Incorporation of porphyrin acetylides into duplexes of the simplified nucleic acid GNA

Article abstract of DOI:10.1039/c0ob00439a

A porphyrin-acetylide-modified GNA (glycol nucleic acid) phosphoramidite building block was synthesized in an economical fashion starting from (S)-glycidyl-4,4′- dimethoxytrityl ether in just 4 steps with an overall yield of 48%. The porphyrin acetylide n

Full text of DOI:10.1039/c0ob00439a

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PAPER
Incorporation of porphyrin acetylides into duplexes of the simplified nucleic
acid GNA†
Hui Zhou,a Andrew T. Johnson,b Olaf Wiestb and Lilu Zhang*a
Received 16th July 2010, Accepted 24th January 2011
DOI: 10.1039/c0ob00439a
A porphyrin-acetylide-modified GNA (glycol nucleic acid) phosphoramidite building block was
synthesized in an economical fashion starting from (S)-glycidyl-4,4¢- dimethoxytrityl ether in just 4
steps with an overall yield of 48%. The porphyrin acetylide nucleotide was incorporated into GNA
duplexes opposite ethylene glycol abasic sites and the duplexes were analyzed by UV-melting, UV-vis,
fluorescence spectroscopy, and circular dichroism. The modified GNA duplexes display lower thermal
stabilities, however, the stabilities of the duplexes can be modulated by the incorporation of Zn2+
(further destabilization) or Ni2+ (stabilization relative to the uncomplexed porphyrin). Uncomplexed as
well as Ni2+-coordinated porphyrins intercalate into the GNA duplex whereas Zn2+-coordinated
porphyrins are most likely located outside the base stack. Adjacent porphyrins in opposite strands of
GNA duplexes show an electronic interaction with each other which might be exploited in the future
for the design of photoelectrical devices.
mated oligonucleotide synthesis enables the solid-phase synthesis
Introduction
of oligonucleotides bearing multiple site-specific modifications.
Chromophores play important roles as imaging tools in the life
Porphyrins have been widely investigated due to their unique
sciences, as components for industrial dyes and pigments, and
photochemical and photophysical properties. Defined multi-
in natural processes such as photosynthesis. The conversion of
porphyrin arrays are attractive for the design of artificial light
harvesting systems and photoelectrical devices.8 With this or
related purposes in mind, DNA has been used as supramolecular
scaffold to arrange porphyrins in a controlled fashion. Porphyrins
have been covalently attached to nucleobases,9–10 the deoxyribose
moiety,11 and internal phosphorus atoms of DNA strands.12
A recent report dealt with the attachment of porphyrins to
deoxyuridine via a rigid acetylene spacer for exploring the use
of DNA as a scaffold to create helical multiporphyrin arrays.9
However, interestingly, there are only very few reports in which
porphyrins are placed in the interior of the DNA as part of the
p-stacking, most likely due to the challenging synthesis of the
necessary porphyrin nucleotide building blocks.13
We recently initiated a research program that aims to use the
simplified nucleic acid GNA (glycol nucleic acid) as a convenient
duplex scaffold for the generation of functional architectures.14
GNA is composed of nucleobases attached to propane-1,2-diol as
nucleoside building blocks that are connected by phosphodiester
bonds (Fig. 1a). GNA duplexes combine high duplex stability
and base pairing fidelity with a structurally simplified acyclic
backbone.15–17 X-ray crystal structures of (S)-GNA duplexes reveal
that the overall GNA double helix is distinguished from canonical
A- and B-form nucleic acids and might be best described as a
helical ribbon loosely wrapped around the helix axis.16,18 (S)-GNA
undergoes extensive zipper-like interstrand interactions and at the
same time possesses a reduced intrastrand base–base stacking
interaction due to a large average slide between neighboring base
sunlight into chemical energy by photosystems I and II relies
on a controlled and defined arrangement of the chlorphyll chro-
mophores for providing efficient light absorption, energy transfer,
and the initiation of charge separation. This example reveals that
the function of chromophore arrays relies on a controlled spatial
organization of the individual chromophores and it is therefore
not surprising that the design of defined chromophore arrays is
receiving an increasing interest.1–4
DNA is currently extensively used as a scaffold for the self-
assembly of structures with defined positioning of multiple
chromophores and other functional moieties.5–7 In this respect,
DNA offers two key advantages: (1) The canonical Watson–Crick
base pairing scheme allows the construction of defined helical
architectures with high predictability. (2) Highly reliable auto-
aFachbereich Chemie, Philipps-Universita¨t Marburg, Hans-Meerwein-
Straße, D-35032, Marburg, Germany. E-mail: zhangl@staff.
uni-marburg.de; Fax: +49-6421-2822189; Tel: +49-6421-2825600
bDepartment of Chemistry and Biochemistry, University of Notre Dame,
Notre Dame, Indiana, 46556-5670, USA
† Electronic supplementary information (ESI) available: Absorption spec-
tra of GNA single strands with and without metals; UV-vis spectra of GNA
duplexes and their corresponding single strands; concentration-dependent
and salt concentration-dependent UV-vis spectra of single strands ON10,
ON11 and their duplex ON10:ON11; temperature-dependent UV-vis
spectra of duplexes at Soret band region; fluorescence spectra of ON12,
ON13 and ON12:ON13, and ON12Zn, ON13Zn and ON12Zn:ON13Zn
Maldi-TOF data of all oligonucleotides. See DOI: 10.1039/c0ob00439a
;
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same time synthetically easily accessible. Based on these two
criteria we selected the phosphoramidite building block 1 as our
target. Accordingly, (S)-glycidyl-4,4¢-dimethoxytrityl ether 2 was
reacted with trimethylsilyl (TMS) acetylene in the presence of n-
BuLi and boron trifluoride etherate to produce in a regioselective
and stereospecific epoxide ring opening the acetylenic alcohol
3 in 70% yield. After removal of the TMS group of 3 with
TBAF (98%), a Pd-catalyzed Sonogashira coupling with 5-bromo-
10,20-diphenylporphinatozinc 5 afforded 6 smoothly in 95%
yield. Further reaction with 2-cyanoethyl-N,N-diisopropylchloro-
phosphoramidite in the presence of Hu¨nig’s base yielded the
phosphoramidite building block 1 (74%). Thus, phosphoramidite
1 was synthesized in a straightforward fashion starting from
epoxide 2 in just four steps with an overall yield of 48% (Scheme 1).
Next, phosphoramidite 1 was used to synthesize GNA strands
with incorporated diphenylporphyrin acetylide nucleotides (P,
Fig. 1b). GNA oligonucleotides were synthesized on CPG sup-
ports with standard protocols for 2-cyanoethylphosphoramidites.
Due to its low solubility in the standard solvent acetonitrile,
phosphoramidite 1 was dissolved in THF for the use in the DNA
synthesizer and the coupling times were extended to 15 min. The
GNA strands were synthesized in trityl-on mode and cleaved
from the resin with concentrated ammonia at 55 ◦C for 12 h.
After cooling to room temperature, the entire solution of crude
tritylated oligonucleotides was applied directly to a Sep–Pak clas-
sic reversed-phase column and subsequently washed, detritylated
with 1.5% aqueous TFA, and then eluted from the column using
30% aqueous acetonitrile. After an additional purification step
with a reversed phase HPLC column, the porphyrin-containing
GNA strands were obtained in high purities and devoid of
the zinc ions, most likely due to the acidic conditions during
the individual detritylation steps. However, zinc and nickel ions
could be readily (re)introduced into the coordination sites of
the porphyrin nucleotides in GNA oligunucleotides to give PM
(Fig. 1b) by mixing the purified single strands with Zn(OAc)2 or
Ni(OAc)2 in Tris-HCl buffered solution.
Fig. 1 (a) Constitution of the (S)-GNA backbone;( b) P:H and PM:H
base pairs used in this study.
pairs. Furthermore, (S)-GNA only displays one large groove,
whereas the canonical major groove is a convex surface. This differ-
ent duplex architecture might offer new opportunities to arrange
functional molecules such as chromophores in a controlled and
unique fashion. Thus, high duplex stabilities exceeding the stabil-
ities of analogous DNA and RNA duplexes, in combination with
an economical and fast synthesis of (modified) phosphoramidite
building blocks render GNA an attractive nucleic acid scaffold for
biotechnology and nanotechnology.
Here we report the first synthesis of a porphyrin-modified GNA
nucleotide, its incorporation into GNA, and the behavior of such
non-metallated and metallated porphyrins in the context of GNA
duplexes.
Results and Discussions
Synthesis of a porphyrin acetylide GNA nucleotide and its
incorporation into GNA oligonucleotides
We were seeking a propane-1,2-diol-modified porphyrin building
block that (a) is capable of stacking in GNA duplexes opposite
an ethylene glycol spacer as shown in Fig. 1b and (b) is at the
Scheme 1 Synthesis of the porphyrin acetylide phosphoramidite 1.
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Table 2 Thermal stabilities of porphyrin-containing 22mer GNA du-
Investigation of duplex stabilities
plexes and Watson–Crick reference duplexesa
We first investigated the stability of GNA duplexes containing a
single non-metallated (P) or metallated porphyrin (PM). Table 1
shows the UV-melting data of duplexes with P and PM incorpo-
rated into the position 8 of 16mer duplexes and two reference
duplexes. It is noteworthy that all GNA duplexes investigated
in this work do only contain A and T nucleotides in order to
keep the GNA scaffold as simple as possible. Pairing P with the
ethylene glycol abasic site H afforded a duplex melting point (Tm)
Entry Sequence
Tm (◦C)
63
1
2
3¢-TTATAAAAATAATAATATTAAT-2¢ (ON12)
2¢-AATATTTTTATTATTATAATTA-3¢(ON13)
3¢-TTATAAAAATTAATATTAAT-2¢ (ON14)
2¢-AATATTTTTAATTATAATTA-3¢(ON15)
3¢-TTATAAAAATPHTAATATTAAT-2¢ (ON16)
2¢-AATATTTTTAHPATTATAATTA-3¢(ON17)
3¢-TTATAAAAATHPTAATATTAAT-2¢ (ON18)
2¢-AATATTTTTAPHATTATAATTA-3¢(ON19)
3¢-TTATAAAAATPZnHTAATATTAAT-2¢ (ON16Zn
61
3
56
4
56
◦
◦
of 48 C (Table 1, entry 3), compared to 54 C for an A:T base
pair at the same position (Table 1, entry 1). Removing one A:T
base pair of the Watson–Crick reference duplex as in ON3:ON4
(Table 1, entry 2) resulted in a melting point of 52 ◦C, thus
demonstrating that the incorporation of a single P:H base pair
has a net destabilizing effect of DTm = 4 ◦C. Replacing the abasic
site H of the P:H base pair in ON6:ON7 (Table 1, entry 3) for the
natural nucleobases T or A, resulting in P:T and P:A base pairs,
reduces the stability by additional 4 and 3 ◦C, respectively (Table 1,
entries 4 and 5). Apparently, these larger natural nucleobases
cannot be accommodated well opposite the space consuming
diphenylporphyrin acetylide nucleobase P, suggesting that the
porphyrin nucleobase is accommodated within the p-stacking of
the GNA duplex. Interestingly, the incorporation of a Zn2+ ion
in the porphyrin results in a significant further destabilization for
5
)
48
2¢-AATATTTTTAHPZnATTATAATTA-3¢(ON17Zn
)
6
3¢-TTATAAAAATPNiHTAATATTAAT-2¢ (ON16Ni) 59
2¢-AATATTTTTAHPNiATTATAATTA-3¢(ON17Ni)
7
3¢-TTATAAAAATHPZnTAATATTAAT-2¢ (ON18Zn
)
49
2¢-AATATTTTTAPZnHATTATAATTA-3¢(ON19Zn
)
8
3¢-TTATAAAAATHPNiTAATATTAAT-2¢ (ON18Ni) 60
2¢-AATATTTTTAPNiHATTATAATTA-3¢(ON19Ni)
9
3¢-TTATAAAAATPZnHTAATATTAAT-2¢ (ON16Zn
2¢-AATATTTTTAHPNiATTATAATTA-3¢(ON17Ni)
)
54
10
3¢-TTATAAAAATPNiHTAATATTAAT-2¢ (ON16Ni) 52
2¢-AATATTTTTAHPZnATTATAATTA-3¢(ON17Zn
)
a Conditions: 10 mM sodium phosphate, 100 mM NaCl, and 2 mM
individual strands.
◦
the PZn:H base pair (Tm = 42 C, Table 1, entry 6), whereas the
pairs. In contrast, Ni2+ prefers square planar coordination and can
therefore be accommodated easily in the base stacking.19
We also investigated duplexes that contain two adjacent por-
phyrin acetylide base pairs. Incorporation of a second adjacent
P:H base pair positioning the porphyrins on opposite strands
(Table 1, entry 9) reduces the duplex melting temperature further
(Tm = 43 ◦C), being 6 ◦C below the melting temperatue of a
reference Watson–Crick duplex that is devoid of the two P:H
base pairs (Table 1 entry 8). Nevertheless the incorporation of
two adjacent P:H base pairs still affords a duplex that is thermally
stable at room temperature. As expected, the introduction of Zn2+
ions into the two porphyrins strongly destabilized the duplex to
Tm = 34 ◦C (Table 1, entry 10), similar to what was observed with
a single Zn2+ porphyrin in GNA (Table 1, entry 6).
Because of this general destabilization trend, we continued to
evaluate duplexes containing two porphyrins in longer (22mer)
and thus more stable duplexes as displayed in Table 2. These
data confirm that two adjacent P:H base pairs in opposite
orientation lead to a destabilization relative to a reference duplex
ON12:ON13 and ON14:ON15 containing only A:T base pairs but
still provide duplexes with robust stabilities (Table 2, entries 1–4)
and this stability can be modulated by the incorporation of Zn2+
(further destabilization) and Ni2+ (stabilization relative to the free
porphyrin) with ◦the nickel(II) containing duplex ON16Ni:ON17Ni
being only by 1 C less stable compared to the reference duplex
(Table 2, entries 5–10).
incorporation of a Ni2+ ion slightly increases the stability compare
to P:H (DTm = + 1 ◦C), and is only by 3 ◦C less stable than
ON3:ON4. These modulations of duplex stabilities by the nature
of the metal ion incorporated into the porphyrin nucleobases
can be interpreted by their different coordination behavior. Zn2+
prefers to coordinate to axial ligands when incorporated into a
porphyrin in an octahedral fashion and needs to dissociate these
ligands if it wants to stack within GNA between neighboring base
Table 1 Thermal stabilities of porphyrin-containing 16mer GNA du-
plexes together with Watson–Crick reference duplexesa
Entry
Sequence
Tm (◦C)
54
1
2
3¢-TAAAAATAATAATATT-2¢ (ON1)
2¢-ATTTTTATTATTATAA-3¢ (ON2)
3¢-TAAAAATATAATATT-2¢ (ON3)
2¢-ATTTTTATATTATAA-3¢ (ON4)
3¢-TAAAAATPATAATATT-2¢ (ON6)
2¢-ATTTTTAHTATTATAA-3¢ (ON7)
3¢-TAAAAATPATAATATT-2¢ (ON6)
2¢-ATTTTTATTATTATAA-3¢ (ON2)
3¢-TAAAAATPATAATATT-2¢ (ON6)
2¢-ATTTTTAATATTATAA-3¢ (ON5)
3¢-TAAAAATPZnATAATATT-2¢ (ON6Zn
2¢-ATTTTTAHTATTATAA-3¢ (ON7)
52
3
48
4
44
5
45
6
)
42
7
3¢-TAAAAATPNiATAATATT-2¢ (ON6Ni)
2¢-ATTTTTAHTATTATAA-3¢ (ON7)
3¢-TAAAAATTAATATT-2¢ (ON8)
49
It is noteworthy that the slopes of the melting curves of GNA
duplexes containing porphyrins are more shallow compared to
the related unmodified duplexes which might be caused by lower
cooperativity among the nucleobases owing to interference by the
porphyrin macrocycle (Fig. 2). In addition, the hyperchromicity of
the GNA duplexes containing porphyrins is reduced, presumably
due to a weaker stacking of base pairs in proximity of P:H base
pairs.
8
49
2¢-ATTTTTAATTATAA-3¢ (ON9)
9
3¢-TAAAAATPHTAATATT-2¢ (ON10)
2¢-ATTTTTAHPATTATAA-3¢ (ON11)
3¢-TAAAAATPZnHTAATATT-2¢ (ON10Zn
43
10
)
)
34
2¢-ATTTTTAHPZnATTATAA-3¢ (ON11Zn
a Conditions: 10 mM sodium phosphate, 100 mM NaCl, and 2 mM
individual strands.
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We next compared the UV-vis spectra of duplexes and their
corresponding single strands in order to obtain information about
the relative orientations of the porphyrin units in the GNA
duplexes. Pronounced changes in the absorption spectra were
observed upon duplex formation as displayed in Fig. 4 and Fig.
S2.† Duplex ON16:ON17, ON16Zn:ON17Zn and ON16Zn:ON17Ni
show a clear peak split of the Soret band. The splitting energies
between the low- and high-energy Soret bands are 920 cm-1 for
duplex ON16:ON17, 962 cm-1 for duplex ON16Zn:ON17Zn and
with the mixed metal porphyrin duplex ON16Zn:ON17Ni having the
highest excition energy (1151 cm-1). The duplex ON16Ni:ON17Ni
exhibits a broadened Soret band instead of two distinct peaks. An
obvious change of the Soret bands of the duplex accompanied
by a blue shift indicates a ground-state interaction of the two
porphyrin moieties. It has been reported that split Soret bands
can be caused by dipole–dipole exciton coupling from slipped-
cofacial interactions between two porphyrins.21 Thus, the two
neighboring porphyrin units in the GNA duplex might adopt a
slipped-cofacial geometry. The Soret band of all duplexes shift
Fig. 2 UV-melting curves of duplexes ON14:ON15, ON16:ON17,
ON16Zn:ON17Zn and ON16Ni:ON17Ni (see Table 2 for the sequences).
Changes in absorbance upon heating as monitored at 260 nm. Conditions:
10 mM sodium phosphate, 100 mM NaCl, pH 7.0, and 2 mM of each
strand.
UV-vis spectroscopy
Due to increased duplex stabilities we investigated the UV-
vis absorption properties of porphyrin GNA strands and the
corresponding duplexes with the 22mer system (Table 2). We began
by analyzing porphyrin-modified single strands without and with
incorporated zinc(II) and nickel(II) ions. The UV-vis spectra of the
single strands ON16, ON16Zn, and ON16Ni are shown in Fig. 3 and
clearly confirm the successful insertion of the metal ions into the
porphyrins. The absorption spectrum of ON16 displays a Soret
band at 426 nm and four Q bands, while ON16Zn and ON16Ni
exhibit one Soret band at 430 nm and 421 nm, respectively, and
just two Q bands. The reduced number of Q bands is typical of
metalloporphyrins.20 In addition, the observed slight red shift of
the Soret band for ON16Zn and blue shift for ON16Ni are consistent
with the well-known red shift of porphyrin Soret bands upon
Zn(II) coordination, and blue shift of Soret bands upon Ni(II)
coordination.20 UV-vis spectra of single strands ON17, ON18,
ON19 and their metal derivatives are provided in the supporting
information.
Fig. 4 UV-vis spectra of GNA duplexes and their corresponding single
strands. (a) ON16, ON17 and ON16:ON17; (b) ON16Ni, ON17Ni and
ON16Ni:ON17Ni The inserts show expanded porphyrin Soret band regions.
Conditions: 10 mM sodium phosphate, 100 mM NaCl, pH 7.0, and 2 mM
of each strand.
Fig. 3 Absorption spectra of ON16, ON16Zn and ON16Ni. The insert
shows the expanded porphyrin Soret band region. Conditions: 10 mM
sodium phosphate, 100 mM NaCl, pH 7.0, and 2 mM of single strands.
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to a shorter wavelengths, indicating that in each duplex two
porphyrin units overlap face-to-face (H-dimer).22,
‡
Next, we measured the influence of temperature on the
UV-vis spectra for the duplexes ON16:ON17, ON16Zn:ON17Zn,
ON16Ni:ON17Ni and ON16Zn:ON17Ni. The results are shown in
Fig. 5 and Fig. S5† and reveal strong spectral changes in the
Soret band of the porphyrin moieties upon heating from 20 ◦C
to 80 ◦C. By increasing the temperature, the peaks gradually shift
to a longer wavelength with disappearance of the splitting of the
peaks and this shift can be associated with a dissociation of the
duplexes into their single strands: two porphyrin moieties in two
strands lose their interaction.
Fig. 5 Temperture-dependent UV-vis spectra of duplexes of ON16:ON17
at the Soret band region. Conditions: 10 mM sodium phosphate, 100 mM
NaCl, pH 7.0 and 2 mM of each strand.
Fig. 6 Fluorescence spectra of GNA duplexes and their corresponding
single strands. (a) ON16, ON17 and ON16:ON17; (b) ON16Zn, ON17Zn
and ON16Zn:ON17Zn. Conditions: 10 mM sodium phosphate, 100 mM
NaCl, pH 7.0 and 2 mM of each strand.
Fluorescence spectroscopy
CD spectroscopy
The fluorescence spectra of ON16, ON17, ON16:ON17, and
ON16Zn, ON17Zn, ON16Zn:ON17Zn are shown in Fig. 6. Ni por-
phyrins could not be investigated since they are not fluorescence.18
ON16 and ON17 which contain free porphyrins display the
characteristic two emission peaks at 660 nm and 725 nm, while
ON16Zn and ON17Zn show features of Zn prophyrin emission
spectra: the emission peaks are situated at 615 nm and 665 nm
which are 50 nm blue shifted compare to ON16 and ON17.20
Apart from the change in intensity, the fluorescent spectra of
duplexes ON16:ON17 and ON16Zn:ON17Zn are rather similar. The
decrease of fluorescence intensity is consistent with the formation
of porphyrin aggregates. In addition, ON16:ON17 shows lower
emission quantity compared to ON16Zn:ON17Zn, which is in
agreement with the fact that the free porphyrins can stack better
within the GNA duplex. Overall, the UV-vis together with the
fluorescence data support interactions between the porphyrins
within the GNA duplex.
In order to learn more about the interaction of the porphyrins
with the GNA duplex, porphyrin-modified GNA duplexes were
investigated by CD spectroscopy as shown in Fig. 7. The CD
signals of porphyrin-GNA duplexes in the GNA absorption
region are almost identical to those of the native GNA duplex,
except for the lower signal intensities. It thus seems that the
porphyrin modifications do not distort the overall GNA duplex
conformation to a significant extent. In the porphyrin absorption
region, induced negative Cotton effects at 406 nm and 400 nm are
observed in the case of ON16:ON17 (Fig. 7a) and ON16Ni:ON17Ni
(Fig. 7c). Typically for DNA-porphyrins, an induced negative
Cotton effect in the Soret region is a signature of the intercalation
of the porphyrin moiety into the DNA duplex.23,24 Together with
the Tm data, we therefore conclude that the porphyrin rings
in duplex ON16:ON17 and ON16Ni:ON17Ni intercalate into the
GNA duplex and stack to neighboring base pairs. However, the
CD spectra of duplexes which contain Zn-porphyrins are different.
In the case of ON16Zn:ON17Zn (Fig. 7b), a clear multisignate
CD spectrum, consisting of two negative bands at 409 and
429 nm and a positive band at 441 nm is observed. The CD
spectrum of ON16Zn:ON17Ni (Fig. 7d) shows a strong negative
‡ Uv-vis spectra of single strands and duplexes are independent of strand
and salt concentrations. Concentration and salt-dependent UV-vis spectra
are shown in the ESI.†
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Fig. 7 CD spectra of GNA duplexes and their corresponding single strands. (a) ON16, ON17, ON16:ON17 and ON14:ON15; (b) ON16Zn, ON17Zn
and ON16Zn:ON17Zn; (c) ON16Ni, ON17Ni and ON16Ni:ON17Ni; (d) ON16Zn, ON17Ni and ON16Zn:ON17Ni. Inserts: Overlap plot of CD and absorption
spectra at the Soret region. Conditions: 10 mM sodium phosphate, 100 mM NaCl, pH 7.0, and 12 mM of each strand.
band at 403 nm and a weak positive band at 439 nm. Bisignate
or multisignate CD spectra were observed in a system with
porphyrins covalently attached to the 5¢-end of DNA duplexes and
such CD spectra were interpreted for the evidence of through space
dipole–dipole electronic interactions between the two porphyrin
chromophores.25 Together with the temperature melting results
(Tables 1 and 2), we therefore assume that the Zn-porphyrins
interact with each other although they are not as close in contact
as the porphyrins in the analogous systems devoid of any metal or
coordinated to nickel ions.
average structures. It is apparent that the porphyrin moieties are
well accommodated within the base stacking with only minor
distortions of the overall duplex structure and with little or no
disruption of hydrogen bonding between the flanking A-T base
pairs. For both systems studied, the average interstrand P–P
˚
˚
distance for the incorporation sites is 17.0 A versus 16.6 A for a
canonical GNA base pair, with the porphyrin slightly pertruding
beyond the band formed by the GNA duplex. Close p-stacking
interactions are observed between the porphyrin base and the
neighboring base pairs. In ON10:ON11, the two porphyrins are
stacked on top of each other as shown in Fig. 8b. The observed
offset of the porphyrins in the calculated structures is in excellent
agreement with the experimental observation of the split Soret
band seen in the UV-Vis spectra of the related duplex ON16:ON17
(Fig. 4). These slipped cofacial geometries allow the phenyl
subsituents of the two adjacent porphyrins to pack efficiently
in a face-to-face or edge-to-face fashion, thus explaining the
experimental observation that the relative destabilization of two
adjacent P:H base pairs is smaller than a single P:H base pair
within GNA (Table 1, entries 3 and 9).
Computational work
In order to gain further insight into the binding mode of
porphyrin acetylide nucleobases within GNA duplexes, we built
models of ON6:ON7 (16mer duplex with single P:H base pair)
and ON10:ON11 (16mer duplex with two adjacent P:H base
pairs) and studied them by molecular dynamics simulations in
analogy to our previously described GNA model.26 Fig. 8 shows
the local environment around the porphyrins of the calculated
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Experimental
Synthesis of porphyrin acetylide phosphoramidite
NMR spectra were recorded on a Brucker AVANCE 300 and
Brucker DRX 500. Low-resolution mass spectra were obtained on
an LC platform from Agilent using ESI technique. High-resolution
mass spectra were obtained on a Thermo Finnigan LTQ FT instru-
ment using APCI ionization. MALDI were obtained on a Ultraflex
from Bruker. Infrared spectra were recorded on a Brucker “Alpha-
P” FT-IR spectrometer. HPLC was performed using an Agilent
technologies 1200 series instrument with fraction collection. All
non-aqueous operations were carried out under dry nitrogen
atmosphere. Solvents and reagents used as supplied from Aldrich,
Alfa Aesar or Acros. 5-Bromo-10, 20-diphenylporphinatozinc 5
was synthesized as reported.27–29
Compound 3. To a solution of ethynyltrimethylsilane (0.47 mL,
3.32 mmol) in dry THF (8 mL) at -78 ◦C was added slowly a
1.6 M hexane solution of n-BuLi (2.1 mL, 3.36 mmol). After
stirring for 10 min, boron trifluoride diethyl etherate (0.42 mL,
3.32 mmol) was added dropwise. After an additional 5 min
◦
at -78 C, a solution of compound 230 (1.25 g, 3.32 mmol)
in anhydrous THF (4 mL) was added slowly. The resulting
solution was stirred for 15 min at -78 ◦C. The reaction was then
quenched with saturated aqueous NaHCO3 (80 mL) at -78 ◦C
and extracted with CH2Cl2 (3 ¥ 60 mL). The combined organic
layers were dried over anhydrous Na2SO4, concentrated by rotary
evaporation, and purified by flash chromatography over silica
gel eluting with hexane–ethyl acetate/triethylamine (10 : 1 : 0.01),
1
affording compound 3 as a colorless oil (1.11 g, 70%). H NMR
(300 MHz, CDCl3): d = 7.45 (m, 2H), 7.16–7.38 (m, 7H), 6.84
(d, J = 8.9 Hz, 4H), 3.92 (m, 1H), 3.79 (s, 6H), 3.24 (m, 2H),
2.52 (d, J = 6.4 Hz, 2H), 0.115 (s, 9H). 13C NMR (75 MHz,
CDCl3): d = 158.65, 144.92, 136.08, 130.18, 128.26, 127.96, 126.95,
113.30, 102.79, 87.34, 86.32, 69.49, 66.10, 55.30, 25.46, 0.14. IR
(thin film): 2956, 2933, 2175, 1607, 1580, 1508, 1462, 1445, 1414,
1335, 1298, 1246, 1174, 1155,1075, 1032, 826, 790, 756, 726, 701,
645, 583 cm-1. HRMS: m/z [M + Na]+ calcd for C29H34O4Si1Na1:
497.2119; found: 497.2120.
Fig. 8 Calculated structures of the porphyrin-containing duplexes
ON6:ON7 (Table 1, entry 3) and ON10:ON11 (Table 2, entry 3). Only
the local environment around the P:H base pairs is shown.
Conclusions
We here reported a straightforward synthetic strategy to em-
ploy GNA as a scaffold to organize porphyrin chromophores.
A diphenylporphyrin acetylide phosphoramidite GNA building
block 1 for automated oligonucleotide synthesis was synthesized in
just four steps with an overall yield of 48%. Incorporated opposite
an ethylene glycol abasic site (H), such porphyrin acetylides (P)
afford duplexes which are thermally destabilized but without
significant perturbation of the overall GNA duplex structures.
Interestingly, the thermal stabilities of such P:H base pairs can
be modulated by the incorporation of zinc(II) and nickel(II) which
lead to a decrease and increase in duplex stabilities, respectively.
This can be rationalized with a flipped-out conformation of
the zinc-porphyrin in contrast to a stacking of the nickel-
porpyhrin within the base stacking of the GNA duplex. Thus,
the conformation of porphyrins in GNA can be modulated by the
nature of the coordinated metal ions. Finally, we demonstrated
that GNA duplexes are a suitable scaffold for bringing two
porphyrins into close contact and to allow the interaction of
porphyrins with different coordinated metal ions. Future work will
investigate applications of such porphyrin-GNA conjugates and
other chromophores for light harvesting and charge transport.
Compound 4. To a solution of compound 3 (1.73 g, 3.65 mmol)
in THF (36.5 mL) was added 1.0 M THF solution of tetra-
butylammonium fluoride (11 mL) and then stirred for 1 h at
room temperature. The reaction was quenched with H2O and
extracted with CH2Cl2 (3 ¥ 50 mL). The combined organic extracts
were dried over anhydrous Na2SO4 and concentrated using rotary
evaporation. The resulting oil was purified by chromatography
over silica gel eluting with hexane–ethyl acetate/triethylamine
(5 : 1 : 0.01), affording compound 4 as a colourless oil (1.45 g, 98%).
1H NMR (300 MHz, CDCl3): d = 7.43 (m, 2H), 7.26–7.34 (m, 6H),
7.19–7.24 (m, 1H), 6.83 (d, J = 8.9 Hz, 4H), 3.91 (m, 1H), 3.79 (s,
6H), 3.24 (m, 2H), 2.47 (ddd, J = 0.8, 2.6, 6.2 Hz, 2H), 2.37 (d, J =
5.0 Hz, 1H), 1.98 (t, J = 2.7 Hz, 1H). 13C NMR (75 MHz, CDCl3):
d = 158.71, 144.88, 136.06, 130.20, 128.28, 128.00, 127.01, 113.32,
86.43, 80.52, 70.67, 69.45, 66.06, 55.37, 24.07. IR (thin film): 3287,
3000, 2836, 2045, 1734, 1607, 1581, 1507, 1462, 1444, 1299, 1245,
1174, 1154, 1072, 1031, 951, 913, 826, 790, 772, 754, 701, 634,
and 582 cm-1. HRMS: m/z [M + Na]+ calcd for C26H26O4Na1:
425.1723; found: 425.1723.
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Compound 6. Compound 527–29 (198 mg, 0.33 mmol),
Pd(PPh3)4 (75.8 mg, 0.07 mmol) and CuI (25 mg, 0.13 mmol)
were placed in a Schlenk flask and the flask was evacuated and
recharged with N2 gas. Dry THF (7 mL) and dry Et3N (6 mL) were
added and the resulting mixture was degassed for 10 min. Then
compound 4 (263.8 mg, 0.66 mmol) in anhydrous THF (3 mL) was
added and the mixture was stirred for 12 h at 65 ◦C. After cooling
to room temperature, the mixture was poured into saturated
aqueous NaHCO3, extracted with CH2Cl2 (3 ¥ 40 mL), dried
over Na2SO4, filtered, and evaporated. The resulting residue was
purified by chromatography over silica gel eluting with hexane–
dichloromethane/triethylamine (1 : 1 : 0.01), affording compound
6 as a purple foam (289 mg, 95%). 1H NMR (300 MHz, CDCl3):
d = 10.05 (s, 1H), 9.41 (d, J = 4.6 Hz, 2H), 9.25 (d, J = 4.5 Hz,
2H), 8.96 (d, J = 4.5 Hz, 2H), 8.91 (d, J = 4.6 Hz, 2H), 8.19 (dd,
J = 1.6, 7.5 Hz, 4H), 7.78 (m, 6H), 7.36 (m, 2H), 7.18–7.26 (m,
6H), 7.10–7.15 (m, 1H), 6.57 (dd, J = 1.3, 8.9 Hz, 4H), 3.74 (m,
1H), 3.38 (m, 1H), 3.33 (d, J = 2.0 Hz, 6H), 3.25 (m, 1H), 2.75 (m,
2H). 13C NMR (125 MHz, CDCl3): d = 158.42, 152.08, 150.42,
149.77, 149.61, 144.99, 142.94, 135.89, 134.90, 132.54, 132.34,
131.78, 130.97, 130.11, 128.18, 128.00, 127.52, 126.93, 126.69,
120.97, 113.29, 107.06, 86.41, 69.92, 66.35, 55.07, 53.55, 29.85,
25.42. IR (thin film): 3052, 2933, 2831, 1597, 1506, 1489, 1459,
1439, 1382, 1298, 1245, 1219, 1173, 1153, 1058, 1031, 989, 904,
825, 790, 749, 727, 716, 619, 580 cm-1. HRMS: m/z [M + H]+
calcd for C58H45N4O4Zn1: 925.2727; found: 925.2726.
(121 MHz, CDCl3): d = 148.05, 147.85. IR (thin film): 2962, 2930,
2866, 2834, 2253, 2227, 2022, 1507, 1490, 1460, 1440, 1382, 1363,
1297, 1248, 1176, 1154, 1124, 1060, 1032, 1002, 992, 908, 827, 792,
780, 751, 728, 717, 700, 658, 582 cm-1. HRMS: m/z [M + H]+ calcd
for C67H62N6O5P1Zn1: 1125.3805; found: 1125.3807.
GNA oligonucleotide synthesis and purification
General protocol30. All oligonucleotides were prepared on an
ABI 394 DNA/RNA synthesizer on a 1 mmole scale. A standard
protocol for 2-cyanoethyl phosphoramidites (0.1 M) was used,
except that the coupling time was extended to 3 min for A and T
nucleotides and 15 min for porphyrin phosphoramidite couplings.
After the trityl-on synthesis, the resin was◦incubated with conc.
aq. NH3 solution (1.5 mL) for 12 h at 55 C. After cooling, the
entire solution was applied directly to a Sep–Pak Classic reversed-
phase column (Waters, 360 mg) and washed sequentially with 3%
NH4OH (15 mL), water (10 mL), 1.5% aqueous TFA (10 mL), and
finally water (10 mL). The oligo was then eluted with 20% aqueous
acetonitrile or 30% for oligonucleotides containing porphyrins and
further purified using a Waters XTerra column (MS C18, 4.6 ¥
50 mm, 2.5 mm) at 55 ◦C with aqueous TEAA and acetonitrile as
the eluent. The identities of all oligonucleotides were confirmed
by MALDI-TOF MS.
Zinc metallation of porphyrin in GNA. To a solution of
porphyrin-GNA single strands (12 nmol GNA single strands in
140 mL of 10 mM Tris-HCl buffer solution, pH 7.6) was added a
solution of Zn(OAc)2 in H2O (10 mM, 12 mL,10 eq.). The solution
was shaken at 25 ◦C and the reaction was followed by absorption
spectroscopy. After around 2 h, the reaction mixture was diluted to
1000 mL with 3% ammonia solution. Excess Zn2+ was removed by
treating the reaction mixture with ion-exchange resins (Chelex 100
sodium form). The strands were purified by HPLC using a Water
XTerra column at 55 ◦C with aqueous TEAA and acetonitrile as
the eluent. The identities of the oligonucleotides was confirmed by
MALDI-TOF MS.
Compound 1. To a solution of compound 6 (93.6 mg,
0.10 mmol) and N,N-diisopropylethylamine (0.1 mL, 0.54 mmol)
in anhydrous CH2Cl2 (4 mL) was added 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite (0.034 mL, 0.15 mmol). After
2 h, the reaction mixture was poured into saturated aqueous
NaHCO3 and extracted by CH2Cl2 (3 ¥ 40 mL). The organic
layer was evaporated to dryness and the resulting residue was
purified by chromatography over silica gel eluting with hexane–
dichloromethane/triethylamine (2 : 1 : 0.01), affording compound
1 as a purple foam (85 mg, 74%). (Compound 1 is a mixture of
two diastermers, the ratio of major and minor isomers is around
Nickel metallation of porphyrin in GNA. Nickel metallation
◦
1
1
was carried out by heating (80 C) under nitrogen atomosphere
2 : 1 according to H NMR.) H NMR (500 MHz, CDCl3): d =
10.04 (s, 2H), 9.64 (d, J = 4.5 Hz, 2H), 9.59 (d, J = 4.6 Hz, 2H),
9.23 (d, J = 4.5 Hz, 4H), 8.93–3.98 (m, 8H), 8.23 (d, J = 7.3 Hz,
8H), 7.77–7.83 (m, 12H), 7.51–7.56 (m, 4H), 7.30–7.38(m, 8H),
7.19–7.25 (m, 4H), 7.09–7.14 (m, 2H), 6.50 (d, J = 8.9 Hz, 2H),
6.47 (d, J = 8.9 Hz, 2H), 6.40 (d, J = 8.9 Hz, 2H) (minor isomer),
6.32 (d, J = 8.8 Hz, 2H) (minor isomer), 4.57–4.69 (m, 2H), 3.63–
3.98 (m, 12H), 3.55–3.60 (m, 2H), 3.38–3.46 (m, 2H), 3.23 (s,
3H), 3.18 (s, 3H), 3.08 (s, 3H) (minor isomer), 2.97 (s, 3H) (minor
isomer), 2.41–2.60 (m, 2H), 2.31–2.37 (m, 2H) (minor isomer),
1.32 (d, J = 6.8 Hz, 6H) (minor isomer), 1.25 (d, J = 6.8 Hz, 6H)
(minor isomer), 1.20 (d, J = 6.8 Hz, 6H), 1.17 (d, J = 6.8 Hz,
6H). 13C NMR (125 MHz, CDCl3): d = 158.22, 158.13, 158.10,
157.94, 152.20, 152.15, 150.43, 150.41, 149.83, 149.70, 149.67,
145.35, 145.32, 142.76, 136.38, 136.29, 136.04, 136.00, 134.72,
132.55, 132.50, 131.85, 131.30, 130.32, 130.13, 130.06, 128.32,
128.28, 127.90, 127.85, 127.62, 126.77, 121.10, 117.80, 117.63,
113.20, 113.17, 113.14, 113.11, 106.98, 86.35, 86.30, 73.12, 72.97,
72.33, 72.20, 65.95, 65.94, 65.79, 65.78, 58.78, 58.74, 58.64, 58.60,
55.10, 54.96, 54.92, 54.84, 43.64, 43.54, 43.49, 25.89, 25.84, 25.81,
24.91, 24.56, 24.81, 24.76, 20.48, 20.42, 20.41, 20.36. 31P NMR
a solution of porphyrin-GNA single strands (2.5 nmol GNA in
325 mL of 10 mM Tris-HCl buffer solution, pH 7.6, and 10 mM
Ni(OAc)2). The reaction was followed by absorption spectroscopy
and found to be completed after heating for 16 h. Workup was
performed in analogy to the zinc(II) complexation.
Analysis of GNA nucleic acids
Thermal denaturation. The melting studies were carried out in
1 cm path length quartz cells (total volumn 325 mL; 200 mL sample
solutions were covered by mineral oil) on a Beckman 800 UV-Vis
spectrophotometer equipped with a thermo-programmer. Melting
curves were monitored at 260 nm with a heating rate of 1 ◦C min-1.
Melting temperatures were calculated from the first derivatives of
the heating curves. Experiments were performed in duplicate and
mean values were taken.
UV-vis spectroscopy. The absorptions of oligonucleotide so-
lutions were measured in a quartz cuvette with a path length
of 1 cm at 260 nm on Beckman 800 UV-Vis spectrophotome-
ter. Temperature-dependent absorption spectra were obtained
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on Beckman 800 UV-Vis spectrophotometer equipped with a
temperature controller. Spectra were obtained every 10 ◦C by
heating from 20 to 80 ◦C. Both single strands and duplexes
(2 mM of each strand) were prepared in 10 mM sodium phosphate,
100 mM NaCl, pH 7.0.
Acknowledgements
We thank the Philipps-University Marburg for financial support.
Notes and references
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Fluorescence spectroscopy. The experiments were performed
in 96-well plates on a Molecular Devices SpectraMax M5 with
excitation at 427 nm and a cutoff filter at 435 nm. The experiments
with single strands or duplexes were performed in 10 mM sodium
phosphate, 100 mM NaCl, pH 7.0, and the concentration of each
strand was 2 mM.
CD spectroscopy. CD measurements were performed on a
JASCO J-810 spectrometer in a 1 mm path length quartz cuvette.
The GNA single strands or duplexes were prepared in 10 mM
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Computational work
Models of ON6:ON7 and ON10:ON11 were built based on a
previously described GNA model.26 In short, crystal structure of
an 8mer duplex of a GNA analog26 was extended by including
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