Toward a Benign Strategy for the Manufacturing of Betulinic Acid

Electronic Supplementary Material (ESI) for Green Chemistry. This journal is © The Royal Society of Chemistry 2016

Toward a benign strategy for the manufacturing of betulinic acid

Anna K. Ressmann,a Thomas Kremsmayr,a Peter Gaertner,a Ronald Zirbs,b and Katharina Bicaa,*

aInstitute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163, 1060 Vienna, Austria.

bGroup for Biologically Inspired Materials, Institute of Nanobiotechnology (DNBT), University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria.

*Corresponding author Katharina Schröder (née Katharina Bica). Email: [email protected], Fax: +43 1 58801 16360; Tel: +43 1 58801 163601.

Materials and methods ...... 2 Figure S1: Biphasic extraction of betulin (2) using hydrophilic ionic liquids ...... 3 Table S1: Recycling of phosphonium hydroxide solution in the biphasic extraction of betulin (2)...... 4 Figure S2: Evaluation of solvents for the oxidation of betulin ...... 5 Table S2-S4: Optimization of oxidation of betulin (2) to betulinic acid (1) ...... 6 Figure S3: Impact of acetic acid on the oxidation of betulin (2) to betulinic acid (1)...... 7 Figure S4-S5: Spectra of isolated betulin (2) (isolated via column chromatography) ...... 8 Figure S6-S7: Spectra of isolated betulin (2) (isolated via crystallization)...... 10 Figure S8-S9: Spectra of betulinic acid (1) obtained via oxidation of pure betulin (2)...... 11 Figure S10-11: Spectra of isolated betulinic acid (1) obtained via oxidation of crude biomass extract (isolated via column chromatography) ...... 13 Figure S12-S13: Spectra of betulinic acid (1) obtained via oxidation of crude biomass extract (isolated via crystallization)...... 15 References...... 17 Materials and methods

Commercially available reagents and solvents from Sigma-Aldrich were used, as received without further purification, unless otherwise specified. 1-Ethyl-3-methylimidazolium acetate ([C2mim][OAc]) was purchased from Iolitec (Heilbronn, Germany). Tetrabutylphosphonium hydroxide solution (40% in H2O) was purchased from TCI Chemicals (Eschborn, Germany), and the exact concentration was determined via titration prior to use. Ionic liquids [C4mim]Cl, [C2mim]Me2PO4, [C4mim]MeSO3 and

[HN(Et)3]HSO4 were synthesized according to standard procedures and analytical data was in accordance with literature.1,2,3,4

All ionic liquids were dried for at least 24-48 h at room temperature or 50 °C and 0.01 mbar before use and stored under argon.

1 13 30 H, C and P NMR spectra were recorded from CDCl3, CD2Cl2, d4-MeOD or d6-DMSO solutions on a Bruker Advance UltraShield 400 (400 MHz) spectrometer and chemical shifts (δ) are reported in ppm using tetramethylsilane as internal standard coupling constants (J) are in Hertz (Hz).

HPLC analysis was performed on a Jasco HPLC unit equipped with a PDA detector.

For the determination of betulin (1) a Maisch ReproSil 100 C18 250 x 4.6, 5 µm was used with

MeOH:H2O (0.1% TFA) = 87:13 as solvent and a flow of 1 ml/min; detection was done at 210 nm, at 30 °C column oven temperature, 25 °C tray temperature. Retention times were 7.3 min for the internal standard and 15.7 min for betulin (1). For the determination of betulin (1), betulinic acid (2) and betulinic aldehyde a Maisch ReproSil 100 C18 250 x 4.6, 5 µm was used with MeOH:H2O (0.1% TFA) = 87:13 as solvent and a flow of 1 ml/min; detection was done at 210 nm, at 30 °C column oven temperature, 25 °C tray temperature. Retention times were 7.3 min for the internal standard, 15.7 min for betulin (1), 16.5 min for betulinic acid (2) and 28.0 min for betulinic aldehyde.

Microwave reactions were performed on a BIOTAGE Initiator™ sixty microwave unit. The reported times are hold times. For this purpose the reaction mixtures were transferred into a glass pressure microwave tube equipped with a magnetic stirring bar, sealed with a Teflon septum and exposed to microwave irradiation at the required temperature. The absorption mode was set to „Very high“.

All reagents were purchased from commercial suppliers and used without further purification unless noted otherwise. Betulin as reference material was isolated from birch bark using extraction with ethanol. It was purified via column chromatography.

Chromatography solvents were distilled prior to use. Column chromatography was performed on a Büchi Sepacore Flash System (2 x Büchi Pump Module C-605, Büchi Pump Manager C-615, Büchi UV Photometer C-635, Büchi Fraction Collector C-660) or standard manual glass columns using silica gel from Merck (40-63 μm) with PE/EtOAc or MeOH/CH2Cl2 mixtures as eluates. Figure S1: Biphasic extraction of betulin (2) using hydrophilic ionic liquids

Several other hydrophilic ionic liquids, either in pure form or as aqueous solution, were also tested for the pretreatment of birch bark. Imidazolium derivatives [C2mim]OAc, [C2mim]Me2PO4, [C4mim]Cl, and [C4mim]MeSO3 as well as the low-prize ammonium protic ionic liquid [HNEt3]HSO4 were evaluated for the biphasic extraction of birch bark using the aqueous solution and n-BuOAc.

Figure S1: Biphasic extraction of betulin (2) using hydrophilic ionic liquids Table S1: Recycling of phosphonium hydroxide solution in the biphasic extraction of betulin (2)

Strategy 1 – Addition of new biomass Strategy 2 – Filtration and evaporation

Cycle Nr. Yield betulin [wt.%] Cycle Nr. Yield betulin [wt.%]

1 26.8 1 26.5

2 29.5 2 10.0

3 32.5 3 n.d.

4 18.6 4 n.d.

Strategy 1: In a 100 ml flask birch bark (1 g) and 9 g [P4444]OH as 40 wt.% solution in water were stirred for 1 h at room temperature. n-BuOAc (30 ml) was added and the mixture was again stirred for 1 h at room temperature. The organic layer was separated and yield was determined via HPLC.Fresh biomass (1 g) was added to the remaining dark slurry. The solution was stirred 1 h at room temperature and n-BuOAc (30 ml) was added. This cycle was repeated two more times until the biomass/IL slurry became unable to stir.

Strategy 2: In a 100 ml flask birch bark (1 g) and 9 g [P4444]OH as 40 wt.% solution in water were stirred for 1 h at room temperature. Water (20 ml) and n-BuOAc (30 ml) were added and the mixture was again stirred for 1 h at room temperature. The organic layer was separated and yield was determined via HPLC. The aqueous phase was diluted with 10 ml ethanol and the obtained precipitate was removed via filtration. The solution was concentrated in vacuo to a volume of 9 ml and reused with 1 g of biomass. Figure S2: Evaluation of solvents for the oxidation of betulin

Several solvents, including dimethylcarbonate (DMC), ethyl acetate (EtOAc), diethylcarbonate (DEC), dichloromethane (CH2Cl2), n-butyl acetate (BuOAc), tert-butanol (t-BuOH) and acetonitrile (ACN) were evaluated in the oxidation of betulin to betulinic acid. Reactions were performed using 10 mg betulin (2) in 1 ml solvent, 1 eq. TEMPO, 3 eq. BAIB for 24 h at room temperature.

conversion betulin (2) yield betulinic acid (1) 100

] 80 % [

d l e i 60 y / n o i s

r 40 e v n o c 20

0 C c C l 2 c H A E C A O N M O D O u C D t H 2 u B A E C B t- solvent Table S2-S4: Optimization of oxidation of betulin (2) to betulinic acid (1)

Table S2: Variation of oxidation reagents

Entry TEMPO [eq.] BAIB [eq.] Conversion betulin (2) [%] Yield betulinic acid (1) [%] 1 1 3 99 26 2 0.5 3 99 38 3 0.2 2 73 16 4 0.2 3 100 41 5 0.2 4 100 43 6 0.1 3 98 45 7 0.1 4 95 38 8 0.05 3 95 37 Performed using 10 mg betulin (2), 50 eq. water, 2 eq. t-BuOH at r.t. for 24 h. Yield was determined via RP-HPLC using 1-methyl-1- cyclohexen as internal standard. Performed in triplicates.

Table S3: Concentration dependency of the starting material in BuOAc

Concentration betulin (2) Conversion betulin (2) Yield betulinic acid (1) Entry [mg/ml] [%] [%] 1 15 100 39 2 10 100 43 3 7.5 88 28 4 5 67 13 Performed using 10 mg betulin (2), 0.2 eq TEMPO, 3 eq BAIB, 50 eq. water, 2 eq. t-BuOH at r.t. for 24 h. Yield was determined via RP-HPLC using 1-methyl-1-cyclohexen as internal standard. Performed in triplicates.

Table S4: Influence of the amount of water for the oxidation

Entry Solvent Water [eq.] Conversion betulin (2) [%]b Yield betulinic acid (1) [%]b 1 DMC 0 100 11.5 2 DMC 10 74 0 3 DEC 0 99 24.3 4 DEC 10 100a 12.3 5 BuOAc 0 95 22.7 6 BuOAc 10 99a 24.2 7 BuOAc 50 99a 26.1 8 BuOAc 250 99a 30.4c 9 BuOAc 2500 10 0 Performed using 10 mg betulin (2) in 1 ml solvent, 1 eq. TEMPO, 3 eq. BAIB. a Addition of 2 eq. t-BuOH. b According to HPLC c unidentified byproducts were detected Figure S3: Impact of acetic acid on the oxidation of betulin (2) to betulinic acid (1) Figure S4-S5: Spectra of isolated betulin (2) (isolated via column chromatography) 4.68 4.58 3.81 3.81 3.79 3.79 3.34 3.34 3.32 3.32 3.20 3.19 3.18 3.17 2.38 1.94 1.85 1.51 1.39 1.28 1.18 1.05 0.98 0.97 0.90 0.82 0.76 0.67 1.00 1.02 1.00 0.96 1.00 0.80 1.81 0.90 12.77 5.35 4.50 2.88 3.20 2.90 2.99 1.25 3.54 3.55 1.30

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 f1 (ppm) 150.83 110.06 79.33 60.91 55.62 50.73 49.09 48.13 43.06 41.25 39.22 39.04 37.64 37.50 34.56 34.32 30.08 29.50 28.34 27.74 27.38 25.53 21.17 19.43 18.65 16.47 16.32 15.72 15.11

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Figure S4: 1H and 13C NMR spectra of isolated betulin (2) of the crude biomass extract (isolated via column chromatography) 2

1 2

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 f1 (ppm)

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 f1 (ppm)

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Figure S5: 1H and 13C NMR spectra of isolated betulin (2) of the crude biomass extract (green, isolated via column chromatography) vs. reference betulin (2) (brown). Figure S6-S7: Spectra of isolated betulin (2) (isolated via crystallization) 1.19 1.19 1.00 0.99 1.29 1.26 3.58 3.97 8.87 13.06 3.64 2.98 3.40 3.27 3.42 1.33

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 f1 (ppm)

Figure S6: 1H NMR spectra of isolated betulin (2) of the crude biomass extract (isolated via crystallization)

1 2

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 f1 (ppm)

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 f1 (ppm)

Figure S7: 1H NMR spectra of isolated betulin (2) of the crude biomass extract (green, isolated via crystallization) vs. reference betulin (2) (brown) Figure S8-S9: Spectra of betulinic acid (1) obtained via oxidation of pure betulin (2) 7.26 1.00 1.01 1.29 0.87 1.23 1.10 1.92 2.93 15.87 5.67 6.81 3.72 3.54 3.49 1.15

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 f1 (ppm) 180.21 150.76 110.06 79.35 56.62 55.68 50.85 49.60 47.23 42.78 41.03 39.23 39.05 38.72 37.55 37.38 34.66 32.50 30.88 30.05 28.34 27.75 25.84 21.20 19.73 18.64 16.49 16.38 15.71 15.05

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Figure S8: 1H and 13C NMR spectra of isolated betulinic acid (1) obtained via oxidation of pure betulin (2) (isolated via crystallization) 2

1 2

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 f1 (ppm)

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 f1 (ppm)

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Figure S9: 1H and 13C NMR spectra of isolated betulinic acid (1) obtained via oxidation of pure betulin (2) (green, isolated via crystallization) vs. commercially available betulinic acid (1) from Sigma-Aldrich (brown) Figure S10-11: Spectra of isolated betulinic acid (1) obtained via oxidation of crude biomass extract (isolated via column chromatography) 4.74 4.61 3.20 3.02 2.28 2.19 1.97 1.96 1.69 1.61 1.38 1.27 1.18 0.97 0.96 0.93 0.82 0.75 0.67 1.00 0.99 1.16 1.05 1.21 1.27 2.41 3.53 15.88 5.42 6.93 3.81 3.41 3.48 1.19

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm) Figure S10: 1H and 13C NMR spectra of isolated betulinic acid (1) obtained after oxidation of crude biomass extract (isolated via column chromatography)

1 2

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 f1 (ppm)

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 f1 (ppm)

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm) Figure S11: 1H and 13C NMR spectra of isolated betulinic acid (1) obtained via oxidation of crude biomass extract (green, isolated via column chromatography) vs. commercially available betulinic acid (1) bought at Sigma- Aldrich (brown)

Figure S12-S13: Spectra of betulinic acid (1) obtained via oxidation of crude biomass extract (isolated via crystallization) 1.00 0.99 1.40 1.02 1.34 1.29 2.48 3.85 19.17 4.91 3.18 3.53 3.65 3.62 3.35 1.58

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 f1 (ppm) 179.95 150.32 109.65 78.96 56.20 55.25 50.43 49.17 46.81 42.36 40.61 38.80 38.63 38.30 37.13 36.96 34.24 32.07 30.46 29.63 27.92 27.33 25.41 20.77 19.31 18.21 16.07 15.96 15.32 15.29 14.63

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Figure S12: 1H and 13C NMR spectra of isolated betulinic acid (1) obtained after oxidation of crude biomass extract (isolated via crystallization, traces of lupeol could not be separated)

1 2

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 f1 (ppm)

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 f1 (ppm) 2

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Figure S13: 1H and 13C NMR spectra of isolated betulinic acid (1) obtained via oxidation of crude biomass extract (green, isolated via crystallization) vs. commercially available betulinic acid (1) bought at Sigma-Aldrich (brown) References

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