Synthesis of masked aldehyde building blocks X-X+2
Supporting Information
Solid-Phase Synthesis of an Apoptosis-Inducing Tetrapeptide Mimicking the Smac Protein
Sebastian T. Le Quement, Mette Ishoey, Mette T. Petersen, Pernille M. Simonsen, Nanna S. Holck and Thomas E. Nielsen*
Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 201, DK-2800 Kgs. Lyngby, Denmark
E-mail address of the corresponding author: ten@kemi.dtu.dk
|Contents |S1 |
|General methods |S2 |
|Solution-phase synthesis of building block 10 and characterization data (Scheme 1) |S4 |
|Solution-phase synthesis of Fmoc-(cis-4-N3)Pro-OH and characterization data |S16 |
|Solid-phase synthesis procedures |S24 |
|Crude RP-HPLC, MS, HRMS and NMR data for tetrapeptide 4·TFA (Scheme 2) |S26 |
|Crude RP-HPLC, MS, HRMS and NMR data for tetrapeptide 16 (Scheme 3) |S28 |
|Crude RP-HPLC, MS, HRMS and NMR data for tetrapeptide 20 (Scheme 4) |S30 |
General methods
Unless otherwise stated, all solution-phase reactions were run under an argon atmosphere. The glassware was dried over a Bunsen flame under vacuum before contact with any of the reactants or solvents. All flasks were equipped with a rubber septum, through which transport of chemicals, from or to the flask, was performed by use of a syringe equipped with a needle. Solvents were typically freshly distilled or dried over molecular sieves. All reactions were monitored by thin-layer chromatography (TLC) and/or reversed-phase high-performance liquid chromatography (RP-HPLC).
Solid-phase reactions were carried out in plastic syringes equipped with polypropylene filters, which allow suction to be applied from below for removal of reagents and solvents. For all solid-supported reactions, PEGA800 resin with a loading of 0.40 mmol/g was used. The PEGA800 resin was purchased from Varian, Inc.
All solvents were of HPLC quality, and commercially available reagents were used without further purification.
Analytical TLC was conducted on Merck aluminium sheets covered with silica (C60). The plates were either visualized under UV-light or stained by dipping in a developing agent followed by heating. Vanillin (15 g in ethanol (250 mL) and conc. H2SO4 (2.5 mL)) and/or p-anisaldehyde (15 g in ethanol (250 mL) and conc. H2SO4 (2.5 mL)) were used as developing agents. Flash column chromatography was performed using a CombiFlash® (Teledyne ISCO) with Matrex 60 Å, 35-70 µ silicagel.
Analytical HPLC was conducted on a Waters Alliance 2695 RP-HPLC system using a Symmetry( C18 column (d 3.5 µm, 4.6 x 75 mm; column temp: 25 °C; flow: 1 mL/min) with detection at 215 nm and 254 nm. Eluents A (0.1% TFA in H2O) and B (0.1% TFA in MeCN) were used in a linear gradient (100% A to 100% B) in a total run time of 13 min.
All new compounds were characterized by 1H NMR, 13C NMR, IR, HPLC, MS (ESI), HRMS (ESI), and optical rotation.
For the recording of 1H NMR and 13C NMR either a Bruker Aspect-3000 spectrometer (operating at 200 MHz for proton and 50 MHz for carbon), a Varian Mercury-300 spectrometer (operating at 300 MHz for proton and 75 MHz for carbon), or a Varian Unity Inova-500 spectrometer (operating at 500 MHz for 1H NMR) were used. The chemical shifts (δ) are reported in parts per million (ppm) and the coupling constants (J) in Hz. Usually DMSO-d6 was used as the solvent and signal positions were measured relative to the signal for DMSO (δ 2.50 ppm for 1H NMR and δ 39.43 for 13C NMR). For spectra recorded in CDCl3, signal positions were measured relative to the signal for CHCl3 (δ 7.26 for 1H NMR and δ 77.0 for 13C NMR).
IR analysis was performed on a Bruker Alpha FT-IR spectrometer.
Analytical LC-MS (ESI) analysis was performed on a Waters AQUITY RP-UPLC system equipped with a diode array detector using an AQUITY UPLC BEH C18 column (d 1.7 µm, 2.1 x 50 mm; column temp: 65 (C; flow: 0.6 mL/min). Eluents A (0.1% HCO2H in H2O) and B (0.1% HCO2H in MeCN) were used in a linear gradient (5% B to 100% B) in a total run time of 2.6 min. The LC system was coupled to a SQD mass spectrometer.
Analytical LC-HRMS (ESI) analysis was performed on an Agilent 1100 RP-LC system equipped with a diode array detector using a Phenomenex Luna C18 column (d 3 µm, 2.1 x 50 mm; column temp: 40 °C; flow: 0.4 mL/min). Eluents A (0.1% HCO2H in H2O) and B (0.1% HCO2H in MeCN) were used in a linear gradient (20% B to 100% B) in a total run time of 15 min. The LC system was coupled to a Micromass LCT orthogonal time-of-flight mass spectrometer equipped with a Lock Mass probe operating in positive electrospray mode.
Measurement of optical rotation was carried out using a Perkin-Elmer polarimeter 341. The temperature for all recordings was approximately 20 °C.
Solution-phase synthesis of building block 10 (Scheme 1)
Compound 6. Carboxylic acid 5 (500 mg, 1.105 mmol) was dissolved in DMF (5 mL) in a round bottomed flask fitted with a magnetic stirrer. NaHCO3 (464 mg, 5.525 mmol) and BnBr (378 mg, 2.210 mmol) were added. The reaction was stirred overnight, and then transferred to a separatory funnel containing ethyl acetate (50 mL), H2O (20 mL) and brine (20 mL). The organic layer was separated and the aqueous phase was extracted with ethyl acetate (3× 30 mL). The combined organic layers were dried over MgSO4 and filtered. The solvent was removed by rotary evaporation and the residue was purified by flash column chromatography on silica gel (heptane:ethyl acetate, 7:3) to give the title compound (6) as a white foam (564 mg, 94%). 1H NMR (300 MHz, CDCl3) δ 7.78-7.73 (m, 2H), 7.60-7.49 (m, 2H), 7.43-7.23 (m, 10H), 5.27 and 5.17 (2 × d, J = 12.3 Hz, 1H), 5.15 and 5.05 (2 × d, J = 12.2 Hz, 1H), 4.48-4.22 (m, 4.5H), 4.00 (t, J = 6.7 Hz, 0.5H), 3.76 (dd, J = 11.2, 5.9 Hz, 1H), 3.54 (d, J = 11.1 Hz, 1H), 2.57-2.43 (m, 1H), 1.99 (t, J = 13.0 Hz, 1H), 1.45 and 1.43 (2 × s, 9H), rotamers; 13C NMR (75 MHz, CDCl3) δ 164.6, 155.1, 154.6, 154.1, 144.0, 143.8, 143.6, 143.4, 141.2, 141.1, 135.1, 134.8, 128.5, 128.4, 128.3, 128.1, 127.6127.5, 127.0, 126.9, 125.0, 124.9, 124.7, 119.9, 119.8, 79.7, 79.6, 67.6, 67.5, 67.3, 58.1, 57.6, 53.6, 53.3, 49.9, 47.0, 28.3, rotamers; IR: 3345, 3065, 3034, 2975, 1743, 1697, 1415, 1350, 1162, 1007, 757, 737, 697 cm-1; Rf = 0.13 (heptane:ethyl acetate, 7:3); [α]589 nm -36.8°; MS (ESI) calcd for C32H34N2NaO6 [M + Na]+ 565.2 found 565.4; HRMS (ESI) calcd C32H35N2O6 [M + H]+ 542.2417 found 542.2504.
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Compound 8. Boc-protected amine 6 (480 mg, 0.885 mmol) was dissolved in 95% TFA (aq) (5 mL) and stirred for approximately 15 min. The reaction was monitored by HPLC. After complete conversion of the substrate the solvent was removed by rotary evaporation. In order to remove traces of water the compound was subjected to repeated additions of toluene (15 mL) and subsequent rotary evaporations. The resulting solid was then dissolved in 1,2-dichloroethane (8 mL) along with cyclohexanecarbaldehyde (99 mg, 0.885 mmol) and the mixture was stirred at room temperature for 10 min, upon which NaCNBH3 (222 mg, 3.540 mmol) was added. The reaction was monitored by HPLC. After completion of the reaction, water (30 mL) was added and then transferred to a separatory funnel with ethyl acetate (30 mL). The organic layer was separated and the aqueous phase was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over MgSO4 and filtered. The solvent was removed by rotary evaporation and the residue was purified by flash column chromatography on silica gel (heptane:ethyl acetate, 1:1) to give the title compound (8) as a sticky white foam (410 mg, 86 %). 1H NMR (300 MHz, CDCl3) δ 7.76 (m, 2H), 7.63-7.57 (m, 1H), 7.57-7.49 (m, 1H), 7.44-7.22 (m, 9H), 5.24 and 5.12 ( 2 × d, J = 12.3 Hz, 1H), 5.14 and 5.03 (2 × d, J = 12.3 Hz, 1H), 4.52-4.42 (m, 1H), 4.41-4.22 (m, 2.5H), 4.01 (t, J = 6.9 Hz, 0.5H), 3.79-3.69 (m, 1H), 3.48-3.27 (m, 2H), 2.49-2.28 (m, 3H), 2.09-1.95 (m, 1H), 1.81-1.57 (m, 5H), 1.43-1.07 (m, 5H), 0.94-0.77 (m, 2H), rotamers; 13C NMR (75 MHz, CDCl3) δ 172.3, 154.7, 154.3, 144.0, 143.9, 143.6, 143.4, 141.1, 141.0, 141.0, 135.4, 135.2, 128.5, 128.4, 128.3, 128.1, 128.0, 127.9, 127.5, 127.4, 126.9, 126.8, 125.0, 124.9, 124.7, 119.8, 67.4, 67.3, 66.8, 66.7, 58.1, 57.7, 57.2, 56.2, 54.5, 54.4, 52.7, 52.4, 47.0, 46.9, 38.0, 37.9, 31.2, 31.1, 26.4, 25.8, rotamers; IR: 3337, 3065, 3035, 2921, 1743, 1703, 1449, 1415, 1348, 1165, 1007, 756, 736, 696 cm-1; Rf = 0.14 (heptane:ethyl acetate, 1:1); [α]589 nm 39°; MS (ESI) calcd for C34H39N2O4 [M + H]+ 539.3 found 539.4; HRMS (ESI) calcd for C34H39N2O4 [M + H]+ 539.2904 found 539.2911.
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Compound 9. Secondary amine 8 (125 mg, 0.232 mmol) was dissolved in toluene (3 mL), Boc2O (152 mg, 0.696 mmol) and NaHCO3 (97 mg, 1.155 mol) were added and the reaction was stirred at room temperature overnight. After completion of the reaction water (20 mL) was added and then transferred to a separatory funnel with ethyl acetate (15 mL). The organic layer was separated and the aqueous phase was extracted with ethyl acetate (3 × 15 mL). The combined organic layers were dried over MgSO4 and filtered. The solvent was removed by rotary evaporation and the residue was purified by flash column chromatography on silica gel (heptane:ethyl acetate, 1:1) to give the title compound (9) as a colourless oil (98 mg, 66%). 1H NMR (300 MHz, CDCl3) δ 7.82-7.71 (m, 2H), 7.63-7.48 (m, 2H), 7.45-7.20 (m, 9H), 5.25 and 5.17 (2 × d, J = 12.2 Hz, 1H), 5.13 and 5.05 (2 × d, J = 12.2 Hz, 1H), 4.54-3.92 (m, 5H), 3.89-3.71 (m, 1H), 3.60-3.49 (m, 1H), 3.14-2.85 (m, 2H), 2.58-2.38 (m, 1H), 2.30-2.10 (m, 1H), 1.83-1.57 (m, 5H), 1.52-1.40 (m, 1H), 1.46 and 1.44 (2 × s, 9H), 1.33-1.10 (m, 3H), 1.00-0.80 (m, 2H), rotamers; 13C NMR (75 MHz, CDCl3) δ 171.7, 171.6, 155.3, 155.2, 154.7, 154.1, 144.0, 143.9, 143.6, 143.4, 141.2, 141.1, 135.4, 135.3, 128.5, 128.4, 128.3, 128.2, 128.2, 128.1, 127.6, 127.5, 126.9, 125.0, 124.9, 124.8, 119.9, 119.8, 80.2, 67.5, 66.9, 66.8, 57.4, 57.1, 54.9, 54.8, 51.9, 47.7, 47.4, 47.0, 38.1, 38.0, 37.8, 30.7, 28.3, 28.2, 26.3, 26.0, 25.9, rotamers; IR: 3010, 2975, 2925, 2851, 1748, 1687, 1450, 1416, 1352, 1166, 1148, 752, 738, 697 cm-1; Rf = 0.51 (heptane:ethyl acetate, 1:1); [α]589 nm -23.5°; MS (ESI) calcd for C39H47N2O6 [M + H]+ 639.3 found 639.5; HRMS (ESI) calcd for C39H47N2O6 [M + H]+ 639.3429 found 639.3439.
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Compound 10. A suspension of Pd/C 10 % (6 mg, 0.0058 mmol) and methanol (1 mL) was added to a round bottomed flask fitted with a magnetic stirrer and filled with H2. A solution of 9 (74 mg, 0.116 mmol) in methanol (2 mL) was then added. The reaction was monitored by RP-HPLC/TLC and left for approximately 2 hours. When RP-HPLC/TLC indicated full conversion of the substrate, methanol (5 mL) was added and the mixture was filtered through a pad of Celite. The solvent was removed by rotary evaporation and the residue was purified by flash column chromatography on silica gel (heptane:ethyl acetate:acetic acid, 50:50:1) to give the title compound (10) as a colourless oil (63 mg, >95%). 1H NMR (300 MHz, CDCl3) δ 7.76 and 7.70 (2 × d, 2H, J = 7.41 Hz), 7.62-7.50 (m, 2H), 7.44-7.25 (m, 4H), 4.54-3.99 (m, 5H), 3.88-3.69 (m, 1H), 3.63-3.45 (m, 1H), 3.13-2.90 (m, 2H), 2.62-2.40 (m, 1H), 2.40-2.23 (m, 1H), 1.87-1.56 (m, 5H), 1.51-1.41 (m, 1H), 1.46 and 1.43 (2 × s, 9H), 1.34-1.11 (m, 3H), 1.00-0.83 (m, 2H), rotamers; 13C NMR (75 MHz, CDCl3) δ 175.2, 155.4, 155.2, 154.3, 144.0, 143.7, 143.5, 141.2, 141.1, 137.8, 128.9, 128.1, 127.7, 127.6, 127.0, 125.2, 125.0, 124.8, 119.9, 119.8, 80.4, 67.8, 57.7, 55.4, 55.3, 52.0, 47.9, 47.6, 47.0, 38.1, 38.0, 30.7, 28.3, 26.3, 26.0, 25.9, rotamers; IR 2975, 2925, 2852, 1685, 1449, 1418, 1364, 1148, 908, 759, 728 cm-1; Rf = 0.27 (heptane:ethyl acetate:acetic acid, 50:50:1); [α]589 nm -20°; MS (ESI) calcd for C32H41N2O6 [M + H]+ 549.3 found 549.4; HRMS (ESI) calcd for C32H41N2O6 [M + H]+ 549.2959 found 549.2958.
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Solution-phase synthesis of Fmoc-(cis-4-N3)Pro-OH and characterization data
Boc-(trans-4-OH)Pro-OMe. Boc-Hyp-OH (5.0 g, 21.6 mmol) was dissolved in dry DMF (75 mL). Cs2CO3 (7.4 g, 22.7 mmol) was added, and the solution was left under magnetic stirring for 15 min. MeI (1.6 mL, 25.6 mmol) was added dropwise and the reaction mixture was left under stirring overnight, whereupon the reaction mixture was filtered through celite. The filtrate was poured into water (250 mL) and extracted with EtOAc (5 ( 120 mL). The combined organic phases were washed with sat. NaHCO3 (aq) (150 mL), brine (130 mL) and dried over Na2SO4. The solvent was removed under reduced pressure, and the yellow oil was purified by flash column chromatography on silica gel (EtOAc:heptane (3:1), Rf = 0.33, KMnO4 stain). The resulting oil was triturated with Et2O to give the title compound (3.2 g, 60%) as a white solid, which was used in the next step without further purification. 1H NMR (300 MHz, DMSO-d6) ( 5.12 and 5.09 (2 ( s, 1H), 4.23-4.17 (m, 2H), 3.65 and 3.62 (2 ( s, 3H), 3.43-3.24 (m, 1H), 2.16-2.06 (m, 1H), 1.94-1.82 (m, 1H), 1.39 and 1.32 (2 ( s, 9H), rotamers; 13C NMR (75 MHz, DMSO-d6) ( 175.3, 175.0, 156.3, 155.8, 81.7, 81.5, 70.7, 70.0, 59.4, 59.0, 55.9, 55.6, 52.6, 40.0, 39.3, 28.7, 28.6, rotamers; MS (ESI) calcd for C11H18NO5 [M - H]- 244.1 found 244.1; HRMS (ESI) calcd C11H20NO5 [M + H]+ 246.1346 found 246.1339; IR (neat) cm-1: 3437, 2989, 2954, 2882, 1738, 1659, 1420, 1197, 773, 573.
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Boc-(cis-4-N3)Pro-OMe. Boc-(trans-4-OH)Pro-OMe (4.0 g, 16.3 mmol) and PPh3 (4.3 g, 16.6 mmol) were dissolved in dry THF (80 mL), and left under magnetic stirring. A solution of DEAD in toluene (40% w/w) (7.1 g, 16.6 mmol) was added dropwise, followed by addition of DPPA (4.6 g, 16.6 mmol) over a period of 15 min. The reaction mixture was left under stirring overnight, whereupon the solvent was removed under reduced pressure. The resulting oil was purified by flash column chromatography on silica gel (EtOAc:heptane (1:1), Rf = 0.48, vanillin stain), to give the title compound (3.3 g, 75%) as a yellow oil. 1H NMR (300 MHz, CD3OD) ( 4.39-4.34 and 4.30-4.25 (2 ( m, 2H), 3.73 and 3.72 (2 ( s, 3H), 3.69-3.62 (m, 1H), 3.39 and 3.35 (2 ( t, J = 2.5 Hz, 1H), 2.59-2.44 (m, 1H), 2.13 and 2.09 (2 ( t, J = 3.3 Hz, 1H), 1.46 and 1.40, (2 ( s, 9H), rotamers; 13C NMR (75 MHz, CD3OD) ( 174.0, 173.7, 155.5, 81.9, 60.7, 59.8, 59.1, 58.8, 52.9, 52.6, 52.0, 36.7, 36.0, 28.7, 28.5, rotamers; MS (ESI) calcd for C11H18N4O4Na [M + Na]+ 293.1 found 293.2; HRMS (ESI) calcd C11H19N4O4 [M + H]+ 271.1406 found 271.1395; IR (neat) cm-1: 2977, 2887, 2102, 1753, 1696, 1392, 1156, 769.
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Boc-(cis-4-N3)Pro-OH. Boc-(cis-4-N3)Pro-OMe (1.5 g, 5.55 mmol) was dissolved in MeOH (15 mL) and 1 M NaOH (aq) (5.9 mL, 5.85 mmol). The reaction mixture was left under magnetic stirring for 20 h, whereupon water was added (50 mL). The MeOH was then removed under reduced pressure and the remaining aqueous phase was acidified with 1 M HCl (aq) to pH 3. The aqueous phase was then extracted with EtOAc (3 ( 50 mL), and the combined organic phases were dried over Na2SO4. Removal of solvent under reduced pressure gave the title compound (0.93 g, 65%) as a slightly yellow oil, which was used in the next step without further purification. 1H NMR (300 MHz, CD3OD) ( 4.23-4.11 (m, 2H), 3.55 and 3.51 (2 ( d, J = 6.0 Hz, 1H), 3.26 and 3.22 (2 ( d, J = 2.9 Hz, 1H), 2.47-2.33 (m, 1H), 2.05-2.0 (m, 1H), 1.33 and 1.29 (2 ( s, 9H), rotamers; 13C NMR (75 MHz, CD3OD) ( 175.3, 155.7, 81.8, 81.7, 60.7, 59.7, 59.1, 58.7, 52.6, 52.0, 36.8, 36.0, 28.7, 28.5, rotamers; MS (ESI) calcd for C10H16N4O4Na [M + Na]+ 279.1 found 279.2; HRMS (ESI) calcd C10H17N4O4 [M + H]+ 257.1250 found 257.1260; IR (neat) cm-1: 2978, 2100, 1697, 1670, 1393, 1367, 1157, 1121, 768.
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Fmoc-(cis-4-N3)Pro-OH. A solution of Boc-(cis-4-N3)Pro-OMe (0.43 g, 1.66 mmol) in CH2Cl2 (3.0 mL) was cooled to 0 oC under magnetic stirring, and TFA (3.0 mL, 39.2 mmol) was added. After stirring for 30 min at 0 oC, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in 10% aqueous Na2CO3 (36 mL) and MeCN (45 mL) under magnetic stirring and cooled to -20 °C. FmocCl (0.48 g, 1.84 mmol) was added in one portion. After stirring for 45 min at -20 °C, the organic solvent was evaporated under reduced pressure. The remaining aqueous phase was acidified to pH 2 with 1 M HCl (aq) and extracted with EtOAc (3 ( 100 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (MeOH:CH2Cl2 (1:19), Rf = 0.2, vanillin stain) to give the title compound (0.42 g, 66%) as a white solid. 1H NMR (300 MHz, CD3OD) ( 7.64 (t, J = 6.0 Hz, 2H), 7.49 (t, J = 7.4 Hz, 2H), 7.25 (dt, J = 7.4, 1.5 Hz, 2H), 7.17 (dt, J = 7.4, 1.2 Hz, 2H), 4.29-4.02 (m, 5H), 3.59 and 3.49 (2 ( dd, J = 11.6, 5.8 Hz, 1H), 3.37-3.28 (m, 1H), 2.45-2.29 (m, 1H), 2.08 (t, J = 13.3 Hz, 1H), rotamers; 13C NMR (75 MHz, CD3OD) ( 218.7, 162.2, 156.5, 156.4, 145.4, 145.1, 142.6, 142.6, 128.9, 128.2, 126.3, 126.2, 121.0, 69.1, 68.8, 60.6, 59.8, 59.7, 59.4, 52.7, 52.3, 43.6, 37.1, 36.0, rotamers; MS (ESI) calcd for C20H18N4O4Na [M + Na]+ 401.1 found 401.2; HRMS (ESI) calcd C20H19N4O4 [M + H]+ 379.1406 found 379.1422; IR (neat) cm-1: 2981, 2104, 1732, 1705, 1423, 1163, 757, 739; [(]589 nm -22(.
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Solid-phase synthesis procedures
Attachment of the Fmoc-protected Rink linker to the amino-functionalized PEGA800 resin was carried out by premixing Fmoc-Rink-OH (3.0 equiv), NEM (4.0 equiv) and TBTU (2.88 equiv) in DMF for 5 min. The volume of DMF was kept to a minimum, just enough for full coverage and swelling of the beads. The resulting solution was added to the resin, and allowed to react for 2 h. After removal of the reaction mixture by suction, the resin was washed with DMF (× 6), CH2Cl2 (× 6) and then dried under vacuum. Completion of the reaction was monitored using the Kaiser test. Three solutions were prepared for the Kaiser test: 1) 500 mg ninhydrin in 10 mL EtOH, 2) 80 g of liquified phenol in 20 mL EtOH, 3) 2 mL 0.001 M KCN (aq) diluted to 100 mL with pyridine. Two drops of each solution were added to 10-20 beads and heated to 120 °C for 2-3 min. A positive test for free primary amino groups was indicated by a blue colouration of the beads.
Peptide synthesis was accomplished by cycles of Fmoc deprotection and TBTU-mediated coupling of Fmoc-protected amino acids. Fmoc deprotection was carried out by treating the resin with 20% piperidine in DMF, first for 2 min, followed by washing with DMF (×1), and then for 18 min. The deprotected resin was then washed with DMF (× 6), CH2Cl2 (× 6) and then DMF (× 6) again. In all cases, the peptide sequence was terminated with a TBTU-mediated coupling of Boc-(N-Me)Ala-OH.
Analysis of all solid-supported tetrapeptides was performed after acidic cleavage of the Rink linker (concomitant Boc deprotection of the N-terminus). A small amount of resin (~5 mg) was treated with 95% TFA (aq) for 2 h. The reaction mixture was transferred to a HPLC vial, and the TFA and water were removed under reduced pressure. MeCN (100 µL) and water (100 µL) were added, and the sample (10 µL) was analyzed by analytical RP-HPLC.
Material sufficient for NMR analyses or biological purposes was obtained by cleaving a resin sample (50-100 mg) as described above, followed by a wash with MeCN (1 mL) and H2O (1 mL). After removal of the solvents on a SpeedVac, the residue was redissolved in a minimum of MeCN and precipitated with Et2O. The typically off-white crystalline peptide was then lyophilized overnight.
For all solid-phase transformations, the volume of solvent was kept to a minimum, just enough for full coverage and swelling of the beads.
The solid-phase azide reduction was carried by treatment of the resin with 0.1 M DTT (4 equiv) and DBU (2 equiv) in DMF for 1 h. After removal of the reaction mixture by suction, the resin was washed with DMF (×6), MeOH (×6), CH2Cl2 (×6) and then dried under vacuum.
The mono-alkylation of the solid supported amine was carried out by addition of cyclohexanecarbaldehyde (10 equiv) in DMF to the resin. The reaction was allowed to proceed for 1 h, whereupon the resin was washed with DMF (×6). To the resin was then added freshly prepared Na(OAc)3BH (5 equiv) in DMF:DMSO:AcOH (50:50:1), and the reaction allowed to proceed for 1 h. After removal of the reaction mixture by suction, the resin was washed with DMF (×6), H2O (×6), DMF (×6), MeOH (×6), CH2Cl2 (×6) and then dried under vacuum.
The di-alkylation of the solid supported amine was carried out by concomitant addition of cyclohexanecarbaldehyde (10 equiv) and freshly prepared Na(OAc)3BH (5 equiv) in DMF:DMSO:AcOH (50:50:1) to the resin. The reaction was allowed to proceed overnight. After removal of the reaction mixture by suction, the resin was washed with DMF (×6), H2O (×6), DMF (×6), MeOH (×6), CH2Cl2 (×6) and then dried under vacuum.
Crude RP-HPLC, HRMS and NMR data for tetrapeptide 4·TFA (Scheme 2)
Compound 4·TFA. 1H NMR (500 MHz, DMSO-d6) δ 9.16-9.06 (m, 1H), 9.06-8.97 (m, 1H), 8.97-8.83 (m, 2H), 8.75 (d, J = 8.1 Hz, 1H), 8.65 (d, J = 7.5 Hz, 1H), 7.47 (s, 1H), 7.30-7.24 (m, 4H), 7.22-7.18 (m, 1H), 7.10 (s, 1H), 4.49 (dd, J = 8.6, 4.9 Hz, 1H), 4.40-4.33 (m, 2H) 4.07-4.02 (m, 1H), 3.94-3.86 (m, 3H), 2.99 (dd, J = 14.0, 5.1 Hz, 1H), 2.91 (dd, J = 14.0, 8.4 Hz, 1H), 2.88-2.76 (m, 2H), 2.50 (br s, 3H), 2.46-2.42 (m, 1H), 2.13-2.06 (m, 1H), 2.05-1.98 (m, 1H), 1.76-1.66 (m, 5H), 1.65-1.59 (m, 1H), 1.33 (d, J = 6.8 Hz, 3H), 1.23-1.13 (m, 3H), 0.92 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.99-0.86 (m, 2H); 13C NMR (50 MHz, DMSO-d6) δ 172.4, 172.2, 169.5, 168.6, 137.7, 129.2, 128.1, 126.3, 57.8, 56.6, 55.8, 54.6, 51.1, 49.5, 37.1, 34.7, 30.7, 30.5, 29.9, 29.7, 29.6, 25.5, 25.0, 18.8, 18.0, 15.8; MS (ESI) calcd for C30H50N6O4 [M + 2H]2+ 279.2 found 279.4; HRMS (ESI) calcd for C30H50N6O4 [M + 2H]2+ 279.1942 found 279.1930.
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Crude RP-HPLC, HRMS and NMR data for tetrapeptide 16 (Scheme 3)
Compound 16. 1H NMR (500 MHz, DMSO-d6) δ 8.93 (br s, 1H), 8.82 (br s, 1H), 8.72 (d, J = 8.1 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.32 (s, 1H), 7.27-7.17 (m, 5H), 7.08 (s, 1H), 4.41-4.37 (m, 3H), 4.35-4.31 (m, 1H), 4.09 (dd, J = 10.4 Hz, J = 6.6 Hz, 1H), 3.91-3.85 (m, 1H), 3.42 (dd, J = 10.2 Hz, J = 6.1 Hz, 1H), 2.97 (dd, J = 13.8 Hz, J = 5.6 Hz, 1H), 2.89 (dd, J = 13.8 Hz, J = 7.7 Hz, 1H), 2.51-2.48 (m, 3H), 2.46-2.41 (m, 1H), 2.04-1.96 (m, 1H), 1.86-1.81 (m, 1H), 1.32 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H); 13C NMR (50 MHz, DMSO-d6) δ 172.3, 170.0, 169.8, 168.6, 137.7, 129.2, 128.0, 126.2, 58.3, 55.9, 53.9, 51.7, 37.3, 33.6, 30.8, 29.9, 19.1, 17.9 15.7; MS (ESI) calcd for C23H35N8O4 [M + H]+ 487.3 found 487.4; HRMS (ESI) calcd for C23H35N8O4 [M + H]+ 487.2776 found 487.2784.
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Crude RP-HPLC, HRMS and NMR data for tetrapeptide 20 (Scheme 4)
Compound 20. 1H NMR (500 MHz, DMSO-d6) δ 8.98 (br s, 1H), 8.89 (br s, 1H), 8.84 (s, 1H), 8.72 (d, J = 8.3 Hz, 1H), 8.37 (d, J = 7.4 Hz, 1H), 7.42 (s, 1H), 7.28-7.23 (m, 4H), 7.21-7.18 (m, 1H), 7.09 (s, 1H), 4.50 (dd app. t, J = 7.4 Hz, 1H), 4.42 (dd app. t, J = 7.2 Hz, J = 7.2 Hz, 1H), 4.39-4.35 (m, 1H), 4.28-4.21 (m, 1H), 4.16-4.08 (m, 1H), 3.95-3.85 (m, 1H), 3.79-3.73 (m, 1H), 3.12-2.94 (m, 4H), 2.97-2.89 (m, 2H), 2.61-2.55 (m, 1H), 2.50 (br s, 3H), 2.13-2.08 (m, 1H), 2.06-1.99 (m, 1H), 1.87-1.60 (m, 12H), 1.32 (d, J = 6.7 Hz, 3H), 1.25-1.10 (m, 6H), 1.08-0.97 (m, 4H), 0.95 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H); 13C NMR (50 MHz, DMSO-d6) δ 172.3, 170.6, 169.4, 168.5, 137.7, 129.2, 128.0, 126.2, 62.9, 59.2, 57.9, 55.9, 54.2, 47.9, 37.2, 33.2, 32.9, 30.8, 30.4, 30.0, 28.5, 25.1, 23.6, 19.0, 17.7, 15.8; MS (ESI) calcd for C37H61N6O4 [M + H]+ 653.5 found 653.8; HRMS (ESI) calcd for C37H61N6O4 [M + H]+ 653.4749 found 653.4742.
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Purity: >95%, Rt = 10.15 min
6
1H NMR of 6
13C NMR of 6
IR of 6
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8
Purity: >95%, Rt = 8.10 min
1H NMR of 8
13C NMR of 8
IR of 8
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Purity: >95%, Rt = 12.00 min
9
1H NMR of 9
13C NMR of 9
IR of 9
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Purity: >95%, Rt = 10.75 min
10
1H NMR of 10
13C NMR of 10
IR of 10
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1H NMR of Boc-(trans-4-OH)Pro-OMe
13C NMR of Boc-(trans-4-OH)Pro-OMe
IR of Boc-(trans-4-OH)Pro-OMe
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1H NMR of Boc-(cis-4-N3)Pro-OMe
13C NMR of Boc-(cis-4-N3)Pro-OMe
IR of Boc-(cis-4-N3)Pro-OMe
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1H NMR of Boc-(cis-4-N3)Pro-OMe
13C NMR of Boc-(cis-4-N3)Pro-OMe
IR of Boc-(cis-4-N3)Pro-OMe
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1H NMR of Fmoc-(cis-4-N3)Pro-OH
13C NMR of Fmoc-(cis-4-N3)Pro-OH
IR of Fmoc-(cis-4-N3)Pro-OH
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4·TFA
Purity: >95%, Rt = 5.26 min
1H NMR of 4·TFA
13C NMR of 4·TFA
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Purity: >95%, Rt = 4.92 min
16
1H NMR of 16
13C NMR of 16
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20
Purity 95%, Rt = 5.91 min
1H NMR of 20
13C NMR of 20
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