Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc

SiO2 nanoparticles as platform for delivery of 30 -triazole analogues of AZT-triphosphate into cells Svetlana V. Vasilyeva a,⇑, Asya S. Levina a, Nikolai S. Li-Zhulanov a, Natalia V. Shatskaya b, Sergei I. Baiborodin b, Marina N. Repkova a, Valentina F. Zarytova a, Natalia A. Mazurkova c, Vladimir N. Silnikov a a b c

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, pr. Lavrent’eva 8, 630090 Novosibirsk, Russia Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia FBRI State Research Centre of Virology and Biotechnology ‘Vector’, Novosibirsk, Russia

a r t i c l e

i n f o

Article history: Received 13 January 2015 Revised 26 February 2015 Accepted 27 February 2015 Available online xxxx Keywords: SiO2 nanoparticles Delivery of triphosphate into cells Substrate properties 30 -Triazole derivatives of thymidine triphosphates AZT ‘Click’ chemistry

a b s t r a c t A system for delivery of analogues of AZT-triphosphates (AZT*TP) based on SiO2 nanoparticles was proposed. For this purpose, a simple and versatile method was developed for the preparation of SiO2dNTP conjugates using the ‘click’-reaction between AZTTP and premodified nanoparticles containing the alkyne groups. The substrate properties of SiO2AZT*TP were tested using Klenow fragment and HIV reverse transcriptase. The 30 -triazole derivatives of thymidine triphosphate being a part of the SiO2AZT*TP nanocomposites were shown to be incorporated into the growing DNA chain. It was shown by confocal microscopy that the proposed SiO2AZT*TP nanocomposites penetrate into cells. These nanocomposites were shown to inhibit the reproduction of POX and Herpes viruses at nontoxic concentrations. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Among numerous nucleoside antivirals, 30 -azidothymidine (AZT) was the first drug approved for the treatment and prophylaxis of HIV/AIDS.1,2 Moreover, azidothymidine is used to treat several virus-associated human cancers, including AIDS-related Kaposisarcoma, Kaposisarcoma-associated primary effusion lymphoma, Epstein–Barr-associated lymphoma, primary central nervous system lymphoma, and adult T cell leukemia.3 As was shown4 in non-viral tumors, AZT was used in phase I and II of clinical trials alone or in combination with other drugs for gastrointestinal cancers, pancreatic cancer, and other advanced malignancies with some tumor regression being reported. Unfortunately, the long term clinical use of AZT is associated with significant side effects including myopathy, cardiomyopathy, and anemia due presumably to the sensitivity of c-DNA polymerase in some cell mitochondria,5 and/or the depletion of thymidine triphosphate6 because AZT can be both a substrate and inhibitor of human thymidine kinases.

⇑ Corresponding author. Tel.: +7 (383)3635183; fax: +7 (383)3635182. E-mail address: [email protected] (S.V. Vasilyeva).

Chemically modification of the 30 -azido group may yield novel classes of nucleoside inhibitors with a distinct binding mode and toxicity profile. A convenient approach to the azido modification would be through click chemistry7 to form 1,2,3-triazoles, which can occupy a much larger binding space with three consecutive nitrogen atoms taking a profoundly different geometry. Unfortunately, it was found in numerous studies in this area that none of the resulting 30 -triazole derivatives of thymidine inhibit HIV or any other DNA or RNA viruses even at very high concentrations (100–500 lM) except for 4-aryl- and 5-aryl-substituted 30 -triazole derivatives of thymidine.8 The lack of the antiviral activity is likely due to inefficient cellular activation because 1,2,3-triazole nucleosides are poor substrates for the cytosolic human thymidine kinase 1 when compared to AZT.9 This problem could be solved by using phosphorylated forms of nucleoside analogues, that is, mono- or, better, triphosphates. In our previous work10, we proposed the system for the delivery of analogues of deoxynucleoside triphosphates (dNTP) based on SiO2 nanoparticles. The goal of this work was to design nanocomposites consisting of 30 -triazole analogues of AZT triphosphate (AZT*TP) immobilized onto SiO2 nanoparticles and to study their substrate properties in reactions catalyzed by DNA polymerases, their ability to penetrate into eukaryotic cells and inhibit DNA viruses.

http://dx.doi.org/10.1016/j.bmc.2015.02.063 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Vasilyeva, S. V.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.02.063

2

S. V. Vasilyeva et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

2. Results and discussion 2.1. Synthesis To use SiO2 nanoparticles as platform for delivery of AZTTP analogues into cells, we proposed a simple and versatile method for their covalent attachment to nanoparticles, that is, the copper (I)catalyzed azide-alkyne cycloaddition (CuAAC) reaction between AZTTP and premodified nanoparticles containing the alkyne groups (Scheme 1). Nanoparticles containing the alkyne groups (1, 2, Scheme 1A) were prepared by the treatment of SiO2–NH2 nanoparticles with pentafluorophenyl ester of 6-(2-propynyloxy)hexanioc acid (1a, Scheme 2A) and N-succinimidyl 5-(2propynyloxy)-5-oxopetanoate (2a, Scheme 2B), respectively. The evaluation of the amount of the amino groups with picric acid11 before and after the reaction showed that 85% of the amino groups were modified. Not all amino groups in SiO2–NH2 are accessible for the reaction because, probably, of steric hindrances. The capacity of the prepared alkyne modified nanoparticles for the alkyne groups was evaluated to be 0.43 lmol/mg. Approximately 8–9-fold excess of 1 or 2 over the amino groups in SiO2–NH2 was shown to be sufficient for the successful conversion of the amino groups into the alkyne groups. The ‘click’-reaction between AZTTP and proposed alkyno-ligands was verified with an example of linker 1a. The reaction product pppAZT*L1 (1b, Scheme 1C) was obtained in a yield of 88% and characterized. The attachment of AZTTP to alkyne-modified nanoparticles 1 and 2 was almost quantitative. The yields of 4, 5 (pppAZT*LSiO2, Scheme 1C) amounted to 77–75%using 4-fold excess of azidothymidine triphosphate over 1 or 2. The capacity of

the prepared nanocomposite for pppAZT (0.32–033 lmol/mg) was evaluated by measuring the optical absorption of resultant product pppAZT*LSiO2 dissolved in 0.2 M NaOH (UV spectrum in 0.2 M NaOH: kmax (e): 267 nm (6650 l mol1 cm1). To study penetration of the proposed pppAZT*LSiO2 nanocomposites into cells, we attached the fluorescein label to either the amino groups of initial SiO2 nanoparticles (6, Scheme 1C) or the amino linker at c-phosphate in NH2 pppAZT*L1SiO2 (7, Scheme 3). Thymidine triphosphate containing the fluorescein label at c-phosphate ((Flu)pppT, 8, Scheme 3) was used as a control to examine whether nucleoside triphosphate itself without nanoparticles can penetrate into cells. The synthesis of compound 6 consisted of three stages. First, SiO2–NH2 nanoparticles were partially modified by the treatment with fluorescein isothiocyanate to introduce the fluorescein residue (3a, Scheme 1B). The capacity of the prepared nanoparticles for fluorescein (80 nmol/mg) was evaluated by measuring the optical absorption of the resulting product dissolved in 0.2 NaOH M (UV spectrum in 0.2 M NaOH: kmax (e): 489 nm (70,000 l mol1 cm1)). The yield of the modification was 16.8%. The remaining amino groups were then acylated by activated ester of 6-(2-propynyloxy)hexanioc acid. The capacity of prepared nanoparticles 3 for the alkyne groups was 0.42 lmol/mg, which were evaluated as for compounds 1 and 2. The next step was the attachment of azidothymidine triphosphate. The capacity of the obtained pppAZT*L1SiO2(Flu) (6) nanocomposite for pppAZT (0.31 lmol/mg) was evaluated by measuring the absorbance of the resulting product dissolved in 0.2 M NaOH (UV spectrum in 0.2 M NaOH: kmax (e): 267 nm (6650 l mol1 cm1)). To prepare nanocomposite 7 containing the fluorescein residue at c-phosphate, we successively treated compound 4 with

Scheme 1. Synthesis of nanocomposites via the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.

Please cite this article in press as: Vasilyeva, S. V.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.02.063

S. V. Vasilyeva et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

3

Scheme 2. Synthesis of carboxyl linkers containing alkyne group.

Scheme 3. Synthesis of fluorescein-labeled thymidine triphosphate (Flu)pppT and (Flu)NHpppAZT*L1SiO2 nanocomposite.

propane-1,3-diamine10 amount of the attached evaluated by measuring NaOH (UV spectrum (70,000 l mol1 cm1)).

and fluorescein isothiocyanate. The fluorescein residue (0.12 lmol/mg) was the absorbance of 7 dissolved in 0.2 M in 0.2 M NaOH: kmax (e): 489 nm

pppAZT⁄ (7) (Fig. 1), while almost no penetration of (Flu)pppT (8) unbound to nanoparticles was observed (data not shown). The data unambiguously indicate the penetration of nucleoside triphosphate bound to the nanoparticles into cells.

2.2. Penetration of pppAZT*LSiO2 nanocomposites into cells

2.3. Substrate properties of the pppAZT*LSiO2 nanobiocomposites

The fluorescein-labelled nanocomposites (3, 6, 7) were found inside cells regardless of whether the label was attached to the amino groups of SiO2 nanoparticles (3, 6) or c-phosphate of

The substrate properties of the synthesized nanocomposites were tested on nylon DNA chips using the model primer/template system and DNA polymerase of E. coli (Klenov fragment) (Fig. 2).

Please cite this article in press as: Vasilyeva, S. V.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.02.063

4

S. V. Vasilyeva et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

Figure 2. (A) Model system for revealing substrate properties of pppAZT*LSiO2 nanocomposite on DNA chips. (B) Results of elongation of primer immobilized on DNA chips in the presence of Klenov fragment and DNA template. 1, Elongation in the presence of pppT and pppCbiot; 2, elongation in the presence of pppAZT*LSiO2 and pppCbiot; 3, repeated elongation in the presence of pppT and pppCbiot; 4, elongation in the presence of pppCbiot without pppT; 5, repeated elongation in the presence of pppT and pppCbiot. PL and biot are polylysine and biotin residues, respectively.

both pppCbiot and pppT. As expected, in the first reaction no product was observed (Fig. 2B, 4), while in the repeated reaction the product containing the biotin residue was formed (Fig. 2B, 5). The results show that the modified AZT residue being attached to nanoparticles retains its terminating property in the polymerization reactions. 2.4. Antiviral properties of pppAZT*LSiO2 nanocomposites

Figure 1. Confocal laser scanning microscope images of Vero cells after incubation with pppAZT*LSiO2(Flu), 6 (A) and (Flu)pppAZT*LSiO2, 7 (B). Laser lines were 405 (for nuclei, DAPI blue), 488 (for fluorescein-labeled nanocomposites, green), and 543 (for cell membranes, red).

The primer was immobilized on nylon strips in the form of polylysine-containing conjugates PL-oligo12, and the polymerization reaction was carried out on chips in the presence of the template, Klenov fragment, and nucleoside triphosphates: pppCbiot and either pppT or pppAZT*LSiO2 (Fig. 2 A). When pppCbiot along with pppT were used as triphosphates, the former was incorporated into the growing chain, so that the biotin residue (biot) became attached to the surface of the chip (Fig. 2B, 1) and was revealed by colorimetric method using streptavidine/alkaline phosphatase conjugate and chromogenic substrates.12 In the case of using pppAZT*LSiO2 instead of pppT, no biotine residue was detected on the chip indicating that pppCbiot was not attached to the surface (Fig. 2B, 2). The repeated polymerization on the same chip using pppCbiot and pppT also showed no attachment of the biotin residue (Fig. 2B, 3). It means that pppAZT*LSiO2 was bound to the primer during the first polymerization reaction and was able to stop the following elongation. To check whether the repeated polymerization reaction can lead to the formation of the product, the first polymerization was carried out in the presence of only pppCbiot and the reaction was then repeated using

Since azidothymidine is well known terminator of DNA polymerases, it can be used as inhibitor of DNA-containing viruses. Antiviral properties of pppAZT*LSiO2 nanocomposites were examined with an example of the inhibition of Herpes simplex and smallpox viruses in the Vero cell culture. In our preliminary experiments, we evaluated the antiviral properties of one of the synthesized nanocomposites, that is, pppAZT*L1SiO2. Cells were pretreated with this nanocomposite at the concentration of 10 lg/mL and control samples (SiO2NH2 nanoparticles, pppAZT, and AZT) prior to infection for 1 h. The cells without a sample were used as controls. The POX and Herpes viruses were then added to the cells, so the concentration of the samples was diluted twice to 5 lg/mL. The cells were incubated for 1 h, and after washing, the incubation continued for 3 days. The virus titers were evaluated by the cytopathic effect and calculated as n-fold inhibition in comparison with the control cells without studied samples. The tentative experiments showed that the pppAZT*LSiO2 nanocomposite at the concentration of 5 lg/mL (0.75 lM for nucleoside) inhibited Herpes simplex virus type I and POX virus by two and one orders of magnitude, respectively. It should be noted that the control samples of SiO2NH2 nanoparticles, AZT, and pppAZT showed almost no antiviral activity even at higher concentrations (25 lg/ml for the particles and 3.7 lM for nucleoside). 3. Conclusions In this work we used the click chemistry to develop the delivery system for AZT triphosphates. Our data confirmed the

Please cite this article in press as: Vasilyeva, S. V.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.02.063

S. V. Vasilyeva et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

successfulness of the Huisgen’s 1,3-dipolar cycloaddition reaction for the preparation of the SiO2dNTP nanocomposites. The fact that polymerase can recognize nucleoside triphosphates in the proposed nanobiocomposites and incorporate them into the growing DNA chain is of great importance. The results demonstrated a possibility of the utilization of SiO2 nanoparticles as vehicles for the delivery of nucleoside triphosphates analogues into cells. It was shown that the proposed SiO2dNTP nanocomposites penetrated into eukaryotic cells. The tentative results showed that these nanocomposites at low concentrations can inhibit the reproduction of POX and Herpes viruses. This makes it possible to use the nanobiocomposites bearing nucleoside triphosphate analogues as promising therapeutic drugs.

4. Experimental section 4.1. General The following reagents were used: 2,20 -dithiodipyridine, fluorescein isothiocyanate isomer I, N-hydroxysuccinimide, butyl acetate, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), copper(II) sulfate pentahydrate, and (+)-Sodium L-ascorbate crystalline, 1,3-diaminopropane, sodium hydride, streptavidin-alkaline phosphatase conjugate (SA-AP), chromogenic substrates (nitro blue tetrazolium chloride, NBT, and 5-bromo-4-chloro-3-indolyl phosphate, BCIP) (Sigma-Aldrich, USA); triphenylphosphine (Fluka Chemie AG, Buchs, Switzerland); propargyl alcohol (Acros Organics, USA); pentafluorophenol; glutaric anhydride; dicyclohexylcarbodiimide (Merck, Germany); SiO2–NH2 nanoparticles (SkySpring Nanomaterials, Inc., USA); methyl 6-bromohexanoate, 50 -gamma-(aminoropyl)amido-triphosphate of thymidine, 20 ,30 deoxy-30 -azidothymidine-50 -triphosphate (Li+ salt) and 1 M TEAAc buffer (pH 7) (NanoTech-S LLC,Novosibirsk, Russia). Oligonucleotides bearing the biotin label were synthesized by the reaction of oligonucleotides containing the amino linker (PL-oligo)12 with succinimidyl D-biotin. Cytidine triphosphate containing the biotin label at the 5 position of cytidine pppCbiot was synthesized according to.13 All other reagents were from Sigma-Aldrich (Milwaukee, WI, USA). Organic solvents were dried and purified by standard procedures. 1H and 31P NMR spectra were recorded on Bruker AV-400 and AV-300, AV-600, Drx 500 spectrometers. The chemical shifts (d) are reported in ppm relative to the residual solvent signals. In case of 31P, an external standard of 85% H3PO4 was used. The coupling constant (J) values are expressed in Hertz (Hz) and spin multiples are given as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), app.t (apparent triplet), and br. s (broad singlet). Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectra were run on Reflex III, Autoflex Speed (Bruker, Germany) with 3-hydroxypicolinic acid as a matrix. UV absorption spectra were recorded on a UV-1800 spectrophotometer (Shimadzu, Japan). Preparative anion exchange chromatography was performed on DEAE Sephadex A-25, 40–120 l, (Pharmacia Fine Chemicals, Sweden). Analytical reverse phase HPLC was performed on a Milichrom A-02 chromatograph (Econova, Russia) using a 2  75 mm column packed with ProntoSIL 120-5C18 AQ (Bischoff, Leonberg, Germany). Preparative reverse phase chromatography was performed on Polygoprep C18, 50–100 l (Macherey-Nagel, Germany). Thin layer chromatography was carried out using Alufolien Kieselgel 60 F254 plates (Merck, Germany) in appropriate solvent mixtures and was visualized by UV irradiation, ninhydrin (for amine groups), or cystein/aqueous sulphuric acid (for nucleoside-containing compounds).

5

4.2. Synthesis 4.2.1. Synthesis of 6-(2-propynyloxy)hexanioc acid pentafluorophenyl ester (1a) Sodium hydride (640 mg, 26.66 mmol) was added to a stirred solution of propargyl alcohol (2.422 mL, 41 mmol) in dry toluene (30 mL). After stirring for 2 hours at 25° C, a solution of methyl 6-bromohexanoate (4 g, 20.5 mmol) was added. The reaction mixture was stirred and refluxed for 7 h. Methyl ester of 6-(2-propynyloxy)hexanioc acid (2.64 g, 70%) was obtained by quenching the cooled reaction mixture in water, extracting by ether, washing with brine, drying over anhydrous magnesium sulphate, followed by evaporation. Methyl ester of 6-(2-propynyloxy)hexanioc acid (2.64 g) was stirred overnight with solution of sodium hydroxide (25 mL). After extracting with ether, the aqueous layer was acidified with concentrated hydrochloric acid. The required acid was obtained by ether extraction. The organic phase was washed with brine and dried over magnesium sulphate. Evaporation gave 6-(2propynyloxy)hexanioc acid (1.58 g) as a colourless oil. The mixture of 6-(2-propynyloxy)hexanioc acid (1.58 g, 9.28 mmol) and pentafluorophenol (1.88 g, 10.2 mmol) in ethyl acetate (10 mL) was cooled on ice. The dicyclohexylcarbodiimide dissolved in ethyl acetate (3 mL) and stirred on ice for 2 h. The reaction mixture was filtered from urine and concentrated by evaporation. The crude product was purified by silica-gel column chromatography using CH2Cl2 as eluent. The appropriate fractions were pooled and evaporated. The yield of target product was 2.5 g, 80%. 1H (300, CDCl3) d: 4.111 (d, 2H, CH„C–CH2–O); 3.51 (t, 2H, O–CH2–CH2–CH2); 2.39 (t, 1H, CH„C–CH2); 2.27 (m, 2H, –CH2–CH2–CH2–C(O)); 1.78 (m, 2H, O–CH2–CH2–CH2); 1.64 (m, 2H, –CH2–CH2–CH2–C(O)); 1.47 (m, 2H, –CH2–CH2–CH2–C(O)). 19F (400, CDCl3) d: 11.83 (d, 2F); 8.47 (t, 2F); 2.25 (t, 2F). 13C (CDCl3, 75 MHz) d: 169.29 (OC(O)); 142.37, 140.66, 139.87, 139.15, 138.14, 136.65 (PhF5); 79.73 (OCH2CCH); 74.04 (OCH2CCH); 69.54 (CH2CH2OCH2); 57.94 (OCH2CCH); 33.10, 33,04 (OC(O)CH2); 28.90 (CH2CH2OCH2); 25.33 (CH2CH2CH2OCH2); 24.37 (C(O)CH2CH2). ESI-QTOF: [M+H]+: calcd 337,08; found 337,09. 4.2.2. Synthesis of N-succinimidyl-5-(2-propynyloxy)-5oxopetanoate (2a) The mixture of glutaric anhydride (10.73 g, 94.0 mmol, 1 equiv) and propargyl alcohol (6.15 g 111.8 mmol, 1.2 equiv) was heated and stirred in a 90 °C oil bath until the mixture became homogeneous (1 h). After homogeneity was reached, the reaction temperature was raised to 105 °C for 30 min. Once the reaction cooled, 50 mL of the saturated sodium bicarbonate solution was added to dissolve the product. The excess of propargyl alcohol was extracted with ether (3  50 mL). The pH value of the aqueous layer was lowered to 3 with diluted HCl, followed by the extraction of the desired product with dichloromethane (3  50 mL). The ether layer was then dried over MgSO4, and the solvent was evaporated under reduced pressure to yield a yellow oil of 5-(2-propynyloxy)-5-oxopetanoic acid 12.04 g (75.3%). 1H NMR (d6-DMSO, 400 MHz) d: 4.62 (d, 2H, CH„C–CH2–O); 3.39 (s, 1H, CH„C– CH2–O); 2.32 (m, 2H, OC(O)CH2CH2CH2C(O)OH); 2.20 (m, 2H, OC(O)CH2CH2CH2C(O)OH); 1.67 (m, 2H, OC(O)CH2CH2CH2 C(O)OH). 13C (d6-DMSO, 75 MHz) d: 174.5 (C(O)OH); 172.2 (C(O)OCH2C„CH); 78.8 (CH2C„CH); 77.6 (CH2C„CH); 51.9 (C(O)OCH2C„CH); 32.8 (CH2CH2CH2); 20.6 (CH2CH2CH2). Dicyclohexylcarbodiimide (4 g, 17.6 mmol) was added to the ice-cold solution of 5-(2-propynyloxy)-5-oxopetanoic acid (3 g, 17.6 mmol) and N-hydroxysuccinimide (2 g, 17.6 mmol) in ethyl acetate (50 mL). The mixture was stirred for 2 h at 0 °C, followed by the incubation overnight in a refrigerator. The precipitate was filtered, and the combined filtrates were concentrated under reduced pressure and crystallized from isopropyl alcohol. The yield

Please cite this article in press as: Vasilyeva, S. V.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.02.063

6

S. V. Vasilyeva et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

of 2a was 3.29 g (70%). 1H NMR (d6-DMSO, 400 MHz) d: 4.66 (d, 2H, CH2C„CH); 3.49 (s, 1H, CH2C„CH); 2.73 (m, 4H, Suc); 2.74 (m, 2H, OC(O)CH2CH2CH2C(O)O-N-Suc); 2.71 (m, 2H, OC(O)CH2CH2 CH2C(O)O-N-Suc); 2.48 (m, 2H, OC(O)CH2CH2CH2C(O)O-N-Suc). MS (DFS High Resolution GC-MS): Calculated for C12H13O6 N m/z 267.0738. Found m/z 212.0552 (C9H10O5 N+). There is a fragment with m/z 55.0 in the spectrum, so we have assumed that it is a breakdown product of the molecular ion. 4.2.3. Synthesis of pppAZT*L1 (1b) Copper(II) sulfate 200 mM in water (600 lL, 120 lmol, 5 eq) was added to a mixture 24 mM solution of ppp-AZT (Li+) in H2O (1 mL, 24 lmol, 1 eq), and 6-(2-propynyloxy)hexanioc acid (4 mg, 24 lmol), followed by the addition of 1 M TEAAc buffer pH 7 (240 lL, 240 lmol 10 equiv) and freshly prepared 1 M sodium ascorbate (120 lL, 120 lmol, 5 equiv). The reaction mixture was stirred at room temperature for 24 h. Crude modified pppAZT was precipitated by 6% LiClO4 in acetone, washed by diethyl ester, and air-dried. The product was purified by chromatography on DEAE Sephadex A-25. The yield of 1b was 16.2 mg (88%). 31P (D2O, 121.5 MHz) d: -9.68 (bd, 1P, Pc); -11.10 (d, 1P, Pa, J 13.26); -22.45 (bt, 1P, Pb). 1H NMR (D2O, 300 MHz) d: 8.2 (s, 1H, H6Thym); 7.84 (s, 1H, H5-Triazole); 6.56 (app.t, 1H, H10 , J 7.2); 5.6 (bd, 1H, H30 ); 4.83 (m, 1H, H40 ); 4.62 (m, 2H, H50 ); 4.27 (m, 2H, O–CH2–Triazole); 3.72 (m, 2H, O–CH2–(CH2)4–); 2.81 (m, 2H, H20 ); 2.15 (m, 2H, HO–C(O)–CH2–); 1.92 (m, 3H, Thym); 1.53 (m, 4H, –CH2–CH2–CH2–CH2–CH2–); 1.26 (m, 2H, –CH2–CH2–CH2– CH2–CH2–). 13C (D2O, 150 MHz) d: 189.30 (C(O)OH); 166.56 (2s, Thym4, Triazole); 151.67 (Thym2); 137.25 (Thym6); 125.12 (Triazole); 111.87 (Thym5); 85.51 (C10 ); 83.32 (C40 ); 70.13 (C50 ) 65.68 (Triazole–CH2–O); 62.47 ((CH2)4CH2OCH2); 60.82 (C30 ); 37.34 (CH2C(O)OH); 35.25 ((CH2)3CH2CH2O); 28.10 (C20 ), 24.75 (2s, C(O)CH2CH2CH2(CH2)2O); 11.84 (CH3 Thym). MALDI TOF: [M+H]+: calcd 678.39.08 (acid); found 678.14. 4.2.4. Synthesis of alkyne modified nanoparticles „L1SiO2 (1) SiO2NH2 nanoparticles (20 mg, 0.5 lmol/mg for the amino groups) were suspended in 0.5 mL of DMSO, followed by the addition of 6-(2-propynyloxy)hexanioc acid pentafluorophenyl ester 1a (22 mg, 80 lmol, 8 equiv) in 0.3 mL DMSO. After stirring the mixture for 30 min at room temperature, it was centrifuged; supernatant was removed, and the particles were washed with aqueous 0.1 M NaCl (3  0.1 mL), water (3  0.1 mL), acetone (3  0.1 mL), and ether (1  0.1 mL) and air-dried. The yield of 1 was 19 mg (90%). The evaluation of the amount of the amino groups with picric acid11 before and after the reaction showed that 85% of the amino groups were modified. The capacity of the prepared alkyne modified nanoparticles for the alkyne groups was evaluated to be 0.43 lmol/mg. 4.2.5. Synthesis of alkyne modified nanoparticles „L2SiO2 (2) SiO2–NH2 nanoparticles (314 mg, 0,157 mmol for the amino groups) were suspended in 3.5 mL of DMSO, followed by the addition of Et3N (55 lL) and 4 portions of the solution N-succinimidyl5-(2-propynyloxy)-5-oxopetanoate 2a (367 mg, 1.37 mmol, 8.73 equiv) in 2 mL of DMSO every 30 min under stirring. After stirring the mixture for 1 h at room temperature, it was centrifuged, the supernatant was removed, and the particles were washed with aqueous 0.1 M NaCl (3  0.1 mL), water (3  0.1 mL), acetone (3  0.1 mL), and ether (1  0.1 mL) and air-dried. The yield was 342 mg (95%). The evaluation of the amount of the amino groups with picric acid11 before and after the reaction showed that 85% of the amino groups were modified. The capacity of the prepared alkyne modified nanoparticles for the alkyne groups was evaluated to be 0.45 lmol/mg.

4.2.6. Synthesis of fluorescein-containing alkyne modified nanoparticles „L1SiO2-Flu (3) SiO2–NH2 nanoparticles (50 mg, 25 lmol for the amino groups) were suspended in 0.5 mL of 0.2 M solution NaHCO3, followed by the addition of fluorescein isothiocyanate (9.7 mg, 25 lmol, 1 equiv) in 100 lL DMSO. After stirring the mixture for 1 h at room temperature, it was centrifuged, the supernatant was removed, and the particles were washed with aqueous 0.1 M NaCl (3  0.5 mL), water (1 mL), acetone (1 mL), and ether (1 mL) and air-dried. The amount of the modified amino-groups in SiO2(Flu)NH2 (3a) was evaluated by measuring the absorbance of the resulting product dissolved in 0.2 M NaOH (UV spectrum: (0.2 M NaOH), kmax/nm (e): 489 (70,000)). The yield of the modification was 16.8%. The capacity of the prepared fluorescein modified nanoparticles for the amine groups was 0.42 lmol/mg. The obtained nanoparticles SiO2(Flu)NH2 (3a) (44.7 mg, 18.78 lmol for the amino groups) were suspended in 0.5 mL of DMSO, followed by the addition of 6-(2-propynyloxy)hexanioc acid pentafluorophenyl ester 1a (42 mg, 80 lmol, 8 equiv) in 0.3 mL of DMSO. The mixture was stirred for 1 h at room temperature, followed by centrifugation. The supernatant was removed, and the particles were washed with aqueous 0.1 M NaCl (3  0.5 mL), water (1 mL), acetone (1 mL), and ether (1 mL) and air-dried. The capacity of the prepared fluorescein and alkyne modified nanoparticles for the alkyne groups was evaluated to be 0.42 lmol/mg. 4.2.7. Synthesis of nanocmposite pppAZT* L1SiO2 (4) Alkyne modified nanoparticles 1 (20 mg, 0.43 lmol/mg for the alkyne groups) were suspended by sonication in 200 lL DMSO. The pppAZT solution (20 mM in H2O, 1.72 mL, 34.4 lmol, 4 equiv) was added to the suspension, followed by the addition of copper(II) sulfate pentahydrate (50 mM stock solution in H2O, 400 lL), 1 M TEAAc buffer (pH 7.0, 40 lL), and freshly prepared 1 M sodium ascorbate (20 lL). The reaction mixture was degassed with argon for 2 min and stirred at room temperature for 4 h. The modified SiO2-bound triphosphate was centrifuged and the supernatant was removed. The nanoparticles were successively washed with aqueous 0.1 M NaCl, water, and diethyl ether, followed by airdrying. The capacity of the prepared nanocomposite for the pppAZT was evaluated by measuring the absorbance of the resulting product SiO2pppAZT dissolved in 0.2 M NaOH (UV spectrum: (0.2 M NaOH), kmax/nm (e): 267 (6650)). It was 0.33 lmol/mg. 4.2.8. Synthesis of pppAZT*L2SiO2 nanocomposite (5) Alkyne modified nanoparticles 2 (20 mg, 0.45 lmol/mg for the alkyne groups) were suspended by sonication in 200 lL of DMSO. The pppAZT solution (20 mM in H2O, 1.8 mL, 36 lmol, 4 equiv) was added to the suspension, followed by the addition of copper(II) sulfate pentahydrate (50 mM stock solution in H2O, 400 lL), 1 M TEAAc buffer (pH 7, 40 lL), and sodium ascorbate (freshly prepared 1 M solution in water, 20 lL). The reaction mixture was degassed with argon for 2 min and stirred at room temperature for 4 h. The modified SiO2-bound triphosphate was centrifuged and the supernatant was removed. The nanoparticles were successively washed with aqueous 0.1 M NaCl, water, and diethyl ether, followed by air-drying. The capacity of the prepared nanocomposite for the pppAZT (0.32 lmol/mg) was evaluated by measuring the absorbance of the resulting product SiO2pppAZT dissolved in 0.2 M NaOH (UV spectrum: (0.2 M NaOH), kmax/nm (e): 267 (6650)). 4.2.9. Synthesis of fluorescein-containing nanocomposite pppAZT*L1SiO2(Flu) (6) The fluorescein-containing alkyne modified nanoparticles 3 (10 mg, 0.42 lmol/mg for the alkyne groups) were suspended by

Please cite this article in press as: Vasilyeva, S. V.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.02.063

S. V. Vasilyeva et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

sonication in 200 lL of DMSO. The pppAZT solution (10 mM in H2O, 1.68 mL, 16.8 lmol, 4 equiv) was added to the suspension followed by the addition of copper(II) sulfate pentahydrate (50 mM stock solution in H2O, 400 lL), 1 M TEAAc buffer (pH 7, 40 lL), and freshly prepared 1 M sodium ascorbate (20 lL). The reaction mixture was degassed with argon for 2 min and stirred at room temperature for 4 h. The modified SiO2-bound triphosphate was centrifuged and the supernatant was removed. The modified nanoparticles were washed with aqueous 0.1 M NaCl, water, and diethyl ether, followed by air-drying. The capacity of the prepared nanocomposite for the pppAZT (0.31 lmol/mg) was evaluated by measuring the absorbance of the resulting product SiO2pppAZT dissolved in 0.2 M NaOH (UV spectrum: (0.2 M NaOH), kmax/nm (e): 267 (6650)). 4.2.10. Synthesis of fluorescein-containing nanocomposite (Flu)pppAZT*L1SiO2– (7) Nanocomposite 4 (2.9 mg, 0.33 lmol/mg for the pppAZT) were ultrasonically suspended in 1 mL of DMSO. (N-Methylimidazole) (8 mg, 7.6 lL, 47.85 lmol, 100 equiv) was added to the suspension, followed by the addition of the 2,20 -dithiodipyridine (2 mg, 95.7 lmol, 100 equiv) and triphenylphosphine (2.3 mg, 100 equiv) solution in 0.2 mL of DMF. After stirring the mixture for 30 min at room temperature, propane-1,3-diamine (7.2 lL, 95.7 lmol, 200 equiv) was added and the mixture was stirred at room temperature for 1 h. The modified nanoparticles were centrifuged, supernatant was removed, and the nanoparticles were successively washed with aqueous 0.1 M NaCl, water, and diethyl ether, followed by air-drying. Prepared nanoparticles 7a (2.9 mg) were ultrasonically suspended in 0.5 mL of a NaHCO3/Na2CO3 buffer. Fluorescein isothiocyanate (3.4 mg) in 0.2 mL of DMSO was added to the suspension. After stirring the mixture for 1 h at room temperature, it was centrifuged, the supernatant was removed, and the particles were washed with aqueous 0.1 M NaCl (3  0.1 mL), water (3  0.1 mL), acetone (3  0.1 mL), and ether (1  0.1 mL) and air-dried. The capacity of the prepared fluorescein modified nanoparticles for the fluorescein groups was 0.12 lmol/mg. It was evaluated by measuring the absorbance of the resulting product 7 dissolved in 0.2 M NaOH (UV spectrum: (0.2 M NaOH), kmax/nm (e): 489 (70,000)). 4.2.11. Synthesis of fluorescein-containing thymidine triphosphate (Flu)pppT (8) Fluorescein isothiocyanate (3.4 mg) was added to the solution of thymidine gamma-(aminoropyl)amido-triphosphate (2.7 mg, 4.8 lmol, 1 equiv) in 10% TEA in water(100 lL). After stirring the mixture for 1 h at room temperature, the product was precipitated by 6% LiClO4 in acetone (10-fold volume). The residue was dissolved in water and diluted by 1% NH4Ac in EtOH. After centrifugation, supernatant was separated and diluted by 10-fold volume of 6% LiClO4 in acetone. The precipitate was separated by centrifuging and dried in vacuum. Rf = 0.15 (PriOH–conc. NH3– H2O, 7:1:2). The yield of 8 was 50%. 31P (121, D2O) d: 0.84 (d, 1P, Pc, J 19.95), 11.20 (d, 1P, Pa, J 19.95), 22.32 (t, 1P, Pb, J 19.95). 1H (300, D2O) d: 7.64 (s, 1H, H(6)); 7.56 (m, 1H, Flu4); 7.50–7.47 (m, 1H, Flu6); 7.24–7.21 (m, 1H, Flu7); 7.01 (m, 2H, Flu40 ,50 ), 6.54–6.50 (m, 2H, Flu10 ,30 ), 6.39–6.33 (m, 2H, Flu70 ,20 ), 6.25 (app.t, 1H, H10 ); 4.57 (m, 1H, H40 ); 4.10 (m, 3H, H50 , H30 ); 3.02 (m, 2H, –CH2–NHC(S)); 2.88 (m, CH2–CH2–NH-P(O); 2.28 (m, 2H, H20 ); 1.8 (m, 5H, CH3(T), CH2–CH2–NH-P(O). MALDI TOF: [M+H]+: calcd 927.10 (acid); found 927.12. 4.3. Visualization of nanocomposites inside cells (penetration of pppAZT*LSiO2 nanocomposites into cells) Vero cells were cultured on glass cover slips (Invitrogen, USA) in RPMI media supplemented with 5% fetal calf serum (5%), penicillin

7

and streptomycin (100 lg/mL, each) at 37 °C in a humidified incubator under ambient pressure air atmosphere containing 5% CO2 to approximately 70% confluence. Before transfection, cells were successively washed with PBS buffer and RPMI medium without serum and antibiotics, followed by the addition of the RPMI medium (500 lL) containing the (Flu)pppT, (Flu)pppAZT* L1SiO2 or pppAZT*L1SiO2(Flu) samples bearing the fluorescein residue either at the c-phosphate or at SiO2 nanoparticles (0.06 mg/mL for nanoparticles and 5 nmol/mL for nucleoside). The cells were incubated with the samples for 24 h, washed with PBS, and fixed with formaldehyde (3.7%) for 10 min. Nuclei and cell membranes were stained with DAPI and Cell Mask Plasma Membrane Stain (Molecular Probes, Invitrogen, USA), respectively, for 10 min. After washing the cells with PBS, they were visualized by confocal laser scanning LSM 510 UV META microscope (Carl Zeiss, Inc., Germany) from the Center for collective use in ICG SB RAS). Laser lines were 405 nm (for nuclei, DAPI blue), 488 nm (for fluorescein-labeled nanocomposites, green), and 543 nm (for cell membranes, red). The thickness of optical sections was 0.21 l. The images are presented in Fig. 1. 4.4. Substrate properties of the pppAZT*LSiO2 nanobiocomposites Substrate properties of the pppAZT*LSiO2 nanobiocomposites were examined using DNA chips, which were prepared by the immobilization of the polylysine-containing primer 0 (ATTCTAGGp5 -PL) and the control polylysine-containing oligonucleotide bearing the biotin label at the 30 -terminal phosphate 0 (biotp-TCCCp5 -PL) on nylon membranes.12 In short, 104 M PL-oligo conjugates were spotted as 0.5 lL droplets on nylon chips (1  3 cm), and after drying spots and washing slides, the amino groups of polylysine were protected by acetylation. Klenow fragment and 10 SE Klenow buffer containing 500 mM Tris–HCl, pH 7.6, 100 mM MgCl2, and 50 mM DTT (Sibenzyme, Russia) were used for the elongation of the primer, immobilized on the nylon chips on the model template 50 CTCCGAAGAAATAAGATCCp. The prepared chip 1 was immersed in 1x Klenow buffer (40 lL) containing pppT (105 M), pppCbiot (105 M), the template (105 M), and Klenov fragment (5103 U), and the extention reaction was carried out at 37 °C for 1 h under stirring. The similar reaction was performed on chips 2 and 3 except that pppAZT*LSiO2 (2 lL, 1 mg/mL, 3104 M for AZT*) was used instead of pppT. The reaction on chips 4 and 5 was performed in the presence of only pppCbiot (in the absence of either pppT or pppAZT*LSiO2). After the completion of the reaction, all chips were washed thoroughly with the buffer (3  300 lL) to remove the unreacted compounds. Chips 3 and 5 were then subjected to repeated extension reaction using pppT and pppCbiot as described for chip 1. The spots containing the biotin label attached to the surface of the chip were visualized after the treatment with SA-AP conjugate and chromogenic substrates (NBT and BCIP).12 The results are presented in Fig. 2. 4.5. Cell viability and antiviral activity of samples Vero cells (FBRI SRC VB ‘Vector’, Russia) were seeded at 100,000 cells/mL in RPMI-1640 nutrient medium in 96-well plates (100 lL/well) and incubated at 37 °C and 5% CO2 to the formation of a continuous monolayer, followed by washing cells with the RPMI-1640 medium (200 lL/well) containing no serum. The sample of the pppAZT*LSiO2 nanocomposite was diluted with the RPMI-1640 medium containing L-glutamine to the concentrations of 10 lg/mL, corresponding to 1.5 lM for nucleoside and added to

Please cite this article in press as: Vasilyeva, S. V.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.02.063

8

S. V. Vasilyeva et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

cells (100 lL/well). After the incubation of cells with the sample at 37°C and 100% humidity in 5% CO2 for 3 days, cells were stained with trypan blue14 and the number of viable cells was counted in a Goryaev chamber. Vero cells without samples were used as a control. To infect Vero cells, we used Herpes simplex type I and POX (smallpox of mice viruses, strain K-1) (FBRI State Research Centre of Virology and Biotechnology ‘Vector’, Russia). The cells were seeded as above and the studied samples, that is, pppAZT*LSiO2 (10 lg/mL, 1.5 lM for nucleoside), AZT (7.5 lM), pppAZT (7.5 lM), and SiO2NH2 (50 lg/mL) in the RPMI-1640 medium containing antibiotics were added to each well (50 lL/well), followed by the incubation for 1 h at 37 °C and 5% CO2. The cells without a sample were used as controls. The serial ten-fold dilutions of POX or Herpes viruses in the RPMI1640 were added to each well (50 lL/well). The concentration of the samples was diluted twice. The initial titers of Herpes and POX viruses were 7.2 and 6.0 TCID50/mL, respectively. The cells were incubated with viruses for 1 h at 37 °C and 5% CO2 and after washing, the new portion of the RPMI-1640 medium (100 lL/well) was added. The incubation continued for three days under the same conditions. The cytopathic effect of the viruses was registered using inverted microscope and expressed as lgTCID50/ml and then calculated as n-fold inhibition as compared with the controls without studied samples.

Acknowledgment The work was supported by RFBR, research project no.14-0400274-a. References and notes 1. Broder, S. Antiviral Res. 2010, 85, 1. 2. Wright, K. Nature 1986, 323, 283. 3. Datta, A.; David, R.; Glennie, S.; Scott, D.; Cernuda-Morollon, E.; Lechler, R. I.; Ridley, A. J.; Marelli-Berg, F. M. Am. J. Transplant. 2006, 6(12), 2871. 4. Gomez, D. E.; Armando, R. G.; Alonso, D. F. Front. Oncol. 2012, 2, 113. 5. Dalakas, M. C.; Illa, I.; Pezeshkpour, G. H.; Laukaitis, J. P.; Cohen, B.; Griffin, J. L. New Engl. J. Med. 1990, 322, 1098. 6. Lynx, M. D.; Mckee, E. E. Biochem. Pharmacol. 2006, 72, 239. 7. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004. 8. Sirivolu, Venkata Ramana; Verneka, Sanjeev Kumar V.; Ilina, Tatiana; Myshakina, Nataliya S.; Parniak, Michael A.; Wang, Zhengqiang J. Med. Chem. 2013, 56, 8765. 9. Lin, J.; Roy, V.; Wang, L.; You, L.; Agrofoglio, L. A.; Deville-Bonne, D.; McBrayer, T. R.; Coats, S. J.; Schinazi, R. F.; Eriksson, S. Bioorg. Med. Chem. 2010, 18, 3261. 10. Vasilyeva, S. V.; Silnikov, V. N.; Shatskaya, N. V.; Levina, A. S.; Repkova, M. N.; Zarytova, V. F. Bioorg. Med. Chem. 2013, 21, 703. 11. Atherton, E.; Sheppard, R. C.; Ward, P. J. Chem. Soc., Perkin Trans. 1 1985, 10, 2065. 12. Levina, A. S. et al Biotechnol. J. 2007, 2, 885. 13. Vasilyeva, S. V.; Budilkin, B. I.; Konevetz, D. A.; Silnikov, V. N. Nucleos. Nucleot. Nucl. Acids 2011, 30, 753. 14. Osano, E.; Kishi, J.; Takahashi, Y. Toxicol. In Vitro 2003, 17, 47.

Please cite this article in press as: Vasilyeva, S. V.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.02.063