Dimeric Calixarenes: A New Family of Major‐Groove Binders

Abstract

A new class of potent DNA binding agents is presented. Dimeric calix[4]arenes with cationic groups at their upper rims and flexible alkyl bridges can be synthesized from triply acyl-protected calix[4]arene tetramines in relatively short synthetic sequences (3-5 steps). The compounds attach themselves to double-stranded nucleic acids in a noncovalent fashion, with micro- to nanomolar affinities. Guanidinium headgroups with their extended hydrogen-bonding "fingers" are more powerful than ammonium groups, and the benzylamine series is superior to the anilinium series (see below). The new ligands easily distinguish between RNA and various DNA types, and produce characteristic changes in UV/Vis, fluorescence, CD, as well as NMR spectra. Especially extended oligonucleotides of more than 100 base pairs are bound with affinities increasing from RNA (10 μM K(d))<AT-rich (1 μM)<GC-rich DNA double strands (100-10 nM). Ethidium bromide displacement studies confirm this order. CE(50) values are remarkably low (1-4 μM), and are more than 300 times lower than that of spermine, which is a typical backbone binder. Stoichiometries are rather high (one calixarene dimer per two BP), suggesting a potential aggregation of bound ligands inside the major groove. Most UV/Vis melting curves display an inverted shape, and start from drastically enhanced absorption intensities for the DNA complexes. DAPI displacement studies prove that up to one equivalent of calixarene dimer can be accommodated in the dye-loaded DNA. RNA complexation by calixarene dimers is accompanied by a drastic CD spectral transition from the typical A-form to a perfect B-signature, providing further experimental evidence for major-groove binding. The orientation of the ligands can be deduced from NMR titrations and is reproduced in Monte-Carlo simulations on 1:1 complexes in water.

Moulding calixarenes for biomacromolecule targeting

“…Gal-3, on the contrary, is a monomeric lectin that tends to self-assemble in a pentameric ensemble. 63,64 Quaternary ammonium, 39,61 N-methylimidazolium, 65 primary ammonium,and guanidinium groups43,66,67 have been since then studied in details. A peculiar binding selectivity was also observed, since different lactocalixarenes show rather different IC 50 values respect to the same galectin and, vice versa, each multivalent ligand has different IC 50 values towards the three different galectins.…”

Biological applications of functionalized calixarenes

“…With the hexaaniline calixarene dimer 98, they introduced a large amphiphilic molecule that selectively binds ds DNA and RNA with submicromolar K D values in buffered aqueous solution; contrary to most other DNA binders, it most likely targets exclusively the major groove and causes no conformational change or destabilization in the DNA duplex. [160][161][162][163] …”

Arginine clustering on calix[4]arene macrocycles for improved cell penetration and DNA delivery

“…1b) could be exploited to enhance their cell-penetrating properties, and we report here the first example of this strategy, applied to DNA delivery and cell transfection. Considering that cyclodextrins18 and calixarenes19202122232425 are non-toxic and have been used as scaffolds for building gene delivery systems, we selected calix[4]arene as the macrocyclic platform and anchored to it single arginine units rather than complex arginine-rich peptides, or linear and long oligoarginines, to limit to a ratio of one to one between amino acids and the lipophilic tails (Fig. 1b).…”