Synthesis of bioconjugated sym -pentasubstituted corannulenes: Experimental and theoretical investigations of supramolecular architectures

Article abstract of DOI:10.1021/bc400408d

Applications of supramolecular architectures span a broad range of fields from medicinal chemistry to materials science and gas storage, making the design and synthesis of such structures a goal of high interest. The unique structural and symmetric proper

Full text of DOI:10.1021/bc400408d

Article
pubs.acs.org/bc
Synthesis of Bioconjugated sym-Pentasubstituted Corannulenes:
Experimental and Theoretical Investigations of Supramolecular
Architectures
Martin Mattarella,† Laura Berstis,† Kim K. Baldridge,*,† and Jay S. Siegel*,†,‡
†Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
‡School of Pharmaceutical Science and Technology, Tianjin University, A203/Building 24, 92 Weijin Road, Nankai District, Tianjin,
300072 P. R. China
S
* Supporting Information
ABSTRACT: Applications of supramolecular architectures span a broad range of fields
from medicinal chemistry to materials science and gas storage, making the design and
synthesis of such structures a goal of high interest. The unique structural and symmetric
properties of corannulene and the recent synthetic developments of C5-symmetric
pentafunctionalized derivatives motivate efforts to synthesize bioconjugated-corannulene
systems and investigate their supramolecular assembly properties. Herein is presented
the synthesis of sym-pentasubstituted corannulenes functionalized with sugar (galactose
and ribose), oligopeptide, nucleosides (thymidine and deoxyadenosine), and palindromic
oligonucleotide strands. Experimental and theoretical results demonstrate capability of
supramolecular assembly formation in these constructs. Ab initio theoretical modeling
enables further evaluation of structure and energetics of oligonucleotide-functionalized
corannulene formation. Results indicate formation of aggregates, although icosahedral
supramolecular formation is not observed. Analyses suggest future improvements to
synthetic routes to achieve icosahedral architectures.
core imparts a relatively high dipole moment to the molecular
and different electronic properties on the concave vs convex π-
face.45 Columnar architectures from derivatives of 1 have been
observed in solid46−50 and liquid-crystalline phases,51 and
shown to respond to an electric field and display ferroelectric
properties.51−55
INTRODUCTION
■
Controlled preparation of three-dimensional (3D) architectures
is an important and long-standing goal in the field of
supramolecular chemistry, with diverse applications as scaffolds
for molecular organization,1 gas storage materials,2 drug
delivery vehicles,3,4 templates for the development of new
materials,5 and tools for encapsulating and controlling reactivity
of guest molecules.6−8 A number of methods for the formation
of noncovalent nanostructure have been developed,9−22 with
promising results from amphiphilic bioconjugates function-
alized with oligopeptides,23−29 nucleosides,30−33 and carbohy-
drates.34−36 Pioneering results reported by Seeman et al.37−40
show the potential of annealing complementary DNA strands
as a high-specificity approach to preparing nano-objects with
desired structures and symmetry.
Molecular building blocks with fivefold symmetry offer the
potential of forming icosahedral supramolecular assemblies.41
Such icosahedra are common in structural biological and in
inorganic clusters;42−44 however, the directed supramolecular
synthesis of such structures from organic building blocks
remains a challenging target. In this regard, the high-symmetry
and geometrical properties of C5-symmetric pentasubstituted
derivatives of corannulene, 1, make supramolecular aggregates
of this class of compounds an attractive option.
Recently, a kilogram-scale synthesis of 1 was developed,56
which allowed the investigation of synthetic routes to sym-
pentachlorocorannulene, 2,57 on a 20 g scale. From 2, a general
strategy via Fe-catalyzed cross coupling chemistry made
available a large family of fivefold symmetric penta-function-
alized corannulene derivatives, on a scale of hundreds of
milligrams, displaying virtually any of the functional groups
common in organic chemistry.58
The synthesis of sym-pentabioconjugated corannulene
derivatives functionalized with lipids59 and (oligo)-
saccharide58,60 has been achieved with a copper-catalyzed
alkyne−azide cycloaddition reaction (CuAAC) on penta-
terminal acetylene corannulene derivative 3 (Figure 1). These
promising results motivated the present elaboration of a general
strategy to sym-pentabioconjugated corannulene derivatives,
and investigation of their assembling behavior. The geometric
properties of single-stranded DNA (ssDNA) conjugated
Besides icosahedral aggregates, the supramolecular chemistry
of corannulene bioconjugates could lead to several new
materials. In particular, the curved geometry of the aromatic
Received: September 5, 2013
Revised: December 3, 2013
Published: December 9, 2013
© 2013 American Chemical Society
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Figure 1. Synthetic route for the synthesis of fivefold symmetric pentabioconjugated corannulene.
Scheme 1. Synthesis of Pentakis-Glycoside Corannulene Derivatives, 4, 5, and 7
corannulene is also computationally characterized with a new
implementation of semiempirical ab initio methodology,
supporting the experimental findings and offering suggestions
for improved molecular design in future constructs.
In the first class of pentakis-carbohydrate corannulene
derivatives, galactoside 458 and riboside 5 were chosen. Two
different moieties in its structure offer possible modes of
interaction: the hydrophobic aromatic core, enabling π−π
interactions, and the five hydrophilic sugar units surrounding
the corannulene, which direct columnar organization in water
via the hydrophobic effect.61
The synthesis of sym-pentariboside corannulene 5 was
achieved with a CuAAC reaction of the alkyne 3 with 2,3,5-
tri-O-acetyl-β-D-ribofuranosyl-1-azide 662 yielding the peracety-
lated corannulene derivative 7 which was subsequently
deprotected with sodium methoxyde (Scheme 1).
Design and Synthesis. Many intermolecular interactions
are involved in the formation of supramolecular aggregates of
amphiphilic molecules, including dipole−dipole, π−π stacking,
hydrogen bonding, nonspecific van der Waals, hydrophobic,
electrostatic, and repulsive steric interactions. Energetic
stabilization from a combination of these forces counterbalance
the entropic cost of desolvation and polymolecular aggrega-
tion.24 Based on these concepts, three classes of C5-symmetric
pentabioconjugated corannulene derivatives functionalized with
carbohydrate, oligopeptide, and nucleoside were designed and
synthesized.
Compound 5 was obtained in good yield and in high purity.
With azides present on the secondary carbon,58−60 such as in 6,
it was observed that the CuAAC reaction required a higher
temperature (80 °C) and longer reaction time (8 h) in order to
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Scheme 2. Synthesis of Pentakis-Oligopeptide Corannulene, 9
achieve full conversion of the starting material, perhaps due to
steric hindrance to accessing the azide group. The protection of
the hydroxyl groups of ribose was necessary for the high-purity
isolation of compound 7 from the crude mixture.
Scheme 3. Synthesis of Azido-Nucleosides Derivatives, 15
and 17
For the second class of bioconjugated pentapods, a
corannulene core was functionalized with the tripeptide H-
Ala-Ala-Ala-OMe, 8. This oligopeptide was selected due to its
capability to form β-strands in solution,63,64 and therefore its
potential to utilize H-bonding networks to form interesting and
intricate supramolecular structures in solution. For instance,
dimeric or polymeric columnar assemblies of the corannulene
core could be stabilized by H-bonds between the oligopeptides
chains.
Corannulene derivative 9 was prepared in good yield and
high purity via the CuAAC reaction of alkyne 3 with the azide
derivative 10, which was prepared by treatment of the amine 8
with trifyl azide prepared in situ (Scheme 2). As for the
synthesis of 7, a higher temperature (80 °C) and longer
reaction time (8 h) was required.
Nucleosides are an important class of biomolecules that
forms strong and predictable intermolecular H-bonding
networks through Watson and Crick base pairing. These
interactions can both drive and stabilize the formation of C5-
symmetric dimers of complementary base-pair sym-pentasub-
stituted corannulene derivatives, utilizing either adenine-
thymine (A-T) or cytosine-guanine (C-G) pairing. For this
third class of bioconjugated corannulene in the present work,
the base-pair A-T was selected, and pentakis-thymidine 11 and
pentakis-deoxyadenosine corannulene 12 derivatives were
synthesized starting from 3 and the azido-nucleosides 17 and
15. Preparation of 5′-azido-2′-deoxy-adenosine 15 was achieved
by nucleophilic substitution of 5′-tosylated-2′-deoxy adenosine
1465 with lithium azide. The relatively low yield obtained for
the synthesis of compound 14 by reaction of 13 with tosyl
chloride was due to the formation of 2′-tosylated and 5′-3′-
ditosylated products. The 5′-azido thymidine 17 was
synthesized following a reported66 one-step procedure, reacting
thymidine 16 with carbon tetrabromide, triphenylphosphine,
and sodium azide (Scheme 3).
The successful synthesis of these bioconjugated penta-
pods58−60 demonstrates the robustness and applicability of
the CuNPS-catalyzed CuAAC procedure for the preparation of
new classes of C5-symmetric pentafunctionalized corannulene
derivatives.
Capitalizing on the bowl-shaped geometry and C5-symmetry
of sym-pentasubstituted corannulene derivatives, the synthesis
of icosahedral supramolecular aggregates was approached using
DNA-based bioconjugation, which offers high specificity for
formation of highly symmetric structures. Four general
strategies for construction of 3D DNA-based architecture
have been developed. The first strategy, “DNA origami”,
involved the folding of a long single-stranded DNA (ssDNA)
into the designed geometry by annealing with short “staple
strands”.67 This procedure enables high geometrical con-
trol,68−73 but requires the use of hundreds of different staple
strands, as well as a long assembly time. The second strategy
involves the use of identical branched DNA building blocks that
assemble into DNA cages that present only dsDNA and a
defined symmetry.74,75 The third strategy, developed by
Sleiman, is based on the assembly of ssDNA polygons with
organic corner units, which are then assembled by ligation into
3D DNA architectures.76−80 This strategy has been improved
and simplified by development of a procedure in which the
Fivefold symmetric pentabioconjugated corannulene deriva-
tives 11 and 12 were synthesized in good yield and high purity
with a CuAAC reaction of alkyne 3 and azides 17 and 15,
respectively (Scheme 4).
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Scheme 4. Synthesis of Azido-Nucleosides Derivatives, 11 and 12
Figure 2. Pentakis-DNA corannulene, 18.
DNA polygon is formed without the separate ligation step.81
The fourth approach, developed by von Kiedrowski, involves
formation of a fully dsDNA 3D-architecture via annealing poly-
DNA-functionalized organic molecules.82,83
In the present work, the fourth strategy was chosen for
formation of icosahedral aggregates, since coupling the
bioconjugation to an organic corannulene core was an
important component. The structure of the pentakis-DNA
corannulene 18 was designed to utilize a 16-base self-
complementary ssDNA sequence (Figure 2), with high
concentration of T and A at the 5′- and 3′-end, to avoid the
formation of loops, and a central C/G region to increase the
stability of the double-stranded structure. As such, the ssDNA
chains may fully anneal with the ssDNA strands of other
molecules in solution into many polymeric structures, including
a supramolecular icosahedral capsid.
Compound 18 was synthesized by a CuAAC “click” reaction
of the alkyne 3 with the 5′-alkylazido-modified oligonucleotide
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Scheme 5. Synthesis of sym-Pentaoligonucleotide Corannulene 18
19. Since the oligonucleotide 19 is not stable under the
conditions of a CuNPS-catalyzed CuAAC reaction, a new
synthetic procedure was developed for the functionalization of
compound 3. After screening of several conditions, the best
results in terms of yield and time were obtained using
copper(I) bromide as the copper source together with tris-
(benzyltriazolymethyl)amine (TBTA)84 as a ligand and
stabilizer of Cu (I) species in a mixture of THF and water
(Scheme 5).
RESULTS AND DISCUSSION
■
Due to the hydrophobic corannulene core of the amphiphilic
sugar pentapods, 4 and 5, combined with the strongly
hydrophilic galactoside and riboside functionalization, these
molecules may form stacked supramolecular aggregates in
aqueous solution. Unfortunately, riboside corannulene 5 did
not show any solubility in water, but was only soluble in
strongly polar solvent such as DMF and DMSO. Therefore,
formation of aggregates in water was investigated only for
galactoside, 4, via 1H NMR, absorption, and emission
spectroscopy.
The thermodynamic behavior of the aggregation phenomena
was evaluated by means of 1H NMR spectral analysis for several
solutions of 4, across a range of concentrations and
temperatures. Upon increasing the temperature of a solution
at constant concentration of 4, a downfield progression of the
chemical shift (δobs) of the five homotopic hydrogens atoms on
corannulene core was observed (Figure 3).
Figure 3. Aromatic region of 1H NMR spectra of a solution 0.28 mM
of 4 at 300 K (red), 310 K ((black), 320 K (blue), 330 K (violet), and
340 K (green); δobs of corannulene hydrogens are shown. Chemical
shifts of the water peak at each temperature were calculated in
previous work.85
The association constants, Ka, associated with the aggrega-
tion of 4 in water were calculated as a function of temperature
by nonlinear least-squares regression based on the equal K or
isodesmic (KE) model of indefinite self-association, as described
by Martin (Figure 4).86
Ka-values (Table 1) were obtained by fitting the experimental
data to eq 1, where δobs is the observed chemical shift, δmon is
the chemical shift of monomer, Ka is the association constant, c
is the molar concentration of the sample, and Δ is the
difference in the chemical shift of the monomer and the
aggregate.
The aggregation number N, i.e., the number of the
monomers included in the aggregates, was determined using
eq 2, where N is the aggregation number. An aggregation
number of 2 was estimated with the assumption that 4 is in
equilibrium between the monomer and the aggregate.87,88
ln[c(δmon − δobs)] = N ln[c(δobs − δ )] + ln Ka + ln N
agg
− (N − 1)ln(δmon − δ )
agg
(2)
The thermodynamic parameters, ΔH (−13 1 kcal mol−1)
and ΔS (−18 4 cal mol−1), associated with the dimerization
process, were determined with a Van’t Hoff plot of the Ka
values calculated at various temperatures (Figure 5). Surpris-
ingly, unlike most amphiphilic molecules, the driving force for
⎧
⎫
⎪
⎪
[1 − (4Kac + 1) ]
2Kac
⎨
⎬
⎪
⎭
δobs = δmon − Δ 1 +
⎪
⎩
(1)
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The hypsochromic shift in the absorption spectra as a
function of increasing temperature (Figure 6, left) and the
quenching of the fluorescence due to the increasing of the
concentration of 4 (Figure 6, right) suggest formation of H-
dimers in solution.89,90 CD analysis of the aggregation process
did not show any significant signal in the range of the
investigated concentration, because of the low signal-to-noise
ratio obtained at these conditions.
Poor solubility of corannulene derivatives 9, 11, and 12 in
nonpolar organic solvents did not permit investigation of
formation of supramolecular assemblies from these compounds
through formation of H-bonding networks. These bioconju-
gated compounds are highly soluble in strong polar solvents
such as DMF, DMSO, and TFE. However, 1H NMR and UV−
vis investigation of solutions of 9, 11, and 12 in these solvents
did not substantiate formation of any aggregates. Most
probably, the absence of supramolecular constructs is due to
the saturation of the H-bonding sites by the solvent.
1
Figure 4. Fitted curves of H NMR spectra of 4.
Table 1. Association Constant Ka for the Aggregation of 4 at
Different Temperatures
Formation of aggregates was further investigated by HR-MS
measurements; the high sensitivity of this technique enables
study of highly diluted solution of compounds 9, 11, and 12 in
methanol. While the pentakis-oligopeptide, 9, did not show
formation of any aggregates, the MS spectrum of a solution
nearly equimolar of 11 and 12 showed a peak corresponding to
the heterodimer (Figure 7).
T (K)
Ka (×104) (M−1
)
300
310
320
330
340
20 10
5
6
3
2
2
1
1.4 0.3
The higher intensity peak for heterodimer over homodimers
in 11−12 is evidence of formation of dimeric aggregates
between the two pentakis-nucleosides corannulenes. However,
neither the conformational details of the dimer nor
intermolecular forces involved could be obtained from the
MS data.
The ability of DNA corannulene derivative 18 to form
ordered supramolecular assemblies was investigated by native
PAGE and ssDNA-selective enzymatic digestion. Successful
icosahedral supramolecular structure formation would require
generation of complete annealing of 12 units of compound 18,
generating 30 dsDNA edges.
Two annealing strategies were pursued utilizing either kinetic
control or thermodynamic control. In the kinetic strategy, a
solution of 18 was first melted of any dsDNA at 95 °C, then
was cooled to 4 °C by immersion in an ice-bath. In the
thermodynamic strategy, the melting step was followed by an
annealing step at a temperature roughly 10 °C below the
Figure 5. Van’t Hoff plot for compound 4.
aggregation of 4 in solution is not the increase in entropy of the
system, but the exothermicity of the process.
Figure 6. Selected region of UV spectra of a solution of 4 at varying temperature (left). Emission spectra of solution of 4 at varying concentrations
(right).
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Figure 7. HR-ESI-MS in MeOH + 0.05% formic acid of 12 (top), mixture of 11 and 12 (middle), and 11 (bottom).
melting point of the double strand for at least an hour, and a
final cooling step at 4 °C. Different outcomes were obtained
from applying these two strategies to the same sample solution
of 18 (1 μM in TE Na+ 100 mM). Formation of small
aggregates, mainly dimers and tetramers, was favored under
thermodynamic control, whereas the kinetically controlled
strategy achieved aggregates up to 12-mers. On the basis of this
promising result, the annealing conditions were optimized,
specifically the concentrations of 18, Na+, and Mg2+ and time of
the annealing step. The conditions maximizing formation of the
12-mer were found with the kinetically controlled protocol with
a solution 2 μM of 18 in TE buffer with Na+ 100 mM and Mg2+
5 mM (Figure 8). By applying the thermodynamically
controlled protocol, optimal conditions for dimer formation
were achieved with a solution 1 μM of 18 in TE buffer with
Mg2+ 10 mM, and an annealing step of 50 h (Figure 8).
Mung bean nuclease (MBN)91,92 is an enzyme that
selectively digests ssDNA, while not disturbing dsDNA.
Therefore, in order to investigate the formation of the fully
double-stranded dimer and 12-mer (icosahedral symmetric
assembly), MBN digestion was carried out on the two annealed
solutions of the dimer and 12-mer of 18 using the above
protocols. The native PAGE of the samples shows all
supramolecular aggregates to be digested by MBN, implying
that neither the fully dsDNA strands associated with the dimer
nor icosahedral supramolecular structures were formed.
Melting temperature (Tm) measurements were carried out
on compound 18 to investigate stability of this annealed dimer
system. Unfortunately, the temperature dependence of the
absorption coefficient of corannulene at 260 nm did not permit
accurate determination of Tm of the dsDNA formed under
either kinetically or thermodynamically controlled conditions.
Computational Analysis. Due to its large size, the RM1
semiempirical Hamiltonian method was used to theoretically
investigate the structural aspects of compound 18, in particular,
to investigate the potential for aggregation into cage-like
supramolecular arrangements. The full theoretical model for
the C5-symmetric compound 18 is composed of 2875 atoms
(8155 basis functions).
Figure 8. Native PAGE (4%) at 4 °C of assembling under kinetic
(left) and thermodynamic control (right). Lanes M: 50 bp DNA
ladder; lanes 1 and 3: assembled samples; lane 2 and 4: results from
enzymatic digestion with MBN.
The RM1 optimized structure of the ssDNA-conjugated
corannulene (Figure 9) is characterized by an arm-length of
75.1 Å, measuring from the center of corannulene to the
oxygen of the final hydroxyl group of the ssDNA. The bowl
depth, a characteristic geometric parameter associated with
corannulene derivatives, was determined for corannulene in
isolation at the RM1 level of theory to be 0.88 Å, comparing
well to the experimentally observed bowl-depth, 0.87 Å.93 The
bowl depth for 18 is slightly more shallow than the isolated
corannulene, at 0.85 Å, and therefore it is expected that this
linker orientation would not be rigidly adopted, but
interconvert as does corannulene.
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Figure 9. RM1 optimized ssDNA-conjugated corannulene.
Figure 10. Supramolecular structures of 18 resulting from (A) 12-mer aggregation into an icosahedron or (B) dimer formation of a pentagonal
prism.
The angle between neighboring arms measured from the
center of corannulene is 71.9°, and 143.7° between non-
neighboring arms. In an idealized icosahedron, these angles
stemming from one vertex of a regular icosahedron are 60° and
108°, respectively, as illustrated in Figure 10, thus substantially
smaller than observed in the optimized structure of 18. This
large discrepancy provides evidence for the lack of icosahedral
12-mer formation, and suggests implementation of a more rigid
linker to the DNA to effectively restrict the angles of the
bioconjugated arms closer to 108°, increasing the probability
for icosahedron formation.
Annealing of the ssDNA arms enabled further investigation
of how changes in arm length impact the side length of an
icosahedral structure. RM1 geometry optimizations on the
paired palindromic dsDNA strands were used to compare the
length and associated interaction energies of the DNA strands.
The binding energy for formation of dsDNA from two single
single ssDNA strands, and the energy of the fixed geometry
determined, resulting in a binding energy of −244 kcal/mol.
Relaxing the ssDNA geometry resulted in a 79 kcal/mol
reduction in energy compared to the fixed geometry, enabling
an estimation of the reorganization energy cost for bringing the
ssDNA together into the dsDNA conformation. Including
effects of reorganization, the final binding energy is determined
to be −87 kcal/mol.
Comparison of the geometric features of ss- versus ds-DNA
shows that the distance between the 3′ and 5′ ends of the
relaxed ssDNA strand are shortened by only 1.3 Å in the
dsDNA conformation. This results in a predicted icosahedral
side length, a in Figure 10A, of 80.2 Å. Provided that the linkers
assume a 108° angle within the constraints of a regular
icosahedron, this would result in a supramolecular structure
with inscribed sphere radius, ri = 60.6 Å, and circumscribed
sphere radius rc = 76.2 Å.
ssDNA strands was determined as Ebinding = EdsDNA − 2EssDNA
.
In contrast to the icosahedral structure, a dimer structure
could easily accommodate a more shallow corannulene-linker
Initially, the optimized dsDNA structure was separated into two
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structure, as calculated for 18, but would necessitate the
bending of the linker-to-DNA junction to an angle, θ in Figure
10, of 98° to properly orient the ssDNA strands for annealing.
This angle in the calculated structure of 18 is a much more
linear 165°, suggesting again a design modification to promote
dimer formation, by rigidifying the linkers to DNA to achieve
this orientation. With future optimization of the structure,
formation of dimer supramolecular aggregate should be
successful, especially considering the greater probability of
two molecules joining in concert, opposed to association of 12
molecules in the correct orientation for icosahedral aggregation.
in a MC Brown solvent system and degassed with N2), using
standard Schlenk techniques, unless otherwise noted. Oligopep-
tide H-Ala-Ala-Ale-OMe acetate salt was purchased from
Bachem, 5′-modified oligonucleotide 19 was purchased from
Metabion, mung bean nuclease was purchased from New
England Biolabs, DNA ladder 50 bp was purchased from
ThermoScientific, Float-A-Lyzer G2 was purchased from
SpectrumLabs, and all the other reagents and solvents were
purchased from Sigma Aldrich or VWR. All reagents and
solvents were used without any further purification. Copper
nanoparticles were prepared following a previously reported
procedure.96 Flash chromatographic purification was performed
using silica gel Merck 60 (particle size 0.040−0.063 mm); the
eluting solvent for each purification was determined by thin-
layer chromatography (TLC). Analytical thin-layer chromatog-
raphy was performed using Merck TLC silica gel 60 F254.
Solvents for chromatography were technical grade and freshly
distilled before use. Microwave reactions were carried out in a
dedicated CEM Discovery microwave oven. The microwave
power was limited by temperature control once the desired
temperature was reached. 1H NMR spectra were recorded on a
Bruker AV2−500 (500 MHz) spectrometer. Solvents for NMR
spectroscopy were purchased from ARMAR chemicals,
degassed with nitrogen, and dried over molecular sieves.
Chemical shifts are reported in parts per million (ppm) relative
to the solvent residual peaks: CDCl3 = 7.26 ppm, d4-MeOD =
3.31 ppm, d6-DMSO = 2.50 ppm, and D2O = 4.79 ppm.
Multiplicities are given as s (singlet), br (broad), d (doublet), t
CONCLUSIONS
■
A powerful and robust procedure for the synthesis of fivefold
symmetric pentasubstituted corannulene derivatives function-
alized with the biomolecules (sugars, oligopeptide, nucleosides,
and oligonucleotide) has been demonstrated. Studies character-
izing aggregation behavior of these new constructs support
formation of supramolecular architectures. Water-soluble
galactoside derivatives of corannulene were observed to form
H-dimers in solution, while dimeric structures can be formed
between pentakis-nucleoside corannulene derivatives most
probably via H-bonding. Particularly interesting and promising
results for formation of corannulene-based supramolecular
architectures were obtained with DNA functionalized penta-
pods. Unfortunately, the formation of fully double-stranded
dimeric or icosahedral supramolecular structures was not
achieved, potentially due to both thermodynamic and geo-
metric factors. Icosahedral aggregates are highly symmetric
structures, and therefore strongly entropically unfavorable, due
to orientation-specific aggregation of 12 molecules in solution.
Theoretical investigations of 18 predict a minimum energy
structure with significant geometric deviations from those
found in an icosahedral orientation. The fully double-stranded
dimer is likely not formed due to sterics, and calculations also
suggest that this molecular geometry is unsupportive of the
annealing of all five dsDNA strands94,95 in a dimeric fashion.
Although formation of the icosahedral aggregate was not
observed in this work, the predicted ∼1.5 nm diameter
nanocapsule offers interesting potential for applications given
both the size and nature of its construction. This cavity size
offers the capacity to enclose large cargo, such as small proteins
or genes. The release of cargo could also be directed by the
same parameters that affect the annealing of the DNA
sequence, namely, temperature, ion and denaturant concen-
trations, and enzymatic activity. With ever-increasing applica-
tions for nanoscale delivery mechanisms in the fields of targeted
medicine and materials science, DNA-based nanocapsules are
an option of growing interest. Specifically, a bioconjugated-
organic construct offers appealing advantages, including
increased stability and rigidity of the organic framework, and
potential for a longer lifetime in vivo due to lower degradation
rates of organic compounds compared to purely biomolecular
constructs. This work encourages the continuation of efforts
toward the synthesis of bioconjugated-corannulene supra-
molecular structures, and together with ab initio structural
analysis, improved design and success of organized dimer or 12-
mer formation is expected.
1
(triplet), q (quadruplet), quint (quintet), m (multiplet). H-
decoupled 13C NMR spectra were obtained on a Bruker AV2−
500 (125 MHz) spectrometer. 13C NMR chemical shifts are
reported relative to the solvent residual peaks: CDCl3 = 77.23
ppm, d4-MeOD = 49.00 ppm, d6-DMSO = 39.52 ppm. UV/vis
measurements were carried out on an Agilent 8453 UV/vis
spectrophotometer using a 1 mm path quartz cuvette. Emission
spectra were recorded on an Edinburgh Instruments FLS920
spectrometer with excitation at 300 nm. [α]D values were
recorded on JASCO P-2000 Polarimeter at 25 °C. IR: Perkin-
Elmer Spectrum One (FT-IR). HR-ESI-MS: Finnigan Mat 900
MS; MALDI-TOF-MS: Bruker Autoflex I using 3-HPA matrix.
Circular dichroism spectra were recorded at room temperature
on a Jasco J-810 spectropolarimeter using 1 mm path quartz
cuvette. A Nanodrop 2000C was used for quantification of 18
in solution. AlphaImager from AlphaInnotech was used for
visualization of gels stained with ethidium bromide.
sym-Penta-2-(1,2,3-triazole-1-(2,3,5-tri-O-acetyl-β-ri-
bofuranosyl)-4-ethyl)-corannulene (7). A mixture of sym-
penta-(1-butyn-4-yl)-corannulene 358 (10.0 mg, 20 μmol),
2,3,5-tri-O-acetyl-β-D-ribofuranosyl-1-azide 662 (62.0 mg, 21
mmol) and copper nanoparticles (13.2 mg, 0.21 mmol) in
DMF (1.0 mL) in a microwave vessel was heated at 80 °C in a
microwave reactor (200 W) for 8 h. The mixture was then
filtrated over Celite and the solvent was evaporated. The
product was purified by column chromatography on silica gel
eluted with a solution of DCM:MeOH 96:4. The solvent was
1
evaporated to yield a yellow solid (21.6 mg, 54%). H NMR
(500 MHz, CDCl3): δ 7.62 (s, 5H), 7.55 (s, 5H), 6.11 (d, J =
3.5 Hz, 5H), 5.81 (t, J = 4.0 Hz, 5H), 5.60 (t, J = 5.0 Hz, 5H),
4.42 (m, 5H), 4.32 (dd, J = 12.5, 3.5 Hz, 5H), 4.19 (dd, J =
12.5, 4.5 Hz, 5H), 3.50 (m, 10H), 3.29 (m, 10H), 2.10 (s,
15H), 2.08 (s, 15H), 1.97 (s, 15H). 13C NMR (125 MHz,
CDCl3): δ 170.5, 169.5, 169.4, 147.7, 140.3, 135.1, 129.7, 123.1,
120.9, 90.0, 80.9, 74.4, 71.0, 63.1, 33.1, 29.8, 28.3, 20.8, 20.6,
EXPERIMENTAL SECTION
■
Synthetic procedures were carried out in an inert atmosphere of
nitrogen, in dry solvents (by passage through alumina columns
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20.5. IR (KBr): ν cm−1 3422, 2927, 2854, 1750, 1445, 1437,
1373, 1231, 1091, 1062, 1041, 923, 900, 811, 730, 602. UV
(DMSO) λmax, nm: 264, 299. [α]D = −17.4 (c = 0.008 in
DMSO). HRMS (ESI) m/z: found 1030.8341 (M + 2Na); calc
(C95H105N15Na2O35) 1030.8341.
= 7.0 Hz, 15H), 1.27 (d, J = 7.5 Hz, 15H), 1.21 (d, J = 7.0 Hz,
15H). 13C NMR (125 MHz, d6-DMSO): δ 173.0, 171.8, 168.4,
146.0, 140.6, 134.1, 129.5, 123.0, 121.4, 57.7, 51.9, 48.0, 47.5,
32.5, 28.1, 18.2, 18.1, 16.8. IR (KBr): ν cm−1 3290, 2930, 1741,
1645, 1551, 1455, 1383, 1329, 1222, 1164, 1052, 998, 953, 684.
UV (DMSO) λmax, nm: 264, 299. [α]D = +3.7 (c = 0.055 in
DMSO). HRMS (ESI) m/z: found 955.9273 (M + 2Na); calc
(C90H115N25Na2O20) 955.9267.
5′-Azido-2′,5′-dideoxy-adenosine (15). A solution of 5′-
O-toluenesulphonyl-2′-deoxy-adenosine 1465 (270 mg, 6.67
mmol) in dry DMF (2.0 mL) was added dropwise to a mixture
of LiN3 (525 mg, 10.7 mmol) in dry DMF (1.0 mL). The
reaction mixture was stirred at room temperature for 2 days;
the solvent was evaporated and the product was purified by
column chromatography on silica gel eluted with a
DCM:MeOH:TEA 9:1:0.25. The solvent was evaporated to
yield a colorless foam (140 mg, 76%). The spectroscopic data
were identical with those reported.97 1H NMR (500 MHz, d4-
MeOD): δ 8.29 (s, 1H), 8.21 (s, 1H), 6.44 (t, J = 7.0 Hz, 1H),
4.57 (m, 1H), 4.08 (m, 1H), 3.64 (dd, J = 13.5, 6.0 Hz, 1H),
3.56 (dd, J = 13.5, 4.0 Hz, 1H), 2.96 (m, 1H), 2.49 (m, 1H).
13C NMR (125 MHz, d4-MeOD): δ 154.0, 141.3, 130.0, 127.1,
87.3, 85.9, 73.0, 53.5, 47.5, 40.4.
sym-Penta-2-(1,2,3-triazole-1-(-β-ribofuranosyl)-4-
ethyl)-corannulene (5). A solution of 7 (9.2 mg, 4.6 μmol)
and MeONa (16.5 mg, 0.31 mmol) in MeOH (1.5 mL) was
stirred under nitrogen at room temperature for 2 days. Dowex
50 was added until pH 6 was reached. The mixture was filtrated
and the solvent was evaporated. The product is a yellow solid
1
(6.0 mg, 94%). H NMR (500 MHz, d6-DMSO): δ 8.25 (s,
5H), 7.86 (s, 5H), 5.91 (d, J = 4.5 Hz, 5H), 5.63 (d, J = 5.5 Hz,
5H), 5.30 (d, J = 3.5 Hz, 5H), 4.99 (t, J = 5.0 Hz, 5H), 4.36 (d,
J = 4.5 Hz, 5H), 4.11 (d, J = 3.5 Hz, 5H), 3.95 (q, J = 4.5 Hz,
5H), 3.61 − 3.48 (m, 20H), 3.21 (br, 10H). 13C NMR (125
MHz, d6-DMSO): δ 146.7, 140.6, 134.1, 129.5, 123.0, 120.8,
109.5, 92.0, 85.7, 75.1, 70.4, 61.4, 32.4, 28.0. IR (KBr): ν cm−1
3359, 2924, 2853, 1595, 1454, 1383, 1331, 1228, 1101, 1047,
863, 824, 755, 102, 621. UV (DMSO) λmax, nm: 264, 300. [α]D
= +255.0 (c = 0.004 in DMSO). HRMS (ESI) m/z: found
1408.5211 (M + Na); calc (C65H75N15NaO20) 1408.5205.
N-(2-Azide-propionyl)-di-L-alanine Methyl Ester (10).
Triflic anhydride (0.65 mL, 3.88 mmol) was added to a solution
of NaN3 (1.2 g, 18.5 mmol) in DCM (3.0 mL) and water (2.0
mL) at 0 °C; the reaction was stirred at room temperature for 2
h. The organic phase was separated and the aqueous extracted
with DCM. The reunited organic phases were washed with a
saturated solution of NaHCO3. The organic phase was then
added to a solution of H-Ala-Ala-Ala-OMe acetate salt (454 mg,
1.49 mmol), CuSO4·5H2O (5.6 mg, 0.02 mmol) and K2CO3
(387 mg, 2.80 mmol) in methanol (6.6 mL) and water (3.2
mL) at room temperature; the mixture was stirred for 22 h. The
organic layer was then separated and the aqueous phase was
extracted with DCM. The reunited organic phases were dried
over Na2SO4 and evaporated to yield the crude product. The
product was purified by column chromatography on silica gel
eluted with a mixture DCM:MeOH 9:1. The solvent was
evaporated to yield a white solid (211 mg, 52%). 1H NMR (500
MHz, CDCl3): δ 6.89 (d, J = 6.5 Hz, 1H), 6.43 (d, J = 6.5 Hz,
1H), 4.56 (quint, J = 7.0 Hz, 1H), 4,42 (quint, J = 7.0 Hz, 1H),
4.07 (q, J = 7.0 Hz, 1H), 3.76 (s, 3H), 1.53 (d, J = 7.0 Hz, 3H),
1.42 (d, J = 7.0 Hz, 3H), 1.41 (d, J = 7.0 Hz, 3H). 13C NMR
(125 MHz, CDCl3): δ 173.2, 171.3, 169.8, 59.1, 52.7, 48.9,
48.4, 18.5, 18.4, 17.2. IR (KBr): ν cm−1 3312, 2972, 2116, 2078,
1740, 1641, 1546, 1450, 1376, 1344, 1325, 1218, 1162, 1106,
1073, 1063, 1008, 984, 956, 855, 663, 572. [α]D = −104.7 (c =
0.0040 in CHCl3). HRMS (ESI) m/z: found 294.1174 (M +
Na); calc (C10H17N5NaO4) 294.1173.
sym-Penta-2-(1,2,3-triazole-1-(5′-yl-2′,5′-dideoxy-ad-
enosine)-4-ethyl)-corannulene (12). A mixture of sym-
penta-(1-butyn-4-yl)-corannulene 358 (10 mg, 19.6 μmol), 5′-
azido-2′,5′-dideoxy-adenosine 15 (65.1 mg, 0.24 mmol) and
copper nanoparticles (10.1 mg, 0.16 mmol) in DMF (1.0 mL)
in a microwave vessel was heated at 60 °C in a microwave
reactor (200 W) for 2 h. The mixture was then filtrated over
Celite and the solvent was evaporated and MeOH was added to
the crude. The solid was then filtrated, washed with cold
MeOH, and recrystallized from water to yield a reddish solid
1
(14.5 mg, 40%). H NMR (500 MHz, d6-DMSO, 373 K): δ
8.18−8.16 (m, 10H), 7.75 (s, 5H), 7.71 (s, 5H), 6.91 (br s,
10H), 6.35 (t, J = 6.5 Hz, 5H), 5.31 (d, J = 5.0 Hz, 5H), 4.70
(dd, J = 14.0, 4.5 Hz, 5H), 4.61 (dd, J = 14.5, 7.0 Hz, 5H), 4.53
(br s, 5H), 4.23 (br s, 5H), 3.42 (t, J = 7.0 Hz, 10H), 3.14 (t, J
= 7.5 Hz, 10H), 2.80 (m, 5H), 2.36 (m, 5H). 13C NMR (125
MHz, d6-DMSO): δ 156.1, 152.6, 149.2, 146.3, 140.4, 139.8,
133.9, 129.4, 128.0, 125.5, 122.8, 84.9, 83.6, 71.1, 67.0, 64.9,
51.4, 38.1, 32.3, 27.7, 25.1. IR (KBr): ν cm−1 3329, 3186, 2928,
1653, 1647, 1640, 1599, 1577, 1474, 1420, 1366, 1331, 1299,
1248, 1217, 1087, 1056, 939, 797, 726, 649. UV (DMSO) λmax
,
nm: 265, 299. [α]D = +25.6 (c = 0.008 in DMSO). HRMS
(ESI) m/z: found 946.3962 (M + 2H); calc (C90H92N40O10)
946.3955.
sym-Penta-2-(1,2,3-triazole-1-(5′-yl-2′-deoxy-ribo-thy-
midine)-4-ethyl)-corannulene (11). A mixture of sym-penta-
(1-butyn-4-yl)-corannulene 358 (9.9 mg, 19.4 μmol), 5′-azido
thymidine 1766 (43.0 mg, 0.16 mmol), and copper nano-
particles (10.9 mg, 0.17 mmol) in DMF (1.0 mL) in a
microwave vessel was heated at 60 °C in a microwave reactor
(200 W) for 2 h. The mixture was then filtrated over Celite and
the solvent was evaporated and MeOH was added to the crude.
The solid was then filtrated and washed with cold MeOH to
sym-Penta-2-(1,2,3-triazole-1-(N-(2-methyl-acetyl)-di-
L-alanine methyl ester)-4-ethyl)-corannulene (9). A
mixture of sym-penta-(1-butyn-4-yl)-corannulene 358 (9.1 mg,
18 μmol), N-(2-azide-propionyl)-di-L-alanine methyl ester 10
(50.3 mg, 0.19 mmol), and copper nanoparticles (13.4 mg, 0.21
mmol) in DMF (0.5 mL) in a microwave vessel was heated at
80 °C in a microwave reactor (200 W) for 8 h. The mixture was
then filtrated over Celite and the solvent was evaporated. The
product was purified by column chromatography on silica gel
eluted with a DCM:MeOH 9:1. The solvent was evaporated to
1
yield a pale yellow solid (24.6 mg, 69%). H NMR (500 MHz,
d6-DMSO): δ 11.30 (s, 5H), 7.98 (s, 5H), 7.77 (s, 5H), 7.31 (s,
5H), 6.15 (t, J = 6.5 Hz, 5H), 5.49 (d, J = 4.5 Hz, 5H), 4.69−
4.58 (m, 10H), 4.27 (m, 5H), 4.06 (m, 5H), 3.45 (br, 10H),
3.17 (d, J = 5.0 Hz, 10H), 2.15−2.07 (m, 10H), 1.75 (s, 15H).
13C NMR (125 MHz, d6-DMSO): δ 163.7, 150.4, 146.4, 140.4,
136.1, 134.1, 129.4, 123.1, 109.8, 84.1, 84.0, 70.7, 51.1, 48.6,
1
yield a yellow solid (19 mg, 57%). H NMR (500 MHz, d6-
DMSO): δ 8.60 (d, J = 7.5 Hz, 5H), 8.39 (d, J = 7.0 Hz, 5H),
8.09 (s, 5H), 7.82 (s, 5H), 5.43 (q, J = 7.0 Hz, 5H), 4.27 (m,
10H), 3.61 (s, 15H), 3.47 (br, 10H), 3.18 (br, 10H), 1.60 (d, J
124
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37.9, 32.4, 27.9, 12.1. IR (KBr): ν cm−1 3421, 2926, 1688, 1472,
1438, 1273, 1221, 1135, 1084, 1056, 958, 874, 766, 558. UV
(DMSO) λmax, nm: 265, 298. [α]D = +108.5 (c = 0.012 in
DMSO). HRMS (ESI) m/z: found 923.8662 (M + 2H); calc
(C90H97N25O20) 923.8665.
chemistry package, GAMESS.98 The RM1 method, developed
by Rocha GB et al.,99 has the ten most common elements for
bioorganic systems, H, C, N, O, F, P, S, Cl, Br, and I, fully
optimized using a training set of 1736 molecules representative
of organic and biochemical systems. This reparameterization
has been demonstrated to achieve greater accuracy in
calculations of enthalpies of formation, dipole moments,
ionization potentials, and interatomic distances, on biosystems,
in comparison to other available semiempirical Hamiltonians.
Full geometry optimization was carried out for the DNA-
conjugated corannulene system 18, as well as several other
components described. Optimization was carried out in gas
phase, and therefore the charge of the DNA backbone was
neutralized by the addition of hydrogen atoms to each
phosphate group. For the purposes of this investigation, effects
of solution environment are not expected to significantly
change the conclusions of the theoretical analysis.
Pentakis-DNA Corannulene (18). Ten μL of a solution of
CuBr (92 μg, 0.64 μmol) and TBTA (0.546 mg, 1.0 μmol) in
water:THF 1:1 (100 μL) was added to a solution of sym-penta-
(1-butyn-4-yl)-corannulene 358 (49 μg, 96 nmol) and
oligonucleotide 19 (1.1 μmol) in THF (5 μL) and water (15
μL) and heated at 60 °C for 2 days and at 80 °C for 4 days. The
reaction mixture was then desalted with Sephadex G-25. The
crude product was then purified by 6% D-PAGE (acrylamide:-
bisacrylamide 19:1, Urea 7 M) at constant power of 40 W,
using TBE buffer (Tris 11 g, boric acid 5.6 g, and EDTA 0.74 g
in 1 L of sterile water). Following electrophoresis, the plate was
wrapped in plastic and placed on a fluorescent TLC plate and
illuminated with UV lamp (254 nm). The band was quickly
excised, and the gel pieces were frozen with liquid nitrogen,
crushed, and incubated overnight in 2 volumes of NaOAc (0.5
M, pH 5.2 with HCl) at 37 °C. The samples were then
centrifuged and the supernatant collected. The gel was the
washed with 1/5 volume of NaOAc 0.5 M, centrifuged, and the
supernatant collected. The aqueous solution was then extracted
with n-butanol to 1/3 of the original volume. 0.16 volume of a
solution of LiCl 4 M and MgCl2 0.125 mM and 3 volumes of
cold ethanol (100%, −80 °C) were added to the aqueous
solution and stored overnight at −80 °C. The sample was then
centrifuged (13200 rpm, 4 °C, 20 min) and the supernatant
was removed. The precipitate was washed 3 times with cold
ethanol (80%, 0 °C), dissolved in sterile Milli-Q water and
desalted with Float-A-Lyzer G2. The amount of 18 was
quantified by UV absorption (300 nm assuming ε = 3.28 ×
104). The sample was then freeze-dried yielding the desired
product as a pale yellow powder (304 μg, 12%). MALDI-TOF
MS m/z found 26483.2 (M + 5Na + 18 H); calc.
(C875H1195N315O490P80Na2Li) 26483.7241.
Assembling Studies. Kinetically Controlled Procedure. A
2 μM solution of 18 in TE buffer (Tris 10 mM, EDTA 1 mM
pH 7.5 with HCl) with NaCl 100 mM and MgCl2 5 mM was
heated for 5 min at 95 °C in a heating block; the sample was
then transferred in an ice-bath, kept at 0 °C for 10 min and
then loaded on the gel.
Thermodynamically Controlled Procedure. A 1 μM
solution of 18 in TE buffer (Tris 10 mM, EDTA 1 mM pH
7.5 with HCl) with MgCl2 10 mM was heated for 5 min at 95
°C in a heating block, then slowly cooled to 55 °C, kept at this
temperature for 50 h, then slowly cooled to 4 °C and loaded on
the gel.
Native PAGE. The samples from assembling studies were
analyzed by 4% native-PAGE (acrylamide:bisacrylamide 19:1)
at 4 °C and at constant current of 10 mA for 1.5 h. The gels
were stained with ethidium bromide.
Enzymatic Digestion. Digestion of single-stranded DNA
with mung bean nuclease was performed for 3 h at 0 °C in
sodium acetate buffer (10 mM, Zn(OAc)2 0.1 mM, cysteine 1
mM, Triton X-100 0.001%, and glycerol 50%) applying 10 units
of mung bean nuclease per μg of 18.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR, 13C NMR, FTIR, and MS spectra for all the pertinent
compounds and MBN digestion test on dsDNA. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*Tel: +41 44 635 4201; E-mail: kimb@oci.uzh.ch.
*Tel: +86-22-87402050; E-mail: dean_spst@tju.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the Swiss National Science
Foundation for support of this work. We are grateful to the
NMR and MS facilities at UZH (O. Zerbe and L. Bigler). We
thank U. Rieder, S. Gallo, J. Marino, and O. Schmidt for the
help and useful discussion. J.S.S. thanks the Synergetic
Innovation Center of Chemical Science and Engineering
(Tianjin University, Tianjin, China).
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