Self-assembled multi-component catenanes: Structural insights into an adaptable class of molecular receptors and [2]-catenanes

Article abstract of DOI:10.1021/ja302345n

Under acidic conditions (50 equiv of TFA), combinations of hydrazide A-B monomers self-assemble into octameric [2]-catenanes with high selectivity for [1₃2]₂, where 1 is a d-Pro-X (X = Aib, Ac₄c, Ac₆c, l-4-Cl-Ph

Full text of DOI:10.1021/ja302345n

Article
pubs.acs.org/JACS
Self-Assembled Multi-Component Catenanes: Structural Insights into
an Adaptable Class of Molecular Receptors and [2]-Catenanes
Mee-Kyung Chung,† Peter S. White,† Stephen J. Lee,‡ Marcey L. Waters,*,† and Michel R. Gagne*,†
́
†Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
‡U.S. Army Research Office, P.O. Box 12211, Research Triangle Park, North Carolina 27709, United States
S
* Supporting Information
ABSTRACT: Under acidic conditions (50 equiv of TFA), combinations of
hydrazide A-B monomers self-assemble into octameric [2]-catenanes with high
selectivity for [132]2, where 1 is a D-Pro-X (X = Aib, Ac4c, Ac6c, L-4-Cl-PhGly)-
derived monomer and 2 is an L-Pro′-L-arylGly (Pro′ = Pro, trans-F-Pro, trans-HO-
Pro, aryl = naphthyl, phenyl)-derived monomer. Five different combinations of
monomers were studied by X-ray crystallography. In each case, the unique aryl
glycine unit is located in the core of the structure where the aryl ring templates a
CH−π−CH sandwich. Analysis of metrical parameters indicates that this core
1
region is highly conserved, while the more peripheral zones are flexible. H NMR spectroscopy indicate that the solid-state
structures are largely retained in solution, though several non-C2-symmetric compounds have a net C2-symmetry that indicates
accessible dynamic processes. Catenane dynamic processes were additionally probed through H/D exchange, with the core being
inflexible relative to the peripheral structure. Mass spectrometry was utilized to identify the constitutional isomerism in the minor
asymmetric [1523] catenanes.
more complex recognition systems than designed host−guest
pairs. A dynamic variant has been recently developed by
Sanders, Lehn, and others as a means of discovering tight-
binding hosts for a broad variety of guests.5 Termed dynamic
combinatorial or constitutional chemistry (DCC), this strategy
enables molecular receptors to be selected from dynamic
libraries of potential hosts without prior knowledge of the
guest’s structure. By its very nature, however, the competitive
selection masks the recognition elements that create the
equilibrium host−guest structure. While much is known about
the constitution and binding properties of these host and host−
guest assemblies, little is known about their molecular
structures.
In the course of our own experiments examining complex
libraries assembled from mixtures of dipeptide monomers,6 we
noted that one specific combination of two monomers led to
the self-assembly of a [2]-catenane composed of 56-membered
macrocyclic tetramers, each of which was composed of a 3:1
mixture of the two monomers (Figure 1).7 While this [2]-
catenane recognizes itself, Sanders has previously demonstrated
that catenated dipeptide trimers will bind quat-salts like
acetylcholine with sub-micromolar affinities, the host−guest
complex of which could be characterized by NMR.8 The lack of
solid-state structural data on these and the other receptors
assembled from peptide monomers8−10 prompted us to utilize
X-ray methods in conjunction with solution-phase studies to
structurally characterize a number of additional [2]-catenanes.
INTRODUCTION
■
The self-assembly of complex structures from simple building
blocks is a general principle that can lead to significant gains in
structure and function.1 As exemplified by the protein folding
problem,2 global minima are achieved through the interplay of
many weak interactions, the quantitation of which is often
challenging. Within this context, it is the balance of non-
covalent forces (H-bonding, electrostatic, π−π, cation−π,
CH−π, and hydrophobic effects) that dictate thermodynamic
minima. Model systems have been widely utilized to investigate
these contributing non-covalent interactions in relative
isolation, with the ease of manipulation and characterization
enabling significant insight into the nature and driving force of
such interactions.3 A key limitation, however, is that few such
model systems simulate the cooperative folding and binding
found in proteins.4
We report herein structural studies of a synthetic self-
assembling system that bridges the complexity/manipulability
gap that exists between small-molecule model systems and
complex biosystem such as proteins. The self-assembled
structure is generated from dipeptide hydrazide monomers
into discrete octameric [2]-catenanes of molecular weight >3
kDa. A structure analysis reveals that the compounds rely on H-
bonding, π−π, and CH−π interactions along with biogenic β-
turns to achieve their minimum-energy structures. The
combination of large size, complexity, and structural manipu-
lability provides the opportunity to gauge how the balance of
forces influences the energetics of this self-assembled structure,
and thus bridges the gap between small molecules and proteins.
Templated synthesis is a long-established strategy for
accessing entropically disfavored compounds and can provide
Received: March 9, 2012
Published: June 11, 2012
© 2012 American Chemical Society
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multivalency and cooperativity in folding and molecular
recognition in these catenanes.
These studies provide insight into the effect of a broad
variety of multivalent non-covalent interactions on the stability
of a self-assembled [2]-catenane in organic solvents. The
intermediate size (>3k Da) and complexity of the [2]-catenane
help to bridge the gap between small-molecule models and
proteins without sacrificing the ability to precisely tune
structure, obtain relative stability data, and structurally
characterize with high-resolution methods in solution, in the
solid state, and in the gas phase (MS).
EXPERIMENTAL SECTION
■
General Methods. Chemicals were purchased from Aldrich, Fisher
Scientific, and Chem-Impex International, Inc. and used as received.
1H and 13C NMR spectra of monomers and 1H, 13C, gradient TOCSY,
ROESY, and HSQC NMR spectra of [2]-catenanes (F3b, HO3c, F3dPh,
F4b) were recorded on a Bruker DRX 500 spectrometer or a Bruker
600 Cryoprobe spectrometer and processed using TOPSPIN Bruker
NMR software (V. 3.0). High-resolution mass spectra were obtained
on an Agilent Accurate LC-TOF mass spectrometer (Agilent Series
6220) operating in positive-ion mode with electrospray ionization
source (fragmentor = 175 V). The data were analyzed using Agilent
MassHunter Workstation Software, Qualitative Analysis (V. B.02.00).
HPLC analysis was performed on a Hewlett-Packard Series 1100
instrument, using a Halo-C18 column (4.6 × 150 mm, 2.7 μm) with
gradient elution (methanol/water) at a flow rate of 0.45 mL/min and
at 55 °C. The injection volume for a 5 mM dynamic self-assembly
(DSA) reaction was typically 3.5 μL. UV absorbance chromatograms
were recorded at wavelengths of 220 and 289 nm. The data were
analyzed using Agilent Chemstation. Accurate Mass LC-TOF analyses
of DSA reactions were performed on an Agilent Series 1200 LC
instrument, using a Halo-C18 column (2.1 × 50 mm, 2.7 μm) with
gradient elution (methanol/water containing 0.1% formic acid) at a
flow rate of 0.5 mL/min and at 50 °C. The eluent was analyzed by an
Accurate Mass LC/MS TOF mass spectrometer (Agilent Series 6220)
in positive-ion mode with an electrospray ionization source
(fragmentor = 375V). The data were analyzed using Agilent
MassHunter Workstation Software, Qualitative Analysis (V.
B.02.00). The isolation of [2]-catenanes was performed on a modified
semi-preparative HPLC (Agilent Series 1200 LC instrument), using an
Agilent Zorbax Eclipse XDB-C18 PrepHT Cartridge column (21.2 ×
250 mm, 7 μm) with isocratic methanol/water elution (no additive) at
a flow rate of 9 mL/min and at 55 °C. The eluent was monitored by
UV absorbance chromatogram at a wavelength of 289 nm, and the
corresponding [2]-catenanes were collected with a fraction collector.
MS/MS analyses of the isolated [2]-catenanes and DSA reactions were
performed on an Accurate Mass LC-QTOF (Agilent Series 6520)
equipped with an Agilent Series 1200 LC instrument without/with a
column (Halo-C18, 2.1 × 50 mm, 2.7 μm) in positive-ion mode with
an electrospray ionization source. X-ray crystallographic analyses of
[2]-catenanes F3dPh, HO3c, F3b, and F4b were performed on a Bruker-
AXS SMART APEX-II system equipped with a graphite mono-
chromator. [Please contact Dr. Peter S. White (pwhite@email.unc.
edu) for correspondence regarding X-ray analyses.]
Figure 1. Cartoon showing the flower-like structure of the [2]-
catenane composed of two interlocked identical tetramers.
Our communicated X-ray structure of [2]-catenane H3a
revealed that the presence of four β-turns in each tetramer
results in collapse of the large macrocycle into a conformer that
converges functionality and enables self-recognition (Figure
1).7 Inter- and intra-macrocycle hydrogen bonds, π−π stacking,
and aliphatic CH−π interactions all appear to contribute to the
high-fidelity recognition of one macrocycle by the other. In
addition to being physically interlocked, two β-turns were
found to create a clip that binds the aromatic portion of the C2-
related macrocycle, using the C−H bonds of one proline ring
and the C−H bonds of one Aib methyl substituent (Aib = 1-
aminoisobutyric acid, or α-methyl-alanine) as the teeth of the
clip, i.e., a CH−π−CH sandwich. Due to the C2-nature of the
catenane, this binding interaction is repeated in the symmetry-
equivalent position. Because the catenane was found to form
only when one monomer contained the phenylglycine or
naphthylglycine side chain that forms the CH−π−CH
sandwich, it appeared that this was a critical element required
for self-templating of the catenane.
To gain further insight into the relative contribution of each
of these non-covalent interactions11−13 to the tightly con-
strained self-complementary structure, we designed a series of
mutated [2]-catenanes and investigated their structure/stability
relationship. This broad study is reported herein in two parts.
The first focuses on structure, principally utilizing X-ray
methods but also supported by solution characterization
tools, including high-resolution NMR spectroscopy to probe
the similarity of solid-state and solution structures, and
solution-phase H/D exchange to probe structural dynamics
and solvent accessibility. These tools are complemented by an
in-depth mass spectrometry fragmentation analysis, which
enables constitutional isomerism to be deconvoluted. In this
first paper, we address the following questions: What
interactions are highly conserved? Are there parts of the
structure that can accommodate variation? Are the features
observed in the solid state maintained in solution? Each of
these questions is addressed with regard to the β-turns,
hydrogen bonds, and π−π stacking interactions in the “aryl
barrel” and the CH−π−CH sandwich. Lastly, the solid-state
and solution-phase structures of two non-symmetric catenanes
made up of two different rings are investigated and compared
to the C2 symmetric catenanes.
Generation and Isolation of 3dPh, HO3c, 3b, and 4b. For
F3dPh, two 5 mM DSA solutions (D-1d/L,L-F2Ph = 1:1) were prepared
on a 20 mL scale. Each was prepared by dissolving the mixture of D-1d
(20.2 mg, 50.0 μmol) and L,L-F2Ph (22.9 mg, 50.0 μmol) in chloroform
(20 mL) and subsequently adding trifluoroacetic acid (50 equiv, 5000
μmol, 371 μL). The solutions were allowed to sit for 6 days, and then
triethylamine (50 equiv, 5000 μmol, 0.7 mL) was added to quench the
dynamics. To remove the precipitates generated during the DSA
process due to the poor solubility of other oligomers in chloroform
and after the addition of triethylamine, the solutions were filtered, and
the two were combined. The volatiles were removed in vacuo, and
chloroform (50 mL) was added. This solution was washed with water
(40 mL) and then dried over anhydrous MgSO4. More chloroform (80
F
F
F
The second paper in this series14 utilizes mutation
experiments to determine the relative contribution of each of
the non-covalent interactions to the structure and the roles of
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mL) was added, and the solution was heated to 50 °C (to mitigate the
poor solubility of F3dPh in CHCl3) and then filtered to remove
MgSO4. The volatiles were removed in vacuo again, and the purity of
Scheme 1. [2]-Catenanes Self-Assembled from Pairs of
Hydrazide/Acetal Monomers
F
the crude product was determined by HPLC. Crude 3dPh contained
F
4% 4dPh, and without further purification, it was recrystallized from
pyridine by slow evaporation at room temperature to generate crystals
suitable for X-ray analysis. For HO3c, two biased monomer mixtures
(D-1c/L,L-HO2 = 3:1) were prepared on a 20 mL scale. Each was
prepared by dissolving D-1c (35.6 mg, 75.0 μmol) and L,L-HO2 (12.7
mg, 25.0 μmol) in 25% acetonitrile/chloroform (20 mL), to which was
subsequently added trifluoroacetic acid (50 equiv, 5000 μmol, 371
μL). The reactions were allowed to sit for 3 days, and then
triethylamine (50 equiv, 5000 μmol, 0.7 mL) was added to neutralize
the acid. The solutions were combined, and the volatiles were removed
in vacuo. Methanol (8 mL) was added to the resulting mixture, which
was then filtered. This solution was eluted on the semi-preparative
HPLC with 87% methanol/water isocratic solution to isolate HO3c.
The collected HO3c was recrystallized from methanol/chloroform by
slow evaporation at room temperature to obtain crystals suitable for X-
ray analysis. For F3b and F4b, three DSA solutions (D-1b/L,L-F2 = 1:1)
were prepared on a 20 mL scale. Each was obtained by dissolving a
mixture of D-1b (27.0 mg, 50.0 μmol) and L,L-F2 (19.1 mg, 50.0 μmol)
in 25% acetonitrile/chloroform (20 mL) and adding trifluoroacetic
acid (50 equiv, 5000 μmol, 371 μL). The reactions were allowed to sit
for 9−10 days, and then triethylamine (50 equiv, 5000 μmol, 0.7 mL)
was added to quench the reaction. All solutions were combined, and
the volatiles were removed in vacuo. Next, 65% methanol/chloroform
(15 mL) was added to the resulting mixture, which was then filtered
prior to semi-preparative HPLC separation (95% isocratic methanol/
water elution). The collected F3b and F4b were recrystallized from
methanol/chloroform by slow evaporation at room temperature to
obtain crystals suitable for X-ray analysis.
F
MS/MS Analysis of Isolated 4b and DSA Reaction Mixtures
Containing F4b and H4a. For the isolated F4b, the sample was
prepared by first dissolving in methanol and then eluting with an
isocratic eluent (50% methanol/water containing 0.1% formic acid) at
a flow rate of 0.2 mL/min for 2 min. Injection volume was 1 μL. The
MS source nebulizer gas and Vcap parameters were set to 35 psig and
3500 V, respectively. The fragmentor parameter was adjusted to either
175.0 or 375.0 V, depending on the experiments. The other MS source
parameters were default settings. The ramped collision energy was
adjusted by slope (typically 2) and offset (0 to 30) values. The MS1
and MS2 data were collected in the range of 100−3200 (m/z) with a
reference ion (m/z = 922.009798). With a 175.0 V fragmentor setting,
the targeted MS1 ion was the abundant doubly charged ion [F4b +
2H]2+ (m/z = 1588.7219) under ESI conditions. In the experiment
with a 375.0 V fragmentor setting, the targeted MS1 ion was the singly
F
charged ion [1b2 22 + H]+ (m/z = 1626.7100). For DSA reaction
mixtures containing F4b and H4a, 0.1 mL of a DSA solution was
diluted with 0.1 mL of 25% acetonitrile/chloroform solution prior to
the injection, and then the mixture was eluted with a gradient profile
(methanol/water containing 0.1% formic acid) at a flow rate of 0.5
mL/min and at 50 °C. The injection volume was typically 2 μL. MS
source parameters, the ramped collision energy, and the MS1 and MS2
data collection ranges were set in the same way. For the DSA solutions
generating F4b, the fragmentor parameters and targeted MS1 ions
respectively] under the acidic conditions necessary for
promoting hydrazone exchange yields a variety of cyclic
oligomers, with dimers, trimers, and tetramers predominating
(traces of pentamers and hexamers are also detected by HPLC
and LC-TOF analyses) (SI1 Figure 1).
Also appearing under these dynamic self-assembly (DSA)
reaction conditions are significantly less polar species that
steadily grow to a maximum after 3−5 days. LC-TOF analyses
showed them to be catenated octamers as judged by the
octamer to tetramer fragmentation pattern, which is character-
istic of an interlocked structure (vide infra) (SI1 Figures 2−
4).15 Two [2]-catenanes dominated, a cyclic octamer (3) made
up of two rings having identical mass, i.e. [1321]2, and an
asymmetric octamer (4) composed of interlocked [1321] and
[1222] rings (Figure 2).
F
were the same as those for the isolated 4b. For the DSA solutions
providing H4a, 175.0 and 420.0 V were chosen as the fragmentor
settings. At 175.0 V, the targeted MS1 ion was the doubly charged
[H4a + 2H]2+ (m/z = 1461.1533), while at 420.0 V, the targeted MS1
ion was the singly charged [1a2H22 + H]+ (m/z = 1510.6581).
RESULTS
■
1. Self-Assembly and Isolation of [2]-Catenanes. As
shown in Scheme 1, the combination of the D-Pro-X monomer
[(D- or D,L-1x; X = Aib, 1-aminocyclobutanecarboxylic acid
(Ac4c), 1-aminocyclohexanecarboxylic acid (Ac6c), L-(4-
chlorophenyl)glycine (L-4-Cl-PhGly)] and L-yPro-L-arylGly
[L,L-y2 or y2Ph; y = H, F or OH; aryl = Naph or Ph,
In some cases, other octamers having a mass of [1424] as well
as constitutional isomers of both 3 and 4 were observed in low
quantities (<10%, Figure 2b). While the amount of catenane 3
was usually improved through the use of biased component
mixtures (i.e., 1:2 = 3:1), the yield of 4 was typically not
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Figure 3. (a) X-ray structure of H3a displaying two identical
interlocked tetramers. (b) View from the bottom of (a) along the
C2 axis showing a dense core. All hydrogen atoms are omitted for
clarity.
of the other macrocycle. The metrical parameters of these units
suggests a nearly perfect arrangement for a beneficial CH−π
interaction.16
Similar features were observed in a series of new [2]-
catenanes (F3b, HO3c, 3dPh, Figure 4) self-assembled from
F
Figure 2. (a) HPLC-UV trace (289 nm) at day 7 of a DSA reaction
with D-1a and L,L-H2 (1:1, 5 mM in MeCN/CHCl3 = 1:3, 50 equiv of
TFA). (b) HPLC-UV trace (289 nm) at day 7 of a DSA reaction with
D-1b and L,L-F2 (1:1, 5 mM in MeCN/CHCl3 = 1:3, 50 equiv of TFA).
Inset: [2]-catenane region.
structurally modified monomer units (Scheme 1). Each is
flower-like with a dense core containing the aryl glycine units
sandwiched between L-proline and a variable unit that includes
1-aminocyclobutanecarboxylic acid (Ac4c), 1-aminocyclohex-
anecarboxylic acid (Ac6c), and L-(4-chlorophenyl)glycine (L-4-
Cl-PhGly). Like H3a, each tetrameric ring is saddle-shaped,
with four β-turns connected by a planar, extended 1,4-
hydrazone/benzamide linkage.
2.1.1. β-Turn Characterization. While proline is well known
to nucleate turn structures in peptides and proteins,10 the
structure adopted by the Pro-X segments (X = variable unit) in
these small-molecule receptors has not previously been
established. In each of the structurally characterized catenanes,
however, it is clear from metrical parameters (Φ, Ψ analysis, SI1
Table 1) that the Pro-X segment adopts a standard suite of β-
turns. For the purpose of this discussion, the β-turns are
identified as β-1−β-4, with β-2 (D-Pro-X unit) and β-4 (L-Pro-L-
arylGly unit) making up the core, and β-1 and β-3 (D-Pro-X
units) creating the turns at the periphery of the central pillar-
like and petal-like structures, respectively (Figure 4). In general
β-2 and β-4 were invariant, adopting a type II′ and type VIII
turn, respectively.17 Depending on the structural modifications,
the peripheral β-1 and β-3 turns were more variable, with type
II′ or type I′ being accessible.17
A comparative analysis of the β-turn metrical parameters of
the four structurally characterized catenanes was informative
(Table 1). Most striking was the invariability of the aryl glycine
unit in the central core (β-4), as almost no dihedral angle
differences (<9°) were noted across the series of structures.
The D-Pro-X unit, which provides the second half of the
molecular clip in the core (β-2), was also tightly constrained,
though the L-4-Cl-PhGly case (HO3c), which positions the 4-Cl-
phenyl unit below the naphthyl core and is non-C2-symmetric,
displayed larger deviations. By contrast, the peripheral β-turns
(β-1, β-3) were considerably more flexible, and were responsive
to changes in the D-Pro-X unit. For example, the Ac6c group in
F3b generated high torsional angle deviation in β-3, while the
Ac4c group in F3dPh and the L-4-Cl-PhGly group in HO3c
caused deviations in β-3 and, more noticeably, in β-1 where the
two loops interact. This analysis shows the turn structure in the
central core, i.e., the point where the CH−π−CH “clip”
interactions occur, to be highly conserved across the series of
sensitive to the feed ratio. The choice of the reaction solvent,
however, strongly affects the [2]-catenane speciation, with pure
chloroform giving 3 exclusively while 25% MeCN/CHCl3
maximized the amount of 4. Using these conditions, a series
of [2]-catenanes were self-assembled and isolated after
neutralizing the reaction solution to halt hydrazone exchange;
additional purification was achieved either by flash column
chromatography or by semi-preparative HPLC. Slow concen-
tration of these products from MeOH/CHCl3 or pyridine led
directly to X-ray-quality crystals.
For the purpose of this and the following paper, 3
corresponds to the C2-symmetric [2]-catenane that is made
up of two interlocked macrocycles assembled from three 1
monomers and one 2 monomer. The Ph superscript
designation refers to the phenyl glycine version of 2 (cf.
naphthylglycine), while the x and y designations correspond to
the D-monomer and naphthylglycine variants described in
Scheme 1, respectively.
2. Structural Analysis of [2]-Catenanes Having Two
Identical Rings (y3x). 2.1. Solid-State Analysis (X-ray).
Previously we communicated the high-resolution solid-state
structural features of H3a.7 Its most striking feature was its
compact structure and interlocked C2-symmetric nature (Figure
3). The structure can be loosely described as flower-like with a
dense core containing the two unique L-proline-L-naphthyl
glycine (L-Pro-L-NaphGly) units, from which radiate four loops
that terminate in β-turns. One loop from each macrocycle is
splayed out in a petal-like arrangement, while the second is
tightly associated with its C2-related pair in a pillar-like central
projection (Figure 3a).
Like the petal-loops, the pillar-like loops terminate in a
proline-induced β-turn. Each of the individual cyclic tetramers
adopts a roughly saddle-shaped conformation, with each of the
four Pro-X sequences adopting a β-turn structure that reverses
the direction of the chain. The fold of the tetramers is such that
the β-turns create a clip-like arrangement that positions a
proline and an Aib methyl above and below the naphthyl ring
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Figure 4. X-ray structure representations with one ring displayed as space filling models and the second interlocked ring as stick figures. (a) Side
view of F3b and (b) bottom view looking up onto the CH−π−CH sandwiches. (c) Side view of F3dPh and (d) top view of (c) highlighting the two
different β-1 turns (type I′ for ring 1 and type II′ for ring 2). (e) Side view of HO3c showing two interlocked tetramers and (f) bottom view of (e)
highlighting the CH−π interactions between L-NaphGly and L-HOPro residues, and the two different π−π stacking interactions between L-NaphGly
and L-4-Cl-PhGly moieties. All hydrogen atoms except those participating in CH−π−CH and CH−π−π sandwiches are omitted for clarity.
H
F
F
Table 1. Direct Comparison of Metrical β-Turn Parameters of 3a, 3b, 3dPh, and HO3c
a
a
ΔF3dPh
ring 1
−
H3a (°)
Δ
HO3c − H3a (°)
a
dihedral angle (deg) of H3a
ΔF3b − H3a (°)
ring 2
ring 1
ring 2
β-1 (loop)
β-2 (core)
β-3 (loop)
β-4 (core)
Φi+1
Ψi+1
Φi+2
Ψi+2
60.1
−145.9
−52.3
−4.6
3.4
−4.1
196.9
147.3
−2.1
2.7
12.6
1.3
6.4
−5.6
12.9
0.1
−24.7
15.9
−20.1
25.2
−10.4
7.5
−35.9
−2.2
Φi+1
Ψi+1
Φi+2
Ψi+2
58.7
−128.9
−62.7
6.0
−3.2
4.7
0.7
−0.4
−3.6
−4.0
3.4
−0.4
−2.4
−6.0
−0.7
−5.6
−17.9
20.2
7.1
7.6
−6.7
1.6
−13.0
−12.6
Φi+1
Ψi+1
Φi+2
Ψi+2
58.5
−126.6
−62.5
−6.0
−11.2
−6.6
−5.5
−1.8
−6.5
7.9
−0.5
−1.4
−9.5
15.9
2.2
−6.0
−10.7
7.1
3.3
−8.9
−5.8
6.6
−21.9
17.6
Φi+1
Ψi+1
Φi+2
Ψi+2
−66.1
−30.7
−159.0
83.0
1.2
−3.2
−3.5
−0.2
−3.4
−1.0
−1.4
−6.8
−5.3
−1.8
0.9
−5.8
−4.8
−1.1
3.2
−6.0
0.8
−1.7
−4.2
−8.6
a
The corresponding dihedral angle differences in H3a vs F3b, F3dPh, and HO3c. Dihedral angle differences of >10° are in bold italics. The rmsd metric
of structural similarity to the parent structure (H3a) obtained by PyMOL pair fitting using 48 arbitrarily chosen backbone atom pairs for F3b vs H3a,
F3dPh vs H3a, and HO3c vs H3a are 0.246, 0.791, and 1.067 Å, respectively. See details in SI1 Table 2.
[2]-catenanes. The tight packing of this region reinforces the
notion of a highly optimized three-dimensional structural motif
which provides significant structural stabilization.
i+3 residue engaged in a conserved inter-ring HB. The series is,
therefore, characterized by a total of six intra-turn hydrogen
bonds for β-1, β-2, and β-3, and six inter-ring hydrogen bonds
(β-2(N)→β-4′(O), β-4(N)→β-2′(O), β-2′(N)→β-4(O), β-
4′(N)→β-2(O), β-4(N)→β-4′(O), and β-4′(N)→β-4(O)).16
Except for catenane F3dPh, the Oi---Ni+3 distances for inter-ring
hydrogen bonds only marginally deviate from the parent
catenane H3a (≤0.063 Å) (SI1 Table 4), indicating that the
skeleton of the [2]-catenane is conserved across the series. As
2.1.2. Hydrogen-Bonding Interactions.18 In the peripheral
turns (β-1 and β-3) of the [2]-catenane, the expected i/i+3
intra-turn hydrogen bonds (HBs) are observed though there
are no inter-macrocycle HBs (SI1 Table 3). In contrast, the β-
turns in the core are distorted to enable inter-ring HBs for β-2
and β-4, with only β-2 containing a typical i/i+3 HB; β-4 has its
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H
F
F
Table 2. Direct Comparison of the Distances (Å) Characterizing CH−π or π−π Stacking Interactions in 3a, 3b, 3dPh, and
HO3c
b,
c
b
ΔF3dPh
−
H3a (Å)
Δ
HO3c − H3a (Å)
a
b
H3a (Å)
ΔF3b − H3a (Å)
ring 1
ring 2
ring 1
ring 2
L-proline and aryl
X and aryl
L-Pro C3
L-Pro C4
L-Pro C5
3.999
3.922
3.916
−0.291
−0.311
−0.163
−0.297
−0.289
−0.074
−0.260
−0.450
−0.317
−0.215
−0.144
−0.136
−0.315
−0.374
−0.310
X = Aib
3.425
X = Ac6c
0.089
X = Ac4c
X = L-4-Cl-PhGly
0.133
0.167
0.120
0.078
d
0.629
a
The distances between an imaginary L-NaphGly plane and the corresponding carbons of three L-Pro CH’s, and an Aib methyl carbon and the L-
b
NaphGly plane in H3a. The differences in the corresponding distances indicating CH−π or π−π stacking interactions in H3a vs 3b, 3dPh, and
F
F
HO3c. The negative values represent the shorter distance compared to those in H3a, implying stronger interactions. For the ring assignment, see
c
d
Figure 4d,f. An imaginary phenyl plane of the L-PhGly unit was used for computing the distances in F3dPh. Following the distance cutoff (X···M <
4.3 Å [Steiner, T.; Koellner, G. J. Mol. Biol. 2001, 305, 535−557], the distance between the L-NaphGly plane and the carbon of the second CH2
group (C2H2) in Ac6c moiety was included (see SI1 Table 5) and compared with the distance between an Aib methyl carbon and the NaphGly plane
in H3a.
will be discussed in more depth in the following paper,14 the
face (∼33°, SI1 Table 5). NMR experiments (vide infra)
indicate that the complex is fluxional, as only a single C2-
symmetric structure is observed in H NMR spectra.
F
Oi---Ni+3 distances for two inter-ring hydrogen bonds in 3dPh
1
(β-2(N)→β-4′(O) and β-2′(N)→β-4(O)) are shorter than
those in the rest of the catenanes. The trigger for the shortening
of these key inter-ring HBs is the replacement of a naphthyl
group for a phenyl group, which initiates a series of changes to
the network of non-covalent interactions.
A comparison of the metrical parameters for these catenanes
F
to H3a was informative (Table 2). In 3b and HO3c, shorter
distances between the naphthyl group and the F- or OH-
substituted proline moieties agreed with the expected enhance-
ment of the CH−π’s electrostatic component, which would be
stabilized by a more positively charged CH interacting with the
arene’s quadrupole.19 Despite the weaker π−donor character-
istics of Ph versus naphthyl,21 the decrease in the CH−π
2.1.3. CH−π Interactions in the Core. CH−π interactions
are well-known stabilizing molecular interactions.19,20 Since an
aryl group in the L,L-2 and 2Ph monomers is required for
catenane formation and the catenane adopts a fold that
sandwiches this arene between an L-proline unit and the X-
moiety of the other macrocycle, CH−π interactions are
reasonably inferred as an important, if not critical, stabilizing
interaction. Metrical parameters from H3a indicate a con-
strained geometry that maximizes the CH−π interaction
between naphthyl and L-Pro on one side (3.9−4.0 Å) and
Aib on the opposite (3.4 Å) (Table 2). Within the new series of
F
H
distances of 3dPh compared to 3a coupled with the stronger
thermodynamic stability of F vs non-F catenanes (see our next
paper14) reinforce the argument that the CH−π interaction can
be electronically manipulated, and that the more acidic CH
bonds of F-substituent prolines enhance the strength. Zondlo
has previously noted this effect in short peptide model systems
in water.19
F
F
catenanes, 3dPh and 3b follow a similar CH−π arrangement,
On the opposite face of the arene clip, the interactions
between the aryl group and Ac4c, Ac6c, and 4-Cl-phenyl
residues (F3dPh, F3b and HO3c) were variable. The combination
of a naphthyl group and the Ac6c moiety was structurally similar
with a geminally substituted cyclohexane replacing the Aib
methyl group of H3a below the naphthyl ring in 3b, and a
F
cyclobutyl group replacing it in 3dPh (see SI1 Table 5 for key
F
H
to 3a, except that the conformationally restricted Ac6c group
metrical parameters).
In the case of catenane HO3c, the naphthyl ring is pinched
between L-Pro and a 4-Cl-phenyl residue, i.e., a CH−π−π
molecular clip. The non-C2-symmetric nature of HO3c is
expressed in the differential π−π interaction between naphthyl
and the 4-Cl-phenyl unit, one being biased toward a nearly
eclipsed face-to-face interaction, while the other is better
described as intermediate between an edge-to-face and face-to-
(cf. CH3) orients a single CH into the aromatic ring, which
shifts this H resonance to higher field than any other CH−π
group in the 1H NMR spectrum, vide infra. The longer distance
H
between the Ph centroid and the Ac4c group in F3dPh (cf. 3a,
Table 2) indicates that the weaker π−donor character of a Ph
group causes a lengthening of the CH−π distances, implying
that the core might be less tightly packed. In the same way,
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catenane HO3c displays lengthened π−π interactions in one of
its two rings, suggesting that the optimum CH−π−π
arrangement cannot be simultaneously achieved in both halves
of the molecule.
2.1.4. Aryl Barrel. The gross features of the catenanes are
established by the connection of the peripheral β-turn loops to
the dense core. This connection is achieved through 1,4-
hydrazone/benzamide linkages that create a barrel-like
structure. The aromatic rings interact with one another through
a combination of slipped face-to-face and edge-to-face arrange-
ments (Figure 5).20,22 Of the eight aromatic rings making up
Other centroid distances informed on the width of the barrel
and hinted at potential for compensating effects in this region.
F
For example, compared to H3a’s barrel, those of 3dPh and
HO3c are smaller; in particular, the A-to-C centroid distances
are considerably shorter, implying that these structures benefit
from greater aromatic stabilization. The barrel size, as estimated
by the product of the A−C and B−D distances support such a
F
compression in 3dPh and HO3c.
2.2. NMR Analysis. 2.2.1. CH−π Interactions. The C2-
symmetric structure of catenane H3a was found to persist in
solution, and despite its size, high-resolution spectra could be
obtained in pyridine-d5, which acted to spread the chemical
7
1
shifts of the H NMR resonances. Close contacts between a
single methyl group of Aib and a proline residue to the
naphthyl ring in the clip was noted by their upfield-shifted
resonances, and appropriate cross-peaks in the ROESY
spectrum.7 Catenane F3b also provided a sharp spectrum
with well-dispersed signals that corresponded to a C2-
symmetric structure (6−8% MeOH-d4/CDCl3) (Figure 6a).
F
Figure 5. X-ray structural representations of 3dPh, emphasizing how
the 1,4-hydrazone/benzamide linkages establish an aryl barrel through
π−π stacking interactions (face-to-face and edge-to-face). The A, B, C,
and D linkages are directly connected to the core L-NaphGly (β-4), D-
Pro (β-2), Ac4c (β-2), and L-Pro (β-4) residues, respectively.
this barrel, a number of the centroid-to-centroid distances were
H
F
illuminating. For catenanes 3a, 3b, and HO3c, each of which
has a type II′ β-1 turn, the A and A′ aryl rings (Figure 5) occupy
positions at the interface of the interlocked region. The arene
rings interact through a slipped face-to-face orientation with a
nearly invariant plane-to-plane distance of 3.86 Å. A similar
geometry but significantly shorter distance (by ∼0.2 Å) was
F
noted for 3dPh, which adopts a single type I′ β-1 turn (F3dPh)
(Table 3 and SI1 Table 6). The combination of a type II′ and a
type I′ in the two interacting β-1 turns establishes an H-bond
between the proline amide carbonyl in ring 2 and the amide
NH of the Ac4c in ring 1 (N−O distance = 3.175 Å).23 It thus
seems reasonable that weaker CH−π interactions in the core
(i.e., Ph vs naphthyl)21 create a more flexible core that can
compensate by optimizing aromatic interactions in the barrel
region.
1
F
Figure 6. 500.1 MHz H NMR spectra at 20 °C of (a) 3b in 7%
F
MeOH-d4/CDCl3; (b) 3dPh in pyridine-d5; (c) HO3c in pyridine-d5.
H
F
F
Table 3. Direct Comparison of the Centroid-to-Centroid Distances (Å) in the Aryl Barrel of 3a, 3b, 3dPh, and HO3c
b
b
ΔF3dPh
ring 1
−
H3a (Å)
Δ
HO3c − H3a (Å)
a
b
H3a (Å)
ΔF3b − H3a (Å)
ring 2
ring 1
ring 2
A to A′ (C2-related ring)
A to C (intra-ring)
B to D (intra-ring)
3.855
8.938
9.121
0.010
0.028
0.249
−0.196
−0.813
0.298
−0.196
−0.775
0.234
−0.044
−1.162
0.683
−0.044
−0.943
0.289
c
d
d
d
d
d
barrel size (Å2)
81.523
2.488
−4.994
−4.971
−5.287
b
−6.290
a
Distances (Å) between imaginary centroids computed for each aryl linkage in the barrel as identified in Figure 5. Differential distances (Å)
describing both close contacts and barrel widths. Two values are reported for F3dPh and HO3c, as these compounds are non-C2-symmetric in the solid
c
state. Negative values described shorter distances. Barrel size is simply obtained from a multiplication of two barrel distances, i.e., (distance of A to
d
C) × (distance of B to D). Differential barrel size (Å2). Negative values described shrunken barrel areas.
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The F-substituted L-proline residues provided a useful handle
for structural analysis as the geminal H* participates in the
CH−π interactions of the clip (Figure 7). The H* was assigned
1
Figure 7. Portion (δ = −0.5 to 5.0 ppm) of 400.0 MHz H NMR
spectra of F3b at 20 °C in 7% MeOH-d4/CDCl3: (a) 19F coupled and
(b) 19F decoupled.
Figure 8. Portion of the 2D 600.1 MHz ROESY spectrum of F3b (F1
δ = −0.5 to 5.0 ppm; F2 δ = 6.5−8.5 ppm; 7% MeOH-d4/CDCl3, 20
°C). Inset: solid-state structure showing CH−π interactions of a L-
proline residue and an Ac6c group to the naphthyl group.
to a doublet at 4.0 ppm (JHF = 53.1 Hz), upfield shifted from
the monomer (δ = 5.1 ppm, JHF = 52.4 Hz). This assignment
was confirmed by comparison to H NMR spectra obtained
1
with 19F decoupling (Figure 7 and SI1 Figure 5). Cross-peaks
indicative of CH−π interactions between the L-FPro CH’s and
the naphthyl group were also observed in the ROESY spectrum
(Figure 8).24 Consistent with its solid-state structure, three
L-FPro CH cross-peaks to the naphthyl group were noted (see
inset Figure 8). The ROESY spectrum of F3b also showed that
one cyclohexyl hydrogen (δ = −0.2 ppm) was significantly
shielded by its close interaction with the opposite face of the
arene (vide supra). Unlike the Aib case, the CH group
participating in the C−H−π interaction does not exchange with
a non-interacting position (CH3 rotation) and is thus more
significantly shifted by its aromatic interaction. The obvious
near-neighbor contacts from the solid-state structures are
clearly maintained in solution.
toward the arene, and both thus exhibit upfield shifting and
cross-peaks to the Naph in the ROESY spectrum.
Despite the degree of spectral overlap in the aromatic region
of HO3c, the 4-Cl-phenyl residue participating in the π−π stack
could be identified on the basis of a combination of TOCSY
and ROESY spectra and its solid-state structure (SI1 Figures
12−14). Indicative were upfield-shifted aryl resonances at δ =
6.74 and 6.80 ppm (the remaining aryl signals resonate from δ
= 7.05 to 7.92 ppm), which additionally showed cross peaks to
the naphthyl group (SI1 Figure 14).
In addition to providing information relevant to dynamics in
the periphery of the catenane, NMR analysis also revealed that
some rotational motion is possible in the core of the 3
1
structure. Since a full assignment of the H NMR spectrum of
F
The remaining catenanes, 3dPh and HO3c, provided high-
F3dPh was achieved (ROESY, COSY, TOCSY, and HSQC, SI1
Figures 9−11), these data combined with spectral integration
indicated that rotation of the templating Ph−CH moiety was
rapid on the NMR time scale as the chemically inequivalent
ortho/ortho′ and meta/meta′ positions were time averaged
(Figure 9, inset). Thus, despite the apparent high density, the
near invariance of the core’s structure, and the tightness of the
CH−π−CH sandwich, solution dynamics can enable even
highly congested groups to rotate on the NMR scale.
resolution 1H NMR spectra in pyridine-d5 solvent (Figure
6b,c). Contrasting their non-C2-symmetric solid-state struc-
tures, however, these spectra were symmetric, suggesting time-
F
averaged dynamic behavior. In the case of 3dPh, this would
require interconversion of either the β-1 type I′ into a type II′
turn or the isomerization of the other loop’s type II′ turn into a
type I′ to regenerate C2 symmetry. A more subtle slipped π/π to
edged π/π exchange is required for macrocycle equilibration in
HO3c.
2.2.2. π−π Interactions in the Aromatic Barrel. In the solid-
state structure of each catenane, the 1,4-hydrazone/benzamide
linkages assemble through a combination of slipped face-to-face
and edge-to-face π−π-stacking interactions into a tightly packed
aromatic barrel (Figure 5).20,22 These close contacts were
Despite representing time-averaged spectra, the proline CH
units participating in the CH−π interactions of both catenanes
are diagnostic and upfield-shifted in their respective ROESY
spectra (SI1 Figures 11a and 13a). The CH−π interacting CH2
F
F
unit of the Ac4c residue in 3dPh are also upfield-shifted, and
investigated spectroscopically in 3dPh due to the simplified
cross-peaks to the Naph group in its ROESY spectrum are
observed. Unlike the Ac6c case, which orients the equatorial
CH to the arene, the Ac4c analogue projects both geminal CH’s
nature of its aromatic region. Spectra in pyridine-d5 generated
well-dispersed amide NH’s, imine CHN, and aromatic
signals. The COSY and TOCSY spectra enabled the aryl barrel
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structure is conserved across the series and is thus an important
contributor to catenane stability.
2.2.3. H/D Exchange. The four hydrazone NH’s (δ = 10.7−
11.3 ppm) and the four amide NH’s of H3a resonate in the
1
downfield portion of the H NMR spectra (pyridine-d5, 20
°C).7 The amide signals were differentiated from the imine CH
signals in the HSQC spectrum by the latter’s correlation to
their imine carbons. The β-4 amide NH was specifically
identified by its COSY cross peak to the adjacent CHα of the
aryl glycine. Since these NH groups (except for the β-1 and β-3
amide NH’s) participate in intra- and inter-macrocyclic
hydrogen-bonding interactions (vide supra), H/D exchange
reactions were utilized to investigate the inherent structural
flexibility of the catenane structure.
In pyridine-d5/D2O, all NH groups of H3a exchanged within
15 h with the exception of the amide NH’s of the β-2 and β-4
turns (Figure 10); these latter amide NH’s were unchanged
1
Figure 10. Portion (δ = 5.8−11.8 ppm) of the 599.8 MHz H NMR
spectra of H3a (pyridine-d5 (0.6 mL) + D2O (0.03 mL), 20 °C),
recorded at different time from mixing.
F
Figure 9. Portion of the 2D 500.1 MHz ROESY spectrum of 3dPh
(F1 δ = 5.9−9.5 ppm; F2 δ = 5.9−9.5 ppm; pyridine-d5, 20 °C).
◆
●
Ar 1,2, Ar 1,2, and ArΔ1,2 represent the remaining aryl ring CH’s (B,
◇
C, and D). Inset: solid-state structure indicating Ar 1,2, neighboring
even after 12 days. Since they are positioned near the Aib and L-
Pro units participating in the CH−π interactions and
additionally form inter-ring HBs (solid state), these data
imply that the core created by the β-2 and β-4 turns is densely
packed and relatively inaccessible. In contrast, the loops
comprised of the β-1 and β-3 turns are relatively flexible and
solvent accessible according to H/D exchange.
imine CH groups in the ring A′ and C (A−CHN, and C−CHN).
CH’s and the phenyl CH’s in the core to be distinguished (SI1
Figures 9 and 10), while the HSQC spectrum differentiated the
amide NH’s from the imine CH’s by the latter’s association to
an imine carbon (NCH) (SI1 Figure 11b). In addition to
signals in the expected spectral region, two resonances were
Catenane HO3c, which adopts a non-symmetric solid-state
1
structure but a C2-averaged H NMR spectrum, was subjected
1
consistently found upfield shifted in the H NMR spectra (δ =
to similar H/D exchange experiments to determine if this
phenomenon was indicative of a more dynamic structure; if the
non-symmetric solid-state structure was simply a crystal
packing effect, then an enhanced H/D exchange rate was not
expected. Additionally hinting at a “looser” arrangement were
the longer CH−π and π−π distances between the NaphGly and
the L-4-Cl-PhGly units. In this case, all NH groups exchanged
within 2.6 days, and the adjacent CHα transformed from a
doublet into a broad singlet lacking the normally observed
HC−NH vicinal coupling (Figure 11a). The contrasting
exchange of the core amide NH’s (β-2 and β-4), in particular,
indicates that the core is less tightly packed in solution and
more accessible than the Aib variant. Since the intermacrocyclic
HB lengths in the core are similar to H3a, this higher reactivity
appears to be driven by a weaker CH−π−π clip as compared to
the CH−π−CH clip. Alternatively, the dynamics that
accompany a structural rocking between the parallel and
edge-to-face aromatic orientations might enhance the cate-
nane’s accessibility to H/D exchange (Figure 4e,f).
5.9−6.5 ppm).
Key to their deconvolution was the strong through-space
correlation between the most upfield of these CH’s (Ar◇1
(2H), δ = 6.1 ppm) and a neighboring imine CH group (A-
CHN (1H), δ = 6.25 ppm) (ROESY, blue boxes in Figure
9), which overlaps with CHα in this compound. In the Figure 9
inset, these correspond to the circled hydrogens, one of which
is being viewed through aromatic ring B (hence the upfield
shift). Each of these resonances could then be correlated to a
network of three symmetry-inequivalent sets of aryl CH’s
(black boxes in Figure 9), thus establishing their close contact
and the spatial proximity of the remaining 1,4-substituted rings
(B, C, and D). Additionally noted was a strong correlation
(green boxes in Figure 9) between two imine CH’s (A−CH
N and C−CHN in Figure 9 inset), consistent with one
particularly close contact in the solid-state structure (3.4 Å).
The high degree of structural order as measured by the
preciseness of inter-aromatic contacts, and homology, as
assessed by the degree to which key ROESY cross-peaks
repeat across the series of catenanes, imply that the aryl barrel
F
For catenane 3dPh, the unique β-1 turn of one ring and the
longer CH−π distances coupled with the symmetric solution
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F
Scheme 2. Possible Daughter Ions for the Isomers of 4b
F
and [1b2 22 + H]+), three trimeric ions, and two dimeric
daughter ions ([1b2 + H]+ and [1b1 21 + H]+) with good
F
amplitude (Figure 12 and SI1 Figure 15). The key dimeric
1
Figure 11. (a) Portion (δ = 5.8−12.5 ppm) of the 500.1 MHz H
NMR spectra of HO3c (pyridine-d5 (0.6 mL) + D2O (0.03 mL), 20
°C), recorded at different time from mixing. The bottom spectrum is
the same portion of HO3c in pyridine-d5 before adding D2O. (b)
1
Portion (δ = 5.6−12.0 ppm) of the 500.1 MHz H NMR spectra of
F3dPh (pyridine-d5 (0.6 mL) + D2O (0.03 mL), 20 °C), recorded at
different time from mixing. The bottom spectrum is the same portion
F
of 3dPh in pyridine-d5 before adding D2O.
structure suggested a compound that was more flexible and
dynamic than the parent H3a. In this case, all hydrazone NH
groups exchanged within 2 h in pyridine-d5/D2O and the H/D
exchange of all amide NH groups was complete within 12 days
(Figure 11b). The rates of H/D exchange rates therefore follow
the trend of HO3c > F3dPh > H3a. For the catenane F3b, its poor
solubility in water-miscible polar solvents precluded its study in
an analogous fashion.
Together these results indicate that H/D exchange reactions
can be used to probe for dynamic processes that perturb the
structure of what is otherwise a highly compact assembly. While
perhaps related to the dynamic processes that symmetrize the
1H NMR spectra, these latter “breathing” dynamics occur on
the sub-millisecond time scale and are thus considerably faster
than H/D exchange. Perturbations in the metrical parameters
thus correlate well to the longer time scale processes that H/D
exchange reveals.
Figure 12. (a) Targeted doubly charged ion in MS1 stage for LC-MS/
MS analyses. LC-QTOF (CID MS/MS) spectra of the isolated F4b at
a collision energy of (b) 31.8, (c) 41.8, and (d) 51.8 V. The y-axis is
normalized.
3. Self-Assembled [2]-Catenanes with Rings of Differ-
F
H
ent Mass (F4b). 3.1. LC-MS/MS Analyses of 4b and 4a by
LC-QTOF. Previous MS/MS analysis of the minor [2]-catenane
H4a implied that it was composed of one [1a·1a·1a·H2] ring
and a symmetric tetramer with alternating 1a and H2 units,
[1a·H2·1a·H2].7 This suggestion was based on the lack of a
[H22+H]+ dimeric daughter ion (Scheme 2). To confirm the
daughter ion [F22 + H]+ (Scheme 2), which only occurs in the
F
non-alternating [1b2 22] structure, was observed in both cases,
but with only trace intensity.
F
structure, we investigated [2]-catenane 4b since it was the
Since we have previously demonstrated that hydrazones are
susceptible to ion−ion crossover in the mass spectrometer, it
was possible that this fragment formed during the electrospray
ionization process.25 An LC-MS/MS analysis of the DSA
sample containing H4a provided the same net result: only traces
of [H22 + H]+ ion (SI1 Figure 16).
major compound in the corresponding DSA mixture. LC-MS/
MS analysis on purified F4b and a DSA solution containing the
catenane F4b (plus others) by LC-QTOF significantly
improved our previous experiments’ resolution, sensitivity,
and mass accuracy.
For both samples, the doubly charged ion ([F4b + 2H]2+, m/
z = 1588.7219) was selected for fragmention on the basis of its
high abundance under the electrospray conditions. Both
To investigate the possibility that the traces of [F22 + H]+
were being generated through ion-crossover pathways, an
alternative MS approach was developed. By manipulating the
MS parameters, the catenanes’s two tetrameric ions could be
F
analyses showed two tetrameric daughter ions ([1b3 21 + H]+
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generated in their singly charged state in MS stage 1 (Scheme
3). The structures of these ions were then ascertained by a
Scheme 3. Possible Daughter Ions for the Isomers of
F
Tetrameric [1b2 22] Macrocycle
secondary fragmention in MS2. If the selected tetrameric ring
was alternating ((ii) in Scheme 3), only the hetero-dimeric
daughter ion would be observed, while structure (i) would
provide all three possible dimeric ions.
Following this protocol, a pure sample of F4b was
decomposed into its two tetrameric singly charged ions
F
F
([1b3 21 + H]+ and [1b2 22 + H]+) in the MS1 stage. The
key tetrameric ion [1b2 22 + H]+ was then selected and
F
additionally fragmented. Since all possible dimeric daughter
ions were detected (Figure 13 and SI1 Figure 17), these data
suggested that the structure of F4b was in fact (i) in Scheme 2.
A similar experiment on H4a, that is, the decomposition of
H
H4a into two tetrameric singly charged ions ([1a3 21 + H]+ and
[1a2 22 + H]+) in the MS1 stage followed by the selection and
H
fragmentation of the key tetrameric ion [1a2 22 + H]+, showed
H
all three possible dimeric daughter ions ([1a2 + H]+, [1a1 21 +
H
H]+, and [H22 +H]+), although the key homodimeric ion [H22 +
H]+ was of low intensity (SI1 Figure 18). We therefore
conclude that the more favorable constitutional isomers of H4a
and F4b are in fact (i) in Scheme 2, and that tetramer
fragmentation to [H22 + H]+ is unfavorable. These new data
support a correction to our previous assignment as (ii) in
Scheme 2.7
F
Figure 13. (a) Tetrameric singly charged ion ([1b2 22 + H]+)
generated in the MS1 stage for LC-MS/MS analyses. LC-QTOF (CID
MS/MS) spectra of the targeted tetrameric ion at a collision energy of
(b) 32.5, (c) 42.5, and (d) 52.5 V. The y-axis is normalized. Two filled
F
circles (green and pink) indicated two trimeric daughter ions ([1b2 21
F
+ H]+ and [1b1 22 + H]+), respectively.
3.2. Solid-State Analysis. The catenane F4b was obtained as
single crystals, and high-quality X-ray diffraction data were
obtained. The octameric [2]-catenane consists of two different
interlocked tetramers: ring 1 is composed of three 1b and one
F2 units, while ring 2 contains two units of 1b and F2 arranged
in a non-alternating manner [1a·1a·F2·F2] (Scheme 2 (i) and
Figure 14). This structure confirmed the MS data and
additionally added that the D-Pro-Ac6c occupied the β-1 and
β-2 turn positions, while L-FPro-L-NaphGly occupied the β-3
and β-4 positions.
adaptability of the β-1 and β-3 turns suggests that if any subunit
were to be replaced by a less optimal version, it would be β-1 or
β-3 (especially); the β-2 and β-4 sites would be the least
accommodating to a non-optimal replacement.
Compared to the identical macrocycles in F3b, ring 1 exhibits
larger torsional angle deviations (SI1 Table 7). While the core
was invariant across the 3 series, the β-4 metrical parameters of
F
F4b deviate from 3b, suggesting significant relay of remote
structural information back to the core, and an alternative
structural solution to minimizing the overall energy. Although
The structure of F4b is similarly flower-like, but compared to
F3b, it has one of its petal-like loops kinked away from the
pillar-like central region. Each tetrameric macrocycle is again
saddle-shaped, with four β-turns connected by an extended 1,4-
hydrazone/benzamide linkage. The metrical parameters of each
β-turn indicate that, while all five D-Pro-Ac6c units adopt type
II′ turns and the two L-FPro-L-NaphGly units in the core adopt
pseudo type VIII or type VIII turns (β-4 turns in ring 1 and ring
2, respectively), the unique L-FPro-L-NaphGly unit adopted a
type I turn (β-3 turn in ring 2) (SI1 Table 7). This latter turn is
that most commonly encountered in L-Pro-L-X structures.26
Conversion of a type I turn to the type VIII required for β-4
requires an ∼130° rotation27 of the L-NaphGly i+2 carbonyl
(Ψi+2). This carbonyl is then positioned to participate in a key
inter-ring HB (SI1 Tables 7 and 9). In hindsight, the greater
F
the turns in the core deviate from those in 3b, the structure
still effectively sandwiches the naphthyl ring between the
D-FPro and Ac6c units of the other macrocycle with CH−π
metrical parameters not unlike those observed in the 3 series
(SI1 Table 8). Based on CH---π distances, the interactions
between proline and NaphGly in each ring are weaker than in
F
the C2-symmetry analogue 3b but still stronger than those
H
observed in 3a. The interactions between NaphGly and Ac6c
in each ring are, however, weaker than those observed in both
F3b and H3a, which is likely the result of the adjacent
accommodating β-3 turn of an unfavorable turn type (type I).
F
F
Like its symmetric analog 3b, 4b has six turn-stabilizing
hydrogen bonds in β-1, β-2, and β-3 and six inter-ring HBs
(between β-2 and β-4 of both rings and between the β-4 turns
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Article
F
Figure 14. (a) X-ray structure representations of 4b, with one ring
displayed in space-filling and the second inter-locked ring in stick. (b)
Bottom view looking down onto the CH−π−CH sandwiches. Among
three L-FPro-L-NaphGly residues, the two core residues form CH−π
interactions, while the β-3 loop unit does not. (c) A single tetrameric
1
Figure 15. Portion (δ = 3.2−5.3 ppm) of the 400.0 MHz H NMR
spectra of F4b at 20 °C in 7% MeOH-d4/CDCl3 (a) coupled with 19
F
and (b) decoupled with 19F. The filled circles (red, blue, and black)
indicate the H* signals of three L-FPro residues described in the insert
picture. A portion (δ = −176 to −183 ppm) of the 400.0 MHz 19F
NMR spectra of F4b at 20 °C in 7% MeOH-d4/CDCl3, (c) 1H coupled
F
ring of F4b composed of three 1b and one F2 units (ring 1, [1b3 21]).
F
F
(d) A single tetrameric ring of 4b composed of two 1b and two 2
F
units (ring 2, [1b2 22]). Only selected hydrogen atoms are displayed.
1
and (d) H decoupled.
of each ring) (SI1 Table 9). The 1,4-hydrazone/benzamide
linkages connecting the β-turns also similarly arrange to form
an aryl barrel (SI1 Table 10). Like catenane F3dPh, which
F
substituted L-Pro units and the Ac6c protons involved in the
CH−π interactions were identified by TOCSY (SI1 Figure
20a). Although spectral complexity made it difficult to identify
the Naph groups involved in CH−π interactions, the TOCSY
spectrum identified six of the eight 1,4-benzamide groups,
which by the process of elimination narrowed the Naph region
(SI1 Figure 20b). The HSQC spectrum identified the imine
CHN (total 8) and three CHα resonances (SI1 Figure 21).
Based on these data, the proline-naphthyl and Ac6c-naphthyl
CH−π interactions could be deduced from the ROESY
spectrum (SI1 Figure 23). While the downfield H* assigned
to β-3 lacked cross-peaks to the naphthyl group, several upfield-
shifted proline CH’s did show cross-peaks. Similarly, only two
sets of upfield cyclohexyl CH resonances corresponding to the
two inequivalent Ac6c CH’s displayed such cross-peaks to the
Naph groups (SI1 Figure 23).
contains weak CH−π interaction units (PhGly-Ac4c), 4b also
displays a shortened inter-ring HBs next to the weak CH−π
moiety (NaphGly-Ac6c). Moreover, this effect also propagates
to the adjacent aryl barrel region where the interfacial (A−A′)
and cross barrel (A−C) distances also shorten. It is clear that
these non-covalent interactions are linked phenomena.
Of course, since 4 is a minor product in most DSA reactions,
the combination of F-proline, naphthylglycine, and Ac6c
appears to be uniquely able to compensate for an unfavorable
unit at β-3.
3.3. NMR Analysis. Like F3b, the catenane F4b also provided
a sharp spectrum with well-dispersed signals in pyridine-d5 (SI1
Figure 19). The eight hydrazone NH’s and three (doublet)
amide NH’s (adjacent to CHα in the L-NaphGly residue) were
well resolved and are consistent with an asymmetric structure
having two different tetrameric macrocycles and three L-Pro-L-
NaphGly residues. The lack of symmetry, however, made for
significant complexity, which could be partially overcome by
direct comparison to the symmetrical and similarly well
Like 3b and 3dPh, uniquely upfield-shifted resonances for
aryl and imine CH’s in the aryl barrel region of ring 1 and ring
2 were also noted in the ROESY spectrum, this time doubled
due to the lack of symmetry (SI1 Figure 24). The conservation
of these and numerous other close contacts across the 3 series
and in F4b thus implies that the solid-state structures are
preserved in solution.
F
F
F
resolved 3b (7% MeOH-d4/CDCl3). Although this solvent
choice caused partial H/D exchange of some NH’s, the spectra
F
were comparable to 3b.
Since catenane F4b has three F-substituted L-proline moieties
and only two are engaged in CH−π interactions, the two
upfield-shifted doublets at 3.9 and 4.1 ppm (4.0 ppm in the 1H
F
The flexibility and dynamics of 4b were also investigated
F
through H/D exchange reactions in pyridine-d5/D2O. Within
3.4 days, all NH groups exchanged and the three CHαs next to
the Naph amide NH’s collapsed into the broad singlets that are
indicative of vicinal H/D exchange (SI1 Figure 25). Overall,
F4b is much less dynamic and/or accessible to H/D exchange
than a non-folded monomer, but still looser and more dynamic
NMR spectrum of 3b) were assigned to the geminal H* in
each of the β-4 turns (Figure 15a). The downfield doublet at
1
F
5.0 ppm (5.1 ppm in the H NMR spectrum of monomer 2)
was assigned to H* in the β-3 turn fluoro-proline of ring 2; 19F-
decoupled H NMR spectra were supportive (Figure 15b).
1
Using the upfield-shifted H* doublets and the upfield-shifted
CH’s in the Ac6c as an anchor point, two sets of the F-
H
than the symmetric catenane 3a.
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Journal of the American Chemical Society
Article
assembly of interwoven non-covalent interactions and that
these interactions combine to maximally stabilize the catenane.
For example, when the core CH−π interactions was weakened
by substituting a naphthyl for an aryl π-donor (PhGly) or an
unfavorable turn (type I β-3 in catenane 4), reinforcing
interactions elsewhere in the structures compensated for the
loss of stabilization (contracted inter-ring hydrogen bonds or
compressed aromatic interactions in the aryl barrel). The
following article examines the interrelated nature of these
interactions.14
High-resolution NMR spectroscopy suggests that the solid-
state structures are largely conserved in solution, and full
assignments were achieved in several cases. H/D exchange in
protic D-solvents were also informative, showing that the loop
regions undergo H/D exchange at relatively fast rates, while the
interior core structures are much less dynamic. In one case, two
key amide NH’s did not exchange over the course of weeks,
even in an aggressive solvent (pyridine/D2O). We take these
data to reflect the degree of motion that is possible in the
structure. The loops which are more flexible and exposed are
readily deuterated, while the interior core NH’s are constrained
and inflexible, and are thus less prone to H/D exchange.
Comparisons across the series of catenanes reveal that where
structural deviations occur from the baseline structure, these
distortions correlate with higher H/D exchange rates.
The β-turns are also important, as their structures act to
project the 1,4-benzamide−hydrazone linkers that create the
aromatic binding pocket. This binding pocket, which can be
viewed as being optimized to bind an identical ring, has an
opening to accommodate the entrance of a suitable guest
(Figure 16). Since a number of turn types are available in a
mixed Pro-X library, one can envision how combinations of
turn geometries can conspire to create an adaptable class of
receptors that through dynamic unit interchange can become
optimized for guest-binding.5
When one of the tetramers is computationally excised from
the interlinked structure previously described, the reversals of
peptide direction achieved by the β-turns become clear. The
view showing these linkers in Figure 16 is additionally
informative. Note how the benzamide groups of the aryl barrel
vertically align to create a deep aromatic groove that is open on
one side and “binds” the threaded benzamide of the other ring.
Such a picture suggests that non-catenated tetramers should be
capable of adopting similar conformers that present deep
aromatically lined pockets for the recognition of flat structures.
Previous work by us and others has shown that non-catenated
tetramers comprised of related monomers bind guests with
aromatic groups like protonated nucleotides and cinchonidine
salts.9c,d The current results suggest a mechanism and structure
for this molecular recognition.
DISCUSSION
■
We have investigated the features of several octameric [2]-
catenane structures derived from dipeptide hydrazide A−B
monomers in the solid state, along with solution character-
ization through high-resolution NMR spectroscopy and H/D
exchange. For catenane class 3, the structures are composed of
two C2-related interlocked rings, each of which is assembled
from three units of 1 and one unit of 2. By contrast, catenane
class 4 comes from the asymmetric assembly of a [1·1·1·2] and
a [1·1·2·2] macrocyclic unit. In all cases (class 3 and 4),
monomer 2 is of the L-Pro-L-ArylGly stereochemistry, and
yields for the catenane were maximized when the aryl group is
naphthyl, though phenyl is sufficient to template self-assembly.
The family of catenanes can be described as flower-like with a
compact core where the aryl of the arylglycine is pinched
between two loops of the saddle-shaped tetrameric ring (Figure
1). The key core formation elements are CH−π interactions
provided by a proline on one side and the methyl of an Aib on
the other. Six inter-ring hydrogen bonds additionally stabilize
the interior core structures. From this highly conserved core
emerge petal-like loops that terminate in β-turns. Each Pro-X
unit adopts a standard suite of β-turns (types I, I′, II′, and VIII)
which have the effect of reversing the chain by ∼180°;
deviations from 180° affect the spatial projection of the flat
extended hydrazone/benzamide aromatic units. The core turns
are invariant and adopt a type II′ turn for β-2 and a type VIII
turn for β-4, while the peripheral turns (β-1 and β-3) are more
variable (types II, I, and I′). These β-turns additionally organize
a tightly packed aryl barrel with little free volume in the static
structure, though transient deformations can accommodate aryl
rotations that are fast on the NMR time scale. It is in this region
of the structure that the two macrocycles thread (Figure 16).
The barrel is characterized by an array of slipped face-to-face
and edge-to-face π-stacking interactions20 and resembles the
aromatic clusters observed in the interior of proteins.28 Analysis
of these structural features reveals that the catenane is an
In addition to displaying a benzamide lined binding pocket,
the peptide turns provide points for hydrogen bond donation
and acceptance at the barrel’s periphery. This display of polar
functionality above and below a non-polar interior has rough
parallels with cyclodextrins, calixarenes, and cucubiturils, all
well-established molecular receptors.29
This analysis therefore suggests that an important
component of the binding capability of these structures
comes from the ability to present an aromatically lined cleft
that is predisposed for binding. One presumes that such a
binding site could accommodate flat arene rings via π−π
interactions as displayed in the [2]-catenane structures
described above, but could also utilize strong cation−π and
F
Figure 16. X-ray structural representations of 3dPh emphasizing how
the benzamide linkages can create an aromatic pocket for binding and
threading a second benzamide chain.
11427
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Journal of the American Chemical Society
Article
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perhaps even CH−π interactions to bind alternative structural
motifs. The display of polar groups above and below this site
could then provide secondary or tertiary points of attachment
as the [2]-catenanes take advantage of herein. Although we
have no direct evidence for this binding mode in any DCC-
selected host−guest complexes, the studies described here
provide our first structural glimpses into the mechanisms of
molecular recognition by this adaptable class of compounds.
CONCLUSION
■
The [2]-catenane structures described herein contain many of
the hallmarks of a protein. (1) They are relatively large in size
(>3 kDa), with a hydrodynamic volume that is large enough to
benefit from NMR pulse sequences designed for macro-
molecules. (2) They have clear components of secondary,
tertiary, and quaternary structure, along with a very strong
sequence-dependent structure/stability relationship. (3) Like
proteins, they additionally contain distinct regions of variable
dynamic behavior (as revealed by H/D exchange). Despite
these many similarities, however, the [2]-catenanes simulta-
neously take on the properties of small-molecule receptors, and
are thus amenable to solid-state, solution-state, and gas-phase
characterization tools. Their structures can be readily
manipulated with non-biogenic amino acids to test the effect
of electronic and/or steric perturbations, they are amenable to
relative stability determinations via their self-assembly proper-
ties, as described in the next paper,14 and they self-assemble in
organic solvents. This balance of physical properties positions
them at a bridge point between proteins and small molecules,
and enables one to begin investigating how diverse non-
covalent interactions acting in a multi-valent format affect
structure, function, and stability in organic solvents, where the
balance of non-covalent forces differ from those in water. These
complex three-dimensional structures provide a promising
scaffold for pre-organization of functional groups needed to
create enzyme-like catalysts.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of monomers and their physical data, solid-state
metrical parameters, physical data, HPLC traces, MS spectra
and various 2D NMR spectra (COSY, TOCSY, HSQC,
(6) Cousins, G. R. L.; Poulsen, S. A.; Sanders, J. K. M. Chem.
Commun. 1999, 1575−1576.
F
F
ROESY) of [2]-catenanes (F3dPh, 3b, HO3C, 4b), LC-MS/
́
(7) Chung, M.-K.; White, P. S.; Lee, S. J.; Gagne, M. R. Angew. Chem.,
Int. Ed. 2009, 48, 8683−8686.
MS analyses of 4b and H4a, crystal data of catenane 3dPh,
F3b, HO3C and F4b. This material is available free of charge via
the Internet at http://pubs.acs.org.
F
F
(8) Lam, R. T. S.; Belenguer, A.; Roberts, S. L.; Naumann, C.;
Jarrosson, T.; Otto, S.; Sanders, J. K. M. Science 2005, 308, 667−669.
(9) (a) Chung, M.-K.; Severin, K.; Lee, S. J.; Waters, M. L.; Gagne,
́
M. R. Chem. Sci. 2011, 2, 744−747. (b) Ingerman, L. A.; Waters, M. L.
AUTHOR INFORMATION
Corresponding Author
mgagne@unc.edu; mlwaters@unc.edu
J. Org. Chem. 2009, 74, 111−117. (c) Chung, M.-K.; Hebling, C. R.;
■
́
Jorgenson, J. W.; Severin, K.; Lee, S. J.; Gagne, M. R. J. Am. Chem. Soc.
2008, 130, 11819−11827. (d) Bulos, F.; Roberts, S. L.; Furlan, R. L.
E.; Sanders, J. K. M. Chem. Commun. 2007, 3092−3093. (e) Nicholas,
K. M.; Masaomi, M. J. Org. Chem. 2007, 72, 9308−9313. (f) Voshell, S.
Notes
The authors declare no competing financial interest.
́
M.; Lee, S. J.; Gagne, M. R. J. Am. Chem. Soc. 2006, 128, 12422−
12423. (g) Roberts, S. L.; Furlan, R. L. E.; Otto, S.; Sanders, J. K. M.
Org. Biomol. Chem. 2003, 1, 1625−1633.
ACKNOWLEDGMENTS
■
(10) (a) Robinson, J. L. Acc. Chem. Res. 2008, 41, 1278−1288.
(b) Francart, C.; Wieruszeski, J. M.; Tartar, A.; Lippens, G. J. Am.
Chem. Soc. 1996, 118, 7019−7027. (c) MacArthur, M. W. J. Mol. Biol.
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Adv. Protein Chem. 1985, 37, 1−109. (e) Kessier, H. Angew. Chem., Int.
Ed. 1982, 21, 512−523.
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6049−6076. (c) Plevin, M. J.; Bryce, D. L.; Boisbouvier, J. Nature
We thank the Defense Threat Reduction Agency (DTRA) for
support (HDTRA1-10-1-0030). S.J.L. thanks the Army
Research Office for support. We thank Dr. Marc ter Horst
for helpful discussions of NMR spectroscopy techniques.
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(15) With a high-energy fragmentor parameter (375.0 V), LC-TOF
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̈
̈
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