, allyl selenolate solution generated from 2c allowed
successful biomimetic conjugate addition (Scheme 2b); LC-MS
analysis revealed >95% conversion of SBL-156Dha (5) to the
desired product SBL-156Seac (6a) after 1 h at room temper-
ature. Even addition to the more sterically demanding26 61 site in
Npβ-61Dha (7) single Dha mutant under slightly elevated
temperature (37 °C) afforded expected Seac-containing protein
with >95% conversion after 2 h (8a, see SI). Importantly,
resulting SBL-derivative proteins retained fold and functional
(peptidase) activity (see SI p S23). Generation of Dha and
addition likely leads to the formation of a 1:1 dr of adducts.26
With model Seac-tagged protein 6a in hand, CM with allyl
alcohol was tested using catalyst 1 under previously optimized
reaction conditions11 (which can be used at pH 4−8). This
reaction was strikingly rapid and was complete after only 15 min
at room temperature (Scheme 3). In comparison, the sulfur-
equivalent protein substrate SBL-156Sac (9a) required 4 h to
reach completion under the same reaction conditions. Rapid
protein modification reactions are rare but powerful.27,28 We
conducted a quantitative comparison of the enhanced rate
observed here for CM of SBL-156Seac with popular
bioconjugation techniques such as Staudinger ligation,29,30
azide−alkyne cycloaddition,31,32 and inverse electron-demand
Diels−Alder cycloaddition,33−36 which have typically been
studied in small-molecule models (“off-protein”). Under
pseudo-first-order conditions with respect to the protein, starting
material and product were monitored by LC-MS at several time
points during reactions (Figure 1a).37 Data analyzed using
nonlinear, single-exponential regression gave pseudo-first-order
rate constants (Figure 1b).
Determination of pseudo-first-order rate constants at various
allyl alcohol concentrations and analysis of the results using linear
regression revealed second-order rate constants for Seac-
mediated CM. The on-protein second-order rate constant for
the CM of allyl alcohol with Seac-protein was determined to be
0.31 0.004 M−1 s−1 (Figure 1c,d), whereas the value for on-
protein Sac-mediated CM in an identical protein context (site
156) was some 10-fold lower (0.031 0.0015 M−1 s−1) under the
same conditions (Figures S21 and S22). Interestingly, Figure 1d
indicated an initially linear increase in observed rate with respect
to allyl alcohol concentration and gradually plateaued at
concentrations >20 mM; this suggests that the catalyst plays a
critical role under these conditions, presumably becoming
“saturated” at high concentrations of allyl alcohol, leading to a
shift in rate-limiting step (see also SI section 2.10 for off-protein
catalyst dependency). Preliminary computations (using methyl
allyl chalcogenides, see SI section 3) suggest a reaction profile in
which the Ru-carbene intermediate after “Se-relay” is unusually
stable and enhanced in stability over “S-relay”; this intermediate
is also accessed through a lowered transition state.
When comparing the second-order rate constants with those
from widely used bioconjugation techniques in chemical biology,
B
dx.doi.org/10.1021/ja403191g | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Scheme 5. Synthesis of H3-9Seac (13)
Scheme 6. CM Reactions on H3-9Seac and the Chemical
“Write−Read−Erase” Cycle via a K9Ac Mimic “Mark” and
Anti-K9Ac Western Blots of H3 Proteins (Also See SI)
Figure 2. Selected rate constants (M−1 s−1) of conjugations. All rate
a
constants are second-order in units of M−1 s−1 with benzyl azide,
c
d
bnorbornenol, bicyclo[6.1.0]non-4-yn-9-ylmethanol, trans-cycloocte-
nol, and eallyl alcohol.
Scheme 4. Cross-Metathesis of SBL 6a and 9a with
Challenging Substrates 10 and 11
selenolate addition to Dha 12 proceeded successfully to yield
Seac protein 13 after 1 h at room temperature (Scheme 5).
With H3-9Seac (13) in hand, we carried out model CM with
allyl alcohol. Coloration implying metal coordination to 13 was
observed; such coordination6,50 can necessitate metal scavenging
prior to MS analysis. Use of 3-mercaptopropionic acid51,50
allowed ready MS monitoring of H3 CM reactions. Pleasingly,
this revealed CM of H3-K9Seac and allyl alcohol with nearly full
conversion to CM-modified H3 14 (Scheme 6a). This ability to
modify site 9 suggested a strategy for chemical recapitulation
(under benign conditions) of a “write−read−erase” cycle that is
observed (enzyme-mediated) in histone epigenetics.48,49 As a
“write” step we used the more challenging N-allyl acetamide
olefin in CM; complete conversion was observed to 15 (Scheme
6a). As a “read” step, CM-modified H3 (15) was analyzed by an
antibody raised to natural epigenetic marker N-acetyl lysine at
position 9 of H3. We were pleased to find that this antibody
successfully cross-reacted and recognized the CM-installed
modification at position 9 as a K9Ac PTM mimic (Scheme
6b). Next, as an “erase” step the CM-installed K9Ac mimic
“mark” was removed with full conversion using Cope-type
elimination through the generation of labile selenoxide using
mild peroxide oxidation to regenerate Dha. Met-containing H3
may be prone to side reaction;52 this was tested and confirmed
through the use of a Met-free H3 variant in which the mark was
cleanly expunged without side reaction (see SI sections 2.11−
2.14). Finally, a full chemical “write−read−erase−rewrite−read”
cycle was performed (SI section 2.12) to demonstrate iterative
histone “switching”.
the rate of on-protein Seac-assisted CM was found to be ∼150-
fold faster than that of off-protein Staudinger ligation (k = 0.002
M−1 s−1)38 and comparable to faster variants of off-protein strain-
promoted azide−alkyne cycloaddition (k = 0.002−0.9 M−1
s−1)39−41 and certain tetrazine-based cycloadditions (k > 0.15
M−1 s−1).27 Notably, however, recent select examples of
catalyzed oxime ligations42 and tetrazine-based reactions are
still more rapid (Figure 2).27,28,43,44 It seems likely that local
environment strongly affects on-protein reactions compared
with off-protein; indeed, here, crowded (e.g., Npβ-61Seac) sites
showed negligible reactivity (see SI). However, detailed on-
protein measurements are rare,42,44 so other relevant direct
comparisons are not possible. Although here directly comparable
reaction conditions could not be achieved, we extrapolate (see SI
section 2.10) off-protein rates for Seac-mediated CM that are at
least comparable and may be enhanced, consistent with CM’s
steric sensitivity.5
Next, having established usefully enhanced on-protein rates,
we examined substrate breadth. Model Seac-tagged protein SBL-
156Seac 6a was tested with challenging metathesis substrate
GlcNAc 10; this olefin is essentially unreactive in sulfur-relayed
CM with SBL-156Sac (9a). Pleasingly, reaction reached 75%
conversion after 1.5 h at 37 °C (Scheme 4). Another biologically
relevant substrate, N-allyl acetamide (11), was used in CM with
protein 6a. Acetamide 11 has been suggested to poison catalyst 1,
and also fails with SBL-156Sac (9a).11,45,46 Despite poisoning,47
after 20 min at 37 °C, LC-MS revealed conversion to desired 6d
(Scheme 4), albeit in 30% yield.
In conclusion, we have demonstrated efficient chemical
incorporation of Seac into proteins. This has facilitated rapid
cross-metathesis for protein modifications; determination of on-
protein rates shows that these outstrip or are comparable to
many of the so-called “click” reactions in chemical biology. Using
such allyl selenide (Seac) proteins, we were able to access a
broader substrate scope in Se-relayed CM than was possible with
After demonstrating CM on SBL-Seac, we tested Se-relayed
CM on more functionally relevant protein systems. Histone
proteins are key chromatin components; post-translational
modifications (e.g. K-Ac) are critical regulators of structure
and function.48,49 First, Seac was installed into key epigenetic site
9 of histone H3. After generation of H3-9Dha,22 subsequent allyl
C
dx.doi.org/10.1021/ja403191g | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
(17) Palioura, S.; Sherrer, R. L.; Steitz, T. A.; Soll, D.; Simonovic, M.
Science 2009, 325, 321.
(18) Levengood, M.; van der Donk, W. A. Nat. Protoc. 2007, 1, 3001.
(19) Wang, J.; Schiller, S. M.; Schultz, P. G. Angew. Chem., Int. Ed. 2007,
46, 6849.
(20) Bernardes, G. J. L.; Chalker, J. M.; Errey, J. C.; Davis, B. G. J. Am.
Chem. Soc. 2008, 130, 5052.
(21) Chalker, J. M.; Gunnoo, S. B.; Boutureira, O.; Gerstberger, S. C.;
̈
allyl sulfides. Direct access to Seac in proteins creates
opportunities for potential PTM mimicry via CM as demon-
strated here: a K9Ac mimic was successfully installed, recognized,
and removed (write−read−erase) in histone H3. Although, to
the best of our knowledge, such a chemical “write−read−erase”
approach has not been explored before, one could envisage a
system based on creation, e.g., of disulfide-linked modification
mimics53 and subsequent reductive cleavage. This synthetic
manipulation of biology also highlights opportunities for new
metathesis catalysts; uses of CM on protein/cell systems may be
enabled by tuned solubilities, compatibilities, and permeabilities.
We note too that this work provides access to a protected
(allylated) Sec in proteins (that could be revealed using, e.g.,
Pd(0))54 and also further motivates developments in genetic
incorporation of allyl chalcogenide amino acids into proteins to
access more generally the wider biological applications of cross-
metathesis.
́ ́
Fernandez-Gonzalez, M.; Bernardes, G. J. L.; Griffin, L.; Hailu, H.;
Schofield, C. J.; Davis, B. G. Chem. Sci. 2011, 2, 1666.
(22) Chalker, J. M.; Lercher, L.; Rose, N. R.; Schofield, C. J.; Davis, B.
G. Angew. Chem., Int. Ed. 2012, 51, 1835.
(23) Riague, E. H.; Guillemin, J.-C. Organometallics 2001, 21, 68.
(24) Crich, D.; Krishnamurthy, V.; Brebion, F.; Karatholuvhu, M.;
Subramanian, V.; Hutton, T. K. J. Am. Chem. Soc. 2007, 129, 10282.
(25) Vetting, M. W.; Hegde, S. S.; Hazleton, K. Z.; Blanchard, J. S.
Protein Sci. 2007, 16, 755.
́ ́
(26) Fernandez-Gonzalez, M.; Boutureira, O.; Bernardes, G. J. L.;
Chalker, J.; Young, M.; Errey, J.; Davis, B. G. Chem. Sci. 2010, 1, 709.
(27) Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin,
J. W. Nat. Chem. 2012, 4, 298.
ASSOCIATED CONTENT
* Supporting Information
Full procedures and protein ESI-MS. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
(28) Lang, K.; Davis, L.; Wallace, S.; Mahesh, M.; Cox, D. J.; Blackman,
M. L.; Fox, J. M.; Chin, J. W. J. Am. Chem. Soc. 2012, 134, 10317.
S
(29) Kohn, M.; Breinbauer, R. Angew. Chem., Int. Ed. 2004, 43, 3106.
̈
(30) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007.
(31) Hong, V.; Steinmetz, N. F.; Manchester, M.; Finn, M. G.
Bioconjugate Chem. 2010, 21, 1912.
AUTHOR INFORMATION
Corresponding Author
ben.davis@chem.ox.ac.uk
■
(32) van Hest, J. C. M.; van Delft, F. L. ChemBioChem 2011, 12, 1309.
(33) Blackman, M. L.; Royzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008,
130, 13518.
(34) Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A. Bioconjugate
Chem. 2008, 19, 2297.
†Present Address
́
́
̀
O.B.: Departament de Quimica Analitica i Organica, Universitat
́
Rovira i Virgili, C/Marcel·li Domingo s/n, Tarragona, Spain.
(35) Schoch, J.; Wießler, M.; Jaschke, A. J. Am. Chem. Soc. 2010, 132,
̈
8846.
Notes
(36) Devaraj, N. K.; Weissleder, R. Acc. Chem. Res. 2011, 44, 816.
(37) Aliquots taken, quenched (Sephadex G-25) prior to LC-MS. No
precipitation observed; % conversion used for concentrations. For
validation of ESI-MS in protein reaction monitoring see ref 11.
(38) Lin, F. L.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.; Bertozzi,
C. R. J. Am. Chem. Soc. 2005, 127, 2686.
(39) Debets, M. F.; van Berkel, S. S.; Dommerholt, J.; Dirks, A. J.;
Rutjes, F. P. J. T.; van Delft, F. L. Acc. Chem. Res. 2011, 44, 805.
(40) Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F. P. J. T.;
van Hest, J. C. M.; van Delft, F. L. Chem. Commun. 2010, 46, 97.
(41) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc. 2010,
132, 3688.
(42) Rashidian, M.; Mahmoodi, M. M.; Shah, R.; Dozier, J. K.; Wagner,
C. R.; Distefano, M. D. Bioconjugate Chem. 2013, 24, 333.
́
(43) Plass, T.; Milles, S.; Koehler, C.; Szymanski, J.; Mueller, R.;
Wießler, M.; Schultz, C.; Lemke, E. A. Angew. Chem., Int. Ed. 2012, 51,
4166.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank EPSRC (Y.A.L., L.L.), CRUK (L.L.), Rhodes Trust
(B.B.), and EU FP7 Marie Curie programme (O.B.) for funding.
B.G.D. is a Royal Society Wolfson Merit Research Award
Recipient.
REFERENCES
■
(1) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974.
(2) Chalker, J. M.; Bernardes, G. J. L.; Davis, B. G. Acc. Chem. Res. 2011,
44, 730.
(3) Takaoka, Y.; Ojida, A.; Hamachi, I. Angew. Chem., Int. Ed. 2013, 52,
4088.
(4) Hoye, T. R.; Zhao, H. Org. Lett. 1999, 1, 1123.
(5) Lin, Y. A.; Davis, B. G. Beilstein J. Org. Chem. 2010, 6, 1219.
(6) Lin, Y. A.; Chalker, J. M.; Floyd, N.; Bernardes, G. J. L.; Davis, B. G.
J. Am. Chem. Soc. 2008, 130, 9642.
(7) Brik, A. Adv. Synth. Catal. 2008, 350, 1661.
(8) Kirshenbaum, K.; Arora, P. S. Nat. Chem. Biol. 2008, 4, 527.
(9) Lin, Y. A.; Chalker, J. M.; Davis, B. G. ChemBioChem 2009, 10, 959.
(10) Ai, H.-W.; Shen, W.; Brustad, E.; Schultz, P. G. Angew. Chem., Int.
Ed. 2010, 49, 935.
(11) Lin, Y. A.; Chalker, J. M.; Davis, B. G. J. Am. Chem. Soc. 2010, 132,
16805.
(12) Foster, S. J.; Kraus, R. J.; Ganther, H. E. Arch. Biochem. Biophys.
1986, 251, 77.
(44) Seitchik, J. L.; Peeler, J. C.; Taylor, M. T.; Blackman, M. L.;
Rhoads, T. W.; Cooley, R. B.; Refakis, C.; Fox, J. M.; Mehl, R. A. J. Am.
Chem. Soc. 2012, 134, 2898.
(45) Furstner, A.; Langemann, K. J. Am. Chem. Soc. 1997, 119, 9130.
̈
(46) Furstner, A.; Seidel, G.; Kindler, N. Tetrahedron 1999, 55, 8215.
̈
(47) The reaction mixture colored consistent with catalyst degradation
and no further reaction was observed (see SI).
(48) Kouzarides, T. Cell 2007, 128, 693.
(49) Bannister, A. J.; Kouzarides, T. Cell Res. 2011, 21, 381.
(50) Spicer, C. D.; Davis, B. G. Chem. Commun. 2011, 47, 1698.
(51) Macdonald, J. E.; Veinot, J. G. C. Langmuir 2008, 24, 7169.
(52) Guo, J.; Wang, J.; Lee, J. S.; Schultz, P. G. Angew. Chem., Int. Ed.
2008, 47, 6399.
(53) Chatterjee, C.; McGinty, R. K.; Fierz, B.; Muir, T. M. Nat. Chem.
Biol. 2010, 6, 267.
(54) Shimizu, I. In Handbook of Organopalladium Chemistry for Organic
Synthesis; Negishi, E.-I., Ed.; John Wiley & Sons, Inc.: New York, 2003;
pp 1981−1994.
(13) Ganther, H. E. Carcinogenesis 1999, 20, 1657.
(14) Stolz, J. F.; Basu, P.; Santini, J. M.; Oremland, R. S. Annu. Rev.
Microbiol. 2006, 60, 107.
(15) Arner
J. Mol. Biol. 1999, 292, 1003.
(16) Aldag, C.; Brocker, M. J.; Hohn, M. J.; Prat, L. H., G.; Plummer,
́
, E. S. J.; Sarioglu, H.; Lottspeich, F.; Holmgren, A.; Bock, A.
̈
̈
A.; Soll, D. Angew. Chem., Int. Ed. 2013, 52, 1441.
̈
D
dx.doi.org/10.1021/ja403191g | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX