Fluorescent 1,2,3-triazole derivative of 3′-deoxy-3-azidothymidine: synthesis and absorption/emission spectra

3′-Deoxy-3-azidothymidine (AZT, zidovudine) is a nucleoside-analog reverse transcriptase inhibitor [1]. Nucleoside analogs are well known as potent antiviral agents used against HIV [1], HSV [2] or HCV [3]. Zidovudine was the first efficient anti-HIV medication, initially used as a ‘stand-alone’ drug and later, as an ingredient of highly-active anti-retroviral therapy (HAART) compositions, together with lamivudine and abacavir. Apart from the usage in HIV therapy, AZT has also found potential application in the treatment of advanced colon cancer [4, 5] and breast cancer [6].

The azide group found in the structure of AZT makes it a ready-to-use click chemistry [7] substrate, able to undergo the copper-catalyzed Huisgen cycloaddition with terminal alkynes to easily yield 1,4disubstituted 1,2,3-triazoles. This reaction has found numerous applications in pharmaceutical sciences and chemical biology, including fluorescence labelling or bioconjugation [8, 9]. Examples of cycloaddition using AZT are known in the literature. The Wang group has developed 1,2,3-triazole derivatives of AZT effective against HIV [10], which upon silylation of the 5′ hydroxyl group became efficient inhibitors of Dengue and West Nile viruses [11]. In 2007 Lee and coworkers developed an efficient AZT ¹⁸F labeling technique for PET studies [12]. The first fluorescent 1,2,3-triazole derivatives of zidovudine were obtained by the Celewicz group in 2011 [13]. This initial study used propargyl derivatives of Cinhona alkaloids as the fluorescent alkyne counterpart and investigated also the cytostatic potency of the triazole products. In 2011, a study by the Park group revealed another fluorescent derivative of AZT, prepared using a custom-made fluorescent probe, and its application for ratiometric analysis of zidovudine incorporation by polymerases and reverse transcriptases [14]. Nevertheless, the efficient synthesis of fluorescent AZT derivatives still remains an open field with numerous potential applications in chemical biology and pharmaceutical sciences.

Herein, we report the synthesis of a 1,2,3-triazole derivative of AZT containing a fluorescent fragment and its UV-VIS absorption and emission spectra. The synthesis of a novel alkyne-containing fluorescent probe, 9H-fluoren-9-ylmethyl prop-2-yn-1-yl carbonate (propargyl-fmoc) and its copper-catalyzed cycloaddition reactions with two model azides, trans-2-azidocyclohexanol, and benzyl azide, are also described. For these reactions, a ligand-assisted protocol, using 2-{4-[(dimethylamino)methyl]-1,2,3-triazol-1-yl}cyclohexan-1-ol (AMTC) as a copper-stabilizing ligand, developed in our research group, was applied [15] and additional solvent composition studies were performed to develop an efficient cycloaddition procedure for propargyl-fmoc (1) and zidovudine. 9H-Fluoren-9-ylmethyl prop-2-yn-1-yl carbonate (propargyl-fmoc, 1) was efficiently prepared from fmoc-chloride and propargyl alcohol (Scheme 1). The detailed synthetic procedure is given in the experimental section.

Scheme 1

Synthesis of propargyl-fmoc (1) and its conjugates with 2-azidocyclohexanol (2), benzyl azide (3) and AZT (4).

To provide initial information on the reactivity of 1 in the copper-catalyzed azide-alkyne cycloaddition, the reactions with two model azides, namely benzyl azide and trans-2-azidocyclohexanol were performed (Scheme 1). A ligand-assisted cycloaddition protocol, developed in our laboratory [15], with a high copper loading (10 mol%) and long reaction time to ensure high conversion was used. The general procedure is given below. The triazoles 2 and 3 were prepared with 66% and 46% yields, respectively.

In spite of satisfactory yields obtained for 2 and 3, the low solubility of 1 in ethanol and water was an inconvenience that pointed towards problems in developing the cycloaddition procedures with AZT. The initial cycloaddition reaction between AZT and 1 performed in the 2:1 water:ethanol system with 10 mol% of copper and 20 mol% of AMTC proceeded with a low yield of 21%. Therefore, it was decided to test several other solvent systems, to find the optimal reaction conditions overcoming the solubility problem. We used water, 2:1 water-isopropanol, 2:1 water-tert-butanol and dichloromethane as solvents. The amounts of copper, ascorbate and AMTC were not changed. The products were isolated by extraction to dichloromethane and chromatographic purification was not necessary. The isolated products were analyzed by HPLC-MS to determine the purity and the presence of the starting material 1; the results are given in Table 1.

The on-water reaction was inefficient – the isolated product contained mostly 1 (78%) and only a small amount (6.5%) of 4 was formed, which is also consistent with the low yield obtained in the water-ethanol system. By contrast, the reactions conducted in the remaining solvents proceeded efficiently, with almost quantitative yield and high purity of the product. The best result was obtained using dichloromethane. The reaction conducted in tert-butanol furnished the product in high yield, but the HPLC purity was slightly lower and the purified product contained a small amount of residual solvent. The reaction in the isopropanol-water mixture was also efficient in terms of isolated yield, but the purity of the product was significantly lower (82%) and small amounts of catalytic ligand and AZT could be detected by HPLC-MS. It is important to note that in all three cases, the conversion of 1 was complete (only trace amounts could be detected by HPLC-MS). A detailed procedure for the synthesis of 4 in dichloromethane is given in the experimental section.

The UV-VIS absorption and emission spectra were recorded for acetonitrile solutions of compounds 1-4 (Figure 1). The absorption spectra display three important bands around 208, 266.5 and 300 nm. The maxima vary only slightly (see Table 2), whereas the absorption intensities for compounds 2 and 3 are greater than the values for 1 and 4. In the case of fluorescence spectra, recorded at 266 nm excitation wavelength, the relative quantum yields (Table 2) obtained for the triazoles 2-4 are significantly lower than that for 1. The emission maxima vary only slightly between the derivatives and are within the range of 2 nm.

Figure 1

UV-VIS absorption (left) and fluorescence (right) spectra measured for 10 μm solutions of 1-4 in acetonitrile.

The studies presented herein provide an efficient protocol for the synthesis of a novel fluorescent 1,2,3triazole derivative 4 of 3′-deoxy-3-azidothymidine as well as its UV-VIS and fluorescence spectra. Water and a water-ethanol mixture proved inefficient for the reaction, whereas almost quantitative yields were obtained using 2:1 water-isopropanol and water-tert-butanol mixtures or dichloromethane. Simple and efficient preparation of fluoren-9-ylmethyl prop-2-yn-1-yl carbonate (propargyl-fmoc, 1) is also presented, together with its UV-VIS absorption and fluorescence spectra and examples of copper-catalyzed azide-alkyne cycloaddition reactions.