Methyl-6-deoxy-6-iodo-3,4-O-isopropylidene-2-O-tertbutyl-

3. Experimental section 95

3.5. Synthesis of queuine

3.5.2. Methyl-6-deoxy-6-iodo-3,4-O-isopropylidene-2-O-tertbutyl-

O O OH OMe

OTBDMS OMe

O O 88%

M: 344.1 g/mol M: 428.3 g/mol TBDMSCl

Imidazol, DCM 16 h, rt

PSfrag replacements 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 69 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138

139 140

141 142 143 144 145

Under argon atmosphere, iodide 139 (10.0 g, 29.1 mmol, 1.0 eq.), imidazole (2.38 g, 34.9 mmol, 1.2 eq.) and TBDMSCl (5.26 g, 34.9 mmol, 1.2 eq.) were dissolved in dry DCM (120 mL) and the solution was stirred for 16 h at ambient temperature. The reaction was quenched with water (100 mL). The layers were separated and the organic layer was washed with water (2×60 mL) and sat. aque-ous NaCl solution (60 mL), dried over MgSO4 and the solvents were evaporated in vacuo. The crude product was purified by column chromatography on silica gel us-ing pentane/EtOAc 95:5. The desired product 140 (11.35 g, 24.8 mmol, 85%) was obtained as clear oil.

1H-NMR (300 MHz, CDCl3): δ= 4.59 (d, J = 3.6 Hz, 1 H, H-1), 4.29 (dd, J = 5.7, 2.4 Hz, 1 H, H-4), 4.19-4.08 (m, 2 H, 3 H, H-3), 3.74 (dd, J = 7.2, 3.6 Hz, 1 H, H-2), 3.44 (s, 3 H, OCH3), 3.41-3.29 (m, 2 H, H-6, H-6’), 1.48 (s, 3 H, C(CH3)2), 1.34 (s, 3 H, C(CH3)2), 0.98 (s, 9 H, SiC(CH3)3), 0.10 (s, 3 H, Si(CH3)2), 0.08 (s, 3 H, Si(CH3)2) ppm.

13C-NMR (125 MHz, CDCl3): δ = 109.2 (C(CH3)2), 100.2 (C-1), 76.9 (C-3), 74.0 (C-4), 71.2 (C-2), 68.6 (C-5), 55.8 (OCH3), 28.1 (C(CH3)2), 26.2 (C(CH3)2), 25.8 (SiC(CH3)3), 18.1 (SiC(CH3)3), 2.85 (C-6), 0.58 (Si(CH3)2), 0.75 (Si(CH3)2) ppm.

MS (ESI): m/z (%) = 481.1 (100) [M+Na]⁺.

HRMS: m/z calc. for C16H31IO5SiNa [M+Na]⁺: 481.0878, found 481.0878.

3. Experimental section

3.5.3. (2R,3S,4S)-3,4-O-Isopropylidene-2-O-tertbutyldimethylsilyl-hex-5-en-1-al (141)

O O

OTBDMS OMe

OTBDMS O

O O 99%

M: 428.3 g/mol M: 300.5 g/mol Zn, Sonication

H2O/THF 1:9 v/v 6 h, rt

140 141

142 143 144 145

Galactopyranoside 140 (32.6 g, 71.2 mmol, 1.0 eq.) and was dissolved in THF/H₂O (9:1, v/v, 600 mL) and zinc powder (46.5 g, 710 mmol, 10.0 eq.) was added. The suspension was stirred at 50 ^◦C for 6 h. After cooling to ambient temperature, the suspension was filtered through a silica plug and washed with Et2O (300 mL). The organic layer was washed with water (200 mL), sat. aqueous NaHCO3 solution (200 mL) and sat. aqueous NaCl solution (200 mL), dried over MgSO4 and the sol-vents were evaporated in vacuo to give the desired product 141 (21.2 g, 70.6 mmol, 99%) as yellowish oil.

1H-NMR (300 MHz, CDCl3): δ = 9.67 (d, J = 0.9 Hz, 1 H, CHO), 5.92 (ddd, J = 17.3, 10.4, 6.8 Hz, 1 H, H-5), 5.35, (dt, J = 17.3, 1.5 Hz, 1 H, H-6), 5.21 (dt, J = 10.4, 1.5 Hz, 1 H, H-6’), 4.72 (t, J = 6.8 Hz, 1 H, H-4), 4.32 (dd, J = 6.8, 5.3 Hz, 1 H, H-3), 4.07 (dd, J = 5.3, 0.9 Hz, 1 H, H-2), 1.51 (s, 3 H, C(CH3)2), 1.35 (s, 1 H, C(CH3)2), 0.92 (s, 9 H, SiC(CH3)3), 0.09 (d, J = 1.8 Hz, 6 H, Si(CH3)2) ppm.

13C-NMR (125 MHz, CDCl3): δ = 201.8 (CHO), 133.8 (C-5), 118.9 (C-6), 109.2 (C(CH3)2), 79.1 (C-3), 78.5 (C-4), 77.4 (C-2), 27.1 (C(CH3)2), 25.9 (SiC(CH3)3), 25.2 (C(CH3)2), 18.4 (SiC(CH3)3), -4.4 (Si(CH3)2), -4.7 (Si(CH3)2) ppm.

MS (ESI): m/z (%) = 323.2 (0.5) [M+Na]⁺.

HRMS: m/z calc. for C15H28O4SiNa [M+Na]⁺: 323.1649, found 323.1648.

3.5. Synthesis of queuine

3.5.4. (2R,3S,4S)-3,4-O-Isopropylidene-2-O-tertbutyldimethylsilyl-hepta-1,5-diene (142)

O O

OTBDMS O

OTBDMS O O

67%

M: 300.5 g/mol M: 398.5 g/mol Tebbe reagent

THF, 15 min, 0 °C

141 142

143 144 145

Under an argon atmosphere, aldehyde 141 (21.2 g, 70.6 mmol, 1.0 eq.) was dis-solved in dry THF (200 mL) and the solution was cooled to 0^◦C. TEBBE REAGENT

(0.5M in toluene, 200 mL, 100 mmol, 1.4 eq.) was added dropwise and the result-ing solution was stirred at 0^◦C for 30 min. Subsequently, Et2O (200 mL) was added and water (12 mL) was added dropwise. The resulting suspension was dried over MgSO4, filtered and the residue was washed with Et2O (100 mL). The solvents of the combined organic layers were evaporated in vacuo and the resulting crude prod-uct was purified by column chromatography on silica gel using pentane/EtOAc 95:5.

The desired product 142 (14.1 g, 47.1 mmol, 67%) was obtained as an orange oil.

1H-NMR (300 MHz, CDCl3):δ = 6.04-5.80 (m, 2 H, H-2, H-6), 5.41-5.12 (m, 4 H, 1, 7), 4.48-4.41 (m, 1 H, 5), 4.22-4.14 (m, 1 H, 4), 4.02-3.95 (m, 1 H, H-3), 1.50 (s, 3 H, C(CH3)2), 1.35 (C(CH3)2), 0.91 (s, 9 H, SiC(CH3)3), 0.09 (s, 3 H, Si(CH3)2), 0.07 (s, 3 H, Si(CH3)2) ppm.

13C-NMR (125 MHz, CDCl3): δ = 136.9 (C-2), 134.8 (C-6), 118.7 (C-7), 116.6 (C-1), 108.7 (C(CH3)2), 82.0 (C-4), 79.2 (C-5), 72.7 (C-3), 28.0 (C(CH3)2), 26.0 (SiC(CH3)3), 25.7 (C(CH3)2), 18.6 (SiC(CH3)3), -4.4 (Si(CH3)2), -4.5 (Si(CH3)2) ppm.

MS (ESI): m/z (%) = 321.2 (14) [M+Na]⁺.

HRMS: m/z calc. for C16H30O3SiNa [M+Na]⁺: 321.1856, found 321.1854.

3. Experimental section

3.5.5. (1S,2S,3S)-1-O-tertbutyldimethylsilyl-2,3-O-isopropylidene-cyclopent-4-ene (145)

O O

OTBDMS

O O 70%

M: 928.5 g/mol M: 270.4 g/mol Grubbs 1st gen.

catalyst, DCM

4h, reflux OTBDMS

144 145

Under an argon atmosphere, diene 142 (942 mg, 3.10 mmol, 1.0 eq.) and G^RUBBS

CATALYST, 1^{ST GEN}. (2.5 mol%, 877.5 µmol, 64 mg) were dissolved in dry DCM (10 mL) and the solution was stirred under reflux for 4 h. After cooling to ambient temperature, the solvent was evaporated in vacuo and the resulting crude product was purified by column chromatography on silica gel using pentane/EtOAc 3:1. The desired product 145 (587 mg, 2.17 mmol, 70%) was obtained as brown oil.

1H-NMR (300 MHz, CDCl3):δ = 6.00 (d, J = 5.7 Hz, 1 H, H-1), 5.88 (dd, J = 5.7, 2.0 Hz, 1 H, H-4), 5.27 (dd, J = 5.6, 1.0 Hz, 1 H, H-3), 4.76 (s, 1 H, H-2), 4.49 (d, J = 5.6 Hz, 1 H, H-5), 1.38 (s, 3 H, C(CH3)2), 1.33 (s, 3 H, C(CH3)2), 0.90 (s, 9 H, SiC(CH3)3), 0.08 (s, 3 H, Si(CH3)2), 0.06 (s, 3 H, Si(CH3)2) ppm.

13C-NMR (125 MHz, CDCl3): δ = 135.5 (C-5), 134.8 (C-4), 111.8 (C(CH3)2), 86.0 (C-3), 84.4 (C-2), 81.1 (C-1), 27.4 (C(CH3)2), 25.8 (C(CH3)2), 18.6 (SiC(CH3)3), -4.4 (Si(CH3)2), -4.5 (Si(CH3)2) ppm.

MS (ESI): m/z (%) = 315.2 (4) [M+HCOO]⁻.

HRMS: m/z calc. for C14H26O3SiHCOOa [M+HCOO]⁻: 315.1640, found 315.1633.

3.5. Synthesis of queuine

3.5.6. (2R,3S,4S)-2-Hydroxy-3,4-O-isopropylidene-hepta-1,5-diene (143)

O O

OTBDMS OH

O O 97%

M: 298.5 g/mol M: 184.2 g/mol TBAF, THF

2h, rt

142 143

144 145

Diene 142 (13.6 g, 45.6 mmol, 1.0 eq.) was dissolved in THF (500 mL) and a solution of TBAF·3 H2O (28.8 g, 91.2 mmol, 2.0 eq.) in THF (100 mL) was added dropwise. The solution was stirred for 2.5 h at ambient temperature. The solvent was evaporated in vacuo and the resulting crude product was purified by column chromatography on silica gel using pentane/EtOAc 5:1. The desired product 143 (8.12 g, 44.1 mmol, 97%) was obtained as yellow oil.

1H-NMR (300 MHz, CDCl3):δ = 6.07-5.93 (m, 1 H, H-2), 5.90-5.77 (m, 1 H, H-6), 5.44-5.17 (m, 4 H, H-1, H-7), 4.63-4.55 (m, 1 H, H5), 4.16-4.04 (m, 2 H, H-4, H-3), 1.52 (s, 3 H, C(CH3)2), 1.38 (s, 3 H, C(CH3)2) ppm.

13C-NMR (125 MHz, CDCl3): δ = 136.8 (C-2), 134.1 (C-6), 119.5 (C-7), 117.2 (C-1), 108.1 (C(CH3)2), 80.8 (C-4), 79.7 (C-5), 70.7 (C-3), 27.5 (C(CH3)2), 25.1 (C(CH3)2) ppm.

MS (ESI): m/z (%) = 207.1 (7) [M+Na]⁺.

HRMS: m/z calc. for C10H16O3Na [M+Na]⁺: 207.0992, found 207.0992.

3. Experimental section

3.5.7. (1S,2S,3S)-1-Hydroxy-2,3-O-isopropylidene-cyclopent-4-ene (130)

O O OH

O O 74%

M: 184.2 g/mol M: 156.2 g/mol Grubbs 2nd gen.

catalyst, DCM

1 h, reflux OH

130 131

132 133 134 135 136 137 138 139 140 141 142 144 143 145

Under an argon atmosphere, diene 143 (8.12 g, 44.1 mmol, 1.0 eq.) and G^RUBBS

CATALYST, 2^{ND GEN}. (2.3 mol%, 848 mg, 1.0 eq.) were dissolved in dry DCM (200 mL) and the solution was stirred under reflux for 1 h. After cooling to ambient temperature, the solvent was evaporated in vacuo and the resulting crude product was purified by column chromatography on silica gel using pentane/EtOAc 2:1. The desired product 130 (5.2 g, 32.7 mmol, 74%) was obtained as brown oil.

13C-NMR (125 MHz, CDCl₃): δ = 135.5 (C-5), 134.8 (C-4), 111.8 (C(CH₃)₂), 86.0 (C-3), 84.4 (C-2), 81.1 (C-1), 27.4 (C(CH3)2), 25.8 (C(CH3)2) ppm.

MS (ESI): m/z (%) = 179.1 (4) [M+Na]⁺.

HRMS: m/z calc. for C8H12O3Na [M+Na]⁺: 179.0679, found 179.0678.

3.5. Synthesis of queuine

3.5.8. (1R,2S,3S)-1-Bromo-2,3-O-isopropylidene-cyclopent-4-ene (122)

O O O O

76%

M: 156.2 g/mol M: 219.1 g/mol Br2, TPP, imidazole

DCM, 45 min, -10 °C Br OH

122 123

124 125 126 127 128 129

130 131

132 133 134 135 136 137 138 139 140 141 142 143 144 145

Triphenylphosphine (16.1 g, 61.4 mmol, 2.0 eq.) was dissolved in DCM (300 mL) and cooled to -10^◦C. A solution of bromine (3.1 mL, 61.4 mmol, 2.0 eq.) in DCM (20 mL) was added dropwise. The resulting solution was added slowly to a solu-tion of alcohol 130 (4.8 g, 30.7 mmol, 1.0 eq.) and imidazole (2.3 g, 33.8 mmol, 1.1 eq.) in DCM (150 mL) at -10^◦C. The reaction mixture was stirred at -10 ^◦C for 45 min, then allowed to reach ambient temperature and Et2O (300 mL) was added.

The resulting organic solution was washed with water (2×300 mL) and sat. aque-ous NaCl solution (300 mL), dried over MgSO4 and the solvents were evaporated in vacuo. The residue was dissolved in DCM (10 mL) and hexane (500 mL) was added. The resulting suspension was filtered, and the filtrate was concentrated in vacuo. The residue was suspended in hexane (150 mL) and filtered. The filtrate was concentrated in vacuo and the resulting crude product purified by column chro-matography on silica gel using pentane/EtOAc 95:5. The desired product 122 (5.1 g, 23.3 mmol, 76%) was obtained as colorless oil.

1H-NMR (300 MHz, CDCl3):δ = 6.01-5.97 (m, 1 H, H-5), 5.97-5.93 (m, 1 H, H-4), 5.32 (d, J = 5.4 Hz, 1 H, H-3), 4.96 (d, J = 5.4 Hz, 1 H, H-2), 4.86-4.82 (m, 1 H, H-1), 1.38 (s, 3 H, C(CH3)2), 1.34 (s, 3 H, C(CH3)2) ppm.

13C-NMR (125 MHz, CDCl3):δ = 135.1 (C-5), 134.2 (C-4), 112.2 (C(CH3)2), 86.0 (C-3), 84.1 (C-2), 54.5 (C-1), 27.5 (C(CH3)2), 26.3 (C(CH3)2) ppm.

MS (ESI): m/z (%) = 241.0 (7) [M+Na]⁺.

HRMS: m/z calc. for C8H11O2BrNa [M+Na]⁺: 240.9835, found 240.9833.

3. Experimental section

3.5.9. 2’,3’-O-Isopropylidene-queuine (144)

O O

41%

M: 219.1 g/mol M: 317.3 g/mol preQ1, DBU, DMF

5 h, 50 °C Br

O O

N N

H NH

H₂N O

122 123

124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142

143 144

145

Under an argon atmosphere, preQ1 (58) (1.0 g, 4.0 mmol, 1.0 eq.) was dissolved in DMF (15 mL). DBU (6.0 mL, 40.0 mmol, 10.0 eq.) was added and the solu-tion was stirred at 50 ^◦C for 15 min. A solution of bromocyclopentene 122 (1.1 g, 4.8 mmol, 1.2 eq.) in DMF (3 mL) was added dropwise and the solution was stirred at 50 ^◦C for 5 h. After cooling to ambient temperature, the solvent was evaporated in vacuo and the residue was purified by column chromatography on silica gel using EtOAc/MeOH/NEt3 3:1:0.1. The resulting crude product was suspended in CHCl3

(100 mL) and stirred for 10 min. The solid components were separated by filtration and suspended in methanol (30 mL). The suspension was filtered, and the filtrate was concentrated in vacuo to yield the desired product 144 (581 mg, 1.64 mmol, 41%) as a slightly yellow solid.

1H-NMR (300 MHz, CD3OD): δ = 6.90 (s, 1 H, H-8), 6.30 (dt, J = 5.9, 1.7 Hz, 1 H, H-5’), 6.01 (d, J = 5.9 Hz, 1 H, H-4’), 5.36 (d, J = 5.8 Hz, 1 H, H-3’), 4.94 (d, J = 5.7 Hz, 1 H, H-2), 4.49-4.29 (m, 3 H, H-1’, H2-10), 1.37 (s, 3 H, C(CH3)2), 1.36 (s, 3 H, C(CH3)2) ppm.

MS (ESI): m/z (%) = 318.2 (100) [M+H]⁺, 340.2 (4) [M+Na]⁺.

HRMS: m/z calc. for C15H20N5O3Na [M+Na]⁺: 318.1561, found 318.1562.

3.5. Synthesis of queuine

Im Dokument Synthesis of rare nucleobases and artificial nucleotides for investigation of catalytic enzyme activity (Seite 135-143)