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common secondary structures of ds-DNA and RNA by sensitive
and biologically applicable methods (e.g., UV/Vis, CD, fluores-
cence, surface-enhanced Raman spectroscopy).[12] The develop-
ment of such single-molecule sensors with several properties
sterical hindrance of the PBIs. Thus, a structure-property rela-
tionship could be established.
could replace the necessity of multiple dyes each being specif- Results and Discussion
ic for one particular DNA or RNA target. The signal selectivity
Synthesis
could be achieved by the fine tuning of the interaction of
chromophores with secondary structures of polynucleotides
which include the introduction of sterically demanding or
binding modulating substituents attached to the dye.[12] In this
regard, one of the most effective approaches seems to be the
variation of selectivity-controlling substituents, which are close-
ly and rigidly attached to the chromophore.
A series of symmetric, homochiral perylene bisimides (denoted
as (N)-l- and (N)-d-Ala-PBI, (N)-l- and (N)-d-Phe-PBI, (O)-l-
and (O)-d-Ala-PBI and (O)-l- and (O)-d-Phe-PBI) that are ap-
pended with a-amino acid derivatives as imide substituents,
bearing spermine or its dioxa analogue as side chains, were
synthesized from commercially available perylene-3,4:9,10-tet-
racarboxylic bisanhydride (PBA) in three steps according to the
route outlined in Scheme 1. The reaction of PBA with homochi-
ral (l- or d-enantiomer) alanine or phenylalanine in imidazole
afforded the corresponding amino acid functionalized symmet-
rical PBIs l-[18] or d-Ala-PBI and l- or d-Phe-PBI in excellent
yields. The successive amidation of the PBI-appended dicarbox-
ylic acids with threefold Boc-protected spermine Boc-(N)[19]
using the activation reagents dicyclohexylcarbodimide (DCC)
and hydroxylbenzotriazole (HOBt) in DMF led to the spermine-
bearing PBI derivatives Boc-(N)-l- or Boc-(N)-d-Ala-PBI and
Boc-(N)-l- or Boc-(N)-d-Phe-PBI in 29 to 72% yield. Deprotec-
tion of the Boc-groups with trifluoroacetic acid (TFA) afforded
the desired products (N)-l- or (N)-d-Ala-PBI and (N)-l- or (N)-
d-Phe-PBI as trifluoroacetic salts in quantitative yields, which
were then lyophilized from water to obtain them in pure form.
For the amidation of the dicarboxylic acids with Boc-protected
4,9-dioxa-1,12-dodecanediamine Boc-(O),[20] however, the acti-
vation reagents N,N-diisopropylethylamine (DIPEA) and O-(7-
azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluroniumhexafluoro-
phosphate (HATU), instead of DCC and HOBt were required.
The deprotection of Boc-groups in the resulted Boc-(O)-l- or
Boc-(O)-d-Ala-PBI and Boc-(O)-l- or Boc-(O)-d-Phe-PBI with
TFA afforded the products (O)-l- or (O)-d-Ala-PBI and (O)-l- or
(O)-d-Phe-PBI in 84 to 97% yield, which were subsequently
purified by lyophilization from water.
During the last decade, self-assembly of a huge variety of
PBI dyes have been investigated and their aggregates have
found various applications as (opto)electronic materials.[13] As
PBI dyes possess a hydrophobic extended p-core, a particular
challenge being the exploration of the aggregation properties
of PBI dyes in water which is, however, required for the appli-
cation of these dyes in biological systems.[14] Beside other ap-
proaches, including ionic self-assembly,[14e,d] the attachment of
polyamine chains at the imide positions of PBIs provides the
necessary solubility of core-planar PBIs in water which make
them interesting for the investigation of interactions with bio-
macromolecules such as DNA.[15] Recently, we have communi-
cated the first examples of spermine-functionalized homochiral
PBIs with unique properties such as strong thermal stabiliza-
tion and high binding affinity towards ds-DNA and RNA.[16]
Here we present our comprehensive studies on the interac-
tion of PBI dyes with a broad variety of polynucleotides by ap-
plying a broad new series of homochiral PBIs (N)-l- and (N)-d-
Phe-PBI, (O)-l- and (O)-d-Ala-PBI and (O)-l- and (O)-d-Phe-
PBI. These PBIs are appended with amino acids possessing
substituents of varied sterical demand at the chirality centers
and incorporate spermine or 4,9-dioxa-1,12-dodecanediamine
side chains at the imide positions (Scheme 1). For the purpose
of comparison, we have included in this study the achiral refer-
ence compound (N)-Gly-PBI[15b] and the previously reported
compounds (N)-l- and (N)-d-Ala-PBI.[16] We have investigated
the spectroscopic response of the PBIs for structurally different
double-stranded DNA and RNA under biologically relevant
conditions. For these studies, we have chosen synthetic poly-
nucleotides instead of short oligonucleotides because the
latter are not suitable for our investigations due to the “cap-
ping” binding of PBIs at the exterior side of alternating base-
pairs which would strongly compete with the few binding
sites along the double strands of oligonucleotides.[8,17] In con-
trast, polynucleotides consisting of more than 100 base pairs
will assure large excess of binding sites along the double helix,
thus the “capping” effect should be negligible. Our detailed
thermal denaturation experiments, fluorimetric titrations and
CD spectroscopic measurements corroborate strong interac-
tions of the homochiral ionic PBIs with the employed polynuc-
leotides and the formation of excitonically coupled dimer ag-
gregates in the grooves of the ds-DNA/RNA. The binding prop-
erties of these PBIs with ds-DNA/RNA are found to be depen-
dent on the number of the positive charges, chirality and
The alanine-functionalized compounds (N)-l- and (N)-d-Ala-
PBI have previously been reported in our communication.[16]
The reference achiral compound (N)-Gly-PBI was prepared ac-
cording to the literature.[15b] All the new compounds were
characterized by 1H NMR spectroscopy, high-resolution mass
spectrometry and, where possible, also by elemental analysis.
The details of the synthesis and characterization data of the
new compounds are given in the Supporting Information.
UV/Vis spectroscopic studies of homochiral PBIs
To ascertain the appropriate conditions for the investigation of
interactions of these homochiral PBIs with double-stranded
(ds)-polynucleotides by different spectroscopic methods such
as UV/Vis, fluorescence and circular dichroism (CD) spectrosco-
py, we have initially performed concentration-dependant UV/
Vis studies of these PBIs in water and DMSO (Figure 1 and S1
in Supporting Information). As a representative example of the
spermine-containing PBIs (denoted as (N)-series), the concen-
Chem. Eur. J. 2015, 21, 7886 – 7895
7887
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