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New NitroG-Grasp Molecules with Enhanced Capture Reactivity for 8-Nitroguanosine in the Aqueous Media
2015 Volume 63 Issue 11 Pages 913-919
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2015 Volume 63 Issue 11 Pages 913-919
8-Nitroguanosine is formed by the nitration of guanosine, and has been conventionally classified as a genotoxic material. Recently, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) has become of great interest due to its biological role as an intracellular signaling molecule. In an attempt to develop recognition molecules for 8-nitroguanosine, we have determined a 1,3-diazaphenoxazine nucleoside derivative (nitroG-grasp) bearing a thiol group with a urea linker, which efficiently displaces the nitro group through the formation of multiple hydrogen-bonded complexes with 8-nitroguanosine. However, a drawback of this capture molecule was that the displacement efficiency was not sufficient to capture 8-nitroguanosine in water. Taking into account that both the flexibility of the linker and the pKa value of the thiol group are determinants of the reactivity, new nitroG-grasp molecules were designed to have a fixed linker structure with different pKa values. Compared to the previous nitroG-grasp, the new compounds with the (2-aminophenyl)methanethiol or the propylene acetal of 3-amino-1-mercaptopropan-2-one unit have exhibited more than 10–100 times faster reactivity in the aqueous media. These compounds may be potential components to construct new nitroG-grasp molecules to capture 8-nitro-cGMP in the biological systems.
A number of oxidizing chemical species exist in cells such as reactive oxygen species (ROS), reactive nitrogen species (RNOS), hypobromous acid (HOBr) and hypochlorous acid (HOCl).1) These oxidants are important for self-defense by oxidation of any extracellular invasion. However, they produce as oxidative stress for cells and cause nucleic acid damages, such as the 8-oxoguanine,2) 8-nitroguanine3,4) or 8-haloguanine derivatives.5) They are highly mutagenic and are regarded as biomarkers of oxidative stress.6) 8-Nitroguanosine (8-nitroG) is frequently generated in inflammatory or infected tissues, and is conventionally classified as a genotoxic materials.7) Recently, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) has become of significant interest because of its role in intracellular signal transduction system.8–11) A metabolic pathway of 8-nitro-cGMP includes its production in response to the inducible nitric oxide synthase (iNOS) activity, reaction with protein sulfhydryl groups to form cGMP adducts (protein S-guanylation), and also transformation to 8-SH-cGMP by the reaction with endogenous H2S/HS−.12) Thus, a new metabolic cycle or signaling pathway has been proposed with regard to 8-nitro-cGMP.13,14) Accordingly, specific molecules for the 8-nitroguanosine derivatives have potentials as chemical sensors for oxidative stress and a lead compound for targeting signaling pathways. Antibodies for the 8-nitroguanosine derivatives have been reported,15) however, it is only recently that small-molecular recognition molecules have been developed by our group.16)
In a series of our studies, we have relied on a 1,3-diazaphenoxaine-2-one skeleton as a platform structure of recognition molecules for the 8-oxidized guanosine derivatives. This skeleton provides a hydrogen bonding site which is complementary to the Watson–Crick face of the guanine base. Differences in the hydrogen bonding mode of the 7- and 8-positions of the 8-oxidized may be differentiated by the additional linker conjugated at the 9-position of the diazaphenoxaine skeleton. Thus oxoG-clamp (1) was developed for the first time as a selective fluorescent probe of 8-oxo-2′-deoxyguanosine (8-oxo-dG), which provides a carbamate oxygen for forming a hydrogen bond with 7N–H of 8-oxo-dG.17–19) Subsequently, the urea NH of the 9-linker was found to form a hydrogen bond with 7N of dG,20) and it has been successfully applied to nitroG-grasp (2). The thiol nucleophile of the urea linker of 2 efficiently displaces the nitro group to form a covalent bond with 8-nitroguanosine (Chart 1). The advantage for the reaction, which is due to the close proximity in the multiple hydrogen-bonded complex, has also been utilized in the design of thioG-grasp (3) for the alkylation of 8-thioguanosine21) (Fig. 1). In our continued effort to apply these compounds in aqueous media, this study has been aimed at improving the reactivity of nitroG-grasp. Our previous studies have established followings: (i) the reaction takes place within the multiple-hydrogen bonded complex, (ii) the reactivity is not proportional to pKa of the thiol group, (iii) that the flexibility of the linker of the thiol group is a determinant of the reactivity.16–21) Taking into consideration these factors, new nitroG-grasp derivatives were designed to have a linker with the 2-hydroxypropanethiol (5), the propanethiol unit fixed by the 1,3-dioxane ring (6) and (2-amonophenyl)methanethiol (7). Among them, the nitroG-grasp derivative (7) has exhibited more than 100 times faster reactivity in aqueous media. We now report the synthesis, evaluation and analysis of the reactivity of the new nitroG-grasp derivative.
The nitro group displacement reaction was postulated to take place through a complex between nitroG-grasp and 8-nitroG as shown in Fig. 1. X represents a linker unit.
dR and R represents protected 2′-deoxyribosyl and ribosyl groups, respectively.
It was determined in a previous study that the propyl linker of nitroG-grasp (2) exhibited the most efficient reactivity for 8-nitroG, and that the reactivity of the thiol group primarily depends on the flexibility of the alkyl linker, and secondly on the pKa values of the thiol group.16) Accordingly, new nitroG-grasp derivatives were designed to make the linker unit (X) more rigid (Chart 1). The 2-hydroxypropyl linker of 5 might have a lower pKa value of the thiol, and the 1,3-dioxane unit of 6 was expected to direct the urea and the thiol unit linker into the same direction against the dioxane ring, and the benzylthiol unit of 7 would have both a rigidity and lower pKa value. The pKa values shown in parenthesis of the compound number in Fig. 2 were the theoretically predicted ones for the corresponding urea part.
The designed nitroG-grasp derivatives (5–7) were synthesized according to a previous method.16–21) The 3′,5′-O-diTBDMS (tert-butyldimethylsilyl)-G-clamp unit as a common intermediate was conjugated with the S-trityl protected amino compounds (10, 12, 15) using carbonyldiimidazole. The hydroxyl amine linker 10 was synthesized from (R)-N-glycidilphthalimide by nucleophilic attack of triphenylmethanethiol, following hydrazinolysis of the phthalimide group. Oxidation of the hydroxyl group of compound 9 gave the intermediate ketone using dimethyl sulfoxide–acetic anhydride (DMSO–Ac2O), which was protected with 2,2-dimethyl-1,3-propanediol forming the dioxane 11. Similarly, the hydrazinolysis of 11 was performed to obtain the 1,3-dioxane linker unit 12 and conjugated with the 3′,5′-O-diTBDMS-G-clamp. The benzyl linker part 15 was synthesized by introduction of the S-trityl group to 2-nitrobenzyl chloride and reduction using hydrogenation of the nitro group in 14. After deprotection of the S-trityl group in 17–19 by trifluoroacetic acid (TFA) in the presence of triisopropylsilane, the designed thiol compounds (5–7) were finally obtained in a moderate yield (Chart 2).
a) n-BuLi, triphenylmethanethiol, THF −78°C, to −10°C, 91%. b) NH2NH2–H2O, EtOH, r.t., 77% for 10, 99% for 12. c) i) Ac2O, DMSO, r.t., 46%. ii) 2,2-Dimethyl-1,3-bis(trimethylsiloxy)propane, TMS-OTf, CH2Cl2, 0°C to r.t., 95%. d) Triphenylmethanethiol, DIPEA, CH3CN, r.t., 82%. e) 5% Pd/C, H2 gas, THF/EtOH=1 : 1, r.t., 74%. f) 10, 12 or 15, carbonyldiimidazole, ClC2H4Cl, r.t. to 80°C, 82% for 17, 96% for 18, 77% for 19. g) TFA, iPr3SiH CH2Cl2, 0°C, 77% for 5, 64% for 6, 69% for 7.
TriAc-nitroG (8-nitro-2′,3′,5′-tri-O-acetylguanosine, 4)10) was used as a target molecule in this study. At first, the nitro group displacement reaction shown in Chart 1 was investigated in acetonitrile (CH3CN) in the presence of triethylamine (TEA). Figure 3A represents an example of HPLC for the reaction using 4 and 7 in CH3CN, indicating that both 4 and 7 were almost consumed to form the adduct as the sole product after 5 min. In CH3CN, all compounds (5–7) rapidly formed the adducts at similar rates with the parent nitroG-grasp (2) (Fig. 3B). The adduct obtained in the reaction with 7 was isolated, and its structure was determined by 1H-NMR, electrospray ionization (ESI)-MS and heteronuclear multiple bond connectivity (HMBC). The HMBC spectrum showed a correlation between the 8-carbon atom of the 8-thioguanine unit and the methylene protons next to the sulfur atom (benzyl position). On the other hand, when the reaction was performed in CH3CN containing 20% water at pH 7.0, the nitroG-grasp (2) formed the adduct at a much slower rate (Fig. 3C, rhombuses), illustrating a drawback of 2 for the reaction in aqueous media. Interestingly, the new nitroG-grasp compounds retained a high reactivity under the same conditions. In particular, the compound with the dioxane linker (6) and that with the benzyl linker (7) exhibited an efficient reactivity (Fig. 3C, squares and triangles). The reaction by 7 proceeded efficiently at a similar rate at concentrations as low as 1 µM, strongly suggesting that the NO2-displacement reaction with 7 takes place within the complexes not by a simple bimolecular reaction. Relatively slow reactions were observed for the compound with the 2-hydroxypropyl linker (5) (Fig. 3C, circles). It should be noted that the new nitroG-grasp molecules (6, 7) have overcome the problem of 2, in that the efficient capture of 8-nitroguanosine can be achieved in aqueous media at pH 7.0. It was found during these experiments that compound 7 formed the homo-disulfide with the half-life of 1 h, which was inhibited in the presence of dithiothreitol (DTT). Compounds 5 and 6 remained unchanged for 24 h under the same conditions. Accordingly, the nitroG-grasp (6, 7) may be potential candidates for further application for targeting 8-nitro-cyclic GMP (8-nitro-cGMP) under physiological conditions.
To reveal the origins of the efficient reactivity of the new nitroG-grasp molecules in the aqueous CH3CN solution, we next evaluated the reaction kinetics by monitoring the reaction by UV-Vis spectroscopy. The absorbance at 368 nm due to the 8-nitroguanine base gradually decreased with desorption of the nitro group.22) Simultaneously, the absorbance at 260 nm increased with formation of the 8-thioguanine base.23) Thus, the absorbance at 400 nm was monitored in the presence of excess nitroG-grasp derivatives to obtain the kinetic parameters of the pseudo-first-order reaction. The obtained kobs values were in the order of 7 (0.0212 s−1)>6 (0.0025 s−1)>5 (0.0008 s−1)>2 (0.0002 s−1), agreeing with the results determined by HPLC (Fig. 3C). The new nitroG-grasp 7 exhibited about a 100-fold higher reactivity compared to the original nitroG-grasp (2) in aqueous CH3CN at pH 7.0. The reaction was performed at different temperatures (30, 35, 40, 45, 50, 55°C), and the kinetic parameters were obtained by Arrhenius plots (Table 1). The new derivatives 5–7 showed lower −TΔS‡ values than the nitroG-grasp 2. Notably, the reaction of 7 exhibited the lowest values of ΔG‡ and ΔS‡. Although its ΔH‡ term was similar to that of 2, the highly favorable ΔS‡ term produced the lowest ΔG‡ value. The favorable ΔS‡ value of 7 may be explained in terms of the fixed conformation of the linker of 7. The conformation of the 2-hydroxyl linker of 5 is more flexible than that of the benzyl linker of 7, but is more restricted compared to the propyl linker of 2 by the gauche effect.24) The dioxane structure of 6 is also preferable for conformational restriction, though its effect is only 3.0 kJ/mol at 300 K.
It has been shown in our previous study that the pKa of the thiol of 2 is reflected in ΔH‡, and the linker length is a determinant of ΔS‡. However, there was no linear correlation between pKa and the ΔH‡ values in this case. Interestingly, the ΔH‡ values showed a linear relationship with the calculated log P (CHCl3/water) values of the linker units (Fig. 4). The ΔH‡ term is proportional to Ea, and may reflect the energy level of the transition complex in the displacement reaction.25) Accordingly, it can be reasonably interpreted that 6 is the most beneficial in terms of the ΔH‡ value due to the hydrophobic nature of the dioxane structure. A plausible intermediate structure of the reaction of 6 and triacetyl 8-nitroG was simulated by MM, suggesting that the acetyl groups of 8-nitroG are in the range of close contact with both the TBDMS groups and the dioxane part of 6 (Fig. 5). Thus, it is concluded that the rate enhancement of 6 is due to its favorable ΔH‡ term (linker lipophilicity) and that of 7 arises from the preferable ΔS‡ term (linker flexibility).
The reaction was performed using 0.4 mM each of nitroG-grasp and triAc-8-nitroG in the presence of 2 mM Et3N in CH3CN at 30°C. The aqueous media constituted of 0.1 M TEAA buffer (pH 7.0 containing 2 mM DTT) : CH3CN=1 : 4 solution containing 0.4 mM each of the reactants. HPLC conditions: column: Xbridge C8 3.5 µm, 3.0×100 mm; solvents: A: 0.1 M TEAA buffer and B: CH3CN, A/B=20 : 80; flow rate: 0.5 mL/min; monitored by UV at 254 nm.
| R-SH | kobs (10−2 s−1)b) | Ea (kJ/mol) | ΔG‡ (kJ) | ΔH‡ (kJ) | −TΔS‡c) (kJ) |
|---|---|---|---|---|---|
| 2 | 0.02 | 27.7 | 97.8 | 25.1 | 69.7 |
| 5 | 0.08 | 33.5 | 94.3 | 30.9 | 60.7 |
| 6 | 0.25 | 24.6 | 91.6 | 22.0 | 66.7 |
| 7 | 2.12 | 28.9 | 86.0 | 26.3 | 57.2 |
a) The pseudo-first order rate constants (kobs) were obtained by monitoring the absorbance at 400 nm using nitroG-grasp (200 µM) and triAc-nitroG (10 µM). The reaction was performed at different temperatures to obtain the kinetic parameters. b) The rate constants were measured at 30°C. c) The T value is 300 K (27°C).
The log P values were computationally calculated using MacroModel.
The 8-nitroguanosine metabolite, such as 8-nitroguanosine 8-nitro-cGMP, has become of significant interest due to its biological role as an intracellular signaling molecule. The 1,3-diazaphenoxazine nucleoside derivatives (nitroG-grasp) have been developed as recognition molecules for the covalent capture of 8-nitroguanosine. In this study, new nitroG-grasp molecules were designed to achieve the efficient reaction in aqueous media, and we have successfully demonstrated that new nitroG-grasp molecules with a rigid linker connecting the thiol and the urea parts exhibited an improved reactivity in aqueous media at pH 7.0. The major effects of the rigid linker were reflected in the favorable activation entropy term compared to the original nitroG-grasp. Considering that the benzylthiol-nitroG-grasp (7) formed the homo-disulfide derivative under aerobic conditions, dioxane-nitroG-grasp (6) may be a good candidate for further study to develop the nitroG-grasp molecule for the capture of 3′,5′-cyclic monophosphate of 8-nitroguanosine in aqueous media. Such studies are now underway in our group along this line, which will be reported in near future.
All solvents were purchased from commercial suppliers and used without purification. The 1H-NMR, and 13C-NMR spectra were recorded by Varian UNITY-400 and Bruker AVANCE-III 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.
(R)-S-Trityl-N-(3-mercapto-2-hydroxypropyl)phthalimide (9)n-BuLi (1.6 M, 0.61 mL, 1 mmol) was dropwise added to a mixture of triphenylmethanethiol (203 mg, 1 mmol) in dry tetrahydrofuran (THF) (10 mL) at −78°C under an argon atmosphere. N-Glycidil phthalimide (276 mg, 1 mmol) in THF was dropwise added to this mixture at −78°C. After 1h, the reaction mixture was warmed to 0°C, stirred for 3 h, quenched with 10% acetic acid (AcOH) in methanol (MeOH) (5 mL) at 0°C and evaporated in vacuo. The residue was extracted with AcOEt and H2O. The organic layer was dried over sodium sulfate (Na2SO4) and evaporated in vacuo. The crude product was purified by flash chromatography (Hex/AcOEt=78/22 to 57/43) to afford compound 10 (437 mg, 91%) as a white foam. 1H-NMR (500 MHz, CDCl3) δ (ppm): 7.83 (2H, dd, J=5.5, 3.5 Hz), 7.72 (2H, dd, J=5.5, 3.5 Hz), 7.41 (6H, d, J=7.5 Hz), 7.25 (6H, t, J=7.5 Hz), 7.16 (3H, t, J=7.5 Hz), 3.63–3.61 (2H, m), 3.56–3.54 (1H, m), 2.49 (1H, dd, J=13.0, 7.5 Hz), 2.37 (1H, dd, J=13.0, 5.0 Hz). 13C-NMR (125 MHz, CDCl3) δ (ppm): 168.6, 144.7, 132.1, 129.7, 128.1, 126.9, 123.5, 69.13, 67.21, 43.25, 37.32. IR (cm−1): 3308, 1771, 1710, 1393. High resolution-electrospray ionization (HR-ESI)-MS (m/z): Calcd for C30H25NO3S (M+Na+) 502.1447. Found 502.1481.
(R)-S-Trityl-3-mercapto-2-hydroxypropylamine (10)Hydrazine monohydrate (13 µL, 0.26 mmol) was dropwise added to a solution of 9 (64 mg, 0.133 mmol) in dry EtOH at room temperature under an argon atmosphere. The reaction mixture was stirred at room temperature for 2 d and filtered. The resulting filtrate was evaporated in vacuo. The crude product was purified by flash column chromatography (CHCl3/MeOH=90/10 to 80/20 linear gradient) to afford compound 7 (36 mg, 77%) as a colorless foam. 1H-NMR (400 MHz, methanol-d4 (CD3OD)) δ (ppm): 7.40 (6H, d, J=7.4 Hz), 7.28 (6H, t, J=7.4 Hz), 7.21 (3H, t, J=7.4 Hz), 3.45–3.39 (1H, m), 2.63 (1H, dd, J=13.2, 3.6 Hz), 2.44 (1H, dd, J=13.2, 8.4 Hz), 2.37 (1H, dd, J=12.4, 6.4 Hz), 2.25 (1H, dd, J=12.4, 6.4 Hz). 13C-NMR (125 MHz, CD3OD) δ (ppm): 146.2, 130.8, 128.9, 127.9, 126.9, 70.9, 67.9, 46.6, 37.7. IR (cm−1): 3143, 1573, 1485, 1444. HR-ESI-MS (m/z): Calcd for C22H23NOS (M+H+) 350.1573. Found 350.1591.
S-Trityl-N-((3-mercapto-2,2-(2,2-dimethyltrimethylenedioxy)propane-1-yl))-phthalimide (11)Ac2O (0.4 mL) was dropwise added to a solution of 9 (48 mg, 0.1 mmol) in dry DMSO (0.6 mL) at room temperature under an argon atmosphere. The reaction mixture was stirred at room temperature for 20 h. The resulting precipitate in the reaction mixture was collected by filtration, and washed with CH3CN to obtain an intermediate ketone as a white solid (22 mg, 46%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.82–7.80 (2H, m), 7.71–7.69 (2H, m), 7.31–7.25 (5H, m), 7.42 (6H, d, J=7.4 Hz), 7.30 (6H, t, J=7.4 Hz), 7.24–7.22 (5H, m), 4.25 (2H, s), 3.18 (2H, s). 13C-NMR (125 MHz, CDCl3) δ (ppm): 197.8, 167.5, 144.1, 134.2, 132.2, 129.7, 128.4, 127.9, 127.3, 123.6, 67.9, 45.5, 40.3. IR (cm−1): 2933, 1776, 1717, 1414. ESI-MS (m/z): Calcd for C30H23NO3S (M+Na+) 500.1291. Found 500.1336.
Trimethylsilyl triflate (TMS-OTf) (0.14 mL, 0.75 mmol) was dropwise added to a mixture of the intermediate ketone (721 mg, 1.5 mmol) and 2,2-dimethyl-1,3-bis(trimethylsiloxy)propane26) (1.88 g, 7.5 mmol) in dry CH2Cl2 (20 mL) at −25°C under an argon atmosphere. The reaction mixture was allowed to warm to room temperature and stirred at room temperature. for 2 d. The reaction mixture was quenched with saturated aqueous sodium bicarbonate (NaHCO3) (40 mL), then extracted with AcOEt. The organic layer was dried over Na2SO4 and evaporated in vacuo. The resulting residue was purified by flash column chromatography (Hex/AcOEt=100/0 to 80/20 linear gradient) to afford 11 (807 mg, 95%) as a white foam. 1H-NMR (500 MHz, CDCl3) δ (ppm): 7.83 (2H, dd, J=5.5, 3.0 Hz), 7.70 (2H, dd, J=5.5, 3.0 Hz), 7.47 (6H, d, J=8.0 Hz), 7.26 (6H, t, J=8.0 Hz), 7.17 (3H, t, J=8.0 Hz), 3.94 (2H, s), 3.40 (2H, d, J=11.5 Hz), 3.25 (2H, d, J=11.5 Hz), 2.53 (2H, s), 0.78 (3H, s), 0.72 (3H, s). 13C-NMR (125 MHz, CDCl3) δ (ppm): 168.1, 144.7, 133.9, 132.2, 129.8, 128.0, 126.7, 123.4, 98.6, 70.7, 66.4, 42.7, 33.6, 29.4, 22.5, 22.4. IR (cm−1): 2951, 1777, 1719, 1390. HR-ESI-MS (m/z): Calcd for C35H33NO4S (M+Na+) 586.2023. Found 586.2048.
S-Trityl-1-amino-3-mercapto-2,2-(2,2-dimethyltrimethylenedioxy)propane (12)12 was synthesized using a procedure similar to that of compound 10. 1H-NMR (500 MHz, CDCl3) δ (ppm): 7.48 (6H, d, J=8.0 Hz), 7.29 (6H, t, J=8.0 Hz), 7.21 (3H, t, J=8.0 Hz), 3.28 (4H, s), 2.82 (2H, s), 2.51 (2H, s), 2.00 (2H, s), 1.04 (3H, s), 0.68 (3H, s). 13C-NMR (125 MHz, CDCl3) δ (ppm): 144.7, 129.8, 128.1, 126.9, 98.9, 70.6, 66.5, 48.2, 30.9, 29.6, 23.0, 22.4. IR (cm−1): 2955, 2336, 1488, 1101. HR-ESI-MS (m/z): Calcd for C27H31NO2S ([M+H]+) 434.2148. Found 434.2188.
S-Trityl-2-nitrobenzylmethanethiol (14)N,N'-Diisopropylethylamine (DIPEA) (1.29 g, 10 mmol) was dropwise added to a solution of 2-nitrobenzyl chloride (860 mg, 5 mmol) and triphenylmethanethiol (1.38 g, 5 mmol) in dry CH3CN (10 mL) under an argon atmosphere. The reaction mixture stirred at room temperature for 16 h, quenched with saturated aqeuous ammonium chloride (NH4Cl) and extracted with ether. The organic layer was washed with brine, dried over Na2SO4 and evaporated. The resulting residue was recrystallized in a Hex/AcOEt (1 : 1) solution to afford compound 14 (1.68 g, 82%) as a pale yellow solid. 1H-NMR (400 MHz, CD3OD) δ (ppm): 7.87 (1H, d, J=8.0 Hz), 7.44 (6H, d, J=8.0 Hz), 7.35 (1H, t, J=7.5 Hz), 7.28 (6H, t, J=8.0 Hz), 7.22 (3H, t, J=8.0 Hz), 6.87 (1H, d, J=7.5 Hz), 3.70 (2H, s). 13C-NMR (125 MHz, CDCl3) δ (ppm): 144.5, 133.3, 133.0, 132.6, 129.8, 128.1, 127.0, 125.0, 68.0, 34.0. IR (cm−1): 1526, 1489, 1348. HR-ESI-MS (m/z): Calcd for C26H21O2NS (M+Na)+ 434.1185. Found 434.1168.
S-Trityl-2-aminobenzylmethanethiol (15)AcOH (20 mg, 0.35 mmol) was dropwise added to a solution of 13 (206 mg, 0.5 mmol) and Pd/C (5%) (104 mg, 0.05 mmol) in THF/EtOH (1 : 1). The reaction mixture was stirred at room temperature under a H2 atmosphere for 2 d and filtered through celite. The resulting filtrate was evaporated in vacuo and purified by flash column chromatography (Hex/CHCl3=100/0 to 39/61, linear gradient) to afford compound 15 (142 mg, 74%) as a pale yellow amorphous material. 1H-NMR (500 MHz, CDCl3) δ (ppm): 7.53 (6H, d, J=8.0 Hz), 7.32 (6H, t, J=8.0 Hz), 7.24 (3H, t, J=8.0 Hz), 7.05–7.01 (2H, m), 6.68 (1H, t, J=7.5 Hz), 6.59 (1H, d, J=7.5 Hz), 3.26 (2H,s). 13C-NMR (125 MHz, CDCl3) δ (ppm): 145.1, 144.7, 131.0, 129.6, 128.8, 128.2, 128.1, 126.9, 120.7, 118.9, 116.3, 67.2, 33.9. IR (cm−1): 1622, 1491, 1444. HR-ESI-MS (m/z): Calcd for C26H23NS (M+Na)+ 404.1443. Found 404.1447.
General Synthesis of S-Trityl Protected NitroG-Grasp DerivativesCarbonyldiimidazole (1.2 eq) was added to a mixture of G-clamp 16 (1.0 eq) in dry dichloroethane at room temperature under an argon atmosphere. The reaction mixture was stirred at room temperature for 1–2 h, and corresponding S-trityl amine linker (10 or 12 or 15) was added to the reaction mixture. This reaction mixture was stirred at 70–80°C overnight, then the solvent was removed by evaporation. The resulting residue was purified by flash column chromatography (CHCl3/MeOH) to afford the protected nitroG-grasp derivatives (17–19).
S-Trityl Protected NitroG-Grasp (17) was synthesized using the G-clamp unit 16 and 10 in 82% yield as a pale yellow foam. 1H-NMR (500 MHz, CDCl3) δ (ppm): 7.53 (1H, s) 7.38 (6H, d, J=7.5 Hz), 7.23 (6H, t, J=7.5 Hz), 7.15 (3H, t, J=7.5 Hz), 6.74 (1H, t, J=7.5 Hz), 6.42 (1H, d, J=7.5 Hz), 6.32 (1H, d, J=7.5 Hz), 6.13 (1H, t, J=5.5 Hz), 4.37 (1H, q, J=5.5 Hz), 4.29 (2H, t, J=6.0 Hz), 3.97 (2H, t, J=4.5 Hz), 3.92 (1H, dd, J=11.5, 2.5 Hz), 3.87 (1H, quint, J=2.0 Hz), 3.77–3.75 (3H, m), 3.55–3.45 (3H, m), 3.28–3.25 (1H, m), 3.06–3.01 (1H, m), 2.45 (1H, dd, J=12.5, 6.5 Hz), 2.34–2.30 (2H, m), 2.05–2.00 (1H, m), 0.96 (9H, s), 0.88 (9H, s), 0.17 (3H, s), 0.15 (3H, s), 0.06 (6H, s). 13C-NMR (125 MHz, CDCl3) δ (ppm): 160.2, 144.9, 144.4, 143.1, 129.8, 129.6, 128.3, 128.0, 127.1, 126.7, 123.7, 115.5, 108.4, 107.6, 87.7, 86.0, 70.8, 70.6, 69.1, 66.7, 62.4, 45.6, 42.1, 39.7, 36.3, 31.1, 26.2, 25.9, 18.6, 18.1, −4.40, −4.72, −5.29, −5.35. IR (cm−1): 2932, 1673, 1556, 1473. HR-ESI-MS (m/z): Calcd for C52H69N5O8SSi2 (M+Na)+ 1002.4298. Found 1002.4306.
S-Trityl Protected NitroG-Grasp (18) was synthesized using the G-clamp unit 16 and 12 in 96% yield as a pale yellow foam. 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.80 (1H, s), 7.45 (6H, d, J=7.4 Hz), 7.27–7.22 (6H, m), 7.15 (3H, t, J=7.4 Hz), 6.89 (1H, t, J=8.4 Hz), 6.52 (1H, d, J=8.4 Hz), 6.35 (1H, d, J=8.4 Hz), 6.20 (1H, t, J=6.4 Hz), 6.03 (1H, br), 4.38–4.36 (1H, m), 4.04 (2H, t, J=4.8 Hz), 3.95–3.93 (2H, m), 3.76 (1H, d, J=10.0 Hz), 3.62 (2H, d, J=4.8 Hz), 3.48 (2H, t, J=6.4 Hz), 2.82 (2H, s), 3.32 (2H, d, J=10.0 Hz), 3.17 (2H, d, J=10.0 Hz), 2.45 (2H, s), 2.39–2.33 (1H, m), 2.08–2.02 (1H, m), 0.94 (12H, s), 0.87 (9H, s), 0.69 (3H, s), 0.15 (3H, s), 0.13 (3H, s), 0.05 (6H, s). 13C-NMR (125 MHz, CDCl3) δ (ppm): 158.7, 144.6, 143.1, 129.7, 128.1, 127.6, 126.8, 123.6, 108.4, 107.2, 98.8, 87.6, 86.1, 70.8, 70.6, 69.2, 66.5, 62.4, 42.1, 39.9, 31.9, 29.6, 26.2, 25.9, 22.8, 22.4, 18.6, 18.1, −4.41, −4.75, −5.31, −5.36. IR (cm−1): 2957, 1723, 1668, 1473. HR-ESI-MS (m/z): Calcd for C57H77N5O9SSi2 (M+Na)+ 1086.4873. Found 1086.4863.
S-Trityl Protected NitroG-Grasp (19) was synthesized using the G-clamp unit 16 and 15 in 77% yield as a pale yellow foam. 1H-NMR (500 MHz, CDCl3) δ (ppm): 7.62 (1H, br), 7.47–7.43 (7H, m), 7.29 (6H, t, J=7.5 Hz), 7.24–7.18 (4H, m), 7.10 (1H, d, J=7.5 Hz), 7.04 (1H, t, J=7.5 Hz), 6.76 (1H, t, J=7.5 Hz), 6.48–6.45 (2H, m), 6.37 (1H, d, J=7.5 Hz), 6.25 (1H, t, J=6.0 Hz), 4.39 (1H, q, J=6.0 Hz), 4.05 (2H, t, J=5.0 Hz), 3.94 (1H, dd, J=11.5, 2.0 Hz), 3.89 (1H, quint, J=2.0 Hz), 3.77 (1H, dd, J=11.5, 2.0 Hz), 3.58–3.54 (2H, m), 3.30 (1H, s), 2.33–2.28 (1H, m), 2.03–1.98 (1H, m), 0.96 (9H, s), 0.88 (9H, s), 0.17 (3H, s), 0.15 (3H, s), 0.06 (6H, s). 13C-NMR (125 MHz, CDCl3) δ (ppm): 156.2, 144.5, 143.2, 137.6, 136.7, 144.5, 143.2, 137.6, 136.7, 131.1, 129.8, 129.6, 128.7, 128.2, 127.7, 127.0, 125.5, 123.7, 119.0, 108.5, 107.3, 87.6, 86.0, 70.9, 68.9, 67.4, 62.4, 48.7, 43.4, 42.1, 39.9, 33.9, 31.1, 29.8, 26.2, 25.9, 18.6, 18.1, 14.3, −4.42, −4.75, −5.31, −5.36. IR (cm−1): 2954, 1674, 1556, 1473. HR-ESI-MS (m/z): Calcd for C56H69N5O7SSi2 (M+Na)+ 1034.4348. Found 1034.4378.
General Synthesis of NitroG-Grasp Derivatives 5–7TFA (2%) was dropwise added to a mixture of S-trityl protected substrates (17–19, 1 eq) and triisopropylsilane (5 eq) in dry CH2Cl2 at 0°C under an argon atmosphere. The reaction mixture was stirred at 0°C for 1 h and quenched with saturated aqueous NaHCO3, then extracted with AcOEt. The organic layer was dried over Na2SO4 and evaporated in vacuo. The resulting residue was purified by flash column chromatography (CHCl3/MeOH) to afford deprotected compounds 5–7.
NitroG-Grasp (5) was synthesized from 17 in 77% yield as a pale yellow amorphous. 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.72 (1H, s), 6.83 (1H, t, J=8.5 Hz), 6.61 (1H, d, J=8.5 Hz), 6.36 (1H, d, J=8.5 Hz), 6.18 (1H, t, J=6.5 Hz), 4.48–4.45 (1H, m), 4.36 (2H, t, J=5.0 Hz), 4.04 (2H, t, J=5.0 Hz), 3.95–3.89 (2H, m), 3.86–3.79 (3H, m), 3.66 (1H, quint, J=5.0 Hz), 3.54 (2H, t, J=5.0 Hz), 3.35 (1H, dd, J=14.0, 6.5 Hz), 3.19 (1H, dd, J=14.0, 5.0 Hz), 2.58 (1H, dd, J=14.0, 5.0 Hz), 2.50 (1H, dd, J=14.0, 6.5 Hz), 2.36–2.31 (1H, m), 2.15–2.10 (1H, m), 0.98 (9H, s), 0.92 (9H, s), 0.19 (3H, s), 0.17 (3H, s), 0.12 (6H, s). 13C-NMR (125 MHz, CD3OD) δ (ppm): 161.4, 156.5, 155.7, 148.1, 144.3, 138.9, 129.6, 129.1, 125.0, 123.5, 120.8, 109.2, 108.6, 89.3, 87.5, 79.5, 73.5, 72.9, 70.1, 63.7, 45.4, 44.7, 42.8, 40.4, 29.3, 26.7, 26.3, 19.4, 18.9, −4.45, −4.65, −5.25, 5.30. IR (cm−1): 2929, 1670, 1560, 1473. HR-ESI-MS (m/z): Calcd for C33H55N5O8SSi2 (M+H)+ 738.3383. Found 738.3392.
NitroG-Grasp (6) was synthesized from 18 in 66% yield as a pale yellow amorphous. 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.67 (1H, s), 6.85 (1H, t, J=8.5 Hz), 6.63 (1H, dd, J=8.5, 1.0 Hz), 6.38 (1H, dd, J=8.5, 1.0 Hz), 6.19 (1H, t, J=6.0 Hz), 4.50–4.48 (1H, m), 4.07 (2H, t, J=5.0 Hz), 3.98–3.94 (2H, m), 3.84 (1H, dd, J=12.5, 3.0 Hz), 3.56–3.54 (4H, m), 3.504 (2H, s), 3.495 (2H, s), 2.79 (2H, s), 2.37–2.32 (1H, m), 2.16–2.011 (1H, m), 0.98 (9H, s), 0.95 (3H, s), 0.93 (9H, s), 0.90 (3H, s), 0.20 (3H, s), 0.18 (3H, s), 0.12 (6H, s). 13C-NMR (125 MHz, CD3OD) δ (ppm): 161.14, 144.32, 129.61, 125.03, 109.16, 108.69, 99.86, 89.32, 87.50, 72.85, 71.47, 70.20, 63.71, 42.80, 40.45, 30.57, 26.60, 26.25, 22.83, 22.66, 19.38, 18.86, −4.46, −4.66, −5.26, −5.32. IR (cm−1): 2927, 1675, 1559, 1473. HR-ESI-MS (m/z): Calcd for C38H63N5O9SSi2 (M+H)+ 822.3958. Found 822.3939.
NitroG-Grasp (7) was synthesized from 19 in 69% yield as a pale yellow amorphous. 1H-NMR (500 MHz, CDCl3) δ (ppm): 7.58 (1H, s), 7.52 (1H, s), 7.19 (2H, t, J=7.5 Hz), 7.24–7.18 (4H, m), 7.10 (1H, d, J=7.5 Hz), 7.01 (1H, t, J=7.5 Hz), 6.74 (1H, t, J=7.5 Hz), 6.45 (1H, d, J=7.5 Hz), 6.31 (1H, d, J=7.5 Hz), 6.17 (1H, t, J=6.0 Hz), 4.35 (1H, q, J=5.0 Hz), 4.08 (2H, br), 3.90 (1H, dd, J=11.0, 2.0 Hz), 3.85–3.84 (1H, m), 3.75 (1H, dd, J=11.0, 2.0 Hz), 3.71 (2H, s), 3.61 (2H, br), 2.27–2.24 (1H, m), 2.04–2.00 (1H, m), 0.95 (9H, s), 0.87 (9H, s), 0.15 (3H, s), 0.13 (3H, s), 0.05 (6H, s). 13C-NMR (125 MHz, CDCl3) δ (ppm): 156.9, 154.03, 153.5, 146.5, 143.0, 136.4, 133.1, 129.3, 128.1, 127.9, 124.7, 123.6, 122.2, 116.2, 108.3, 107.4, 87.5, 85.8, 70.7, 69.0, 62.3, 41.9, 39.6, 30.9, 29.7, 26.1, 25.7, 25.6, 18.5, 18.0, −4.55, −4.88, −5.50, −5.72. IR (cm−1): 2957, 1723, 1668, 1473. HR-ESI-MS (m/z): Calcd for C37H55N5O7SSi2 (M+H)+ 770.3434. Found 770.3450.
Covalent Adduct (8)Et3N (2.2 mg, 0.02 mmol) and dithiothreitol (1.5 mg, 0.01 mmol) were added to a solution of 4 (4.6 mg, 0.01 mmol) and 7 (7.7 mg, 0.01 mmol) in dry CH3CN (2 mL) under an argon atmosphere. The reaction mixture stirred at room temperature for 2.5 h, quenched with H2O and extracted with AcOEt. The organic layer was washed with brine, dried over Na2SO4 and evaporated in vacuo. The resulting residue was purified by flash column chromatography (CHCl3) to afford 8 (8.0 mg, 68%) as a white amorphous. 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.48 (1H, s), 7.41 (1H, d, J=7.5 Hz), 7.32 (1H, d, J=7.5 Hz), 7.19 (1H, d, J=7.5 Hz), 7.06 (1H, d, J=7.5 Hz), 6.66 (1H, t, J=7.5 Hz), 6.36 (1H, d, J=7.5 Hz), 6.15 (1H, d, J=7.5 Hz), 6.13 (1H, t, J=5.0 Hz), 6.05 (1H, t, J=6.0 Hz), 5.84 (1H, d, J=5.0 Hz), 5.76 (1H, t, J=6.0 Hz), 4.55–4.49 (2H, m), 4.42–4.39 (3H, m), 4.35–4.30 (2H, m), 4.00–3.95 (2H, m), 3.82–3.80 (2H, m), 3.74–3.62 (3H, m), 2.29–2.24 (1H, m), 2.12–2.09 (4H, m), 2.04 (3H, s), 2.02 (3H, s), 0.92 (9H, s), 0.90 (9H, s), 0.13 (3H, s), 0.11 (3H, s), 0.08 (6H, s). IR (cm−1): 2933, 1669, 1557, 1473. 13C-NMR (125 MHz, CD3OD) δ (ppm): 172.4, 171.4, 171.2, 160.0, 159.2, 156.2, 155.9, 155.2, 155.1, 148.6, 147.5, 144.2, 138.4, 132.1, 129.2, 126.9, 125.4, 123.7, 117.0, 115.8, 108.7, 107.7, 89.2, 88.3, 87.4, 81.0, 75.0, 73.2, 72.7, 71.8, 70.8, 64.1, 63.6, 42.6, 41.2, 33.7, 30.8, 30.5, 26.7, 26.3, 20.9, 20.5, 19.4, 18.9, −4.25, −4.50, −5.11. IR (cm−1): 2926, 1748, 1652, 1562. HR-ESI-MS (m/z): Calcd for C53H72N10O15SSi2 (M+H)+ 1177.4511. Found 1177.4562.
We are grateful for the support provided by a Grant-in-Aid for Scientific Research (B) (No. 15H04633) and a Grant-in-Aid for Challenging Exploratory Research (No. 26670004) from the Japan Society for the Promotion of Science (JSPS). Y.F. also acknowledges a Grant-in-Aid for Young Scientists (B) (No. 15K18831) from JSPS.
The authors declare no conflict of interest.
