Supporting Information

Supporting Information Stereoselective cascade to C3-methylated strictosidine derivatives employing transaminases and strictosidine synthases Eva-Maria Fischereder, Desiree Pressnitz, and Wolfgang Kroutil* Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria Email: Wolfgang.Kroutil@uni-graz.at Content 1 Experimental... 2 1.1 Supporting Biotransformation results... 2 1.1.1 Supporting transaminase screening results... 2 1.1.2 Cascade transformation... 3 1.2 Codon optimized DNA sequences for E. coli of strictosidine synthases... 4 1.3 Primer sequences for strictosidine synthases... 6 1.4 Determination of strictosidine synthase activity... 6 1.5 Determination of transaminase activity... 7 1.6 Synthesis of indolylpropanones (1a-e)... 8 1.6.1 General procedure for the synthesis of 2-nitropropene... 8 1.6.2 General procedure for the preparation of indolylnitropopanes 4b-4e... 8 1.6.3 General procedure for the preparation of indolylpropanones 1b-e... 10 2 GC- and HPLC-methods... 11 3 NMR-spectra... 19 4 Abbreviations... 39 5 References... 39 1

1 Experimental 1.1 Supporting Biotransformation results 1.1.1 Supporting transaminase screening results Table S1. Conversions for the asymmetric amination of ketones 1a-1e using (R)/(S)- selective TAs transaminase 2a [%] 2b [%] 2c [%] 2d [%] 2e[%] Bacillus megaterium 24 32 10/98 b <1 38/54 b Paracoccus denitrificans <1 <1 <1 <1 <1 Pseudomonas putida <1 8 <1 <1 <1 Pseudomonas fluorescens 49/81 b 26 3 <1 5 Arthrobacter citreus <1 12 n.d. <1 n.d. Silibacter pomeroyi 74/98 b 98 5 <1 41/98 b Chromobacterium violaceum 37 94 8/21 b <1 54/64 b Vibrio fluvialis n.d. n.d. n.d. <1 n.d. Arthrobacter sp. (R)-selective 95/98 b 98 45/>99 b 84/98 b 54/>99 b Aspergillus terreus n.d. n.d. 25 <1 52 Reaction conditions: substrate 1a-1e (50 mm), lyophilized E. coli cells containing the overexpressed (S)/(R)-selective TAs (20 mg), D or L-alanine (250 mm), ammonium formate (150 mm), potassium phosphate buffer (100 mm, ph 7), PLP (1 mm), NAD + (1 mm), FDH (11 U), L-AlaDH (1.4 U), DMSO (10% v/v), 24 h, 30 C, reaction volume = 1 ml. Determination by GC-FID. b Conversions after 48 h. Table S2. Ee-values for the asymmetric amination of ketones 1a-1e using (R)/(S)-selective TAs transaminase 2a [%] 2b [%] 2c [%] 2d [%] 2e [%] Bacillus megaterium n.d. n.d. >98 (S) n.d. 96 (S) Paracoccus denitrificans n.d. n.d. n.d. n.d. n.d. Pseudomonas putida n.d. n.d. n.d. n.d. n.d. Pseudomonas fluorescens >98 (S) n.d. n.d. n.d. n.d. Arthrobacter citreus n.d. n.d. n.d. n.d. n.d. Silibacter pomeroyi >98 (S) 97 (S) n.d. n.d. >98 (S) Chromobacterium violaceum n.d. 88 (S) 95 (S) n.d. 98 (S) Vibrio fluvialis n.d. n.d. n.d. n.d. n.d. Arthrobacter sp. (R)-selective >98 (R) >98 (R) >98 (R) >98 (R) >98 (R) Aspergillus terreus n.d. n.d. >98 (R) n.d. n.d. The enantiomeric excess was determined after derivatization of the corresponding amines to acetoamides. n.d. = not determined. 2

1.1.2 Cascade transformation In this section the results of the cascade with the two other active strictosidine synthases Rauvolfia serpentina (RsSTR) and its corresponding variant (RvSTR) are shown. 70 3b 3e 3a 60 50 product 3 [%] 40 30 20 22 22 21 33 26 10 9 4 4 8 9 4 0 (1S,3R) RsSTR (1S,3R) RvSTR (1S,3S) RsSTR (1S,3S) RvSTR Scheme S1. Conversion for strictosidine derivatives in the one-pot, one-step cascade. Reaction conditions: lyophilized E. coli cells containing the overexpressed (S)/(R)-selective TAs (10 mg)/str from Rauvolfia serpentina (RsSTR) (90 mu)/ variant of Rauvolfia serpentina (RvSTR) (12 mu), D or L-alanine (250 mm), ammonium formate (150 mm), potassium phosphate buffer (100 mm, ph 7), PLP (1 mm), NAD + (1 mm), FDH (11 U), L- AlaDH (1.4 U), DMSO (5% v/v), ketone (1a-1e; 2 mm), secologanin (4 mm), 24 h, 30 C, reaction volume = 1 ml. Reactions were analyzed by HPLC-UV. All experiments were performed in triplicate. The de-values in all cases were >98%. 3

70 3b 3e 3a 60 50 product 3 [%] 40 30 20 10 4 21 4 5 12 39 9 29 28 10 40 0 (1S,3R) RsSTR (1S,3R) RvSTR (1S,3S) RsSTR (1S,3S) RvSTR Scheme S2. Conversion for strictosidine derivatives in the one-pot, two-step cascade. Reaction conditions: lyophilized E. coli cells containing the (S)- or (R)-selective TAs (10 mg), D or L -alanine (250 mm), ammonium formate (150 mm), potassium phosphate buffer (100 mm, ph 7), PLP (1 mm), NAD + (1 mm), FDH (11 U), L-AlaDH (1.4 U), DMSO (5% v/v), ketone (1a-c,1e; 2 mm), 24 h, 30 C. Removal of TAs by centrifugation (20 min., 13000 rpm). Addition of lyophilized E. coli cells containing overexpressed STR from Rauvolfia serpentina (RsSTR) (90 mu)/ variant of Rauvolfia serpentina (RvSTR) (12 mu) and secologanin (4 mm), 24 h, 30 C. Reactions were analyzed by HPLC-UV. All experiments were performed in triplicate. The de-values in all cases were >98%. 1.2 Codon optimized DNA sequences for E. coli of strictosidine synthases Restriction sites are underlined BamHI (GGATCC), SacI (GAGCTC), NcoI (CCATGG), XhoI (CTCGAG) Rauvolfia serpentina RsSTR ggatccggcaaaactgagcgatagccagaccatggcactgtttaccgtttttctgctgtttctgagcagcagcctggcactgagcagcccgattct GAAAGAAATTCTGATTGAAGCACCGAGCTATGCACCGAATAGCTTTACCTTTGATAGCACCAACAAAGGCTTTTATACCAGCG TTCAGGATGGTCGTGTTATCAAATATGAAGGTCCGAATAGCGGCTTTGTGGATTTTGCCTATGCAAGCCCGTATTGGAATAAA GCCTTTTGTGAAAATAGCACCGATGCCGAAAAACGTCCGCTGTGTGGTCGTACCTATGATATTAGCTATAATCTGCAGAACAA CCAGCTGTATATCGTGGATTGTTATTATCATCTGAGCGTTGTTGGTAGCGAAGGTGGTCATGCAACCCAGCTGGCAACCAGCG TTGATGGTGTTCCGTTTAAATGGCTGTATGCAGTTACCGTTGATCAGCGTACCGGTATTGTGTATTTTACCGATGTTAGCACCC TGTATGACGATCGTGGTGTGCAGCAGATTATGGATACCAGCGATAAAACCGGTCGTCTGATTAAATACGATCCGAGCACCAA AGAAACCACCCTGCTGCTGAAAGAACTGCATGTTCCGGGTGGTGCAGAAGTTAGCGCAGATAGCAGCTTTGTTCTGGTTGCCG AATTTCTGAGCCATCAGATTGTGAAATATTGGCTGGAAGGTCCTAAAAAAGGCACCGCAGAAGTTCTGGTTAAAATTCCGAA TCCGGGTAACATTAAACGTAATGCCGATGGTCATTTTTGGGTTAGCAGCAGCGAAGAACTGGATGGTAATATGCATGGTCGC GTTGATCCGAAAGGCATTAAATTCGATGAATTTGGCAACATCCTGGAAGTTATTCCGCTGCCTCCGCCTTTTGCCGGTGAACA TTTTGAGCAGATTCAAGAACATGATGGCCTGCTGTATATTGGCACCCTGTTTCATGGTAGCGTTGGTATTCTGGTGTATGATAA AAAAGGTAACAGCTTTGTGAGCAGCCACTAAgagctc 4

Rauvolfia serpentina variant V208A RvSTR ggatccgcaaaactgagcgatagccagaccatggcactgtttaccgtttttctgctgtttctgagcagcagcctggcactgagcagcccgat TCTGAAAGAAATTCTGATTGAAGCACCGAGCTATGCACCGAATAGCTTTACCTTTGATAGCACCAACAAAGGCTTTTAT ACCAGCGTTCAGGATGGTCGTGTTATCAAATATGAAGGTCCGAATAGCGGCTTTGTGGATTTTGCCTATGCAAGCCCGT ATTGGAATAAAGCCTTTTGTGAAAATAGCACCGATGCCGAAAAACGTCCGCTGTGTGGTCGTACCTATGATATTAGCTA TAATCTGCAGAACAACCAGCTGTATATCGTGGATTGTTATTATCATCTGAGCGTTGTTGGTAGCGAAGGTGGTCATGCA ACCCAGCTGGCAACCAGCGTTGATGGTGTTCCGTTTAAATGGCTGTATGCAGTTACCGTTGATCAGCGTACCGGTATTGT GTATTTTACCGATGTTAGCACCCTGTATGACGATCGTGGTGTGCAGCAGATTATGGATACCAGCGATAAAACCGGTCGT CTGATTAAATACGATCCGAGCACCAAAGAAACCACCCTGCTGCTGAAAGAACTGCATGCACCGGGTGGTGCAGAAGTT AGCGCAGATAGCAGCTTTGTTCTGGTTGCCGAATTTCTGAGCCATCAGATTGTGAAATATTGGCTGGAAGGTCCTAAAA AAGGCACCGCAGAAGTTCTGGTTAAAATTCCGAATCCGGGTAACATTAAACGTAATGCCGATGGTCATTTTTGGGTTAG CAGCAGCGAAGAACTGGATGGTAATATGCATGGTCGCGTTGATCCGAAAGGCATTAAATTCGATGAATTTGGCAACATC CTGGAAGTTATTCCGCTGCCTCCGCCTTTTGCCGGTGAACATTTTGAGCAGATTCAAGAACATGATGGCCTGCTGTATAT TGGCACCCTGTTTCATGGTAGCGTTGGTATTCTGGTGTATGATAAAAAAGGTAACAGCTTTGTGAGCAGCCACTAAgagct c Ophiorriza pumila OpSTR ccatgggcagtccggaattttttgaatttattgaagcaccgagctatggtccgaatgcatatgcctttgatagtgatggtgaac TGTATGCAAGCGTTGAAGATGGTCGCATCATCAAATATGATAAACCGAGCAACAAATTTCTGACCCATGCAGTTGCAAGCCC GATTTGGAATAATGCACTGTGTGAAAATAACACCAACCAGGATCTGAAACCGCTGTGTGGTCGTGTTTATGATTTTGGCTTTC ATTATGAAACCCAGCGCCTGTATATTGCCGATTGTTATTTTGGTCTGGGTTTTGTTGGTCCGGATGGTGGTCATGCAATTCAGC TGGCAACCAGCGGTGATGGTGTTGAGTTTAAATGGCTGTATGCACTGGCAATTGATCAGCAGGCAGGTTTTGTTTATGTTACC GATGTTAGCACCAAATATGACGATCGTGGTGTTCAGGATATCATTCGCATTAATGATACCACCGGTCGCCTGATTAAATACGA TCCGAGCACCGAAGAGGTTACCGTTCTGATGAAAGGTCTGAATATTCCGGGTGGCACCGAAGTTAGCAAAGATGGTAGCTTT GTTCTGGTGGGTGAATTTGCAAGCCATCGTATTCTGAAATATTGGCTGAAAGGTCCGAAAGCAAATACCAGCGAATTTCTGCT GAAAGTTCGTGGTCCGGGTAACATTAAACGTACCAAAGATGGCGATTTTTGGGTTGCAAGCAGCGATAATAATGGTATTACC GTTACACCGCGTGGTATTCGCTTTGATGAATTTGGTAATATTCTGGAAGTTGTGGCAATTCCGCTGCCGTATAAAGGTGAACA TATTGAACAGGTGCAAGAACATGATGGTGCCCTGTTTGTTGGTAGCCTGTTTCATGAATTTGTGGGCATTCTGCACAACTATA AAAGCAGCGTTGATCACCACCAAGAAAAAAACAGCGGTGGTCTGAATGCAAGCTTTAAAGAATTTAGCAGCTTTGGCAGCCA TCACCATCATCATCATTAGctcgag Ophiorriza japonica OjSTR ggatccgggtagcagcgaagcaatggttgttagcattctgtgtgcaatttttctgagcagcctgagcctggttagcagcagt CCGGAATTTTTTCAGTTTATTGAAGCACCGAGCTATGGTCCGAATGCCTATGCATTTGATAGTGATGGTGAACTGTATGC CAGCGTTGAAGATGGTCGTATCATCAAATATGACAAACCGAGCAAAAAATTCCTGAATCATGCAGTTGCAAGCCCGATT TGGAATAATGCACTGTGTGAAAATAACACCAACCAGGATCTGAAACCGCTGTGTGGTCGTGTTTATGATTTTGGCTTTC ATTATGAAACCCAGCGCCTGTATATTGCCGATTGTTATTTTGGTCTGGGTTTTGTTGGTCCGGATGGTGGTCGTGCAATT CAGCTGGCAACCAGCGCAGATGGTGTGAAATTTATGTGGCTGTATGCACTGGCAATTGATCAGCAGACCAGCTTTGTTT ATGTTACCGGTGTTAGCACCAAATACGATGATCGTGGTGTGCAAGAAATTATCCGCATTAATGATACCACCGGTCGCCT GATTAAATACGATCCGAGCACCAAAGAAGTTACCGTTCTGATGAAAGGTCTGAATATTCCGGGTGGCACCGAAGTTAGC AAAGATGGTAGCTTTGTTCTGGTTGCAGAATTCTATAGCCATCGGATTCTGAAATATTGGCTGAAAGGTCCGAAAGCAA ATACCAGCGAATTTCTGCTGAAAGTTCGTGGTCCGGGTAACATTAAACGTACCAAAGATGGCGATTTTTGGGTTGCAAG CAGCGATAATAATGGTATTACCGTTACACCGCGTGGTATTCGCTTTGATGAATTTGGTAATATTCTGGAAGTTGTGGCAA TTCCGCTGCCGTATAAAGGTGAACATATTGAACAGGTGCAAGAACATAATGGTGCACTGTTTGTTGGTAGCCTGTTTCA TGAATTTGTGGGCATTCTGCACAACTATAAAAGCAGCGTTGATCATCACCACCAAGAAAAAAATCAGGGTGGTCTGAAT GATGCCAGCTTTAAAGAATTTAGCAGCTTTTAAgagctc Catharantus roseus CrSTR ggatccggccaattttagcgagagcaaaagcatgatggcagtgttctttatgttttttctgctgctgctgagcagctcactg GCACTGAGCAGCCCGATTCTGAAAAAAATCTTTATTGAAAGCCCGAGCTATGCACCGAATGCATTTACCTTTGATAGCA CCGATAAAGGCTTTTATACCAGCGTTCAGGATGGTCGTGTTATCAAATATGAAGGTCCGAATAGCGGCTTTACCGATTTT GCCTATGCAAGCCCGTTTTGGAATAAAGCCTTTTGTGAAAATAGTACCGACCCGGAAAAACGTCCGCTGTGTGGTCGTA CCTATGATATTAGCTATGATTATAAAAACAGCCAGATGTATATCGTGGATGGCCATTATCATCTGTGCGTTGTTGGTAAA GAAGGTGGTTACGCAACCCAGCTGGCAACCAGCGTGCAGGGTGTTCCGTTTAAATGGCTGTATGCAGTTACCGTTGATC AGCGTACCGGTATTGTGTATTTTACCGATGTTAGCAGCATCCATGATGATAGTCCGGAAGGTGTTGAAGAAATTATGAA TACCAGCGATCGTACCGGTCGTCTGATGAAATATGATCCGAGCACCAAAGAAACCACCCTGCTGCTGAAAGAACTGCAT GTTCCGGGTGGTGCAGAAATTAGCGCAGATGGTAGCTTTGTTGTTGTTGCAGAATTTCTGAGCAACCGCATTGTGAAAT ATTGGCTGGAAGGTCCTAAAAAAGGTAGTGCCGAATTTCTGGTTACCATTCCGAATCCGGGTAACATTAAACGTAATAG CGACGGTCATTTTTGGGTGAGCAGCAGCGAAGAACTGGATGGTGGTCAGCATGGTCGCGTTGTTAGCCGTGGCATTAAA TTCGATGGTTTTGGTAATATCCTGCAGGTTATCCCGCTGCCTCCGCCTTATGAAGGTGAACATTTTGAGCAGATTCAAGA ACATGATGGCCTGCTGTATATTGGTAGCCTGTTTCATAGCAGCGTTGGTATTCTGGTTTATGATGATCATGATAACAAAG GCAACAGCTATGTGAGCAGCTAAgagctc 5

1.3 Primer sequences for strictosidine synthases Table S3: Construct design for strictosidine synthases STR Plasmid Restriction sites N-terminus C-terminus His-tag RsSTR pet-28a(+) BamHI SacI N- terminal RvSTR pet-28a(+) BamHI SacI N- terminal CrSTR pet-28a(+) BamHI SacI N- terminal OpSTR pet-28a(+) NcoI a XhoI a C- terminal OjSTR pet-28a(+) BamHI SacI N- terminal a Restriction sites were already included in ordered DNA sequence. Table S4: Primer sequences for the corresponding STR plasmids STR Primer Sequence (5-3 ) Forward GGATCCGCAAAACTGAGCGATAGCCA RsSTR Reverse GAGCTCTTAGTGGCTGCTCACAAAGCT RvSTR CrSTR OjSTR Forward GGATCCGCAAAACTGAGCGATAG Reverse GAGCTCTTAGTGGCTGCTCACAAA Forward GGATCCGCCAATTTTAGCGAGAG Reverse GAGCTCTTAGCTGCTCACATAGCT Forward GGATCCGGTAGCAGCGAAGCA Reverse GAGCTCTTAAAAGCTGCTAAATTCTTTAA 1.4 Determination of strictosidine synthase activity The activity of whole cell E. coli strictosidine synthase (STR) batches was assayed for the condensation reaction between tryptamine and secologanin to (S)-strictosidine over 30 minutes. A corresponding amount of lyophilized cells containing the respective STRs were rehydrated 6

in PIPES buffer (50 mm, ph 6.8, 225 µl) at 28 C for 30 minutes. Afterwards aliquots of substrate stock solution (10 mm tryptamine, 20 mm secologanin, 25µL) were added. Samples were shaken at 28 C and 800 rpm. Samples were taken after 5, 10, 20 and 30 minutes. The reactions were terminated by addition of 250 µl methanol, followed by centrifugation (10 min, 13000rpm). Conversions were determined on HPLC-UV. Table S5. Specific activities [mu/mg cells] of the overexpressed strictosidine synthases Enzyme mg cells/ml Average n [nmol/min] Average relative activity [mu/mg] OpSTR 1.24 0.05 1.77 RsSTR 0.60 1.40 9.33 RvSTR 15.0 1.84 0.49 CrSTR 0.60 1.23 8.23 OjSTR 20.0 - - a a no activity towards the natural reaction detected. 1.5 Determination of transaminase activity The activity of whole cell E. coli/transaminase batches was assayed for the deamination of (S)- or (R)-methylbenzylamine to acetophenone over 3 minutes. A corresponding amount of lyophilized cells containing the respective transaminase were rehydrated in a solution of PLP (1 mm) and sodium pyruvate (100 mm) at 30 C for 30 minutes. (R)/(S)-Methylbenzylamine (100 mm) was added and the reaction was further incubated at 30 C and 800 rpm. After 1, 2 and 3 minutes, the reaction was stopped by addition of 10 N NaOH (100 µl) followed by extraction with ethyl acetate (2 x 500 µl). The combined organic phases were dried over Na2SO4 and the conversion was determined on GC-FID. Table S6: Specific activities [U/mg cells] of the overexpressed transaminases Enzyme mg cells/ml Average n [µmol/min] Average relative activity [U/mg] BM-TA 5 0.71 0.14 PF-TA 2.5 7.50 3.00 CV-TA 2.5 9.70 3.88 AB-TA 5 3.00 0.60 SiLi-TA 2.5 14.70 5.88 AT-TA 5 2.13 0.42 7

1.6 Synthesis of indolylpropanones (1a-e) For the synthesis of starting materials a two-step literature procedure was adapted. 1 1.6.1 General procedure for the synthesis of 2-nitropropene Phthalic anhydride (29.6 g, 200 mmol) and 2-nitro-1-propanol (10.5g, 100 mmol) were combined in a round bottom flask equipped with a distilling head and heated until a homogeneous solution was formed (174 C). Under gentle application of vacuum the pale-green product was distilled. The co-distilled water was removed via CaCl2 filtration affording dry 2- nitropropene (4.8 g; 55%). The material was stored in a benzene solution (1 g/10 ml) over CaCl2. 1 H-NMR-spectrum is in accordance to literature. 1 1.6.2 General procedure for the preparation of indolylnitropopanes 4b-4e The selected indole (1 eq.) was suspended in dry benzene (20 ml). Then 2-nitropropene as a 10%-benzene solution (2 eq.) was added and the reaction mixture was refluxed under inert conditions until the whole starting material was consumed. The dark reaction mixture was cooled to room temperature and the solvent was evaporated. Column chromatography yielded the corresponding indolylnitropropane. 8

1-(5-hydroxyindol-3-yl)-2-nitropropane 4b Column chromatography (silica; CHCl3/MeOH = 99/1; Rf: 0.40). After purification 4b was obtained as brown oil (1.57 g; 62 %). 1 H-NMR (d6-aceton, 300 MHz): δ = 1.55 (3H, d, J = 6 Hz, CH3), 3.17 (1H, dd, J1 = 6 Hz, J2 = 9 Hz, CH2), 3.26 (1H, dd, J1 = 6 Hz, J2 = 9 Hz, CH2), 4.94 (1H, sext, J = 6 Hz, CH), 6.72 (1H, dd, J1 = 3 Hz, J2 = 7 Hz, aryl), 6.97 (1H, d, J = 3 Hz, aryl), 7.12 (1H, d, J = 3 Hz, CH), 7.21 (1H, d, J = 9 Hz, aryl), 7.67 (1H, s, OH), 9.90 (1H, bs, NH). 13 C-NMR (d6-aceton,75 MHz): δ =18.29, 31.14, 83.98, 102.2, 108.4, 111.7, 111.8, 124.3, 128.0, 131.4, 150.9. MS (EI, 70 ev): m/z = 220 (M, 70), 175 (60), 159 (35), 146 (100), 133 (34), 117 (9), 73 (18). 1-(5-methylindol-3-yl)-2-nitropropane 4c Column chromatography (silica; CHCl3/petrol ether = 1/1). TLC (silica; CHCl3; Rf: 0.61). After purification 4c was obtained as a brown oil (1.66 g; 50 %). 1 H-NMR (CDCl3, 300 MHz): δ = 1.59 (3H, d, J = 6 Hz, CH3), 2.49 (1H, s, CH3-aryl), 3.17 (1H, dd, J1 = 7 Hz, J2 = 15 Hz, CH2), 3.47 (1H, dd, J1 = 6 Hz, J2 = 15 Hz, CH2), 4.90 (1H, sext, J = 6 Hz, CH), 6.98 (1H, d, J = 3 Hz, CH), 7.05 (1H, d, J = 9 Hz, CH-Aryl), 7.26 (1H, d, J = 9 Hz, aryl), 7.36 (1H, s, aryl), 7.98 (1H, bs, NH). 13 C-NMR (CDCl3,75 MHz): δ = 18.98, 21.54, 31.31, 83.83, 109.4, 11.1, 117.1, 123.1, 124.1, 127.2, 129.2, 134.5. MS (EI, 70 ev): m/z = 218 (M, 62), 172 (65), 157 (40), 144 (100), 131 (25), 115 (16), 72 (16). 1-(6-methoxyindol-3-yl)-2-nitropropane 4d Column chromatography (silica; CHCl3/petrol ether = 1/1). TLC (silica; CHCl3; Rf: 0.45). After purification 4d was obtained as brown oil (1.67 g; 70 %). 1 H-NMR-spectrum is in accordance to literature. 1 13 C-NMR (CDCl3,75 MHz): δ = 18.97, 31.35, 55.66, 55.70, 83.87, 94.79, 109.9, 118.8, 121.3, 121.7, 137.9, 156.7. MS (EI, 70 ev): m/z = 234 (M, 100), 188 (89), 160 (74), 117 (18), 80 (11). 1-(7-methylindol-3-yl)-2-nitropropane 4e Column chromatography (silica; CHCl3/petrol ether = 1/1). TLC (silica; CHCl3; Rf: 0.74). After purification 4e was obtained as a brown oil (1.06 g; 35 %). 1 H-NMR (CDCl3, 300 MHz): δ = 1.59 (3H, d, J = 6 Hz, CH3), 2.48 (1H, s, CH3-aryl), 3.20 (1H, dd, J1 = 6 Hz, J2 = 15 Hz, CH2), 3.50 (1H, dd, J1 = 6 Hz, J2 = 15 Hz, CH2), 4.90 (1H, sext, J = 6 9

Hz, CH), 7.03-7.12 (3H, m, CH & aryl), 7.42 (1H, d, J = 6 Hz, aryl), 8.03 (1H, bs, NH). 13 C- NMR (CDCl3, 75 MHz): δ =16.55, 18.98, 31.41, 83.87, 110.4, 115.9, 120.0, 120.6, 122.7, 122.9, 126.5, 135.7. MS (EI, 70 ev): m/z = 234 (M, 100), 188 (89), 160 (74), 117 (18), 80 (11). MS (EI, 70 ev): m/z = 218 (M, 82), 172 (72), 157 (44), 144 (100), 131 (25), 115 (16), 72 (9). 1.6.3 General procedure for the preparation of indolylpropanones 1b-e To a stirred solution of the corresponding indolylnitropropanes 4b-e (1 eq.) in dry MeOH (30 ml), sodium methoxide (2 eq.) was added and the reaction mixture was stirred for 1h under inert conditions. Afterwards TiCl3 (powder; 5 eq.) was suspended in 4 M ammonium acetate buffer which finally was adjusted to ph 4.3. The suspension was added to the reaction mixture which was allowed to stir at room temperature overnight. Upon completion of the reaction, the mixture was extracted with diethyl ether (5 x 50 ml) and ethyl acetate (3 x 30 ml). The combined organic phase was then washed with 10%-NaHCO3-solution (100 ml), dried over Na2SO4 and evaporated under reduced pressure. Column chromatography and recrystallization yielded the corresponding indolylpropanons. 1-(5-hydroxyindol-3-yl)-2-propanone 1b Column chromatography (silica; CHCl3). TLC (silica; CHCl3/MeOH = 95/5; Rf: 0.3). The product obtained from column chromatography was further purified via recrystallization from toluene. 1b was obtained as yellowish solid (370 mg; 32 %). Mp = 80-82 C (lit. 78-81 C). 1 1 H-NMR-spectrum is in accordance to literature. 1 13 C-NMR (d6-aceton,75 MHz): δ = 27.55, 40.53, 102.5, 107.5, 111.6, 111.7, 124.4, 128.3, 131.4, 150.8, 205.8. MS (EI, 70 ev): m/z = 189 (M, 23), 146 (100), 117 (7). 1-(5-methylindol-3-yl)-2-propanone 1c Column chromatography (silica; CHCl3/petrol ether: 80/20). TLC (silica; CHCl3; Rf: 0.20). 1c was obtained as reddish solid (250 mg; 18 %). Mp = 59-61 C 10

1 H-NMR (CDCl3, 300 MHz): δ = 2.18 (3H, s, CH3), 2.46 (3H, s, CH3-aryl), 3.80 (2H, s, CH2), 7.03 (1H, d, J = 1.2 Hz, aryl), 7.06 (1H, s, CH) 7.25 (1H, d, J = 9 Hz, CH), 7.33 (1H, s, aryl), 8.13 (1H, bs, NH). 13 C-NMR (CDCl3,75 MHz): δ = 21.51, 28.85, 40.94, 108.2, 111.0, 118.2, 123.3, 123.9, 127.5, 129.1, 134.5, 207.8. MS (EI, 70 ev): m/z = 187 (M, 21), 144 (100), 115 (11). 1-(6-methoxyindol-3-yl)-2-propanon 1d Column chromatography (silica; CH2Cl2/MeOH/TEA = 98/1/1; Rf: 0.28). The product obtained from column chromatography was further purified via recrystallization from toluene. 1d was obtained as dark yellowish needles (300 mg; 20 %). Mp = 137-139 C (lit. 138-140 C). 1 1 H- NMR-spectrum is in accordance to literature. 13 C-NMR (CDCl3,75 MHz): δ = 28.85, 40.95, 55.49, 55.50, 94.75, 108.7, 109.8, 119.3, 121.6, 121.9, 137.0, 156.7, 207.6. MS (EI, 70 ev): m/z = 187 (M, 21), 144 (100), 115 (11). 1-(7-methylindol-3-yl)-2-propanone 1e Column chromatography (silica; CHCl3/petrol ether = 80/20) followed by (silica; CHCl3; Rf: 0.20). 1e was obtained as brownish solid (142 mg; 21 %). Mp = 59-61 C 1 H-NMR (CDCl3, 300 MHz): δ = 2.18 (3H, s, CH3), 2.49 (3H, s, CH3-aryl), 3.83 (2H, s, CH2), 7.02-7.13 (3H, m, aryl & CH), 7.41 (1H, d, J = 6 Hz, aryl), 8.15 (1H, bs, NH). 13 C-NMR (CDCl3,75 MHz): δ = 16.57, 28.91, 40.99, 109.2, 116.4, 120.0, 120.5, 122.8, 122.9, 126.8, 135.8, 207.6. MS (EI, 70 ev): m/z = 187 (M, 21), 144 (100), 115 (11). 2 GC- and HPLC-methods Conversion of amines 2a,c-e was measured by achiral GC-FID equipped with a HP-5 column (30 m x 0.32 mm). Conversion of amine 2b as well as strictosidine derivatives 3a,b and 3e were measured on achiral HPLC-UV equipped with a Luna C18 column (250 mm x 4.6 mm). All eevalues of amines 2a-e were recorded on HPLC-UV equipped with a chiracel OJ column (250 mm x 4.6 mm) after derivatization to the corresponding acetoamides. Method GC-measurements: carrier gas = Helium; flow = 2 ml/min; temperature program = from 100 C to 300 C/rate 10 C per minute. 11

Method achiral HPLC-UV-measurements: eluent = NH4COOH-buffer (30 mm, ph 2.8)/acetonitrile (+ 0.1% v/v TFA); gradient = 90/10 to 50/50 within 8 minutes to 20/80 within 3 minutes; flow = 1 ml/min. Method A chiral HPLC- UV-measurements: isocratic eluent = n-heptane/i-propanol = 85/15; flow = 0.8 ml/min; oven temperature = 39 C. Method B chiral HPLC- UV-measurements: isocratic eluent = n-heptane/i-propanol = 80/20; flow = 0.8 ml/min; oven temperature = 39 C. NH2 N H 2a Conversion determined on GC-FID: amine 2a: 10.39 min., ketone 1a: 10.20 min.; ee (method A): (R)-2a: 23.3 min., (S)-2a: 25.8 min. Figure S1. Rac-2a Figure S2. (S)-2a from biotransformation with SiLi-TA Figure S3: (R)-2a from biotransformation with ArR-TA 12

HO NH2 N H 2b Conversion determined on HPLC-UV: amine 2b: 6.0 min., ketone 1b: 8.8 min.; ee (method B): (R)-2b: 22.2 min., (S)-2b: 26.1 min. Figure S4. Mixture of bioaminated products obtained by enantiocomplementary enzymes Figure S5. (S)-2b from biotransformation with SiLi-TA Figure S6. (R)-2b from biotransformation with ArR-TA NH2 N H 2c Conversion determined on GC-FID: amine 2c: 11.0 min., ketone 1c: 10.3 min.; ee (method A): (R)-2c: 16.9 min., (S)-2c: 26.0 min. 13

Figure S7. Mixture of bioaminated products obtained by enantiocomplementary enzymes Figure S8. (S)-2c from biotransformation with BM-TA Figure S9. (R)-2c from biotransformation with ArR-TA NH2 O N H 2d Conversion determined on GC-FID: amine 2d: 12.8 min., ketone 1d: 12.9 min.; ee (method A): (R)-2d: 16.6 min., (S)-2d: 25.4 min. Figure S10. Rac-2d 14

Figure S11. (R)-2d from biotransformation with ArR-TA NH2 N H 2e Conversion determined on GC-FID: amine 2e: 10.9 min., ketone 1e: 11.1 min.; ee (method A): (R)-2e: 19.7 min., (S)-2e: 15.7 min. Figure S12. Mixture of bioaminated products obtained by enantiocomplementary enzymes Figure S13. (S)-2e from biotransformation with SiLi-TA Figure S14. (R)-2e from biotransformation with ArR-TA 15

3a Conversion determined on HPLC-UV: amine 2a: 8.0 min., product 3a: 9.4 min., ketone 1a: 12.3 min. Figure S15. (1S/3S)-3a from cascade reaction employing SiLi-TA & OpSTR abundance [mau] 1000000 800000 600000 400000 200000 0 9.43 9.46 9.49 9.53 9.56 9.59 9.62 9.65 9.69 9.72 9.75 9.78 9.81 9.85 9.88 9.91 9.94 9.97 10.01 10.04 10.07 10.10 10.13 10.17 retention time [min] (1S,3S)-3a (1S,3R)-3a Figure S16. Overlay of chromatography traces of (1S,3S)- and (1S,3R)-3a obtained from two individual cascades. 3b Conversion determined on HPLC-UV: amine 2b: 8.0 min., product 3b: 7.7 min., ketone 1c: 8.8 min. 16

Figure S17. (1S/3R)-3b from cascade reaction between SiLi-TA & OpSTR abundance [mau] 700000 600000 500000 400000 300000 200000 100000 0 7.19 7.24 7.30 7.35 7.40 7.46 7.51 7.56 7.62 7.67 7.72 7.78 7.83 7.88 7.94 7.99 8.04 8.10 8.15 8.20 8.26 8.31 8.36 retention time [min] (1S,3S)-3b (1S,3R)-3b Figure S18. Overlay of chromatography traces of (1S,3S)- and (1S,3R)-3b obtained from two individual cascades. 3e Conversion determined on HPLC-UV: amine 2e: 8.3 min., product 3e: 9.9 min., ketone 1e: 13.5 min. Figure S19. (1S/3R)-3e from cascade reaction between SiLi-TA & OpSTR 17

abundance [mau] 900000 800000 700000 600000 500000 400000 300000 200000 100000 0 9.76 9.81 9.87 9.92 9.97 10.03 10.08 10.13 10.19 10.24 10.29 10.35 10.40 10.45 10.51 10.56 10.61 10.67 10.72 10.77 10.83 10.88 10.93 retention time [min] (1S,3S)-3e (1S,3R)-3e Figure S20. Overlay of chromatography traces of (1S,3S)- and (1S,3R)-3e obtained from two individual cascades. Figure S21. Isomeric composition of derivative 3e obtained by chemical Pictet-Spengler (PS) reaction (A, B and D) and from biotransformation (C). A: Substrate: (S)-2e; B: Substrate (R)- 2e; D: substrate: 1:1 mixture of (S)/(R)-2e. C: Substrate: (R)-2e (formed by -TA from Arthrobacter sp. in the cascade) and transformed with OpSTR. Reaction conditions for chemical transformation: (S)/(R)-2e (2 mm), DMSO (5% v/v), maleic acid buffer (10 mm, ph 2.8, 475 µl), secologanin (4 mm), 24 h at 60 C. 18

3 NMR-spectra wb_emf_hydroxyinole-nitro 1h 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Figure S22. 1 H-spectrum of 1-(5-hydroxyindol-3-yl)-2-nitropropane 4b Figure S23. 13 C-spectrum of 1-(5-hydroxyindol-3-yl)-2-nitropropane 4b 19

Figure S24. 1 H-spectrum of 1-(5-methylindol-3-yl)-2-nitropropane 4c Figure S25. 13 C-spectrum of 1-(5-methylindol-3-yl)-2-nitropropane 4c 20

Figure S26. 1 H-spectrum of 1-(6-methoxyindol-3-yl)-2-nitropropane 4d Figure S27. 13 C-spectrum of 1-(6-methoxyindol-3-yl)-2-nitropropane 4d 21

Figure S28. 1 H-spectrum of 1-(7-methylindol-3-yl)-2-nitropropane 4e Figure S29. 13 C-spectrum of 1-(7-methylindol-3-yl)-2-nitropropane 4e 22

wb_emf_4hyroxyino 1h 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 f1 (ppm) 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 Figure S30. 1 H-spectrum of 1-(5-hydroxyindol-3-yl)-2-propanone 1b Figure S31. 13 C-spectrum of 1-(5-hydroxyindol-3-yl)-2-propanone 1b 23

Figure S32. 1 H-spectrum of 1-(5-methylindol-3-yl)-2-propanone 1c Figure S33. 13 C-spectrum of 1-(5-methylindol-3-yl)-2-propanone 1c 24

Figure S34. 1 H-spectrum of 1-(6-methoxyindol-3-yl)-2-propanone 1d Figure S35. 13 C-spectrum of 1-(6-methoxyindol-3-yl)-2-propanone 1d 25

Figure S36. 1 H-spectrum of 1-(7-methylindol-3-yl)-2-propanone 1e Figure S37. 13 C-spectrum of 1-(7-methylindol-3-yl)-2-propanone 1e 26

Figure S38. 1 H-spectrum of (S)-1-(indol-3-yl)-2-aminopropane 2a Figure S39. 13 C-spectrum of (S)-1-(indol-3-yl)-2-aminopropane 2a 27

Figure S40. 1 H-spectrum of (R)-1-(5-hydroxy-indol-3-yl)-2-aminopropane 2b Figure S41. 13 C-spectrum of (R)-1-(5-hydroxy-indol-3-yl)-2-aminopropane 2b 28

Figure S42. 1 H-spectrum of (R)-1-(5-methyl-indol-3-yl)-2-aminopropane 2c Figure S43. 13 C-spectrum of (R)-1-(5-methyl-indol-3-yl)-2-aminopropane 2c 29

Figure S44. 1 H-spectrum of (R)-1-(6-methoxy-indol-3-yl)-2-aminopropane 2d Figure S45. 13 C-spectrum of (R)-1-(6-methoxy-indol-3-yl)-2-aminopropane 2d 30

Figure S46. 1 H-spectrum of (R)-1-(7-methyl-indol-3-yl)-2-aminopropane 2e Figure S47. 13 C-spectrum of (R)-1-(7-methyl-indol-3-yl)-2-aminopropane 2e 31

Figure S48. 1 H-spectrum of (1S,3S)-3-methyl strictosidine 3a Figure S49. 13 C-spectrum of (1S,3S)-3-methyl strictosidine 3a 32

Figure S50. COSY-spectrum of (1S,3S)-3-methyl strictosidine 3a Figure S51. HSQC-spectrum of (1S,3S)-3-methyl strictosidine 3a 33

C3 wb_emf_stric-un 1 2 3 4 5 f1 (ppm) 6 7 8 9 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure S52. NOESY-spectrum of (1S,3S)-3-methyl strictosidine 3a Figure S53. 1 H-spectrum of (1S,3R)-3-methyl-8-hydroxy strictosidine 3b 34

Figure S54. 13 C-spectrum of (1S,3R)-3-methyl-8-hydroxy strictosidine 3b Figure S55. COSY-spectrum of (1S,3R)-3-methyl-8-hydroxy strictosidine 3b 35

Figure S56. HSQC-spectrum of (1S,3R)-3-methyl-8-hydroxy strictosidine 3b Figure S57. NOESY-spectrum of (1S,3R)-3-methyl-8-hydroxy strictosidine 3b 36

Figure S58. 1 H-spectrum of (1S,3R)-3,10-methyl strictosidine 3e Figure S59. 13 C-spectrum of (1S,3R)-3,10-methyl strictosidine 3e 37

Figure S60. COSY-spectrum of (1S,3R)-3,10-methyl strictosidine 3e Figure S61. HSQC-spectrum of (1S,3R)-3,10-methyl strictosidine 3e 38

Figure S62. NOESY-spectrum of (1S,3R)-3,10-methyl strictosidine 3e 4 Abbreviations Sext = sextett, MeOH = methanol, NaOH = sodium hydroxyd, HCl = hydrochloric acid, TEA = triethylamine, TFA = trifluoro acetic acid, FA = formic acid, TLC = thin layer chromatoraphy. 5 References (1) Nichols, D. E.; Lloyd, D. H.; Johnson, M. P.; Hoffmann, A. J. J. Med. Chem. 1988, 31, 1406-1412. 39