A Fluorogenic Screening for Enantio- and Diastereoselectivity of 2-Deoxy-d-ribose-5-phosphate Aldolases

Carolin Bisterfeld; Irene Küberl née Kullartz; Markus Dick; Jörg Pietruszka

doi:10.1055/s-0035-1560723

Synlett, Inhaltsverzeichnis

Synlett 2016; 27(01): 11-16
DOI: 10.1055/s-0035-1560723

letter

A Fluorogenic Screening for Enantio- and Diastereoselectivity of 2-Deoxy-d-ribose-5-phosphate Aldolases

Carolin Bisterfeld

^aInstitute of Bioorganic Chemistry, Heinrich-Heine-University at Forschungszentrum Jülich, and Bioeconomy Science Center (BioSC), 52426 Jülich, Germany

,

Irene Küberl née Kullartz

^aInstitute of Bioorganic Chemistry, Heinrich-Heine-University at Forschungszentrum Jülich, and Bioeconomy Science Center (BioSC), 52426 Jülich, Germany

,

Markus Dick

^aInstitute of Bioorganic Chemistry, Heinrich-Heine-University at Forschungszentrum Jülich, and Bioeconomy Science Center (BioSC), 52426 Jülich, Germany

,

Jörg Pietruszka*

^aInstitute of Bioorganic Chemistry, Heinrich-Heine-University at Forschungszentrum Jülich, and Bioeconomy Science Center (BioSC), 52426 Jülich, Germany

^bInstitute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany eMail: j.pietruszka@fz-juelich.de

› Institutsangaben

Abstract

Alle Artikel dieser Rubrik

Dedicated to Prof. Steven V. Ley on the occasion of his 70^th birthday

Abstract

A highly sensitive, fluorescence-based selectivity screening system for 2-deoxy-d-ribose-5-phosphate aldolases was realized by installing short, straightforward syntheses to fluorophore-coupled carbohydrates as d-ribose, l-ribose, and d-xylose. The substrates allow the simultaneous determination of enantioselectivity and diastereoselectivity of DERA-catalyzed aldol reactions.

Key words

2-deoxy-d-ribose-5-phosphate aldolase - fluorogenic screening - enantioselectivity - diastereoselectivity - extremophilic aldolases

Volltext

Referenzen

References and Notes
1 Authors contributed equally.

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14 (3S,4R)-4-(Hydroxymethyl)-1-methoxytetrahydrofuran-3-ol (d-12) and (3R,4S)-4-(Hydroxymethyl)-1-methoxytetrahydrofuran-3-ol (l-12) 2-Deoxy-d-ribose (d-11, 0.5 g, 3.7 mmol) were dissolved in methanolic HCl (10 mL, 0.1%) and stirred at r.t. for 12 min. The reaction was controlled by rotary power and after full conversion neutralized with Ag₂CO₃. The mixture was filtrated, and the solvent was evaporated. The crude product was purified by Kugelrohr distillation; 506 mg (92%, dr A/B = 0.6:0.4) d-12 were isolated as a colorless oil. Analogously 100 mg (0.75 mmol) 2-desoxy-l-ribose (l-11) were converted into 116 mg (0.78 mmol, quantitative yield, dr A/B = 0.6:0.4) l-12. R_f = 0.06 (PE–EtOAc, 40:60). IR (film): ν = 3383, 2918, 1444, 1206, 1050 cm^–1. GC–MS (EI, +, 70 eV): m/z (%) = 147 (1) [M + H]⁺, 117 (100) [C₅H₉O₃]⁺, 88 (52) [C₅H₁₀O]⁺, 71 (26) [C₄H₈O]⁺. ¹H NMR (600 MHz, CDCl₃): δ = 2.00 (m, 1 H, 2b-H_A), 2.08–2.14 (m, 3 H, 2b-H_B, 2a-H_A, 3-OH_A), 2.26 (ddd, J = 13.9, 6.9, 2.0 Hz, 1 H, 2a-H_B), 2.50 (d, J = 5.7 Hz, 1 H, 3-OH_B), 2.78 (m, 1 H, 5-OH_B), 2.90 (d, J = 10.23 Hz, 1 H, 5-OH_A), 3.38 (m_c, 6 H, OMe_A, OMe_B), 3.58–3.73 (m, 4 H, 5a-H_A, 5a-H_B, 5b-H_A, 5b-H_B), 4.05 (ddd, J = 3.5 Hz, 1 H, 4-H_B), 4.10–4.17 (m, 2 H, 4-H_A, 3-H_A), 4.51 (ddd, J = 8.4, 6.9, 3.5 Hz, 1 H, 3-H_B), 5.10 (dd, J = 4.5, 0.2 Hz, 1 H, 1-H_A), 5.11 (dd, J = 5.7, 2.0 Hz, 1 H, 1-H_B) ppm. ¹³C NMR (151 MHz, CDCl₃): δ = 41.5 (C-2_A), 42.5 (C-2_B), 54.9 (OMe_A), 55.4 (OMe_B), 63.1 (C-5_A), 63.5 (C-5_B), 72.1 (C-3_B), 72.8 (C-3_A), 87.4 (C-4_A), 87.5 (C-4_B), 105.5 (C-1_A), 105.6 (C-1_B) ppm. HRMS (ESI, +): m/z (%) calcd for C₆H₁₂O₄Na [M + Na]⁺: 171.06333; found: 171.06275. d-12: [α]_D +39.4 (c 1.0, CHCl₃); lit.¹³ [α]_D +38.4 (c 0.6, CH₃COOH). l-12: [α]_D –40 (c 0.93, CHCl₃)
15 [(3S,4R)-3-Hydroxy-1-methoxytetrahydrofuran-4-yl]methyl p-Tosylate (d-13) and [(3R,4S)-3-Hydroxy-1-methoxy-tetrahydrofuran-4-yl]methyl p-Tosylate (l-13) Under an argon atmosphere d-12 (4.20 g, 28.4 mmol) was dissolved in pyridine (100 mL) and cooled to –5 °C. A solution of p-TsCl (5.22 g, 27.4 mmol, 1.00 equiv) in dry CH₂Cl₂ (40 mL) was added dropwise. The temperature of the reaction was maintained between –5 °C and 0 °C. The reaction was stirred for 1 h at 0 °C, warmed up to r.t. and stirred overnight. The conversion was controlled by TLC. Ice-cold H₂O (130 mL) and EtOAc were added, and the reaction was extracted with a sat. CuSO₄ solution. The aqueous phases were combined and extracted with CH₂Cl₂. The combined organic phases were dried over MgSO₄ and filtered. The solvent was evaporated, and the crude product was purified by column chromatography (PE–EtOAc, 70:30); 5.60 g (18.5 mmol, 65%, dr A/B = 0.7:0.3) of d-13 were isolated as a colorless oil. Analogously 1.00 g (8.75 mmol) of l-12 were converted into 1.40 g (4.63 mmol, 68%, dr A/B = 0.7:0.3) of the colorless oil l-13. R_f = 1.9 (PE–EtOAc, 40:60). IR (film): ν = 3454, 2928, 1598, 1444, 1180, 1096, 665 cm^–1. GC–MS (EI, +, 70 eV): m/z (%) = 301 (1) [M – H]⁺, 271 (1) [M – OCH₃]⁺, 91 (71) [C₇H₈]⁺. ¹H NMR (600 MHz, CDCl₃): δ = 2.03–2.10 (m, 3 H, 2a-H_B, 2b-H_A, 2b-H_B), 2.21 (ddd, J = 13.4, 6.8, 1.7 Hz, 1 H, 2a-H_A), 2.45 (s, 6 H, Me_A, Me_B), 3.23 (s, 3 H, OMe_B), 3.34 (s, 3 H, OMe_A), 4.02–4.13 (m, 6 H, 5-H_A, 5-H_B, 4-H_A, 4-H_B), 4.20 (ddd, J = 4.06, 4.02, 1.7 Hz, 1 H, 3-H_A), 4.43 (ddd, J = 6.6, 6.5 Hz, 3.4 Hz, 1 H, 3-H_B), 5.01–5.04 (m, 2 H, 1-H_A, 1-H_B), 7.33–7.37 (m, 4 H, 3′-H_A, 3′-H_B), 7.76–7.83 (d, J = 8.3 Hz, 4 H, 2′-H_A, 2′-H_B) ppm. ¹³C NMR (151 MHz, CDCl₃): δ = 21.7 (Me), 41.0 (C-2_A/B), 41.4 (C-2_A/B), 55.0 (OMe_A/B), 55.1 (OMe_A/B), 69.4 (C-1′_A/B), 70.1 (C-1′_A/B), 72.7 (C-3_A/B), 72.8 (C-3_A/B), 83.0 (C-5/C-4), 84.6 (C-5/C-4), 106.4 (C-1_A/B), 106.7 (C-1_A/B), 128.0 (C-2′), 130.0 (C-3′), 145.1 (C-4′) ppm. HRMS (ESI, +): m/z (%) calcd for C₁₃H₁₈O₆SNa [M + Na]⁺: 325.07218; found: 325.07163. d-13 [α]_D +30 (c 0.6, CHCl₃). l-13 [α]_D –31.5 (c 0.9, CHCl₃)
16 7-{[(3S,4R)-1,3-Dihydroxytetrahydrofuran-4-yl]methoxy}-4-methyl-2H-chromen-2-one (d-5) and 7-{[(3R,4S)-1,3-Dihydroxytetrahydrofuran-4-yl]methoxy}-4-methyl-2H-chromen-2-one (l-5) Compound d-13 (7.00 g, 23.2 mmol) was dissolved in DMF (70 mL). K₂CO₃ (7.00 g, 50.6 mmol) and 4-methylumbelliferone (5.30 g, 30.0 mmol, 1.3 equiv) were added. The reaction was stirred for 16 h at 75 °C, then quenched with H₂O (70 mL), extracted with EtOAc and washed with 0.1 M NaOH. The organic phase was dried over MgSO₄ and filtered. The solvent was evaporated. The crude product was dissolved in MeCN–H₂O (150 mL, 1:3), mixed with DOWEX 50WX 8-100 (4.5 g), stirred for 1.5 h, and stored for 2 d at r.t. Resulting MeOH was evaporated. The solution was filtered, and the solvent was evaporated. The crude product was purified by column chromatography (gradient PE–EtOAc, 70:30 → 40:60 → 10:90 → 0:100) yielding 6.40 g (21.9 mmol, 88%, dr A/B 0.8:0.2) d-5. Analogously 115 mg (0.38 mmol) of l-13 was converted into 97 mg (0.33 mmol, 87%, dr A/B 0.8:0.2) l-5. R_f = 0.09 (n-pentane–EtOAc, 20:80). IR (film): ν = 3450, 2928, 1736, 1611, 1366, 1216 cm^–1. GC–MS (EI, +, 70 eV): m/z (%) = 293 (100) [M + H]⁺, 246 (10) [C₁₄H₁₄O₄]⁺. ¹H NMR (600 MHz, CDCl₃): δ = 2.17–2.21 (m, 1 H, 2′a-H_B), 2.24 (ddd, J = 13.9, 5.9, 4.6 Hz, 1 H, 2′a-H_A), 2.32–2.37 (m, 2 H, 2′b-H_A,_B), 2.39 (d, J = 1.5 Hz, 6 H, Me_A,_B), 3.99 (dd, J = 10.0, 4.7 Hz, 1 H, 5′a-H_A), 4.06 (dd, J = 10.0, 4.8 Hz, 1 H, 5′b-H_A), 4.18 (dd, J = 5.3, 5.3 Hz, 1 H, 5′a-H_B), 4.26 (dd, J = 5.3, 4.9 Hz, 1 H, 5′b-H_B), 4.42 (ddd, J = 5.9, 1.5, 1.4 Hz, 1 H, 3′-H_A), 4.47 (ddd, J = 4.6, 4.5, 2.1 Hz, 1 H, 3′-H_B), 4.59 (ddd, J = 4.8, 4.7, 1.5 Hz, 1 H, 4′-H_A), 4.64 (ddd, J = 5.3, 4.9, 4.6 Hz, 1 H, 4′-H_B), 5.66 (dd, J = 4.6, 0.2 Hz, 1 H, 1′-H_A), 5.67 (dd, J = 5.6, 2.5 Hz, 1 H, 1′-H_B), 6.14 (d, J = 1.5 Hz, 2 H, 3-H_A,_B), 6.79 (d, J = 2.6 Hz, 2 H, 8-H_A,_B), 6.85 (dd, J = 8.8, 2.6 Hz, 1 H, 6-H_A), 6.89 (dd, J = 8.8, 2.6 Hz, 1 H, 6-H_B), 7.48 (d, J = 8.8 Hz, 2 H, 5-H_A,_B) ppm. ¹³C NMR (151 MHz, CDCl₃): δ = 18.8 (Me), 41.6 (C-2′), 68.8 (C-5′), 73.5 (C-4′), 85.4 (C-3′), 99.8 (C-1′), 101.8 (C-8), 112.4 (C-3), 112.6 (C-6), 114.1 (C-4a), 125.8 (C-5), 152.7 (C-8a), 155.3 (C-7), 161.4 (C-2), 161.6 (C-4) ppm. HRMS (ESI, +): m/z (%) calcd for C₁₅H₁₇O₆ [M + H]⁺: 293.10251; found: 293.10190. d-5 [α]_D +30 (c 0.6, CHCl₃). l- 5 [α]_D –42.4 (c 0.23, CHCl₃)
17 7-[(3S,4R)-3-Hydroxy-1-methoxytetrahydrofuran-4-yl]methoxy-4-methyl-2H-chromen-2-one (14) Compound d-13 (10 g, 33.1 mmol) was dissolved in DMF (25 mL). K₂CO₃ (2.5 g) and 4-methylumbelliferone (7.58 g, 43 mmol, 1.3 equiv) were added. The reaction was stirred for 16 h at 75 °C, then quenched with H₂O, extracted with EtOAc, and washed with 0.1 M NaOH. The organic phase was dried over MgSO₄, and the solvent was evaporated. The crude product was purified by column chromatography (n-pentane–EtOAc, 50:50), yielding 8.33 g (27.2 mmol, 82%, dr A/B = 0.4:0.6) of 14 as a colorless oil. R_f = 0.33 (n-pentane–EtOAc, 20:80). IR (film): ν = 3586, 3322, 3093, 2923, 2838, 1709, 1613, 1389, 1369, 1294, 1150, 1069, 1027, 971, 838 cm^–1. GC–MS (EI, +, 70 eV): m/z (%) = 133 (10) [C₅H₉O₄]⁺, 147 (8) [C₆H₁₁O₄]⁺. ¹H NMR (600 MHz, CDCl₃): δ = 2.15 (ddd, J = 13.4, 6.4, 5.4 Hz, 1 H, 2′a-H_A), 2.22 (ddd, J = 13.8, 6.4, 4.6 Hz, 2 H, 2′a-H_B, 2′b-H_B), 2.32 (ddd, J = 13.4, 6.4, 1.8 Hz, 1 H, 2′b-H_A), 2.39 (d, J = 1.3 Hz, 3 H, Me_A,_B), 3.33 (s, 3 H, OMe_A), 3.42 (s, 3 H, OMe_B), 4.03 (dd, J = 10.0, 4.5 Hz, 1 H, 5′a-H_B), 4.07–4.13 (m, 3 H, 5′a-H_A, 5′b-H_A, 5′b-H_B), 4.26 (ddd, J = 6.4, 6.4, 4.4 Hz, 1 H, 3′-H_A), 4.30 (ddd, J = 6.4, 1.6, 1.5 Hz, 1 H, 3′-H_B), 4.43 (ddd, J = 4.6, 4.5, 1.6 Hz, 1 H, 4′-H_B), 4.57 (ddd, J = 6.7, 6.6, 4.4 Hz, 1 H, 4′-H_A), 5.13 (dd, J = 5.4, 1.8 Hz, 1 H, 1′-H_A), 5.16 (dd, J = 4.6, 0.2 Hz, 1 H, 1′-H_B), 6.13 (d, J = 1.3 Hz, 2 H, 3-H_A,_B), 6.81 (d, J = 2.5 Hz, 1 H, 8-H_B), 6.84 (d, J = 2.5 Hz, 1 H, 8-H_A), 6.86 (dd, J = 8.8, 2.5 Hz, 1 H, 6-H_B), 6.89 (dd, J = 8.8, 2.5 Hz, 1 H, 6-H_A), 7.48 (d, J = 8.8 Hz, 1 H, 5-H_B), 7.49 (d, J = 8.8 Hz, 1 H, 5-H_A) ppm. ¹³C NMR (151 MHz, CDCl₃): δ = 18.8 (Me), 41.2/41.7 (C-2′), 55.2/55.3 (OMe), 68.8/69.9 (C-5′), 73.0/73.3 (C-4′), 83.8/85.4 (C-3′), 101.7/101.8 (C-1′), 105.5/105.8 (C-8), 112.3/112.4 (C-3), 112.7 (C-6), 114.0/114.1 (C-4a), 125.7 (C-5), 152.6/152.7 (C-8a), 155.2/155.3 (C-7), 161.3/161.4 (C-2), 161.7/161.8 (C-4) ppm. HRMS (ESI, +): m/z (%) calcd for C₁₆H₂₀O₆ [M + H]⁺: 307.11761; found: 307.11762. [α]_D +29.8 (c 1.0, CDCl₃)
18 Dess DB, Martin JC. J. Am. Chem. Soc. 1991; 113: 7277
19 7-[(4R)-1-Methoxy-3-oxotetrahydrofuran-4-yl]methoxy-4-methyl-2H-chromen-2-one (15) To a solution of DMP (10.4 g, 24.5 mmol, 1.5 equiv) in CH₂Cl₂ (300 mL) compound 14 (5 g, 16.3 mmol) dissolved in CH₂Cl₂ (25 mL) were added. The reaction was stirred for 2 h at r.t. before it was quenched with 1 M Na₂S₂O₃ solution (100 mL). Sat. NaHCO₃ solution (100 mL) was added, and the reaction was stirred until both phases were clear. The mixture was diluted with CH₂Cl₂ and H₂O, the phases were separated, and the aqueous layer was extracted with CH₂Cl₂. The organic phases were combined, dried over MgSO₄, and the solvent was evaporated. The crude product was purified by column chromatography (PE–EtOAc, 60:40) yielding 3.97 g (13.1 mmol, 80%, dr A/B 0.7:0.3) of product 15. R_f = 0.49 (PE–EtOAc, 50:50). IR (film): ν = 3079, 2939, 2359, 1752, 1707, 1610, 1391, 1268, 1087, 1063, 1016, 982, 953, 833, 806 cm^–1. GC–MS (EI, +, 70 eV): m/z (%) = 58 (100) [C₃H₆O]⁺. ¹H NMR (600 MHz, CDCl₃): δ = 2.38 (d, J = 1.19 Hz, 3 H, Me_B), 2.39 (d, J = 1.28 Hz, 3 H, Me_B), 2.50 (dd, J = 18.21, 0.32 Hz, 1 H, 2′a-H_B), 2.52 (dd, J = 18.14, 1.12 Hz, 1 H, 2′a-H_A), 2.74 (dd, J = 18.21, 5.61 Hz, 1 H, 2′b-H_B), 2.83 (dd, J = 18.14, 5.58 Hz, 1 H, 2′b-H_A), 3.42 (s, 3 H, OMe_A), 3.48 (s, 3 H, OMe_B), 4.15 (dd, J = 10.3, 7.27 Hz, 1 H, 4′-H_A), 4.26–4.34 (m, 4 H, 5′a-H_A, 5′’a-H_B, 5′’b-H_B, 4′-H_B), 4.44 (dd, J = 7.27, 3.17 Hz, 1 H, 5′b-H_A), 5.40 (dd, J = 5.58, 1.12 Hz, 1 H, 1′-H_A), 5.43 (dd, J = 5.61, 0.32 Hz, 1 H, 1′-H_B), 6.14 (m_c, 2 H, 3-H_A,_B), 6.83 (d, J = 2.88 Hz, 1 H, 8-H_B), 6.84 (d, J = 2.42 Hz, 1 H, 8-H_A), 6.84 (dd, J = 8.67, 2.88 Hz, 1 H, 6-H_B), 6.90 (dd, J = 8.84, J = 2.42, 1 H, 6-H_A), 7.48 (d, J = 8.67 Hz, 1 H, 5-H_B), 7.50 (d, J = 8.84 Hz, 1 H, 5-H_A) ppm. ¹³C NMR (151 MHz, CDCl₃): δ = 18.7 (Me), 43.6 (C-2′_A), 43.7 (C-2′_B), 55.2 (OMe_A), 55.3 (OMe_B), 67.2 (C-4′_B), 69.5 (C-4′_A), 76.1 (C-5′_B), 78.0 (C-5′_A), 101.6 (C-8_B), 101.9 (C-8_A), 101.9 (C-1′_B), 102.7 (C-1′_A), 112.3 (C-3_A), 112.4 (C-3_A), 112.5 (C-4a_A,_B), 114.1 (C-6_A), 114.2 (C-6_B), 125.6 (C-5_B), 125.7 (C-5_A), 152.5 (C-4_A,_B), 155.1 (C-8a_B), 155.2 (C-8a_A), 161.2 (C-7_A,_B), 161.3 (C-2_B), 161.4 (C-2_A), 210.6 (C-3′_A), 210.8 (C-3′_B) ppm. HRMS (ESI, +): m/z (%) calcd for C₁₆H₁₈O₆ [M + H]⁺: 305.10196; found: 305.10199. [α]_D +44.5 (c 1.1, MeCN).
20 7-[(3R,4R)-3-Hydroxy-1-methoxytetrahydrofuran-4-yl]methoxy-4-methyl-2H-chromen-2-one (16) Compound 15 (3.97 g 13.1 mmol) was dissolved in EtOH–H₂O (1:1) and cooled to 0 °C. NaBH₄ (1.23 g, 32.7 mmol, 2.5 equiv) was added, and the reaction was stirred for 1 h at r.t. The reaction was quenched with a sat. NH₄Cl solution and extracted with CHCl₃. The organic phases were washed with H₂O, dried over MgSO₄, and the solvent was evaporated. The diastereomers (dr 1:1) could be separated by column chromatography (PE–EtOAc, 50:50); 1.45 g (4.73 mmol, 36%) of the desired diastereomer 16 were isolated. R_f = 0.17 (PE–EtOAc, 50:50). IR (film): ν = 3395, 3060, 2946, 1716, 1609, 1370, 1208 cm^–1. GC–MS (EI, +, 70 eV): m/z (%) = 133 (19) [(C₅H₉O₄)⁺], 147 (8) [(C₆H₁₁O₄)⁺]. ¹H NMR (600 MHz, CDCl₃): δ = 2.13–2.21 (m, 2 H, 2′-H), 2.37 (d, J = 1.2 Hz, 3 H, Me), 2.96 (d, J = 10.9 Hz, 1 H, 3′-OH), 3.38 (s, 3 H, OMe), 4.19 (dd, J = 9.7, 6.8 Hz, 1 H, 5′a-H), 4.34 (ddd, J = 6.8, 4.1 Hz, 1 H, 4′-H), 4.36–4.41 (m, 2 H, 5′b-H, 3′-H), 5.11 (dd, J = 4.0 Hz, 1 H, 1′-H), 6.10 (q, J = 1.2 Hz, 1 H, 3-H), 6.87 (d, J = 2.5 Hz, 1 H, 8-H), 6.90 (dd, J = 8.8, 2.5 Hz, 1 H, 6-H), 7.48 (d, J = 8.8 Hz, 1 H, 5-H) ppm. ¹³C NMR (151 MHz, CDCl₃): δ = 18.8 (Me), 41.5 (C-2′), 55.3 (OMe), 69.0 (C-5′), 71.6 (C-3′), 82.5 (C-4′), 101.9 (C-8), 105.5 (C-1′), 112.2 (C-3), 112.6 (C-6), 113.9 (C-4a), 125.7 (C-5), 152.6 (C-4), 155.3 (C-8a), 161.4 (C-7), 161.9 (C-2) ppm. HRMS (ESI, +): m/z (%) calcd for C₁₆H₂₀O₆ [M + H]⁺: 307.11761; found: 307.11769. [α]_D –67.4 (c 1.0, CHCl₃).
21 7-{[(3R,4R)-1,3-Dihydroxytetrahydrofuran-4-yl]methoxy}-4-methyl-2H-chromen-2-one (6) Compound 15 (980 mg, 3.2 mmol) was dissolved in MeCN–H₂O (3:1). DOWEX 50WX 8-100 (130 mg) was added, and the reaction was stirred for 1.5 h at r.t. Resulting MeOH was evaporated, and the mixture was stored at r.t. for 2 d. The solution was filtered, and the solvent was evaporated. The crude product was purified by column chromatography (PE–EtOAc, 70:30) yielding 544 mg (1.86 mmol, 58%, dr A/B 0.7:0.3) of product 6 as a white solid. R_f = 0.06 (PE–EtOAc, 50:50). IR (film): ν = 3356, 1705, 1609, 1364, 1207 cm^–1. GC–MS (EI, +, 70 eV): m/z (%) = 293 (100) [M + H]⁺, 246 (12) [C₁₄H₁₄O₄]⁺. ¹H NMR (600 MHz, CDCl₃): δ = 2.19 (ddd, J = 13.8, 4.6, 4.6 Hz, 1 H, 2′a-H_A), 2.23–2.32 (m, 3 H, 2′b-H_A, 2′a-H_B, 2′b-H_B), 2.40 (d, J = 1.3 Hz, 6 H, Me_A,_B), 4.28 (dd, J = 10.0 Hz, J = 6.2 Hz, 1 H, 5′a-H_B), 4.30–4.34 (m, 3 H, 5′a-H_B, 5′a-H_A, 5′b-H_A), 4.35 (dd, J = 6.9, 3.9 Hz, 1 H, 3′-H_B), 4.42 (d, J = 8.6, 4.6 Hz, 1 H, 3′-H_A),4.50–4.54 (m, 1 H, 4′-H_A), 4.69 (ddd, J = 6.2, 3.9, 3.0 Hz, 1 H, 4′-H_B), 5.61 (dd, J = 4.6, 0.3 Hz, 1 H, 1′-H_A), 5.78 (dd, J = 5.4, 3.3 Hz, 1 H, 1′-H_B), 6.14 (q, J = 1.3 Hz, 2 H, 3-H_A,_B), 6.89 (d, J = 2.5 Hz, 2 H, 8-H_A,_B), 6.91 (dd, J = 8.7, 2.5 Hz, 2 H, 6-H_A,_B), 7.50 (d, J = 8.7 Hz, 2 H, 5-H_A,_B) ppm. ¹³C NMR (151 MHz, CDCl₃): δ = 18.8 (Me), 42.1 (C-2′), 68.8 (C-5′), 71.9 (C-4′), 82.3 (C-3′), 99.4 (C-1′), 102.0 (C-8), 112.4 (C-3), 112.5 (C-6), 114.1 (C-4a), 125.8 (C-5), 152.7 (C-8a), 155.3 (C-7), 161.4 (C-2), 161.8 (C-4) ppm. HRMS (ESI, +): m/z (%) calcd for C₁₆H₂₀O₆ [M + H]⁺: 293.10196; found: 293.10218. [α]_D +11.5 (c 0.2, MeCN).
22 Protocol of the Fluorogenic Assay Triethanolamine buffer (120 μL, 0.1 M, pH 7.0), the specific substrate (10 μL of a 10 mM solution), and BSA (10 μL of a solution 40 mg/mL in H₂O) were pipetted in a 96-well microtiterplate. Cell-free crude lysate (60 μL) was added and then directly measured with a GENios Microtiterplate Reader (Tecan, Switzerland) for 1–2 h, applying an excitation wavelength of 340 nm and an emission wavelength of 460 nm with a band-width of 5 nm. For calibration a solution of 4-methylumbelliferone in MeCN was prepared and measured in triethanolamine buffer (using 0–2.5 nmol 4-methylumbelliferone). The calibration curve is shown in the Supporting Information.
23 Protein Expression Genes, coding for DERA (de °C) from P. aerophilum, T. maritima and C. psychrerythraea were ordered as synthetic genes from GenScript, followed by cloning into the pET-21a(+)-vector. For S. halifaxensis, the corresponding gene could be isolated from genomic DNA, whereas de °C genes from E. coli and R. erythropolis were isolated previously.²⁴ The proteins were expressed in E. coli BL21(DE3) strain, using TB medium. Expression was started by applying 0.1 mM IPTG, and cells were harvested after 16 h (24 h for psychrophilic enzymes) incubation at 25 °C (18 °C); 1 g cells were resuspended in potassium phosphate (KP_i) buffer (5 mL, 20 mM, pH 7). The cells were disrupted via sonification, and after centrifugation (15 min at 12000 × g) the supernatant was taken for kinetic measurements. A SDS-gel of all used proteins is shown in the Supporting Information.
24 Kullartz I, Pietruszka J. J. Bioltechnol. 2012; 161: 174
25 Sakuraba H, Yoneda K, Yoshihara K, Satoh K, Kawakami R, Uto Y, Tsuge H, Takahashi K, Hori H, Ohshima T. Appl. Environ. Microbiol. 2007; 73: 7427
26 Egorova K, Antranikian G. Curr. Opin. Microbiol. 2005; 8: 649
27 Feller G. Scientifica 2013; 28

28a Chen L, Dumas DP, Wong CH. J. Am. Chem. Soc. 1992; 114: 741
28b DeSantis G, Liu J, Clark DP, Heine A, Wilson IA, Wong C.-H. Bioorg. Med. Chem. 2003; 11: 43

Zusatzmaterial

Supporting Information