Chemical Synthesis

General Methods

Chemicals were purchased from Aldrich, Acros Organics, Fluka, TCI and Dextra and used without further purification. Technical solvents were distilled prior to use. Thin layer chromatography was performed on silica gel 60 F254 coated aluminum sheets (Merck) with detection by UV light (λ = 254 nm or 366 nm). Additionally, the sheets were stained by dipping in acidic ethanolic p-anisaldehyde solution, basic KMnO4 solution or ninhydrin solution followed by gentle heating.

Preparative flash column chromatography (FC) was performed with an MPLC-Reveleris system from Grace. Nuclear magnetic resonance (NMR) spectra were recorded at room temperature on Avance III 400 and Avance III 600 instruments from Bruker. Chemical shifts are reported relative to solvent signals (CDCl3: δH = 7.26 ppm, δC = 77.16 ppm; D2O: δH = 4.79 ppm; DMSO-d6: δH = 2.50 ppm, δC = 39.52 ppm). Signals were assigned by first-order analysis and, when feasible, assignments were supported by two-dimensional ¹H,¹H and ¹H,¹³C correlation spectroscopy (COSY, HSQC). High-resolution mass spectrometry (HRMS) was carried out on a micrOTOF II instrument from Bruker Daltonics. CHN-analysis was performed at the facility of the university of Konstanz with a CHNS-analyzor vario EL from elementar. Analytical RP-HPLC-MS was performed on a Shimadzu system LCMS2020 (pumps LC-20 AD, autosampler SIL-20AT HAS, column oven CTO-20AC, UV-Vis detector SPD-20A, fluorescence detector RF-20A, controller CBM-20, ESI detector, LCMS Software Solution). Column: Nucleodure C18 Gravity 3 μm from Macherey-Nagel (125 × 4 mm), flow 0.4 mL min^–1, mobile phase: gradient of acetonitrile with 0.1% formic acid (solvent B) in water with 0.1% formic acid (solvent A).

Fluorescence was measured using a Tecan infinite M200 reader. For UV-irradiation a hand-held UV-lamp (UVM-18EI Series UV Lamp, 8 Watt, 302 nm) from UVP was used.

Kinetic measurements

10 mM solutions of tetrazine 22^[94] and alkenes were prepared in acetate buffer (100 mM, pH 4.7, 20°C) and mixed in a quartz cuvette immediately before the measurement. The reactions were monitored over time by the decreasing absorption of the tetrazine at 522 nm and the tetrazine concentration was calculated using Beer-Lambert law, with ε = 437.45 M^-1cm^-1. The second-order rate constant was determined by plotting the inverse tetrazine concentration versus time followed by linear regression analysis as reported earlier.^[94]

Plate reader assay

Stock solutions of tetrazoles (mainly 2 mM) and alkenes in ethanol were mixed 1:1 in a 96-well plate (Corning, 96 flat bottom black polystyrol). The plate was irradiated (302 nm) with a hand

68 7. Experimental Section

held UV-lamp which was directly placed on top of the plate for the depicted time. Fluorescence was measured using a Tecan infinite M200 reader (ex: 360 nm, em (tetrazole 41): 445 nm, em (tetrazole 42): 485 nm). Standard error was calculated of at least two replicates using Sigma plot 13.

1,2:3,4-Di-O-isopropylidene-α-D-galactopyranose

1,2:3,4-Di-O-Isopropylidene-α-D-galactopyranose was prepared according to Chan et al.^[196] in 93%.

1,2:3,4-Di-O-isopropylidene-α-^L-galactopyranose (6)

In the same way as Chan et al.^[196]L-galactose (3 g, 1 eq, 16.6 mmol) and ZnCl2 (2.25 g, 1 equiv, 16.6 mmol) were powdered and degassed. 60 mL acetone and 60 µL conc. H2SO4 were added and the mixture was stirred at rt for two days. To neutralize the reaction 1 M K2CO3-solution was added (15 mL) and the reaction mixture was filtered through Celite and washed with acetone. The filtrate was concentrated in vacuo and extracted with DCM (3x). The organic layers were dried (NaSO4) and concentrated. The crude product was purified by flash column chromatography (PE:EtOAc, 1:1) to give 6 as a colorless oil (3.73 g, 86%). Analytical data match to literature.^[147]

Rf (PE:EtOAc, 1:1) = 0.3; ¹H NMR (400 MHz, CDCl3): δ = 5.57 (d, J = 5.0 Hz, 1H, H-1), 4.61 (dd, J = 8.0, 2.4 Hz, 1H, H-3), 4.33 (dd, J = 5.0, 2.4 Hz, 1H, H-2), 4.27 (dd, J = 8.0, 1.5 Hz, 1H, H-4), 3.91 – 3.80 (m, 2H, H-5, H-6a), 3.77 – 3.70 (m, 1H, H-6b), 1.53, 1.45 (2x s, 3H, CH3), 1.33 (s, 6H, 2x CH3) ppm; ¹³C NMR (101 MHz, CDCl3): δ = 109.6, 108.8 (2x C(CH3)2), 96.4 (C-1), 71.7, 70.9, 70.7, 68.2 (C-2, C-3, C-4, C-5), 62.5 (C-6), 26.2, 26.1, 25.1, 24.4 (4x CH3) ppm; HRMS (ESI-IT):

m/z calcd for C12H21O6: 261.13326 [M + H]⁺; found: 261.13398; calcd for C12H20O6 +Na⁺: 283.11521 [M + Na]⁺; found: 283.11600.

7. Experimental Section 69

1,2:3,4-Di-O-isopropylidene-α-D-galacto-hexodialdo-1,5-pyranose

1,2:3,4-di-O-isopropylidene-α-D-galacto-hexodialdo-1,5-pyranose was prepared through a Swern oxidation according to Midland et al.^[149] yielding 84%.

1,2:3,4-Di-O-isopropylidene-α-L-galacto-hexodialdo-1,5-pyranose (7)

A solution of oxalyl chloride (1.86 mL, 1.5 eq, 21.5 mmol) in 35 mL dry DCM was cooled to -55°C (CHCl3/liquid nitrogen bath) under nitrogen conditions. DMSO (3.26 mL, 3.2 equiv, 45.8 mmol) was added slowly at -55°C. Generation of gas was observed. The reaction mixture was stirred for 30 min at -55°C. The sugar 6 (3.73 g, 1 equiv, 14.3 mmol) was coevaporated with toluene, dissolved in 3 mL dry DCM and added slowly. After stirring for 40 min, NEt3 (13 mL, 6.5 equiv, 88 mmol) was added and the reaction mixture was allowed to stir for 30 min at -55°C. The cold bath was removed, and the resulting milky mixture was allowed to warm to rt (1 h). Water (20 mL) was added and the organic layer was removed. The aqueous layer was extracted with DCM (3x) and the combined organic layers were washed with NaHCO3, dried (MgSO4) and concentrated in vacuo. Purification by column chromatography (PE:EtOAc, 3:1) afforded 2.7 g of the aldehyde 7 (10.6 mmol, 74%) as an oil.

Rf (PE:EtOAc, 3:1) = 0.1 (smear); ¹H NMR (400 MHz, CDCl3): δ = 9.62 (s, 1H, H-6), 5.67 (d, J = 4.9 Hz, 1H, H-1), 4.68 – 4.57 (m, 2H, H-3, H-4), 4.38 (dd, J = 4.9, 2.5 Hz, 1H, H-2), 4.19 (d, J = 2.2 Hz, 1H, H-5), 1.51, 1.44, 1.35, 1.32 (4x s, 3H, CH₃) ppm; ¹³C NMR (101 MHz, CDCl₃):

δ = 200.5 (C-6), 110.2, 109.2 (2x C(CH3)₂), 96.4 (C-1), 73.4 (C-5), 71.9, 70.6, 70.5 (C-2, C-3, C-4), 26.2, 25.9, 25.0, 24.4 (4x CH3) ppm; HRMS (ESI-IT): m/z calcd for C12H19O6: 259.11761 [M + H]⁺; found: 259.11865.

70 7. Experimental Section

6-C-Methylidene-1,2:3,4-di-O-isopropylidene-α-D-fucose

The sugar was synthesized through a Wittig reaction starting from the aldehyde as described for the L-derivative 8, to get 72%. Analytical data agree with literature^[197] in which the same sugar was prepared using a different procedure.

6-C-Methylidene-1,2:3,4-di-O-isopropylidene-α-L-fucose (8)

A suspension of methyltriphenylphosphonium bromide (5.5 g, 2.7 equiv, 15.4 mmol) in dry THF (16 mL) was cooled to 0°C. A hexane solution of n-BuLi (1.6 M, 2.3 equiv, 8 mL, 12.9 mmol) was added, turning the mixture yellow. After stirring for 5 min the ice bath was removed and the stirring was continued at rt for 1 h. The reaction mixture was cooled to -78°C and in 3 mL dry THF dissolved sugar 6 (1.46 g, 1 equiv, 5.6 mmol) was added slowly. After stirring for 30 min at -78°C the mixture was allowed to stir at rt for another 3 h. Sat. NH4Cl solution (9 mL) was added and the layers were separated. The aquous layer was extracted with ether (4x). The combined organic layers were dried (MgSO4), concentrated and purified by flash column chromatography (PE:EtOAc, 6:1) to yield 8 (0.55 g, 38%) as a gum. The procedure is published in a similar way by Alley et al.^[150] Analytical data are in good agreement.

Rf (PE:EtOAc, 6:1) = 0.35; ¹H NMR (400 MHz, CDCl3): δ = 5.93 (ddd, J = 16.9, 10.6, 6.0 Hz, 1H, H-6), 5.58 (d, J = 5.0 Hz, 1H, H-1), 5.37 (dt, J = 17.4, 1.6 Hz, 1H, CH2a), 5.27 (dt, J = 10.6, 1.5 Hz, 1H, CH₂b), 4.62 (dd, J = 7.9, 2.4 Hz, 1H, H-3), 4.32 (dd, J = 5.0, 2.4 Hz, 1 H, H-2) 4.29 (dd, J = 6.0, 1.5 Hz, 1H, H-5), 4.22 (dd, J = 7.9, 2.0 Hz, 1H, H-4), 1.54, 1.47 (2x s, 3H, CH3), 1.34 (s, 6H, 2x CH3) ppm; ¹³C NMR (101 MHz, CDCl3): δ = 134.0 (C-6), 117.5 (CHCH2), 109.4, 108.6 (2x C(CH3)2), 96.6 (C-1), 73.6 (C-4), 71.0 (C-3), 70.6, 69.1 (C-2, C-5), 26.3, 26.1, 25.1, 24.5 (4x CH3) ppm; HRMS (ESI-IT): m/z calcd for C13H20O5 +Na⁺:279.12029 [M + Na]⁺; found: 279.12128.

7. Experimental Section 71

6-C-Methylidene-1,2,3,4-tetra-O-acetyl-α/β-D-fucose

The sugar was prepared from 6-C-Methylidene-1,2:3,4-di-O-isopropylidene-α-^D-fucose according to standard procedures as depicted for the L-derivative. Analytical data agree with the literature.^[197]

6-C-Methylidene-1,2,3,4-tetra-O-acetyl-α/β-L-fucose (Ac4Fuc6CH2, 1)

For isopropylidene deprotection sugar 8 (550 mg, 1 equiv, 2.15 mmol) was stirred in 70% acetic acid (13.8 mL) at 90°C for 6 h. The solvent was removed in vacuo (nitrogen evaporator) and coevaporated once with toluene. Without further purification Ac2O (2.3 mL, 10 equiv, 21.5 mmol) and pyridine (1.6 mL, 10 equiv, 21.5 mmol) were added and the mixture was stirred at rt overnight. The yellowish solution was evaporated in vacuo and the residue was dissolved in DCM. The organic layer was extracted with 1 M HCl (3x) followed by sat. NaHCO3 solution (3x) and water (1x). The organic layers were combined, dried (MgSO4) and purified by column chromatography (PE:EtOAc, 3:1) followed by crystallization (EtOAc, PE) to give 454 mg 1 as α/β mixture of 1:10 (61 %). The compound was previously synthesized by Alley et al.^[150] not giving analytical data.

Rf (PE:EtOAc, 3:1) = 0.2; α anomer: ¹H NMR (400 MHz, CDCl3): δ = 6.43 (d, J = 2.5 Hz, 1H, H-1), 5.75 – 5.64 (m, 1H, CHCH2), 5.47 (s, 1H, H-3 or H-4), 5.44 – 5.32 (m, 3H, CH2a, H-2, H-3 or H-4), 5.27 (d, J = 10.6 Hz, 1H, CH2b), 4.64 (d, J = 5.1 Hz, 1H, H-5), 2.15, 2.11, 2.02, 2.00 (4x s, 3H, CH3) ppm; ¹³C NMR (101 MHz, CDCl3): δ = 170.3, 170.1, 169.5, 169.1 (4x CO), 131.8 (CHCH2), 118.6 (CH2), 90.0 (C-1), 71.9 (C-5), 70.0, 67.8, 66.7 (C-2, C-3, C-4), 21.1, 20.8, 20.74, 20.71 (4x CO) ppm; β anomer: ¹H NMR (400 MHz, CDCl3): δ = 5.74 (d, J = 8.3, Hz, 1H, H-1), 5.75 – 5.64 (m, 1H, CHCH2), 5.44 – 5.32 (m, 3H, CH2a, H-3, H-4), 5.27 (d, J = 10.6 Hz, 1H, CH2b), 5.11 (dd, J = 10.3, 3.0 Hz, 1H, H-2), 4.32 (dt, J = 5.1, 1.7 Hz, 1H, H-5), 2.14, 2.12, 2.04, 1.99 (4x s, 3H, CH3) ppm; ¹³C NMR (101 MHz, CDCl3): δ = 170.4, 170.1, 169.6, 169.2 (4x CO), 131.3 (CHCH2), 119.1 (CH2), 92.3 (C-1), 74.8 (C-5), 71.2 (C-2), 69.4, 68.1 (C-3, C-4), 21.0, 20.81, 20.77, 20.7 (4x CH3) ppm; Anal. Calcd: C, 52.32; H, 5.85 Found: C, 52.28; H, 8.85; HRMS (ESI-IT): m/z calcd for C15H20O9 +Na⁺:367.09995 [M + Na]⁺; found: 367.09987.

72 7. Experimental Section

6-C-Methylidene-α/β-L-fucose (Fuc6CH2, 9)

Peracetylated sugar 1 (30 mg, 1 equiv, 0.087 mmol) was dissolved in dry MeOH (2.5 mL) and NEtMe2 (0.5 mL, 50 equiv, 4.6 mmol) was added. The mixture was stirred for 3 days at rt before the solvent was evaporated. The crude product was coevaporated three times with toluene (1 mL) and purified by flash column chromatography (DCM:MeOH = 6:1). In order to remove in MeOH dissolved silica, the free sugar 9 was dissolved in water and filtered after 48 h. Lyophilizing gave 57% of Fuc6CH2 (9) in a α:β mixture of 1:2. The analytical data are in good agreement with the literature^[198] were it is enzymatically prepared.

Rf (DCM:MeOH, 6:1) = 0.23; α anomer: ¹H NMR (400 MHz, D2O): δ = 6.04 – 5.89 (m, 1H, H-6), 5.50 – 5.33 (m, 2H, CH2), 5.31 (d, J = 3.8 Hz, 1H, H-1), 4.63 – 4.58 (m, 1H, H-5), 4.01 – 3.98 (m, 1H, H-3), 3.95 – 3.90 (m, 1H, H-4), 3.84 (dd, J = 10.2, 3.9 Hz, 1H, H-2) ppm; ¹³C NMR (101 MHz, D2O): δ = 133.9 (C-6), 118.0 (C-7), 92.4 (C-1), 71.5, 71.2, 69.1, 68.1 (C-2, C-3, C-4, C-5) ppm;

β anomer: ¹H NMR (400 MHz, D2O): δ = 6.04 – 5.89 (m, 1H, H-6), 5.50 – 5.33 (m, 2H, CH2), 4.65 (d, J = 7.9 Hz, 1H, H-1), 4.24 (dd, J = 5.7, 1.4 Hz, 1H, H-5), 3.95 – 3.90 (m, 1H, H-4), 3.72 (dd, J = 9.9, 3.5 Hz, 1H, H-3), 3.53 (dd, J = 9.9, 7.9 Hz, 1H, H-2) ppm; ¹³C NMR (101 MHz, D2O):

δ = 133.5 (C-6), 118.0 (C-7), 96.4 (C-1), 75.6 (C-5), 72.8, 71.7, 71.0 (C-2, C-3, C-4) ppm; HRMS (ESI-IT): m/z calcd for C7H12O5 +Na⁺:199.0588 [M + Na]⁺; found: 199.0579.

(6R,S)-6-C-Vinyl-1,2:3,4-di-O-isopropylidene-α-D-galactose

The sugar was synthesized through a Grignard reaction according to the literature^{[199, 200]} yielding 71%.

7. Experimental Section 73

(6R,S)-6-C-Vinyl-1,2:3,4-di-O-isopropylidene-α-L-galactose (10)

Magnesium (0.33 g, 3 equiv, 13.6 mmol) was dried in a flask and dry THF (6 mL) was added under nitrogen conditions. Vinylbromide (1 M in THF, 13.6 mL, 3 equiv, 13.6 mmol) was added slowly and catalytic amounts of iodide were added to the stirring mixture in order to start the reaction. After refluxing for 3 h sugar 7 (1.3 g, 1 equiv, 4.5 mmol, dissolved in 6 mL dry THF) was added and the reaction mixture was refluxed for 4 h. Water (12 mL) was added slowly, followed by 1 M HCl (15 mL, to get acidic pH). Layers were separated and the aqueous layer was extracted with EtOAc (3x). The combined organic layers were dried (MgSO4) and evaporated.

Crude product 10 was purified by column chromatography (DCM:MeOH, 95:5) to give 0.76 g (60%) as diastereomer mixture (ratio 3:2) of 10 as a yellow solid.

Rf (DCM:MeOH, 95:5) = 0.38; Major isomer: ¹H NMR (400 MHz, CDCl3): δ = 6.03 (ddd, J = 17.2, 10.6, 5.3 Hz, 1H, CHCH2), 5.57 (d, J = 4.9 Hz, 1H, H-1), 5.43 (dt, J = 17.3, 1.6 Hz, 1H, CH2a), 5.30 – 5.22 (m, 1H, CH2b), 4.62 (dd, J = 8.0, 2.4 Hz, 1H, H-3), 4.47 (dd, J = 8.0, 1.9 Hz, 1H, H-4), 4.40 – 4.33 (m, 1H, H-6), 4.32 (dd, J = 5.0, 2.4 Hz, 1H, H-2), 3.66 (dd, J = 7.0, 1.9 Hz, 1H, H-5), 1.51, 1.49, 1.36, 1.33 (4x s, 3H, CH3) ppm; Minor isomer: ¹H NMR (400 MHz, CDCl3): δ = 5.92 (ddd, J = 17.2, 10.6, 5.9 Hz, 1H, CHCH2), 5.59 (d, J = 4.8 Hz, 1H, H-1), 5.49 (dt, J = 17.3, 1.6 Hz, 1H, CH2a), 5.30 – 5.22 (m, 1H, CH2b), 4.58 (dd, J = 8.0, 2.4 Hz, 1H, H-3), 4.40 – 4.33 (m, 1H, H-6), 4.32 (dd, J = 5.0, 2.4 Hz, 1H, H-2), 4.26 (dd, J = 8.0, 1.8 Hz, 1H, H-4), 3.57 (dd, J = 7.0, 1.8 Hz, 1H, H-5), 1.51, 1.47, 1.33, 1.32 (4x s, 3H, CH3) ppm; Both isomers: ¹³C NMR (101 MHz, CDCl3): δ = 138.0, 135.6 (CHCH2), 117.8, 116.4 (CHCH2), 109.6, 109.6, 108.9, 108.8 (2x C(CH3)2), 96.7, 96.6 (C-1), 72.0, 71.9, 71.6, 71.3, 71.2, 71.0, 71.0, 70.9, 70.6, 69.5 (C-2, C-3, C-4, C-5, CHOH), 26.2, 26.14, 26.08, 26.07, 25.12, 25.06, 24.5, 24.4 (4x CH3) ppm.

(6R,S)-6-C-Vinyl-6-O-imidazole-1-carbonothioyl-1,2:3,4-di-O-isopropylidene-α-D-galactose

The sugar was synthesized as described for the L-derivative with the same analytical data.

74 7. Experimental Section

(6R,S)-6-C-Vinyl-6-O-imidazole-1-carbonothioyl-1,2:3,4-di-O-isopropylidene-α-L-galactose

Sugar 10 (765 mg, 1 equiv, 2.7 mmol) and 1,1’-thiocarbonyl-diimidazole (675 mg, 1.4 equiv, 3.8 mmol) were refluxed in toluene (30 mL) for 4 h. The solvent was evaporated and the residue was dissolved in DCM and washed with NaHCO3. The aqueous layer was extracted with DCM (3x) and the combined, yellow organic layers were dried (MgSO4) and evaporated. The activated sugar was purified by flash column chromatography, yielding the major isomer in 36% (370 mg).

Rf (PE:EtOAc, 1:1) = 0.3; ¹H NMR (400 MHz, CDCl3): δ = 8.23 (t, J = 0.9, 1H, NCHN), 7.47 (t, J = 1.5 Hz, 1H, NCHCH), 7.12 (dd, J = 1.8, 0.9 Hz, 1H, NCHCH), 5.97 – 5.80 (m, 2H, CHCH2, H-6), 5.56 (d, J = 5.0 Hz, 1H, H-1), 4.61 (dd, J = 7.9, 2.4 Hz, 1H, H-3), 4.32 (dd, J = 5.0, 2.4 Hz, 1H, H-2), 4.29 (dd, J = 5.1, 2.1 Hz, 1H, H-5), 4.19 (dd, J = 7.9, 2.0 Hz, 1H, H-4), 3.87 – 3.79 (m, 2H, CH2CH), 1.53 (s, 3H, CH3), 1.45 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.33 (s, 3H, CH3) ppm;

13C NMR (101 MHz, CDCl3): δ = 135.5 (CAr), 131.3 (C-6/CHCH2), 130.6 (CAr), 126.5 (C-6/CHCH2), 116.2 (CAr), 109.6, 108.8 (2x C(CH3)2), 96.6 (C-1), 73.3 (C-5), 71.0, 70.5, 68.2 (C-2, C-3, C-4), 32.7 (CH2CH), 26.3, 26.1, 25.0, 24.6 (4x CH3) ppm.

6-C-Vinyl-1,2:3,4-di-O-isopropylidene-α-^D-fucose

6-C-vinyl-1,2:3,4-di-O-isopropylidene-α-D-fucose was synthesized as described for the L-derivative 12 with the same analytical data.

6-C-Vinyl-1,2:3,4-di-O-isopropylidene-α-L-fucose (12)

Bu3SnH (1.2 mL, 2.5 equiv, 4.4 mmol), AIBN (0.2 M in toluene, 2 mL, 0.25 equiv, 0.44 mmol) and toluene (12 mL) were combined under nitrogen conditions and heated. At 80°C the activated sugar (670 mg, 1 equiv, 1.76 mmol), dissolved in 6 mL toluene was added slowly. The mixture was refluxed for 3 h, evaporated and dissolved in DCM. The organic layer was washed with water (2x) which was subsequently extracted with DCM (2x). Combined organic layers were dried

7. Experimental Section 75

(MgSO4) and evaporated. Crude 12 was purified by flash column chromatography (PE:EtOAc, 20:1) yielding 38 mg (8%) desired product 12 and 193 mg (40%) of the rearranged byproduct 13.

Rf (PE:EtOAc, 6:1) = 0.4; ¹H NMR (400 MHz, CDCl3): δ = 5.86 (ddt, J = 17.1, 10.3, 6.7 Hz, 1H, with toluene. Flash column chromatography (DCM:MeOH, 6:1) gave a α:β mixture (1:1) of pure product 14 (37 mg, 73%).

76 7. Experimental Section

2.54 – 2.31 (m, 2H, H-6) ppm; ¹³C NMR (101 MHz, D2O): δ = 134.3 (CHCH2), 117.5 (CHCH2), 96.4 (C-1), 74.2, 73.0, 71.9, 69.9 (C-2, C-3, C-4, C-5), 34.3 (C-6) ppm.

6-C-Vinyl-1,2,3,4-tetra-O-acetyl-α/β-^L-fucose (Ac₄Fuc6Vin, 2)

To free sugar 14 (27 mg, 1 equiv, 0.14 mmol) pyridine (200 µL, 18 equiv, 2.5 mmol) and Ac2O (200 µL, 15 equiv, 2.1 mmol) were added and the mixture was stirred at rt for two days. The solvent was removed and the mixture was dissolved in DCM and washed with 10% KHSO4 (2x) and sat. NaHCO3 (2x). The organic layer was dried (MgSO4) and evaporated. The crude product was purified by flash column chromatography (PE:EtOAc, 3:1) yielding Ac4Fuc6Vin (2, 43 mg, 85%, α/β mixture of 1:2).

Rf (PE:EtOAc, 3:1) = 0.2; α anomer: ¹H NMR (400 MHz, CDCl3): δ = 6.35 (d, J = 2.1 Hz, 1H, H-1), 5.73 – 5.63 (m, 1H, CHCH2), 5.39 (s, 1H, H-4), 5.35 – 5.26 (m, 2H, H-2, H-3), 5.13 – 4.99 (m, CH2CH, 2H), 4.10 (q, J = 5.9, 4.6 Hz, 1H, H-5), 2.47 – 2.19 (m, 2H, H-6), 2.17, 2.11, 2.03, 1.98 (4x s, 3H, CH3) ppm; ¹³C NMR (101 MHz, CDCl3): δ = 170.4, 170.3, 170.0, 169.2 (4x CO), 132.5 (CHCH2), 118.6 (CHCH2), 90.1 (C-1), 71.0, 69.2, 68.0, 66.7 (C-2, C-3, C-4, C-5), 34.8 (C-6), 21.0, 21.0, 20.8, 20.7 (4x CH3) ppm; β anomer: ¹H NMR (400 MHz, CDCl3): δ = 5.73 – 5.63 (m, 1H, CHCH2), 5.66 (d, J = 8.2 Hz, 1H, H-1), 5.35 – 5.26 (m, 2H, H-4, H-2 or H-3), 5.13 – 4.99 (m, 3H, CH2CH, H-2 or H-3), 3.79 (t, J = 7.0 Hz, 1H, H-5), 2.47 – 2.19 (m, 2H, H-6), 2.16, 2.14, 2.00, 1.99 (4x s, 3H, CH3) ppm; ¹³C NMR (101 MHz, CDCl3): δ = 170.3, 170.1, 169.6, 169.2 (4x CO), 132.2 (CHCH2), 118.9 (CHCH2), 92.4 (C-1), 74.0, 71.4, 68.5, 68.1 (C-2, C-3, C-4, C-5), 34.7 (C-6), 21.0, 20.8, 20.7 (4x CH3) ppm; HRMS (ESI-IT): m/z calcd for C16H22O9 +Na⁺: 381.1156 [M + Na]⁺; found: 381.1138.

(6R,S)-6-C-Allyl-1,2:3,4-di-O-isopropylidene-α-D-galactose

The sugar was synthesized through a metalorganic reaction according to literature^[201] yielding 64%. Analytical data are in agreement with literature.^[201]

7. Experimental Section 77

(6R,S)-6-C-Allyl-1,2:3,4-di-O-isopropylidene-α-L-galactose (18)

To a solution of aldehyde 7 (2.74 g, 1 equiv, 10.6 mmol) in 23 mL THF, allyl bromide (2 mL, 2 equiv, 21.2 mmol) and zinc dust (1.4 g, 2 equiv, 21.2 mmol) were added, slowly followed by sat.

NH4Cl solution (8.6 mL). After stirring for 3 h at rt sat. NH4Cl solution (11 mL) was added and the

(6R,S)-6-C-allyl-1,2:3,4-di-O-isopropylidene-α-D-galactose (100 mg, 0.37 mmol, 1 equiv) was coevaporated with toluene before it was dissolved in dry toluene (3.7 mL). 1,1’-thiocarbonyldiimidazole (92.3 mg, 0.52 mmol, 1.4 equiv) was added and the mixture was refluxed for 5 h. The solvent was evaporated and the product purified with flash column chromatography (PE:EtOAc, 2:1) yielding 65% (100 mg).

Rf (PE:EtOAc, 3:1) = 0.3; ¹H NMR (400 MHz, CDCl3): δ = 8.39 (s, 1H, NCHN), 7.65 (t, J = 1.4 Hz, 1H, NCHCH), 7.07 (s, 1H, NCHCH), 5.89 – 5.72 (m, 2H, CHCH2, H-6), 5.56 (d, J = 5.0 Hz, 1H,

78 7. Experimental Section

H-1), 5.20 – 5.09 (m, 2H, CHCH2), 4.63 (dd, J = 7.9, 2.5 Hz, 1H, H-3), 4.35 (dd, J = 5.0, 2.5 Hz, 1H, H-2), 4.26 (dd, J = 7.9, 1.9 Hz, 1H, H-4), 4.14 – 4.07 (m, 1H, H-5), 2.93 – 2.82 (m, 1H, CH2a), 2.68 (ddd, J = 15.2, 7.8, 6.0 Hz, 1H, CH2b), 1.49, 1.45, 1.33, 1.30 (4x s, 3H, CH3) ppm; ¹³C NMR (101 MHz, CDCl3): δ = 132.1, 130.0, 119.6 (3x CAr), 118.3 (CHCH2), 109.9, 109.0 (2x C(CH3)2), 96.6 (C-1), 81.3, 71.0, 70.8, 70.7, 67.0 (C-2, C-3, C-4, C-5, C-6), 33.5 (CH2), 26.2, 26.1, 25.0, 24.6 (4x CH3) ppm.

(6R,S)-6-C-Allyl-6-C-iodo-1,2:3,4-di-O-isopropylidene-α-D-fucose

(6R,S)-6-C-allyl-6-C-iodo-1,2:3,4-di-O-isopropylidene-α-D-fucose was synthesized as described for the L-derivative 19 with the same analytical data.

(6R,S)-6-C-Allyl-6-C-iodo-1,2:3,4-di-O-isopropylidene-α-L-fucose (19)

Sugar 18 (2.52 g, 1 equiv, 8.4 mmol), PPh3 (7.9 g, 3.6 equiv, 30 mmol), imidazole (1.9 g, 3.3 equiv, 28 mmol) and I2 (3.4 g, 1.6 equiv, 13.4 mmol) were degassed. Dry toluene (80 mL) was added and the mixture was refluxed under N2 for 2 h. The reaction mixture was treated with 1 M Na2S2O3 solution (50 mL) and extracted with toluene (4x). The combined organic layers were dried (MgSO4), concentrated and purified by column chromatography (PE:EtOAc, 15:1) to give 780 mg (23%) of iodide 19.

Rf (PE:EtOAc, 9:1) = 0.3; ¹H NMR (400 MHz, CDCl3): δ = 5.91 (dddd, J = 16.3, 10.5, 8.0, 5.5 Hz, 1H, CHCH2), 5.60 (d, J = 5.0 Hz, 1H, H-1), 5.22 (q, J = 1.4 Hz, 1H, CHCH2a), 5.18 (dt, J = 8.8, 1.6 Hz, 1H, CHCH2b), 4.58 (dd, J = 7.9, 2.5 Hz, 1H, H-3), 4.38 (dd, J = 7.8, 1.9 Hz, 1H, H-4), 4.32 (dd, J = 5.0, 2.5 Hz, 1H, H-2), 4.22 (ddd, J = 9.7, 7.5, 3.6 Hz, 1H, H-6), 3.82 (dd, J = 9.8, 1.9 Hz, 1H, H-5), 2.81 (dddt, J = 15.3, 5.3, 3.4, 1.7 Hz, 1H, CH2a), 2.62 (dt, J = 15.5, 7.8 Hz, 1H, CH2b), 1.54, 1.44, 1.34, 1.33 (4x s, 3H, CH3) ppm; ¹³C NMR (101 MHz, CDCl3): δ = 136.1 (CHCH2), 118.2 (CHCH2), 109.8, 109.1 (2x C(CH3)2), 97.0 (C-1), 72.0 (C-5), 71.2, 71.0, 70.9 (C-2, C-3, C-4), 39.2 (CH2), 34.1 (C-6), 26.1, 26.0, 25.1, 24.7 (4x CH3) ppm; HRMS (ESI-IT): m/z calcd for C₁₅H₂₄O₅: 411.06629 [M + H]⁺; found: 411.06589; calcd for C₁₅H₂₃O₅ +Na⁺:433.04824 [M + Na]⁺; found: 433.04852.

7. Experimental Section 79

6-C-Allyl-1,2:3,4-di-O-isopropylidene-α-D-fucose

6-C-allyl-1,2:3,4-di-O-isopropylidene-α-D-fucose was synthesized as described for the L-derivative 20. Alytical data are identical to literature^[202] were they synthesize the compound using a different procedure.

6-C-Allyl-1,2:3,4-di-O-isopropylidene-α-^L-fucose (20)

AIBN (78 mg, 0.25 equiv, 0,47 mmol) and nBu₃SnH (1.2 ml, 2 equiv, 4.6 mmol) were refluxed in dry toluene (30 mL) under nitrogen conditions. The iodide 19 (780 mg, 1 equiv, 1.9 mmol) was dissolved in dry toluene (6 mL) and added slowly. The mixture was allowed to reflux overnight.

DCM (20 mL) was added and the organic layer was washed with brine. After phase separation the aqueous layer was extracted with DCM (5x). Combined organic layers were dried (MgSO4), concentrated in vacuo and purified by column chromatography (PE:EtOAc, 15:1) to yield sugar 20 (475 mg, 88%) as a colorless oil.

R_f (PE:EtOAc, 15:1) = 0.25; ¹H NMR (400 MHz, CDCl₃): δ = 5.82 (ddt, J = 17.0, 10.2, 6.6 Hz, 1H, CHCH2), 5.53 (d, J = 5.1 Hz, 1H, H-1), 5.06 (dt, J = 17.1, 1.8 Hz, 1H, CHCH2a), 4.98 (ddt, J = 10.2, 2.2, 1.3 Hz, 1H, CHCH2b), 4.58 (dd, J = 7.9, 2.3 Hz, 1H, H-3), 4.29 (dd, J = 5.1, 2.3 Hz, 1H, H-2), 4.12 (dd, J = 7.9, 1.9 Hz, 1H, H-4), 3.74 (ddd, J = 8.8, 4.6, 1.8 Hz, 1H, H-5), 2.31 – 2.06 (m, 2H, CH2), 1.80 (dtd, J = 14.2, 8.4, 5.9 Hz, 1H, H-6a), 1.69 – 1.60 (m, 1H, H-6b), 1.51, 1.46, 1.35, 1.32 (4x s, 3H, CH3) ppm; ¹³C NMR (101 MHz, CDCl3): δ = 138.2 (CHCH2), 115.3 (CHCH2), 109.1, 108.5 (2x C(CH3)2), 96.7 (C-1), 73.0, 71.1, 70.7 (C-2, C-3, C-4), 66.7 (C-5), 29.8 (C-7), 29.4 (C-6), 26.2, 26.1, 25.1, 24.5 (4x CH3) ppm; HRMS (ESI-IT): m/z calcd for C15H24O5 +H⁺: 285.1697 [M + H]⁺; found: 285.1697.

6-C-Allyl-1,2,3,4-tetra-O-acetyl-α/β-D-fucose

6-C-allyl-1,2,3,4-tetra-O-acetyl-α/β-D-fucose was synthesized as described for the L-derivative 3 with the same analytical data.

80 7. Experimental Section

6-C-Allyl-1,2,3,4-tetra-O-acetyl-β-L-fucose (Ac4Fuc6All, 3)

For deprotection sugar 20 (475 mg, 1 equiv, 1.67 mmol) was stirred in 70% acetic acid at 90°C for 6 h. The solvent was removed in vacuo (nitrogen evaporator) and coevaporated once with toluene. Without further purification Ac2O (1.8 mL, 10 equiv, 16.7 mmol) and pyridine (1.25 mL, 10 equiv, 16.7 mmol) were added and the mixture was stirred at rt overnight. The yellowish solution was evaporated in vacuo and the residue was dissolved in DCM. The organic layer was extracted with 1 M HCl (3x) followed by sat. NaHCO3 solution (3x) and water (1x). The organic layers were combined, dried (MgSO4) and purified by column chromatography (PE:EtOAc, 3:1) followed by crystallization (EtOAc,PE) to give the β-anomer of 3 (215 mg, 35%) as white needles.

Rf (PE:EtOAc, 3:1) = 0.2; ¹H NMR (400 MHz, CDCl3): δ = 5.73 (ddt, J = 17.0, 10.2, 6.8 Hz, 1H, NEtMe2 (0.5 mL, 50 equiv, 4.6 mmol) was added. The mixture was stirred for 3 days at rt before the solvent was evaporated. The crude product was coevaporated with toluene (3x, 1 mL) and purified by flash column chromatography (DCM:MeOH = 6:1). In order to remove in MeOH dissolved silica, the free sugar 21 was dissolved in water and filtered after 48 h. Lyophilizing gave 78% of Fuc6All (21) in an α:β mixture of 1:2.

7. Experimental Section 81 1.35 mmol, 5 equiv) and Pd/C (40 mg, 40 w%) were added. The flask was rinsed two times with H2 and the mixture was allowed to stir at room temperature for 1.5 h. The mixture was filtered over Celite and the solvent removed. The crude amine 35 was coevaporated with toluene and used without further purification. The sugar was dissolved in DMF (3 mL) and DIPEA (138 µL, 0.81 mmol, 3 equiv) was added. After stirring at room temperature for 10 min NHS-activated cyclopropene 36^[102] (60 mg, 0.32 mmol, 1.2 equiv, dissolved in 1 mL DMF) was added dropwise.

The clear solution was stirred at room temperature for 3 h and 45 min before the solvent was

82 7. Experimental Section

4-Nitrophenyl (4-(2-phenyl-2H-tetrazol-5-yl)phenyl) carbonate (44)

Tetrazole 43 (456 mg, 1.91 mmol, 1 equiv) and para-nitrophenyl choroformate (1.01 g, 4.97 mmol, 2.6 equiv) were dissolved in dry pyridine (15 mL) and cooled to 0°C under nitrogen conditions. The mixture was stirred for 22 h while warming up to rt. The solvent was removed and the crude mixture was purified by column chromatography (PE:EtOAc, 1:1 to EtOAc) yielding the product 44 (620 mg, 1.54 mmol, 81%) as light yellow solid.

Rf (PE:EtOAc, 1:1) = 0.6; ¹H NMR (400 MHz, DMSO-d6): δ = 8.42 – 8.35 (m, 2H), 8.32 – 8.27 (m, 2H), 8.21 – 8.15 (m, 2H), 7.78 – 7.60 (m, 7H) ppm; ¹³C NMR (101 MHz, DMSO-d6): δ = 163.9 (CO), 139.6 (NCN), 130.2, 128.2, 126.1, 125.5, 122.72, 122.67, 122.4, 120.0, 115.8 ppm.

4-Nitrophenyl (4-(5-phenyl-2H-tetrazol-2-yl)phenyl) carbonate

Tetrazole-OH (178 mg, 0.75 mmol, 1 equiv) and para-nitrophenyl chloroformate (392 mg, 1.94 mmol, 2.6 equiv) were dissolved in dry pyridine (6 mL) and cooled to 0°C under nitrogen conditions. The mixture was stirred for 19 h while warming up to rt. The solvent was removed and the crude mixture was purified by column chromatography (PE:EtOAc, 2:1 to 1:2) yielding the activated tetrazole (230 mg, 0.57 mmol, 76%) as light yellow solid.

Rf (PE:EtOAc, 1:1) = 0.7; ¹H NMR (400 MHz, DMSO-d6): δ = 8.38 (dd, J = 9.4, 2.8 Hz, 2H), 8.33 – 8.26 (m, 2H), 8.19 (dd, J = 7.4, 2.2 Hz, 2H), 7.81 – 7.71 (m, 4H), 7.66 – 7.58 (m, 3H) ppm;

13C NMR (101 MHz, DMSO-d6): δ = 154.9 (CO), 145.5 (NCN), 129.4, 126.7, 126.1, 125.5, 123.1, 122.70, 122.66, 121.6, 115.8 ppm.

7. Experimental Section 83

13-biotinylamido-4,7,10-trioxatridecan-1-(4-(2-phenyl-tetrazol-5-yl)phenoxy)carbamate (tetrazole-biotin, 45)

Activated tetrazole 44 (100 mg, 0.25 mmol, 1 equiv) and biotin-PEG-NH2 [192]

(133 mg, 0.3 mmol, 1.2 equiv) were dissolved under nitrogen conditions in dry DMF (5 mL) and pyridine (0.5 mL). The mixture was stirred at rt for 64 h and evaporated. The oily yellow residue was dissolved in DCM (30 mL) and washed with water (2x 15 mL). The combined aqueous layers were extracted with DCM (2x 15 mL) and the combined organic layers were dried (MgSO₄) and evaporated. The resulting yellow oil was purified by column chromatography (DCM:MeOH, 8:1) to give tetrazole-biotin 45 as colorless oil (32.2 mg, 0.045 mmol, 18%).

Rf (DCM:MeOH, 10:1) = 0.1; ¹H NMR (400 MHz, DMSO-d6): δ = 8.21 – 8.13 (m, 4H, H-2, H-6, H-3‘ and H-5‘ or H-2‘ and H-6‘), 7.87 (t, J = 5.7 Hz, 1H, NHCO), 7.76 – 7.66 (m, 3H, NHCO, H-3, H-5), 7.66 – 7.59 (m, 1H, H-4), 7.39 – 7.32 (m, 2H, H-2‘ and H-6‘ or H-3‘ and H-5‘), 6.40 (s, 1H, NH), 6.34 (s, 1H, NH), 4.32 – 4.26 (m, 1H, NHCHCH2), 4.15 – 4.09 (m, 1H, NHCHCH), 3.56 – 3.43 (m, 10H, 5x CH2O), 3.40 (q, J = 6.3 Hz, 2H, CH2O), 3.18 – 3.12 (m, 2H, CONHCH2), 3.11 – 2.98 (m, 3H, CHS, CONHCH2), 2.81 (dd, J = 12.4, 5.1 Hz, 1H, CHexo), 2.57 (d, J = 12.4 Hz, 1H, CHendo), 2.04 (t, J = 7.4 Hz, 2H, CH2), 1.73 (p, J = 6.6 Hz, 2H, CH2), 1.66 – 1.54 (m, 4H, 2x CH2), 1.54 – 1.38 (m, 2H, CH2), 1.30 (dq, J = 15.3, 8.3, 7.5 Hz, 2H, CH2) ppm; ¹³C NMR (101 MHz, DMSO-d6): δ = 171.9, 164.1, 162.7, 153.9, 153.1, 136.2 (Cquart), 130.3, 130.2, 127.8 (5x CAr), 123.0 (Cquart), 122.6, 120.0 (4x CAr), 69.8, 69.6, 69.55, 68.1, 67.9 (5x CH2), 61.1 (NHCHCH), 59.2 (NHCHCH2), 55.4 (CHS), 40.3 (SCH2), 37.9, 35.7 (2x CH2NH), 35.2, 29.4, 28.2, 28.0, 25.3 (5x CH₂) ppm; HRMS (ESI-IT): m/z calcd for C₃₄H₄₆N₈O₇S+H⁺: 711.3283 [M + H]⁺; found: 711.3266.

84 7. Experimental Section

13-biotinylamido-4,7,10-trioxatridecan-1-(4-(5-phenyl-tetrazol-2-yl)phenoxy)carbamate (tetrazole-biotin, 46)

Activated tetrazole (766 mg, 1.9 mmol, 1 equiv) and biotin-PEG-NH2 [192]

(1.1 g, 2.46 mmol, 1.2 equiv) were dissolved under nitrogen conditions in dry DMF (45 mL) and pyridine (4 mL). The mixture was stirred at rt for 41 h and evaporated. The oily yellow residue was dissolved in DCM (50 mL) and washed with water (5x 50 mL). The combined aqueous layers were extracted with DCM (5x 50 mL) and the combined organic layers were dried (MgSO4) and evaporated. The resulting yellow oil was purified by column chromatography (DCM:MeOH, 10:1) to give tetrazole-biotin 46 as colorless solid (307 mg, 0.44 mmol, 23%).

Rf (DCM:MeOH, 10:1) = 0.6; ¹H NMR (400 MHz, DMSO-d6): δ = 8.24 – 8.09 (m, 4H, H-2, H-6 and H-2‘, H-6‘ or H-3‘, H-5‘), 7.91 (t, J = 5.7 Hz, 1H, NHCO), 7.73 (t, J = 5.6 Hz, 1H, NHCO), 7.68 – 7.51 (m, 3H, H-3, H-5, H-4), 7.49 – 7.40 (m, 2H, H-2‘, H-6‘ or H-3‘, H-5‘), 6.40 (s, 1H, NH), 6.34 (s, 1H, NH), 4.35 – 4.24 (m, 1H, NHCH), 4.11 (ddt, J = 7.9, 5.2, 2.7 Hz, 1H, NHCH), 3.61 – 3.43 (m, 10H, 5x CH2CO), 3.38 (td, J = 6.3, 2.0 Hz, 2H, CH2CO), 3.20 – 3.12 (m, 2H, NHCH2), 3.07 (dq, J = 7.3, 5.6 Hz, 3H, CHS, CNCH2), 2.81 (dd, J = 12.4, 5.0 Hz, 1H, CH2Sexo), 2.57 (d, J = 12.5 Hz, 1H, CH2Sendo), 2.04 (t, J = 7.4 Hz, 2H, CH2), 1.73 (p, J = 6.6 Hz, 2H, CH2), 1.66 – 1.54 (m, 4H, 2x CH2), 1.53 – 1.39 (m, 2H, CH2), 1.29 (hept, J = 8.0, 7.3 Hz, 2H, CH2) ppm;

13C NMR (101 MHz, DMSO-d6): δ = 171.9, 164.5, 162.7, 153.8, 152.2, 131.0 (6x Cquart), 129.4, 126.7 (4x CAr), 126.5 (Cquart), 123.3, 121.2 (5x CAr), 69.8, 69.58, 69.55, 68.1, 67.9 (6x CH2O), 61.1, 59.2 (2x NHCH), 55.4 (CHS), 40.5 (CH2S), 37.9, 35.7 (2x CH2NH), 35.2, 29.4, 28.2, 28.1, 25.3 (6x CH2) ppm; HRMS (ESI-IT): m/z calcd for C34H46N8O7S+H⁺: 711.3283 [M + H]⁺; found:

711.3263.

7. Experimental Section 85

3-(4-Methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazole-4-carboxamide (pyrazoline 47)

Tetrazole 41 (50 mg, 0.2 mmol, 1 equiv) and acrylamide (17.5 mg, 0.3 mmol, 1.5 equiv) were dissolved in EtOH (15 mL). The mixture was stirred and irradiated (302 nm) from the top for 2 h at room temperature. The formed yellow precipitate was filtered off to yield crude pyrazoline 47 (26.7 mg, 45%). Solvent was removed from the filtrate and purified by column chromatographie (PE:EtOAc = 1:3) yielding another 20% product (12.2 mg).

Rf (PE:EtOAc, 1:3) = 0.3; ¹H NMR (400 MHz, DMSO-d6): δ = 7.76 – 7.71 (m, 1H, NH), 7.70 – 7.64 (m, 2H, HAr), 7.27 (s, 1H, NH), 7.26 – 7.18 (m, 2H, HAr), 7.06 – 6.95 (m, 4H, HAr), 6.77 (tt, J = 7.3, 1.1 Hz, 1H, HAr), 4.58 (dd, J = 12.6, 7.7 Hz, 1H, CHCH2), 3.80 (s, 3H, CH3), 3.71 (dd, J = 17.4, 12.6 Hz, 1H, CH2a), 3.21 (dd, J = 17.4, 7.8 Hz, 1H, CH2b) ppm; ¹³C NMR (101 MHz, DMSO-d6):

δ = 173.4 (CO), 149.2, 147.9, 145.5 (3x Cquart), 129.4, 127.8, 119.0, 114.6, 112.8 (5x CAr), 62.8 (CHCH2), 55.7 (CH3) ppm; HRMS (ESI-IT): m/z calcd for C17H17N3O2 +H⁺: 296.1394 [M + H]⁺; found: 296.1383.

1-(4-Methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-4-carboxamide (pyrazoline 48)

Tetrazole 42 (50 mg, 0.2 mmol, 1 equiv) and acrylamide (17.5 mg, 0.3 mmol, 1.5 equiv) were dissolved in EtOH (15 mL). The mixture was stirred and irradiated (302 nm) from the top for 2 h at room temperature. The formed precipitate was filtered off to yield pyrazoline 48 (29 mg, 49%).

Solvent was removed from the filtrate and purified by column chromatographie (PE:EtOAc = 1:2) yielding another 20% product.

Rf (PE:EtOAc, 1:1) = 0.2; ¹H NMR (400 MHz, DMSO-d6): δ = 7.71 (dd, J = 7.1, 1.8 Hz, 2H, HAr), 7.42 (dd, J = 8.3, 6.7 Hz, 2H, HAr), 7.38 – 7.32 (m, 1H, HAr), 7.26 (d, J = 2.1 Hz, 1H, NH2), 7.02 – 6.94 (m, 2H, HAr), 6.92 – 6.80 (m, 2H), 4.54 (dd, J = 12.6, 8.4 Hz, 1H, CHCH2), 3.77 – 3.65 (m, 1H, CH2a), 3.70 (s, 3H, CH3), 3.21 (dd, J = 17.4, 8.5 Hz, 1H, CH2b) ppm; ¹³C NMR (101 MHz, DMSO-d6): δ = 173.4 (CO), 153.4, 147.1, 139.8, 132.6 (4x Cquart), 129.1, 129.0, 126.0, 115.0, 114.4 (5x CAr), 63.9 (CHCH2), 55.8 (CH2), 39.0 (CH3) ppm; HRMS (ESI-IT): m/z calcd for C17H17N3O2 -H⁺: 294.1237 [M - H]⁺; found: 294.1227.

86 7. Experimental Section

1,3,4,6-Tetra-O-acetyl-N-Boc-mannosamine (Ac4ManNBoc, 50)

Boc protection was performed according to Zamora et al.^[195] Mannosamine hydrochloride (560 mg, 2.6 mmol, 1 equiv) was dissolved in 40% EtOH (13 mL) and NaHCO3 (640 mg, 7.7 mmol, 3 equiv) was added. The suspension was sonicated and Boc2O (630 mg, 2.9 mmol, 1.1 equiv), dissolved in EtOH (1.2 mL), was added slowly. The mixture was sonicated for 3 h, turning yellow. The solvent was removed and the remaining yellow oil dissolved in water (15 mL), washed with hexane (3x 20 mL) and extracted with 1-butanol (4x 20 mL). The combined butanol layers were washed with water (2x 5 mL) before the solvent was evaporated. The boc-protected mannosamine was used without further purification and dissolved in Ac2O (3 mL) and pyridine (3 mL) and stirred at room temperature overnight. The solvent was evaporated and the remaining crude dissolved in DCM (10 mL) and extracted with KHSO4 (2x 10 mL) and NaHCO3 (2x 10 mL).

The combined organic layers were dries (MgSO4) and evaporated. The product was purified using the Grace Reveleris System (PE:EtOAc = 10 – 50%) yielding an α/β-mixture (3:1) Ac4ManNBoc

7. Experimental Section 87

1,3,4,6-Tetra-O-acetyl-N-acryl-α/β-D-mannosamine (Ac4ManNAcryl, 52)

Ac4ManNH2*TFA (51, 150 mg, 0.43 mmol, 1 equiv) was dissolved in dry DCM (4 mL) and DIPEA (160 µL, 0.94 mmol, 2.2 equiv) was added. The mixture was stirred at room temperature for 5 min and cooled to 0°C. Acryloyl chloride (60 µL, 0.74 mmol, 1.7 equiv) was added drop wise and the mixture was stirred. After 75 min sat. NaHCO3 (3 mL) was added. The aqueous layer was extracted with DCM (2x 5 mL) and the combined organic layers were dried (MgSO4). The crude product was purified using the Grace Reveleris System (PE:EtOAc = 10 – 50%) yielding 25% over two steps as α/β mixture (4:1).

(1S,2S,4S)-Bicyclo[2.2.1]hept-5-en-2-ylacetic acid (400 mg, 2.63 mmol, 1 eq) and NHS (423.5 mg, 3.7 mmol, 1.4 eq) were dissolved in anhydrous THF (14 mL). DCC (652 mg, 3.2 mmol,1.2 eq) dissolved in THF (8 mL) was added and the mixture was stirred at room temperature overnight. The mixture was filtered to remove the urea and the filtrate was evaporated. The crude product was purified by column chromatography (PE:EtOAc = 2:1) to yield the activated ester as a white solid (500 mg, 2 mmol, 76%).

88 7. Experimental Section

Mannosamine hydrochloride (360 mg, 1.7 mmol, 1 equiv) was dissolved in dry methanol (10 mL) and neutralized by addition of NaOMe (0.5 M, 3.5 mL) and stirred at rt for 75 min. NHS-activated endo-norbornene (498 mg, 2 mmol, 1.2 equiv) was suspended in MeOH (10 mL) and added. The mixture was allowed to stir overnight before the solvent was evaporated. The crude product was dissolved in pyridine (1.6 mL), Ac2O (1.6 mL, 17 mmol, 10 equiv) was added and the mixture was stirred overnight at room temperature. The mixture was concentrated, diluted with DCM (50 mL) and washed with 10% potassium bisulfate solution (50 mL), saturated sodium bicarbonate solution (50 mL) and brine (50 mL). The organic layer was dried (MgSO4) and the solvent was evaporated. The crude product was purified by flash column chromatography (PE:EtOAc = 1:1) to yield Ac4ManNNorb (53) as a white foam (0.24 mmol, 120 mg, 15%).

7. Experimental Section 89

45.8, 42.9, 41.9, 35.8, 32.0 (C-1’, C-2’, C-4’, C-7’, C-8’), 21.0, 20.94, 20.90, 20.88, 20.85, 20.82, 20.80, 20.7 (8x CH3) ppm; HRMS (ESI-IT): m/z calcd for C23H31NO10 +H⁺: 482.2021 [M + H]⁺; found: 482.2008.

90 7. Experimental Section

7. Experimental Section 91

Im Dokument Metabolic Glycoengineering with Alkene-Functionalized Carbohydrates (Seite 83-107)