4-amino-1H-benzo[g]quinazoline-2-one: a fluorescent analog of cytosine to probe protonation sites in triplex forming oligonucleotides
Frdric Godde
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Jean-Jacques Toulm
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Serge Moreau
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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.
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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)