In vitro conversion of chanoclavine-i aldehyde to the stereoisomers festuclavine and pyroclavine was controlled by the second reduction step

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1 1 Electronic Supporting Information to: In vitro conversion of chanoclavinei aldehyde to the stereoisomers festuclavine and pyroclavine was controlled by the second reduction step Marco Matuschek a, Christiane Wallwey a, Beate Wollinsky a, Xiulan Xie b and ShuMing Li a * a PhilippsUniversität Marburg, Institut für Pharmazeutische Biologie und Biotechnologie, Deutschhausstraße 17A, D35037 Marburg, Germany. shuming.li@staff.unimarburg.de, Tel: , Fax: b PhilippsUniversität Marburg, Fachbereich Chemie, HansMeerweinStraße, Marburg, Germany.

2 2 Figure S1 HPLC analysis of the cultural extract of P. commune NRRL 2033 (B) by using ergot alkaloids or precursors as standards (A). The substances were detected with a Photodiode Array detector and illustrated for absorption at 282 nm. Table S1 1 HNMR and 13 CNMR data of (8R,9S)fumigaclavine A in protonated form (CD 3 OD) Position δ C δ H, multi., J in Hz HMBC correlation α 4 β 5 β 7 α 7 β 8 β 9 α 10 α , d, , ddd, 1.2, 11.7, , m 3.80, m 3.49, d, , dd, 12.9, , m 5.78, br s 3.73, m 6.76, d, , t, , d, , d, , s 1.88, s C2 to H14 C3 to H2 C5 to H18 C7 to H17, H18 C8 to H17 C9 to H17, H7 α C11 to H13 C12 to H14 C13 to H14 C14 to H12 C15 to H2 C16 to H2, H12, H14 C17 to H7 β C19 to H20 Table S2 NOE contacts for proving the stereochemistry of (8R,9S)fumigaclavine A Protons Strength H5 β to H7 β Medium H10 α to H4 α Medium H9 α to H17 Medium H9 α to H10 α Medium H17 to H10 α Medium H17 to H7 α Medium

3 3 Figure S2.1 1 HNMR spectrum of (8R,9S)fumigaclavine A in protonated form in CD 3 OD

4 4 Figure S2.2 NOESY spectrum of (8R,9S)fumigaclavine A in protonated form in CD 3 OD

5 5 Figure S2.3 Enhanced parts of the HSQC spectrum of (8R,9S)fumigaclavine A in protonated form in CD 3 OD

6 6 Figure S2.4 HMBC spectrum of (8R,9S)fumigaclavine A in protonated form in CD 3 OD

7 7 Figure S3 Analysis of the overproduction and purification of His 6 FgaOx3 pc (lanes 15) and FgaFS pc (lanes 711). The proteins were separated on a 15% SDSpolyacrylamide gel and stained with Coomassie Brilliant Blue R250. Lane 1: Total proteins before induction; 2: Total proteins after induction; 3: Soluble proteins after induction; 4: NiNTA elution of His 6 FgaOx3 pc ; 5: Coresin elution of His 6 FgaOx3 pc ; 6: Molecular weight standard; Lane 7: Total proteins before induction; 8: Total proteins after induction; 9: Soluble proteins after induction; 10: Coresin elution of His 6 FgaFS pc ; 11: NiNTA elution of His 6 FgaFS pc ; 12: Molecular weight standard

8 8 Table S3 1 HNMR and 13 CNMR data of pyroclavine in protonated form (CD 3 OD) Position δ C δ H, multi., J in Hz HMBC correlation α 4 β 5 β 7 α 7 β 8 β 9 α 9 β 10 α , d, , ddd, 14.0, 11.6, , dd, 14.2, **, td, 11.0, , dt, 12.8, *, dd, 12.8, , m 2.63, ddt, 13.7, 3.6, , td, 13.1, *, dt, 4.2, , dd, 7.2, , dd, 8.2, , d, , d, , s C2 to H4 α, H4 β, H12 C3 to H2, H4 α, H4 β C4 to H5, H18 C5 to H4 α, H4 β, H7 α, H9 β, H12, H18 C7 to H17, H18 C8 to H7 α, H7 β, H9 β, H17, H18 C9 to H7 α, H17 C10 to H4 β, H9 β, C11 to H9 β, H10 α, H13 C12 to H2, H13, H14 C13 to H12 C14 to H12 C15 to H2, H13 C16 to H2, H4 β, H12, H14 C17 to H7 β, H9 β * = overlapped signals, J measured from DQFCOSY cross peaks, ** = under the solvent signal Figure S4 NOE contacts (red arrows) for proving the stereochemistry of pyroclavine

9 9 Figure S5.1 1 HNMR spectrum of 4 in protonated form in CD 3 OD

10 10 Figure S CNMR spectrum of 4 in protonated form in CD 3 OD Figure S5.3 DQFCOSY spectrum of 4 in protonated form in CD 3 OD

11 11 Figure S5.4 NOESY spectrum of 4 in protonated form in CD 3 OD

12 12 Figure S5.5 Enhanced parts of the HSQC spectrum of 4 in protonated form in CD 3 OD

13 13 Figure S5.6 HMBC spectrum of 4 in protonated form in CD 3 OD

14 14 Figure S6 HPLC chromatograms of dichloromethane extracts of the incubation mixtures with different enzyme combinations. The reaction mixtures contained 5 mm of the cofactors NAD +, FMN, NADPH, 1 mm chanoclavinei and 5 µg of FgaDH, one old yellow enzyme and one imine reductase and were incubated at 30 C for 15 h. The substances were detected with a Photodiode Array detector and illustrated for absorption at 282 nm. 1: chanoclavinei; 2: chanoclavinei aldehyde; 3: festuclavine; 4: pyroclavine; 5: shunt product Table S4 Conversion of chanoclavinei aldehyde to festuclavine and pyroclavine in the presence of NADPH or NADH. The assays contained 10 µg of the recombinant enzymes each and were incubated at 30 C for 15 h. enzyme combination FgaOx3 pc + FgaFS FgaOx3 pc + FgaFS pc FgaOx3 pc + EasG chanoclavinei consumption (%) incubation with NADPH ratio of products festuclavine pyroclavine (3) (4) chanoclavinei consumption (%) incubation with NADH ratio of products festuclavine pyroclavine (3) (4) FgaOx3 + FgaFS FgaOx3 + FgaFS pc FgaOx3 + EasG