Supplementary Information. Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds

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1 Supplementary Information Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds Yu-Chen Liu 1,2,5, Yi-Shan Li 1, Syue-Yi Lyu 1,4, Li-Jen su 1, Yu-ou Chen 1,3, Yu-Ting uang 1, siu-chien Chan 1, Chuen-Jiuan uang 1, Gan-ong Chen 1, Chia-Cheng Chou 6, Ming-Daw Tsai 1,3,5 & Tsung-Lin Li 1,7* 1 Genomics Research Center, Academia Sinica, Taipei 115, Taiwan. 2 Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Biochemistry, Academia Sinica, Taipei 115, Taiwan. 3 Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan. 4 Department of Microbiology and Immunology, ational Yang-Ming University, Taipei 112, Taiwan. 5 Institute of Biochemical Science, ational Taiwan University, Taipei 115, Taiwan. 6 ational Synchrotron Radiation Research Center, sinchu 3, Taiwan. 7 Biotechnology Center, ational Chung sing Univerisity, Taichung 42, Taiwan. * To whom correspondence should be sent. tlli@gate.sinica.edu.tw This PDF file includes: Supplementary Methods Supplementary Results (Supplementary Figures 1-19, Supplementary Tables 1-4) Supplementary References 1

2 Supplementary Methods Cloning and protein purification. Protein expression, purification and confirmation of purity were performed as described previously 1-4. The dbv29 gene was amplified from genomic DA by PCR using the first two primers shown in the Primer list below. The products were each ligated according to the manufacturers' instruction (ovagene) into the pet-28a(+) expression vector which provides an -terminal is 6 -tagged protein. E. coli cells were grown at 37 C in 1 L of LB medium until an A 6 of about.7 was reached. Protein expression was induced with.2 mm IPTG at 16 C, and, after 12 h, the cells were harvested by centrifugation, resuspended in 1 mm imidazole-cl buffer, p 7.8 (binding buffer), and ruptured using a microfluidizer. The cell-free extract prepared by centrifugation was applied to an i 2+ -TA resin column, which was then washed successively with binding buffer and wash buffer before eluting the target protein with 2 mm imidazole-cl. Gel filtration was performed using an Ä kta FPLC system equipped with an S-2 Superdex column (Amersham Bioscience) under isocratic conditions (2 mm Tris, p 7.6, 1 mm KCl). The buffer was exchanged for 5 mm EPES buffer, p 7.2 using Millipore centrifugal filters. Protein concentrations were estimated using the Bradford assay. The purified protein was confirmed by SDS-PAGE, Western blotting, and electrospray mass spectrometry (ESI-MS). Primer list (primers used in this study) Mutants Dbv29-f Dbv29-r Dbv29-C26A-f Dbv29-C26A-r Dbv29-C92A-f Dbv29-C92A-r Dbv29-C151A-f Dbv29-C151A-r Dbv29-C161A-f Dbv29-C161A-r Dbv29-91A-f Dbv29-91A-r Dbv29-I41A-f Primer AAA TTT CAT ATG ACC GGC GGC ACC GGA GCG GGG CCC CTC GAG TCA GGG CCG GAT CGA CAA CGC CCG GAG CTG CGG GGC GAG CGC GCC TTA CCG CCG GCC GGC CCG GTC GAC CGG GCC GGC CGG CGG TAA GGC GCG CTC GCC CCG CAG CTC CGG GCC GTC CGC AGC GGT GGG CAC GCT TTC GAG GAC TTC GTC GAC AAC GTT GTC GAC GAA GTC CTC GAA AGC GTG CCC ACC GCT GCG GAC GGC GTG ACC ATA CCG GGT GGG GTC GCC GGC GGG GTC GGC GTC GGC GGA TCC GCC GAC GCC GAC CCC GCC GGC GAC CCC ACC CGG TAT GGT CAC GTC GGC GTC GGC GGA CAC ATC GCC GGA GGC GGG TAC GGC CCG CTG CAG CGG GCC GTA CCC GCC TCC GGC GAT GTG TCC GCC GAC GCC GTC GTC CGC AGC GGT GGG GCC TGT TTC GAG GAC TTC GAA GTC CTC GAA ACA GGC CCC ACC GCT GCG GAC GGC GCC GTC TGG CTG GCC GGC TAC GGC GGG AAG 2

3 Dbv29-I41A-r Dbv29-I41W-f Dbv29-I41W-r Dbv29-44A-f Dbv29-44A-r Dbv29-R41A-f Dbv29-R41A-r Dbv29-R36E-f Dbv29-R36E-r Dbv29-R36L-f Dbv29-R36L-r Dbv29-S364K-f Dbv29-S364K-r Dbv29-S364R-f Dbv29-S364R-r Dbv29-T366A-f Dbv29-T366A-r Dbv29-T366E-f Dbv29-T366E-r Dbv29-T366L-f Dbv29-T366L-r Dbv29-W399A-f Dbv29-W399A-r Dbv29-W399F-f Dbv29-W399F-r Dbv29-Y135F-f Dbv29-Y135F-r Dbv29-Y165F-f Dbv29-Y165F-r Dbv29-Y165W-f Dbv29-Y165W-r Dbv29-Y37F-f Dbv29-Y37F-r CTT CCC GCC GTA GCC GGC CAG CCA GAC GGC GCC GGC GCC GTC TGG CTG TGG GGC TAC GGC GGG AAG CTT CCC GCC GTA GCC CCA CAG CCA GAC GGC GCC GAC CCG CGC TAC CTC GCC CTG AAG CTG CGT GGC GCC ACG CAG CTT CAG GGC GAG GTA GCG CGG GTC ACT CCG GAC GAC CCG GCT TAC CTC AAC CTG AAG CTT CAG GTT GAG GTA AGC CGG GTC GTC CGG AGT CCG GGG CGA GGA GGC GAA GGC CCG GCG TCG AAG CTT CGA CGC CGG GCC TTC GCC TCC TCG CCC CGG CCG GGG CGA GGA GGC CTG GGC CCG GCG TCG AAG CTT CGA CGC CGG GCC CAG GCC TCC TCG CCC CGG GGC AGG GGC CCG GCG AAA AAG ACG AAA GCC GGC GCC GGC TTT CGT CTT TTT CGC CGG GCC CCT GCC GGC AGG GGC CCG GCG AGG AAG ACG AAA GCC GGC GCC GGC TTT CGT CTT CCT CGC CGG GCC CCT GCC GGC CCG GCG TCG AAG GCC AAA GCC GGC TAC CTG CAG GTA GCC GGC TTT GGC CTT CGA CGC CGG GCC GGC CCG GCG TCG AAG GAA AAA GCC GGC TAC CTG CAG GTA GCC GGC TTT TTC CTT CGA CGC CGG GCC GGC CCG GCG TCG AAG CTG AAA GCC GGC TAC CTG CAG GTA GCC GGC TTT CAG CTT CGA CGC CGG GCC GAC TAC GGC GCC GTC GCC CTG ATC GGC TAC GGC GCC GTA GCC GAT CAG GGC GAC GGC GCC GTA GTC GAC TAC GGC GCC GTC TTC CTG ATC GGC TAC GGC GCC GTA GCC GAT CAG GAA GAC GGC GCC GTA GTC ACG CTC TCA GAG GTG TTC GAA AAG CTC TAC CTG CAG GTA GAG CTT TTC GAA CAC CTC TGA GAG CGT TGC GGA GGC GGG TTC GGC CCG CTG TCA TGA CAG CGG GCC GAA CCC GCC TCC GCA ATC TGC GGA GGC GGG TGG GGC CCG CTG TCA CGG CCG TGA CAG CGG GCC CCA CCC GCC TCC GCA GAT ACG AAA GCC GGC TTC CTG CGC AAG CGG CTG CAG CCG CTT GCG CAG GAA GCC GGC TTT CGT 3

4 Dbv29-Y43F-f Dbv29-Y43F-r Dbv29-Y428F-f Dbv29-Y428F-r Dbv29-Y453F-f Dbv29-Y453F-r Dbv29-Y47F-f Dbv29-Y47F-r Dbv29-Y473F-f Dbv29-Y473F-r Dbv29-Y473E-f Dbv29-Y473E-r GTC TGG CTG ATC GGC TTC GGC GGG AAG GTG AAC GTT CAC CTT CCC GCC GAA GCC GAT CAG CCA GAC ATA CTC AAG GTG AAC TTC ATC ACC GGT TGG GCG CGC CCA ACC GGT GAT GAA GTT CAC CTT GAG TAT CTC TAT GCC GAT GTG TTC GCC GAG ACC GGC GGG CCC GCC GGT CTC GGC GAA CAC ATC GGC ATA GAG GTC AGC GAT GGG GCG TTC ATC AAT TAC CCC GAC GTC GGG GTA ATT GAT GAACGC CCC ATC GCT GAC GGG GCG TAC ATC AAT TTC CCC GAC AGC GAC CTC GAG GTC GCT GTC GGG GAA ATT GAT GTA CGC CCC GGG GCG TAC ATC AAT GAA CCC GAC AGC GAC CTC GAG GTC GCT GTC GGG TTC ATT GAT GTA CGC CCC Synthetic conditions for new analogs. Teicoplanin (a mixture of five analogs) was purchased from AAPI Chemicals Ltd (UK). The mixture was subjected to PLC purification to obtain C 1 -Tecicoplanin with purity >95%. 5-Azide pentylamine (99%) was obtained from Chung-Yi Wu (Academia Sinica) as a gift. All other chemicals were obtained from Sigma/Aldrich Chemical Co. (St. Louis, M, USA) without further purification unless otherwise stated. Teicoplanin analogs (2-6) were enzymatically synthesized as described previously 1-4. In brief, compound 2 was prepared by adding Dbv21 or rf2* (1 g) into a typical buffer solution (5 mm EPES, p 7.2, 1 mm acl, 1 mm DTT) containing Tei (1, 1 mm) for 5 h at 37 C; compounds 3-6 were prepared by adding Dbv8 (1 g) into the same buffer solution but containing compound 2 and butanoyl-, hexanoyl-, octanoyl-, or decanoyl-coa for compounds 3-6, respectively, at 25 C overnight. For the final reaction, Teicoplanin analogs (9, 1, 11, 25 and 32, which showed relatively higher yields from initial tests) were enzymatically synthesized using the optimized conditions described below. Enzyme stability (and, indirectly, the extent of exposure of the aldehyde for functionalization) was tested in an array of organic solvents (MeC, Me, Et, DF, DMF, DCM, DMS etc.); DMS turned out to be the most appropriate solvent as Dbv29 is highly stable in up to 9% DMS. 5% DMS, however, is included in the current protocol as it gives relatively higher yields. ther conditions, such as the amounts of alkylamine and reductant 5, were also optimized one at a time against the fixed concentration of Tei (.5 mm) for a better yield (below). In general, 1-fold of alkylamine and the reducing agent versus Tei was determined to be most appropriate for the current protocol, which is summarized as follows: Tei (.5 mm), alkylamine (5 mm; carbon number >6), and a(c)b 3 (5 mm) in 5% DMS buffer solution at 4

5 mau mau 37 C overnight incubation. 1 Fixed acb 3 (5 mm) and Tei (.5 mm) 2 Fixed acb 3 (5 mm) and Tei (.5 mm) mau (1x Tei) mau (1x Tei) octylamine (mm) benzylamine (mm) 8 Fixed octylamine (5 mm) and Tei (.5 mm) 2 Fixed benzylamine (5 mm) and Tei (.5 mm) 6 4 (1x Tei) 15 1 (1x Tei) acb 3 (mm) acb 3 (mm) A 1-fold ratio of alkylamine (5 mm) and acb 3 (5 mm) to Tei (.5 mm) proved sufficiently optimized for the octylamine-tei derivative (left) and the benzylamine-tei derivative (right), although higher the concentrations of either reagent provided marginally higher yields. Compound characterization. All compounds were characterized by MS and PLC, which can be found in Supplementary Figure 11. For select compounds, MR analyses were performed on a Bruker Avance 6 spectrometer equipped with CryoProbe, with tetramethylsilane (TMS) as an internal standard. Compounds were dissolved in deuterated dimethyl sulfoxide (DMS-d 6 ) if not otherwise stated, and spectra were recorded at room temperature. MR peaks are listed below; original spectra can be found in Supplementary Figures Teicoplanin (1). RMS ES(+): [M++a] 2+, calc. for C Cl a [M++a] 2+ (Supplementary Figure 19). 1 -MR (6 Mz, DMS-d 6 ) (Supplementary Figure 12): 8.43 (s, 1), 7.75 (m, 1), 7.5 (m, 1), 7.34 (m, 2), 7.21 (m, 1), 7.13 (d, J 8.3, 1), 7.5 (d, J 8.3, 1), 6.91 (m, 2), 6.84 (m, 1), 6.77 (m, 1), 6.68 (m, 2), 6.43 (s, 1), 6.23 (s, 1), 5.7 (s, 1), 5.55 (m, 1), 5.46 (m, 2), 5.36 (m, 1), 5.28 (d, J 8.4, 1), 5.21 (s, 1), 4.5 (m, 2), 4.2 (s, 1), 4.5 (m, 1), 3.9 (m, 2), 3.7 (m, 8), 3.52 (m, 2), 3.41 (m, 4), 3.7 (m, 1), 2.17 (m, 2), 2. (s, 2), 1.55 (m, 2), 1.42 (m, 1), 1.15 (m, 8),.77 (m, 6). 13 C MR (6 Mz, Methanol-d 4 ): 178.1, 177.9, , , , , 5

6 171.9, 17.93, , 16.77, , 158.5, , 155.4, , 153.9, , 15.99, 15.14, , , , , , , , , 13.94, , 129.5, 128.7, , , 126.5, , , 123.2, , 121., , , 11.98, 11.34, 19.31, 17.11, 15.82, 14.25, 12.97, 11.25, 97.82, 78.92, 77.48, 77.37, 74.81, 74.24, 71.66, 71.5, 71.6, 67.41, 63.63, 62.9, 61.71, 6.6, 58.78, 57.53, 57.27, 56.66, 55.33, 49.71, 49.58, 49.44, 49.3, 49.15, 49.1, 48.87, 48.72, 46.83, 39.8, 37.59, 37.1, 3.21, 3.9, 28.74, 27.98, 26.55, 23.4, 23.21, An expanded version of SQC and MBC spectra of the reference compound is shown in Supplementary Figure 12. Compound 2. RMS ES + : [M+2] 2+, calc. for C Cl [M+2] 2+ (Supplementary Figure 19). 1 -MR (6 Mz, DMS-d 6 ) (Supplementary Figure 13): 9.66 (m, 2), 9.32 (m, 1), 8.7 (m, 1), 7.91 (m, 2), 7.77 (m, 2), 7.6 (m, 1), 7.31 (m, 1), 7.15 (m, 6), 6.97 (m, 2), 6.71 (m, 4), 6.45 (m, 3), 6.31 (m, 1), 5.26 (m, 6), 5.8 (m, 4), 4.91 (m, 1), 4.64 (m, 1), 4.38 (m, 7), 3.78 (m, 4), 3.4 (m, 5), 2.87 (m, 4), 1.89 (m, 3), 1.26 (m, 1), 1.15 (m, 1). 13 C MR (6 Mz, DMS-d 6 ): , , , , , 168.9, , , , 158.8, , 158.6, , , , , , , , , , , 15.1, , 149.2, , , , , , 126.2, 125.7, , 12.88, 12.84, 12.22, 12.5, , , 16.6, 98.8, 97.46, 96.58, 78.1, 77.9, 77.1, 76.92, 75.71, 73.77, 73.62, 72.99, 72.9, 72.53, 7.71, 7.64, 7., 69.9, 69.81, 66., 65.94, 63.1, 61.23, 61.17, 6.98, 6.85, 6.76, 59.32, 57.66, 57.42, 56.89, 56.3, 56.1, 55.99, 54.74, 45.57, 23.24, An expanded version of SQC, MBC and 1 CSY spectra of the reference compound is shown in Supplementary Figure 13. Compound 9. RMS ES (+): [M++a] 2+, calc. for C Cl a [M++a] 2+ (Supplementary Figure 19). 1 -MR (6 Mz, DMS-d 6 ) (Supplementary Figure 14): 8.45 (s, 1), 7.72 (s, 1), 7.51 (m, 1), 7.33 (m, 2), 7.21 (m, 2), 7.7 (m, 2), 6.93 (m, 3), 6.84 (d, J 8.4, 1), 6.77 (m, 2), 6.62 (m, 3), 6.42 (s, 1), 6.19 (s, 1), 5.71 (s, 1), 5.55 (s, 1), 5.46 (m, 4), 5.3 (s, 1), 5.18 (s, 2), 4.47 (m, 4), 4.2 (s, 1), 4.9 (m, 1), 3.91 (m, 1), 3.7 (m, 1), 3.51 (m, 2), 3.41 (m, 3), 3.11 (m, 1), 2.22 (m, 2), 2.3 (m, 3), 1.55 (m, 2), 1.41 (m, 1), 1.27 (m, 3), 1.14 (m, 5), 1.4 (m, 2),.87 (m, 1),.77 (m, 6). 13 C MR (6 Mz, Methanol-d 4 ): 22.76, 22.2, , , , , 172.2, , 171.9, 171.3, 17.87, , 16.82, , 158.2, , 155.2, , , 152.3, 15.97, 15.47, , , , 139.2, 6

7 136.5, , , , 132.8, 13.84, , 129.6, , 127.9, , , 125.3, , 123.1, , , , , 116.2, 111.8, 11.31, 19.87, 17.17, 15.77, 13.38, 12.95, 11.17, 97.8, 78.83, 77.35, 74.85, 74.22, 73.75, 71.62, 71.51, 71.1, 67.41, 63.59, 61.73, 58.79, 57.68, 57.59, 57.32, 57.13, 56.8, 56.56, 55.29, 39.81, 37.59, 37.3, 3.27, 3.15, 3.1, 29.99, 28.77, 28.5, 26.67, 23.42, 23.21, 23.18, An expanded version of SQC and MBC spectra of the reference compound is shown in Supplementary Figure 14. Compound 1. RMS ES (+): [M+2] 2+, calc. for C Cl [M+2] 2+ (Supplementary Figure 19). 1 MR (6 Mz, DMS-d 6 ): 7.71 (m, 1), 7.16 (m, 3), 5.32 (m, 1), 4.9 (m, 1), 4.26 (m, 1), 4.13 (m, 1), 3.67 (m, 1), 3.57 (m, 1), 3.43 (m, 1), 3.33 (m, 3), 3.6 (m, 3), 2.88 (m, 1), 2.52 (m, 4), 2.1 (m, 2), 1.88 (m, 1), 1.26 (m, 23),.82 (m, 9). 13 C MR (6 Mz, DMS-d 6 ): , 158.8, , , , , , 13.13, 13.6, , , 12.37, , , , 96.92, 92.25, 76.87, 76.78, 74.85, 73.79, 73.1, 72.52, 72.39, 72., 7.69, 7.6, 7.31, 69.8, 67.44, 63.1, 61.23, 61.5, 6.96, 56.6, 38.1, 35.14, 31.31, 31.25, 3.93, 3.1, 29.82, 29.15, 29.12, 29.6, 28.91, 28.86, 28.75, 28.72, 28.61, 28.59, 28.4, 27.41, 26.72, 26.63, 26.59, 25.14, 25.7, 22.59, 22.44, 22.13, 18.59, 14., An expanded version of SQC and MBC spectra of the reference compound is shown in Supplementary Figure 15. Compound 25. RMS ES(+): [M+2] 2+, calc. for C Cl [M+2] 2+ (Supplementary Figure 19). 1 -MR (6 Mz, DMS-d 6 ) (Supplementary Figure 16): 9.67 (m, 2), 8.61 (m, 1), 8.4 (m, 1), 7.93 (m, 2), 7.77 (m, 2), 7.18 (m, 14), 6.71 (m, 3), 6.48 (m, 2), 6.23 (m, 1), 5.24 (m, 7), 4.96 (m, 1), 4.62 (m, 1), 4.39 (m, 5), 4.8 (m, 1), 3.7 (m, 2), 3. (m, 4), 2.14 (m, 1), 1.99 (m, 1), 1.86 (m, 3), 1.48 (m, 2), 1.37 (m, 1), 1.15 (m, 12),.84 (d, J 6.5, 6). 13 C MR (6 Mz, DMS-d 6 ): , , , , , , , , , , , , , , , 158., 157.8, , , , , , , , , , , 13.21, 13.9, , , 129.2, , , 128.8, , 125.2, 12.79, 12.74, 12.2, , , , 19.2, 98.91, 96.62, 76.95, 76.83, 73.8, 73.73, 72.85, 72.8, 72.71, 72.56, 72.44, 72.2, 7.76, 7.68, 7.9, 69.81, 66.2, 63.13, 61.3, 61.2, 61.8, 6.76, 56.58, 56.38, 56.19, 55.77, 54.57, 51.74, 51., 5.16, 35.98, 31.37, 29.23, 29.1, 29.2, 28.97, 28.92, 28.87, 28.8, 27.47, 27.4, 26.76, 26.71, 26.67, 25.11, 24.97, 23.25, 23.17, 22.65, 22.18,14.9. An expanded version of SQC, MBC and 1 CSY spectra of 7

8 the compound 25 is shown in Supplementary Figure 16. For selected 1 MR assignment see Supplementary Figure 18 and Supplementary Table 4. Compound 32 (de-r4-tei). RMS ES(-): [M-2] -2, calc. for C Cl [M-2] -2 (Supplementary Figure 19). 1 -MR (6 Mz, DMS-d 6 ) (Supplementary Figure 17): 9.68 (m, 2), 9.27 (m, 1), 8.65 (m, 1), 8.53 (s, 1), 8.42 (s, 1), 7.92 (m, 2), 7.8 (m, 1), 7.26 (m, 3), 7.18 (m, 2), 7.1 (m, 4), 6.97 (m, 1), 6.75 (m, 3), 6.46 (m, 2), 6.23 (m, 1), 5.27 (m, 2), 5.13 (m, 2), 4.42 (m, 2), 4.34 (m, 1), 4.28 (d, J 11.5, 1), 4.12 (m, 1), 3.6 (m, 2), 2.95 (m, 1), 1.88 (m, 2), 1.26 (m, 1). 13 C MR (6 Mz, DMS-d 6 ): , , , , , , , , , , , , , , , 158.8, , , , , 155.9, 15.27, 15.23, 149.6, , , , , , , , , , , 126.2, , , , 12.71, 12.19, 12.8, , 116.3, 15.66, 99.26, 99.13, 97.52, 96.65, 76.91, 76.81, 73.8, 73.66, 73., 72.55, 7.59, 7., 69.92, 69.81, 65.99, 65.92, 63.11, 61.13, 6.75, 59.25, 56.12, 56.7, 55.86, 55.79, 55.17, 54.59, 23.21, An expanded version of SQC, MBC and 1 CSY spectra of the reference compound is shown in Supplementary Figure 17. Antibiotic assays. MIC assays were performed as described previously 1. In vivo study. 6-8 ICR female mice were purchased from the ational Laboratory Animal Breeding and Research Center, Taipei, Taiwan. Mice with average body weight of 27 to 3 g were subjected to infection via intravenous (i.v.) injection with cfu/mouse of E. faecalis (ATCC 51559) at day. For treatment study, mice were randomized into four groups at the start of the experiment and administered 1 mg/kg of either vancomycin, teicoplanin, benzylamine-teicoplanin (25) or saline (control) by i.v. twice a day for three days (from day 1 to day 3, a total of 6 doses). Mice were subjected to anesthesia; whole blood was then sampled from orbital sinus on day 1, day 2, and day 3. The whole blood underwent serial dilutions with PBS, which were plated on Brain eart infusion agar (BI agar; Difico, Detroit, MI, USA) for enumeration of cfu (colony formation unit). The graphs and statistical analyses were performed using SigmaPlot and SigmaStat. Differences were considered significant if the P value was <.5. 8

9 Supplementary Results Supplementary table 1. Data collection and refinement statistics. Data collection Dbv29 Dbv29/Teicoplanin Space group P P2 1 Cell dimensions a, b, c (Å ) 66.9, 66.9, , , ( ) 9., 9., , 91.3, 9. Resolution (Å ) ( ) * ( ) R merge 8.3 (16.) 8.8 (63.) I / I 24.8 (17.) 22.5 (2.5) Completeness (%) 95.7 (92.) 98.9 (96.7) Redundancy 9.7 (9.8) 7.3 (6.4) Refinement Resolution (Å ) o. reflections R work / R free.237/ /.252 o. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å ).5.15 Bond angles ( ) The number of crystals is indicated in parentheses for the following data sets: Dbv29 (2) and Dbv29/Teicoplanin (1). *Values in parentheses are for highest-resolution shell. 9

10

11 Supplementary figure 1. Multiple sequence alignments for Dbv29 and homologues. Residues that covalently link to FAD are highly conserved; 91 and C151 of Dbv29 are marked on the top of sequences. Residues corresponding to Tyr-473 are conserved in Dbv29, Aknx (Tyr-45) and GX (Tyr-429). otes: 1) In GX 9, Tyr-429 initiates the catalytic activity by proton abstracton that is also facilitated by Asp-355. In Aknx 1, this second residue is Tyr-144 that may coordinate with Tyr-45. In BBE 11, the proton abstraction step is carried out by Glu-417). 2) Accession codes for Dbv29 (Q7WZ62; onomuraea sp. ATCC39727), Aknx (QPCD7; Streptomyces galilaeus), GX (Q6PW77; Acremonium strictum) and BBE (P3986; Eschscholzia califomica) are designated by the UniProtKB databank ( 3) Identical residues are colored in red, similar residues are colored in yellow. 11

12 12 F-domain S-domain Subdomain 1 Subdomain 2 B1 B2 B3 B4 B5 B6 B7 B8 B9 B1 B11 B12 B13 B14 B15 B a

13 9 12 B13 1 B1 B12 13 B11 11 B15 B16 B B6 B5 B9B7 B8 7 2 B3 B4B2 B1 2 b 13

14 FAD 91 c C151 14

15 d 15

16 e 16

17 f 17

18 Dbv29 Aknx C RMSD.734 g Supplementary figure 2. verall structure of Dbv29 and comparisons to Aknx. (a) Dbv29 can be dissected into two domains, F and S. The F and S domains consist of residues 1 23 and , respectively, in which the F domain can be further divided into two subdomains. (b) For the F domain, the -terminal subdomain (subdomain 1, residues 1-15) consists of a central four-stranded -sheet (B1-B4 with the strand order of 1, 2, 4, 3, wherein B1 runs antiparallel to others), sandwiched on each side by one -helix (1 and 2); the second subdomain (subdomain 2, resides and ) consists of a -sheet of five antiparallel strands (B5-B9 with the strand order of 5, 6, 9, 7, 8) that faces against five irregular -helices (3, 4, 6, 7 and 2). For the S domain, this domain is composed of an extended antiparallel seven-stranded -sheet (B1-B16 with the strand order of 13, 1, 12, 11, 15, 16, 14), faced by four major -helices in a row (12, 1, 13, and 16). These two domains are joined by two long loop regions (residues and ). (c) The 2F o -F c electron density map of FAD contoured at 1.. (d) The isoalloxazine ring of FAD is situated at the interface of the flavin- and substrate-binding domains, while the ADP-ribosyl part is packed into a deep pocket between the two subdomains 18

19 of the F-domain. (e) The lipid cavity mainly is formed by residues Phe-93, Ser-364, Tyr-395, Trp-399 and Ile-429. (f) The residues for FAD binding are highly conserved (framed in blue), while the residues for substrate binding account for the major variations between the proteins (framed in black). The tyrosine pair (Y165/Y473) is conserved in both Dbv29 and Aknx, while Aknx also need a second general base pair (W399 and I41). (g) Dbv29 and Aknx were superimposed with the rmsd of.734 Å for 48 Cα, which suggests both tertiary structures are similar but with variations in the substrate-binding domain framed in red. 19

20 Dimeric form a 2

21 Monomeric form b 21

22 Dbv29 (dimer) FAD c 22

23 Tei-bound Dbv29 (monomer) FAD diol-tei (8) d 23

24 native complex Ca RMSD.291 e 24

25 f L Å R Å 3.2 Å 3.81 Å 3.72 Å 44 Y139 R Å L351 Supplementary figure 3. Interfaces for and structural analysis of dimeric and monomeric Dbv29. Analyses of native (a) and ligand-bound (b) structures of Dbv29 with PISA 12 showed that the interfaces for the native and complex structures are 135 and 3 Å, respectively. These results agree with that the native and complex structures are dimeric and monomeric, respectively. The analysis statistics for each protein is demonstrated on bottom panel. (c) The dimer of Dbv29 in a P space group (two polypeptide chains in an asymmetric unit); FAD cofactors are represented as stick models. (d) Monomer of Dbv29 in complex with a water-coordinated-diol intermediate (also see Fig. 2c) in a P2 1 space group (an asymmetric unit consists of 25

26 four polypeptide chains in which there are no typical protein-protein interactions with one another); the FAD cofactors and the Tei intermediate are represented as stick models in orange and blue, respectively. (e) Superposition of the ligand-free and ligand-bound structures. Substrate-free and -bound structures were superimposed with the rmsd of.291 Å for 498 Cα, which suggests binding of ligand does not induce substantial conformational changes. (f) Residues Arg41 (forming two hydrogen bonds) and Asn44 (four hydrogen bonds) were identified as potentially significant residues in the dimer interface, and were investigated further by site-directed mutagenesis (Supplementary Table 2) and AUC analysis (Supplementary figure 7 m, n). (Protein interfaces, surfaces and assemblies were analyzed using the PISA server at the European Bioinformatics Institute ( 26

27 a 27

28 diol-tei (8) b 28

29 Y395 W399 M34 G35 E36 Q294 Q314 Q348 R357 W35 P355 S353 L351 c 29

30 R357 G356 P355 S353 M34 S364 Y395 C151 V15 L351 Q348 F93 W399 Q294 G149 P167 Q314 W35 Y473 Y165 W292 L168 d 3

31 Y165 FAD I41 Y473 W399 F93 T366 I429 S364 Y395 e 31

32 Y135 Y43 Y165 Y Å Y47 Y37 Y37 Y473 f g Y428 T Å R36 Y453 h i Supplementary figure 4. Substrate binding site. (a) Residues that line the ligand-binding site are located in different loops highlighted in orange (residues , , 3-38, , and ). (b) The 2F o -F c electron density map of the diol intermediate contoured at 1.. (c,d) Surface (c) and ball-and-stick (d) illustrations of hydrophobic interactions (residues and surface are colored according to their hydrophobicity; blue: positive charge; red: negative charge; white: neutral) and hydrogen bonds (yellow dashed line) with the heptapeptide backbone, indicating the major interacting forces between the aglycone and substrate-binding-site residues. (e) View of the lipid tunnel in Dbv29, in which the -acyl moiety of the aminosugar at r4 is inserted into a hydrophobic cavity that is mainly composed of residues Phe93, Ser364, Thr366, Tyr395, Trp399 and Ile429 (electrostatic surface potentials of negative potential (78 KT) and positive potential (78 KT) are colored in red and blue, respectively; neutral surface potential regions are displayed in white). In particular, Trp399 is nearby the reducing end of the sugar, and, together with Ile41, stands above the shoulder of the r4 sugar head and poises the head facing the isoalloxazine ring. Ile41 stands on the other side of the sugar head, nearby the C6 carbon. Thr366, 32

33 situated opposite Trp399 at the entry of the lipid cavity, may help the lipid chain insert into the lipid cavity (panel d). Ser364, which lines the bottom of the cavity, may force the lipid to fold in a spiral manner. (f-h) Tyrosine residues near the active site that could play a role in enzyme structure or function. (f) Tyr135, Tyr37, Tyr43 and Tyr47 are shown in addition to the previously identified Tyr165 and Tyr473. (g,h). Close up view of two tyrosine pairs, Tyr43/Tyr37 (4.1 Å ) (g) and Tyr428/Tyr453 (2.8 Å ) (h), that are positioned within approximate hydrogen-bond distances of each other. (i) Close up view of two additional residues (Arg36 and Thr366) postulated to play a role in Dbv29 function. Thr366 gates the lipid cavity along with bulky and charged Arg36 that sits on the same long loop. Specific residues in proximity to the ligand, as shown in panels c-i, were subject to mutagenesis to explore these interactions further (see Supplementary Table 2). 33

34 Supplementary Table 2. Relative enzymatic activities of Dbv29 mutants Mutants Expression Flavinylation/ Relative Rationale utcome Source of result FAD binding activity a C26A Putative point of active Role disproven Ref. 2 site general base C92A Putative point of active Role disproven Ref. 2 site general base Putative point of active Role disproven/ Ref. 2 C151A site general base/ Role proven Putative point of covalent attachment C161A Putative point of active Role disproven Ref. 2 site general base 91A Putative point of Role proven Ref. 2 covalent attachment I41A Potential Role proven This work ligand-binding residue I41W Potential Role proven This work ligand-binding residue 44A Potential interface Role proven This work interacting residue R41A Potential interface Role proven This work interacting residue R36E Putative point affecting Role disproven This work FAD attachment R36L Putative point affecting Role disproven This work FAD attachment Potential Role proven/ This work S364K ligand-binding residue/ Role disproven Putative point affecting FAD attachment Potential Role proven/ This work S364R ligand-binding residue/ Role disproven Putative point affecting FAD attachment Potential Role proven/ This work T366A ligand-binding residue/ Role disproven Putative point affecting FAD attachment Potential Role proven/ This work T366E ligand-binding residue/ Role disproven Putative point affecting FAD attachment Potential Role proven/ This work T366L ligand-binding residue/ Role disproven Putative point affecting FAD attachment W399A Potential Role proven This work ligand-binding residue W399F Potential Role proven This work ligand-binding residue Y135F Putative point affecting ot conclusive This work FAD attachment Y165F Putative point of active Role proven Ref. 2 site general base Y165W Putative point of active Role proven This work site general base 34

35 Y37F Y43F Y428F Y453F Y47F Y473F Y473E A/C151A R41A/44A R36E/T366E R36L/T366A R36L/T366E Y165F/Y473F Y428F/Y453F Y37F/Y43F Putative point affecting FAD attachment Putative point affecting FAD attachment Putative point affecting FAD attachment Putative point affecting FAD attachment Putative point affecting FAD attachment Putative point of active site general base Putative point of active site general base Putative point of covalent attachment Potential interface interacting residue Putative point affecting FAD attachment Putative point affecting FAD attachment Putative point affecting FAD attachment Putative point of active site general base Putative point affecting FAD attachment Putative point affecting FAD attachment Role disproven Role disproven Role disproven Role disproven Role disproven This work This work This work This work This work Role proven Ref. 2 Role proven This work Role proven Ref. 2 Role proven Role disproven Role disproven Role disproven This work This work This work This work Role proven Ref. 2 Role disproven Role disproven a. The products of mutants were analyzed by PLC; peak areas were integrated and averaged for 3 triplicate points in 1.5 hours; the reaction rates were calculated using linear regression equation; relative activities were determined by dividing individual reaction rate with that of WT; the relative activity of WT is 1.. This work This work 35

36 a 1* 3 6* * 9 19 i ii iii iv v vi vii viii ix x b min 36

37 c k cat (s -1 ) K m ( M -1 ) k cat / K m (s -1 M -1 ) C 2 -pseudo-tei C 1 -pseudo-tei Time (min) KCal/Mole of Injectant cal/sec µcal/sec Model: nesites Chi^2/DoF = 1.634E Sites K 9.42E5 2.24E5 M-1 D E cal/mol DS -219 cal/mol/deg Molar Ratio d Supplementary figure 5. Substrate preferences, kinetics, and thermodynamics for Dbv21, Dbv29, and rf2* (a Dbv21 ortholog in Tei (1) producing strain 3,13,14 ). (a) The reactions catalyzed by each protein. Compounds 9, 16, and 19 are products of Dbv29 when compounds 1, 3, and 6 serves as substrates, respectively. (b) PLC traces for enzymatic reactions of Dbv21 and/or Dbv29 in given conditions (described below; note that compounds 1/6 and 9/19 have the same retention time). i) LC trace of C 4 -Tei (3) that was enzymatically synthesized by Dbv8. ii) LC trace of the reaction catalyzed by Dbv29 in the presence of C 4 -Tei (3). iii) LC trace of the reaction catalyzed by Dbv21 in the presence of 3. iv) LC trace of C 1 -Tei (6) that was enzymatically synthesized by Dbv8. v) LC trace of the reaction catalyzed by Dbv29 in the presence of C 1 -Tei (6). vi) LC trace of the reaction catalyzed by Dbv21 in the presence of C 1 -Tei (6). vii) LC trace of the reaction catalyzed by both Dbv21 and 29 in the presence C 1 -Tei (6); the ratio of products 19 to 2 is about 1, indicating Dbv29 is more efficient than 37

38 Dbv21 when both are included in the same reaction. viii) LC trace of the reaction catalyzed by Dbv29 in the presence of substrates C 4 -Tei (3) and C 1 -Tei (6); the ratio of products 19 to 16 is about 1. ix) LC trace for compound 1 in a solution with no acid* added. x) LC trace for compound 9 and compound 1 in a solution with no acid. Comparing traces ii and v, we see that Dbv29 favors a substrate with a longer acyl side chain (6, C 1 ) to that with a shorter chain (3, C 4 ) by 1-fold. Similarly, comparing traces iii and vi, and the data in panel c, we conclude that Dbv21 also favors longer acyl substrates. The data in traces ix and x explore the role of glycosylation in increasing compound solubility. In acidic conditions (which will protonate carboxylic acid), Tei (1) elutes later than when no acid is added (2.7 min vs. 2.4 min). The solubility of oxo-tei (9) changes more drastically in the absence of acid, eluting at 21.2 minutes with acid (after Tei) and 16.8 minutes with no acid (before Tei). (c) Kinetic data of rf2* against short (C 2 ) and long (C 1 ) lipid chain substrates. The results show C 1 is about 1-fold higher than C 2 in terms of enzyme specificity (k cat /K m ), in which k cat is about the same for both while C 1 has higher substrate affinity. Thus rf2* also prefers a longer chain. (d) ITC thermogram for Dbv29 and Teicoplanin. Each exothermic heat pulse corresponds to injection of 2 l of Tei (1 mm) into the protein solution (.22 mm); integrated heat areas constituted a differential binding curve that was fit to a standard single-site binding model (rigin 7., MicroCal itc 2 ) to give the stoichiometry of binding 15, = 1.5, binding affinity, KA = M -1 (KD = 1 M) and enthalpy of binding, D = -75 kcal mol

39 Supplementary Table 3. Product yields of oxidized, aminated or amidated teicoplanin analogs synthesized by Dbv29. Product Coenzyme A derivative (Carbon o.) 1 Amine added Product type: amide or amine Yield (%) Amine 25 - Amide 1 - Amide <1 - Amide 1 - Amine 25 - Amine 17 C

40 C C C C6 Amide 1 C8 Amide 1 C6 Amine 3 C8 Amine 3 - Amide <1 - Amine 46 4

41 - Amine <5 - Amine <5 - Amine <5 - Amide <5 - Amine <1 - Amine Dbv8 was used to add various lengths of acyl side chains from corresponding coenzyme A derivatives to the C2 amine group. If no CoA derivative is listed, Teicoplanin was used directly. The product yields were determined by PLC; yield = (peak area of aminated or amidated product)/(peak areas of aminated and amidated and oxidized products) 1%. 41

42 Log E.faecium cfu/ml whole blood Van 1 25 Saline Day a 1 Log E.faecium cfu/ml whole blood Van 1 25 Saline b Supplementary figure 6. Mice infection test for analog 25 in blood bacterial clearance. (a) The plot shows the whole blood bacterial counts of mice infected by E. faecalis (ATCC 51559) and treated with vancomycin (Van, in black) or teicoplanin (1, in red) or analog 25 (in green) or saline (control, in blue) for three days. The bacterial counts for the control are steadily increased over time, while the counts for groups treated with drugs are all suppressed but in various extents. Teicoplanin and analog 25 are more prominent than vancomycin in terms of overall drug efficacy. (b) The bar chart shows the bacterial counts at day 3 (end of treatment). In general, these three drugs all show positive effects against the infection, while only analog 25 displays significant. The asterisk (*) indicates significant difference (P <.5); data were expressed as mean ± SD for a group of three mice) 42

43 WT f r =1.33 Dimer s=5.97 Mcal=13.9 (kda) Y165F f r =1.44 Dimer s=5.92 Mcal=19.7 (kda) Y473F f r =1. Dimer s=6. Mcal=98.1 (kda) Y165F/Y473F Monomer s=3.44, 4.12 Mcal=68.9, 64.1 (kda) f r =

44 91A/C151A Dimer s=6.28 Mcal=126.2 (kda) f r =1.44 WT + Tei (1) Monomer s=4.17 Mcal=79.7 (kda) 1 m n R41A/44A Mix of monomer and dimer s=3.9, 4.4 Mcal=67.7, 58.8 (kda) s=6.4 Mcal=95.7 (kda) f r =1.22 Supplementary figure 7. Analytical ultracentrifugation (AUC) analyses for Dbv29 and mutants 16. (a-n) The raw experimental data were analyzed by Sedfit ( and the plots were generated by MATLAB (MathWork, Inc.). The spectra in left panels (a, c, e, g, i, k, m) are the calculated c(s, f r ) distributions that are shown as two-dimensional distribution with grid lines representing the s and f r grid in the thermograph. Below this c(s, f r ) surface a contour plot of the distribution is projected into the s-f r plane, where the magnitude of c(s, f r ) is indicated by contour lines at constant c(s, f r ) in equidistant intervals of c. Contour plots in the right panels (b, d, f, h, j, l, n) are transformed from 44

45 the calculated c(s, f r ) distribution and are shown as c(s, M) distribution. The dotted lines (colored in blue) indicate lines of f r (frictional ratio). The signal of the c(s, M) distribution is indicated by the color temperature. The insert grayscale bars in the right panels indicate the residuals bitmap of each fit. (a, b) Dbv29 WT (dimer, s=5.97 (red dashed line), f r =1.33 (blue dashed line)); (c, d) Dbv29 Y165F (dimer, s=5.92, f r =1.44); (e, f) Dbv29 Y473F (dimer, s=6, f r =1.); (g, h) Dbv29 Y165F/Y473F (monomer, s=3.44, f r =1.66); (i, j) Dbv29 91A/C151A (dimer, s=6.28, f r =1.44); (k, l) Dbv29 WT in the presence of 1 mm Tei (1) (monomer, s=4.17, f r =1.44); (m, n) Dbv29 R41A/44A (monomer, s=3.9, f r =1.22). The wild-type enzyme and the two single mutants (Y165F and Y473F) (a-f) remain associated as dimers as opposed to the double mutant that dissociates into monomers (g, h). The mutant also has a larger frictional ratio (f r ), suggesting its molecular shape has been altered considerably. 45

46 a b Dbv29 Y165F Dbv29 WT ml ml Dbv29 Y473F c ml d Dbv29 Y165F/Y473F e f ml Supplementary figure 8. Gel filtration analyses for wild-type and mutant Dbv29. The wild-type enzyme (a), single mutations Y165F (b) and Y473F (c), and the double mutant 91A/C151A (d) all display sharp and symmetric peaks. Mutations at the interface (R41A/44A) cause a destabilization of dimeric structure, yielding a bimodal distribution (e). The broadened and distorted peak for the Y165F/Y473F double mutant (f) suggests structural heterogeneity. 46

47 4 Mean Residue Ellipticity Wavelength (nm) WT Y165F Y473F Y165F/Y473F 91A/C151A R41A/44A elix Strand Turns Unordered (%) (%) (%) (%) WT Y165F Y473F Y165F/Y473F A/C151A R41A/44A Supplementary figure 9. Circular dichroism (CD) analyses for Dbv29 and mutants. CD spectra of WT and mutants (Y165F, Y473F, Y165F/Y473F, 91A/C151A and R41A/44A), in which Y165F/Y473F and R41A/44A double-mutations are somewhat deviated from others as shown in secondary structure distributions. 47

48 a b Supplementary figure 1. CD analysis of protein stability. (a) The denaturation temperature (T m ) for WT was determined by CD to be 84.7 C, meaning Dbv29 is unusually stable. (b) The denaturation temperature (T m ) for the Y165F/Y473F mutant was determined to be 5.4 C; the protein was significantly destabilized by lacking the two oxygen atoms on the tyrosine pair. Therefore, residues Y165 and Y473 have considerable influences on protein stability. ( T m = 34.3 C) 48

49 RT: uau (2) 2 C Time (min) Cl 2 Cl Chemical Formula: C Cl Exact Mass: L: 2.3E6 Channel B UV orf2-p orf2-P # RT: AV: 395 L: 3.25E5 T: ITMS + c ESI Full ms [5.-2.] (2) i m/z RT: C 4 -Tei (3) L: 1.4E6 Channel B UV CP uau Time (min) 49

50 8619-4CP # RT: AV: 69 L: 2.8E6 T: ITMS + c ESI Full ms [5.-2.] C 4 -Tei (3) ii m/z RT: C 6 -Tei (4) L: 2.12E6 Channel B UV CP uau Time (min) CP # RT: AV: 82 L: 6.19E6 T: ITMS + c ESI Full ms [5.-2.] C 6 -Tei (4) iii m/z 5

51 RT: C 8 -Tei (5) L: 7.47E5 Channel B UV CP uau Time (min) CP # RT: AV: 86 L: 2.35E6 T: ITMS + c ESI Full ms [5.-2.] C 8 -Tei (5) iv m/z RT: C 1 -Tei (6) L: 5.88E5 Channel B UV cp uau Time (min) 51

52 8619-1cp # RT: AV: 58 L: 2.82E6 T: ITMS + c ESI Full ms [5.-2.] C 1 -Tei (6) v m/z RT: aglycone (7) L: 3.46E6 Base Peak m/z= MS teicohydrolysistest Time (min) 9327-teicohydrolysis-test3 # RT: AV: 28 L: 1.6E6 T: ITMS + c ESI Full ms [2.-2.] aglycone (7) vi m/z 52

53 RT: L: 5.56E5 Channel B UV 9714-tei+ dbv29+ dmso+ butylamine uau Time (min) 9714-tei+dbv29+dmso+nacnbh3+butylamine # RT: AV: 8 SB: , L: 4.15E4 F: ITMS + c ESI Full ms [1.-2.] ( a) m/z 9714-tei+dbv29+dmso+butylamine # RT: AV: 9 SB: , L: 9.78E3 F: ITMS + c ESI Full ms [1.-2.] vii m/z 53

54 RT: SM: 15B Tei+Dbv29+ DMS+aCB3+anline Time (min) 977-tei+dbv29+dmso+nacnbh3+anline # RT: AV: 49 L: 1.28E4 T: ITMS + c ESI Full ms [2.-2.] ( a) m/z 977-tei+dbv29+dmso+nacnbh3+anline # RT: AV: 49 L: 6.86E3 T: ITMS + c ESI Full ms [2.-2.] viii m/z 54

55 RT: uau Time (min) L: 8.29E4 Channel B UV 9511-tei+ dbv29+dmso+ nacnbh3+ octyalmine- 1mm-25mm 9612-Tei+Dbv29+DMS+aCB3+octylamine-1mM-ms # RT: AV: 12 SB: , L: 2.85E5 F: ITMS + c ESI Full ms [1.-2.] m/z 9612-Tei+Dbv29+DMS+aCB3+octylamine-1mM-ms # RT: AV: 15 SB: , L: 7.63E3 F: ITMS + c ESI Full ms @pqd2. [5.-2.] ix m/z 55

56 RT: SM: 7G Tei+Dbv29+DMS+ acb3+dodecylamine Time (min) tei+dbv29+dmso+nacnbh3+dodecylamine # RT: AV: 21 SB: , L: 4.45E3 T: ITMS + c ESI Full ms [2.-25.] m/z 977-tei+dbv29+dmso+nacnbh3+dodecylamine # RT: AV: 59 L: 2.1E4 T: ITMS + c ESI Full ms [2.-2.] Chemical Formula: C Exact Mass: x m/z 56

57 uau Time (min) CP+Dbv29 # RT: AV: 16 L: 3.15E5 T: ITMS + c ESI Full ms [5.-2.] xi m/z uau Time (min) 57

58 8619-6cp+dbv29 # RT: AV: 195 L: 3.16E6 T: ITMS + c ESI Full ms [5.-2.] xii C Cl Cl Chemical Formula: C Cl Exact Mass: m/z uau Time (min) cp+dbv29 # RT: AV: 275 L: 1.1E6 T: ITMS + c ESI Full ms [5.-2.] xiii m/z 58

59 uau Time (min) cp+dbv29 # RT: AV: 297 L: 8.25E5 T: ITMS + c ESI Full ms [5.-2.] xiv m/z RT: SM: 5G C Tei+Dbv29+ DMS+aCB3+ hexylamine Time (min)

60 9721-6C-Tei+Dbv29+DMS+aCB3+hexylamine # RT: AV: 4 L: 1.3E4 F: ITMS + c ESI Full ms [1.-2.] m/z C-Tei+Dbv29+DMS+aCB3+hexylamine # RT: AV: 4 SB: , L: 3.15E3 F: ITMS + c ESI Full ms [1.-2.] xv m/z RT: SM: 5G C Tei+Dbv29+ DMS+aCB3+ hexylamine Time (min)

61 9721-8C-Tei+Dbv29+DMS+aCB3+hexylamine # RT: AV: 5 SB: , L: 5.89E3 F: ITMS + c ESI Full ms [1.-2.] m/z C-Tei+Dbv29+DMS+aCB3+hexylamine # RT: AV: 5 SB: , L: 2.69E3 F: ITMS + c ESI Full ms [1.-2.] xvi m/z RT: SM: 5G C Tei+Dbv29+ DMS+aCB3+ octylamine Time (min)

62 9721-6c-tei+dbv29+dmso+nacnbh3+octylamine # RT: AV: 8 SB: , L: 9.66E3 F: ITMS + c ESI Full ms [1.-2.] m/z c-tei+dbv29+dmso+nacnbh3+octylamine # RT: AV: 8 SB: , L: 3.3E3 F: ITMS + c ESI Full ms [1.-2.] xvii m/z RT: SM: 5G C Tei+Dbv29+ DMS+aCB3+ octylamine Time (min) 62