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have shown similar results with no AGTA recognition.[6c] Addi-
tionally, as evident with TTGA at the 5’ end when A is replaced
by T, cooperative binding of 1 is present, but decreased. These
results suggest that because the base pair composition is very
well maintained (GC and AT content), it is the stacking of the
base pairs AA·TT versus AT·AT that influences changes in minor
groove microstructure and affects the affinity and binding
mode of 1. Further investigations are necessary to identify
minor groove microstructures for sequences with similar struc-
tures to ATGA.
In the spectra shown, peaks of the systems correspond well
to their expected molecular weights (i.e., m/z) for free DNA
and DNA–ligand complexes. The ionization process of ESI-MS
results in multiply charged species and for the raw data, every
system shows multiple, charge states (Supporting Information,
Figure S4). Due to the nature of the analyte and negative
mode analysis, the most abundant charge states range be-
tween ꢀ3 and ꢀ6. These lower net charges indicate the DNA
backbone becomes partially neutralized during the electro-
spray process during which ammonium ions transfer a proton
to the phosphate backbone and the ammonia ions evaporate.
The amount of neutralization occurred depends on the size of
the DNA, concentration of ammonium ions, and instrument
parameters used.[8a,19] Positively charged dicationic diamidines
help in neutralizing the backbone, however, the presence of
ligand does not affect the overall charge after forming a com-
plex. For instance, peaks remain the most abundant in ꢀ4 and
ꢀ5 charge states for both free AAATTT and AAATTT+(1) 1 com-
plexes. The spectral peaks are transformed by deconvolution—
the ability to transform multiple charge peaks into the single
peak, zero charge molecular ion species. Deconvolution greatly
simplifies the spectra for optimum visualization and is ach-
ieved by multiplying the charge of the species by its respective
m/z.
Based on the ESI-MS studies of 1 with several DNAs, we can
now see that it binds as a highly cooperative dimer to ATGA-
like sequences but as a monomer to A-tract sequences. Based
on the structural similarity of benzimidazole and indole
groups, we expected the indole analogue of 1, 6, to bind as
a similar cooperative dimer. With a few exceptions, however,
dimerization among minor groove binders containing an
indole system is rare.[11b,14] Most indole-containing minor
groove binders recognize AT sequences strictly as a monomer.
For instance, DAPI, the most thoroughly studied indole-con-
taining compound, binds AT sequences as a monomer only.[12]
More interestingly, however, is the higher affinity of 6 over
1 for ATGA which is unexpected since the curvature and con-
formation of the benzimidazole and indole systems are essen-
tially the same (Figure 6). Biosensor SPR studies (data not pub-
lished) have shown that 6 binds as a strong dimer to ATGA
with a higher affinity over 1 which is in agreement with the
thermal melting studies, however, the results from ESI-MS are
not completely consistent. In the mass spectra, the 6–ATGA rel-
ative peak abundances are not as high as one would anticipate
based on the results with 1. At this time, it is not completely
understood why the 6–DNA peaks, which includes 6 with
ATGA and both AT sequences, are less than expected. This is
especially surprising since there has been excellent correlation
between ESI-MS and thermal melting with 1 and the other an-
alogues. One possible explanation may be technique related in
which the compound interacts with the injection tubing so
that the total concentration of 6 in the sample solution de-
creases below the expected amount. A lower concentration of
6 would then result in less 6 complex formed and lower abun-
dances of 6–DNA complexes detected.
To examine competition for DNA sites by 1 and analogues
using ESI-MS, proper care must be taken to ensure that the
molecular weights of the small molecules and their complexes,
and all possible stoichiometries, are distinguishable. On the
other hand, another approach is to examine the binding of
a single compound with an array of target sequences and their
mutations. Different DNA sequences can be examined simulta-
neously in this way as long as the molecular weights of the
DNAs and complexes are distinguishable. A combination of an
ATGA cognate sequence, ATGA-mutant sequences, and a refer-
ence DNA (R2) were screened with 1. To obtain different mo-
lecular weights for the variants, such as ATGA and AGTA which
have the same stem molecular weights, the hairpin loops of
the DNAs were altered with different numbers of thymidine
and cytidine or by incorporation of a deoxyuridine so that the
flanking base pairs were preserved.
Lower DNA concentrations such as 2.5 mm have been tested
and not surprisingly, there is little difference in the peak inten-
sities when comparing 2.5 mm of DNA versus 5 mm of DNA. The
level of cooperativity is still observed, and is in agreement with
earlier reports from our group demonstrating the cooperative
binding of 1 to ATGA by ESI-MS using 5 mm of DNA.[10] For our
systems, there is a general preference for using 5 mm of DNA
since it results in a larger signal for the DNA and/or complexes
over using 2.5 mm. A spectrum using 2.5 mm of DNA with com-
pound 1 can be found in the Supporting Information (Fig-
ure S5). Due to the nature of compounds 1–8 and other dicat-
ionic diamidines, an unknown amount of ligand is often lost
during the injection process. At times, the ligand will presuma-
bly become stuck and remain fixed to the inside of the injec-
tion tubing, therefore reducing the total ligand concentration.
This phenomenon has been experienced on multiple occasions
and requires thorough cleanings of the instrument between
different samples. Samples containing DNA only (no com-
pound) are routinely injected before beginning any new analy-
sis to check for and remove residual ligand through binding of
free DNA. Results can be successfully quantified using ESI-MS,
as long as the specific response sensitivity and the concentra-
tions are accurately known. It is possible to determine an equi-
librium binding constant for DNA and small molecule systems
and there are examples in the literature demonstrating
this.[18,20] The ability to determine binding constants for dicat-
ionic diamidines is primarily limited to the loss of ligand
during injection and response factors for the DNA and com-
plexes, and these limitations influence our preference to use
ESI-MS for qualitative purposes only.
Other methods can also be used, with or without ESI-MS, to
efficiently screen for DNA-binding compounds. For instance,
thermal melting studies are commonly used to screen for bind-
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Chem. Eur. J. 2015, 21, 1 – 13
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ÝÝ These are not the final page numbers!