2016 Volume 64 Issue 9 Pages 1315-1320
Cross-linking is a widely-used technology in the studies of DNA, RNA and their complexes with proteins. Intrinsically active alkylating moieties and photo-activated agents are chemically or enzymatically incorporated into nucleic acids. Thionucleobases resemble the corresponding natural bases, and form cross-links by UVA irradiation. They form cross-links only with a site in close contact, thereby allowing identification of the contacts within the nucleic acids and/or between the nucleic acids and proteins in complex nucleoprotein assemblies. On the other hand, the thionucleobase forms a cross-link less efficiently for the reaction with the opposite natural base in the DNA duplex. In this study, 6-thioguanine was connected to 2′-deoxyribose through an ethylene linker at the 1′-position (Et-thioG). The linker was expected to bring the 6-thio group close to the nucleobase in the opposite strand. In a duplex in which the 2′-deoxy-6-thioguanosine (6-thio-dG) did not form a crosslink, Et-thioG efficiently formed crosslink with a high selectivity for T by UVA irradiation, but with a much lower efficiency for dA, dG, dC, 5-methyl-dC or dU. Interestingly, the yield of the photo-crosslinked product with dT was effectively improved in the presence of dithiothreitol or sodium hydrosulfide (NaSH) at a low UVA irradiation dose. The efficient and selective cross-link formation at a low UVA dose may be beneficial for the biological application of Et-thioG.
The intra- and interstrand cross-linking of nucleic acids is a widely used technology for a variety of purposes; i.e., the induction of DNA damage, regulation and manipulation of genes, fixation of DNA nano-structure, etc. DNA cross-linking agents are chemically or enzymatically incorporated into nucleic acids, which include psoralen, 3-cyanovinylcarbazole, aziridine, quinone methide, aldehyde, abasic site, disulfide, benzophenone, bisamide, alkylating anticancer agents, etc. The 2-amino-6-vinylpurine derivative and 5-methyl-4-vinylpyrimidine-2-one have been developed by our group as unique cross-linking agents.1–4) 6-Thioguanosine, 4-thiothymine and 4-thiouridine are also cross-linking agents, and are characteristic in that they are activated by UVA irradiation to form a cross-link only with the close contact-site either at the nucleic acid or the protein in the complex.5) Because of this unique property, despite little knowledge about the photo-adducts, the thionucleobases have been extensively used to identify the close contact-sites of the nucleic acid. We have applied 6-thio-2′-deoxyguanosine (6-thio-dG) as a platform for the functionality-transfer reaction in the site-specific chemical modification of nucleic acids.6–8) In the meantime, we became interested in the photo-induced interstrand cross-link of 6-thio-dG in the duplex DNA. However, the literature9) and our own experiments indicated that 6-thio-dG in the oligonucleotide generates little photo-cross-linking with normal bases in the opposite DNA strand. The absorbed energy of 6-thio-dG might be consumed for other reactions such as the activation of molecular oxygen. In this study, we found that the newly designed 6-thioguanine derivative (Et-thioG, 1) connected with the 2′-deoxyribose unit via the ethylene linker efficiently generated a photo-induced interstrand crosslink with a high selectivity to the thymidine base in the opposite strand.
In designing a new 6-thioguanine derivative, we took into account that the cross-linking reaction of 6-thio-dG requires close contact with the target base. Thus, the 6-thioguanine base was designed to connect to the 2′-deoxyribose unit through the ethylene spacer (Et-thioG, 2). It was expected from molecular modeling that the distance between the thiocarbonyl part and the 5,6-double bond of the thymine base became closer in the duplex (Fig. 1).
The synthesis of ODN1 incorporating 1 is summarized in Chart 1. The transformation from 2-deoxyribose to the key intermediate 2 was performed as described in previous reports.10,11) The chloride atom of 2 was displaced by cyanoethanethiol, and the protecting groups were removed, then the 2-amino group was protected by the Pac group to afford the diol derivative (3). The phosphoroamidite precursor (4) was synthesized from 3, which was applied to the DNA synthesizer. The synthesized ODN1 was cleaved from the resin using NaSH in a 1 M NaOH solution, then purified by HPLC. Deprotection of the 4,4′-dimethoxytrityl (DMTr) group was shortly done in 10% AcOH for 5 min, because a prolonged treatment in 10% AcOH caused desulfurization. The obtained ODN1 was finally purified by HPLC, and its structure was determined by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)/MS and quantified by UV absorbance.
a) 1) HS(CH2)2CN, N-methylpyrrolidine, CH2Cl2, 2) phenoxyacetyl chloride, 1-HBT, pyridine, CH3CN, 3) BF3–Et2O, Me2S, b) 1) DMTrCl, Pyridine, 2) DIPEA, (i-Pr)2NP(Cl)OC2H4CN, CH2Cl2, c) 1) DNA Synthesizer, 2) 0.01 M NaSH, 1 M NaOH, 3) HPLC purification, 4) 10% AcOH, 5) HPLC purification.
The photo-cross linking was investigated using ODN1 and its complementary sequence ODN2 (5′-d(AGA AAG GAG AAYAAA G)-3′), which contains the natural nucleotide Y on the side opposite to Et-thioG of ODN1 (Fig. 2). The ODN1 formed stable duplexes with ODN2 with the melting temperature of 41.5°C (Y=dT), 41.2°C (Y=dA), 43.2°C (Y=dG), 44.5°C (Y=dC), 44.7°C (Y=d5mC) and 41.7°C (Y=dU) under the reaction conditions. At first, the solution of ODN1 and ODN2 in the buffer (50 mM Tris–HCl, 100 mM NaCl, 10 mM MgCl2, dA as the internal standard, pH 7.0) was irradiated by a high-pressure mercury lamp (500 W, 365 nm) for 10 min at ambient temperature, then the mixture was analyzed by HPLC. When ODN2 with Y=T is on the site opposite to Et-thioG, the new peak ODN3 appeared, while ODN1 disappeared and ODN2 decreased after irradiation for 10 min. ODN3 was determined to be the cross-linked product by the MALDI-TOF/MS measurement ([ODN3]− calcd 9815.64, found 9814.63). The chemical yield of ODN3 was calculated based on the dA peak as the internal standard. Figure 2A illustrates the HPLC charts before and after the UVA irradiation. In this case, the yield of the cross-linked ODN3 was calculated to be 54%. The other natural bases formed little cross-linked product (Fig. 2B), demonstrating the selective cross-linking of Et-thioG to the opposing T. As ODN1 incorporating 6-thio-dG did not form a cross-link with any natural base at Y in ODN2, the ethylene spacer is the major contributor for this selective cross-linking to T. In the case of 2′-deoxyuridine at Y of ODN2, the yield of the photo-cross-link was significantly reduced, showing the importance of the 5-methyl group of the thymine base for the cross-link formation.
5-Methyl-2′-deoxycytidine is represented as d5mC. The photo-cross-link reaction was performed using 10 µM of ODN1 and 10 µM of ODN2 in the buffer containing 50 mM Tris–HCl, 100 mM NaCl, 10 mM MgCl2 and dA as the internal standard at pH 7.0 and ambient temperature. UVA was irradiated at 365 nm for 10 min by a 500 W high-pressure mercury lamp. HPLC conditions; column: SHISEIDO C18, 4.6×250 mm; solvent: A: 0.1 M TEAA buffer, B: CH3CN, B: 10% to 30%/20 min, 30% to 100%/25 min, linear gradient; flow rate: 1.0 mL/min; UV 254 nm.
A part of ODN2 remained unreacted after ODN1 was consumed (Fig. 2A), mostly because ODN1 suffered from a side reaction such as the photo-oxidation of the 6-thioguanine unit.12) Therefore, we next checked the effect of a reducing agent to improve the cross-linking yield. When the 500W high-pressure mercury lamp was used, contrary to expectation, reducing agents, such as dithiothreitol (DTT), reduced the yield (Fig. 3). Interestingly, although UVA irradiation at 365 nm using a 4W UV lamp resulted in a lower efficiency for the photo-cross linking, DTT and NaSH drastically improved the yield up to 66 and 45%, respectively (Fig. 3). An efficient and selective cross-linking to T at a low UVA irradiation dose may be beneficial for the application of Et-thioG.
The photo-cross-link reaction was performed in the absence or presence of the additive (5 mM) and analyzed in the same way as described in the caption of Fig. 2. DTT: dithiothreitol, GSH: glutathione, TCEP: tris(2-carboxyethyl)phosphine, NaSH: sodium hydrosulfide. H: UVA irradiation by a 500 W high-pressure mercury lamp, L: UVA irradiation by a 4 W handy UV lamp.
The initial cross-linked product ODN3 was found to give multiple HPLC peaks after isolation and storing in the buffer (Fig. 4A). The peak change was faster either at pH 9 or 5 than at pH 7. As their MS spectra were not different from ODN3, the initial ODN3 might be transformed into a mixture of isomers.
The discussion about this photo-crosslink formation is summarized in Chart 2. The UVA irradiation activates 6-thioguanine (I), which generates O2− by Type I and 1O2 by Type II photoreactions.5,13) When the thymine base is not close to the activated 6-thioguanine (I), these reactive oxygen species (ROS) react with the 6-thioguanine part.8) When the thymine base is within close contact with the activated 6-thioguanine (I), the 2+2 photo-cycloaddition can occur to form the interstrand cross-link (III).14) The reducing agents, such as DTT, prevent the oxidation of the 6-thioguanine part by reacting with ROS, and increase the yield of the crosslinked adduct. In the case of a high UVA irradiation dose by a 500 W lamp, the initially formed adduct might suffer from photo-degradation. Instability of the crosslinked adduct is shown in a HPLC chart of the photo-reaction (Fig. 2A). The 5-methyl group of the thymine base can stabilize the biradical intermediate (II), thereby T forms a cross-link (III) more efficiently than dU. The formed thietane structure is generally unstable and undergoes C–S bond cleavage via ionic and/or diradical mechanisms, leading the latter to the open form (IV) by subsequent hydrogen abstraction. The MS values of the cross-linked products (III, IV) accord with the observed ones. The thiol group was hydrolyzed to give the hydroxylated structure (V). Aromatized purine structures are also possible for IV and V. The electrospray ionization (ESI)-MS data of the degradation product by partial hydrolysis suggests a plausible structure to be as shown in VI. The transformations from III to IV and V are reflected by the HPLC changes to multiple peaks as shown in Fig. 4A; the major peak at 0 h and that at 6 h are thought to correspond to the initial product III and the isomerized product IV, respectively, and other minor peaks might contain further isomerized products including V.
ODN3 was subjected to enzymatic hydrolysis using BAP and VPDE for 1 h in the buffer at pH 9 containing 50 mM Tris–HCl and 1 mM MgCl2.
To confirm the cross-linked structure, the mixture of the cross-linked ODN3 was subjected to enzymatic hydrolysis using bacterial alkaline phosphatase (BAP) and Viper’s venom phosphodiesterase (VPDE) for 1 h in the buffer at pH 9 containing 50 mM Tris–HCl and 1 mM MgCl2 and analyzed by LC/ESI-MS. In a partially hydrolyzed mixture, a new peak was detected at 34 min in addition to T, dA, dG and dC (Fig. 4B), which showed a peak at m/z=698.15 in the extracted ion chromatogram (XIC). This MS value is consistent with the expected value for VI ([M−H]− m/z 698.1594). Although the cross-linked adduct was not directly confirmed, the formation of VI strongly support that the photo-cross linking took place with the thymine base. The remaining 5′-phosphate groups of the fragment VI may be rationalized in terms of inhibition of the dephosphorylation due to its non-natural nucleotide structure.15) Prolonged degradation reaction resulted in the formation of a complex mixture, probably because of instability of the cross-linked adduct.
In conclusion, Et-thioG was designed to connect the 6-thioguanine base to 2′-deoxyribose through an ethylene linker at the 1′-position (Et-thioG) to bring the 6-thio group close to the nucleobase in the opposite strand, and it has been successfully demonstrated that 6-thio-dG forms cross-links by UVA radiation only with a site in close contact. Thus, Et-thioG has overcome a drawback of 6-thio-dG as a photo-cross linking agent in that it forms a cross-link less efficiently with natural nucleobases in the DNA duplex. Interestingly, the yield of the photo-crosslinked product with dT was effectively improved in the presence of dithiothreitol or NaSH at a low UVA irradiation dose. The efficient and selective cross-link formation at a low UVA dose may be beneficial for the biological application of Et-thioG.
All solvents were purchased from commercial suppliers and used without purification. The 1H-NMR spectra were recorded by Varian UNITY-400 spectrometers. The IR spectra were recorded on a Perkin-Elmer Spectrum One FTIR spectrometer. The mass spectra were recorded by an Applied Biosystems Mariner System 5299 spectrometer.
The Ethyl Linker Derivative of S-(2-Cyanoethyl)-6-thioguanine Connected with β-D-Ribofuranose Part (3)A solution of 27) (1.21 g, 2.02 mmol), 2-cyanoethanethiol ((2.06 mL, 20.2 mmol) and N-methylpyrrolidine (1.82 mL, 20.2 mmol) in dry CH2Cl2 (40 mL) was stirred for 24 h at room temperature under argon. The mixture was diluted with CH2Cl2, washed with aqueous 1 M KH2PO4 solution, dried over Na2SO4, and evaporated. The residue was purified by silica gel column chromatography (Kanto 60 N, hexane–AcOEt=1 : 3) to give the 5′-O-trity-3′-O-MOM protected derivative of 3 as a colorless caramel (1.08 g, 83%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.68 (1H, s), 7.46 (6H, dd, J=7.8, 1.2), 7.32−7.21 (9H, m), 4.90 (2H, br s), 4.59 (2H, s), 4.31-4.03 (5H, m), 3.53 (2H, t, J=7.4), 3.28 (3H, s), 3.17 (2H, d, J=3.2), 2.87 (2H, t, J=7.4), 2.21–2.17 (1H, m), 2.04–1.95 (2H, m), 1.73–1.66 (1H, m). 13C-NMR (125 MHz, CDCl3) δ (ppm): 159.01, 151.21, 144.06, 141.17, 128.88, 127.97, 127.21, 125.99, 118.48, 95.46, 86.82, 84.38, 78.98, 75.43, 64.61, 55.55, 41.06, 38.74, 35.46, 24.30, 18.97. IR (cm−1): 3340 (w), 3197 (w), 2933 (w), 2249 (w), 1593 (m), 1550 (s), 1449 (m), 1403 (m). High resolution (HR)-ESI-MS (m/z): Calcd for C36H39 N6O4S [M+H]+ 651.2748. Found 651.2738.
The above compound (630 mg, 0.97 mmol) was dried by azeotrope with CH3CN and dissolved in dry CH3CN (3 mL) and dry pyridine (1.5 mL), followed by the addition of 1-hydroxybenzotriazole (327 mg, 2.4 mmol) and molecular sieves 4 Å, then the mixture was stirred for 3 h, followed by the addition of phenoxyacetyl chloride (0.66 mL, 4.8 mmol). The mixture was stirred for 59 h at room temperature, diluted with AcOEt, filtered through a celite pad, and the filtrate was washed with brine, dried over Na2SO4, and evaporated. The residue was purified by silica gel column chromatography (Kanto 60 N, CHCl3–AcOEt=2 : 1) to give the 5′-O-trity-3′-O-MOM protected 2-phenoxyacetyl amino derivative of 3 as a colorless caramel (744 mg, 98%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.87 (1H, s), 7.94 (1H, s), 7.45 (6H, d, J=7.6), 7.37–7.21 (9H, m), 7.08–7.00 (3H, m), 6.94 (2H, d, J=8.4), 4.68 (2H, s), 4.58 (2H, s), 4.39–4.22 (3H, m), 4.03–3.91 (2H, m), 3.58 (2H, t, J=7.0), 3.27 (3H, s), 3.18 (2H, t, J=5.0), 3.14 (2H, t, J=5.0), 2.25–2.00 (4H, m). 13C-NMR (125 MHz, CDCl3) δ (ppm): 171, 28, 165.99, 160.06, 157.19, 151.04, 149.97, 144.03, 143.60, 130.02, 128.90, 128.84, 127.96, 127.23, 122.60, 118.64,115.07, 95.42, 86.83, 84.43, 78.90, 75.26, 68.02, 64.57, 55.54, 48.42, 41.54, 38.73, 35.34, 21.18, 18.72. IR (cm−1): 3396 (w), 2946 (w), 2331 (w), 1721 (m), 1578 (s). ESI-MS (m/z): Calcd for C44H45N6O6S [M+H]+ 785.31. Found 785.54.
BF3·Et2O (1.89 mL, 15 mmol) was added to a solution of the above compound (520 mg, 0.66 mmol) in Me2S (5.8 mL) at 0°C under argon. The mixture was stirred for 30 min, quenched with a saturated aqueous NaHCO3 solution, extracted with CHCl3, dried over Na2SO4, and evaporated. The residue was purified by silica gel column chromatography (Kanto 60 N, CHCl3–MeOH=19 : 1) to give 3 as a colorless caramel (180 mg, 55%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 9.13 (1H, s), 7.95 (1H, s), 7.37 (2H, t, J=8.0), 7.10–7.04 (3H, m), 4.70 (2H, s), 4.44–4.20 (6H, m), 3.85–3.74 (1H, m), 3.64–3.58 (1H, m), 3.08 (2H, t, J=6.8), 2.17–2.07 (4H, m). 13C-NMR (125 MHz, CDCl3) δ (ppm): 166.17, 160.01, 157.05, 150.91, 149.72, 143.34, 129.89, 128.63, 122.50, 118.37, 114.93, 87.06, 75.25, 72.98, 67.91, 62.89, 41.26, 35.67, 25.02, 18.61. IR (cm−1): 3395 (br), 2923 (w), 1698 (m), 1578 (s), 1494 (s). HR-ESI-MS (m/z): Calcd for C23H27N6O5S [M+H]+ 499.1758. Found 499.1771.
5′-O-Dimethoxytrity-3′-O-2-cyanoethyl Diisopropylphosphoramidite Precursor (4)The diol derivative 3 (50 mg 0.10 mmol) was dried by azeotrope with CH3CN and pyridine and dissolved in dry pyridine (0.7 mL), then the solution was dried over molecular sieves 4 Å for 30 min. Dimethoxytrityl chloride (84.7 mg, 0.25 mmol) was added to this solution. The mixture was stirred for 6 h, diluted with AcOEt, washed successively with water, brine and water, dried over Na2SO4, then evaporated. The residue was purified by silica gel column chromatography (Kanto 60 N, CHCl3–0.5%pyridine) to give the 5′-O-dimethoxytrity derivative of 3 as a yellow oil (64 mg, 80%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.92 (1H, s), 7.89 (1H, s), 7.42–6.96 (14H, m), 6.79 (4H, d, J=9.2), 4.65 (2H, s), 4.34–4.26 (3H, br), 4.11–4.08 (1H, br), 3.92–3.90 (1H, br), 3.74 (6H, s), 3.53 (2H, t J=7.2), 3.21–3.02 (4H, m), 2.21–1.73 (4H, m). IR (cm−1): 2917 (w), 2249 (w), 1718 (w), 1577 (m), 1508 (m), 1495 (m). HR-ESI-MS (m/z): Calcd for C44H44N6O7S [M+H]+ 801.3065. Found 801.3061.
The above product (52.4 mg 0.062 mmol) was dried by azeotrope with CH3CN and dissolved in CH2Cl2 (1 mL). Diisopropylamine (65 µL) was added to this solution and the mixture was stirred for 30 min at 0°C under argon, then 2-cyanoethyl diisopropylchlorophosphoramidite (42 µL, 0.19 mmol) was added to the solution, and the mixture was stirred for 1 h. The reaction mixture was quenched by the addition of a saturated aqueous NaHCO3 solution, extracted with AcOEt, dried over Na2SO4, and evaporated. The residue was purified by flash chromatography (Kanto 60 N, hexane–AcOEt=1 : 1) to give the phosphoramidite precursor (4) as a gray oil (50 mg), which was re-solidified in a hexane solution at −80°C to give 4 as a gray solid (45 mg, 73%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.84 (1H, s), 7.91 (1H, s), 7.45–6.78 (18H, m), 4.65 (2H, s), 4.40–4.31 (3H, br), 4.11–4.03 (2H, br), 3.75 (6H, d, J=2.0), 3.73–3.46 (4H, m), 3.18–3.08 (4H, m), 2.54 (1H, t, J=6.4), 2.41 (1H, t, J=6.4), 2.24–1.768 (6H, m), 1.27–1.03 (12H, m). 31P-NMR (162 MHz, CDCl3) δ (ppm): 148.51, 148.48. IR (cm−1): 2966 (w), 2929 (w), 1720 (w), 1577 (s), 1508 (s) 1495 (m). HR-ESI-MS (m/z): Calcd for C53H61N8O8PSNa [M+Na]+ 1023.3908. Found 1023.3963.
DNA SynthesisThe oligonucleotides were synthesized by the automated DNA synthesizer according to the conventional amidite chemistry and the standard synthesis protocol. A bottle containing a solution of 4 in dry CH3CN was installed in the synthesizer together with the amidite precursors of the natural nucleotides, and 4 was incorporated at position X of ODN1 (5′-d(CTT TXTTC TCC TTT CT)-3′). After the synthesis, the resins were stored for 4 h at room temperature in a 1 M aqueous NaOH solution containing of 0.01 M NaSH. The reaction mixture was filtered though a membrane filter, and the filtrate was purified by HPLC (column: Cosmosil 5C18-MS-II, 10×250 mm, solvent: A: 0.1 M triethylammonium acetate (TEAA) buffer, B: CH3CN, B: 10 to 40%/20 min, linear gradient; flow rate: 1.0 mL/min; UV 254 nm). The HPLC chart showed that DMTr-protected ODN1 was synthesized in about 33% yield. The peak at 18.5 min was collected and freeze-dried. The residue was treated for 5 min with a 10% aqueous AcOH solution, and purified again by HPLC to give ODN1. The UV spectrum of ODN1 was measured for quantification. Analysis and purification were done by HPLC equipped with a ODS column (Cosmosil 5C18-MS-II, 10×250 mm, solvent: A: 0.1 M TEAA buffer, B: CH3CN, B: 10 to 30%/20 min, linear gradient; flow rate: 1.0 mL/min; UV 254 nm). The structure of ODN1 was confirmed by MALDI-TOF/MS [ODN1−H]−: Calcd for 4795.7. Found 4795.4. The UV spectrum of ODN shows an absorbance at λmax of 267 nm for the natural nucleotides and λmax of 338 nm for the 6-thioguanine part.
The UV Melting TemperatureUV Absorbance melting curves were obtained using 1.0 µM of each ODN1 and ODN2 in the buffer containing 50 mM Tris–HCl, 100 mM NaCl, 10 mM MgCl2. The temperature was raised at the rate of 1.0°C/min and the UV change was monitored at 260 nm.
Photo-irradiation for Interstrand Crosslink FormationA solution of ODN1 (10 µM) and ODN2 (10 µM)) was prepared in the buffer containing 50 mM Tris–HCl, 100 mM NaCl, 10 mM MgCl2 and dA as the internal standard, and adjusted to pH 7.0. High UVA irradiance at 365 nm was done using a ultra-high-pressure mercury lamp (500 W, Ushio Optical Modulex SX-UI500HQ) and a low UVA irradiance at 365 nm was done using a handy-type UV lamp (4 W, Funakoshi UVGL-25). The reaction vessel was irradiated by the UVA-Light for 10 min using the 500 W lamp or for 20 min using the 4 W Handy lamp at ambient temperature. The reaction mixture was analyzed by HPLC (column: SHISEIDO C18, 4.6×250 mm; solvent: A: 0.1 M TEAA buffer, B: CH3CN, B: 10 to 30%/20 min, 30 to 100%/25 min, linear gradient; flow rate: 1.0 mL/min; UV 254 nm). The newly appeared peak shown in Fig. 2A was isolated and confirmed to be the interstrand cross-linked product ODN3 by MALDI-TOF/MS [ODN3−H]−: Calcd for 9813.6. Found 9815.03.
Analysis of the Adduct Structure of ODN3 by Enzymatic HydrolysisThe cross-linked product ODN3 (ca. 200 pmol) obtained using 400 pmol each of ODN1 and ODN2 was hydrolyzed using BAP (bacterial alkaline phosphatase, 1.6 u) and VPDE (Viper’s venom phosphodiesterase, 0.4 u) in a buffer (20 µL) containing 50 mM Tris–HCl and 1 mM MgCl2 at pH 9. The reaction mixture after 1 h reaction was analyzed by LC/MS under the following HPLC conditions; XBD-C8 column (4.6×150 mm), solvent A: 5 mM HCOONH4, sovent B: CH3CN, a linear gradient of 10–40%B/40 min, flow rate of 0.2 mL/min, the column oven: 35°C. Peaks were monitored at 254 nm and the ESI/MS data were collected in the negative mode. Figure 4B shows the HPLC charts monitored at 254 nm and the extracted-ion chromatogram (XIC) at 698.15±0.3 for the analysis of the 1 h reaction mixture.
We are grateful for the support provided by a Grant-in-Aid for Scientific Research (B) (No. 15H04633) from the Japan Society for the Promotion of Science (JSPS).
The authors declare no conflict of interest.
