4-amino-1H-benzo[g]quinazoline-2-one: a fluorescent analog of cytosine to probe protonation sites in triplex forming oligonucleotides

Nucleic Acids Research, Aug 2000

We developed a new fluorescent analog of cytosine, the 4-amino-1H-benzo[g]quinazoline-2-one, which constitute a probe sensitive to pH. The 2′-O-Me ribonucleoside derivative of this heterocycle was synthesized and exhibited a fluorescence emission centered at 456 nm, characterized by four major excitation maxima (250, 300, 320 and 370 nm) and a fluorescence quantum yield of Φ = 0.62 at pH 7.1. The fluorescence emission maximum shifted from 456 to 492 nm when pH was decreased from 7.1 to 2.1. The pKa (4) was close to that of cytosine (4.17). When introduced in triplex forming oligonucleotides this new nucleoside can be used to reveal the protonation state of triplets in triple-stranded structures. Complex formation was detected by a significant quenching of fluorescence emission (∼88%) and the N-3 protonation of the quinazoline ring by a shift of the emission maximum from 485 to 465 nm. Using this probe we unambiguously showed that triplex formation of the pyrimidine motif does not require the protonation of all 4-amino-2-one pyrimidine rings.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://nar.oxfordjournals.org/content/28/15/2977.full.pdf

4-amino-1H-benzo[g]quinazoline-2-one: a fluorescent analog of cytosine to probe protonation sites in triplex forming oligonucleotides

Frdric Godde 0 Jean-Jacques Toulm 0 Serge Moreau 0 0 INSERM U-386, IFR Pathologies Infectieuses, 146 rue Lo Saignat, Universit Victor Segalen , 33076 Bordeaux We developed a new fluorescent analog of cytosine, the 4-amino-1H-benzo[g]quinazoline-2-one, which constitute a probe sensitive to pH. The 2-O-Me ribonucleoside derivative of this heterocycle was synthesized and exhibited a fluorescence emission centered at 456 nm, characterized by four major excitation maxima (250, 300, 320 and 370 nm) and a fluorescence quantum yield of = 0.62 at pH 7.1. The fluorescence emission maximum shifted from 456 to 492 nm when pH was decreased from 7.1 to 2.1. The pKa (4) was close to that of cytosine (4.17). When introduced in triplex forming oligonucleotides this new nucleoside can be used to reveal the protonation state of triplets in triple-stranded structures. Complex formation was detected by a significant quenching of fluorescence emission (88%) and the N-3 protonation of the quinazoline ring by a shift of the emission maximum from 485 to 465 nm. Using this probe we unambiguously showed that triplex formation of the pyrimidine motif does not require the protonation of all 4-amino-2-one pyrimidine rings. - Triple helix formation can be used to target double-stranded DNA through the binding of a short oligonucleotide to homopurinehomopyrimidine sequences via Hoogsteen (or reverse Hoogsteen) hydrogen bonding (1,2). A triple helical approach can also be used to target a single-stranded DNA or RNA sequence using an oligonucleotide able to form both Watson Crick and Hoogsteen hydrogen bonds (3,4). Thus in triple helices of the pyrimidine motif a third pyrimidine strand interacts with the WatsonCrick double strand forming CG*C+ and TA*T base triplets (in this notation the third strand is written in the last position and * indicates Hoogsteen type interaction). However, the requirement for the protonation of cytosine residues in the third strand results in a pH-dependent stability of PyPu*Py triplexes with an optimum stability at non-physiological pH 5.06.0 (5). In recent studies on triplexes, we developed a new fluorescent analog of thymine which enabled us to probe triple helix formation, the benzo[g]quinazoline-2,4-(1H,3H)-dione 1 (Fig. 1) (6,7). Taking into account the luminescent properties of this heterocycle, we surmised that 2 (4-amino-1H-benzo[g]quinazoline-2-one), an analog of cytosine could exhibit similar properties and that N-3 protonation would induce variations of these fluorescence capacities. In this communication we show that 2 constitutes a fluorescent probe sensitive to pH, able to detect complex formation. It can be used to reveal easily the protonation state of triplets in triple-stranded structures. The benzo[g]quinazoline heterocycles 1 and 2 were introduced on a 2-O-methylribose backbone. We report the complete synthetic routes for the 2-O-Me-ribonucleosides of 1 and 2 (1N and 2N), as well as the corresponding phosphoramidites allowing the synthesis of modified oligonucleotides. MATERIALS AND METHODS Thin layer chromatography (TLC) was performed on Merck silica gel 60F254 aluminium-backed plates; visualization was done by UV illumination and staining with 10% perchloric acid solution. Flash chromatography refers to column chromatography performed with Merck silica gel 60 (0.040.063 mm). The NMR spectra were recorded on a Brker AC 200 spectrometer working at 200 MHz for 1H, 50.32 MHz for 13C and 81.02 MHz for 31P. The ROESY, HMQC and HMBC experiments were performed on a Brker AMX 500 spectrometer. The chemical shifts are expressed in p.p.m. using TMS as an internal standard (1H and 13C data) and 85% H3PO4 as an external standard (31P data). The IR data and UV spectra were recorded on a Brker UFS-25 spectrofluorimeter and on a Kontron Uvikon 940 spectrophotometer, respectively. All chemical reagents were obtained from Aldrich (St Quentin Fallavier, France) except ammonium sulfate (Fluka, St Quentin Fallavier, France) and sodium Fontenay Sous Bois, France). 1-(2,3,5-tri-O-benzoyl--D-ribofuranosyl)-benzo[g]quinazoline-2,4-(3H)-dione (4). A mixture of benzo[g]quinazoline2,4-dione 1 (7) (20.6 g, 97 mmol), a few crystals each of ammonium sulfate and acetamide and a few drops of trimethylsilylchloride were refluxed in 500 ml hexamethyldisilazane (HMDS) for 3 days with exclusion of moisture. Excess HMDS was removed in vacuo by co-evaporation with toluene. The residue was dissolved in a dry mixture of acetonitrile/toluene (200/50 ml) then 1-O-acetyl-2,3,5-tri-O-benzoyl--D-ribofuranosyl (49 g, 97 mmol) was added. The mixture was cooled to 0C and then 1.3 equivalents of trimethylsilyl triflate (24.4 ml) were added (30 min). The reaction mixture was then stirred at room temperature (RT) for 4 h. The solution was poured on a saturated aqueous NaHCO3 solution and the product extracted into toluene (3 100 ml). The organic phase was washed with saturated aqueous NaCl and then dried over anhydrous MgSO4. The (...truncated)


This is a preview of a remote PDF: https://nar.oxfordjournals.org/content/28/15/2977.full.pdf

Frédéric Godde, Jean-Jacques Toulmé, Serge Moreau. 4-amino-1H-benzo[g]quinazoline-2-one: a fluorescent analog of cytosine to probe protonation sites in triplex forming oligonucleotides, Nucleic Acids Research, 2000, pp. 2977-2985, 28/15, DOI: 10.1093/nar/28.15.2977