Synthesis of novel modified uracil for dual-purpose: Quenching and photoswitching

Hybridization of nucleic acids is considered the cornerstone for many biomedical applications as antigene and antisense. Recently, much effort has been devoted to regulate nucleic acid hybridization using external stimuli as light, pH, electric field and heat. Light is preferred over all other external stimuli due to many reasons such as efficiency, clean and does not produce any contaminants to the biological system under study.


Introduction
Hybridization of nucleic acids is considered the cornerstone for many biomedical applications as antigene and antisense. Recently, much effort has been devoted to regulate nucleic acid hybridization using external stimuli as light, pH, electric field and heat. Light is preferred over all other external stimuli due to many reasons such as efficiency, clean and does not produce any contaminants to the biological system under study.
Photoregulation of nucleic acids can be achieved via attaching a photoresponsive molecule to the single-stranded DNA. Azobenzene one of photochromic molecules that extensively has been widely used for photoregulation of nucleic acid through geometrical structural change from trans to cis isomer and reverse with response of optical excitation with light at suitable wavelength. -Irradiation with UV light led to trans-to-cis isomerization while irradiation with visible light or thermally recovers the trans isomer.
Incorporation of azobenzene to oligonucleotides was obtained via two approaches (i) as a side chain on the oligonucleotide phosphate backbone or (ii) as a linker between two oligonucelotide segments in the main chain of an oligonucleotide. In principle, introduction of several azobenzene residues to oligonucleotides led to an increase in melting temperature difference Tm corresponding to the photo-induced isomerization.
Despite the efficiency of azobenzenes in reversible photoswitching of nucleic acid-related activities, a desire to overcomer the non-nucleosidic nature of the attached azobenzene moiety by designing azo-nuclobase analogue that able to maintain the H-binding ability and thusly enhance the stabilization of duplex formation. 4-(dimethylamino)azobenzene-4'-carboxylic acid (DABCYL), universal quencher, one of azobenzene derivatives that widely used as a quencher for nucleic acid detection using fluorescent probe technique. Here we report the synthesis of novel azo-based uracil derivatives with potential dual-purpose as photo trigger and/or as a quencher that mimics the structure of the universal quencher DABCYL. The modified uracil is designed to have 4-(dimethylamino)phenylazo moiety at C5 position due to at this position there is no interference with the hydrogen bonding sites of uracil and therefore retain the base pairing ability with adenine. During the progress of this work, two examples of azobenzene-modified 2'-deoxyuridine at C5 position have been reported.

Results and discussions
We are interested in the synthesis of 4-(dimethylamino)phenylazo derivative as azobenzene analogue for several reasons: 1) 4-(dimethylamino)phenylazo moiety is able to induce a conformational changes by photoisomerization. 2) The presence of dimethylamino group which is susceptible to protonation facilitates the investigation of pH effect. 3) The structure of azo-based nucleoside is structurally mimics the universal quencher. 4-(dimethylamino)azobenzene-4`-carboxylic acid (DABCYL), therefore, may have potential quenching effect as shown in figure 1.

Figure 1: Comparison between dapcyl and the modified azo-based uracil
The synthesis of azo-based uracil , was obtained by treatment of 5-aminouacil with NaNO in the presence of HCl to afford the corresponding diazonium salt followed by coupling with dimethylaniline to give the desired product in 80% yield. The synthetic approach to synthesize the phosphoramidite for DNA synthesis purpose is depicted in scheme 1. Azo-modified uracil then reacted with 1`-chloro-3,5-di-O-p-toluoyl-1,2dideoxy--D-ribofuranose under basic condition to give the corresponding deoxyriboside followed by removal of the toluoyl protecting groups using sodium methoxide in methanol giving the azo-based 2-deoxyriboside in 79% yield. Standard methods were used to convert the unprotected nucleosides to 5`-dimethoxytrityl-protected derivative by using dimethoxytrityl chloride (DMTCl) in pyridine giving in 63% yield followed by conversion to the corresponding cyanoethyl phosphoramidite derivative in 81 % yield.
To get an access for RNA chemistry, ribonucleoside was synthesized following Vorbrüggen procedure starting with refluxing azo-modified uracil with hexamethyldisilazane (HMDS) and trimethylsilyl chloride (TMSCl) to form the corresponding silylated nucleoside followed by coupling with 1-O-acetyl-2,3,5-tribenzoyl ribofuranose in dimethylformamide (DMF) in the presence of SnCl to afford in 71 %.
Removal of benzoyl groups was carried out using ammonia in methanol to afford azobased riboside in 86 % yield.

Photoisomerization studies
Azobenzene is well known to isomerize from its mostly thermally stable planar transisomer to cis-isomer upon exposure to UV-light irradiation (300nm ~ 400nm), and the reverse process takes place corresponding to irradiation with visible light (> 400nm) or thermally (Fig. 2). This process is completely reversible under UV and visible irradiations. From this vein, the synthesized nucleosides carrying azophenyl moiety is expected to follow the same behavior that azobenzene follows. To assess the photochromic behavior of the synthesized nucleosides and by monitoring the changes that may occur at their absorption with response of photoirradiation. As shown in figure 3, the absorption spectrum of trans isomer has a major peak centered at 454 nm (in dichloromethane for ) and at 443 nm (in acetonitrile for ) attributed to strong -* transition which overlapped with the weak peak attributed to weak n-* transition. Upon irradiating with monochromic light at 365 nm, a decrease in the intensity of the absorption band was observed along with the irradiation time due to trans-to-cis isomerization, while the reverse cis-to-trans isomerization was achieved via thermal relaxation.

pH-Sensitivity
Azobenzenes especially aminoazobenzene and dimethylaminoazobenzene attracted much attention due to their ability to change colour in solution with change of the pH. It is well established that protonation of aminoazobenzene derivatives led to the formation of two tautomeric azonium and ammonium forms. As illustrated in figure 5, Azonium ion is obtained by protonation on azo-nitrogen leading to delocalized of the lone-pair electrons of the aromatic ring and as consequence a red shift in absorption maximum was occurred.

UV-Vis measurments:
All UV-Vis spectra were measured with a Varian Cary 300 Bio spectrophotometer using quartz cuvette cells of 1 mm of optical path.

Photoisomerization.
Trans-to-cis photoisomerization of nucleosides and were monitored by UV-Vis spectroscopy. A solution of the nucleosides at 1.06 x10 -M concentration ( in dichloromethane) and ( in acetonitrile) were irradiated with UV-light at room temperature using a UVGL-58 hand held lamp (6 watt, 366 nm) at A distance of approximately 5 cm. The spectral change for absorption maximum peak at 455 nm (for ) and 436 nm (for ) were monitored with the time course of irradiation. While the cis-totrans isomerization was achieved and confirmed by retrieving the maximum absorption bands at 455 nm (for ) and 436 nm (for ) thermally by leaving the samples at the dark at room temperature.

Effect of acids
The spectral change of the synthesized photochromic nucleosides at concentration 6.2x10 -M (in dichloromethane for ) and 1.5x 10 -M (in ethanol for ) were monitored upon addition of small aliquots of TFA solution (0.005 M).