Control of Amphiphile Self-Assembly via Bioinspired Metal Ion Coordination

Article abstract of DOI:10.1021/jacs.7b11005

Inspired by marine siderophores that exhibit a morphological shift upon metal coordination, hybrid peptide-polymer conjugates that assemble into different morphologies based on the nature of the metal ion coordination have been designed. Coupling of a peptide chelator, hexahistidine, with hydrophobic oligostyrene allows a modular strategy to be established for the efficient synthesis and purification of these tunable amphiphiles (oSt(His)₆). Remarkably, in the presence of different divalent transition metal ions (Mn, Co, Ni, Cu, Zn, and Cd) a variety of morphologies were observed. Zinc(II), cobalt(II), and copper(II) led to aggregated micelles. Nickel(II) and cadmium(II) produced micelles, and multilamellar vesicles were obtained in the presence of manganese(II). This work highlights the significant potential for transition metal ion coordination as a tool for directing the assembly of synthetic nanomaterials.

Full text of DOI:10.1021/jacs.7b11005

Article
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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Control of Amphiphile Self-Assembly via Bioinspired Metal Ion
Coordination
Abigail S. Knight,*,† Josefin Larsson,† Jing M. Ren,† Raghida Bou Zerdan,† Shay Seguin,† Remy Vrahas,†
Jianfang Liu,§ Gang Ren,§ and Craig J. Hawker*,†,‡
†California NanoSystems Institute and Materials Research Laboratory and ‡Materials Department and Department of Chemistry and
Biochemistry, University of California, Santa Barbara, California 93106, United States
§The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
S
* Supporting Information
ABSTRACT: Inspired by marine siderophores that exhibit a
morphological shift upon metal coordination, hybrid peptide−
polymer conjugates that assemble into different morphologies
based on the nature of the metal ion coordination have been
designed. Coupling of a peptide chelator, hexahistidine, with
hydrophobic oligostyrene allows a modular strategy to be
established for the efficient synthesis and purification of these
tunable amphiphiles (oSt(His)6). Remarkably, in the presence of
different divalent transition metal ions (Mn, Co, Ni, Cu, Zn, and
Cd) a variety of morphologies were observed. Zinc(II), cobalt(II),
and copper(II) led to aggregated micelles. Nickel(II) and
cadmium(II) produced micelles, and multilamellar vesicles were
obtained in the presence of manganese(II). This work highlights the significant potential for transition metal ion coordination as
a tool for directing the assembly of synthetic nanomaterials.
have demonstrated that these siderophores, composed of a
hydrophilic peptide-based chelator and a fatty acid tail, can
undergo an unprecedented metal-induced morphology tran-
sition. In the absence of metal ions, assembly occurs to give
micelles, while coordination with an excess of Fe(III), Zn(II),
Cd(II), or La(III) results in dimerization of the amphiphile,
inducing a substantial shift in the packing parameter and
formation of new morphologies (vesicles or multilamellar
vesicles).
INTRODUCTION
■
Living organisms exploit a variety of stimuli to create nanoscale,
responsive materials leading to complex macroscale functions.
Structural change in response to a stimulus is critical for such
functions (e.g., the release of a signaling molecule in a cascade
or active transport across a membrane). Significantly, these
dynamic responses allow for an array of structures/functions to
be derived from a limited set of building blocks. In the pursuit
of synthetic nanomaterials with comparable functionality, a
toolbox of strategies including materials that respond to light,1
pH,2 temperature,3 and chemical4 stimuli has been developed.
These strategies provide a valuable foundation for the
development of responsive materials, but thus far they are
largely limited to generating structures that interchange
between two morphologies in response to various stimuli.
Transition metal ions are a unique tool for engineering
responsive character based on their various binding stoichio-
metries and geometries, allowing a single material to have a
diverse set of responses to different metal ions. This allows
structural control in small molecules5−8 and proteins,9−11 with
the dynamic nature of the coordination bonds being utilized to
develop responsive films,12,13 self-healing soft materials,14−16
subcomponent self-assembly of polymeric materials,17−21 and
hierarchical assemblies of nanoparticles.22,23 However, the only
example of metal ion coordination leading to a controllable
change in the morphology in an aqueous amphiphilic self-
assembled system is demonstrated with natural siderophores
that are secreted by marine bacteria.24,25 Butler and co-workers
RESULTS AND DISCUSSION
■
Inspired by these natural siderophore systems, we sought to
design a synthetic amphiphile that forms tunable morphologies
in the presence of different transition metal ions (Figure 1). A
modular synthetic approach was therefore devised based on a
tunable macromolecular hydrophobic unit26−28 and a peptide
chelator (Scheme 1).29,30 Oligostyrene was selected as the
hydrophobic tail due to its high hydrophobicity, chemical
inertness, and glass transition temperature above room
temperature. These features impart chemical and kinetic
stability to the assembled structures. Hexahistidine, known to
dimerize in the presence of transition metal ions,9,31,32 was
chosen as the hydrophilic chelating domain. Key features of this
design are the high degree of structural control over
functionality and molecular weight with the peptide component
Received: October 18, 2017
© XXXX American Chemical Society
A
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Figure 2. Synthesis of maleimide-terminated oligostyrene. (a)
Synthetic scheme; (b) 1H NMR spectrum of the protected-maleimide
oligomer demonstrating chain end fidelity.
Figure 1. Schematic illustrating a single amphiphilic material that can
be transformed into a variety of unique morphologies in response to
the presence of different divalent transition metal ions.
Scheme 1. Modular Design Strategy for the Synthesis of
oSt(His)6
being discrete and the synthetic oligomer with a narrow
distribution (Đ = 1.2). This allows accurate tuning of assembly
and dynamic behavior.
Figure 3. (a) Synthetic scheme for the coupling of oligostyrene to
hexahistidine peptide via maleimide−cysteine reaction; (b) MALDI
MS confirming synthesis of the peptide−polymer amphiphile. The
arrow indicates the difference of a single styrene monomer unit.
To probe the impact of metal ions on the assembly, a metal-
free synthesis of the amphiphile was required. Therefore,
efficient and metal-free chemistry (thiol−maleimide reaction)
was selected to couple the peptide and oligostyrene.33 To
increase the modularity of this system, polymerization from a
preformed, protected maleimide initiator was employed with
atom transfer radical polymerization (ATRP),34 allowing for a
more controlled polymerization, noncoordinating chain ends,
and limited in situ deprotection of the initiator (Figure 2a).
Oligomers (degree of polymerization (DP) < 10) of styrene
were targeted to provide a hydrophobic block of comparable
molecular weight to drive the self-assembly of a peptide
containing a hexahistidine unit with a terminal cysteine for
conjugation to the maleimide. A narrow molecular weight
distribution centered at a pentamer and high retention of the
furan-protected chain end was confirmed by size exclusion
coupling reaction was optimized to ensure high yields and
minimization of side products. Efficient purification of the
desired amphiphile from residual peptide and disulfide dimer
was achieved by reverse-phase chromatography eluting with 3:1
water/acetonitrile containing 0.1% TFA to give pure oSt(His)6,
which was fully characterized by mass spectrometry (Figure 3b)
and HPLC (Figure S5).
The critical aggregation concentration (CAC) and assembled
morphology of oSt(His)6 were initially studied in the absence
of transition metals. The CAC was determined to be less than
10 μM by incubating oSt(His)6 (concentrations of 0.3−600
μM) with solvatochromic Nile Red using a procedure similar to
that described by Stupp and co-workers35 (Figure S6). To
ensure complete self-assembly of the amphiphiles, 600 μM was
then selected as the concentraction of oSt(His)6 for all
subsequent experiments. The assembly proceeded in a
noncoordinating buffer,36 HEPES (100 mM, pH 7), at 80 °C
for 30 min to allow equilibration above the glass transition
temperature of the styrene oligomers. Following imaging with
transmission electron microscopy (TEM; cryogenic, Figure 4;
negative staining, Figure 5 and Figure S7), vesicle formation
was observed in the absence of any divalent metal ions.
Additional characterization by dynamic light scattering (DLS,
1
chromatography (SEC) and H NMR analysis (Figure 2b,
Figure S3). The reactive maleimide was then exposed by
heating the oligostyrene to 120 °C to release the furan followed
by coupling to the peptide in DMF with HEPES buffer salt.
Quenching with 0.1% trifluoroacetic acid (TFA) then gave the
crude peptide−oligomer amphiphile (Figure 3a), which was
initially characterized by high-performance liquid chromatog-
raphy (HPLC). Isolation and purification of amphiphiles is
traditionally challenging, and therefore the stoichiometry of the
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Figure 4. Schematic of the oSt(His)6 and assembled structures in the absence (left) and presence (right) of Zn(II) (15 mM) in addition to cryo-
TEM images. Scale bars represent 200 nm (larger image) and 20 nm (inset image).
Figure 5. Varying concentrations of Zn(II) lead to different degrees of aggregation. Negative-stained TEM images of oSt(His)6 (600 μM) assembled
with 0−15 mM Zn(II). Negative staining was performed with uranium formate, and all scale bars represent 200 nm. All assembly was performed in
buffered solution (HEPES, 100 mM, pH 7).
Figure S15) confirmed vesicle formation with only minimal
aggregation. This low level of aggregation is presumably due to
secondary interactions between the noncoordinating zwitter-
ionic buffer and the positively charged peptide corona.
into the aggregated network was observed to increase with
increasing temperature (Figure 6, Figure S6). For each 20 °C
decrease in temperature, approximately 4 times longer was
required for full incorporation into aggregated networks.
Samples heated to 40 °C do not appear to assemble fully,
Zinc has been previously demonstrated to dimerize
hexahistidine peptides31 and was initially investigated for
directing amphiphile assembly. An excess of Zn(II) (15 mM)
was therefore added to a buffered oSt(His)6 solution (600 μM
oSt(His)6 and 100 mM HEPES) at a neutral pH (confirmed
with bromothymol blue) and assembly conducted by heating
the mixture to 80 °C and slowly cooling it to room
temperature. Imaging with TEM (cryogenic, Figure 4; negative
staining, Figure 5 and Figure S7) revealed a definitive change in
morphology. In direct contrast to the vesicles formed in the
absence of transition metals, zinc induced the formation of
networks of aggregated micelles. It is particularly noteworthy
that the metal ion was able to both change the size of the
spherical assembly and lead to network-like aggregates.
The impact of zinc concentration, temperature, ionic
strength, and salt counterion on the assembly was then
examined to probe the factors controlling the formation of the
aggregated micelle networks. Incubating oSt(His)6 with
increasing concentrations of ZnCl2 from 120 μM (∼0.1
equiv, with respect to oSt(His)6) to 15 mM (∼25 equiv)
results in an observable transition from vesicles to micelles to
aggregated micelles (Figure 5 and Figure S8). Further
characterization was performed at 15 mM Zn(II), as this
provides the clearest difference in morphology when compared
to the metal-free assemblies. To monitor formation of these
networks, samples were heated at 40, 60, and 80 °C, all above
the Tg of the oligostyrene (34 °C), and the respective
morphologies characterized via TEM after different incubation
times (Figure S6e). Significantly, the incorporation of micelles
Figure 6. TEM images of oSt(His)6 assembled in the presence of
Zn(II) with different incubation times and temperatures. Samples
negatively stained with uranium formate. All scale bars represent 200
nm.
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likely due to this temperature being close to the reported Tg.
Additionally, networks that were assembled at 80 °C (30 min)
were cycled again through the same assembly conditions and
demonstrated to be unaffected by reheating (Figure S8a),
confirming the robustness of the final morphology. The impact
of diluting the structure postassembly was tested via imaging a
10-fold dilution of the original assembled solution (Figure
S8b), revealing a similar aggregated network morphology.
PIPES, as an alternative to HEPES (Figure S8c), and
Zn(NO3)2 and Zn(OAc)2 (Figure S8e) as replacements for
ZnCl2, also led to the assembly of a network of aggregated
micelles as visualized by TEM. Finally, when Zn(II) was
replaced by noncoordinating Mg(II), spherical assemblies
similar to those formed under metal-free conditions were
observed (Figure S8d), reinforcing that specific interactions
between the peptide domain of the amphiphile and Zn(II) lead
to the network morphology.
Encouraged by the morphological change induced by Zn(II),
additional transition metal ions were selected to evaluate how
ions with different properties (e.g., ionic radius and
coordination stoichiometry/geometry) impact assembly.
Under identical conditions, assembly of oSt(His)6 in the
presence of Mn(II), Co(II), Ni(II), Cu(II), Cd(II), and Fe(III)
was conducted, leading to a diverse array of morphologies
(Figure 7, Figure S14, DLS Figure S15, negatively stained TEM
Figures S9−13). Co(II)-directed assembly led to aggregated
micelle networks similar to those formed in the presence of
Zn(II), as visualized by cryogenic TEM. Comparable
aggregation was also observed in the presence of Cu(II) with
preliminary cryo-TEM visualization showing a more organized
network. In contrast, Mn(II)-, Ni(II)-, and Cd(II)-directed
assembly led to isolated particles as characterized by TEM and
DLS, indicating that these metal ions did not induce secondary
aggregation (Figure 7). Interestingly, micelles were observed in
the presence of Ni(II) and Cd(II), whereas multilamellar
vesicles were formed in the presence of Mn(II). A hollow core
and thick corona with faint rings can be observed in the
cryogenic images. Assembly in PIPES buffer allowed for clear
visualization of multiple layers (Figure S13d); the particles
formed in this buffer are larger, likely due to the higher ionic
strength, as has been observed with phospholipids.37 On
progressing to the Fe(III) ion, only unstructured aggregates
that separated from the aqueous solution were observed
(Figure S14). The wide range of morphologies obtained clearly
indicate that the nature of the transition metal ion and
associated atomic-scale differences in coordination significantly
affects nanoscale assembly, further highlighting the promise of
metal binding in the development of responsive materials.
Intrigued by the observation that Ni(II) and Cd(II), ions
with relatively large differences in ionic radius, both assembled
into micelles, we turned our attention to the role of
coordination geometry. With the exception of Mn(II), all of
the ions have been demonstrated to dimerize hexahistidine;31
therefore, we hypothesized that the structures are composed of
amphiphiles binding in a 2:1 oSt(His)6:metal ion ratio.
Different binding geometries of these 2:1 coordination
complexes may lead to the distinct assemblies visualized by
cryo-TEM. For proteins engineered to contain imidazole-based
binding sites, X-ray crystallography38 has shown a tetrahedral
binding geometry for Zn(II),39 square planar for Cu(II), and
octahedral for Ni(II).40 On the basis of these reported assembly
profiles,41 we propose coordination modes for each of the
morphologies (Figure 7) with square-planar (Cu(II)) and
Figure 7. Assembly of oSt(His)6 in the presence of divalent transition
metals. Cryo-TEM images show oSt(His)6 assembled in the presence
of Mn(II), multilamellar vesicles; Co(II)/Cu(II), aggregated micelles;
and Ni(II)/Cd(II), micelles. Scale bars represent 200 nm (larger
image) and 20 nm (inset image).
tetrahedral (Zn(II) and Co(II)) leading to aggregated micelle
networks, whereas an octahedral geometry (Ni(II) and Cd(II))
with coordinated water (common in proteins41 and observed in
subcomponent assembly20) leads to isolated micelles. Unlike
the prior transition metal ions, Mn(II) does not dimerize
hexahistidine1 and assembly is dictated by binding stoichiom-
etry instead of geometry. To further evaluate this difference,
oSt(His)6 was assembled in the presence of differing
stoichiometries of Mn(II) and Ni(II) with the size of the
resulting nanostructures being characterized by DLS. As shown
in Figure 8, only 0.5 equiv of Ni(II) are needed for micelle
formation, while approximately 5.0 equiv are required for
Mn(II) to form the final multilamellar vesicle. We therefore
conclude that multiple Mn(II) ions are bound to each
oSt(His)6.
CONCLUSIONS
■
In conclusion, we describe the formation of tunable nanoscale
morphologies directed by metal ion coordination. A peptide
polymer amphiphile, oSt(His)6, was synthesized with a modular
strategy and observed to assemble into vesicles in the absence
D
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ACKNOWLEDGMENTS
■
This work was supported primarily by the National Science
Foundation (MRSEC program DMR 1720256). Work at the
Molecular Foundry was supported by the Office of Science,
Office of Basic Energy Sciences, of the U.S. Department of
Energy under Contract No. DE-AC02-05CH11231. A.S.K. was
supported by a postdoctoral fellowship from the Arnold and
Mabel Beckman Foundation. J.M.R. thanks the Victorian
Endowment for Science, Knowledge and Innovation (VESKI)
for a postdoctoral fellowship.
REFERENCES
■
(1) Coleman, A. C.; Beierle, J. M.; Stuart, M. C.; Macia,
G.; Mika, J. T.; van Dijken, D. J.; Chen, J.; Browne, W. R.; Feringa, B.
L. Nat. Nanotechnol. 2011, 6, 547−552.
́
B.; Caroli,
(2) Jang, S. G.; Audus, D. J.; Klinger, D.; Krogstad, D. V.; Kim, B. J.;
Cameron, A.; Kim, S.-W.; Delaney, K. T.; Hur, S.-M.; Killops, K. L.;
Fredrickson, G. H.; Kramer, E. J.; Hawker, C. J. J. Am. Chem. Soc.
2013, 135, 6649−6657.
(3) Moughton, A. O.; O’Reilly, R. K. Chem. Commun. 2010, 46 (7),
1091−1093.
(4) Yan, Q.; Zhao, Y. Angew. Chem., Int. Ed. 2013, 52, 9948−9951.
(5) Sato, S.; Murase, T.; Fujita, M. Supramolecular Chemistry: From
Molecules to Nanomaterials; John Wiley & Sons, Ltd: Hoboken, 2012.
(6) Geng, Y.; Wang, X.-J.; Chen, B.; Xue, H.; Zhao, Y.-P.; Lee, S.;
Tung, C.-H.; Wu, L.-Z. Chem. - Eur. J. 2009, 15, 5124−5129.
(7) Li, L.; Ke, C.-F.; Zhang, H.-Y.; Liu, Y. J. Org. Chem. 2010, 75,
6673−6676.
Figure 8. Proposed binding modes of oSt(His)6 to divalent transition
metals. (a) Illustration of the proposed coordination of oSt(His)6 to
divalent ions. Number distributions from DLS for oSt(His)6 assembled
in the presence of different numbers of equivalents of (b) Ni(II) and
(c) Mn(II).
(8) Xiao, Z.-Y.; Zhao, X.; Jiang, X.-K.; Li, Z.-T. Chem. Mater. 2011,
23, 1505−1511.
(9) Van Eldijk, M. B.; Schoonen, L.; Cornelissen, J. J. L. M.; Nolte, R.
J. M.; Van Hest, J. C. M. Small 2016, 12, 2476−2483.
(10) Salgado, E. N.; Radford, R. J.; Tezcan, F. A. Acc. Chem. Res.
2010, 43, 661−672.
of transition metals and aggregated particles in the presence of
Zn(II). The aggregation was determined to be due to the
coordination of the Zn(II) ion. Aggregated particles were also
observed in the presence of Co(II) and Cu(II), isolated
micelles directed by Ni(II) and Cd(II) were observed, and
multilamellar vesicles were formed when oSt(His)6 coordinated
to Mn(II). Proposed models for binding geometry and
stoichiometry illustrate the control over nanoscale morpholo-
gies dictated by small differences in the nature of the transition
metal ion. This design principle illustrates the wide variety of
nanoscale materials that can be assembled from a well-defined
oligomer−peptide amphiphile and provides an attractive
avenue for the development of multistimuli-responsive nano-
materials. Future directions of this work are aimed at
continuing to probe the mechanism and limitations of the
role of metal ions in directing the assembly of amphiphiles.
(11) Nepal, M.; Sheedlo, M. J.; Das, C.; Chmielewski, J. J. Am. Chem.
Soc. 2016, 138, 11051−11057.
(12) Kumpfer, J. R.; Rowan, S. J. ACS Macro Lett. 2012, 1, 882−887.
(13) Michal, B. T.; McKenzie, B. M.; Felder, S. E.; Rowan, S. J.
Macromolecules 2015, 48, 3239−3246.
(14) Mozhdehi, D.; Ayala, S.; Cromwell, O. R.; Guan, Z. J. Am. Chem.
Soc. 2014, 136, 16128−16131.
(15) Grindy, S. C.; Learsch, R.; Mozhdehi, D.; Cheng, J.; Barrett, D.
G.; Guan, Z.; Messersmith, P. B.; Holten-Andersen, N. Nat. Mater.
2015, 14, 1181−1292.
(16) Whittell, G. R.; Hager, M. D.; Schubert, U. S.; Manners, I. Nat.
Mater. 2011, 10, 176−188.
(17) Ott, C.; Hoogenboom, R.; Hoeppener, S.; Wouters, D.; Gohy,
J.-F.; Schubert, U. S. Soft Matter 2008, 5, 84−91.
(18) Gao, H.; Liu, G.; Chen, X.; Hao, Z.; Tong, J.; Lu, L.; Cai, Y.;
Long, F.; Zhu, M. Macromolecules 2010, 43, 6156−6165.
(19) Chen, X.; Xu, N.; Li, N.; Lu, L.; Cai, Y.; Zhao, Y.; Wang, D. Soft
Matter 2013, 9, 1885−894.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b11005.
■
S
(20) Wu, X.; Xu, N.; Zhu, Z.; Cai, Y.; Zhao, Y.; Wang, D. Polym.
Chem. 2014, 5, 1202−1209.
(21) Xu, N.; Han, J.; Zhu, Z.; Song, B.; Lu, X.; Cai, Y. Soft Matter
2015, 11, 5546−5553.
(22) Wang, Y.; Hollingsworth, A. D.; Yang, S. K.; Patel, S.; Pine, D. J.;
Weck, M. J. Am. Chem. Soc. 2013, 135, 14064−14067.
(23) Lunn, D. J.; Gould, O. E. C.; Whittell, G. R.; Armstrong, D. P.;
Mineart, K. P.; Winnik, M. A.; Spontak, R. J.; Pringle, P. G.; Manners,
I. Nat. Commun. 2016, 7, 12371.
(24) Martinez, J. S.; Zhang, G. P.; Holt, P. D.; Jung, H.-T.; Carrano,
C. J.; Haygood, M. G.; Butler, A. Science 2000, 287, 1245−1247.
(25) Butler, A.; Theisen, R. M. Coord. Chem. Rev. 2010, 254, 288−
296.
Full experimental procedures and additional character-
ization data (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*aknight@berkeley.edu
*hawker@mrl.ucsb.edu
ORCID
Craig J. Hawker: 0000-0001-9951-851X
Notes
(26) Chen, C.; Wylie, R. A. L.; Klinger, D.; Connal, L. A. Chem.
The authors declare no competing financial interest.
Mater. 2017, 29, 1918−1945.
E
DOI: 10.1021/jacs.7b11005
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(27) Roy, D.; Cambre, J. N.; Sumerlin, B. S. Prog. Polym. Sci. 2010,
35, 278−301.
(28) Stuart, M. A. C.; Huck, W. T. S.; Genzer, J.; Muller, M.; Ober,
̈
C.; Stamm, M.; Sukhorukov, G. B.; Szleifer, I.; Tsukruk, V. V.; Urban,
M.; Winnik, F.; Zauscher, S.; Luzinov, I.; Minko, S. Nat. Mater. 2010,
9, 101−113.
(29) Cui, H.; Webber, M. J.; Stupp, S. I. Biopolymers 2010, 94, 1−18.
(30) Mikhalevich, V.; Craciun, I.; Kyropoulou, M.; Palivan, C. G.;
Meier, W. Biomacromolecules 2017, 18, 3471−3480.
(31) Evers, T. H.; Appelhof, M. A. M.; Meijer, E. W.; Merkx, M.
Protein Eng., Des. Sel. 2008, 21, 529−536.
(32) Valenti, L. E.; De Pauli, C. P.; Giacomelli, C. E. J. Inorg. Biochem.
2006, 100 (2), 192−200.
(33) Velonia, K.; Rowan, A. E.; Nolte, R. J. M. J. Am. Chem. Soc.
2002, 124, 4224−4225.
(34) Mantovani, G.; Lecolley, F.; Tao, L.; Haddleton, D. M.; Clerx, J.;
Cornelissen, J. J. L. M.; Velonia, K. J. Am. Chem. Soc. 2005, 127, 2966−
2973.
(35) Moyer, T. J.; Finbloom, J. A.; Chen, F.; Toft, D. J.; Cryns, V. L.;
Stupp, S. I. J. Am. Chem. Soc. 2014, 13, 14746−14752.
(36) Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa,
S.; Singh, R. M. M. Biochemistry 1966, 5, 467−477.
(37) Kodama, M.; Miyata, T. Thermochim. Acta 1995, 267, 365−372.
(38) Bailey, J. B.; Subramanian, R. H.; Churchfield, L. A.; Tezcan, F.
A. Methods Enzymol. 2016, 580, 223−250.
(39) Salgado, E. N.; Faraone-Mennella, J.; Tezcan, F. A. J. Am. Chem.
Soc. 2007, 129, 13374−13375.
(40) Salgado, E. N.; Lewis, R. A.; Mossin, S.; Rheingold, A. L.;
Tezcan, F. A. Inorg. Chem. 2009, 48, 2726−2728.
(41) Harding, M. M.; Nowicki, M. W.; Walkinshaw, M. D.
Crystallogr. Rev. 2010, 16, 247−302.
F
DOI: 10.1021/jacs.7b11005
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX