Oligopyrenotides: Abiotic, polyanionic oligomers with nucleic acid-like structural properties

Article abstract of DOI:10.1021/ja102042p

Oligopyrenotides, abiotic oligomers that exhibit significant structural analogies to the nucleic acids, are described. They are composed of achiral, phosphodiester-linked pyrene building blocks and a single chiral 1,2-diaminocyclohexane unit. These oligomers form stable hybrids in aqueous solution. Hybridization is based on stacking interactions of the pyrene building blocks. They show thermal denaturation/renaturation behavior that closely resembles DNA and RNA hybridization. In addition, oligopyrenotides display salt-concentration-dependent structural polymorphism. Thus, they possess a number of structural attributes that are typical of nucleic acids and therefore may serve as model systems for the design of artificial self-replicating systems.

Full text of DOI:10.1021/ja102042p

Published on Web 05/11/2010
Oligopyrenotides: Abiotic, Polyanionic Oligomers with
Nucleic Acid-like Structural Properties
Robert Ha¨ner,* Florian Garo, Daniel Wenger, and Vladimir L. Malinovskii
Department of Chemistry and Biochemistry, UniVersity of Bern, Freiestrasse 3,
CH-3012 Bern, Switzerland
Received March 10, 2010; E-mail: robert.haener@ioc.unibe.ch
Abstract: Oligopyrenotides, abiotic oligomers that exhibit significant structural analogies to the nucleic acids,
are described. They are composed of achiral, phosphodiester-linked pyrene building blocks and a single
chiral 1,2-diaminocyclohexane unit. These oligomers form stable hybrids in aqueous solution. Hybridization
is based on stacking interactions of the pyrene building blocks. They show thermal denaturation/renaturation
behavior that closely resembles DNA and RNA hybridization. In addition, oligopyrenotides display salt-
concentration-dependent structural polymorphism. Thus, they possess a number of structural attributes
that are typical of nucleic acids and therefore may serve as model systems for the design of artificial self-
replicating systems.
Introduction
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10.1021/ja102042p  2010 American Chemical Society

Oligopyrenotides
A R T I C L E S
Figure 1. Oligomers 3-8 composed of achiral pyrene building blocks and a chiral 1,2-diaminocyclohexane unit as well as the respective monomeric
building blocks (PAM ) phosphoramidite, as shown in (R,R)-1; DMT stands for 4,4′-dimethoxytrityl).
On the other hand, organized supramolecular assemblies that
are primarily based on aromatic stacking interactions play an
important role in the development of novel types of materials.50,51
These systems are based either on the interactions of monomers
to form discotic stacks52-54 or on oligomers with an uncharged
backbone.55 The importance of the polyanionic backbone for
the proper functioning of DNA as a genetic material has been
emphasized previously.56 Fully artificial oligomers composed
of non-hydrogen-bonding aromatic stacking moieties assembled
on a polyanionic backbone, however, have not been reported.
We report here that a simple, aromatic oligophosphate exhibits
structural features typically observed in nucleic acids. Polyan-
ionic pyrene oligomers form water-soluble helical hybrids
displaying salt-dependent structural polymorphism. In contrast
to nucleic acids, however, the interaction of individual strands
does not involve complementary hydrogen bonding but instead
relies on interstrand stacking of pyrenes.
in water is ensured by hydrophilic phosphodiester groups. Since
the pyrene components are achiral, a single chiral building block,
either a (1S,2S)- or (1R,2R)-1,2-diaminocyclohexane unit,57 is
introduced into the oligomer. This unit serves as the source of
chiral information according to which the oligopyrene strands
adopt a preferred helical sense (see below). The corresponding
phosphoramidites [1; see the Supporting Information (SI)] and
the pyrene building block (2)58-60 are shown in Figure 1.
Oligomerization was performed on a DNA synthesizer by
phosphoramidite chemistry61 using a pyrene-functionalized
polystyrene support (see the SI). Oligomers 3- 8 were obtained
after reversed-phase HPLC purification.
Spectroscopic Investigation. Initial analysis of the oligomers
by absorption and circular dichroism (CD) spectroscopy showed
no signs of defined structures at low sodium chloride concentra-
tions (<0.75 M). Above this concentration, however, structural
organization became evident (see the SI). Up to ∼2 M NaCl,
the magnitude of the Cotton effects grew with increasing salt
concentration. Further augmentation of the salt concentration
(up to 4.0 M NaCl) resulted in a major structural change, as
evidenced by CD spectroscopy. This behavior is illustrated in
Figure 2a, which shows the CD spectrum of oligomer 5. The
spectra taken at 1.5 and 3.0 M NaCl are approximately mirror-
inverted, indicating a switch in overall helicity.62,63 Vibronic
structures in the UV-vis spectra (Figure 2a) indicate well-
defined pyrene conformations. The relative intensities of the
vibronic bands were affected by ionic strength. The fluorescence
spectra (Figure 2b) show the expected broad pyrene excimer
band. The maximum of this band was blue-shifted with
increasing oligomer concentration (e.g., from 512 to 502 nm
for 0.25 and 6.0 µM oligomer, respectively, at 1.5 M NaCl). A
Results and Discussion
Design of Oligomers. As in nucleic acids, individual units
are linked by phosphodiester groups. The nucleobases are
replaced by pyrene units, and flexible aliphatic linkers are used
in place of the natural ribose or deoxyribose (Figure 1). The
aromatic nature of the pyrenes favors stacking interactions
between the building blocks, while the propyl linkers possess
enough flexibility to allow unhindered self-assembly. Solubility
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A R T I C L E S
Ha¨ner et al.
Figure 2. Spectroscopic data for (a, b) 5 and (c, d) 8 [10 mM phosphate buffer (pH 7.0), 20 °C]: (a, c) (top) CD and (bottom) UV-vis absorbance spectra
at various salt concentrations (oligomer concentration: 5.0 µM for CD and 2.0 µM for UV-vis); (b, d) fluorescence spectra at various oligomer and salt
concentrations.
blue shift reflects an increase in the twisting angle of the stacked
pyrenes.38,64,65 In combination with the observed CD and UV
effects, this strongly supports the conclusion that oligopyrene
5 adopts a helical structure. In comparison, oligomer 3, which
has only one oligopyrene chain linked to the diaminocyclohex-
ane, showed no vibronic bands in the UV absorbance spectra
nor Cotton effects in the CD spectra (see the SI).
Oligomer 8, which differs from 5 in the length of the pyrene
chains, also revealed a high degree of structural organization
(Figure 2c). The CD spectra again disclosed a strong structural
dependence on ionic strength. As in the case of 5, the curves at
lower and higher ionic strength differed significantly, being
Figure 3. Temperature-dependent CD spectra of oligomer 8 [5 µM
oligomer concentration, 10 mM phosphate buffer (pH 7), 3.0 M NaCl].
The arrow indicates increasing temperature (10 °C intervals).
almost mirror images. UV-vis absorbance supports pyrene
done with nucleic acids or oligonucleotides. This is illustrated
stacking by salt-dependent changes in the vibronic band
by the CD and absorption curves of the same sample of oligomer
intensities, indicating reduced flexibility in the pyrene stack with
8 before and after heating (Figure 4). It is important to note
that the increase in the CD signal intensities was accompanied
by a decrease in the corresponding UV-vis absorption bands.
This reflects tighter stacking interactions between the chro-
increasing salt concentration (Figure 2d). At all concentrations
tested, excimer emission showed a maximum at ∼495 nm. This
corresponds closely to the emission observed for the helical form
of oligomer 5 and suggests that 8 adopts a well-ordered, helical
mophores as the structural organization process proceeds.
conformation at all oligomer concentrations tested. In addition,
Oligomer 6 differs from 8 in that it bears only one pyrene
chain instead of two. Duplex formation of this oligomer was
also supported by UV-vis, CD, and fluorescence spectroscopy
(see the SI). Salt-concentration-dependent changes in the
vibronic band intensities indicated pyrene stacking for 6 also.
The intensity of the CD signals gradually increased with rising
isosbestic points observed in temperature-dependent CD spec-
troscopy provide evidence for a two-state model (Figure 3).
Taken together, these data support a model in which pyrene
oligomers self-associate to form helical structures with inter-
strand stacked pyrenes.
Consistent and reproducible spectra of oligopyrenes were
salt concentration, but in contrast to the behavior of oligomers
ensured by including a preannealing step, similar to what is
8 and 5, the overall forms of the curves were not significantly
affected by the ionic strength. The concentration-dependent
excimer blue shift resulting from pyrenyl twisting was present
at 1.5 M NaCl concentration but not at 3.0 M NaCl.
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Figure 4. (top) CD and (bottom) UV-vis spectra of oligomer 8 [3.0 M
NaCl, 10 mM phosphate (pH 7.0), 20 °C]. Spectra were recorded before
heating (black curves) and after heating to 90 °C and cooling back to room
temperature (red curves).
Figure 6. CD spectra of oligomer 5 taken before and after repeated heating
to 90 °C. Spectra were taken after equilibration for 30 min at each
temperature.
Figure 5. Thermal denaturation of oligomers 5, 6, and 8 at 1 µM
concentration [conditions: 10 mM phosphate (pH 7.0), 3.0 M NaCl;
absorbance monitored at 282 nm]. For clarity, only the third ramp of a
cooling-heating-cooling cycle is shown for each oligomer.
Figure 7. van’t Hoff plots of 1/Tm vs ln(C) for oligomers 4-8.
The changes in the CD spectra of the oligomers are remark-
able. Two markedly different structural conformations exist at
medium (0.5-1.5 M NaCl) and high (2-4 M NaCl) salt
concentrations for oligomers 5 and 8. The same behavior was
observed for the enantiomeric oligomers 4 and 7 (see the SI).
Generally, all of the oligomers with (S,S)-linkers gave rise to
an intensive negative band at 240 nm at high salt concentration
(e.g., oligomers 5 and 8; Figure 2). This band changed to a
positive signal at medium salt concentrations for oligomers 5
and 8, but oligopyrene 6 did not exhibit this polymorphic
behavior.
from these plots are given in Table 1.67 Hybridization is
enthalpy-driven. The van’t Hoff enthalpy values (∆HvH) range
between approximately -2 and -3 kcal/mol, while the entropies
∆SvH are between -4.5 and -7.5 cal mol-1 K-1 per interstrand
stacking interaction (also see the SI). These values are smaller
than the average stacking enthalpies and entropies per base pair
observed for double-stranded oligonucleotides of comparable
length, which are on the order of -8 kcal/mol and -20 cal
mol-1 K-1, respectively.68 The difference can be attributed, at
least in part, to a reduction in the aromatic surface area involved
in stacking interactions and by the absence of hydrogen bonds.
Formation of other helical hybrids is a further possibility. In
particular, triple helices based on pyrene interstrand stacking
are imaginable (see the SI). Model considerations show that
four-stranded hybrids are unlikely to form via interstrand
stacking, at least with the type and length of interpyrenotide
linkers used in the present oligomers.
Denaturation Experiments. The hybridization process of the
oligomers was monitored at different wavelengths (245, 282,
and 365 nm). Cooperative melting was observed for all hybrids
with exception of 3, for which no transition was observed. Figure
5 shows the denaturation curves at 282 nm for oligomers 5, 6,
and 8. The folding process was entirely reversible. No differ-
ences could be observed by CD during several heating/cooling
cycles. As can be seen in Figure 6, the spectra recovered
completely after denaturation at 90 °C and re-equilibration at
ambient temperature. For all of the oligomers, the denaturation
curves and corresponding melting temperatures (Tm) were
dependent on oligomer concentration (see the SI), which rules
out monomolecular structures. Thus, the oligomers form mul-
timeric hybrids under the conditions tested.
Control of Helicity. Pyrene components are achiral. The 1,2-
diaminocyclohexane unit serves as the chiral point of reference
according to which the pyrene double strand adopts its
(66) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601–1620.
(67) van’t Hoff analysis is generally based on the assumption that ∆H is
temperature-independent (e.g., see ref 66). As in the case of nucleic
acid hybridization, this assumption may not be entirely correct. For
example, see: (a) Rouzina, I.; Bloomfield, V. A. Biophys. J. 1999,
77, 3242–3251. (b) Mikulecky, P. J.; Feig, A. L. Biopolymers 2006,
82, 38–58. Nevertheless, van’t Hoff analysis renders a first approxima-
tion of the thermodynamic data for the formation of a double-stranded
oligopyrenotide hybrid.
The formation of double-stranded helices in chimeric
DNA-pyrene oligomers has been shown.38,60,65 Such types of
double-helical structures are most likely also to be formed by
the present oligopyrenotides. The obtained Tm values show good
correlations in van’t Hoff plots [1/Tm vs ln(C)],66 as displayed
in Figure 7. Values of the thermodynamic parameters derived
(68) SantaLucia, J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1460–1465.
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Ha¨ner et al.
Table 1. Thermodynamic Parameters for Bimolecular Hybrid Formation of Oligopyrenotides
d
]
d e
,
d e
,
oligomer
sequencea
Tm [°C]b
∆H [kcal mol-1
T∆S [kcal mol-1
]
∆G [kcal mol-1
]
c
3
4
5
6
7
8
(S,S)-(Py)4
-
-
-
-
(Py)4-(R,R)-(Py)4
(Py)4-(S,S)-(Py)4
(S,S)-(Py)7
(Py)7-(R,R)-(Py)7
(Py)7-(S,S)-(Py)7
41
42
56
68
71
-38.4
-35.3
-41.1
-55.6
-51.1
-27.7
-24.8
-28.5
-39.6
-35.6
-10.7
-10.4
-12.6
-16.0
-15.4
a (S,S) and (R,R) stand for the respective isomers of 1,2-diaminocyclohexane and Py for the pyrene building block (see Figure 1). b Melting
temperatures at an oligomer concentration of 1 µM (exptl error (1 °C). Conditions: 10 mM phosphate (pH 7.0), 3.0 M NaCl; absorbance was measured
at 282 nm. c No Tm was observed. d Enthalpies, entropies, and free energies obtained from van’t Hoff plots [1/Tm vs ln(C)] for self-complementary
oligomers forming a bimolecular hybrid (also see the SI).66 e Using T ) 293 K.
Figure 8. CD spectra of the enantiomeric pair of oligomers 7 and 8 at 3.0
M NaCl [5.0 µM oligomer concentration; 10 mM phosphate (pH 7.0)].
helicity.7,69 The formation of enantiomorphic structures by pairs
of two mutually enantiomeric oligopyrenes is shown in Figure
8. The CD spectra of 7 and 8 (as well as those of 4 and 5; see
the SI) are mirror-symmetric. Thus, control of extended left-
or right-handed helices is effectively achieved by a single chiral
building block. On the other hand, helicity can also be influenced
by external factors, as evidenced by the remarkable change in
the CD spectra at various salt concentrations (Figure 2). The
origin of the apparent change in handedness induced by
increasing the NaCl concentration from 1.5 to 3.0 M is difficult
to assign. The salt concentration may have a direct effect on
the stacking interactions of the pyrenes, or it may change the
local structure of the glycol amide linkers. Furthermore, the
pyrene carboxamide groups are also potentially involved in
hydrogen bonding, and their conformations may be salt-
dependent. In any case, a direct involvement of the chiral 1,2-
diaminocyclohexane unit in the helical structure is likely, as
inversion of helicity was observed in the oligopyrenotides in
which this building block is located at an internal position (5
and 8) but not in 6, in which it occupies a terminal, conforma-
tionally much less constrained position.70
Structural Parallels between Oligopyrenes and DNA. DNA
is highly polymorphic. Depending on external factors, DNA of a
given primary sequence can exist in different forms. In solution,
the most common types are A-, B-, and Z-DNA. Interconversion
between the different forms can be effected by changing various
parameters, such as the degree of hydration (A-to-B transition)22
or the ionic strength (B-to-Z transition).71 Other secondary and
tertiary structures include the G-quadruplex72,73 and H-DNA.74
The oligomers presented here are composed of repeating units
Figure 9. AMBER-minimized molecular model80 of a right-handed
homodimeric duplex formed by oligopyrenotide 5. Alternating interstrand-
stacked pyrenes are shown in blue and orange space-filling representations,
and linkers between pyrenes are shown as sticks. (S,S)-1,2-Diaminocyclo-
hexane units are shown in dark-green.
of phosphodiester-linked pyrenes. Because of the obvious
resemblance of this pattern to natural oligonucleotides, we refer
to the units as pyrenotides (or, more generally, arenotides) and
their oligomers as oligopyrenotides (oligoarenotides). The
resemblance between oligopyrenotides and natural DNA goes
beyond the formal similarity of the repetitive units, however.
Just as in the DNA duplex, hybrids of oligopyrenotides are
stabilized by hydrophobic and stacking interactions among the
pyrenes, while the hydrophilic phosphate groups ensure water
solubility. Figure 9 shows a minimized model as an illustration
of a right-handed, homodimeric duplex structure of oligopy-
renotide 5. Hybrid formation and structural stability of oligopy-
renotides is salt-dependent, as is the case for nucleic acids.
Maybe most importantly, oligopyrenotides exhibit remarkable
polymorphism, which is strongly reminiscent of DNA. A drastic
change in helicity, comparable to the B-to-Z transition, is
effected by altering the salt concentration. Because of these
structural similarities to nucleic acids, oligoarenotides represent
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was elongated to 2 min. Cleavage from the pyrene-modified
polystyrene solid support and final deprotection were achieved by
treatment with 30% ammonium hydroxide at 55 °C overnight. The
crude oligomers were purified by reversed-phase HPLC (LiChro-
spher 100 RP-18, 5 µm, Merck; Bio-Tek Instruments Autosampler
560) using eluent A ) (Et3NH)OAc (TEAAc) buffer (0.1 M, pH
7.0) and eluent B ) 80% acetonitrile/20% TEAAc buffer; elution
was performed at 50 °C. Mass spectrometry (MS) data for the
purified oligomers were obtained by electrospray ionization (ESI)
MS in negative-ion mode (Applied Biosystems Sciex QTrap). For
a simplistic model of a self-replicating system. It is conceivable
that primitive self-replicating systems could be based on
hybridization through aromatic stacking interactions without
mutual recognition of complementary bases via hydrogen
bonding. Sequence information may be provided in mixed
sequences, since the electronic and structural diversity present
in different aromatic building blocks should favor certain types
of stacking interactions.75,76 Mixed sequences might therefore
lead to sequence-specific hybridization even in the absence of
specific hydrogen-bonding patterns.77 In addition, oligopy-
renotides possess a phosphodiester backbone similar to that of
DNA and RNA. While the phosphodiester groups ensure water
solubility of the oligomers, they may also be crucial for the
formation of multistranded helices in polar environments. The
importance of the polyanionic backbone for the proper function-
ing of DNA as a genetic molecule was pointed out by Benner
and Hutter.56 The polyelectrolyte backbone controls the degree
of folding of the single strand and thereby ensures that the single
strand can act as a template by formation of multistranded
hybrids. Thus, although simple oligopyrenotides such as the ones
presented here have little sequence information, they may serve
as model compounds for the design of artificial, self-replicating
systems.27,78,79
oligomer concentration determination, a value of ε260
) 9000
nm
M-1 cm-1 per pyrene unit was used.
Thermal Denaturation Experiments. A Varian Cary 100 Bio-
UV/vis spectrophotometer equipped with a Varian Cary block
temperature controller and Varian WinUV software was used to
determine the melting curves at 245, 282, and 365 nm (cooling-
heating-cooling cycles over the temperature range 10-90 °C,
temperature gradient 0.5 °C min-1, optical path length 10 mm).
Melting temperatures (Tm) were determined from the second cooling
ramp of the cooling-heating-cooling cycle. The data were
collected and analyzed with Kaleidagraph software from Synergy
Software.
UV-Vis Spectra. UV-vis spectra were collected in quartz
cuvettes with an optical path of 10 mm over the range 200-500
nm on a Varian Cary 100 Bio-UV/vis spectrophotometer equipped
with a Varian Cary block temperature controller and Varian WinUV
software.
Conclusions
We have described abiotic, polyanionic oligomers called
oligopyrenotides that exhibit remarkable structural analogies to
DNA. They are composed of achiral, non-nucleosidic pyrene
building blocks linked by phosphodiester groups. A single 1,2-
diaminocyclohexane unit provides the chiral information leading
to formation of homochiral helices.7,62,69 The oligomers form
stable hybrids in aqueous solution. Oligopyrenotides display salt-
concentration-dependent structural polymorphism. Despite their
chemical simplicity, oligopyrenotides possess a number of
structural attributes typically observed in nucleic acids and
therefore may serve as model compounds for the design of
artificial self-replicating systems.
Circular Dichroism Spectra. CD spectra were recorded on a
JASCO J-715 spectropolarimeter equipped with a JASCO PFO-
350S temperature controller. The samples were scanned at 50 nm
min-1 with a bandwidth of 1 nm and a response time of 1 s over
the 200-500 nm range at constant temperature. Each spectrum was
taken as an average of three scans using a 10 mm cell. Values of
∆ε were determined by dividing the ratio of the measured ellipticity
Θ (mdeg) and 32980 by the total oligomer concentration [i.e., ∆ε
) {Θ [mdeg]/32980}/(total oligomer concentration)].
Fluorescence Experiments. Variable-temperature fluorescence
data were collected on a Varian Cary Eclipse fluorescence spec-
trophotometer equipped with a Varian Cary block temperature
controller (excitation at 354 nm; excitation and emission slit width
of 5 or 2.5 nm, detector sensitivity of 600 or 800 V). Varian Eclipse
software was used to investigate the fluorescence of the different
oligomers over the wavelength range 365-680 nm. The data were
collected and analyzed with Kaleidagraph software from Synergy
Software.
Experimental Section
General. Reagents used for synthesis of pyrene and chiral 1,2-
diaminocyclohexane phosphoramidites were purchased from Fluka,
Sigma-Aldrich, or Acros and used without further purification. Full
experimental details and spectroscopic characterization data are
provided in the SI.
Preparation of Oligomers. Oligomers were synthesized on a
394 DNA/RNA Synthesizer (Applied Biosystems) according to
standard phosphoramidite chemistry (“trityl-off” mode). The pyrene
and 1,2-diaminocyclohexane phosphoramidite building blocks were
used as 0.1 M solutions in dichloromethane. The coupling time
Molecular Modeling. The model of a duplex composed of
oligomer 5 (Figure 9) was generated using HyperChem 7.5.80
Geometry optimization (in vacuum) was carried out using the
AMBER force field. After solvation of the structure (geometry
optimization in solution with the AMBER force field) and use of
molecular dynamics to anneal the system, reoptimization of the
geometry using the AMBER force field was performed.
(75) Hunter, C. A.; Sanders, J. K. M. J. Am. Chem. Soc. 1990, 112, 5525–
5534.
Acknowledgment. This work was supported by the Swiss
National Foundation (Grant 200020-117617).
(76) Meyer, E. A.; Castellano, R. K.; Diederich, F. Angew. Chem., Int.
Ed. 2003, 42, 1210–1250.
Supporting Information Available: Synthetic procedures;
analytical details; melting curves; and UV-vis, fluorescence,
and CD spectra. This material is available free of charge via
the Internet at http://pubs.acs.org.
(77) Langenegger, S. M.; Ha¨ner, R. Bioorg. Med. Chem. Lett. 2006, 16,
5062–5065.
(78) Wu, Y. Q.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.;
Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 7621–7632.
(79) Krueger, A. T.; Lu, H. G.; Lee, A. H. F.; Kool, E. T. Acc. Chem. Res.
2007, 40, 141–150.
(80) HyperChem, release 7.5; Hypercube, Inc.: Gainesville, FL, 2003.
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