pH-Dependent Switching of Base Pairs Using Artificial Nucleobases

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Article Cite This: J. Org. Chem. 2018, 83, 1320−1327

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pH-Dependent Switching of Base Pairs Using Artificial Nucleobases with Carboxyl Groups Tanasak Kaewsomboon, Shuhei Nishizawa, Takashi Kanamori, Hideya Yuasa, and Akihiro Ohkubo* Department of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan S Supporting Information *

ABSTRACT: In this study, we report the synthesis of modified oligonucleotides consisting of benzoic acid or isophthalic acid residues as new nucleobases. As evaluated by UV thermal denaturation analysis at different pH conditions (5.0, 6.0, 7.0, and 8.0), these modified oligonucleotides exhibited pH-dependent recognition of natural nucleobases and one is first found to be capable of base pair switching in response to a pH change. The isophthalic acid residue incorporated into the oligonucleotide on a D-threoninol backbone could preferentially bind with adenine but with guanine in response to a change in the pH conditions from pH 5 to pH 7 (or 8) without significant difference in duplex stability. These findings would be valuable for further developing pH-responsive DNA-based molecular devices.

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INTRODUCTION

Functional residues capable of changing structure in response to external stimuli such as heat and light have been used to create very useful molecular devices in the field of nanotechnology.1−7 One reported application of such functional residues is their incorporation into oligonucleotides to regulate duplex formation with a complementary strand.8−11 For example, the hybridization behavior of azobenzene-modified DNA can be changed by irradiation with UV or visible light.12−15 Wada et al. have reported that the addition of boric acid can strongly inhibit duplex formation in peptide ribonucleic acids.16 However, there are few reports on the use of external stimuli to change the strand to which an artificial oligonucleotide binds.9 Therefore, in this study, we have designed novel artificial nucleobases, the base recognition patterns of which can be modified by external stimuli. Perumalla et al. previously reported that the carboxyl group of benzoic acid can form two hydrogen bonds with the exocyclic amino group and N1 of adenine in the solid state.17 Additionally, it is well-known that carboxyl group-modified oligonucleotides can form a duplex with a complementary oligonucleotide containing a pyridine nucleobase at the corresponding site.18,19 However, these base pairs are formed by copper coordination, rather than hydrogen bonding. Therefore, it is possible that a benzoic acid residue incorporated into an oligonucleotide might form a base pair with adenine through hydrogen bonding under acidic conditions, and with guanine under neutral or basic conditions, as shown in Figure 1. Thus, we anticipated that incorporation of a benzoic acid or similar residue could be used to create oligonucleotides with the ability to preferentially bind with different base pairs in response to a pH change. © 2018 American Chemical Society

Figure 1. Chemical structures of base pairs containing a carboxyl group(s) under acidic and neutral or basic conditions.

In the present study, we synthesized oligonucleotides consisting of a benzoic acid residue (X1) with the canonical 2′-deoxyribose backbone, and of a benzoic acid residue (X2) or isophthalic acid residue (X3) with a D-threoninol20 backbone (Figure 2). An oligonucleotide sequence consisting of a phenyl

Figure 2. Chemical structures of modified oligonucleotides containing the X1, X2, X3, and X4 residues.

residue with a D-threoninol backbone (X4)21 was also synthesized for use as a sterically similar structure without a carboxyl group. The binding and nucleobase recognition abilities of the modified oligonucleotides with complementary strands containing different canonical nucleobases at the Received: November 8, 2017 Published: January 11, 2018 1320

DOI: 10.1021/acs.joc.7b02828 J. Org. Chem. 2018, 83, 1320−1327

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The Journal of Organic Chemistry Scheme 1. Synthesis of X1 Phosphoramidite Unit 6

Scheme 2. Synthesis of X2 and X3 Phosphoramidite Units 14a−b

study because its preparation from thymidine is much more efficient than the preparation of the mono-3′-TBDMSprotected glycal.25 The deprotection of the bis-TBDMS ethers of adduct 3 was carried out using TBAF (Bu4NF) in combination with acetic acid as a buffer to avoid the cleavage of a methyl ester protecting group of the benzoic acid moiety.35 The 3′-carbonyl group was then stereoselectively reduced to a hydroxyl group using NaBH(OAc)3.30,36 The nucleoside 4 was obtained in high yield (85%) and its β-anomeric conformation was confirmed by NOE 1H NMR spectroscopy37 (see the Supporting Information for details). Subsequent dimethoxytritylation and phosphitylation were performed to yield phosphoramidite 6.

opposite site were examined using UV thermal denaturation analysis at different pH conditions (5.0, 6.0, 7.0, and 8.0).

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RESULTS AND DISCUSSION Synthesis of X1 Phosphoramidite Unit. For the incorporation of benzoic acid residue on a deoxyribose backbone (X1),22,23 bis-TBDMS-protected ribofuranoid glycal 124,25 was coupled with methyl 3-iodobenzoate (2)26 by a Pd0mediated C−C coupling reaction27−32 to afford adduct 3 with an isolated yield of 48%, as shown in Scheme 1. Although previous studies recommend the use of a mono-3′-TBDMSprotected ribofuranoid glycal for more efficient cross-coupling reaction,33,34 bis-protected-TBDMS glycal 1 was used in this 1321

DOI: 10.1021/acs.joc.7b02828 J. Org. Chem. 2018, 83, 1320−1327

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The Journal of Organic Chemistry Synthesis of X2 and X3 Phosphoramidite Units. The synthetic route of X2 and X3 phosphoramidites 14a−b is shown in Scheme 2. For the synthesis of the benzoic acid derivatives, esterification of benzoyl chloride derivatives 7a−b with 2cyanoethanol was carried out to give compounds 8a−b. After selective deprotection of 8a−b using 1 equiv of NaOH in THF−H2O (1:1, v/v) for 10 min, the benzoic acid derivatives 9a−b were obtained. For the synthesis of a D-threoninol backbone, the 3′-hydroxyl group of D-threoninol 1038 was protected with TBDMS and, after subsequent deprotection of the 2′-amino group, D-threoninol 11 was obtained. The benzoic acid derivatives 9a−b were then condensed with D-threoninol 11 using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) to afford adducts 12a−b in good yields (96% and 86% for 12a and 12b, respectively). After deprotection of TBDMS with triethylamine trihydrofluride (Et3N-3HF)39 in the presence of Et3N,40 phosphitylation was performed to obtain phosphoramidites 14a−b. Oligonucleotide Synthesis and Postsynthetic Deprotection. The chain elongation of modified oligonucleotides (ODN 1−4, as shown in Figure 3) containing X1−X4 was

Figure 4. Comparison of the Tm values for the duplexes formed between ODN 1 and ODN 5−6. The measurements were performed in a buffer containing 10 mM sodium phosphate (pH 5.0−8.0), 150 mM NaCl, 0.1 mM EDTA, and 2 μM of the duplex.

was relatively unstable, especially under basic conditions. The C1′−C1′ distances in the noncanonical X1-A or X1-G pairs are larger than the corresponding distance in the canonical pairs (T-A or C-G pairs). Because of the low flexibility, a deoxyribose backbone would not be able to accommodate the expanded C1′−C1′ distance in the expected X1-A or X1-G pairs. Therefore, it may be hypothesized that, in order to form a base pair with X1, the 2′-deoxyadenine would rotate into synconformation, imposing a reduced C1′−C1′ distance, and form an X1-A pair through Hoogsteen base pairing. In this manner, the carboxyl group of the benzoic acid residue could accept a hydrogen bond from the exocyclic amino group of A and donate one to N7 of A.17 However, this hydrogen bonding mode is not possible in the case of an X1-G pair, especially under higher pH, thus causing instability of an X1-G pair, compared to an X1-A pair. Additionally, it was found that the Tm values of the duplexes containing X1-A or X1-G were lower than that (46.8 °C at pH 7) of the duplex containing a T-A base pair at the X-Y position. Subsequently, we studied the thermal stabilities of the duplexes containing X2 or X3, which have a more flexible backbone, D-threoninol.44 The results are shown in Figures 5

Figure 3. Sequences of ODN 1−8.

carried out in a DNA synthesizer using the standard phosphoramidite method. After the chain elongation, ODN 1 was deprotected and released from the CPG resin by treatment with 0.4 M NaOH in MeOH−H2O (4:1, v/v)41 at room temperature for 24 h. For the deprotection of the 2-cyanoethyl (CE) groups of the oligonucleotides containing X2 and X3 residue (with D-threoninol backbone), the CPG resins were treated with 10% DBU/pyridine−BSA (1:1, v/v) at room temperature for 3 h,42,43 followed by treatment with a saturated aqueous ammonia solution at room temperature for 6 h to obtain ODN 2−3. The ODN 4 was deprotected and released from the CPG resin by treatment with a saturated aqueous ammonia solution at room temperature for 2 h. ODN 1−4 were then characterized by MALDI-TOF mass spectrometry (see the Supporting Information). UV Thermal Denaturation Analysis. To study the effect of pH on the binding and nucleobase recognition abilities of the carboxyl group-modified oligonucleotides, the thermal stabilities of DNA duplexes containing X1−X4 with complementary strands containing different conventional bases were examined. Figure 4 shows the Tm values of the DNA duplexes formed between ODN 1 and ODN 5 or 6. At pH 5.0, the Tm value of the DNA duplex containing X1-A (ODN 1-ODN 5) was 5.0 °C higher than that of the duplex containing X1-G (ODN 1-ODN 6). The Tm value of the DNA duplex containing X1-A decreased by 8.1 °C when the pH was raised from pH 5 to pH 8. A similar decrease of Tm values was also observed upon raising the pH value from 5 to 8 in the case of X1-C (ODN 1-ODN 7) or X1-T (ODN 1-ODN 8), as shown in Figure S1. These results indicate that X1 can form a stable base pair with adenine under acidic conditions, but not in its deprotonated form under neutral or basic conditions. On the contrary, the Tm values of the DNA duplex containing X1-G unexpectedly remained constant or slightly decreased when the pH was increased. These results suggest that the DNA duplex containing X1-G

Figure 5. Comparison of the Tm values of the duplexes formed between ODN 2 and ODN 5−6. The measurements were performed in a buffer containing 10 mM sodium phosphate (pH 5.0−8.0), 150 mM NaCl, 0.1 mM EDTA, and 2 μM duplex. 1322

DOI: 10.1021/acs.joc.7b02828 J. Org. Chem. 2018, 83, 1320−1327

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The Journal of Organic Chemistry and 6, respectively. The Tm value of the DNA duplex containing X2-A (ODN 2-ODN 5) at pH 5 was 3.9 °C higher

Figure 7. Comparison of the Tm values of the duplexes formed between ODN 4 and ODN 5−6. The measurements were performed in a buffer containing 10 mM sodium phosphate (pH 5.0−8.0), 150 mM NaCl, 0.1 mM EDTA, and 2 μM duplex.

Figure 6. Comparison of the Tm values of the duplexes formed between ODN 3 and ODN 5−6. The measurements were performed in a buffer containing 10 mM sodium phosphate (pH 5.0−8.0), 150 mM NaCl, 0.1 mM EDTA, and 2 μM duplex.

as that of the one containing X4-G at the same pH, despite the hydrogen bonds formed in the X3-G base pair. These results imply that the duplex formation of the modified oligonucleotides containing a carboxyl group might be inhibited by electrostatic repulsion between the negatively charged phosphate skeleton and the negatively charged carboxyl groups. Therefore, MgCl2 was added to the phosphate buffer containing the DNA duplexes to increase their stabilities by reducing the electrostatic repulsion, as shown in Figure 8. It

than that at pH 8 because of the formation of an X2-A base pair under acidic conditions. For the case of the DNA duplex containing X3-A (ODN 3-ODN 5), the Tm difference between pH 5 and pH 8 was 3.4 °C greater than that in the X2-A containing duplex. Additionally, as expected, it was observed that the Tm values of the DNA duplexes containing X2-G or X3G were higher than those of the duplex containing X1-G, especially under neutral or basic conditions, owing to increased backbone flexibilities. The Tm differences between the DNA duplexes containing X2-G or X3-G (ODN 2-ODN 6 and ODN 3-ODN 6, respectively) at pH 5 and pH 8 were greater than that of the duplex containing X1-G. Particularly, when compared at the same pH, the Tm differences between the DNA duplexes containing X3-A and X3-G for each pH were all greater than those between the duplexes containing X2-A and X2-G. Moreover, the Tm value of the DNA duplex containing X3-A at pH 5 was nearly the same as those of the duplexes containing X3-G at pH 7 and 8. These results indicate that the oligonucleotide containing X3 (ODN 3) more preferentially forms duplexes with ODN 5 at pH 5, but with ODN 6 at pH 7 or 8 without a dramatic decrease in the stabilities of the duplexes. In other words, X3 incorporated into the oligonucleotide is able to change its preferential base pair from X3-A to X3G in response to an external stimulus, a change in the pH from 5 to 7 (or 8), without significantly changing the thermal stability of the DNA duplexes. To our knowledge, this is the first example in which the artificial nucleobases incorporated into the oligonucleotide have been demonstrated to be capable of base pair switching in response to a pH change without a significant change in duplex stability. Next, to further confirm whether the pH-responsive Tm change was produced by the carboxyl group or not, the thermal stability of the DNA duplexes containing X4, which do not contain a carboxyl group, was studied as shown in Figure 7. For all tested pH conditions, the Tm values of DNA duplexes containing X4-A or X4-G (ODN 4-ODN5 and ODN 4-ODN 6) showed almost no difference, indicating that the carboxyl group modification was responsible for the pH-responsive Tm change. However, as seen in Figures 6 and 7, the Tm value of the DNA duplex containing X3-G at pH 8 was nearly the same

Figure 8. Effect of the addition of MgCl2 on the Tm values of the duplexes formed between ODN 2−4 and ODN 6. The measurements were performed in a buffer containing 10 mM sodium phosphate, (pH 7.0), 150 mM NaCl, 0.1 mM EDTA, and 2 μM duplex in the presence of 10 mM MgCl2.

was observed that the stabilizing effect on the DNA duplexes due to the addition of 10 mM MgCl2 became greater as the number of carboxyl groups incorporated into the DNA duplex increased. The increase in the Tm values of the DNA duplex containing X3-G due to the addition of MgCl2 at pH 7 was 7.0 °C.

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CONCLUSION In summary, we have successfully synthesized oligonucleotides containing benzoic acid or isophthalic acid residues as nucleobases. We also reported the pH dependence of the base recognition ability of the modified oligonucleotides and found for the first time that the artificial nucleobase is capable of base pair switching in response to a pH change. In particular, 1323

DOI: 10.1021/acs.joc.7b02828 J. Org. Chem. 2018, 83, 1320−1327

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The Journal of Organic Chemistry

0.5 °C/min, and kept at 5 °C for 5 min. The melting profiles were then recorded at 260 nm using a UV spectrometer (Pharma Spec UV1700, Shimadzu) by increasing the temperature at a rate of 0.5 °C/min from 5 to 90 °C. The Tm values were calculated as the temperature that exhibited the maximum of the first derivative of the recorded melting profile. The final values were determined by averaging three independent measurements with a typical standard deviation of around 1.0 °C. Synthesis of Compound 3. A solution of bis(dibenzylideneacetone)palladium(0) (41.7 mg, 72.5 μmol) and tris(pentafluorophenyl)phosphine (77.2 mg, 145 μmol) in argon-bubbled acetonitrile (29 mL) was stirred at room temperature for 30 min under an argon atmosphere to make a Pd−ligand mixture. Then methyl 3-iodobenzoate26 (0.42 g, 1.59 mmol), ribofuranoid glycal 124,25 (0.50 g, 1.45 mmol), and N,N-diisopropylethylamine (0.5 mL, 2.9 mmol) were added into the Pd−ligand mixture. The reaction mixture was heat, refluxed, and stirred at 96 °C under an Ar atmosphere for 64 h. Then the reaction mixture was allowed to cool down to ambient temperature and diluted with CH2Cl2. The organic layer was washed twice with water and once with brine, before being dried over Na2SO4. The residue was purified further by neutral silica 60N gel column chromatography (0−10% EtOAc in hexane) to obtain compound 3 as colorless syrup (0.34 g, 48%). 1H NMR (500 MHz, CDCl3) δ 0.02 (s, 6H), 0.21 (d, J = 9.1 Hz, 6H), 0.87 (s, 9H), 0.95 (s, 9H), 3.75−3.80 (m, 1H), 3.88−3.93 (m, 4H), 4.60 (s, 1H), 4.80 (s, 1H), 5.75 (d, J = 2.8 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.92− 7.99 (m, 2H); 13C NMR (120 MHz, CDCl3) δ −5.3, −4.9, −4.9, 18.1, 18.6, 25.6, 26.1, 52.0, 64.4, 84.3, 101.8, 128.1, 128.4, 129.0, 129.9, 132.2, 143.6, 151.3, 167.1. HR-ESI-TOF MS calcd for C25H42NaO5Si2+ [M + Na]+ 501.2463, found 501.2461. Synthesis of Compound 4. A mixture of compound 3 (2.50 g, 5.22 mmol), CH3COOH (2.38 mL, 41.76 mmol), and tetra-nbutylammonium fluoride (5.46 g, 20.88 mmol) in THF (52.2 mL) was stirred at room temperature for 12 h under an argon atmosphere.45 After being co-evaporated with toluene, the residue was purified by C200 silica gel column chromatography (40−80% EtOAc in hexane). The fractions were collected and concentrated under reduced pressure to obtain a colorless syrup of crude (1.49 g). Then 1.21 g of that crude was co-evaporated with CH3CN before being added into the mixture of CH3CN (242.5 mL) and CH3COOH (80 mL). After the mixture was cooled to −40 °C, sodium triacetoxyborohydride (1.13 g, 5.33 mmol) was cautiously added. The reaction mixture was stirred at −20 °C for 1.5 h under an argon atmosphere. After the reaction was complete, the reaction mixture was evaporated under reduced pressure, diluted with CH2Cl2, and washed with water. Repeated reverse extraction was also carried out because the product migrated to the aqueous layer. The residue was purified by C200 silica gel column chromatography (0−3% MeOH in CH2Cl2). The fractions were collected and concentrated under reduced pressure to obtain compound 4 as colorless syrup (1.12 g, 85% for two steps). 1H NMR (500 MHz, CDCl3) δ 1.90−2.04 (m, 1H), 2.21−2.31 (m, 1H), 3.11 (br, 1H), 3.70−3.83 (m, 2H), 3.89 (s, 3H), 3.98−4.10 (m, 1H), 4.36−4.48 (m, 1H), 5.16−5.23 (m, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.93 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H); 13C NMR (120 MHz, CDCl3) δ 44.0, 52.3, 63.4, 73.7, 79.7, 87.7, 127.2, 128.7, 129.0, 130.4, 130.8, 142.1, 167.3. HR-ESI-TOF MS calcd for C13H16NaO5+ [M + Na]+ 275.0890, found 275.0894. Synthesis of Compound 5. Compound 4 was co-evaporated with toluene, CH2Cl2, and pyridine to get rid of the remaining CH3COOH or moisture. After that, a mixture of compound 4 (1.00 g, 3.96 mmol), 4,4′-dimethoxytrityl chloride (1.48 g, 4.36 mmol) in pyridine (39.6 mL) was stirred at room temperature for 12 h under an argon atmosphere. Then 4,4′-dimethoxytrityl chloride (0.15 g, 0.11 mmol) was added more to promote product formation, and the reaction was stirred at the same condition for another 2 h. After that, the reaction mixture was diluted with CH2Cl2, and washed with saturated NaHCO3 aq. twice and water once, before being dried over Na2SO4. The residue was purified by C200 silica gel column chromatography using CH2Cl2 containing 0.5% pyridine (by volume) as eluent. The fractions were collected and concentrated under reduced pressure to obtain

X3 incorporated into the oligonucleotide can change its preferential base pair from X3-A to X3-G in response to a change in the pH value from pH 5 to 7 (or 8), respectively, without significant difference in duplex stability. In this sense, these pH-responsive properties would be very worthwhile and relevant for applications in gene therapy and nanotechnology. Further studies are currently in progress in those directions.

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EXPERIMENTAL SECTION

General Experimental Procedure. All chemical reagents and dry solvents were purchased from Glen Research and Tokyo Chemical Industry, and used as received. The dry solvents were stored over molecular sieves (4 Å) after use. Reactions were monitored using TLC plate silica gel 60 F254. Spots on TLC were identified by using UV, ninhydrin, or anisaldehyde. Column chromatography was performed with silica gel C-200 (Wako Chem. Co. Ltd.), NH-type (Wako Chem. Co. Ltd.), or 60N (Kanto Chem. Co., Inc.) columns. A mini-pump for a goldfish bowl was used for rapid chromatographic separation. Recycling preparative HPLC was performed by using an apparatus from Japan Analytical Industry Co. Ltd. Chemical reagents for DNA synthesis and unmodified DNA phosphoramidite units (dA, dT, dG, dC) including a CPG resin column were purchased from Glen Research. Sep-pak C18 cartridges for DNA purification were purchased from Waters. The unmodified oligonucleotides (ODN 5− 8) were purchased from Sigma-aldrich. 1H, 13C, and 31P NMR spectra were recorded at 500, 120, and 203 MHz, respectively. Peak multiplicity was expressed as follows: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad. The chemical shifts were expressed in ppm relative to residual solvents as internal standard, CDCl3 (7.26 ppm) for 1H NMR spectra and CDCl3 (77 ppm) for 13C NMR spectra. ESI mass was performed using a Mariner (PerSeptive Biosystems Inc.). MALDI-TOF mass was performed using a Bruker Daltonics [Matrix: 3-hydroxypicolinic acid (100 mg/mL) in H2O− diammonium hydrogen citrate (100 mg/mL) in H2O (10:1, v/v)]. Oligonucleotide Synthesis. The modified oligonucleotides were synthesized on a 1 μmol scale in an automated DNA/RNA synthesizer (ABI 392 DNA/RNA synthesizer) according to the standard phosphoramidite protocol of ultramild DNA synthesis (activator: 0.25 M 5-benzylthio-1H-tetrazole in anhydrous MeCN, capping reagents: 5% phenoxyacetic anhydride in THF−pyridine and 10% 1methylimidazole in THF, deblocking reagent: 3% trichloroacetic acid in CH2Cl2, and oxidizing reagent: 0.02 M I2 in THF−pyridine−water). For the postsynthetic deprotection, ODN 1 was treated and cleaved (from CPG resin) with 0.4 M NaOH in MeOH−H2O (4:1, v/v)41 at room temperature for 24 h. ODN 2−3 were treated with 10% DBU/ pyridine−BSA (1:1, v/v) at room temperature for 3 h,42,43 followed by treatment with a saturated aqueous ammonia solution at room temperature for 6 h for their cleavage from CPG resin. ODN 4 was treated and cleaved (from CPG resin) with a saturated aqueous ammonia solution at room temperature for 6 h. The elution was removed by evaporation under reduced pressure, and the remaining was then purified on a Sep-pak C18 cartridge. The 5′-DMTr protecting group was removed in this cartridge using 2% trifluoroacetic acid (aq.). Then, the resulting oligonucleotide mixture was eluted by using 25−30% acetonitrile in water and further purified by anion exchange HPLC (DNAPac PA100 9 × 250 mm). The purity analysis was performed on a Waters Alliance system with a Waters 3D UV detector and a Gen-PakTM FAX column (Waters, 4.6 × 100 mm). A linear gradient (0−60%) of Solvent B (1 M NaCl in 25 mM phosphate buffer (pH 6.0) and 10% CH3CN) in solvent A (25 mM phosphate buffer (pH 6.0) and 10% CH3CN) was used at 50 °C with a flow rate of 1.0 mL/min for 45 min. UV Thermal Denaturation Experiment (Tm Measurements). Oligonucleotides ODN 1, 2, 3, and 4 (2 μM) and their complementary ODN 5, 6, 7, and 8 (2 μM) were dissolved in a buffer consisting of 150 mM NaCl, 10 mM sodium phosphate, and 0.1 mM EDTA adjusted to four different pH conditions (5.0, 6.0, 7.0, and 8.0). The solution was kept at 90 °C for 5 min for complete dissociation of the duplex to single strands, then cooled at the rate of 1324

DOI: 10.1021/acs.joc.7b02828 J. Org. Chem. 2018, 83, 1320−1327

Article

The Journal of Organic Chemistry compound 5 as a pale yellow solid (1.55 g, 71%). 1H NMR (500 MHz, CDCl3) δ 2.03−2.13 (m, 1H), 2.25−2.32 (m, 1H), 3.29−3.38 (m, 2H), 3.69−3.85 (m, 9H), 4.05−4.10 (m, 1H), 4.41−4.46 (m, 1H), 5.20−5.25 (m, 1H), 6.82 (d, J = 8.4 Hz, 4H), 7.14−7.51 (m, 10H), 7.59 (d, J = 7.6 Hz, 1H), 7.95 (d, J = 7.7 Hz, 1H), 8.10 (s, 1H); 13C NMR (120 MHz, CDCl3) δ 44.2, 52.1, 52.2, 55.3, 55.4, 64.5, 74.8, 79.6, 79.7, 86.4, 86.6, 86.7, 113.2, 126.9, 127.3, 128.0, 128.3, 128.6, 128.9, 130.2, 130.4, 130.7, 136.1, 142.5, 145.0, 158.6, 167.1. HR-ESITOF MS calcd for C34H34NaO7+ [M + Na]+ 577.2197, found 577.2190. Synthesis of Compound 6. Compound 5 (0.3 g, 0.54 mmol) was co-evaporated with anhydrous CH3CN 5 times and dissolved in anhydrous CH3CN (5.4 mL). N,N-Diisopropylamine (45 uL, 0.32 mmol), 1H-tetrazole (23 mg, 0.32 mmol), and 2-cyanoethyl-N,N,N,Ntetraisopropyl-phosphordiamidite (262 uL, 0.83 mmol) were added, and the reaction mixture was stirred at room temperature for 6 h under an argon atmosphere. Then the mixture was diluted with CH2Cl2 and the organic layer was washed twice with saturated NaHCO3 aq. and water once, before being dried over Na2SO4. The residue was purified by C200 silica gel column chromatography with CH2Cl2 containing triethylamine 1% while increasing EtOAc 0−2% as eluent. Then the residue was further purified by recycle chromatography using CH2Cl2 to obtain compound 6 as a white solid (0.34 g, 82%). 1H NMR (500 MHz, CDCl3) δ 1.01−1.22 (m, 12H), 2.02−2.11 (m, 1H), 2.33−2.50 (m, 2H), 2.62 (t, J = 6.4 Hz, 1H), 3.22−3.28 (m, 1H), 3.33−3.42 (m, 1H), 3.52−3.88 (m, 13H), 4.22−4.27 (m, 1H), 4.48−4.55 (m, 1H), 5.19−5.25 (m, 1H), 6.77−6.85 (m, 4H), 7.15− 7.99 (m, 12H), 8.15 (s, 1H); 13C NMR (120 MHz, CDCl3) δ 20.3, 24.5, 43.1, 43.2, 43.5, 52.0, 55.2, 58.3, 64.1, 75.7, 75.8, 76.1, 76.2, 79.9, 85.9, 86.1, 86.3, 113.1, 117.5, 117.6, 126.7, 127.2, 127.8, 128.2, 128.5, 128.8, 130.1, 130.3, 130.6, 135.9, 136.0, 136.1, 142.3, 144.9, 158.4, 167.0; 31P NMR (203 MHz, CDCl3) δ 148.9, 149.1. HR-ESI-TOF MS calcd for C43H51N2NaO8P+ [M + Na]+ 777.3275, found 777.3258. Synthesis of Compound 11. Compound 1038 was co-evaporated with pyridine 3 times and toluene 2 times. Then a mixture of compound 2 (4.51g, 8.96 mmol), 1H-imidazole (1.48 g, 21.5 mmol), and TBDMS chloride (2.43 g, 16.1 mmol) in DMF (9 mL) was stirred at room temperature overnight under an argon atmosphere. The reaction mixture was diluted with CH2Cl2, and the organic layer was washed with saturated NaHCO3 aq. and dried over Na2SO4. The residue was purified by NH-type silica gel column chromatography and concentrated under reduced pressure to obtain compound 3 which was then co-evaporated with pyridine and toluene. Next, 71 mL of 40% methylamine in methanol solution (100 equiv) was added to compound 3 (4.67 g, 7.56 mmol). The reaction mixture was stirred at 50 °C in an oil bath for 2 days under an argon atmosphere. The reaction mixture was concentrated under reduced pressure and purified by NH-type silica gel column chromatography. Then it was concentrated under reduced pressure again to obtain compound 4 as yellow syrup (3.25 g, 69%). 1H NMR (500 MHz, CDCl3) δ −0.09 (s, 3H), 0.00 (s, 3H), 0.77 (s, 9H), 1.07 (d, J = 6.0 Hz, 3H), 1.61 (br, 2H), 2.69−2.75 (m, 1H), 2.96 (t, J = 7.9 Hz, 1H), 3.09−3.16 (m, 1H), 3.76 (s, 6H), 6.80 (d, J = 8.6 Hz, 4H), 7.14−7.21 (m, 1H), 7.23−7.33 (m, 6H), 7.42 (d, J = 8.1 Hz, 2H); 13C NMR (120 MHz, CDCl3) δ −4.8, −4.0, 18.1, 20.8, 25.9, 55.3, 57.5, 66.2, 69.1, 86.1, 113.2, 126.8, 127.9, 128.3, 130.2, 136.4, 136.5, 145.2, 158.5. HR-ESI-TOF MS calcd for C31H43NNaO4Si+ [M + Na]+ 544.2854, found 544.2864. Synthesis of Compound 12a. The commercially available 7a (isophthaloyl chloride or 1,3-benzenedi-carbonyl dichloride) (5.08 g, 25 mmol) was dissolved in pyridine (125 mL). Six equivalents of 2cyanoethanol (10.2 mL, 150 mmol) was added, and the reaction mixture was stirred overnight. Then the mixture was diluted with CH2Cl2 and the organic layer was washed with water before being dried over Na2SO4. The mixture was then co-evaporated with toluene, followed by CH2Cl2 to obtain crude mixture 8a (4.9 g), which was then dissolved in a 0.1 M mixture of THF (90 mL) and NaOH (0.72 g, 18 mmol) dissolved in 90 mL of water (1:1, v/v). The reaction mixture was stirred at room temperature for 10 min under an argon atmosphere. According to the TLC, 8a still remained; therefore, 0.18 g of NaOH (0.25 equiv) dissolved in water (22.5 mL) was added more

into the reaction mixture. After being stirred for 10 min, the reaction mixture was diluted with CH2Cl2 and the organic layer was washed once with water and once with 1 M HCl (aq). The organic layer was collected, dried over Na2SO4, and concentrated under reduced pressure to obtain compound 9a as a light yellow solid (1.81 g, 33% (two steps from compound 7a)). Then, a mixture of compound 11 (1.71 g, 3.28 mmol), triethylamine (1.14 mL, 8.20 mmol)), HBTU (1.87 g, 4.92 mmol), and compound 9a (0.86 g, 3.93 mmol) in DMF (16.4 mL) was stirred at room temperature for 6 h under an argon atmosphere. The reaction mixture was washed with saturated NaHCO3 aq. 3 times and dried over Na2SO4. The residue was purified by NH-type silica gel column chromatography (0−40% EtOAc in hexane). The fractions were collected and concentrated under reduced pressure to obtain compound 12a as a white solid (2.28 g, 96% (one step from compound 9a)). 1H NMR (500 MHz, CDCl3) δ −0.12 (s, 3H), 0.04 (s, 3H), 0.78 (s, 9H), 1.21 (d, J = 6.2 Hz, 3H), 2.83 (t, J = 6.5 Hz, 2H), 3.06 (t, J = 8.2 Hz, 1H), 3.31−3.37 (m, 1H), 3.78 (s, 6H), 4.08−4.31 (m, 2H), 4.54 (t, J = 6.4 Hz, 2H), 6.47 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 7.2 Hz, 4H), 7.20 (t, J = 7.2 Hz, 1H), 7.27− 7.35 (m, 5H), 7.43 (d, J = 7.5 Hz, 2H), 7.55 (t, J = 7.8 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.38 (s, 1H); 13C NMR (120 MHz, CDCl3) δ −4.9, −4.0, 18.0, 18.2, 21.5, 25.9, 55.0, 55.4, 59.5, 63.2, 66.8, 76.9, 77.2, 77.4, 86.4, 113.3, 116.7, 126.9, 128.0, 128.3, 129.2, 129.7, 130.1, 130.2, 132.1, 132.6, 135.5, 136.2, 136.3, 145.0, 158.6, 165.3, 166.0. HR-ESI-TOF MS calcd for C42H50N2NaO7Si+ [M + Na]+ 745.3285, found 745.3301. Synthesis of Compound 12b. Commercially available 7b (1,3,5benzenetricarbonyl trichloride) (5.31 g, 20 mmol) was dissolved in pyridine (100 mL). Eight equivalents of 2-cyanoethanol (3hydroxypropionitrile) was added, and the reaction mixture was stirred overnight. The mixture was diluted with CH2Cl2, and the organic layer was washed with water before being dried over Na2SO4. Then the residue was co-evaporated with toluene and CH2Cl2 to obtain crude mixture 8a (6.83 g), which was then dissolved in THF (92.5 mL) and 0.2 M NaOH in water (92.5 mL). After being stirred at room temperature for 10 min under an argon atmosphere, the reaction mixture was diluted with CH2Cl2 and the organic layer was washed with water one time and 1 M HCl (aq) one time. The organic layer was collected, dried over Na2SO4, and concentrated under reduced pressure to obtain compound 9b as a white solid (2.11 g, 2 steps 34% (two steps from compound 7b)). Then a mixture of compound 11 (1.32 g, 2.53 mmol), triethylamine (0.88 mL, 6.33 mmol), HBTU (1.44 g, 3.79 mmol), and compound 9b (0.96 g, 3.04 mmol) in DMF (12.6 mL) was stirred at room temperature for 4 h under an argon atmosphere. The reaction mixture was washed with saturated NaHCO3 aq. and dried over Na2SO4. The residue was purified by NH-type silica gel column chromatography (0−70% EtOAc in hexane). The fractions were collected and concentrated under reduced pressure to obtain compound 12b as a white solid (1.78 g, 86% (one step from compound 9b)). 1H NMR (500 MHz, CDCl3) δ −0.11 (s, 3H), 0.05 (s, 3H), 0.79 (s, 9H), 1.22 (d, J = 6.1 Hz, 3H), 2.86 (t, J = 6.3 Hz, 4H), 3.08 (t, J = 8.1 Hz, 1H), 3.28−3.39 (m, 1H), 3.78 (s, 6H), 4.29 (q, J = 7.0, 6.3 Hz, 2H), 4.57 (t, J = 6.3 Hz, 4H), 6.54 (d, J = 8.7 Hz, 1H), 6.82 (d, J = 7.7 Hz, 4H), 7.15−7.36 (m, 6H), 7.43 (d, J = 7.8 Hz, 2H), 8.62 (s, 2H), 8.80 (s, 1H); 13C NMR (120 MHz, CDCl3) δ −3.9, 18.1, 18.3, 21.6, 26.1, 55.3, 55.4, 60.1, 63.3, 66.8, 86.5, 113.4, 116.6, 127.0, 128.0, 128.3, 130.2, 130.6, 132.8, 133.6, 136.2, 136.3, 158.7, 164.4. HR-ESI-TOF MS calcd for C46H53N3NaO9Si+ [M + Na]+ 842.3443, found 842.3425. Synthesis of Compound 13a. A mixture of compound 12a (2.28 g, 3.15 mmol), triethylamine (1.32 mL, 9.46 mmol), and triethylamine trihydrofluoride (1.54 mL, 9.46 mmol) in THF was stirred at room temperature overnight under an argon atmosphere. Then the reaction mixture was diluted with CH2Cl2 and the organic layer was washed with saturated NaHCO3 aq. 3 times and dried over Na2SO4. The residue was purified by NH-type silica gel column chromatography (0−70% EtOAc in hexane). The fractions were collected and concentrated under reduced pressure to obtain compound 13a as a white solid (1.48 g, 77%). 1H NMR (500 MHz, CDCl3) δ 1.22 (d, J = 6.3 Hz, 3H), 2.83 (t, J = 6.4 Hz, 2H), 3.02 (s, 1H), 3.33−3.42 (m, 1325

DOI: 10.1021/acs.joc.7b02828 J. Org. Chem. 2018, 83, 1320−1327

Article

The Journal of Organic Chemistry

NMR (203 MHz, CDCl3) δ 148.9, 149.2. HR-ESI-TOF MS calcd for C49H56N5NaO10P+ [M + Na]+ 928.3657, found 928.3631.

1H), 3.50−3.62 (m, 1H), 3.76 (d, J = 3.4 Hz, 6H), 4.07−4.27 (m, 2H), 4.56 (t, J = 6.4 Hz, 2H), 6.74−6.85 (m, 5H), 7.17−7.41 (m, 8H), 7.59 (t, J = 7.7 Hz, 1H), 8.04 (d, J = 7.7 Hz, 1H), 8.22 (d, J = 7.7 Hz, 1H), 8.45 (s, 1H); 13C NMR (120 MHz, CDCl3) δ 18.3, 20.4, 54.5, 55.4, 59.7, 65.2, 68.5, 87.0, 113.5, 127.3, 128.1, 128.3, 128.4, 129.3, 129.8, 130.1, 132.3, 132.8, 135.3, 135.6, 135.7, 144.6, 158.8, 165.4, 166.8. HR-ESI-TOF MS calcd for C36H36N2NaO7+ [M + Na]+ 631.2415, found 631.2408. Synthesis of Compound 13b. A mixture of compound 12b (1.78 g, 2.17 mmol), triethylamine (0.91 mL, 6.51 mmol), and (1.01 mL, 6.51 mmol) triethylamine trihydrofluoride in THF was stirred at room temperature overnight under an argon atmosphere. Then the reaction mixture was diluted with CH2Cl2 and the organic layer was washed with saturated NaHCO3 aq. 3 times and dried over Na2SO4. The residue was purified by NH-type silica gel column chromatography (0−90% EtOAc in hexane). The fractions were collected and concentrated under reduced pressure to obtain compound 13b as a white solid (1.49 g, 91%). 1H NMR (500 MHz, CDCl3) δ 1.21 (d, J = 6.3 Hz, 3H), 2.85 (t, J = 6.4 Hz, 4H), 2.93 (d, J = 2.5 Hz, 1H), 3.38− 3.44 (m, 1H), 3.50−3.55 (m, 1H), 3.75 (d, J = 3.2 Hz, 6H), 4.13−4.19 (m, 1H), 4.22−4.28 (m, 1H), 4.57 (t, J = 6.4 Hz, 4H), 6.77−6.92 (m, 5H), 7.17−7.44 (m, 8H), 8.64 (s, 2H), 8.80 (s, 1H); 13C NMR (120 MHz, CDCl3) δ 18.2, 20.4, 54.8, 55.5, 60.0, 64.9, 68.3, 77.4, 87.0, 113.5, 116.8, 127.3, 128.2, 130.2, 130.6, 133.0, 133.6, 135.7, 144.6, 158.8, 164.4. HR-ESI-TOF MS calcd for C40H39N3NaO9+ [M + Na]+ 728.2578, found 728.2558. Synthesis of Compound 14a. Compound 13a (1.48 g, 2.42 mmol) was co-evaporated with anhydrous CH3CN 5 times and dissolved in anhydrous CH3CN (24.2 mL). N,N-Diisopropylethylamine (0.21 mL, 1.46 mmol), 1H-tetrazole (0.10 g, 1.46 mmol), and 2cyanoethyl-N,N,N,N-tetraisopropylphosphordiamidite (0.92 mL, 0.29 mmol) were added and the reaction mixture was stirred at room temperature for 6 h under an argon atmosphere. Then the mixture was diluted with CH2Cl2 and the organic layer was washed with saturated NaHCO3 aq. 2 times before being dried over Na2SO4. The residue was purified by C200 silica gel column chromatography with eluent (0− 60% EtOAc in hexane) containing triethylamine 1%. Then the residue was further purified by recycle chromatography using CH3CN to obtain compound 14a as a white solid (1.44 g, 75%). 1H NMR (500 MHz, CDCl3) δ 0.98−1.18 (m, 12H), 1.25−1.35 (m, 3H), 2.36−2.59 (m, 2H), 2.73−2.83 (m, 2H), 3.19−3.63 (m, 5H), 3.65−3.85 (m, 7H), 4.33−4.47 (m, 2H), 4.47−4.55 (m, 2H), 6.45−6.84 (m, 5H), 7.12− 7.47 (m, 8H), 7.51−7.59 (m, 1H), 8.00−8.21 (m, 2H), 8.43 (d, J = 22.5 Hz, 1H); 13C NMR (120 MHz, CDCl3) δ 18.3, 18.3, 20.0, 20.5, 20.6, 24.6, 24.8, 25.0, 43.4, 54.9, 55.4, 58.1, 58.4, 59.7, 62.9, 63.4, 69.2, 86.3, 113.3, 117.0, 118.1, 127.0, 128.1, 128.2, 128.3, 128.4, 129.2, 129.7, 130.3, 132.4, 132.5, 132.7, 135.5, 136.2, 145.0, 158.7, 165.5, 166.2; 31P NMR (203 MHz, CDCl3) δ 149.1, 149.2. HR-ESI-TOF MS calcd for C45H53N4NaO8P+ [M + Na]+ 831.3499, found 831.3474. Synthesis of Compound 14b. Compound 13b (1.49 g, 2.11 mmol) was co-evaporated with anhydrous CH3CN 5 times and dissolved in anhydrous CH3CN (21 mL). N,N-Diisopropylethylamine (0.18 mL, 1.27 mmol), 1H-tetrazole (89 mg, 1.27 mmol), and 2cyanoethyl-N,N,N,N-tetraisopropyl-phosphordiamidite (0.81 mL, 2.54 mmol) were added, and the reaction mixture was stirred at room temperature for 6 h under an argon atmosphere. Then the mixture was diluted with CH2Cl2 and the organic layer was washed twice with saturated NaHCO3 aq., before being dried over Na2SO4. The residue was purified by C200 silica gel column chromatography with an eluent (0−60% EtOAc in hexane) containing triethylamine 1%. Then the residue was further purified by recycle chromatography using CH3CN to obtain compound 14b as a white solid (1.52 g, 81%). 1H NMR (500 MHz, CDCl3) δ 0.95−1.23 (m, 12H), 1.23−1.37 (m, 3H), 2.35− 2.63 (m, 2H), 2.73−2.92 (m, 4H), 3.19−3.66 (m, 5H), 3.67−3.87 (m, 7H), 4.30−4.49 (m, 2H), 4.50−4.62 (m, 4H), 6.54−6.92 (m, 5H), 7.14−7.37 (m, 6H), 7.43 (d, J = 7.7 Hz, 2H), 8.67 (d, J = 20.7 Hz, 2H), 8.80 (s, 1H); 13C NMR (120 MHz, CDCl3) δ 2.0, 18.2, 20.1, 20.6, 24.5, 24.6, 24.7, 24.8, 24.9, 25.0, 43.4, 55.4, 55.7, 58.2, 58.4, 60.1, 62.8, 63.4, 69.2, 86.32, 113.3, 117.0, 118.3, 127.1, 128.1, 128.3, 130.3, 130.6, 133.0, 133.5, 136.1, 136.3, 145.0, 158.68, 164.5, 165.3; 31P

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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.joc.7b02828. Figure S1, Table S1, and characterization data of all new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Akihiro Ohkubo: 0000-0002-5676-6094 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Izumi Science and Technology Foundation.

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REFERENCES

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