Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 695 Weinheim, 7

2 Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 695 Weinheim, 7 Supporting Information for SorF, A Glycosyltransferase with Promiscuous Donor Substrate Specificity in vitro Maren Kopp, Carsten Rupprath, Herbert Irschik, Andreas Bechthold, Lothar Elling,* and Rolf Müller* TLC analysis The formed sorangiosids were monitored by TLC-analysis. After solid-phase extraction, µl of product sample and µl sorangicin A ( mm, acceptor substrate), respectively, were separated on a RP8 W/UV 5 plate ( x cm, from Macherey- Nagel, Düren, Germany) with 5% (v/v) ammonium acetate (.65 M, ph 6.7), 35% (v/v) acetonitrile and 5% (v/v) methanol as solvent. After 3 h of incubation, the plate was dried by using hot air and then sprayed with Ekkert s reagent (85 ml ethanol, ml conc. acetic acid and.5 ml p-anisaldehyde) and coloured spots were developed for 5 min at C.

3 sorangicin A SorA t h t h Figure S. TLC of the synthesis of with dtdp-glc as donor substrate for SorF. From left to right: sorangicin A, reaction after h and reaction after h incubation time. All samples were purified by solid-phase extraction.

4 sorangicin A SorA t h t 8h Figure S. TLC of the synthesis of using dtdp-rha as donor substrate for SorF. From left to right: sorangicin A, reaction after h and reaction after 8 h incubation. All samples were purified by solid-phase extraction. In Figure S, it is evident that some acceptor substrate remains after 8 h, which agrees with the CE results (9% conversion to, see Figure S). In the case of the (Figure S) a new spot appeared and the substrate spot completely disappeared. When using as substrate dtdp-6-deoxy--keto-α-d-glucose, a new spot appeared while the sorangicin A spot remained visible (5% conversion from CE analysis). The

5 product formed by transfer of galactose showed only a very weak new spot in TLC analysis. The R f values are summarized in Table S. Table S. R f values of different sorangiosids (the R f value for sorangicin A is.8). compound 3 5 R f CE analysis The reactions of SorF with different nucleotide sugars and sorangicin A were monitored by CE via the production of NDP. UDP-D-Glucose dtdp-d-glucose 5 Incubation time / min? Conversion / %? Figure S3. Percentage conversion for synthesis of by SorF with dtdp-glc and UDP-Glc as donor substrates, respectively.

6 9 8 Conversion / %? dtdp-l-rhamnose UDP-D-xylose dtdp-6-deoxy--keto-d-glucose Incubation time / h? Figure S. Percentage conversion for the synthesis of glycosyl-sorangiosids by SorF with different nucleotide sugars, as indicated. The conversion of dtdp-6-deoxy--keto-α-d-glucose stopped at 5% while the conversion to and reached ~9%. HPLC-MS analysis of alternative substrates for SorF Lane numbering for Figures S5 S8: DAD trace, UV absorption at 9 nm (or as indicated in chromatogram) MS, BPC base peak chromatogram, m/z range 3 MS, EIC extracted ion chromatogram (EIC) revealing product peak in sample MS, EIC detection of residual starting material, sorangicin A, in sample 5 MS, EIC authentic sorangicin A reference (m/z [M H] - = 85.6) 6 DAD trace, UV detection of sorangicin A reference compound Note for Tables S S6: MS and MS spectra were recorded in negative ionization mode. Parent ion m/z [M H] values are shown in green. Fragmentation ions shown in red are present in the MS spectra of both starting material and product. Numbers given in black indicate less abundant ions fragments.

7 R-S Intens. 5 MK_3.D: UV Chromatogram, -9 nm x 7 MK_3.D: BPC - -All MS.5. x 7. MK_3.D: EIC All MS.5. x 7 3 MK_3.D: EIC All MS.5. x 7 36 MK_6.D: EIC All MS 5 MK_6.D: UV Chromatogram, 9 nm Time [min] SorA Figure S5. HPLC/MS results for (R-S, m/z 95.7) and sorangicin A (SorA, m/z 85.6). Table S. Fragment ions detected in MS measurements indicated in the chromatograms from Figure S5. MS fragments sorangicin (SorA ) MS fragments (R-S)

8 Intens. Gl-S MK_.D: UV Chromatogram, 9 nm x 6 6 MK_.D: BPC - -All MS x 7 MK_.D: EIC All MS..5. x 5 3 MK_.D: EIC All MS x 7 36 MK_6.D: EIC All MS Time [min] SorA Figure S6. HPLC/MS results for (Gl-S, m/z 967.7) and sorangicin A (SorA, m/z 85.6). Table S3. Fragment ions detected in MS measurements indicated in the chromatograms from Figure S6. MS fragments sorangicin (SorA) MS fragments (Gl-S)

9 6dkG-S Intens. MK_.D: UV Chromatogram, 9 nm 35 3 x 6 MK_.D: BPC - -All MS x 6 MK_.D: EIC All MS 3 3a x 6 3 MK_.D: EIC All MS 3b x 6 MK_.D: EIC All MS 6 x 7 36 MK_6.D: EIC All MS 5 MK_6.D: UV Chromatogram, 9 nm Time [min] SorA Figure S7. HPLC/MS results for 3 (6dkG-S, m/z 99.7) and sorangicin A (SorA, m/z 85.6). Table S: Fragment ions detected in MS measurements indicated in the chromatograms from Figure S7. MS fragments sorangicin (SorA ) MS fragments 3 (6dfkG-S)

10 Ga-S Intens x 6 6 x 5 x MK_.D: UV Chromatogram, 9 nm MK_.D: BPC - -All MS MK_.D: EIC All MS MK_.D: EIC All MS. x 7 36 MK_6.D: EIC All MS MK_6.D: UV Chromatogram, 9 nm Time [min] SorA Figure S8. HPLC/MS results for 5 (Ga-S, m/z 967.7) and sorangicin A (SorA, m/z 85.6). Table S5. Fragment ions detected in MS measurements indicated in the chromatograms from Figure S8. MS fragments sorangicin (SorA ) MS fragments 5 (Ga-S)

11 Xy-S Intens. XYL-SR.D: UV Chromatogram, -9 nm 3 x7 XYL-SR.D: BPC - +All MS.5..5 x6.5 XYL-SR.D: EIC All MS x7 XYL-SR.D: EIC All MS Time [min] Figure S9. HPLC/MS results for (Xy-S, m/z 937.5, lane, and ) and sorangicin A (lane 3, SorA, m/z 85.6). SorA Table S6. Fragment ions detected in MS measurements indicated in the chromatograms from Figure S9. MS fragments sorangicin (SorA ) MS fragments (Xy-S)

12 H H H H H R R : H H H H H H H H H H H H 5 3 H H H H Scheme S. glycosyl-sorangiosids produced in vitro using the promiscuous glycosyltransferase SorF.