1‐, 2‐, and 4‐Ethynylpyrenes in the Structure of Twisted Intercalating Nucleic Acids: Structure, Thermal Stability, and Fluorescence Relationship

Abstract

A postsynthetic, on-column Sonogashira reaction was applied on DNA molecules modified by 2- or 4-iodophenylmethylglycerol in the middle of the sequence, to give the corresponding ortho- and para-twisted intercalating nucleic acids (TINA) with 1-, 2-, and 4-ethynylpyrene residues. The convenient synthesis of 2- and 4-ethynylpyrenes started from the hydrogenolysis of pyrene that has had the sulfur removed and separation of 4,5,9,10-tetrahydropyrene and 1,2,3,6,7,8-hexahydropyrene, which were later converted to the final compounds by successive Friedel-Crafts acetylation, aromatization by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and a Vilsmeier-Haack-Arnold transformation followed by a Bodendorf fragmentation. Significant alterations in thermal stability of parallel triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in homopyrimidine TINAs. Thus, for para-TINAs the bulge insertion of an intercalator led to high thermal stability of Hoogsteen-type parallel triplexes and duplexes, whereas Watson-Crick-type duplexes were destabilized. In the case of ortho-TINA, both Hoogsteen and Watson-Crick-type complexes were stabilized. Alterations in the thermal stability were highly influenced by the ethynylpyrene isomers used. This also led to TINAs with different changes in fluorescence spectra depending on the secondary structures formed. Stokes shift of approximately 100 nm was detected for pyren-2-ylethynylphenyl derivatives, whereas values for 1- and 4-ethynylpyrenylphenyl conjugates were 10 and 40 nm, respectively. In contrast with para-TINAs, insertion of two ortho-TINAs opposite each other in the duplex as a pseudo-pair resulted in formation of an excimer band at 505 nm for both 1- and 4-ethynylpyrene analogues, which was also accompanied with higher thermal stability.

Triplex‐Forming Twisted Intercalating Nucleic Acids (TINAs): Design Rules, Stabilization of Antiparallel DNA Triplexes and Inhibition of G‐Quartet‐Dependent Self‐Association

“…In the emission spectra of phenylethynylpyren-1-yl moiety two characteristic bands at 400 nm and 425 nm are detected (Figure 4). [23,30] Due to TINA-TINA interactions, a strong exciteddimer (excimer) band appears at 495 nm for ONs 4 and 9 in native conditions. In contrast, the intensity of the excimer band is significantly lower for ONs 3 and 8, which form fewer high-order structures on the native gel.…”

“…The remaining GA TINA-TFOs were able to bind to the DNA duplex in an antiparallel fashion even in the presence of K + . [26,27] We [21][22][23][28][29][30] and others [31] have undertaken several studies on the ability of TINA moiety to stabilize pH-dependent parallel CT-triplexes, while very little is known about the effects of TINA on antiparallel GA or GT-containing TFOs. Herein we synthesized several triplex-forming 16-mer GT oligonucleotides with 2-4 TINA insertions to target the polypurine tract of HIV-1 proviral DNA [32] in different orientations.…”

Rapid genotyping using pyrene−perylene locked nucleic acid complexes

“…Moreover bright fluorescence of the complexes containing monomers M 2 /M 3 -M 4 contradicts intercalation typically leading to quenched PAH fluorescence due to interaction with nucleobases. 32 The quantum yields and, hence, fluorescence brightness values FB of double-labeled probes ON5-ON8 were very high (Φ f 0.72-1.00 and FB up to 52.0), however without sensitivity of the fluorescence to single-base mismatches (Fig. S1).…”

“…First, owing to lower thermodynamic stability short fluorescent oligonucleotides generally display higher sensitivity of hybridization toward mismatched DNA/RNA targets than their longer analogs. [29][30][31][32][33] Second, flexibility of the terminally attached PAHs substitution in the pyrene core from 1 to 4, since this modification has already showed promising results in labeling of nucleic acids (monomer M 2 , Fig. 1).…”

“…1). [31][32][33][34] We demonstrate that this modification significantly improved photophysical and spectral characteristics of the pyrene with a strong effect on its interactions with the commonly used PAH acceptor FRET perylene (monomers M 3 and M 4 , Fig. 1).…”