Use of a Compact Tripodal Tris(bipyridine) Ligand to Stabilize a Single-Metal-Centered Chirality: Stereoselective Coordination of Iron(II) and Ruthenium(II) on a Semirigid Hexapeptide Macrocycle

Article abstract of DOI:10.1021/acs.inorgchem.8b00416

Fe(II)-coordinating hexapeptides containing three 2,2′-bipyridine moieties as side chains were designed and synthesized. A cyclic hexapeptide having three [(2,2′-bipyridin)-5-yl]-d-alanine (d-Bpa5) residues, in which d-Bpa5 and Gly are alternately arranged with 3-fold rotational symmetry, coordinated with Fe(II) to form a 1:1 octahedral Fe(II)-peptide complex with a single facial-? configuration of the metal-centered chirality. NMR spectroscopy and molecular dynamics simulations revealed that the Fe(II)-peptide complex has an apparent C₃-symmetric conformations on the NMR time scale, while the peptide backbone is subject to dynamic conformational exchange between three asymmetric β/γ conformations and one C₃-symmetric γ/γ/γ conformation. The semirigid cyclic hexapeptide preferentially arranged these conformations of the small octahedral Fe(II)-bipyridine complex, as well as the Ru(II) congener, to underpin the single configuration of the metal-centered chirality.

Full text of DOI:10.1021/acs.inorgchem.8b00416

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Use of a Compact Tripodal Tris(bipyridine) Ligand to Stabilize a
Single-Metal-Centered Chirality: Stereoselective Coordination of
Iron(II) and Ruthenium(II) on a Semirigid Hexapeptide Macrocycle
Yuka Kobayashi,† Masaru Hoshino,† Tomoshi Kameda,‡ Kazuya Kobayashi,§ Kenichi Akaji,§
Shinsuke Inuki,† Hiroaki Ohno,† and Shinya Oishi*,†
†Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
‡Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi,
Koutou-ku, Tokyo 135-0064, Japan
§Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
S
* Supporting Information
ABSTRACT: Fe(II)-coordinating hexapeptides containing
three 2,2′-bipyridine moieties as side chains were designed
and synthesized. A cyclic hexapeptide having three [(2,2′-
bipyridin)-5-yl]-D-alanine (D-Bpa5) residues, in which D-Bpa5
and Gly are alternately arranged with 3-fold rotational
symmetry, coordinated with Fe(II) to form a 1:1 octahedral
Fe(II)−peptide complex with a single facial-Λ configuration of
the metal-centered chirality. NMR spectroscopy and molecular
dynamics simulations revealed that the Fe(II)−peptide
complex has an apparent C3-symmetric conformations on
the NMR time scale, while the peptide backbone is subject to
dynamic conformational exchange between three asymmetric β/γ conformations and one C3-symmetric γ/γ/γ conformation. The
semirigid cyclic hexapeptide preferentially arranged these conformations of the small octahedral Fe(II)−bipyridine complex, as
well as the Ru(II) congener, to underpin the single configuration of the metal-centered chirality.
ions.22 Three bipyridine ligands coordinate with Fe(II) with
INTRODUCTION
■
high affinity to form an octahedral [FeII(bpy)3] complex. This
A number of biological processes and organic transformations
complex exists in a rapid equilibrium between two inseparable
are driven by chiral metal complexes having stereogenic metal
center(s).1−5 Metal-centered chirality also contributes to the
construction of a variety of supramolecular architectures.3,6−8
For example, desferrioxamine B is an achiral trihydroxamic acid
siderophore that forms an Fe(III)−siderophore complex,
ferrioxamine B.9 The two mirror-image isomers (Λ and Δ
forms) of ferrioxamine B are stereoselectively recognized by the
iron-siderophore transport proteins, FhuD from Escherichia coli
and FhuD2 from Staphylococcus aureus, respectively.10,11
Similarly, other complexes of siderophores adopt a variety of
characteristic shapes with distinct configurations with metal-
centered chirality, which are favorable for uptake into the
microorganisms.12 Additionally, there have also been several
reports on the applications of chiral metal complexes to
medicinal chemistry research,13−15 including kinase inhib-
itors16,17 and TNF-α inhibitors.18,19 Investigations of the
effective scaffolds to stabilize metal-centered chirality, as well
as to enhance understanding of the underlying mechanism(s)
that generate the metal-centered chirality, facilitate the design
of molecular architectures for novel organometallic catalysts
and medicinal applications.20,21
mirror-image Δ and Λ isomers of the propeller-like helical
arrangements of the bipyridine ligands because of the
configurational lability of Fe(II) (Figure 1). When enantiomeri-
cally pure chiral bipyridine derivatives are employed for the
Fe(II)-coordination, the dynamic metal-centered chirality (Δ
and Λ) may be controlled, leading to preferential formation of
Figure 1. Dynamic equilibrium of the Fe(II)−tris(2,2′-bipyridine)
complex between the Λ and Δ forms.
2,2′-Bipyridine (bpy) is one of the most well-known
bidentate ligands that coordinates with a variety of metal
Received: February 20, 2018
© XXXX American Chemical Society
A
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the Fe(II) complex(es) with a thermodynamically favorable
configuration.23−28 For example, Fe(II)-coordination with
valabipy L1, in which L-valine moieties are attached at the
bipyridine 5- and 5′-positions, afforded a 2:1 ratio of
diastereomer complexes (Δ-[FeII(L-L1)3] and Λ-[FeII(L-L1)3])
initially, while only the thermodynamically preferred Δ form
was present after overnight standing (Figure 2).24 C2-symmetric
in which a single Fe(II) ion was captured by a tripodal
tris(bipyridine) ligand on a cyclic hexapeptide scaffold in a
stereoselective manner. The solution structures of this chiral
octahedral complex, as well as the congeners, were probed
using NMR spectroscopy and circular dichroism (CD) analysis,
which demonstrated the unusual formation of an apparent C3-
symmetric mononuclear complex. We also conducted molec-
ular dynamics (MD) simulations to investigate the conforma-
tions of the scaffold that supports the metal-centered chirality.
These structural analyses, accompanied by theoretical calcu-
lations, provide insight into the chiral cage-like architecture of
Fe(II)−bipyridine complex. Investigation of the Ru(II)−
bipyridine complex congener was also conducted.
RESULTS AND DISCUSSION
■
Design and Synthesis of Cyclic Hexapeptides with
Three Bipyridine Moieties. Cyclic hexapeptides are a useful
scaffold for host−guest chemistry32 and pharmaceutical
applications.33 Previously, we have investigated the structure−
function relationships of cyclic hexapeptide siderophores and
derivative peptides for their Fe(III)- and Fe(II)-coordinating
properties.34,35 The functional groups, linker lengths, and
arrangements of the component amino acids considerably
altered the coordination preferences and affinities for Fe(III)
and Fe(II). For example, appropriate spatial arrangement of
three 2-(1H-1,2,3-triazol-4-yl)pyridines on the cyclic hexapep-
tide allowed the Fe(II) coordination to provide a mononuclear
Fe(II)−peptide complex with moderate affinity.35 On the basis
of these findings, we hypothesized that three bipyridine ligands
on a cyclic hexapeptide could capture a single Fe(II) ion and
induce highly preferential metal-centered chirality. From this
perspective, two cyclic hexapeptides (1 and 2) having [(2,2′-
bipyridin)-4-yl]alanine (Bpa4) and [(2,2′-bipyridin)-5-yl]-
alanine (Bpa5) were designed, in which Bpa (Bpa4 or Bpa5)
and Gly are alternately arranged to give 3-fold rotational
symmetry (Figure 3). The spatial dispositions of the three
bipyridine ligands were varied depending on the substituted
positions (bipyridine 4- and 5-positions) to investigate possible
different coordination modes with Fe(II). The open-chain
congeners (1L and 2L) were also designed as control peptides
to verify the contribution of the macrocyclic structure to the
Fe(II)-coordination in peptides 1 and 2, respectively.
Initially, bipyridine-containing amino acids were prepared as
the building blocks for Fmoc-based solid-phase peptide
synthesis (Schemes S1 and S2). In the key process, alkylation
of (diphenylmethylene)glycine ester with the appropriate
(bipyridinyl)methyl bromide and subsequent separation using
chiral acids were employed to provide protected Bpa (Fmoc-D-
Bpa4-OH and Fmoc-D-Bpa5-OH) with D-configurations. Using
the resulting Bpa amino acids, cyclic hexapeptides 1 and 2 and
linear peptides 1L and 2L were synthesized by the standard
protocol (for details, see the Supporting Information). All
peptides were obtained with satisfactory purity (>95%), and
had the correct m/z values for [M + H]+ by matrix assisted
laser desorption/ionization-time-of-flight (MALDI-TOF) MS
and electrospray ionization (ESI)-MS analyses.
Figure 2. Examples of bipyridine-containing chiral ligands.
pinene-bipyridine-type ligand L2 is an alternative chiral
bipyridine ligand that forms two diastereomeric Fe(II)
complexes with a slight preference for the Δ configuration.26
The resulting isomers Δ-[FeII((−)-L2)3] and Λ-[FeII((−)-L2)3]
were discernible as two independent signals by NMR
spectroscopy. The tripodal ligand L3 containing three pinene-
bipyridine moieties coordinates with a single Fe(II) ion to form
a 1:1 complex with only the Λ configuration.25
The metal-centered chirality of bipyridine ligands can be also
preferentially induced by peptide secondary structures. For
example, Fe(II) coordination with three polyproline II-type
helices with a bipyridine ligand at the N-terminus led to folding
of a collagen triple helix.29 The associated bipyridine moieties
predominantly formed the Δ isomer at the Fe(II) center
because of the right-handed supercoils of the polyproline II-
type helices. In addition, chiral metalloproteins have been
designed in which one or more bipyridine motifs were
incorporated into the peptide backbone. When bipyridine
ligands were arranged via short peptide spacers, Ru(II), Co(II),
and Ni(II) were able to be coordinated to form chiral
octahedral complexes.30,31 Assembly of three C2-symmetric
helical peptides with a single bipyridine moiety in the presence
of Fe(II) and Co(II) also induced metal-centered chirality by
remote control via the helical chirality.27 These approaches
have provided unprecedented and unique molecular architec-
tures with preferential chirality; however, relatively large
peptide structures were required for stabilization of the
metal-centered chirality.
Fe(II) Coordination of Bipyridine-Containing Cyclic
Hexapeptides. The formation of the complexes between
Fe(II) and the Bpa-containing peptides was assessed by UV−
vis spectral analysis (Figure 4). The [FeII(bpy)3] complex
shows an unique absorption band in the 450−650 nm range,
which is derived from the metal-to-ligand charge transfer
(MLCT) transition.25,36 When each of the cyclic hexapeptides
In this article, we describe the successful characterization of a
small Fe(II)−bipyridine complex with metal-centered chirality,
B
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Figure 4. UV−vis absorption spectra of peptides and the Fe(II)
complexes (0.05 mM) in CH3CN/H2O (1:1).
In contrast, the Fe(II) complexes of Bpa5-peptides 2 and 2L
displayed a single peak by HPLC (Figure S1b). The m/z values
for [M + Fe]2+ (Fe(II)−2: 451.4; Fe(II)−2L: 480.6) and [M +
Fe]+ (Fe(II)−2: 902.37; Fe(II)−2L: 961.41) were detected by
ESI-MS and MALDI-TOF MS analyses, respectively (Figure
S2), suggesting that 1:1 complexation between Fe(II) and each
peptide led to a single species for each of Fe(II)−2 and Fe(II)−
2L. As such, the three Bpa4 residues in hexapeptides 1 and 1L
preferred intermolecular association with other peptides via
Fe(II), while the Bpa5 residues in peptides 2 and 2L favored
intramolecular assembly with a single Fe(II) ion to form a
mononuclear complex. These results suggested that the
position of the bipyridine attachment on the hexapeptide side
chains had a large effect on the coordination geometry, thereby
contributing to the mode of assembly between Fe(II) and the
bipyridines on the hexapeptides.
Investigation of the Possible Stereoisomers of Fe(II)−
Peptide Complexes by NMR Spectroscopy. To determine
the solution structure of the Fe(II)−peptide complexes, NMR
analyses were carried out in CD3CN/D2O (1:1). An octahedral
metal complex composed of three asymmetric bipyridine
ligands can form four stereoisomers by the combination of
two optical isomers (Δ and Λ) and two geometrical isomers
[facial (fac) and meridional (mer)].37−39 These possible
interchangeable stereoisomers (Δ/Λ and fac/mer) of the
complexes can be detected as separate signals in NMR
analysis,23,26−28 according to the previous reports of the Fe(II)
complexes of three chiral substituted-bipyridine ligands.
Figure 3. Structures of 2,2′-bipyridine-containing D-amino acid (D-
Bpa) residues and the sequences of D-Bpa-containing hexapeptides.
(1 and 2) was mixed with an equimolar amount of Fe(II) ions
(as FeSO4) in CH3CN/H2O (1:1), the mixtures exhibited
UV−visible spectra characteristic of the [FeII(bpy)3] complex
with a broad absorption band with maximum absorbance
around 520−530 nm. The open-chain congeners (1L and 2L)
also had spectra similar to those of cyclic peptides 1 and 2,
respectively. These results indicated that three bipyridine
moieties in the side chains of all four peptides coordinated
with Fe(II) to form some sort of Fe(II)−peptide complex,
although the stoichiometry remains unknown.
Next, we investigated the component species in these
Fe(II)−peptide complexes using several separation techniques.
The HPLC chromatograms of Fe(II)−1 and Fe(II)−1L, which
contain three Bpa4 residues, showed a large number of peaks
(Figure S1a). In the ultrafiltration experiment of Fe(II)−1 and
Fe(II)−1L mixtures using an ultrafilter (100 kDa cutoff), the
UV−visible absorbance of the filtrates was significantly
decreased (Figure S3). These results suggested that the Fe(II)
complexes of Bpa4-peptides 1 and 1L consisted of multiple
components, in which the molecular size was estimated to be
more than 100 000 Da. These species are likely to be derived
from the heterogeneous aggregation of Fe(II) and Bpa4
residues on the peptides into large supramolecular assemblies.
The 1H NMR spectrum of a mixture of peptide 2 and Fe(II)
suggested that the complex appeared to consist of a single
species. The single sets of D-Bpa5 and Gly signals indicated that
the Fe(II)−2 complex is present as a single C3-symmetric
isomer with fac configuration (Figure 5b). Indeed, a mer
geometrical configuration of Fe(II)−2 would be unlikely,
because the short methylene tethers between the bipyridine
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1
Figure 5. H NMR spectra of peptide 2 (a) and its Fe(II) complex (b) in CD3CN/D2O (1:1).
ligands and the peptide macrocyclic scaffold to favor the edge
configuration 83.37,40 Compared with the 1H NMR spectrum of
uncoordinated peptide 2 (Figure 5a), a remarkable upfield shift
of the bipyridine 6-H and 6′-H signals on addition of Fe(II)
was observed, which confirms the coordination between Fe(II)
and the side-chain bipyridines.25 Upfield shifts of the bipyridine
4-H, 4′-H, and 5′-H, as well as downfield shifts of the
bipyridine 3-H and 3′-H signals in the spectrum of 2 were also
observed. 1H-diffusion ordered spectroscopy (1H-DOSY)
spectra were recorded to estimate the molar ratio of the
Fe(II)−2 complex (Figure S4). The diffusion coefficient value
of the Fe(II)−2 complex was essentially identical with that of
the uncoordinated cyclic peptide 2 [D[Fe(II)−2]: 10−9.55 (m2/
sec); D(2): 10−9.57 (m2/sec)], suggesting that the molecular
size was not changed upon Fe(II)-coordination indicating a 1:1
Fe(II)−peptide 2 complex.
Metal-Centered Chirality of the Fe(II)−Peptide Com-
plexes. Next, we focused on the metal-centered chirality of the
Fe(II)−peptide complexes. It is known that the two stereo-
isomers (Δ and Λ configurations) of Fe(II)−tris(bipyridine)
ligand complexes can be distinguished using CD analysis. The
CD spectrum of the Fe(II)−2 complex showed the character-
istic sign of the exciton couplet of π−π* transitions (250−350
nm) and CD signals in the MLCT region (450−600 nm)
(Figure 6), indicating that the complex predominantly existed
The 1H NMR spectrum of a mixture of linear peptide 2L and
Fe(II) was considerably different from that of Fe(II)−2 (Figure
S5). The observed multiple signals indicated that the Fe(II)−
2L mixture included multiple components, which appeared
inconsistent with the single peak observed in HPLC analysis. A
comparison of the 1H-DOSY spectra of Fe(II)−2L and
uncoordinated peptide 2L suggested that most of the peptides
formed a 1:1 complex with Fe(II), while a small but significant
amount of molecules were present as higher oligomeric forms
of Fe(II)−2L complexes (Figure S6). These minor compo-
nent(s) might reflect the lower affinity and/or lower specificity
of peptide 2L, compared with cyclic peptide 2, in the formation
of the 1:1 complex with Fe(II). The 1H NMR spectra of the D-
Bpa4-containing peptides 1 and 1L were also measured
(Figures S7 and S9). In the absence of Fe(II), the line shape
and overall complexity of the spectra of 1 and 1L were very
similar to those of the isomeric counterparts 2 and 2L,
respectively. However, substantial line broadening was observed
on addition of Fe(II), suggesting the formation of species with
a large molecular weight. This is supported by the results of
ultrafiltration experiments (Figure S3, >100 000 Da) and the
1H-DOSY spectra of Fe(II)−1 and Fe(II)−1L (Figures S8 and
S10).
Figure 6. CD spectra of D-Bpa5-containing peptide−Fe(II) complexes
in H2O/CH3CN (1:1).
in the Λ configuration.23−28,41 Taken with the results of the
NMR investigations, Fe(II)−2 should only form a complex
with the fac-Λ configuration. An identical CD signal was
observed in the mixture of linear peptide 2L and Fe(II),
suggesting that peptide 2L also formed Fe(II)-complexes with
an Λ configuration, although both fac and mer isomers might be
present. The observed common metal-centered chirality of
Fe(II)−2 and Fe(II)−2L was in contrast with that of the
tripodal tris(triazolylpyridine) ligand, in which different Fe(II)-
centered chirality was observed depending on the scaffold
peptide structure.35 The Fe(II) complexes of D-Bpa4-
containing peptides 1 and 1L showed significantly weaker
CD intensity compared with the Fe(II)−2 complex (Figure
S11a). While the D-Bpa4-containting peptides (1 and 1L) had
favorable coordination ability with Fe(II), as shown by the
similar UV−vis absorbance to that of Fe(II)−2 (Figure 4), the
arrangements of the three bipyridine ligands is not conducive
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Figure 7. Conformation analysis of fac-Λ-Fe(II)−2 by MD simulations. PCA map (a); peptide backbones of representative structures in each cluster
(b); and graphical image of conformation exchange (c).
Figure 8. Representative structures in clusters A1 and B and overlay of representative structures in A1−A3.
for the predominant formation of either Δ or Λ configurational
stereoisomers. These results suggested that the Fe(II)-centered
chirality of the Fe(II)−2 complex can be attributed to the
coordinating ligand structure, the substitution position, and the
peptide scaffold. To the best of our knowledge, the Fe(II)−2
complex is the smallest-sized octahedral Fe(II)−ligand complex
with a single configuration at the chiral metal center.
Conformational Exchange of the Cyclic Hexapeptide
Scaffold with Maintenance of Metal-Centered Chirality.
Taking into account the C3-symmetric structure of the Fe(II)−
2 complex with the fac-Λ configuration suggested by NMR and
CD analysis, the conformation(s) in solution was further
investigated using Amber 10:EHT in Molecular Operating
Environment (MOE). Since the 1H NMR spectrum of Fe(II)−
2 showed only single sets of signals for D-Bpa5 and Gly, C3
symmetrical conformation(s), in which both the sets of three D-
Bpa5 and three Gly residues are chemically identical, were
anticipated to be most stable. However, contrary to our
expectations, some of calculated structures after the conforma-
tional search were not C3 symmetrical (Figure S12). We
suspect that multiple asymmetric conformations of the Fe(II)−
2 complex might be chemically exchanging in fast regime on
the NMR time scale.42
To explore the conformational exchange of the fac-Λ-
Fe(II)−2 complex, a replica exchange molecular dynamics
(REMD) simulation was performed (see the Experimental
E
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Figure 9. Dihedral angles of fac-Λ-Fe(II)−2 by MD simulation. Ramachandran plots (a); and χ1/χ2 plots (b).
Section for details of the procedure).43−45 Because the
conformational space of the Fe(II)−2 complex is too large to
intuitively describe its characteristics, principle component
analysis (PCA) can be useful to classify the sampled
conformations.46−48 PCA classifies conformations using as
few principal component (PC) axes as possible, and the PC
coordinates correspond to the conformations of the molecules.
The contributions of PC-1, PC-2, and PC-3 were 34.7, 31.1,
and 3.5%, respectively, which showed that PC-1 and PC-2
mainly contributed to the conformational space description.
Thus, we described the free energy landscape by the two-
dimensional PCA. The free energy values (F) in the PC space
are related to the population density of the conformations. All
structures obtained from MD simulations were classified into
four conformational clusters: A1, A2, A3, and B (Figure 7a). The
superimposed structures showed that the conformations within
the same clusters were similar to each other (Figure S13),
suggesting the effectiveness of the two-dimensional PCA
analysis. The most representative structures from each cluster
are shown in Figures 8 and S14.
In the structure belonging to cluster A1, a characteristic
intramolecular hydrogen bond in the cyclic hexapeptide scaffold
was observed between the Gly carbonyl oxygen (CO) and
the D-Bpa5 amide hydrogen (N−H), which indicated the
presence of a β-turn substructure with Gly and D-Bpa5 at the i
and i+3 positions, respectively. The conformations in clusters
A2 and A3 also contained a β-turn but between a different set of
Gly and D-Bpa5 residues than in the structure belonging to A1.
When these macrocyclic structures of A1−A3 were turned 120°,
the structures were completely overlapped (Figure 8, right).
The conformation in cluster B had a single C3-symmetric
structure. The projections of the three Gly carbonyl groups
(CO) in B are all directed away from the Fe(II) center.
While no hydrogen bond to form a β-turn was observed, three
γ-turn-like structures appeared in the cage space of the
conformation in cluster B, in which the carbonyl groups of D-
Bpa5 (i) were located in close proximity to the amide
hydrogens (N−H) of the next D-Bpa5 (i+2). One of these γ-
turn-like structures was preserved in the conformations of A1−
A3, in which one D-Bpa5 residue occupies both the β-turn i+3
position and the γ-turn i position.
These conformational characteristics of clusters A1−A3 and B
were also supported by Ramachandran plots, which were
calculated from the component structures in highly populated
regions in PCA. In the conformations belonging to cluster A1 as
representative of clusters A1−A3, three separate clusters of φ
and ψ dihedral angles for each Gly or D-Bpa5 residue were
observed (Figure 9a). Among them, the φ/ψ plots around
−37.3 8.7°/−55.5 8.3° for D-Bpa5(2) and −79.3 11.2°/
3.7 13.2° for Gly(3) in A1 suggested that these sequential
residues occupied the i+1 and i+2 positions of the mirror-image
type I′ β-turn. Additionally, the φ/ψ dihedral angles of Gly(5)
[φ: −68.7
18.5°; ψ: 57.1
25.3°] were close to the
theoretical values for a mirror-image γ-turn i+1 position. The
φ/ψ charts of conformations in A2 and A3 were similar in shape
to A1, but the arrangements of the component amino acids
were shifted, indicating that the conformations in A1−A3 were
well-overlapped, but independent, conformations. In contrast,
the φ and ψ plots of the three Gly residues of structures in
cluster B overlapped to form one cluster in the region of −68.9
12.2°/37.1 23.8°, indicating that the three Gly residues are
at the i+1 positions of three mirror-image γ-turn-like structures.
The φ and ψ plots of the three D-Bpa5 residues in the
conformations in cluster B also converged to one cluster in the
region of 119.1 26.5°/−83.2 18.6°. These uniform dihedral
angles for the component amino acids support the presence of
C3-symmetrical structures in B. Interestingly, the χ1 and χ2 plots
for D-Bpa5 were well overlapped in all the conformations in
A1−A3 and B (Figure 9b), indicating that restricted χ1 and χ2
dihedral angles contribute to stabilization of Fe(II)-centered
F
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chirality with the Λ configuration, even over the relatively
flexible cyclic peptide conformations. This apparently para-
doxical induction of metal-centered chirality with a single
configuration using a flexible scaffold is similar to that observed
in the Fe(III)-coordination of enterobactin, in which the
Fe(III)-centered chirality is dictated by the relatively flexible
trilactone scaffold.49
These results suggest that the structure of Fe(II)−2 exists in
rapid equilibrium between three asymmetric β/γ conformations
in A1−A3 via one apparently C3-symmetric γ/γ/γ conforma-
tions in B (Figure 7b,c and Table S1). In the interchange
process, the interaction of the D-Bpa5 N−H group with the Gly
CO group in the β-turn of conformations in A1−A3 was
switched to an interaction of the D-Bpa5 N−H group with the
D-Bpa5 CO group in a γ-turn of the conformations in B.50−52
We speculated that the observation of only one set of Gly and
1
D-Bpa5 signals in the H NMR spectrum of the Fe(II)−2
complex is derived from the averaged signals through
conformational exchange between these conformations. This
1
theory is supported by several NMR observations. In the H
NMR spectrum of Fe(II)−2, several protons (D-Bpa5 α-H, D-
Bpa5 β-H, bipyridine 6-H, and bipyridine 6′-H) appeared as
broad signals at room temperature, whereas they became much
sharper at a high temperature (Figure S15). Similarly, in the
13C NMR spectra, all of peptide backbone (α-C, CO) and
several side-chain (D-Bpa5 β-C and bipyridine 6-C) signals
were detected only at a high temperature (Figure S16). We
assumed that these temperature dependencies of the line shape
in the Fe(II)−2 complex were caused by chemical exchange
line broadening, in which the environment around the nuclei
(and thus the chemical shift values) varied at the intermediate
rate of the NMR chemical shift time scale (micro- to
millisecond). Furthermore, in the relaxation dispersion experi-
ments, chemical exchange was observed for the signals from the
D-Bpa5 β-H and bipyridine 6-H (Figure S17).53 On the basis of
these NMR analyses, chemical exchange occurred over several
microseconds or longer, which is far beyond the time scale
accessible by conventional MD simulations. In fact, when we
performed 1 μs standard MD simulations, conformational
exchange to other clusters in the PC space was not observed
during the simulation (data not shown).
Chiral Ru(II) Complex with a Bpa5-Containing Cyclic
Hexapeptide. Three 2,2′-bipyridines coordinate with Ru(II)
to form a [RuII(bpy)3] complex. This type of ruthenium(II)
complexes can be used in luminophore materials,54 as well as
visible-light-mediated photoredox catalysis.55 Because the
Ru(II) complex is substitutionally inert, two mirror-image
isomers of Δ-[RuII(bpy)3] and Λ-[RuII(bpy)3] can be
isolated.56 This is in contrast to the interconvertible Δ-
[FeII(bpy)3] and Λ-[FeII(bpy)3] complexes. We next inves-
tigated the chemical properties of a chiral Ru(II) complex with
cyclic peptide 2.
Figure 10. Spectral data of Ru(II)−2 complex. UV−vis absorption
spectrum (a); fluorescence spectrum (b); and CD spectrum (c).
data support the formation of an octahedral Ru(II)−2 complex.
The metal-centered chirality in Ru(II)−2 was determined to be
the Λ configuration based on the characteristic intense CD
exciton couplet around 290 nm in the CD spectrum (Figure
10c).58,59 As the Λ configuration is common to both the
Fe(II)−2 and Ru(II)−2 complexes, this induction of metal-
centered chirality regardless of the metal ion species can be
attributed to the bipyridine arrangements on the macrocyclic
scaffold of peptide 2. Although irradiation of Λ-Ru(II)−(bpy)3
with light-emitting diode (LED) light led to racemization of the
complex,60 the metal-centered chirality of the Ru(II)−2
complex was stable even after 6.5 W LED light irradiation for
24 h in CH3CN/H2O (1:1) at room temperature (Figure
S20).61
The 1H NMR and 1H-DOSY spectra of the Ru(II)−2
complex were recorded to estimate the impact of the
substitution of Fe(II) with Ru(II) on the structure of the
complexes (Figures S21 and S22). Overall, the spectra of
Ru(II)−2 were essentially similar to those of Fe(II)−2,
including the characteristic downfield shift of the bipyridine
6-H and 6′-H signals.25 Interestingly, broad D-Bpa5 α-H and D-
Bpa5 β-H signals were found in both the Fe(II)−2 and
Ru(II)−2 complexes. These results indicated that the Fe(II)−2
and Ru(II)−2 complexes exhibit similar overall conformations.
Considering that the metal-centered chirality of inert Ru(II)−
bipyridine complexes cannot be converted to the other
The reaction of peptide 2 with RuCl2(dmso)4 in MeOH/
H2O (1:1) at 80 °C provided the expected 1:1 complex of
orange-colored Ru(II)−2 as a major product (Figure S18),
which was characterized by ESI-MS (m/z: 474.4 for [M +
Ru]2+) and MALDI-TOF MS (m/z: 947.40 for [M + Ru −
H]+) analyses (Figure S19). In the UV−vis spectrum, an
absorption maxima around 450 nm was observed, which is
characteristic of MLCT bands of the [RuII(bpy)3] complex
(Figure 10a).57 Additionally, the fluorescence spectrum of
Ru(II)−2 had an identical emission maximum around 625 nm
to that of [RuII(bpy)3] (Figure 10b).57 These spectroscopic
G
DOI: 10.1021/acs.inorgchem.8b00416
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
the force field. The exterior dielectric constant was set to the dielectric
constant of CH3CN/H2O (1:1, ε = 60).63 To simulate the Fe(II)
coordination, Fe(II) and each N atom of the bipyridines were
restricted to be arranged within 1.8−2.3 Å.
configuration, these peak broadenings of D-Bpa5 residues in the
1H NMR spectra of Fe(II)−2 and Ru(II)−2 are likely to be
derived from conformational exchange, especially in the
relatively flexible peptide backbone, in the NMR time scale.
Replica Exchange MD Simulation. The peptide was solvated
with 589 H2O molecules, 210 CH3CN molecules, 1 Fe(II) ion, and 2
chloride ions, which corresponded to the conditions when the peptide
was dissolved in CH3CN/H2O (1:1). Peptide 2 was described by the
AMBERff14SB force field,64 CH3CN molecules were described by the
general amber force field (GAFF),65 and water molecules were
described by the TIP3P model.66 The partial charges of D-Bpa5 and
CH3CN molecules were assigned as restrained electrostatic potential
(RESP) charges (Figure S23).67 The chloride ion parameters were
from the Joung and Cheatham model,68,69 and Li/Merz ion
parameters were used for the Fe(II) ion.70 The bonded interaction
between the Fe(II) ion and the side chain of D-Bpa5 was modeled by
the parameters for the heme molecule.71 We simulated the system with
dodecahedron periodic boundary conditions and used the particle-
mesh Ewald method.72 The bond lengths that include hydrogen atoms
in the peptide were constrained by the p-LINC73 and SETTLE was
used for the bond lengths of H2O molecules.74 The cutoff distance for
nonbonded interactions were 10 Å.
To enhance sampling, we used the Replica Exchange MD method
in the NPT ensemble.43−45 We used 32 replicas; the temperatures of
replicas were distributed in the range of T = 300−550 K (T = 300.0,
305.1, 310.4, 315.7, 321.2, 326.9, 332.6, 338.6, 344.4, 350.5, 356.9,
363.2, 369.9, 376.8, 383.7, 390.8, 398.0, 406.3, 414.5, 423.2, 432.1,
441.4, 450.9, 460.7, 471.0, 481.5, 492.2, 502.9, 514.6, 525.9, 538.2, and
550.0 K). The pressure of each replica was 1.0 atm. Exchange attempts
were made after every 1 ps. The acceptance ratios were between 29
and 36%, which showed the conformational space of the system was
well sampled. The replica temperatures were maintained by the
Langevin dynamics, and the pressures were maintained by the
Parrinello−Rahman barostat.75 As an initial structure for simulation,
the structure of the 1:1 complex of Fe(II)−2 fixed to the fac-Λ form
was used (Figure 3).
CONCLUSIONS
■
We have designed and synthesized an optically pure Fe(II)-
coordinated, cage-shaped complex of tripodal tris(bipyridine)
ligands. The alternating arrangement of D-Bpa5 and Gly in a
cyclic hexapeptide scaffold predominantly formed a 1:1 Fe(II)−
peptide 2 complex with only a single configuration at the chiral
metal center. In contrast, D-Bpa4-containing congener 1
preferred a heterogeneous assembly via intermolecular Fe-
(II)−bipyridine coordinations, suggesting that the three
bipyridine moieties in 2 complexed with a single Fe(II) ion
because of the suitable spatial dispositions of the cyclic
hexapeptide scaffold, as well as the bipyridine substitution
position. Spectroscopic analyses revealed that the Fe(II)−
peptide 2 complex was a single isomer of a C3-symmetric
structure with the fac-Λ configuration. MD simulations of the
Fe(II)−2 complex suggested that the cyclic peptide backbone
undergoes dynamic conformational changes between three β-
turn conformations and one C3-symmetric conformation.
Peptide 2 also coordinated with an inert Ru(II) ion to form
a photoluminescent complex with a C3-symmetric fac-Λ
configuration, which may be useful as a chiral photocatalyst.
In total, this semirigid cyclic hexapeptide is a suitable small
scaffold for the facile induction of metal-centered chirality with
a single configuration, even with the labile Fe(II) ion.
EXPERIMENTAL SECTION
■
UV−Vis Spectra, CD Spectra, and LC-MS Analysis. Peptide
(0.05 mM) [or 2,2′-bipyridine (0.15 mM)] and FeSO4 (0.05 mM)
were dissolved in CH3CN/H2O (1:1). Ru(II)−peptide 2 (0.05 mM)
was also dissolved in CH3CN/H2O (1:1). UV−vis spectra were
recorded on a Shimadzu UV-2450 UV−vis spectrophotometer at
room temperature. CD spectra were recorded on a JASCO J-720
circular dichroism spectrometer at 20 °C. After recording the spectra,
the samples were analyzed with a Waters 2795 Separations Module.
Fluorescence Spectra. Each Ru(II) complex (5 μM) was
dissolved in H2O. The excitation light was set to 450 nm and the
emission spectra were recorded on a Shimadzu RF-5300PC
spectrofluorophotometer at 25 °C. The quantum yield of Ru(II)−2
was calculated to be Φ = 0.030 using Ru(II)−(bpy)3 as a standard.62
Ultrafiltration Experiment. A mixture of Fe(II) and peptide
[0.05 mM each in CH3CN/H2O (1:1)] was diluted five times with
H2O. The solution was centrifuged through a 100 kDa ultrafilter
(Microcon centrifugal filter unit YM-100) for 3 min at 7000 × g. The
UV−vis spectra of the solutions before and after ultrafiltration were
measured.
The initial state was prepared by the steepest descent minimization,
followed by a 200 ps MD simulation at 300 K/1 atm with the
constraint for the solute heavy atoms and continuing 200 ps MD
simulation without constraint. The REMD simulation time was 500 ns
for each replica for all simulations. The total simulation time was
32 000 ns. The simulation was carried out by GROMACS 2016
simulator.76,77
PCA was employed to analyze the free energy landscape.46−48 First,
we selected 5000 configurations of the peptide and the Fe(II) ion from
a snapshot of the 500 ns of the simulation from the trajectory at 300 K.
These conformations were superimposed onto a reference con-
formation. Subsequently, the variance−covariance matrix of the
ensemble was diagonalized to obtain the eigenvectors (PC axes) and
eigenvalues (λi) (which are the standard deviations of the conforma-
tional distribution along the ith PC axis). Here, λi/Σiλi was regarded as
the relative contribution of the distribution along the ith PC.
Selection of the Representative Structures. First, the
coordinates of every atom, including hydrogen and side-chain atoms,
were averaged over all structures within the same group classified by
the PCA analysis (800, 1018, 978, and 259 structures, for A1, A2, A3,
and B, respectively) to calculate the four “averaged” coordinates. Next,
the root-mean-square deviations from these “averaged” coordinates
were calculated for each structure, and the coordinate with the smallest
deviation was selected as the most representative structure for that
group.
1
1H-DOSY Spectra. H-DOSY experiments were performed on a
Bruker Avance I 600 spectrometer using a standard stimulated echo
pulse sequence with bipolar gradient pulses at 25 °C. The pseudo-2D
spectra were analyzed by a DOSY analysis module integrated in
topspin 2.1 software.
Relaxation Dispersion Experiments. Relaxation dispersion
experiments were performed using a standard Carr−Purcell−
Meiboom−Gill (CPMG) spin−echo sequence with presaturation for
water-suppression. The spectra were recorded on a Bruker Avance I
600 spectrometer at 25 °C. The total relaxation period during CPMG
pulsing was implemented in a constant time manner with T = 80 ms.
Peak intensities were compared between the spectra recorded with a
different set of τcp intervals, the time between successive 180° pulses of
80, 180, 480, and 980 μs.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b00416.
■
S
Experimental procedures, characterization data, and
NMR spectra of newly synthesized compounds; 1H
Conformational Search by MOE. Amber 10:EHT in MOE
(Chemical Computing Group Inc., Montreal, Canada) was selected as
H
DOI: 10.1021/acs.inorgchem.8b00416
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
1
NMR, 13C NMR, and H-DOSY NMR spectra, mass
spectra, UV−vis absorption, CD spectra, and HPLC
chromatograms of peptides, peptide−Fe(II) complexes,
and peptide−Ru(II) complexes; structures obtained by
simulation experiments; charges and atom types for MD
simulation (PDF)
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Accession Codes
CCDC 1824844−1824845 contain the supplementary crystal-
lographic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by email-
ing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
■
Corresponding Author
(16) Debreczeni, J. E.; Bullock, A. N.; Atilla, G. E.; Williams, D. S.;
Bregman, H.; Knapp, S.; Meggers, E. Ruthenium Half-Sandwich
Complexes Bound to Protein Kinase Pim-1. Angew. Chem., Int. Ed.
2006, 45, 1580−1585.
(17) Maksimoska, J.; Feng, L.; Harms, K.; Yi, C.; Kissil, J.;
Marmorstein, R.; Meggers, E. Targeting Large Kinase Active Site
with Rigid, Bulky Octahedral Ruthenium Complexes. J. Am. Chem. Soc.
2008, 130, 15764−15765.
*Tel.: +81-75-753-4561. Fax: +81-75-753-4570. E-mail:
soishi@pharm.kyoto-u.ac.jp.
ORCID
Tomoshi Kameda: 0000-0001-9508-5366
Hiroaki Ohno: 0000-0002-3246-4809
Shinya Oishi: 0000-0002-2833-2539
(18) Leung, C. H.; Zhong, H. J.; Yang, H.; Cheng, Z.; Chan, D. S. H.;
Ma, V. P. Y.; Abagyan, R.; Wong, C. Y.; Ma, D. L. A Metal-Based
Inhibitor of Tumor Necrosis Factor-α. Angew. Chem., Int. Ed. 2012, 51,
9010−9014.
(19) Kang, T. S.; Mao, Z.; Ng, C. T.; Wang, M.; Wang, W.; Wang, C.;
Lee, S. M. Y.; Wang, Y.; Leung, C. H.; Ma, D. L. Identification of an
Iridium(III)-Based Inhibitor of Tumor Necrosis Factor-α. J. Med.
Chem. 2016, 59, 4026−4031.
(20) Crassous, J. Transfer of Chirality from Ligands to Metal
Centers: Recent Examples. Chem. Commun. 2012, 48, 9684−9692.
(21) Meggers, E. Exploiting Octahedral Stereocenters: From Enzyme
Inhibition to Asymmetric Photoredox Catalysis. Angew. Chem., Int. Ed.
2017, 56, 5668−5675.
(22) Kaes, C.; Katz, A.; Hosseini, M. W. Bipyridine: The Most
Widely Used Ligand. A Review of Molecules Comprising at Least Two
2,2’-Bipyridine Units. Chem. Rev. 2000, 100, 3553−3590.
(23) Ahn, D. R.; Kim, T. W.; Hong, J. I. Induction of
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Grants-in-Aid for Scientific
Research from JSPS, Japan (24659004 and 26-7879); the
Platform Project for Supporting Drug Discovery and Life
Science Research funded by Japan Agency for Medical Research
and Development (AMED), Japan; and Takeda Science
Foundation. Y.K. is grateful for JSPS Research Fellowships
for Young Scientists. In this research work, we used the
supercomputer of ACCMS, Kyoto University. We are grateful
to Dr. Yoshiaki Yano and Dr. Toru Nakatsu (Kyoto University)
for their generous supports of this research.
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