Building‐Block Approach for the Straightforward Incorporation of a New FRET (Fluorescence‐Resonance‐Energy Transfer) System into Synthetic DNA

Abstract

The synthesis of the two new phosphoramidites 5 and 8 bearing a carbostyril (¼ quinolin-2(1H)-one) chromophore used as donor entity in our recently developed new FRET (fluorescence-resonance-energy transfer) system is described (Schemes 1 and 2) The high stability of the chromophore to basic conditions enables the incorporation of the phosphoramidites directly into DNA during solid-phase synthesis (Schemes 3 and 4). Since this is also possible for the (bathophenanthroline)ruthenium(II) complex used as acceptor (Scheme 4, Steps d and e), the whole labelling procedure to insert the FRET system into synthetic DNA is straightforward and represents a major improvement to our previous strategy.Introduction. -Fluorescently labelled nucleic acids are particularly important tools in molecular biology, diagnostics, and structural studies [1] [2]. In this context, DNA with incorporated dyes representing fluorescence-resonance-energy transfer (FRET) systems have proven to be important tools for the elucidation of distance-dependent interactions on the molecular level and in real time mode. Any changes in donoracceptor distance will affect the rate of energy transfer which can be measured either by steady-state or time-resolved fluorescence studies. The latter approach requires labels with a long lifetime of their fluorescence, and this is usually achieved via the use of lanthanide chelates as acceptor in FRET systems [3] [4]. As alternative labels, we have developed, some time ago, (bathophenanthroline)ruthenium(II) complexes (bathophenanthroline ¼ 4,7-diphenyl-1,10-phenanthroline) which show, after excitation of their metal-to-ligand charge transfer, a fluorescence lifetime in the low msec range [5]. In combination with a quinolinone derivative as donor, this yielded a highly efficient and robust new FRET system, of which the feasibility was first demonstrated in peptides in the course of the development of a homogeneous assay for the serine protease thrombin [6] [7]. Later, we were also able to demonstrate the efficiency of the system in DNA [8]. In this study, both constituents, the (bathophenanthroline)ruthenium(II) complex as well as the carbostyril (¼ quinolin-2(1H)-one) chromophore were attached to aminomodified DNA after deprotection following solid-phase assembly of the oligonucleotide chain. The described method was time-consuming. Hence, we sought for possibilities to improve this labelling procedure.

An Efficient and Facile Methodology for Bromination of Pyrimidine and Purine Nucleosides with Sodium Monobromoisocyanurate (SMBI)

“…of SMBI for 1 h followed by addition of 0.5 equiv of the reagent. Reaction was stopped after stirring for another 1 h when compound 24 [10] was obtained in 93% yield. Similarly, 2'- O -Methyladenosine ( 25 ) was treated with SMBI (1.25 equiv.…”

“…Halonucleosides have been used extensively as starting material for these reactions. 8-Bromopurine nucleosides have been used as key intermediates for synthesizing fluorescently labeled nucleosides, which have promising applications in the field of molecular biology [9,10]. In view of this relevance, development of efficient methodology for the synthesis of 5-bromopyrimidine and 8-bromopurine nucleosides is always of immense interest.…”

“…Bromination at C-5 of pyrimidine moieties was achieved by reaction of Br 2 /water [15], Br 2 /DMF [16], Br 2 /CCl 4 [17], Br 2 /CCl 4 in solvent mixture of anhydrous acetic acid and pyridine [18], N -bromosuccinimide (NBS) in DMF [19], NBS/NaN 3 in DME [20], NBS in ionic liquids [21], m -chloroperbenzoic acid (MCPBA)/HBr in DMA or DMF [22], ceric ammonium nitrate (CAN)/LiBr in AcOH or MeCN [23], KBr/potassium monoperoxysulfate under aqueous conditions [24]. Bromination of C-8 of purine moieties has been attained by reaction of Br 2 in glacial AcOH/NaOAc [25], NBS in DMF [19], Br 2 in NaOAc buffer/dioxane [10] and Br 2 in NaOAc buffer [26,27]. A thorough study of the literature procedures for bromination of nucleosides shows that most of these methods have some disadvantages, such as highly acidic reaction condition, handling of toxic reagents, longer reaction time, complicated work up procedure and low yield of the products.…”

Electronic Relaxation Dynamics in 2-Quinolinones with Extended Conjugation

“…Quinolinones have been proposed as potential drugs for treatment of cancers, − in addition to other medical applications. − They have also been explored as high-energy antennae for fluorescence resonance energy transfer (FRET) and luminescence resonance energy transfer (LRET) measurements, − and as anchoring groups in dye-sensitized solar cells . Additionally, the photochemical synthesis of quinolinone derivatives has received significant attention. , The correlation of structure and function is critical in all of these applications, and outside of medical applications the behavior electronic excited states is of particular interest.…”

Novel Three-Color FRET Tool Box for Advanced Protein and DNA Analysis

“…Recently, we have reported on two different alternative FRET systems of which the first one was based on a carbostyril donor and a Ru(II)-bathophenanthroline complex as acceptor . We were able to establish the FRET system in peptides as well as in oligonucleotides and it was used for a new assay format for the transcription factor NF-κB. − The second system consisted of the Ru-complex as donor in combination with an anthraquinone as acceptor . During the insertion into peptides and DNA-fragments, the spectroscopic properties of the three different chromophores used in these systems remained largely unaffected by the labeling to peptides or oligonucleotides, and both systems turned out to be very robust, efficient, and sensitive.…”