Synthesis and Fluorescence Quenching Study of the Novel Cationic Probe Derived from Luminarosine

Abstract

Dedicated to Professor Andre¬ M. Braun on the occasion of his 60th birthday A simple and efficient transformation of the zwitterionic luminarosine into a brightly fluorescent cationic analogue, namely 1-amino-9-methoxy-2,4,10-triaza-4b-azoniaphenanthrene (3), is reported. The fluorescence quenching of 3 by common nucleotides, calf-thymus (CT) DNA, and halide ions was investigated by means of spectrophotometric and spectrofluorometric methods. Intermolecular static and dynamic fluorescencequenching constants for quenching of 3 by nucleotides and halide ions were determined in aqueous solution. Evidence for formation of nonfluorescent ground-state complexes of 3 with nucleotides and CT-DNA is presented. Scatchard analysis of the CT-DNA quenching data resulted in a binding constant of 2.8 Â 10 4 m À1 and a number of binding sites per base pair of 0.049., which has excitation and emission maxima at 430 and 530 nm in aqueous solution, belongs to a rather limited number of modified nucleosides that absorb and emit light in the visible region. The attractive photophysical properties of this compound [2], as well as its chemical stability under oligonucleotide-synthesis conditions, make it an excellent candidate for a fluorescent probe of nucleic acids and other biological systems. Sequence-specific introduction of this fluorophore into oligodeoxynucleotides has already been achieved [3]. Recent studies on fluorescence quenching of the parent de-O-acetylated nucleoside (luminarosine) and the aglycone (luminarine) by I À ions in living cells revealed the great potential of the former as a long-wavelength iodidesensitive fluorescent indicator for measurements of the cystic fibrosis transmembraneconductance regulator (CFTR) gene expression in cells [4]. The high intracellular fluorescence of the luminarosine, combined with its red-shifted emission and I À selectivity, make it superior to quinolinium-based halide indicators and indicates its possible application in the high-throughput screening of compounds for correction of the cystic fibrosis phenotype. Encouraged by these findings, we have undertaken the synthesis and detailed photophysical studies of some analogues of the luminarosine modified within the chromophoric part in the hope of obtaining fluorophores of different and possibly improved photophysical properties. In this communication, we present the simple and efficient transformation of the zwitterionic luminarosine into another very bright fluorophore, namely its cationic analog, 1-amino-9-methoxy-2,4,10-triaza-4b-azoniaphenanthrene (3). The spectral properties as well as the results of a fluorescence-quenching study of 3 by common nucleotides and halide ions is discussed.

Interaction of 9-Methoxyluminarine with Different G-Quadruplex Topologies: Fluorescence and Circular Dichroism Studies

“…The calculated K b is a few times higher for c- MYC in comparison with other G4s and the lowest binding constant was obtained between 9-MeLM and double stranded DNA. The K b for 9-MeLM/ds26 (2.4 × 10 4 M −1 ) is in good agreement with the K b value (2.8 × 10 4 M −1 ) estimated previously for 9-MeLM/CT-DNA using a Scatchard plot [ 53 ]. Additionally, the Job plot, based on the changes in the emission at 495 nm, was constructed for 9-MeLM/c- MYC ( Figure 3 B).…”

“…9-Methoxyluminarine 9-MeLM is a cationic derivative of highly fluorescent nucleoside 7-(b’-D-ribofuranosylamino)-pyrido [2,1-h]pteridin-ll-ium-5-olate, termed luminarosine, bearing tricyclic betaine as an aglycone [ 51 , 52 , 53 ]. 9-MeLM also exhibits bright and stable fluorescence in the visible region as well as high quantum yield ( Table 1 ).…”

“…Among the halide ions, only I − and, to a lesser extent, Br − ions quench the fluorescence of 9-metoxyluminarine. So far, it has been shown that 9-MeLM showed moderate binding to calf thymus DNA with K b value 2.8 × 10 4 M −1 in 0–3.6 mM in the bp concentration range [ 53 ]. Taking into account the structural and photophysical properties of 9-methoxyluminarine, we assumed that 9-MeLM may indicate higher binding affinity to the G-quadruplexes than to double-stranded DNA.…”

“…Undeniably, the solubility, fluorescence, quantum yield, luminescence lifetime and photostability of 9-MeLM are also important factors that encouraged us to undertake investigations towards verifying its ability to serve as a selective G4s ligand. 9-Methoxyluminarine 9-MeLM is a cationic derivative of highly fluorescent nucleoside 7-(b'-D-ribofuranosylamino)-pyrido [2,1-h]pteridin-ll-ium-5-olate, termed luminarosine, bearing tricyclic betaine as an aglycone [51][52][53]. 9-MeLM also exhibits bright and stable fluorescence in the visible region as well as high quantum yield (Table 1).…”

“…Table 1. Absorption and emission parameters for aqueous solution of 9-methoxyluminarine [53]. The human telomeric sequence d[AGGG(TTAGGG) 3 ] (22HT) has been found to form different types of G-quadruplex structures.…”