Synthesis and Characterization of Cell-Permeable Oligonucleotides

Article pubs.acs.org/bc

Synthesis and Characterization of Cell-Permeable Oligonucleotides Bearing Reduction-Activated Protecting Groups on the Internucleotide Linkages Hisao Saneyoshi,*,† Koichi Iketani,† Kazuhiko Kondo,† Takeo Saneyoshi,‡ Itaru Okamoto,† and Akira Ono*,† †

Department of Material and Life Chemistry, Faculty of Engineering, Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku, Yokohama 221-8686, Japan ‡ Brain Science Institute RIKEN, 2-1 Hirosawa, Wako City, Saitama 351-0198, Japan S Supporting Information *

ABSTRACT: Cell-permeable oligodeoxyribonucleotides (ODNs) bearing reduction-activated protecting groups were synthesized as oligonucleotide pro-drugs. Although these oligonucleotides were amenable to solid-phase DNA synthesis and purification, the protecting group on their phosphodiester moiety could be readily cleaved by nitroreductase and NADH. Moreover, these compounds exhibited good nuclease resistance against 3′-exonuclease and endonuclease and good stability in human serum. Fluoresceinlabeled ODNs modified with reduction-activated protecting groups showed better cellular uptake compared with that of naked ODNs.

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INTRODUCTION Synthetic oligonucleotides such as antisense oligonucleotides and siRNAs can be used as biological tools and therapeutic agents to develop a better understanding of pharmacological processes and treat various human diseases.1−5 However, the application of naked oligonucleotides as therapeutic agents has been limited by their polyanionic and hydrophilic properties, which can prevent them from being adsorbed into cells. Furthermore, naked oligonucleotides are rapidly degraded in the biological fluid. The development of an efficient strategy capable of delivering oligonucleotides to a specific target site is therefore highly desirable for the therapeutic application of these systems. Various delivery systems including liposomes, micelles, and nanoparticles have been developed to date to allow for the efficient delivery of oligonucleotides.6−10 The direct conjugation of oligonucleotides with carbohydrates, peptides, vitamins, lipids, and antibodies has also been used to facilitate the efficient delivery of these agents to specific target sites via receptor-mediated mechanisms or direct penetration.11−18 An alternative prooligonucleotide approach was developed by Imbach and coworkers in the 1990s.19−24 This particular strategy is based on the use of biodegradable protecting groups for the phosphodiester moieties between the internucleotide linkages of the oligonucleotides. The resulting pro-oligonucleotides (pro-oligos) are bioreversible phosphotriester-type oligonucleotides, which can be readily adsorbed into the cytoplasm without the need for a transfection reagent. Following their uptake into © XXXX American Chemical Society

the cytoplasm, the protecting groups on these pro-oligos can be deprotected by endogenous esterases releasing the active oligonucleotides. The main advantages of this strategy are as follows: (i) there is no need for transfection reagents; (ii) these systems are typically resistant to nucleases; and (iii) several protecting groups are amenable to this approach with different deprotection triggers.25−47 We are particularly interested in using the hypoxic conditions found in the tumor microenvironment to develop targeted prooligo systems as therapeutic agents that can be activated by the hypoxic conditions found in advanced solid tumors.48 Several hypoxia-activated prodrugs based on a nitro reduction mechanism have been reported in the literature.49 Furthermore, nitrothienyl and nitrofuranyl protecting groups have been applied to nucleic acids to protect their internucleotide linkages, leading to improved cellular uptake and nuclease resistance.38 However, this strategy has only ever been applied to oligo thymidylates. Mixed-sequence oligonucleotides bearing bioreductively sensitive protecting groups would be valuable tools for biological research, with a wide range of potential applications in cancer biology and medicinal studies. Furthermore, these systems could be used without the need for a transfection reagent. In this study, we used a 3-(2-nitrophenyl)propyl Received: July 7, 2016 Revised: August 20, 2016

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DOI: 10.1021/acs.bioconjchem.6b00368 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Schematic representation of the behavior expected of the pro-oligos.

phosphate protecting groups were performed under standard conditions (e.g., NH4OH, rt, 2 h or 55 °C, 4 h). The oligothymidylates (ODN 1, 2, and 5−7) and oligonucleotides with mixed sequences incorporating 2 or 3 (ODN 4 and 5) were successfully synthesized according to the route shown in Scheme 2. Typical reverse-phase high-performance liquid chromatography (HPLC) profiles are shown in Figure 2 (upper: before purification; bottom: after purification). These data showed that the NPP groups were stable to the sequential deprotection and purification steps involved in the synthesis of the oligonucleotide, with the desired products providing double peaks that are derived from diastereoisomers. It is noteworthy that purified ODN 1 remained unchanged after being treated with NH4OH at 55 °C for 6 h (Figure S1). As a model, an experiment was conducted under bioreductive conditions to evaluate the deprotection of the NPP groups in the ODNs (Scheme 3). ODN 1 (5′-TTTXTTT-3′; X = modified T unit) was treated with nitroreductase (from Escherichia coli) in the presence of NADH, and the reaction was monitored by HPLC. Time-course HPLC chromatograms are shown in Figure 3A. A peak corresponding to ODN 1 was observed in the HPLC chromatogram prior to the addition of the enzyme along with a peak corresponding to NADH. After incubation with the enzyme for 30 min, the intensity of the peak corresponding to ODN 1 decreased considerably. As the reaction proceeded, so too did the reduction in the intensity of the peak corresponding to ODN 1 until it had been finally converted to the deprotected product (as indicated by the black arrow in the figure). The conversion yields for each time line are shown in Figure 3B. An oligonucleotide bearing PP groups on its internucleotide linkages (ODN 2) was also synthesized as a noncleavable substrate. This oligonucleotide was also treated with nitroreductase and NADH under the same conditions as those described above for the NPP protected oligonucleotide. The results revealed that ODN 2 was completely stable under the enzymatic conditions for 6 h. The stability of this system was attributed to the absence of a nitro group on the protecting group (Figure 3C,D).

(NPP) group as a new hypoxia-labile protecting group for the phosphodiester bonds of oligonucleotides. From a structural perspective, this protecting group contains aliphatic and aromatic groups with no acyl groups, and it was therefore envisaged that this group would be stable for standard chemical synthesis. It was also envisaged that this group would remain stable in biological fluids except for those found in hypoxic environments. The most likely mechanism for the deprotection of the NPP group is an intramolecular cyclization, which would be triggered by the bioreduction of the nitro group to release the active oligonucleotides (Figure 1). In this study, we synthesized pro-oligos bearing an NPP group and investigated the deprotection of these systems under model bioreductive conditions. We also evaluated the nuclease resistance, duplex formation, and cellular uptake properties of these NPP-protected pro-oligos.

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RESULTS AND DISCUSSION For the synthesis of the monomer unit, we coupled the known phosphorodiamidite derivative 136 with 3-(2-nitrophenyl)propan-1-ol50,51 and 3-phenylpropan-1-ol to give the desired coupling products 2 and 3, respectively (Scheme 1). A series of oligodeoxyribonucleotides (ODN 1−7) incorporating 2 or 3 was synthesized on a DNA synthesizer system using standard methods. The release of the oligonucleotide from the CPG support and the reactions for the deprotection of the base and Scheme 1. Synthesis of Nucleoside Phosphoroamidites Bearing NPP and 3-Phenylpropyl Groups

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Bioconjugate Chemistry Scheme 2. Synthesis of ODNs Bearing NPP and 3-Phenylpropyl Groups

Introduction of protecting groups at the internucleotide linkage induced destabilization of duplex formation. Next, the susceptibility of the synthesized ODNs to nucleolytic enzymes was examined. A total of two kinds of nucleases, Crotalus adamanteus venom phosphodiesterase (CAVP) and DNase I, were used as models for a 3′exonuclease and an endonuclease, respectively. The stability of the ODNs in human serum was also tested. The ODN 5 containing four residues of each nucleotide analogue were labeled with fluorescein at the 5′- end and incubated with CAVP, DNase I, or 50% human serum. The reactions were then analyzed by polyacrylamide gel electrophoresis under denaturing conditions. The results for the denature gel analysis of the ODNs treated with CAVP are shown in Figure 5. The control oligonucleotides were completely converted to a series of short nucleotide fragments after being incubated with CAVP for 30 min. Conversely, ODN 5 remained unchanged after being incubated with enzyme for 120 min. ODN 5 also exhibited good nuclease resistance against endonuclease. Furthermore, ODN 5 remained intact after being incubated with DNase 1 for 72 h, whereas naked ODN was completely consumed after 3 h under the same conditions (Figure 6). ODN 5 also exhibited much greater stability in 50% human serum than did the naked ODN. Taken together, these results showed that ODN 5 was resistant to the effects of several nucleases, including human serum (Figure 7). Next, we investigated the cellular uptake properties of the ODNs in HeLa cells. ODNs 5−7 were labeled with fluorescein at their 5′-end containing phosphotriester. HeLa cells were treated with individual solutions of the different ODNs (10 μM) for 1 h and washed with PBS before being fixed with paraformaldehyde and dyed with Alexa594-phalloidin and Hoechst 33258. The fixed cells were observed by confocal microscopy (Figure 8A). The intensity of the fluorescence of the labeled pro-oligos in the cells increased as the number of protecting groups increased. The maximum intensity was observed for ODN 7, which contained 67% phosphotriester moieties (Figure 8B). This result indicated that the pro-oligos bearing reductionresponsive protecting groups were cell-permeable.

Figure 2. Crude reverse-phase HPLC profiles of a typical ODN with a mixed sequence after DNA synthesis. The purities of the synthesized oligonucleotides were determined by HPLC and their structures were confirmed by MALDI-TOF mass spectroscopy (Table S1).

Scheme 3. Bioreductive Deprotection of the 3-(2Nitrophenyl)propyl Groups in the ODNs

ODN 1 was found to be stable in buffer, both with and without NADH or nitroreductase, for at least 6 h. HPLC profiles for all of the control experiments are shown in Figure S1. Next, hybridization affinities of pro-oligonucleotides with mixed sequences were investigated. The sequences used in this study and thermal denaturation profiles are shown in Figure 4. Each ODN has three or four phosphotriester linkages. C


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Figure 3. Reverse-phase HPLC chromatograms for the nitroreductase-mediated deprotection of the ODNs. Panel A: reverse-phase HPLC profiles showing the time course for the deprotection reaction; panel B: deprotection yield; panel C: reverse-phase HPLC for the deprotection of NPP after 6 h; panel D: reverse-phase HPLC profile for the deprotection of PP after 6 h. The asterisk indicates a contamination peak from the enzyme solution. The yields were estimated by HPLC analysis based on peak area.

Figure 4. Thermal denaturation profiles of pro-oligos. Conditions: duplex (2 μM), 100 mM NaCl in 10 mM MOPS (pH 7.0).

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CONCLUSIONS In summary, we have designed and synthesized a series of new pro-oligos, which were activated under bioreductive conditions. We also succeeded in the synthesis of pro-oligos consisting of mixed sequences, which were evaluated in terms of their thermal stabilities. The protecting group in these pro-oligos was readily cleaved by nitroreductase and NADH. These pro-oligos exhibited good nuclease resistance against 3′-exonucleases and endonucleases and good stability in human serum. Notably, ODN 7 showed much better cellular uptake in HeLa cells compared with that of naked ODN, as well as slightly better

uptake than ODN 5 and ODN 6. These results therefore indicate that the pro-oligos described in this study could be used as pro-drugs for the delivery of oligonucleotide-based therapeutics in hypoxic cells.

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EXPERIMENTAL SECTION

General Procedures. Chemicals were purchased from Wako Pure Chemicals (Osaka, Japan), Sigma-Aldrich (St. Louis, MO), Tokyo Chemical Industry (Tokyo, Japan), and Glen Research (Sterling, VA) and used without further purification. NMR spectra were recorded on a JEOL (Tokyo,

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Figure 5. Polyacrylamide gel electrophoresis of 5′-fluorescein labeled ODNs hydrolyzed by CAVP (a 3′-exonuclease). (a) Unmodified oligonucleotide. (b) ODN 5. ODNs were incubated with snake venom phosphodiesterase for 0 min (lane 1), 5 min (lane 2), 15 min (lane 3), 30 min (lane 4), 60 min (lane 5), 90 min (lane 6), and 120 min (lane 7). The experimental conditions are described in the Experimental section.

Figure 7. Polyacrylamide gel electrophoresis of the 5′-fluorescein labeled ODNs treated with 50% human serum. (a) Unmodified oligonucleotide. (b) ODN 5. ODNs were incubated with 50% human serum for 0 min (lane 1), 1 h (lane 2), 2 h (lane 3), 3 h (lane 4), 4 h (lane 5), 5 h (lane 6), and 6 h (lane 7). Experimental conditions are described in the Experimental section.

phosphoramidite 2 and 3 are used as 0.1 M solutions in dry acetonitrile. Synthesized ODNs were released from CPG support and deprotected by NH4OH at room temperature for 2 h or 55 °C for 4 h. The CPG solid support was filtered off, and the filtrate was concentrated in vacuo. Crude ODNs were purified by C-18 cartridge. Each sample was further purified by using reverse-phase HPLC. The structures of each ODN were confirmed by measurement of matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry on the AXIMA-CFR plus (Shimadzu) by using refectionnegative mode. (Matrix for ionizing samples was used as a mixture (10:1:1; saturated 3-hydroxy-2-picolic acid, 2-picolic acid−H2O (50 mg/mL), and ammonium citrate−H2O (50 mg/ mL)). Thermal Denaturation. Each sample containing a duplex (2 μM) in a buffer of 10 mM MOPS (pH 7.0) with 100 mM NaCl was used this study. The thermally induced transitions of the duplexes were monitored at 260 nm on a UV-1650PC spectrophotometer (Shimadzu) with the Tm analysis accessory (TMSPC-8). The temperature was ramped at 1.0 °C min−1. Enzymatic Deprotection of Protecting Groups at the Internucleotide Linkage. ODN 1 (6 μM), NADH (10 mM), and nitroreductase (from E. coli, 160 μg) in 200 μL of 50 mM sodium phosphate buffer (pH 7.0) was incubated at 37 °C. Aliquots of sample solution were analyzed by reverse-phase HPLC at appropriate times. HPLC conditions: A buffer (0.1 M TEAA containing 5% CH3CN), B buffer (0.1 M TEAA containing 50% CH3CN); gradient (B) 5% → 60% (30 min). Column: Intersil ODS-3 (4.6 × 250 mm) (GL Sciences). Hydrolysis of ODN 5 with CAVP. ODN 5 labeled with fluorescein at the 5′-end (10 μM) was incubated with phosphodiesterase I (0.1 μg, C. adamanteus venom; SIGMA) in a buffer containing 50 mM Tris−HCl (pH 8.0) and 10 mM MgCl2 (total volume of 500 μL) at 37 °C. At the appropriate time, aliquots (20 μL) of the reaction mixture were separated, and the mixture was heated for 5 min at 90 °C. The solutions were analyzed by electrophoresis on 20% polyacrylamide gel containing 7 M urea. The gels were visualized by an AE-9000 EGraph (ATTO). Hydrolysis of ODN 5 with DNase 1. ODN 5 labeled with fluorescein at the 5′-end (10 μM) was incubated with

Figure 6. Polyacrylamide gel electrophoresis of 5′-fluorescein labeled ODNs hydrolyzed by DNase I (an endonuclease). (a) Unmodified oligonucleotide. (b) ODN 5. ODNs were incubated with DNase I for 0 min (lane 1), 3 h (lane 2), 6 h (lane 3), 12 h (lane 4), 24 min (lane 5), 48 h (lane 6), and 72 h (lane 7). The experimental conditions are described in the Experimental section.

Japan) instrument at 600 MHz for 1H NMR, 125 MHz for 13C NMR, and 172 MHz for 31P NMR. Chemical shifts were measured from tetramethylsilane for 1H NMR spectra, CDCl3 (77.0 ppm) for 13C NMR spectra, and 85% phosphoric acid (0.0 ppm) for 31P NMR spectra. The coupling constant (J) was reported in hertz. Abbreviations for multiplicity were: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad. Column chromatography was carried out with a silica gel C-60 (Kanto, Japan) or NH silica gel (Fuji Silysia Chemical; Kasugai, Japan). Thin-layer chromatography (TLC) analyses were carried out on Kieselgel 60-F254 plates (Merck). Reversephase HPLC was carried out with Intersil ODS-3 (4.6 × 250 mm; GL Sciences). The UV−VIS spectrum was recorded on a UV-1650PC Spectrophotometer (Shimadzu, Kyoto, Japan). The HPLC system consisted of a controller, a pump, a UV monitor (SPD-10AVP; Shimadzu), and a recorder (CR6A; Shimadzu). Oligonucleotide Synthesis. Oligonucleotides synthesis was carried out on the Applied Biosystems 394 DNA/RNA synthesizer according to the manufacturer′s recommendations. 2-Cyanoethyl phosphoramidite (dT, dC, dA, and dG) and E


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Cell Culture, Oligonucleotide Treatment, and Imaging Analysis. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen), 10% fetal bovine serum, and penicillin−streptomycin at 37 °C in 5% CO2 and 95% air. Cells were plated on glass coverslip in a 24 well culture plate at 2 × 104 cells per well overnight. The following day, cells were washed once with 400 μL of OptiMEM I (Invitrogen) and preincubated in OptiMEM I for 20 min at 37 °C and 5% CO2. Cells were treated with the pro-oligo solution at the 10 μM concentration in OptiMEM I for 60 min. After being washed three times with 400 μL of OptiMEM I for 5 min at 37 °C, 5% CO2 cells were fixed for 20 min in 4% parafolmaldehyde in PHEMS buffer (60 mM PIPES, 25 mM HEPES (pH 7.4), 5 mM EGTA, 1 mM MgCl2, and 3% sucrose) at room temperature, washed three times for 5 min in PBS, and stained with Alexa594 phalloidin (1:100) and Hoechst 33258 (10 μg.mL) in TBST for 20 min at room temperature. After being washed with PBS, coverslips were mounted on glass slides with Prolong gold (Invitrogen) and imaged using a confocal microscope (FV1200; Olympus). The amount of fluoresceinlabeled pro-oligos in the cells was analyzed with Atto Image Analysis Software CS Analyzer 4.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00368. Figures showing reverse-phase HPLC profiles of synthesized ODNs and HPLC profiles of control experiments of ODN 1 and ODN 2. A table showing the oligonucleotide sequence and MALDI-TOF mass analysis of synthesized oligonucleotides. Additional details on synthetic procedures for all compounds and copies of 1H, 13C, and 31P NMR spectra. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

Figure 8. Confocal images showing the cellular uptake of the 5′fluorescein labeled pro-ODNs in HeLa cells (panel A) and analysis of the amount of fluorescein-labeled pro-oligos in the cells (panel B). The numbers of protecting groups increased from the left side to the right side (ODN 5−7).

*E-mail: [email protected]. *E-mail: akiraono@ kanagawa-u.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Strategic Research Base Development Program for Private Universities (Kanagawa University, 2012−2016) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (A.O.) and Takahashi Industrial and Economic Research Foundation (H.S.).

recombinant DNase I (100 units; Takara) in a buffer containing 100 mM sodium acetate (pH 6.0) and 5 mM MgCl2 (total volume of 100 μL) at 37 °C. At the appropriate time, aliquots (10 μL) of the reaction mixture were separated, and the mixture was added to 5 mM EDTA and heated for 5 min at 90 °C. The solutions were analyzed by electrophoresis on 20% polyacrylamide gel containing 7 M urea. The gels were visualized by an AE-9000 E-Graph. Stability of ODN 5 in the 50% Human Serum. ODN 5 labeled with fluorescein at the 5′-end (10 μM) was incubated with human serum (Sigma)/PBS (1:1, v/v, total volume of 100 μL) at 37 °C. At the appropriate time, aliquots (10 μL) of the reaction mixture were separated, and the mixture was added to 10 M urea and heated for 5 min at 90 °C. The solutions were analyzed by electrophoresis on 20% polyacrylamide gel containing 7 M urea. The gels were visualized by an AE-9000 E-Graph.

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