Resolution of Mixed Site DNA Complexes with Dimer‐Forming Minor‐Groove Binders by Using Electrospray Ionization Mass Spectrometry: Compound Structure and DNA Sequence Effects

Abstract

Small molecule targeting of the DNA minor groove is a promising approach to modulate genomic processes necessary for normal cellular function. For instance, dicationic diamindines, a well-known class of minor groove binding compounds, have been shown to inhibit interactions of transcription factors binding to genomic DNA. The applications of these compounds could be significantly expanded if we understand sequence-specific recognition of DNA better and could use the information to design more sequence-specific compounds. Aside from polyamides, minor groove binders typically recognize DNA at A-tract or alternating AT base pair sites. Targeting sites with GC base pairs, referred to here as mixed base pair sequences, is much more difficult than those rich in AT base pairs. Compound 1 is the first dicationic diamidine reported to recognize a mixed base pair site. It binds in the minor groove of ATGA sequences as a dimer with positive cooperativity. Due to the well-characterized behavior of 1 with ATGA and AT rich sequences, it provides a paradigm for understanding the elements that are key for recognition of mixed sequence sites. Electrospray ionization mass spectrometry (ESI-MS) is a powerful method to screen DNA complexes formed by analogs of 1 for specific recognition. We also report a novel approach to determine patterns of recognition by 1 for cognate ATGA and ATGA-mutant sequences. We found that functional group modifications and mutating the DNA target site significantly affect binding and stacking, respectively. Both compound conformation and DNA sequence directionality are crucial for recognition.

The Thiophene “Sigma‐Hole” as a Concept for Preorganized, Specific Recognition of G⋅C Base Pairs in the DNA Minor Groove

“…Reaction of the readily available bromoheteroaryl aldehydes 1 with 4-cyanophenylboronic acid under standard Suzuki coupling [49] conditions conveniently provided the 5-(4-cyanophenyl)-2-formyl 5-ring heterocycles 2, which could be used to prepareb otht he tri-a nd tetra-heteroaryl target molecules 4 and 6.O xidative condensation and cyclization, mediated by sodium metabisulfite, of the aldehydes 2 with 3-amino-4-(methylamino) benzonitrile gave the bis-nitriles 3 in acceptable yields. [50] These could be readily converted into the desired diamidines 4 using LiN(TMS) 2 in THF. [51] In as imilar sequence, the aldehydes 2 were allowedt or eact with 3'amino-4'-(methylamino) [1,1'-biphenyl]-4-carbonitrile, again mediated by sodium metabisulfite, to produce the bis-nitriles 5.T hese weret hen converted to the diamidines 6 as previously described.…”

“…Mass spectrometry is an excellent method for the evaluation of relative binding affinities, [26,30,40] stoichiometry,a nd the validation of experimental results obtained from other methods, such as SPR, in which macromolecule-ligand stoichiometry is obtained by fitting the signal at different concentrations. [50,58] In this case the N-MeBI-thiophene compounds are far from ideal and ac onsiderable amount of the compounds are adsorbedo nt he surface of the injector tube. Satisfactory results were obtainedw ith the DB2429 complex to evaluate stoichiometry with the AAAGTTT sequence (SupportingI nformation, Figure S4).…”

Mixed up minor groove binders: Convincing A·T specific compounds to recognize a G·C base pair

“…41,42 The use of multiple DNA sequences simultaneously mixed with a small molecule creates a competitive binding environment for comparison of DNA-ligand interactions. Since the free ligand concentration for all of the DNAs is the same, peak intensities of the free, unbound DNA and the DNA-small molecule complexes offer insight into the binding mode and preferred binding site(s).…”

Small Sequence‐Sensitive Compounds for Specific Recognition of the G⋅C Base Pair in DNA Minor Groove

“…Competition ESI‐mass spectrometry (MS) provides a direct analysis of relative binding affinity, stoichiometry, and specificity for small molecule DNA complexes . The use of some DNA sequences simultaneously mixed with a ligand creates a competitive binding environment for direct comparison of DNA interactions.…”