Synthesis and Properties of 2'-deoxyuridine Analogues Bearing Various Azobenzene Derivatives at the C5 Position

Nucleic acids that change their properties upon photo-irradiation could be powerful materials for molecular sensing with high spatiotemporal resolution. Recently, we reported a photo-isomeric nucleoside bearing azobenzene at the C5 position of 2'-deoxyuridine (dU Az), whose hybridization ability could be reversibly controlled by the appropriate wavelength of light. In this paper, we synthesized and evaluated dU Az analogues that have various para-substitutions on the azobenzene moiety. Spectroscopic measurements and HPLC analyses revealed that the para-substitutions of the azobenzene moiety strongly affect the photo-isomerization ability and thermal stability of the cis-form. The results suggest that proper substitution of the azobenzene moiety can improve the properties of dU Az as a light-responsive nucleic acid probe.


Introduction
Much attention has been focused on chemically modified nucleic acids that can alter their properties by some external stimuli [1][2][3].Photo-responsive nucleic acids have been realized by regulation of hydrogen bonding [4] and stacking effects [5] between nucleobases.Light is considered to be a promising external stimulus due to the possibility of accurately controlling the location, dosage, and time of the irradiation.Various photochromic derivatives have been attached to nucleic acids to photo-control their properties [6].For example, diarylethene-modified oligonucleotides (ONs) showed changes in their optical properties [7,8] and hybridization abilities [8] by photo-isomerization.However, the changes in hybridization abilities were moderate and the diarylethene modification itself destabilized the DNA duplex in both forms.To obtain a significant melting temperature difference (Tm) upon photo-isomerization, ONs containing cis-trans type photochromic derivatives have been reported.Owing to their dynamic structural changes upon photo-irradiation, cis-trans type photochromic derivatives could greatly influenced the stability of nucleic acid duplexes.Although stilbene-type derivative-modified ONs have been utilized for photo-control of duplex formation, G-quadruplex formation, and gene expression, they require short wavelength photo-irradiation for cis to trans isomerization [9][10][11].This would be disadvantageous for biomolecules due to photo-damaging reactions including the formation of pyrimidine dimers [12].Azobenzene derivative-modified ONs are quite promising photochromic ONs because they can rapidly photo-isomerize by longer wavelengths of light [13,14].Asanuma et al. have demonstrated that ONs modified with azobenzene moieties could be utilized as photo-responsive tweezers, RNA scission, and engines [15].Recently, we reported that the ON containing C5-azobenzene-substituted 2'-deoxyuridine (dU Az ) could be photo-isomerized from trans to cis with an efficiency of 60% by UV light (365 nm) and from cis to trans with an efficiency of 80% by visible light (450 nm) [16].The ON containing dU Az showed interesting hybridization properties, namely, the Tm values of the duplexes formed between dU Az -modified ON and complementary DNA or RNA were higher after UV irradiation than before irradiation.This may be because the hydrophobic azobenzene in the trans form extends to the outside of the groove and interferes with hydration and the formation of interstrand cation bridges to stabilize the duplexes.Meanwhile, cis-dU Az did not affect the duplex stability due to the compact conformation of the azobenzene moiety.These results indicated that dU Az could be a potential building block to control nucleic acid hybridization with high spatiotemporal resolution.
In this study, we describe the synthesis and properties of dU Az analogues bearing various para-substituted azobenzene derivatives (Figure 1).The isomerization properties of photochromic compounds have been proposed to be strongly influenced by substituents [17].Thus, the introduction of electron-donating or -withdrawing substituents into the azobenzene moiety could improve the properties of dU Az and create unique photo-sensors for various biomolecules.

General
Reagents and solvents were purchased from commercial suppliers and were used without purification unless otherwise specified.All experiments involving air-and/or moisture-sensitive compounds were carried out under N2 or Ar atmosphere.All reactions were monitored with analytical TLC (Merck Kieselgel 60 F254; Merck, Darmstadt, Germany).Column chromatography was carried out using FL-100D silica gel (Fuji Silysia, Aichi, Japan).Physical data were measured as follows.NMR spectra were recorded on JNM-ECS-300, JNM-ECS-400, and JNM-ECS-500 spectrometers (JEOL, Tokyo, Japan) using CDCl3 or DMSO-d6 as solvents with tetramethylsilane as an internal standard.IR spectra were recorded on a FT/IR-4200 spectrophotometer (JASCO, Tokyo, Japan).Optical rotations were recorded on a JASCO P-2200 instrument.FAB mass spectra were measured on a JEOL JMS-700 mass spectrometer.Solid-phase ON synthesis was performed on an nS-8 Oligonucleotide Synthesizer (GeneDesign, Osaka, Japan).MALDI-TOF mass spectra were recorded on an ultrafleXtreme mass spectrometer (Bruker Daltonics, Billerica, MA, USA) for oligonucleotides and on a JMS-S3000 (JEOL, Tokyo, Japan) for small molecules.Photo-irradiation experiments were conducted with a Xenon lamp (MAX-303; Asahi Spectra, Tokyo, Japan) using HQBP 450-VIS ø 25 and 365-VIS ø 25 as the optical filters.UV/Vis absorption measurements and UV melting experiments were performed using a UV-1650PC UV-Vis spectrophotometer equipped with a TMSPC-8 Tm analysis accessory (Shimadzu, Kyoto, Japan).ITC experiments were performed using a Microcal iTC200 (Malvern Instruments, Worcestershire, UK).

Synthesis of dU Az -Modified Oligodeoxynucleotides
Solid-phase oligonucleotide synthesis was performed using commercially available reagents and phosphoramidites with activator 42 (Sigma-Aldrich) as the activator.para-Substituted-dU Az phosphoramidites were chemically synthesized as described above.All of the reagents were assembled, and the ONs were synthesized according to the standard synthesis cycle (trityl on mode).Cleavage from the solid support and deprotection were accomplished with concentrated ammonium hydroxide solution at 55 °C for 12 h.The crude oligonucleotides were purified with Sep-Pak Plus C18 cartridges (Waters, MA, USA) followed by RP-HPLC on a XBridge TM OST C18 column, 2.5 μm, 10 × 50 mm (Waters) using MeCN in 0.1 M triethylammonium acetate buffer (pH 7.0).The purified oligonucleotides were quantified by UV absorbance at 260 nm and confirmed by MALDI-TOF mass spectrometry (Table 1).

UV Melting Experiments
Equimolecular amounts of the target DNA/RNA and ONs were dissolved in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl to give a final strand concentration of 4.0 µM.The melting samples were denatured at 100 °C and annealed slowly to room temperature.Absorbance was recorded in the forward and reverse directions at temperatures of 5-90 °C at a rate of 0.5 °C/min.

Photo-Isomerization of dU Az Analogues
ONs were dissolved in a 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl to give a final strand concentration of 4.0 µM.The ON solution was exposed to the appropriate wavelength of monochromic light for 10 seconds and subsequently analyzed by RP-HPLC.The ratio of cis/trans isomers was obtained from the HPLC peak areas at 260 nm.

Thermal Isomerization of cis-dU Az Analogues
ONs were dissolved in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl to give a final strand concentration of 4.0 µM.The oligonucleotide solution was exposed to the appropriate wavelength of monochromic light for 10 seconds and subsequently heated to 60 °C.The change of absorbance at 365 nm or 400 nm was monitored by a UV-Vis spectrophotometer and plotted to calculate the half-life time of cis-dU Az analogues.

Isothermal Titration Calorimetry
Prior to experiments, all solutions (titrant and titrand) were denatured at 100 °C and annealed slowly to room temperature.Titrations were carried out by injecting 2 µL portions of the complementary RNA strand into 250 µL of the dU Az -modified ON solution in the calorimetric cell.The dU Az -modified ON concentrations in the cell were 0.1 µM, and the solutions in the syringe were 1 µM.The equilibration period after each injection was 360 s.The measurements were performed with and without the photo-isomerization of dU Az -modified ONs.

Isomerization Properties of Para-Substituted dU Az Analogues
The influence of para-substitution on the efficiency of the dU Az cis-trans photo-isomerization in ONs was investigated by UV spectroscopy and HPLC analysis.The photo-isomer ratio was measured at 260 nm because ONs 27-32 have the same extinction coefficient before and after irradiation at this wavelength (Figure S1).The wavelengths of light used for photo-isomerization and their efficiencies are shown in Table 3.Ten seconds of irradiation was confirmed to be enough for reaching photostationary state (PSS); the cis/trans ratios of ONs 27-32 after longer irradiation times (30 and 120 min) were the same as that after 10 s of irradiation.Compared with ON 27 containing dU Az , ONs 28 and 29 containing electron-donating substituted dU Az were photo-isomerized from trans to cis more effectively.Irradiation of ON 28 (X = dU Az −OMe) using 365 nm afforded a mixture with 79% of the cis-form.In contrast, ON 30 bearing the electron-withdrawing CF3 group showed a lower trans to cis isomerization efficiency.However, the cis to trans isomerization efficiency was higher than that of ON 27 containing dU Az .ONs 31 and 32 containing dU Az -NO 2 and dU Az -pyridyl were barely photo-isomerized.The low efficiency of photo-isomerization was due to the low electron density at the azo-structure [23].In case of dU Az -pyridyl, electrons tend to be delocalized at the nitrogen of the pyridine ring [24].ONs 28-31 were photo-isomerized trans to cis and vice versa for at least three cycles without any attenuation of efficiency.The wavelength of monochromic light corresponds to π-π * transition (trans to cis) or n-π * (cis to trans).
Photo-irradiation was performed for 10 s at room temperature; d The ratio of isomers is calculated from the peak area of HPLC traces (260 nm).
Next, the thermo-stability of ONs with various para-substituted dU Az in the cis-form was investigated [25].The ONs modified with dU Az analogues were cis-isomerized by the appropriate wavelength of light and subsequently heated to 60 °C.The change in absorbance at 365 nm was monitored by a UV-Vis spectrophotometer and plotted to calculate the half-life of the cis-isomers (Table 4).With regard to ONs 28 and 29 containing electron-donating substituted dU Az , thermo-stabilities of the cis-isomer were significantly decreased.This phenomenon could be attributed to the para-electron-donating substituent and ethynyl linker creating a push-pull configuration of the azobenzene moiety, which is well known in easily thermo-isomerized azobenzene derivatives [18].On the other hand, ONs 30 and 31 containing electron-withdrawing substituted dU Az showed long half-life times.The cis-isomer of ON 31 (X = dU Az −NO 2 ) scarcely isomerized from cis to trans even when heated to 60 °C for more than 10 h.In the case of ONs 30 and 31, both the ethynyl linker and para-substitution work as 4,4'-di-electron-withdrawing moieties.The 4,4'-di-electron-withdrawing substitution of azobenzene strengthens the thermal stability of the cis-form [26].The para-substitution would sterically and electronically affect the stability of the duplexes formed by dU Az analogue-modified ONs with complementary strands.Therefore, we investigated the Tm values of the duplexes formed between ON 28-32 and full match DNA or RNA (Tables 5 and 6).It was found that para-substitution of dU Az slightly destabilized the DNA/DNA duplexes but had little influence on the DNA/RNA duplexes.Modulating the rate of the thermal relaxation of the switched state is also very important because it realizes the spatiotemporal control of nucleic acid properties [27].The thermal stability of the cis-isomer influences the method of utilization of photo-switches.cis-ON 28 (X = dU Az -OMe) showed the shortest thermal half-life among the dU Az analogue-modified ONs that we evaluated.This property is crucial for a photo-switch that thermally relaxes the hybridization ability.This would enable repeated activation with spatiotemporal resolution.On the other hand, ONs 30 and 31 (X = dU Az -CF 3 and dU Az -NO 2 ) have a long half-life of the cis-form.In this case, it is possible to turn the hybridization property of dU Az on and off using the appropriate wavelength of light.
The para substitutions decreased the affinity of ONs modified with dU Az analogues against DNA but had little influence on that against RNA.These results suggest that para substitutions influence the duplex stability not electronically but sterically.The difference in the stability between DNA/DNA duplexes and DNA/RNA duplexes is attributed to the difference in the duplex structures.DNA/DNA duplexes tend to have B-form structures whereas DNA/RNA duplexes tend to have more A-form structures.Thus, the DNA/DNA duplex has a comparatively shallow major groove and para-substitution of the azobenzene moiety may sterically destabilize the duplex.The destabilization of duplexes in the trans-form may lead to an increase in the difference of hybridization ability between trans-and cis-dU Az and improve the property of dU Az for photo-switching hybridization.
We introduced various para substitutions into dU Az and modulated the photo-and thermo-sensitivities for trans-cis isomerization.Our results represent an important strategy for optimization of the properties of photo-switches.For example, dU Az -OMe showed better trans to cis isomerization efficiency and lower thermal stability of the cis-isomer than dU Az .This analogue could be utilized as a useful photo-switch for situations in which a fast drop in activity is desired.dU Az -modified ON shows an interesting hybridization property, namely the affinity of the cis-form against complementary strands is higher than that of the trans-form, different from most other photo-switches [16].Para-substituted dU Az analogues have the potential for unique nucleic acid probes and drugs that reversibly capture the target DNA and RNA with spatiotemporal control.

Conclusions
We synthesized photo-isomeric dU Az analogues bearing various para-substituted azobenzene derivatives and investigated the influence of the para substitution on the photo-isomerization properties, thermal stabilities of the cis-isomer, and hybridization abilities.Electron-withdrawing substituents on dU Az lowered the efficiency of photo-isomerization while improving the thermal stability of the cis-isomer.On the other hand, electron-donating substituents improved the efficiency of photo-isomerization while decreasing the thermal stability of the cis-isomer.According to UV-melting experiments, para-substitution of dU Az tended to destabilize DNA/DNA duplexes but had little effect on the stabilities of the DNA/RNA duplexes regardless of the nature of the substituents.ITC experiments revealed that the duplex formed between dU Az -OMe-modified ON and complementary RNA are more stable after photo-irradiation than that before photo-irradiation.This trend was observed in the duplex formed between dU Az -Me-modified ON and complementary RNA.These results indicated that dU Az -OMe and dU Az -Me could regulate the stability of the DNA/RNA duplex by photo-irradiation similar to dU Az and appropriate substitution could optimize the properties of dU Az as light-responsive nucleic acid probes.

Figure 1 .
Figure 1.Para-substituted dU Az analogues used in this study.

Table 1 .
Yields and MALDI-TOF MS data of dU Az -modified ONs.

Table 2 .
The oligonucleotides used in this study.

Table 3 .
Photo-isomerization properties of para-substituted dU Az a .

Table 4 .
Half-life time of the cis-form of para-substituted-dU Az at 60 °C a .

Table 5 .
Tm values of duplexes formed by dU Az analogue-modified ONs with complementary DNA a .Conditions: 10 mM Na 2 HPO 4 (pH 7.0), 100 mM NaCl, 4 μM ON.The T m values given are the average of at least three data points.The T m value of natural DNA 26/DNA 33 duplex is 52 °C; b The ΔT m indicates the difference from ON 27/DNA 33 duplex.