Supporting Information © Wiley-VCH 2005 69451 Weinheim, Germany

First Total Synthesis of a Bacillus Anthracis

Tetrasaccharide Antigen –

Creation of an Anthrax Vaccine Candidate**

Daniel B. Werz and Peter H. Seeberger*

[*] Prof. Dr. P. H. Seeberger, Dr. Daniel B. Werz Laboratory for Organic Chemistry Swiss Federal Institute of Technology Zürich ETH Hönggerberg HCI F 315 Wolfgang-Pauli-Str. 10 CH-8093 Zürich Switzerland E-mail: [email protected]

General Methods. All chemicals used were reagent grade and used as supplied except where noted. Dichloromethane (CH2Cl2) was purchased from JT Baker and purified by a Cycle-

Tainer Solvent Delivery System. Pyridine and acetonitrile were refluxed over calcium hydride and distilled prior to use. Analytical thin-layer chromatography was performed on E. Merck silica gel 60 F254 plates (0.25 mm). Compounds were visualized by dipping the plates in a cerium sulfate ammonium molybdate solution or a sulfuric acid/methanol solution followed by heating. Liquid chromatography was performed using forced flow of the indicated solvent on Sigma H-type silica (10–40 mm). 1H NMR spectra were obtained on a Varian VXR-300

(300 MHz), Bruker-600 (600 MHz) and are reported in parts per million (δ) relative to CHCl3

(7.26 ppm) or in the case of CD3OD as solvent relative to TMS (0.00 ppm). Coupling constants (J) are reported in Hertz. 13C NMR spectra were obtained on a Varian VXR-300 (75

MHz), Bruker-600 (150 MHz) and are reported in δ relative to CDCl3 (77.0 ppm) as an internal reference or to TMS (0.00 ppm).

4-Methoxyphenyl α-D-fucopyranoside (4). D-Fucose (3) (4.95 g, 30.14 mmol) was dissolved in 30 mL of acetic anhydride and 30 mL of pyridine and stirred for 12 h. The solvent was removed in vacuo. Column chromatography (2:1 hexane/EtOAc) yielded 10.15 g

(quant.) of 1,2,3,4-tetraacetyl-D-fucose (as a mixture of α- and β-anomer).

1,2,3,4-tetraacetyl-D-fucose (20.0 g, 60.2 mmol) and p-methoxyphenol (MPOH) (10.46 g,

• 84.3 mmol) were dissolved in 220 mL of dichloromethane and cooled to 0 °C. BF3 OEt2 (21.4 g, 19.0 mL, 150.5 mmol) was added and the mixture stirred overnight. The reaction mixture was quenched by addition of saturated NaHCO3 solution (Be careful: Much foam is generated!), washed 2 times with water, dried over Na2SO4, concentrated and the residue was purified by column chromatography (3:1 hexane/EtOAc) yielding 16.8 g (71%) of 4- methoxyphenyl 1,2,3,4-tetraacetyl-α-D-fucose (Only the β-anomer reacts, α-anomer could be recovered!). 4-methoxyphenyl 1,2,3,4-tetraacetyl-α-D-fucose (3.90 g, 9.85 mmol) was dissolved in 150 mL of methanol and 6 mL of 0.5 M NaOMe solution were added. The mixture was stirred for 6 h. The solvent was removed in vacuo. Column chromatography (3:1 dichloromethane/methanol) afforded 2.63 g (99%) as a colorless solid: [α]D: +201.6 (c = 0.32,

1 CH3OH). H NMR (CD3OD, 300 MHz) δ 1.18 (d, J = 6.6 Hz, 3H), 3.74 (s, 3H), 3.89 (m, 2H),

4.09 (quart., J = 6.0 Hz, 1H), 5.31 (d, J = 3.6 Hz, 1H), 6.82 (d, J = 9.3 Hz, 2H), 7.04 (d, J = 13 9.3 Hz, 2H). C NMR (CD3OD, 75 MHz) δ 16.3, 55.7, 67.9, 69.5, 71.2, 73.2, 100.0, 115.1,

119.0, 152.2, 156.0. MALDI-HRMS: m/z [M + Na]+ calcd 293.0996, obsd 293.0994.

4-Methoxyphenyl 3,4-O-Isopropylidene-2-O-levulinoyl-α-D-fucopyranoside (5). 4-

Methoxyphenyl α-D-fucopyranoside (4) (500 mg, 1.85 mmol) was suspended in 15 mL of acetone. 2,2-dimethoxypropane (327 mg, 0.385 mL, 3.15 mmol) was added in one portion and

• the mixture cooled to 0 °C, then BF3 OEt2 (40 mg, 0.28 mmol) was added dropwise. The mixture was stirred for 12 h. Afterwards the reaction was quenched by addition of 0.2 ml of pyridine. The volatiles were removed in vacuo. The resulting crude product was purified by flash chromatography (3:1 hexane/EtOAc) to afford 551 mg (96%) of 4-Methoxyphenyl 3,4-

Isopropylidene-D-fucopyranoside as a colorless oil.

4-Methoxyphenyl 3,4-O-Isopropylidene-α-D-fucopyranoside (3.30 g, 10.65 mmol), levulinic acid (1.36 g, 1.20 mL, 11.71 mmol), DMAP (1.43 g, 11.71 mmol) and diisopropyl carbodiimide (1.34 g, 1.65 mL, 10.65 mmol) are dissolved at 0 °C in 35 mL of dichloromethane and stirred for 3 h. The reaction mixture is diluted with 80 ml of hexane/EtOAc (3:1) and flushed through a 3 cm plug of silica gel. The filtrate was concentrated and purified by column chromatography (3:1 → 2:1 hexane/EtOAc) yielding

4.01 g (92%) of 5 as a colorless oil: [α]D: +162.4 (c = 0.95, CHCl3). IR (thin film, CHCl3):

-1 1 3008, 1740, 1719, 1506, 1384, 1158, 1078 cm . H NMR (CDCl3, 300 MHz) δ 1.31-1.38 (m,

6H), 1.55 (s, 3H), 2.15 (s, 3H), 2.65-2.78 (m, 4H), 3.77 (s, 3H), 4.15 (dd, J = 5.4 Hz, J =

2.4 Hz, 1H), 4.27 (m, 1H), 4.49 (dd, J = 8.1 Hz J = 5.1 Hz, 1H), 5.01 (dd, J = 8.1 Hz, J =

3.9 Hz, 1H), 5.44 (d, J = 3.9 Hz 1H), 6.83 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H). 13C

NMR (CDCl3, 75 MHz) δ 13.4, 26.5, 28.0, 28.1, 29.8, 37.9, 55.6, 63.9, 71.8, 73.3, 76.0, 95.6,

109.4, 114.5, 117.7, 150.5, 154.9, 172.1, 205.9. MALDI-HRMS: m/z [M + Na]+ calcd

431.1676, obsd 431.1683.

4-Methoxyphenyl 3-O-Benzyl-2-O-levulinoyl-α-D-fucopyranoside (6). 4-Methoxyphenyl

3,4-O-Isopropylidene-2-O-levulinoyl-α-D-fucopyranoside (5) (449 mg, 1.10 mmol) was dissolved in 5 mL of methanol and 0.5 mL of water. An aqueous solution of 3.7% hydrochloric acid was added (some drops) until pH 3 was reached. The mixture was heated to

50 °C and stirred for 18 h until the TLC showed complete conversion. The mixture was extracted with EtOAc, washed 2 times with NaHCO3, dried over Na2SO4 and was concentrated. The resulting crude product was purified by flash chromatography (2:1 → 1:1

→ 1:2 hexane/EtOAc) to afford 345 mg (85%) of 4-Methoxyphenyl 2-O-levulinoyl-α-D- fucopyranoside as a colorless oil.

4-Methoxyphenyl 2-O-levulinoyl-α-D-fucopyranoside (2.37 g, 6.46 mmol) and dibutyl tin oxide (1.61 g, 6.46 mmol) are dissolved in 70 mL of toluene. The mixture was refluxed with a

Dean-Stark trap for 2.5 h. Then it was cooled to 60 °C, benzylbromide and tetrabutylammonium iodide were added and refluxed for 2 h. After cooling to room temperature the reaction mixture was diluted with water and extracted 3 times with EtOAc, dried over Na2SO4 and concentrated. The crude product was purified by column chromatography (3:2 hexane/EtOAc) yielding 2.82 g (95%, 2 steps) of 6 as a colorless oil:

[α]D: +140.8 (c = 0.35, CHCl3). IR (thin film, CHCl3): 3015, 1739, 1713, 1503, 1362,

-1 1 1050cm . H NMR (CDCl3, 300 MHz) δ 1.29 (d, J = 6.6 Hz, 3H), 2.14 (s, 3H), 2.62 (m, 3H),

2.72 (m, 2H), 3.76 (s, 3H), 3.92 (s, 1H), 4.09 (m, 2H), 4.74 (s, 2H), 5.28 (dd, J = 10.2 Hz J =

3.9 Hz, 1H), 5.50 (m, 1H), 6.82 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 7.33 (m, 5H).

13 C NMR (CDCl3, 75 MHz) δ 16.2, 28.1, 29.9, 37.9, 55.7, 66.2, 69.8, 70.0, 72.4, 75.6, 96.2,

114.5, 118.0, 118.2, 127.6, 127.7, 127.9, 128.5, 137.7, 150.7, 155.0, 172.0, 206.0. MALDI-

HRMS: m/z [M + Na]+ calcd 481.1833, obsd 481.1824.

4-Methoxyphenyl 4-Azido-3-O-benzyl-6-desoxy-2-O-levulinoyl-α-D-glucopyranoside (7).

4-Methoxyphenyl 3-O-Benzyl-2-O-levulinoyl-α-D-fucopyranoside (6) (971 mg, 2.12 mmol) was azeotroped 2 times with toluene and dissolved in 20 mL of dichloromethane. Pyridine

(1.84 g, 1.87 mL, 23.3 mmol) was added and the mixture cooled to 0 °C. Triflic anhydride was added and the mixture stirred for 90 min at 0 °C. The mixture was quenched by addition of diluted NaHCO3 solution, the phases were separated and the organic layer extracted 3 times with 3% hydrochloric acid, once with water, dried over Na2SO4 and was concentrated. The resulting crude product was azeotroped 3 times with toluene to remove all traces of pyridine and dried for 1 h in vacuo. The yellow product was dissolved in 25 mL of DMF and sodium azide (3.58 g, 55.1 mmol) was added, and the reaction mixture stirred overnight. The mixture was diluted with water and EtOAc, the phases were separated. The organic phase was washed

3 times with brine, dried over Na2SO4 and concentrated. Column chromatography (3:1 hexane/EtOAc) afforded 816 mg (80%, 2 steps) of 7 as a slightly yellow oil: [α]D: +192.6 (c =

1.25, CHCl3). IR (thin film, CHCl3): 3015, 2103, 1739, 1713, 1503, 1400, 1359, 1159, 1034

-1 1 cm . H NMR (CDCl3, 300 MHz) δ 1.31 (d, J = 6.3 Hz, 3H), 2.14 (s, 3H), 2.58 (m, 2H), 2.69

(m, 2H), 3.26 (t, J = 9.9 Hz, 1H), 3.77 (s, 3H), 3.83 (m, 1H), 4.08 (m, 1H), 4.84 (d, J =

10.8 Hz, 1H), 4.90 (d, J = 10.8 Hz, 1H), 4.96 (dd, J = 9.9 Hz, J = 3.6 Hz, 1H), 5.48 (d, J = 3.3

Hz, 1H), 6.83 (d, J = 9.0 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 7.31-7.42 (m, 5H). 13C NMR

(CDCl3, 75 MHz) δ 18.4, 28.0, 29.8, 37.8, 55.7, 66.8, 67.9, 73.6, 75.4, 76.6, 78.1, 95.7, 114.6,

118.2, 127.8, 127.8, 128.3, 137.8, 150.4, 155.2, 171.9, 205.7. MALDI-HRMS: m/z [M + Na]+ calcd 506.1898, obsd 506.1889.

4-Azido-3-O-benzyl-6-desoxy-2-O-levulinoyl-α-D-glucopyranosyl Trichloroacetimidate

(8). 4-Methoxyphenyl 4-Azido-3-O-benzyl-6-desoxy-2-O-levulinoyl-α-D-glucopyranoside (7)

(2.02 g, 4.18 mmol) was dissolved in a mixture of 50 mL of acetonitrile and 50 mL of water. Cerium ammonium nitrate (6.87 g, 12.55 mmol) was added and the mixture stirred for 2 h at room temperature. After TLC showed complete removal of the MP group, the mixture was diluted with EtOAc, washed 2 times with brine, then with water, dried over Na2SO4 and was concentrated. The resulting crude product was purified by column chromatography (1:1 →

1:2 hexane/EtOAc) to afford 1.47 g (93%) of 4-Azido-3-O-benzyl-6-desoxy-2-O-levulinoyl-

α-D-glucopyranoside as a slightly yellow solid. This compound (300 mg, 0.796 mmol) was dissolved in 5 mL of tricloroacetonitrile and 4 mL of dichloromethane. A catalytic amount of sodium hydride (5 mg) was added, the solution turned brownish and was stirred for 45 min.

TLC indicated complete conversion. The solvent was removed and the residue purified by flash column chromatography (3:1 hexane/EtOAc) to afford 322 mg (78%, 2 steps) of 8 (α/β

4:3) as a yellow oil: IR (thin film, CHCl3): 2924, 2112, 1747, 1720, 1674, 1455, 1360, 1290,

-1 1 1156 cm . H NMR (CDCl3, 300 MHz) δ 1.34 (d, J = 6.3 Hz, 3H, α), 1.40 (d, J = 6.0 Hz, 3H,

β), 2.15 (s, 3H), 2.46 (m, 2H), 2.68 (m, 2H), 3.29 (quart, J = 9.6 Hz, 1H), 3.46 (m, 1H, β),

3.63 (t, J = 9.3 Hz, 1H, β), 3.81 (m, 1H, α), 3.96 (t, J = 9.6 Hz, 1H, α), 4.71-4.87 (m, 2H),

5.03 (dd, J = 9.9 Hz, J = 3.6 Hz, 1H, α), 5.26 (t, J = 9.0 Hz, 1H, β), 5.71 (d, J = 8.1 Hz, 1H,

β), 6.40 (d, J = 3.3 Hz, 1H, α), 7.26-7.37 (m, 5H), 8.60 (s, 1H, α), 8.64 (s, 1H, β). 13C NMR

(CDCl3, 75 MHz, α/β mixture) δ 18.6, 18.6, 27.8, 27.9, 30.0, 37.8, 37.9, 67.5, 69.4, 72.1,

72.6, 72.9, 75.1, 75.5, 77.6, 81.2, 93.7, 95.8, 128.2, 128.4, 128.7, 137.6, 137.7, 161.0, 161.4,

171.2, 172.1, 206.1. MALDI-HRMS: m/z [M + Na]+ calcd 543.0575, obsd 543.0567.

4-Methoxyphenyl α-L-Rhamnopyranoside (10). L-Rhamnose (as monohydrate) (9) (25.0 g,

137.2 mmol) was dissolved in acetic anhydride (154 g, 1.51 mol) and pyridine (160 mL) and stirred for 12 h. The solvent was removed in vacuo. Column chromatography (2:1 hexane/EtOAc) yielded 45.3 g (quant.) of 1,2,3,4-tetraacetyl-L-rhamnose. 1,2,3,4-Tetraacetyl-L-rhamnose (19.2 g, 57.8 mmol) and p-methoxyphenol (MPOH) (12.2 g,

• 98.3 mmol) were dissolved in 150 mL of dichloromethane and cooled to 0 °C. BF3 OEt2 (23.0 g, 20.5 mL, 162.0 mmol) was added and the mixture stirred overnight while warming to room temperature. The reaction mixture was quenched by addition of saturated NaHCO3 solution, washed 2 times with water, dried over Na2SO4, concentrated and the residue was purified by column chromatography (3:1 hexane/EtOAc) yielding 18.3 g (80%) of 4-methoxyphenyl

1,2,3,4-tetraacetyl-α-L-rhamnose. 4-Methoxyphenyl 1,2,3,4-tetraacetyl-α-L-rhamnose (18.0 g,

45.4 mmol) was dissolved in 300 mL of methanol and 9 mL of 0.5 M NaOMe solution were added. The mixture was stirred for 12 h, the solvent was removed in vacuo. Column chromatography (4:1 dichloromethane/methanol) afforded 2.63 g (96%) as a colorless solid.[13]

4-Methoxyphenyl 4-O-Benzyl-2,3-O-isopropylidene-α-L-rhamnoyranoside (11). 4-

Methoxyphenyl α-L-rhamnopyranoside (9) (11.7 g, 43.3 mmol) was dissolved in 120 mL of acetone. 2,2-Dimethoxypropane (7.7 g, 9.1 mL, 73.7 mmol) was added in one portion and the

• mixture cooled to 0 °C, then BF3 OEt2 (916mg, 818 µL, 6.50 mmol) was added dropwise. The mixture was stirred for 12 h while warming to room temperature. Afterwards the reaction was quenched by addition of 1.0 mL of pyridine. The volatiles were removed in vacuo. The resulting crude product was purified by flash chromatography (3:1 hexane/EtOAc) to afford

11.25 g (84%) of 4-Methoxyphenyl 2,3-Isopropylidene-α-L-rhamnopyranoside as a colorless oil.

4-Methoxyphenyl 2,3-Isopropylidene-α-L-rhamnopyranoside (7.50 g, 24.2 mmol) was dissolved in 75 mL of DMF and cooled to 0 °C. Benzylbromide (8.28 g, 48.4 mmol) and sodium hydride (60% in oil) (1.94 g, 48.4 mmol) were added carefully. The mixture was stirred for 12 h while warming to room temperature. The solution was quenched with water and diluted with EtOAc. The phases were separated and the organic layer was washed 3 times with water, dried over Na2SO4 and concentrated. The crude product was purified by column chromatography (4:1 hexane/EtOAc) yielding 10.03 g (quant.) of 11 as a colorless oil: [α]D: -

-1 1 91.4 (c = 1.07, CHCl3). IR (thin film, CHCl3): 3007, 2937, 1507, 1454, 1388 cm . H NMR

(CDCl3, 300 MHz) δ 1.25 (d, J = 6.3 Hz, 3H), 1.44 (s, 3H), 1.56 (s, 3H), 3.30 (m, 1H), 3.77

(s, 1H), 3.87 (m, 1H), 4.37 (d, J = 5.1 Hz, 1H), 4.44 (t, J = 6.0 Hz, 1H), 4.67 (d, J = 11.4 Hz,

2H), 4.94 (d, J = 11.4 Hz, 2H), 5.62 (s, 1H), 6.84 (d, J = 9.0 Hz, 2H), 6.99 (d, J = 9.0 Hz,

13 2H), 7.28-7.40 (m, 5H). C NMR (CDCl3, 75 MHz) δ 17.8, 26.3, 27.9, 55.5, 65.3, 72.9, 75.9,

78.5, 80.9, 96.0, 109.4, 114.5, 117.6, 127.6, 127.9, 128.2, 138.2, 150.1, 154.8. MALDI-

HRMS: m/z [M + Na]+ calcd 423.1785, obsd 423.1777.

4-Methoxyphenyl 2-O-Acetyl-4-O-benzyl-α-L-rhamnopyranoside (12). 4-Methoxyphenyl

2,3-O-Isopropylidene-4-O-benzyl-α-L-rhamnopyranoside (11) (9.38 g, 23.45 mmol) was dissolved in 100 mL of methanol and 10 mL of water. An aqueous solution of 3.7% hydrochloric acid was added until pH 3 was reached. The mixture was heated to 45 °C and stirred for 2 d until the TLC showed complete conversion. The mixture was extracted with

EtOAc, washed 2 times with NaHCO3, dried over Na2SO4 and was concentrated. The resulting crude product was purified by flash chromatography (2:1 → 1:1 hexane/EtOAc) to afford 7.52 g (89%) of 4-Methoxyphenyl 4-O-benzyl-α-L-rhamnopyranoside as a colorless powder.

4-Methoxyphenyl 4-O-Benzyl-α-L-rhamnopyranoside (4.06 g, 11.28 mmol) and 1,1,1- triethoxyethane (2.20 g, 2.47 mL, 13.53 mmol) are dissolved in 75 mL of DMF and stirred for

50 min at 50 °C. DMF was removed in vacuo. The residue was dissolved in AcOH/H2O (4:1, v/v) and stirred for 10 min at room temperature. The acetic acid is removed in vacuo and the crude product was purified by column chromatography (3:1 hexane/EtOAc) yielding 4.51 g (98%, 2 steps) of 12 as a colorless oil: [α]D: -69.4 (c = 1.25, CHCl3). IR (thin film, CHCl3):

-1 1 3005, 2933, 1739, 1503, 1374, 1126, 1041 cm . H NMR (CDCl3, 300 MHz) δ 1.34 (d, J =

6.0 Hz, 3H), 2.19 (s, 3H), 2.41 (b, 1H), 3.44 (t, J = 9.3 Hz, 1H), 3.76 (s, 3H), 3.92 (m, 1H),

4.31 (d, J = 9.0 Hz, 2H), 4.74 (d, J = 12.6 Hz, 2H), 4.87 (d, J = 12.6 Hz, 2H), 5.29 (m, 1H),

5.36 (s, 1H), 6.82 (d, J = 9.0 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 7.33 (m, 5H). 13C NMR

(CDCl3, 75 MHz) δ 18.1, 21.2, 55.7, 68.1, 70.0, 72.6, 75.3, 81.6, 96.3, 114.5, 117.6, 127.8,

127.8, 128.4, 138.0, 149.9, 154.9, 170.6. MALDI-HRMS: m/z [M + Na]+ calcd 425.1571, obsd 425.1565.

2-O-Acetyl-4-O-benzyl-3-O-fluorenylmethoxycarbonyl-α-L-rhamnopyranosyl

Trichloroacetimidate (13). 4-Methoxyphenyl 2-O-Acetyl-4-O-benzyl-α-L- rhamnopyranoside (12) (4.57 g, 11.36 mmol) was dissolved in 60 mL of pyridine. FmocCl

(5.88 g, 22.73 mmol) was added in one portion and the mixture stirred for 2 h at room temperature. Pyridine was removed in vacuo and the residue adsorbed on silica gel. 6.21 g

(88%) of pure 4-Methoxyphenyl 2-O-Acetyl-4-O-benzyl-3-O-fluorenylmethoxycarbonyl-α-L- rhamnopyranoside were obtained by column chromatography (5:1 → 4:1 → 3:1 hexane/EtOAc).

4-Methoxyphenyl 2-O-Acetyl-4-O-benzyl-3-O-fluorenylmethoxycarbonyl-α-L- rhamnopyranoside (3.10 g, 4.96 mmol) was dissolved in a mixture of 55 mL of toluene,

80 mL of acetonitrile and 55 mL of water. Cerium ammonium nitrate (21.7 g, 39.7 mmol) was added and the mixture was vigorously stirred for 80 min at room temperature. After TLC showed complete removal of the MP group, the phases of the mixture were separated, the organic phase was washed 2 times with water, saturated aqueous NaHCO3, then again once with water, dried over Na2SO4 and was concentrated. The resulting crude product was adsorbed on silica gel and purified by column chromatography (3:1 → 2:1 hexane/EtOAc) to afford 1.95 g (76%) of 2-Acetyl-4-benzyl-3-fluorenylmethoxycarbonyl-L-rhamnopyranoside as a slightly yellow solid. This compound (675 mg, 1.30 mmol) was dissolved in 6 mL of tricloroacetonitrile and 7 mL of dichloromethane. A catalytic amount of sodium hydride

(10 mg) was added and the mixture stirred for 60 min. The solvent was removed in vacuo and the residue purified by flash column chromatography (2:1 hexane/EtOAc) to afford 810 mg

(94%) of 13 (only traces of β product) as a slightly yellow oil: [α]D: -17.3 (c = 0.51, CHCl3),

-1 1 IR (thin film, CHCl3): 3026, 1754, 1672, 1451, 1380, 1262, 1067 cm . H NMR (CDCl3,

300 MHz) δ 1.41 (d, J = 6.0 Hz, 3H), 2.22 (s, 3H), 3.69 (t, J = 9.3 Hz, 1H), 4.11 (m, 1H), 4.33

(m, 2H), 4.52 (m, 1H), 4.68 (d, J = 11.1 Hz, 1H), 4.86 (d, J = 11.1 Hz, 1H), 5.26 (dd, J = 12.3

Hz, J = 3.3 Hz, 1H), 5.61 (m, 1H), 6.23 (s, 1H), 7.28 (m, 7H), 7.39 (m, 2H), 7.60 (m, 2H),

13 7.77 (d, J = 6.6 Hz, 2H), 8.71 (s, 1H). C NMR (CDCl3, 75 MHz) δ 18.1, 21.0, 46.7, 68.4,

70.3, 70.6, 75.5, 75.6, 77.8, 94.7, 120.0, 124.9, 125.1, 127.1, 127.7, 127.8, 127.9, 128.4,

137.4, 141.1, 141.2, 142.9, 143.4, 154.0, 160.0, 169.6. MALDI-HRMS: m/z [M + Na]+ calcd

684.0929, obsd 684.0939.

n-Pentenyl 3,4-O-Benzyl-α-L-rhamnopyranosyide (15). Rhamnosyl trichloroacetimidate 14

(220 mg, 0.42 mmol) was azeotroped 3 times with toluene and dried for 1 h in vacuo. The compound was dissolved in dichloromethane (5 mL) and cooled to -40 °C. n-Pentenol

(71 mg, 85 µL, 0.83 mmol) and TMSOTf (11 mg, 9 µL, 0.04 mmol) were added, the solution was stirred for 45 min while warming to -20 °C. Then it was quenched by addition of some drops of pyridine. The solvent was removed and the resulting crude product was purified by column chromatography (6:1 → 5:1 hexane/EtOAc) to afford 149 mg (79%) of a colorless oil. This compound (125 mg, 0.28 mmol) was dissolved in methanol (10 mL) and a solution of 0.1 M sodium methoxide (0.1 mL) was added and the solution stirred for 4 h. The solvent was removed in vacuo. The product was purified by flash column chromatography (3:1 hexane/EtOAc) to afford 109 mg (96%) of 15 as a colorless oil: [α]D: -36.0 (c = 0.53, CHCl3),

-1 1 IR (thin film, CHCl3): 3005, 2923, 1451, 1385, 1082 cm . H NMR (CDCl3, 300 MHz) δ

1.31 (d, J = 6.0 Hz, 3H), 1.66 (quint., J = 7.5 Hz, 2H), 2.10 (q, J = 6.9 Hz, 2H), 2.48 (d, J =

1.8 Hz, 1H), 3.36-3.48 (m, 2H), 3.63-3.76 (m, 2H), 3.85 (dd, J = 9.0 Hz, J = 3.6 Hz, 1H),

4.02 (m, 1H), 4.64 (d, J = 11.1 Hz, 1H), 4.71 (s, 2H), 4.79 (s, 1H), 4.89 (d, J = 10.8 Hz, 1H),

13 4.96-5.06 (m, 2H), 5.78 (m, 1H), 7.27-7.38 (m, 10H). C NMR (CDCl3, 75 MHz) δ 18.0,

28.7, 30.3, 66.9, 67.2, 68.6, 72.0, 75.4, 80.0, 80.1, 98.8, 114.8, 127.6, 127.7, 127.8, 127.9,

128.3, 128.4, 137.8, 137.9, 138.2. MALDI-HRMS: m/z [M + Na]+ calcd 435.2142, obsd

435.2142.

n-Pentenyl 2-O-Acetyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→2)-3,4-O-benzyl-α-L- rhamnopyranoside (16). Compound 15 (291 mg, 0.71 mmol) and rhamnose trichloroacetimidate 13 (620 mg, 0.94 mmol) were azeotroped 3 times together with toluene and dried for 1 h in vacuo. The mixture was dissolved in dichloromethane (7 mL) and cooled to 0 °C. TMSOTf (24 mg, 19 µL, 0.11 mmol) was added, the solution stirred for 1 h and quenched by addition of some drops of pyridine. The solvent was removed and the resulting crude product was purified by column chromatography (3:1 Hexane/EtOAc) to afford 583 mg

(91%) of a colorless oil. This compound was dissolved in dichloromethane (10 mL) and DMF

(3 mL), triethylamine (0.5 mL) was added at room temperature. After 5 h the solvent was removed in vacuo. The crude product was purified by column chromatography (3:1 hexane/EtOAc) to afford 390 mg (89%) of 16 as a colorless highly viscous oil: [α]D: -37.7

-1 1 (c = 0.35, CHCl3), IR (thin film, CHCl3): 2912, 1733, 1451, 1369, 1221, 1082 cm . H NMR

(CDCl3, 300 MHz) δ 1.30 (d, J = 6.3 Hz, 3H), 1.36 (d, J = 6.0 Hz, 3H), 1.66 (quint., J =

7.2 Hz, 2H), 2.11 (q, J = 6.0 Hz, 2H), 2.16 (s, 3H), 2.21 (d, J = 4.5 Hz, 1H), 3.38 (m, 2H),

3.47 (t, J = 9.6 Hz, 1H), 3.66 (m, 2H), 3.87 (m, 2H), 3.96 (m, 1H), 4.20 (m, 1H), 4.61-4.74 (m, 5H), 4.88 (dd, J = 14.1 Hz, J = 10.8 Hz, 2H), 4.97-5.07 (m, 3H), 5.34 (m, 1H), 5.81 (m,

13 1H), 7.26-7.39 (m, 15H). C NMR (CDCl3, 75 MHz) δ 18.1, 18.2, 21.2, 28.7, 30.4, 66.8,

67.9, 67.9, 70.2, 72.1, 72.6, 75.0, 75.2, 75.4, 79.7, 80.2, 81.5, 98.7, 99.0, 114.9, 127.4, 127.5,

127.6, 127.8, 128.0, 128.3, 128.4, 137.9, 138.1, 138.3, 138.3, 170.5. MALDI-HRMS: m/z [M

+ Na]+ calcd 713.3296, obsd 713.3284.

As side product 57 mg (11%) of n-Pentenyl 3-O-Acetyl-4-O-benzyl-α-L-rhamnopyranosyl-

(1→2)-3,4-O-benzyl-α-L-rhamnopyranoside (16’) were obtained as a colorless highly viscous

-1 oil: [α]D: -43.2 (c = 0.21, CHCl3), IR (thin film, CHCl3): 2918, 1740, 1450, 1375, 1080 cm .

1 H NMR (CDCl3, 300 MHz) δ 1.31 (d, J = 6.3 Hz, 3H), 1.35 (d, J = 6.0 Hz, 3H), 1.65 (quint.,

J = 7.2 Hz, 2H), 2.09 (m, 6H), 3.37 (m, 2H), 3.47-3.60 (m, 2H), 3.63 (m, 2H), 3.86 (dd, J =

12.= Hz, J = 2.1 Hz, 1H), 3.93 (m, 2H), 4.22 (m, 1H), 4.67-4.74 (m, 5H), 4.87 (d, J = 10.8 Hz,

2H), 5.00 (m, 2H), 5.32 (dd, J = 9.0 Hz, J = 1.9 Hz, 1H), 5.80 (m, 1H), 7.26-7.36 (m, 15H).

13 C NMR (CDCl3, 75 MHz) δ 18.0, 18.1, 21.3, 28.7, 30.4, 66.7, 68.0, 68.1, 69.7, 72.5, 73.6,

74.8, 75.5, 75.6, 79.1, 80.0, 80.4, 98.8, 101.2, 114.8, 127.6, 127.7, 127.7, 127.8, 128.0, 128.3,

128.3, 137.9, 138.2, 138.4, 169.7. MALDI-HRMS: m/z [M + Na]+ calcd 713.3296, obsd

713.3283.

4-Methoxyphenyl 4-Azido-3-O-benzyl-6-desoxy-2-O-methyl-β-D-glucopyranosyl-(1→3)-

2-O-acetyl-4-O-benzyl-α-L-rhamnopyranoside (17). Compound 12 (94 mg, 0.233 mmol) and anthrose trichloroacetimidate 8 (146 mg, 0.280 mmol) were azeotroped 3 times together with toluene and dried for 30 min in vacuo. The mixture was dissolved in dichloromethane

(3.5 mL) and cooled to 0 °C. TMSOTf (8 mg, 7 µL, 0.035 mmol) was added, the solution stirred for 1 h and quenched by addition of some drops of pyridine. The solvent was removed and the resulting crude product was purified by column chromatography (4:1 → 3:1 hexane/EtOAc) to afford 160 mg (90%) of a colorless oil. This compound (265 mg, 0.348 mmol) was dissolved in dichloromethane (10 mL) and hydrazinium acetate in 2 mL of methanol was added at room temperature and stirred for 12 h. The solvent was removed by rotary evaporation and the crude product was purified by column chromatography (3:1 hexane/EtOAc) to afford 231 mg (quant.) of a colorless oil. This compound (450 mg, 0.68 mmol) was dissolved in THF (8 mL) and methyl iodide (3.84 g, 2.51 mL, 27.1 mmol).

Silver(I)oxide (2.36 g, 10.2 mmol) was added as well as 5 drops of dimethyl sulfide as catalyst. The flask was wrapped in aluminium foil to exclude daylight. The mixture was stirred for 8 h. After filtration over a pad of celite to remove the silver salts a column chromatography (3:1 hexane/EtOAc) was performed to yield 335 mg (73%) of 17 as a colorless highly viscous oil: [α]D: -21.7 (c = 0.3, CHCl3), IR (thin film, CHCl3): 2934, 2110,

-1 1 1742, 1601, 1507, 1248, 1093 cm . H NMR (CDCl3, 300 MHz) δ 1.30 (d, J = 6.3 Hz, 3H),

1.34 (d, J = 6.3 Hz, 3H), 2.17 (s, 3H), 3.08-3.21 (m, 3H), 3.39 (t, J = 9.0 Hz, 1H), 3.16 (t, J =

7.5 Hz, 1H), 3.16 (s, 3H), 3.77 (s, 3H), 3.92 (m, 1H), 4.35 (dd, J = 9.6 Hz, J = 3.3 Hz, 1H),

4.60 (d, J = 7.5 Hz, 1H), 4.66 (d, J = 10.5 Hz, 1H), 4.87 (quart., J = 10.9 Hz, 2H), 5.00 (d, J =

10.5 Hz, 1H), 5.32 (m, 1H), 5.34 (m, 1H), 6.82 (d, J = 12.0 Hz, 1H), 6.95 (d, J = 12.0 Hz,

13 1H), 7.26-7.44 (m, 10H). C NMR (CDCl3, 75 MHz) δ 18.1, 18.5, 21.2, 55.7, 60.7, 67.5,

68.3, 70.5, 72.2, 75.1, 75.4, 76.6, 80.5, 82.8, 84.4, 96.2, 103.0, 114.5, 117.7, 127.8, 127.9,

128.3, 128.4, 137.8, 138.0, 149.9, 154.9, 170.1. MALDI-HRMS: m/z [M + Na]+ calcd

700.2841, obsd 700.2828.

4-Azido-3-O-benzyl-6-desoxy-2-O-methyl-β-D-glucopyranosyl-(1→3)-2-O-acetyl-4-O- benzyl-α-L-rhamnopyranosyl Trichloroacetimidate (18). Compound 17 (125 mg, 0.185 mmol) was dissolved in a mixture of acetonitrile and water (6 mL/ 6 mL), cerium ammonium nitrate (354 mg, 0.646 mmol) was added in one portion and the mixture stirred for 90 min.

TLC indicated complete conversion into the hemiacetal. The solution was poured into brine and the aqueous phase was extracted 2 times with EtOAc. The combined organic phases were washed 2 times with water, dried over Na2SO4 and concentrated. The resulting crude product was purified by column chromatography (2:1 hexane/EtOAc). The product containing fractions were concentrated and the residue dissolved in dichloromethane (2 mL) and trichloroacetonitrile (2 mL). A catalytic amount of sodium hydride (3 mg) was added, stirred for 60 min, concentrated and purified by column chromatography (3:1 hexane/EtOAc) to afford 125 mg (95%, 2 steps) of 18 as a slightly yellow oil: [α]D: 3.3 (c = 0.25, CHCl3), IR

-1 1 (thin film, CHCl3): 2923, 2103, 1744, 1672, 1605, 1364, 1231 cm . H NMR (CDCl3,

300 MHz) δ 1.23 (d, J = 5.4 Hz, 3H), 1.38 (d, J = 6.3 Hz, 3H), 2.17 (s, 3H), 3.06-3.17 (m,

3H), 3.38 (t, J = 8.7 Hz, 1H), 3.61 (m, 1H), 3.62 (s, 3H), 3.95 (m, 1H), 4.24 (dd, J = 9.6 Hz, J

= 3.3 Hz, 1H), 4.57 (d, J = 8.1 Hz, 1H), 4.63 (d, J = 10.2 Hz, 1H), 4.86 (quart., J = 10.8 Hz,

2H), 4.98 (d, J = 10.2 Hz, 1H), 5.32 (m, 1H), 6.19 (d, J = 1.8 Hz, 1H), 7.31-7.44 (m, 10H),

13 8.68 (s, 1H). C NMR (CDCl3, 75 MHz) δ 18.0, 18.4, 21.0, 60.6, 67.5, 70.5, 70.7, 71.2, 75.3,

75.3, 75.6, 79.9, 82.7, 84.4, 90.9, 94.6, 103.1, 127.7, 128.0, 128.1, 128.2, 128.3, 128.4, 137.7,

137.8, 159.9, 169.6. MALDI-HRMS: m/z [M + Na]+ calcd 737.1518, obsd 737.1504.

n-Pentenyl 4-Azido-3-O-benzyl-6-desoxy-2-O-methyl-β-D-glucopyranosyl-(1→3)-2-O- acetyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2-O-acetyl-4-O-benzyl-α-L- rhamnopyranosyl-(1→2)-3,4-O-benzyl-α-L-rhamnopyranoside (19). Disaccharide 16

(98 mg, 0.140 mmol) and disaccharide trichloroacetimidate 18 (121 mg, 0.169 mmol) were azeotroped 3 times together with toluene and dried for 30 min in vacuo. The mixture was dissolved in dichloromethane (4.5 mL) and cooled to 0 °C. TMSOTf (5 mg, 4 µL,

0.021 mmol) was added, the solution stirred for 70 min and quenched by addition of some drops of pyridine. The solvent was removed and the resulting crude product was purified by column chromatography (3:1 hexane/EtOAc) to afford 154 mg (73%) of 19 as a slightly yellow highly viscous oil: [α]D: -11.5 (c = 0.60, CHCl3), IR (thin film, CHCl3): 2932, 2105, -1 1 1750, 1670, 1359 cm . H NMR (CDCl3, 300 MHz) δ 1.10 (d, J = 6.0 Hz, 3H), 1.27 (m, 6H),

1.31 (d, J = 6.0 Hz, 3H), 1.62 (m, 2H), 2.08 (m, 2H), 2.10 (s, 3H), 2.14 (s, 3H), 2.92-3.13 (m,

3H), 3.24-3.36 (m, 2H), 3.40-3.53 (m, 3H), 3.57-3.68 (m, 2H), 3.58 (s, 3H), 3.79-3.89 (m,

4H), 4.16 (t, J = 3.0 Hz, 1H), 4.19 (t, J = 3.0 Hz, 1H), 4.46 (d, J = 7.8 Hz, 1H), 4.54-4.71 (m,

6H), 4.76-4.98 (m, 6H), 5.02 (m, 1H), 5.06 (m, 1H), 5.27 (m, 1H), 5.32 (m, 1H), 5.77 (m,

13 1H), 7.23-7.42 (m, 25H). C NMR (CDCl3, 75 MHz) δ 17.8, 18.0, 18.0, 18.3, 21.1, 21.2,

28.7, 30.3, 60.5, 66.8, 67.4, 67.9, 68.2, 68.6, 70.3, 71.9, 72.2, 72.2, 74.8, 75.2, 75.4, 75.5,

75.5, 75.9, 77.2, 79.4, 80.1, 80.2, 80.6, 82.6, 84.4, 98.6, 98.7, 99.1, 102.7, 114.7, 127.3, 127.4,

127.5, 127.7, 127.7, 127.9, 128.1, 128.2, 128.3, 137.8, 137.9, 138.1, 138.4, 138.4, 169.6,

169.7. MALDI-HRMS: m/z [M + Na]+ calcd 1266.5720, obsd 1266.5700.

n-Pentenyl 4,6-Dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-β-D- glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L- rhamnopyranoside (2). Tetrasaccharide 19 (37 mg, 0.029 mmol) was dissolved under an Ar atmosphere in THF (6 mL) and ammonia was condensed into the reaction vessel at -78 °C until the mixture reached a volume of about 20 mL. Small pieces of carefully washed sodium were added to the reaction vessel affording a solution with a deep blue color. After 30 min the reaction was quenched by adding some drops of methanol. Again, small pieces of sodium were added until the blue color persists for at least 30 min. The reaction was quenched by adding several mL of methanol. The dry ice bath was removed, the reaction mixture was allowed to warm to room temperature in order to evaporate the ammonia. Dowex 50-X8 acidic resin (washed and dried) was added until pH 7 was reached. The mixture was filtered, rinsed and concentrated. The crude product was purified by column chromatography using silica gel (dichloromethane/methanol 3:1). The product containing fractions were combined, concentrated and further purified by using a Sephadex column (ethanol/water 1:1). The product containing fractions were combined and concentrated affording 12 mg (60%) of a colorless film. This compound (7 mg, 0.010 mmol) was dissolved in DMF (2.5 mL) and

3-hydroxy-3-methylbutyric acid (1.8 mg, 0.015 mmol) in 0.5 mL of DMF were added. HATU

(5 mg, 0.015 mmol), followed by N,N,-diisopropylethylamine (2 mg, 2.5 µL, 0.015 mmol) was added yielding a slightly yellow solution. The mixture is stirred at room temperature for

2 h. ESI-MS analysis showed complete conversion (m/z 806, [M + Na]+). All volatiles were removed in vacuo. The residue was purified by column chromatography using silica gel

(dichloromethane/methanol 4:1) and further purified by using a Sephadex column

(ethanol/water 1:1) yielding 5 mg (75%) of 2 as a colorless film: [α]D: -7.9 (c = 0.18, CHCl3),

1 H NMR (CD3OD, 600 MHz) δ 1.21 (d, J = 6.2 Hz, 3H), 1.24 (d, J = 6.2 Hz, 3H), 1.25 (d, J =

6.2 Hz, 3H), 1.29 (m, 9H), 1.68 (m, 2H), 2.15 (m, 2H) 2.36 (pquart., 2H), 3.02 (dd, J =

9.1 Hz, J = 7.9 Hz, 1H), 3.38 (m, 1H), 3.40-3.44 (m, 3H), 3.51 (t, J = 9.6 Hz, 1H), 3.55-3.60

(m, 2H), 3.66 (s, 3H), 3.67 (m, 1H), 3.70-3.78 (m, 3H), 3.80 (m, 2H), 3.83 (m, 1H), 3.90 (dd,

J = 9.4 Hz, J = 3.3 Hz, 1H), 4.05 (dd, J = 3.2 Hz, J = 1.9 Hz, 1H), 4.18 (dd, J = 3.3 Hz, J =

1.7 Hz, 1H), 4.62 (d, J = 7.8 Hz, 1H), 4.77 (d, J = 1.6 Hz, 1H), 4.91 (d, J = 1.8 Hz, 1H), 4.96

13 (m, 1H), 5.03 (m, 1H), 5.05 (d, J = 1.6 Hz, 1H), 5.83 (m, 1H). C NMR (CD3OD, 150 MHz)

δ 18.0, 18.1, 18.2, 18.5, 29.6, 29.7, 30.0, 31.5, 49.8, 58.2, 61.2, 68.0, 70.0, 70.2, 70.6, 70.8,

71.8, 71.9, 72.2, 72.3, 73.2, 73.4, 74.4, 75.1, 79.2, 80.2, 82.0, 85.7, 100.4, 103.6, 104.0, 105.5,

115.4, 139.4, 174.7. MALDI-HRMS: m/z [M + Na]+ calcd 806.3786, obsd 806.3797.