Synthesis of DNA with green perylene bisimides as DNA base substitutions

Article abstract of DOI:10.1002/ejoc.201100519

Perylene-3, 4:9, 10-tetracarboxylic acid bisimide represents a particularly attractive building block for DNA-based multichromophores and nanoassemblies due to its unique combination of optical and electronic properties. Despite the broad applicability of the perylene bisimide dye for DNA-based assemblies, significant fluorescence quenching by photoinduced charge-transfer processes to guanines in DNA represents a major disadvantage. In order to develop a redox actively and optically tuned perylene bisimide derivative with fluorescence that does not photooxidize guanines in DNA, we chose to attach two N-pyrrolidinyl substituents as strongly electron-donating substituents at the 1-and 7-positions of the bay area ("APBI"). The corresponding phosphoramidite 1 was synthesized and incorporated syntheti cally into oligonucleotides by using the automated DNA building block chemistry. The 2'-deoxyribofuranoside of natural nucleosides was replaced by (S)-1-minopropane-2, 3-diol as an acyclic linker between the phosphodiester bridges that is tethered to one of the imide nitrogen atoms of the APBI dye. Electrochemical and optical characterization of the isolated APBI dye shows that photoinduced charge transfer with guanines is indeed very unlikely. Single APBI DNA base substitutions show an exciplex-type emission with pH sensitivity. The interstrand APBI dimer in DNA could be regarded as a hydrophobically interacting base pair that stabilizes the thermal stability of the double strand. The APBI dimer behaves like an H aggregate, and the fluorescence is quenched quantitatively.

Full text of DOI:10.1002/ejoc.201100519

FULL PAPER
DOI: 10.1002/ejoc.201100519
Synthesis of DNA with Green Perylene Bisimides as DNA Base Substitutions
Florian Menacher[a] and Hans-Achim Wagenknecht*[a][‡]
Keywords: Chromophores / DNA / Fluorescence / Oligonucleotides / Nitrogen heterocycles
Perylene-3,4:9,10-tetracarboxylic acid bisimide represents a
particularly attractive building block for DNA-based multi-
chromophores and nanoassemblies due to its unique combi-
nation of optical and electronic properties. Despite the broad
applicability of the perylene bisimide dye for DNA-based as-
semblies, significant fluorescence quenching by photoin-
duced charge-transfer processes to guanines in DNA repre-
sents a major disadvantage. In order to develop a redox ac-
tively and optically tuned perylene bisimide derivative with
fluorescence that does not photooxidize guanines in DNA,
we chose to attach two N-pyrrolidinyl substituents as
strongly electron-donating substituents at the 1- and 7-posi-
tions of the bay area (“APBI”). The corresponding phos-
phoramidite 1 was synthesized and incorporated syntheti-
cally into oligonucleotides by using the automated DNA
building block chemistry. The 2Ј-deoxyribofuranoside of nat-
ural nucleosides was replaced by (S)-1-aminopropane-2,3-
diol as an acyclic linker between the phosphodiester bridges
that is tethered to one of the imide nitrogen atoms of the
APBI dye. Electrochemical and optical characterization of the
isolated APBI dye shows that photoinduced charge transfer
with guanines is indeed very unlikely. Single APBI DNA base
substitutions show an exciplex-type emission with pH sensi-
tivity. The interstrand APBI dimer in DNA could be regarded
as a hydrophobically interacting base pair that stabilizes the
thermal stability of the double strand. The APBI dimer be-
haves like an H aggregate, and the fluorescence is quenched
quantitatively.
blocks were developed to modify oligonucleotides with per-
ylene bisimides.[6–9] Among these are two phosphoramidites
that allow capping of DNA duplexes or hairpins with the
perylene bisimide dye,[6–8] but only one phosphoramidite for
modification of DNA with perylene bisimide as an internal
base substitution.[9] Additionally, two special perylene bis-
imide building blocks based on active esters were developed
for the DNA-directed assembly of this dye in thermophilic
foldamers and for single molecule measurements.[10,11] Es-
pecially, two of the mentioned perylene bisimide phos-
phoramidites were extensively applied to construct excep-
tionally stable DNA triplexes,[12,13] for electron-transfer
studies,[14] for fluorescence quenching in highly sensitive
molecular beacons,[15] and for forced base-flipping.[16] We
showed that perylene bisimide stacks as a chiral oligochro-
mophore inside the double helical framework.[17] Moreover,
we[9,17,19] and others[20] have shown that perylene bisimide
triggers the reversible aggregation of complete DNA du-
plexes, hairpins, and dumbbells, yielding electronically con-
jugated nanostructures.
Despite the great potential of the common perylene bis-
imide dye for DNA-based assemblies, it is important to
point out that the nearly quantitative fluorescence quench-
ing that is observed with the dye in the vicinity of guanines
due to photoinduced electron-transfer processes limits the
applicability.[17,21] None of the above-mentioned examples
of DNA assemblies applied perylene bisimide modified at
the core of the chromophore, the so-called bay area. This
is surprising because both the optical and redox properties
of perylene bisimide chromophores can be tuned signifi-
Introduction
The helical organization of organic chromophores as
base substitutions inside the DNA duplex or as base modi-
fications outside the DNA double helix allows the construc-
tion of multichromophore architectures based on corre-
sponding building blocks. The double helix provides a sup-
ramolecular scaffold that controls the photophysical inter-
actions in such a way that unique fluorescence properties
are obtained which are significantly different from the chro-
mophore monomers.[1] Among the various applied fluoro-
phores, perylene-3,4:9,10-tetracarboxylic acid bisimide rep-
resents a particularly attractive building block for DNA-
based multichromophores owing to its unmatched combi-
nation of tinctorial strength, high fluorescence quantum
yield, photostability, and accessibility of radical anionic
states.[2,3] The noncovalent interactions of perylene bisim-
ides with DNA were studied with derivatives that were
modified at the imide substituents with positively charged
amino functionalities.[4,5] Moreover, synthetic protocols for
at least three different phosphoramidites as DNA building
[a] Institute for Organic Chemistry, University of Regensburg,
93040 Regensburg, Germany
Fax: +49-941-943-4804
E-mail: achim.wagenknecht@chemie.uni-regensburg.de
[‡] Current address: Institute for Organic Chemistry,
Karlsruhe Institute of Technology,
Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
Fax: +49-721-608-47486
E-mail: wagenknecht@kit.edu
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100519.
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Synthesis of DNA with Green Perylene Bisimides as DNA Base Substitutions
cantly by introduction of substituents at the central carbo- hairpins[14] revealed that the photoexcited chromophore is
cyclic scaffold. In order to develop a green and thereby re- able to inject electron holes into adjacent purine, but not
dox-tuned perylene bisimide derivative we chose to attach pyrimidine bases. Electron holes that were injected primar-
two N-pyrrolidinyl substituents as strongly electron-donat- ily into adenine tracts are trapped efficiently on guanines.
ing substituents at the bay-area (“APBI”). Such amino- However, it is important to point out that in the absence of
modified and therefore green perylene bisimide derivatives adjacent guanines the fluorescence intensity of PBI is still
were synthesized and characterized previously,[21–26] as ana- sufficient to obtain clear fluorescence readout for DNA as-
logs of chlorophyll a[27,28] and as water-soluble, potential semblies[18–20] and bioanalysis.[16,17] With respect to the
noncovalent DNA binders,[29–31] but they have not yet been power of perylene bisimide in DNA-based nanostructur-
used for DNA. In order to extend the toolbox of available ing[6–28] it looked promising to design a new DNA base sub-
chromophores and dyes as building blocks for DNA we stitution based on the perylene bisimide scaffold that over-
present herein the synthesis of the green perylene bisimide comes the problem of photoinduced guanine oxidation by
derivative 1, its electrochemical characterization, its syn- maintaining the fluorescence quantum yield of PBI in guan-
thetic incorporation into oligonucleotides, and the optical ine-poor sequences. Two strongly electron-donating substit-
properties of several DNA duplexes that contain one or two uents at the core of the chromophore should reduce the
APBI base substitutions of this type.
electron deficiency significantly and thus may prevent pho-
toinduced guanine oxidation. According to the broad vari-
ety of synthetically available modified perylene bisimide de-
rivatives,[2,3] it looked reasonable to attach two N-pyrrolid-
inyl substituents at the so-called bay area of the chromo-
phore. Furthermore, previous work suggests using the 1-
and 7-positions to anchor the two amino substituents.
Results and Discussion
Synthesis of the APBI DNA Building Block
The first available covalent DNA–perylene bisimide con-
jugates have indicated clearly that significant fluorescence
The development of the synthetic procedure for the cor-
quenching is observed by the attached DNA bases.[6,9] responding APBI phosphoramidite 1 is based on our ex-
More detailed studies by time-resolved methods using per- perience in the preparation of perylene bisimide building
ylene bisimide as an internal DNA base substitution in blocks for DNA,[9] combined with the knowledge obtained
Scheme 1. Synthesis of APBI DNA building block 1 and deprotected APBI 7. Reagents and conditions: (a) Br2, I2, H2SO4, 95 °C; 18 h,
quant.; (b) Zn(OAc)2, 2-ethylhexylamine, pyridine, 75 °C; 15 h, 25%; (c) pyrrolidine, 55 °C; 24 h, 82%; (d) 2-cyanoethyl-N,N-diiso-
propylchlorophosphoramidite, NEt3, CH2Cl2, r.t., 1.5 h; 74%; (e) dichloroacetic acid, CH2Cl2, r.t., 10 min; 93%.
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F. Menacher, H.-A. Wagenknecht
FULL PAPER
from literature for perylene bisimide modifica- to its better solubility, 6 was used to determine the redox
tions.[21,23,24,30,31] According to the latter publications, the potentials in dichloromethane by cyclic voltammetry (Sup-
synthesis starts with perylene bisanhydride 2, which was porting Information, Figure S1). Two oxidation potentials
brominated in the presence of a catalytic amount of iodine at 0.24 and 0.37 V (vs. ferrocene) are well separated, revers-
(Scheme 1). In contrast to the published pro- ible (50–60 mV difference of the half-wave potentials), and
cedures,[21,24,30,31] 6 equiv. of bromine had to be added to in agreement with literature values for similar N-pyrrolid-
achieve a sufficient amount of product mixture consisting inyl-substituted perylene bisimide derivatives.[2,23,27] The ap-
of desired 1,7-dibrominated product 3 and the correspond- parent single reduction potential at –1.29 V is the result of
ing 1,6-disubstituted isomer. Due to the low solubility of two half-wave potentials that show a difference of 0.13 V.
both compounds, separation was impossible at this stage. Such a large value indicates two not well-separated poten-
For the later incorporation as a DNA base substitution into tials at approximately –1.25 and –1.33 V. In fact, according
oligonucleotides, the 2Ј-deoxyribofuranoside of natural nu- to the literature, two reduction potentials are expected at
cleotides was replaced by (S)-1-aminopropane-2,3-diol as –1.28 and –1.46 V (based on ferrocene oxidation potential
an acyclic linker. This approach is extensively used by our of +0.52 V vs. SCE).[2,23,27] The value of E00 was deter-
group not only for perylene bisimide[9,17–19] but also for mined (Supporting Information, Figure S2) to be 1.7 eV (at
other chromophores,[32] and by other groups for the prepa- 730 nm). If this value is added to the ground state potential
ration of glycol nucleic acids (GNA)[33] and twisted intercal- of ca. –1.3 V, the excited state of 6 exhibits a reduction po-
ating nucleic acids (TINA).[34] Accordingly, dibrominated tential of +0.4 V. On the other hand, the oxidation potential
perylene bisanhydride 3 was treated with 2 equiv. of DMT- of guanine is +1.3 V (vs. NHE), which corresponds to a
protected (S)-1-aminopropane-2,3-diol 4 and 1 equiv. of 2- potential of +0.7–0.9 V (vs. ferrocene).[35] According to the
ethylhexylamine in the presence of ZnII.[9] The DMT pro- Rehm–Weller equation (neglecting the Coulomb energy),
tecting group supports the solubility of product 5, which the calculation ΔG = Eox – Ered – E00 gives a positive driv-
was obtained in a yield of 25% over the first two synthetic ing force of 0.3–0.5 eV and thereby clearly excludes photo-
steps. It is important to note that the temperature of 75 °C oxidation of guanines in DNA by APBI.
and the reaction time of 12 h are critical; further increase
in the reaction temperature or enhancement in the reaction
time leads to destruction of desired product 5. After this
reaction, the separation of the 1,6- and 1,7-isomers was
achieved by normal column chromatography. Subsequently,
the pyrrolidinyl groups were introduced into 5 by nucleo-
philic aromatic substitutions in pyrrolidine,[21,23] and per-
ylene bisimide 6 was obtained in 82% yield. Finally, DNA
building block 1 could be prepared in 74% yield only by a
modified phosphoramidite procedure using 20 equiv. of
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite and
10.5 equiv. of triethylamine. An analytical sample of APBI
dye 7 without any protecting group was prepared by treat-
ment of 6 with dichloroacetic acid.
Synthesis and Optical Characterization of the APBI
Modification in Single-Stranded DNA
Perylene bisimide derivative 1 represents the DNA build-
ing block for the APBI modification and was used for auto-
mated oligonucleotide synthesis on the 1 μmole scale. To
avoid precipitation of the phosphoramidite during the
DNA synthesis, APBI phosphoramidite 1 was dissolved in
CH2Cl2, and the coupling methodology was changed to
CH2Cl2/acetonitrile mixtures. Single-stranded DNA1
(Scheme 2) bears a single APBI base surrogate and was pre-
pared to characterize the optical properties of this modifi-
cation in an oligonucleotide, in comparison with the dye
without DNA. Tritylated perylene bisimide derivative 6 was
used for this measurement due to its good solubility. The
UV/Vis absorption and fluorescence spectra of single-
Electrochemical Characterization
In order to verify our proposal that the amino-modified stranded DNA1 or DNA2 in aqueous buffer with 6 in
perylene bisimide chromophore of 7 is potentially unable to DMSO are very similar (Supporting Information, Fig-
photoinduce guanine oxidation, we estimated the excited- ures S3–S6). Most importantly, the fluorescence intensity is
state reduction potential and compared it with the oxi- almost the same if the optical density of both samples at
dation potential of guanine according to Rehm–Weller. Due the excitation wavelength (650 nm) is identical. The latter
Scheme 2. Sequences of the modified DNA double strands (DNA1Y and DNA2) synthesized in this study; (X = APBI, Y = A, G, T,
C).
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Synthesis of DNA with Green Perylene Bisimides as DNA Base Substitutions
observation supports strongly the idea that guanine oxi- modified single-stranded DNA1 at pH 7 is approximately
dation as the major fluorescence quenching mechanism of 2%. This is comparable to the quantum yields of unsubsti-
conventional perylene bisimide is prevented because the tuted perylene bisimide in DNA (1–2%),[19] but only in the
APBI modification in DNA1 is embedded in a G environ- absence of adjacent guanines. In the latter case, quantitative
ment. This was expected based on the electrochemical data fluorescence quenching is observed with unsubstituted per-
as described above and is supported by our corresponding ylene bisimide.
experiments with the DNA building block without the
amino substituents.[9,17,18] In the latter case, the fluores-
cence intensity is strongly quenched in comparison to that
of the building block alone.
APBI as a Monomeric and Dimeric DNA Base Substitution
in Double-Stranded DNA
For optical characterization it is important to point out
that at neutral pH it is not certain whether the two amino
substituents are completely protonated or deprotonated.
Based on literature, the pKa of pyrrolidines can be some-
where between 3.6 (N-phenyl pyrrolidine) and 10.5 (N-al-
kylated pyrrolidines). Hence, we measured the UV/Vis ab-
sorption and fluorescence spectra of single-strand DNA1
not only at pH 7 but additionally at pH 3 and at pH 13
(Figure 1). The UV/Vis absorption spectra show remark-
able similarity. On the other hand, the fluorescence inten-
sity drops from pH 3 to pH 13. These changes do not reflect
simply the different extinction coefficients. Concomitantly,
the emission maxima are shifted from 767 (at pH 3) to
787 nm (at pH 13). The latter observation indicates a more
pronounced charge-transfer character of the APBI excited
state at a higher pH value of the buffer. If we assume that
at pH 13 both amino groups are not protonated, they repre-
sent strongly electron-donating groups. Together with the
electron-poor core of the perylene bisimide chromophore,
an intramolecular exciplex is formed in the excited state that
is characterized by a broad and redshifted emission band
without any perylene bisimide-typical fine structure. At
pH 3, the N-pyrrolidinyl substituents are protonated and
thus turn into electron-withdrawing groups. The result is a
less-pronounced redshift, but more intense fluorescence.
The fluorescence of DNA1 at pH 7 shows contributions
Single-stranded DNA1 was hybridized with four dif-
ferent unmodified counterstrands to corresponding du-
plexes DNA1Y (Y = A, G, T, or C) in order to explore any
potential preference for the DNA base opposite to the
APBI base surrogate. The measured Tm values at pH 7 can
be found in a small range between 63.8 and 65.8 °C
(Table 1). The latter value is that of DNA1A, indicating a
slight preference of the APBI modification for adenine as
“counterbase”. Representatively, the Tm value of the un-
modified duplex corresponding to DNA1C (meaning APBI
is replaced by G) is 70.7 °C.[36] The observed difference of
–5.7 °C is typical for chromophore modifications based on
the (S)-1-aminopropane-2,3-diol linkage.[32]
Table 1. Melting temperatures (Tm) and UV/Vis absorption ratios
of APBI-modified single-stranded DNA1 and double-stranded
DNA1Y and DNA2.
Tm [°C]
A704/A766
DNA1
DNA1A
DNA1C
DNA1G
DNA1T
DNA2
–
0.6
0.4
0.5
65.8
65.0
63.8
63.7
78.0
0.6
0.5
1.5[a]
[a] A687/A734
.
Based on our experience with perylene bisimide[17,18] and
from both types of exciplex emissions, indicating a partially thiazole orange[37] as dimeric DNA base substitutions, we
protonated/deprotonated state of the APBI chromophore. explored also the interstrand-type aggregation of the APBI
The experimentally determined quantum yield of APBI- base surrogate inside DNA. We prepared DNA2, which
carries a diagonal arrangement of two adjacent APBI chro-
mophores, and characterized this artificial “base pair” by
optical spectroscopy. The base sequence complementarily
places the two APBI chromophores in close proximity. The
UV/Vis spectra of DNA2 at pH 7 (Figure 2) exhibit re-
markable differences compared to the single modified refer-
ences, for example, DNA1A. The absorption of the single
APBI chromophore exhibits an absorption band at 766 nm
and a side band at ca. 704 nm, which represent the typical
0Ǟ1 and 0Ǟ0 vibronic transitions of this dye. The two
major absorption bands of the APBI dimer in DNA2 that
occur at 687 and ca. 734 nm are not only blueshifted by 20–
30 nm but show remarkably changed intensities. The ab-
sorption ratio A687/A734 of DNA2 is 1.5, whereas the ratios
A704/A766 of the single-modified DNA duplexes lie in the
range 0.4–0.6. These altered ratios are indicative of a strong
Figure 1. UV/Vis absorption spectra (top) and fluorescence spectra
excitonically coupled APBI chromophore pair.[28] The fluo-
(bottom) of single-stranded DNA1 at pH 3, 7, and 13 (inset shows
rescence of the APBI dimer in DNA2 is quenched quantita-
normalized fluorescence); all measurements were performed with
2.5 μm DNA in 10 mm Na-Pi-buffer, 250 mm NaCl.
tively. Together with the hypsochromic shift of the absorp-
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F. Menacher, H.-A. Wagenknecht
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tion (from 766 to 687 nm), these results indicate the forma- toolbox of available chromophores and dyes as functional
tion of an H-type aggregate between two adjacent APBI building blocks for DNA. Hence, these results could be
chromophores. H-aggregates are typically nonemissive. An- promising to develop new bioanalytical tools based on
other indication for ground state-chromophore interactions APBI or this chromophore could be used in nanostructur-
is provided by the melting temperature of DNA2, which is ing DNA aggregates that are pH sensitive.
78.0 °C. Compared with the Tm value of 74.5 °C for the
corresponding completely unmodified duplex (G instead of
APBI), the APBI dimer yields a strong stabilizing effect to
Experimental Section
the hybrid. This is a remarkable result, as single DNA
Materials and Methods: All chemicals and solvents were purchased
modifications that carry a glycol-type linker system instead
from Alfa Aesar, Fluka, Merck, Riedel-de Haen, and Sigma Ald-
of the natural 2Ј-deoxyribofuranoside between the phos-
rich and were used without further purification; the unmodified
oligonucleotides were purchased from Metabion. All reactions were
phodiesters are typically strongly destabilizing.[32,33,36] In
case of larger chromophores, like perylene bisimide, some
destabilization can be regained by hydrophobic interactions
with the DNA base stack. Moreover, in the case of DNA2
a clear stabilization of 3.5 °C is observed, which can be at-
tributed to the interaction of the two APBI chromophores
inside this duplex. Hence, the APBI dimer can be regarded
as a hydrophobically interacting base pair like our pre-
viously published perylene bisimide and thiazole orange
pairs[17,18,37] and the biphenyl, phenanthrenyl, and bi-
naphthyl base pairs recently published by other
groups.[38–40]
accomplished under an inert atmosphere. The NMR spectra and
the molecular weight were recorded by the Center for Chemical
Analysis of the faculty. 1H, 13C, and 31P NMR spectra were re-
corded with a Bruker Avance 600-Kryo with CD2Cl2 as deuterated
solvent; CI-MS were recorded with a Finnigan MAT SSQ 710 A,
ESI-MS were recorded with a ThermoQuest Finnigan TSQ 7000,
and EI-MS were recorded with a Finnigan MAT SSQ 710 A. TLC
was performed on Fluka silica gel 60 F254 coated aluminum foil.
Column chromatography was carried out with silica gel 60 from
Aldrich (60–43 μm). Spectroscopic measurements were recorded in
Na-Pi buffer solution (10 mm) using quartz glass cuvettes (10 mm).
Absorption spectra and the melting temperatures (2.5 μm DNA,
250 mm NaCl, 10–90 °C, 0.7 °C/min, step width 1 °C) were re-
corded with a Varian Cary 100 spectrometer equipped with a 6ϫ6
cell changer unit. Fluorescence was measured with a Jobin–Yvon
Fluoromax 3 fluorimeter with a step width of 1 nm and an integra-
tion time of 0.2 s. All spectra were recorded with an excitation and
emission bandpass of 8 nm and are corrected for Raman emission
from the buffer solution.
Preparation of DNA: Oligonucleotides were prepared with an Expe-
dite 8909 Synthesizer from Applied Biosystems (ABI) using stan-
dard phosphoramidite chemistry. For APBI phosphoramidite 1 the
coupling time was enhanced from 96 to 1517 s. To avoid precipi-
tation during the DNA synthesis, 1 was dissolved in CH2Cl2. Rea-
gents and CPG (1 μmol) were purchased from ABI and Glen Re-
search. After preparation, the trityl-off oligonucleotide was cleaved
off the resin and deprotected by treatment with conc. NH4OH at
room temperature for 24 h. The oligonucleotide was dried and puri-
fied by HPLC on a RP-C18 Column using the following condi-
tions: A = NH4OAc buffer (50 mm; pH 6.5); B = acetonitrile, flow
rate 2.5 mL/min, UV detection at 260 nm (DNA), at 435 and
704 nm (APBI). The oligonucleotides were lyophilized and quanti-
fied by their absorbance at 435 nm with a Varian Cary 100 spec-
trometer.
Figure 2. UV/Vis absorption spectra (top) and fluorescence spectra
(bottom) of double-stranded DNA1Y (Y = A, G, C, or T) and
DNA2; all measurements were performed with 2.5 μm DNA in
10 mm Na-Pi-buffer, pH 7.0, 250 mm NaCl.
Conclusions
1,7-Dibromoperylene-3,4:9:10-Tetracarboxylic Acid Dianhydride (3):
Perylene-3,4:9,10-tetracarboxylic
acid
dianhydride
(5.00 g,
A redox-tuned and therefore green perylene bisimide de-
rivative (APBI) was synthesized and developed as a DNA
modification based on phosphoramidite building block
strategy. Two N-pyrrolidinyl substituents of APBI as
strongly electron-donating substituents at the bay area pre-
vent fluorescence quenching by photoinduced charge trans-
fer to guanines. Single APBI DNA base substitutions in
oligonucleotides show an exciplex-type emission with pH
sensitivity. The interstrand APBI dimer stabilizes the DNA
duplex and thus could be regarded as a hydrophobically
interacting base pair. Optically, the APBI dimer behaves
like an H aggregate, and accordingly, the fluorescence is
12.7 mmol, 1 equiv.) was suspended in conc. sulfuric acid (150 mL)
and stirred 1 h at room temperature. Iodine (0.26 g, 1.0 mmol,
0.08 equiv.) was added, and the mixture was warmed up to 85 °C
over 45 min. Finally, bromine (3.92 mL, 12.2 g, 76.5 mmol,
6 equiv.) was added, and the mixture was stirred overnight at 95 °C.
After cooling, the intensive red raw product was precipitated by the
addition of water. The residue was washed with water until the
wash solution had a neutral pH value. A rust-colored product
(6.91 g, 12.6 mmol, 99%) was isolated as a mixture of the 1,7- and
1,6-isomers. MS (CI): m/z (%) = 550.3 (100) [M–]. MS (EI): m/z
(%) = 549.9 (100) [M+].
N-(2-Ethylhexyl)-NЈ-[3-O-trityl-2-(S)-hydroxypropyl]-1,7-dibromo-
quenched quantitatively at neutral pH. APBI extends the perylene-3,4:9,10-tetracarboxylic Acid Dianhydride (5): Compound
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Synthesis of DNA with Green Perylene Bisimides as DNA Base Substitutions
3 (1.00 g, 1.8 mmol, 1 equiv.) and zinc acetate dihydrate (200 mg,
0.9 mmol, 0.5 equiv.) were suspended in pyridine (200 mL) and
warmed up to 75 °C. After reaching this temperature, a mixture of
racemic 2-ethylhexylamine (0.30 mL, 236 mg, 1.8 mmol, 1 equiv.)
and 4[41] (1.44 g, 3.7 mmol, 2 equiv.) in pyridine (30 mL) was added
dropwise over 3 h, and the mixture was stirred for another 12 h.
The solvent was evaporated, and the dark red residue was purified
twice by column chromatography (1st: CH2Cl2/acetone, 20:1; 2nd:
hexane/acetone, 3:1). A dark red solid was yielded (480 mg,
0.5 mmol, 25%). Rf (CH2Cl2/acetone, 15:1) ≈ 0.63; Rf (hexane/ace-
tone, 3:1) ≈ 0.27. HRMS (EI): m/z = 1034.1746 [M–]. For 1H NMR
and 13C NMR data, see the Supporting Information.
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Financial support by the Deutsche Forschungsgemeinschaft (Wa
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Eur. J. Org. Chem. 2011, 4564–4570
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Received: April 13, 2011
Published Online: June 28, 2011
4570
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 4564–4570