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1 doi: /nature Supplementary Methods 2. Supplementary Text 2.1 Physico-Chemical Properties and Structural Elucidation of Compound Physico-Chemical Properties and Structural Elucidation of Compound Physico-Chemical Properties and Structural Elucidation of Compound Physico-Chemical Properties and Structural Elucidation of Compound Physico-Chemical Properties and Structural Elucidation of Compound Physico-Chemical Properties and Structural Elucidation of Compound Physico-Chemical Properties and Structural Elucidation of PPL. 3. Supplementary Figures Supplementary Figure 1. NMR spectra of compound 1. Supplementary Figure 2. NMR spectra of compound 2. Supplementary Figure 3. NMR spectra of compound 3. Supplementary Figure 4. NMR spectra of compound 4. Supplementary Figure 5. NMR spectra of compound 5. Supplementary Figure 6. NMR spectra of compound 6. Supplementary Figure 7. NMR spectra of PPL. Supplementary Figure 8. NMR spectra of GlcN-Ins. Supplementary Figure 9. NMR spectra of compound 10. Supplementary Figure H NMR spectra of compound 11. Supplementary Figure H NMR spectra of MSH. 4. Supplementary Tables Supplementary Table 1. NMR spectroscopic data for compound 1. Supplementary Table 2. NMR spectroscopic data for compound 2. Supplementary Table 3. NMR spectroscopic data for compound 3. Supplementary Table 4. NMR spectroscopic data for compound 4. Supplementary Table 5. NMR spectroscopic data for compound

2 Supplementary Table 6. NMR spectroscopic data for compound 6. Supplementary Table 7. NMR spectroscopic data for PPL. 5. Supplementary References 2

3 1. Supplementary Methods Chemical Synthesis of MSH The synthesis of MSH was carried out according to the procedures described previously 1,2, making use of the available synthetic intermediate GlcN-Ins (described above). Compound 9 was commercially available, and compounds GlcN-Ins, 10, 11 and MSH have been characterized previously. For GlcN-Ins, 1 H NMR (500 MHz, D 2 O) δ 5.41 (d, J = 3.3 Hz, 1H), 4.19 (app s, 1H), 3.92 (t, J = 10.1 Hz, 1H), (m, 2H), (m, 2H), 3.67 (dd, J = 10.0 Hz, 1.9 Hz, 1H), 3.67 (t, J = 9.7 Hz, 1H), 3.52 (dd, J = 10.1, 2.1 Hz, 1H), 3.48 (t, J = 9.6 Hz, 1H), 3.34 (dd, J = 10.7, 3.4 Hz, 1H), 3.29 (t, J = 9.4 Hz, 1H); 13 C NMR (125 MHz, D 2 O) δ 97.2, 78.9, 74.1, 72.7, 71.9, 71.8, 71.6, 70.9, 69.5, 69.4, 60.2, ESI-HR-MS Calcd. for C 12 H 25 NO [M+H] +, found Preparation of the Synthetic Intermediate 10. A solution of 283 mg (0.64 mmol) of N,N -di-tert-butoxycarb-onyl-l-cysteine (compound 9) in 4.5 ml of acetic acid was treated with 417 mg (6.42 mmol) of zinc dust in four portions over a 1-h period. The solution was then filtered and concentrated after additional stirring for 2 h. The oily residue was then dissolved in 1.4 ml of acetic anhydride and 2.0 ml of a 1.0 N solution of cold aqueous potassium hydrogen carbonate. After stirring for 0.5 h at 2 C, the solution was diluted with water and washed with dichloromethane. The aqueous solution was then acidified to ph 2.0 with 6 N sulfuric acid, and extracted with dichloromethane. The organic layer was concentrated and subjected to further separation on silica gel using 15:1 dichloromethane/methanol as the eluent. For the purified 3

4 compound 10, 1 H NMR (500 MHz, CDCl 3 ) δ 8.70 (br s, 1H), 5.34 (d, J = 7.4 Hz, 1H), (m, 1H), 3.44 (dd, J = 14.2, 3.1Hz, 1 H), 3.29 (dd, J = 14.0, 6.5Hz, 1H), 2.34 (s, 3H), 1.42 (s, 9H); 13 C NMR (125 MHz, CDCl 3 ) δ 195.5, 174.1, 155.6, 80.6, 54.0, 53.2, 31.1, 31.0, 30.5, 28.2; ESI-HR-MS Calcd. for C 10 H 17 NO 5 SNa [M+Na] +, found Preparation of the Synthetic Intermediate 11. A solution of 4.0 mg (0.012 mmol) of GlcN-Ins and 6.0 mg (0.023 mmol) of 10 in 0.5 ml of DMF was treated at 0 C with 8.7 mg (0.023 mmol) of HATU and 6.1 µl (0.035 mmol) of diisopropylethylamine. After stirring at room temperature for 12 h, the reaction mixture was concentrated under reduced pressure, and further purification of product 11 was carried out on an Aglient Zorbax column (SB-C18, 5 µm, mm, Agilent Technologies Inc., USA) by gradient elution of solvent A (H 2 O) and solvent B (CH 3 CN) at a flow rate at 2 ml/min (mau at 230 nm) over a 30-min period as follows: T = 0 min, 5% B; T = 5 min, 5% B; T = 20 min, 50% B; T = 25 min, 50% B; T = 28 min, 5% B; and T = 30 min, 5% B. For the purified compound 11, 1 H NMR (500 MHz, D 2 O) δ 5.17 (d, J = 2.4Hz, 1H), 4.36 (dd, J = 7.8, 4.9Hz, 1H), 4.22 (app s, 1H), 3.98 (dd, J = 10.6, 2.9 Hz, 1H), (m, 2 H), (m, 3 H), 3.64 (t, J = 9.8 Hz, 1H), (m, 1H), 3.54 (dd, J = 9.9, 2.6 Hz, 1H), 3.50 (t, J = 9.6 Hz, 1H), 3.44 (dd, J = 13.9, 3.9 Hz, 1H), 3.30 (t, J = 9.4 Hz, 1H), 3.15 (dd, J = 14.3, 8.5 Hz, 1H), 2.42 (s, 3H), 1.46 (s, 9H); ESI-HR-MS Calcd. for C 22 H 39 N 2 O 14 S [M+ H] +, found Preparation of MSH. A solution of 1.0 mg (1.7 µmol) of 11 in 0.75 ml of cold trifluoroacetic acid was stirred for 15 min and then concentrated under reduced pressure. The colorless residue was then treated with 60 µl of pyridine. After stirring for 30 min, the mixture was concentrated, and then azeotroped several times with water and toluene. For the purified compound MSH, 1 H NMR (500 MHz, D 2 O) δ 5.16 (d, J = 3.6 Hz, 1H), 4.55 (t, J = 6.4, 5.8 Hz, 1H), 4.21 (t, J = 2.5 Hz, 1H), 4.00 (dd, J = 10.7, 3.6 Hz, 1H), (m, 2H), (m, 1H), 3.64 (t, J = 10.5 Hz, 1H), 3.61 (dd, J = 10.2, 2.5 Hz, 1H), 3.54 (dd, J = 9.4, 2.6 Hz, 1H), 3.50 (t, J = 9.6 Hz, 1H), 3.31 (t, J = 9.4 Hz, 1H), 2.97 (dd, J = 14.1, 5.3 Hz, 1H), 2.92 (dd, J = 14.1, 6.9 Hz, 1H), 2.09 (s, 3H); ESI-HR-MS Calcd. for C 17 H 31 N 2 O 12 S [M+H] +, found

5 2. Supplementary Text 2.1 Physico-Chemical Properties and Structural Elucidation of Compound 1 Compound 1 was purified as yellowish white amorphous solid: UV (H 2 O) λ max 210 nm; 1 H and 13 C NMR(500 and 125 MHz, respectively, D 2 O) see Supplementary Table 1; ESI-HR-MS Calcd. for C 33 H 59 N 4 O 18 S [M+H] +, found The molecular formula of 1 was established to be C 33 H 58 N 4 O 18 S by analysis of its HR-ESI-MS, 13 C NMR and DEPT spectra. The 13 C NMR and DEPT spectra of 1 indicated that the molecular has three amide carbon atoms, three methyl carbon atoms, six methylene carbon atoms and twenty one methine carbon atoms. The planar structure of 1 was established by detailed analysis of its 1D and 2D NMR spectra ( 1 H, 13 C, DEPT, HSQC and HMBC), and by comparison of the 1 H and 13 C NMR spectroscopic data of 1 with those of lincomycin A and MSH 1-4 (Supplementary Table 1). The moieties A and B of 1 shared the similar NMR spectra with Lincomycin A and MSH, respectively. The major difference in moiety A compared with lincomycin A was the absence of N-methyl and S-methyl group signals. The absence of a S-methyl group signal and the obvious downfield shift of C3'' in 13 C NMR spectra (34.6 ppm in B moiety and 28.5 ppm in MSH) suggested the attachment of moiety B onto moiety A via a C-S bond linkage between C3'' and the sulfur atom. This S-linkage was further supported by HMBC couplings of H3'' (moiety B) to C1 (moiety A). The anomeric configuration of C1 (moiety A) of 1 was determined to be α-orientation, according to the observed coupling constant ( 3 J H,H = 5.6 Hz) similar to that in lincomycin A ( 3 J H,H = 5.8 Hz). The absolute configuration of 1 was suggested according to those of lincomycin A and MSH. 2.2 Physico-Chemical Properties and Structural Elucidation of Compound 2 5

6 Compound 2 was purified as yellowish white amorphous solid: UV (H 2 O) λ max 210 nm; 1 H and 13 C NMR (500 and 125 MHz, respectively, D 2 O) see Supplementary Table 2; ESI-HR-MS Calcd. for C 21 H 38 N 3 O 9 S [M+H] +, found The molecular formula C 21 H 37 N 3 O 9 S was established by analysis of the ESI-HR-MS, 13 C NMR and DEPT spectra, which supported that 2 was the N-Acetyl-cysteine S-conjugate. The planar structure and absolute configuration of 2 were established by detailed analysis of its 1D and 2D NMR spectra ( 1 H, 13 C, DEPT, HSQC and HMBC), and by comparison of its 1 H and 13 C NMR spectroscopic data with those of 1 (Supplementary Table 2). 2.3 Physico-Chemical Properties and Structural Elucidation of Compound 3 Compound 3 was purified as white amorphous solid: UV (H 2 O) λ max 253 nm; 1 H and 13 C NMR (500 and 100 MHz, respectively, D 2 O) see Supplementary Table 3; 31 P NMR (162 MHz, D 2 O) δ (d, 19.4 Hz), (d, 20.6Hz); ESI-HR-MS Calcd. for C 18 H 31 N 6 O 16 P [M+H] +, found The molecular formula C 18 H 30 N 6 O 16 P 2 was established by analysis of the ESI-HR-MS and 13 C NMR spectra, which was consistent with the expectation that 3 was the GDP-octose. The planar structure and absolute configuration of 3 were established by detailed analysis of its 1D and 2D NMR spectra ( 1 H, 13 C, HSQC and HMBC), and by comparison of its 1 H and 13 C NMR spectroscopic data with those of 5 and GDP 5 (Supplementary Table 3). 6

7 2.4 Physico-Chemical Properties and Structural Elucidation of Compound 4 Compound 4 was purified as yellowish white amorphous solid: UV (H 2 O) λ max 243 nm; 1 H and 13 C NMR (500 and 125 MHz, respectively, D 2 O) see Supplementary Table 4; ESI-HR-MS Calcd. for C 25 H 44 N 5 O 8 S [M+H] +, found The molecular formula of 4 was established to be C 25 H 43 N 5 O 8 S by analysis of its ESI-HR-MS, 13 C NMR and DEPT spectra. The 13 C NMR and DEPT spectra of 4 indicated that the molecule has four quaternary carbon atoms (two carbonyls and two olefinic carbon atoms), eleven methine carbon atoms (including one olefinic carbon atoms), five methylene carbon atoms and three methyl carbon atoms. The 13 C NMR spectra of 4 showed only twenty three carbon signals, indicating that this chemical has three identical carbons, which were subsequently identified as methyl groups [δ H 3.2(9H, s)] attached to electron-withdrawing groups. The planar structure of 4 was established by detailed analysis of its 1D and 2D NMR spectra ( 1 H, 13 C, DEPT, HSQC and HMBC), and by comparison of the 1 H and 13 C NMR spectroscopic data of 4 with those of lincomycin A and EGT 6 (Supplementary Table 4). The moieties A and B of 4 shared similar NMR spectra with Lincomycin A and EGT, respectively. The major difference in moiety A compared to Lincomycin A was the absence of signals for the N-methyl and S-methyl groups. The absence of a S-methyl group signal and the marked HMBC correlations of H1 (A moiety) to C2'' (B moiety) suggested the attachment of moiety B onto moiety A via a C-S bond between C2'' and the sulfur atom. The anomeric configuration of C1 (A moiety) of 4 was determined to be β-orientation, according to the observed coupling constant ( 3 J H,H = 9.3 Hz) larger than that in lincomycin A ( 3 J H,H = 5.8 Hz), and the presence of obvious NOESY correlations of H1to H3 and H5. The absolute configuration of 4 was suggested according to those of lincomycin A and EGT. 2.5 Physico-Chemical Properties and Structural Elucidation of Compound 5 7

8 Compound 5 was purified as yellowish white amorphous solid: UV (H 2 O) λ max 243 nm; 1 H and 13 C NMR (500 and 125 MHz, respectively, D 2 O) see Supplementary Table 5; ESI-HR-MS Calcd. for C 17 H 31 N 4 O 7 S [M+H] +, found The molecular formula C 17 H 30 N 4 O 7 S was established by analysis of its ESI-HR-MS, 13 C NMR and DEPT spectral data. The 13 C NMR and DEPT spectra of 5 indicated that the molecule has three quaternary carbon atoms (one carbonyl and two olefinic carbon atoms), nine methine carbon atoms (including one olefinic carbon atom), one methylene carbon atom and two methyl carbon atoms. The planar structure of 5 was established by detailed analysis of its 1D and 2D NMR spectra ( 1 H, 13 C, DEPT, HSQC and HMBC), and by comparison of the 1 H and 13 C NMR spectroscopic data of 5 with those of 4 (Supplementary Table 5). The major difference of 5 compared with 4 was the absence of NMR signals for PPL (moiety A). The anomeric configuration of C1 (moiety A) of 5 was determined to be β-orientation, according to the observed coupling constant ( 3 J H,H = 9.8 Hz) as that in 4 ( 3 J H,H = 9.3 Hz), and the presence of obvious NOESY correlations of H1 to H3 and H5. The absolute configuration of 5 was suggested according to those of lincomycin A and EGT. 2.6 Physico-Chemical Properties and Structural Elucidation of Compound 6 Compound 6 was purified as yellowish white amorphous solid: UV (H 2 O) λ max 210 nm; 1 H and 13 C NMR (500 and 125 MHz, respectively, D 2 O) see Supplementary Table 6; ESI-HR-MS Calcd. for C 25 H 46 N 3 O 17 S [M+H] +, found The molecular formula was established to 8

9 be C 25 H 45 N 3 O 17 S by analysis of its ESI-HR-MS, 13 C NMR and DEPT spectral data. The 13 C NMR and DEPT spectra of 6 indicated that the molecule has two amide carbon atoms, nineteen methine carbon atoms, two methylene carbon atom and two methyl carbon atoms. The planar structure of 6 was established by detailed analysis of its 1D and 2D NMR spectra ( 1 H, 13 C, DEPT, HSQC and HMBC), and by comparison of the 1 H and 13 C NMR spectroscopic data of 6 with those of 1 (Supplementary Table 6). The major difference of 6 compared with 1 was the absence of NMR signals for PPL (A moiety). The anomeric configuration of C1 (moiety A) of 6 was determined to be -orientation, according to the observed coupling constant ( 3 J H,H = 5.6 Hz) as the same as that of 1. The absolute configuration of 6 was suggested according to those of lincomycin A and MSH. 2.7 Physico-Chemical Properties and Structural Elucidation of PPL Compound PPL was purified as yellowish white amorphous solid: UV (H 2 O) λ max 210 nm; 1 H and 13 C NMR(400 and 100 MHz, respectively, D 2 O) see Supplementary Table 7; ESI-HR-MS Calcd. + for C 8 H 16 NO [M+H] +, found The molecular formula was established to be C 8 H 15 NO 2 by analysis of its ESI-HR-MS, 13 C NMR and DEPT spectral data. The planar structure of PPL was elucidated by detailed analysis of its 1D and 2D NMR spectra( 1 H, 13 C, DEPT, HSQC and HMBC) (Supplementary Table 7). This compound has been previously characterized

10 3. Supplementary Figures Supplementary Figure 1. NMR spectra of compound 1. (a) 1 H NMR spectrum. (b) 13 C and DEPT 135 spectrum. (c) 1 H- 1 H COSY spectrum. (d) HSQC spectrum. (e) HMBC spectrum. (f) NOESY spectrum. a 10

11 b 11

12 c d 12

13 e f 13

14 Supplementary Figure 2. NMR spectra of compound 2. (a) 1 H NMR spectrum. (b) 13 C NMR spectrum. (c) 1 H- 1 H COSY spectrum. (d) HSQC spectrum. (e) HMBC spectrum. (f) NOESY spectrum. a b 14

15 c d 15

16 e f 16

17 Supplementary Figure 3. NMR spectra of compound 3. (a) 1 H NMR spectrum. (b) 13 C NMR spectrum. (c) 1 H- 1 H COSY spectrum. (d) HSQC spectrum. (e) HMBC spectrum. (f) NOESY spectrum. (g) 31 P NMR spectrum. a b 17

18 c d 18

19 e f 19

20 g 20

21 Supplementary Figure 4. NMR spectra of compound 4. (a) 1 H NMR spectrum. (b) 13 C and DEPT 135 spectrum. (c) 1 H- 1 H COSY spectrum. (d) HSQC spectrum. (e) HMBC spectrum. (f) NOESY spectrum. a 21

22 b 22

23 c d 23

24 e f 24

25 Supplementary Figure 5. NMR spectra of compound 5. (a) 1 H NMR spectrum. (b) 13 C and DEPT 135 spectrum. (c) 1 H- 1 H COSY spectrum. (d) HSQC spectrum. (e) HMBC spectrum. (f) NOESY spectrum. a 25

26 b 26

27 c d 27

28 e f 28

29 Supplementary Figure 6. NMR spectra of compound 6. (a) 1 H NMR spectrum. (b) 13 C and DEPT 135 spectrum. (c) 1 H- 1 H COSY spectrum. (d) HSQC spectrum. (e) HMBC spectrum. (f) NOESY spectrum. a 29

30 b 30

31 c d 31

32 e f 32

33 Supplementary Figure 7. NMR spectra of PPL. (a) 1 H NMR spectrum. (b) 13 C and DEPT 135 spectrum. (c) 1 H- 1 H COSY spectrum. (d) HSQC spectrum. (e) HMBC spectrum. (f) NOESY spectrum. a 33

34 b 34

35 c d 35

36 e f 36

37 Supplementary Figure 8. NMR spectra of GlcN-Ins. (a) 1 H NMR spectrum. (b) 13 C and DEPT 135 spectrum. a b 37

38 Supplementary Figure 9. NMR spectra of compound 10. (a) 1 H NMR spectrum. (b) 13 C NMR spectrum. a b 38

39 Supplementary Figure H NMR spectra of compound

40 Supplementary Figure H NMR spectra of MSH. 40

41 4. Supplementary Tables Supplementary Table 1. NMR spectroscopic data for compound 1 a. position δ H (mult., J in Hz) δ C mutl. position δ H (mult., J in Hz) δ C mutl. Moiety A Moiety B (1H, d, 5.6) 89.8 CH (1H, d, 3.5) CH (1H, m) 70.2 CH (1H, dd, 3.8, 56.7 CH 10.4) (1H, m) 72.9 CH (1H, m) 73.5 CH (1H, d, 2.2) 71.0 CH (1H, t, 9.5) 72.7 CH (1H, m) 72.3 CH (1H, m) 75.2 CH (1H, dd, 4.4, 56.4 CH 6a 3.91 (1H, m) 63.3 CH 2 9.2) (1H, m) 69.1 CH 6b 3.83 (1H, m) (3H, d, 6.3) 18.8 CH 3 1' 3.63 (1H, m) 81.8 CH 2' 4.54 (1H, dd, 4.8, 62.3 CH 2' 4.24 (1H, m) 74.4 CH 9.0) 2'-C=O C 3' 3.57 (1H, m) 73.7 CH 3'a 2.35 (1H, m) 38.4 CH 2 4' 3.67 (1H, m) 74.7 CH 3'b 2.17 (1H, m) 5' 3.35 (1H, t, 9.4) 76.9 CH 4' 2.42 (1H, m) 39.4 CH 6' 3.82 (1H, m) 74.8 CH 5'a 3.66 (1H, m) 53.7 CH 2 1'' C=O 5'b 3.00 (1H, m) 2'' 4.64 (1H, t, 6.8) 56.4 CH 6' 1.47 (2H, m) 36.1 CH 2 3''a 3.11 (1H, dd, 6.6, 34.6 CH ) 7' 1.39 (2H, m) 23.2 CH 2 3''b 3.03 (1H, m) 8' 0.94 (3H, t, 7.3) 15.8 CH 3 4'' C=O 5'' 2.17 (3H, s) 24.5 CH 3 a In D 2 O, 500MHz for 1 H and 125MHz for 13 C NMR; Chemical shifts are reported in ppm. All signals are determined by 1 H - 1 H COSY, HSQC, HMBC and NOESY correlation. 41

42 Supplementary Table 2. NMR spectroscopic data for compound 2 a. position δ H (mult., J in Hz) δc mutl (1H, d, 5.7) 89.9 CH (1H, dd, 5.9, 10.4) 70.3 CH (1H, m) 72.8 CH (1H, d, 2.9) 71.1 CH (1H, d, 9.3) 72.2 CH (1H, dd, 4.8, 9.5) 56.4 CH (1H, m) 69.0 CH (3H, d, 6.5) 18.6 CH 3 2' 4.54 (1H, dd, 4.5, 9.4) 62.2 CH 2'-C=O C 3'a 2.34 (1H, m) 38.3 CH 2 3'b 2.18 (1H, m) 4' 2.42 (1H, m) 39.4 CH 5'a 3.68 (1H, m) 53.6 CH 2 5'b 3.01 (1H, m) 6' 1.47 (2H, m) 36.1 CH 2 7' 1.39 (2H, m) 23.2 CH 2 8' 0.94 (3H, t, 7.2) 15.8 CH 3 1'' C 2'' 4.45 (1H, dd, 5.3, 6.9) 57.5 CH 3''a 3.11 (1H, dd, 4.4, 12.8) 35.6 CH 2 3''b 3.06 (1H, dd, 7.1, 13.5) 4'' C 5'' 2.10 (1H, s) 24.6 CH 3 a In D 2 O, 500MHz for 1 H and 125MHz for 13 C NMR; Chemical shifts are reported in ppm. All signals are determined by 1 H - 1 H COSY, HSQC, HMBC and NOESY correlation. 42

43 Supplementary Table 3. NMR spectroscopic data for compound 3 a. position δ H (mult., J in Hz) δc (J in Hz) mutl. Moiety A [1H, dd, 3.6 (J H,H ), 6.7 (J H,P )] 98.4 [d, 5.9 (J C,P )] CH (1H, m) 70.5 [d, 8.3 (J C,P )] CH (1H, dd, 3.0, 9.6) 71.6 [d, 8.2 (J C,P )] CH (1H, m) 71.8 [d, 5.4 (J C,P )] CH (1H, d, 5.9) 70.0 CH (1H, t, 5.7) 59.4 CH (1H, m) 67.3 CH (3H, d, 6.5) 20.7 CH 3 Moiety B C C C C (1H, s) CH 1' 5.98 (1H, d, 6.1) 89.4 CH 2' 4.81 (1H, m) 76.3 CH 3' 4.56 (1H, t, 4.0) 73.1 [d, 8.0 (J C,P )] CH 4' 4.40 (1H, brs) 86.4 [d, 8.7 (J C,P )] CH 5' 4.26 (2H, m) 68.0 [d, 5.5 (J C,P )] CH 2 a In D 2 O, 500MHz for 1 H and 100MHz for 13 C NMR; Chemical shifts are reported in ppm. All signals are determined by 1 H - 1 H COSY, HSQC, HMBC and NOESY correlation. 43

44 Supplementary Table 4. NMR spectroscopic data for compound 4 a. position δ H (mult., J in Hz) δc mutl. Moiety A (1H, d, 9.3) 89.8 CH (1H, m) 72.3 CH (1H, m) 76.4 CH (1H, app s) 70.4 CH (1H, d, 10.0) 79.6 CH (1H, dd, 3.9, 9.9) 55.1 CH (1H, m) 68.7 CH (3H, d, 6.5) 17.8 CH 3 2' 4.49 (1H, dd, 4.4, 9.2) 61.9 CH 2'-C=O C 3'a 2.30 (1H, m) 38.2 CH 2 3'b 2.13 (1H, m) 4' 2.38 (1H, m) 39.2 CH 5'a 3.63 (1H, m) 53.3 CH 2 5'b 3.00 (1H, dd, 9.8, 10.9) 6' 1.43 (2H, m) 35.9 CH 2 7' 1.35 (2H, m) 23.0 CH 2 8' 0.91 (3H, t, 7.2 ) 15.7 CH 3 Moiety B 2'' C 4'' C 5'' 7.17 (1H, s) CH 6'' 3.27 (2H, m) 27.8 CH 2 7'' 3.93 (1H, m) 80.7 CH 8'' C 9'' 3.30 (3H, s) 54.6 CH 3 a In D 2 O, 500MHz for 1 H and 125MHz for 13 C NMR; Chemical shifts are reported in ppm. All signals are determined by 1 H - 1 H COSY, HSQC, HMBC and NOESY correlation. 44

45 Supplementary Table 5. NMR spectroscopic data for compound 5 a. position δ H (mult., J in Hz) δc mutl. Moiety A (1H, d, 9.8) 89.8 CH (1H, t, 9.6) 71.8 CH (1H, dd, 3.2, 9.5 ) 76.1 CH (1H, d, 2.7) 70.9 CH (1H, d, 7.4) 76.8 CH (1H, m) 57.9 CH (1H, m) 66.8 CH (3H, d, 6.6) 19.3 CH 3 Moiety B 2' C 4' C 5' 7.07(1H, s) CH 6' 3.17(2H, m) 27.9 CH 2 7' 3.84(1H, dd, 4.2, 11.1) 80.9 CH 8' C 9' 3.21(3H, s) 54.7 CH 3 a In D 2 O, 500MHz for 1 H and 125MHz for 13 C NMR; Chemical shifts are reported in ppm. All signals are determined by 1 H - 1 H COSY, HSQC, HMBC and NOESY correlation. 45

46 Supplementary Table 6. NMR spectroscopic data for compound 6 a. position δ H (mult., J in Hz) δc mutl. Moiety A (1H, d, 5.6) 90.8 CH (1H, m) 69.8 CH (1H, dd, 3.2, 10.4) 72.2 CH (1H, d, 2.8) 71.4 CH (1H, d, 5.6) 69.6 CH (1H, t, 6.0) 59.5 CH (1H, m) 67.1 CH (3H, d, 6.4) 20.7 CH 3 Moiety B (1H, d, 3.8) CH (1H, dd, 3.7, 10.7) 56.6 CH (1H, m) 73.3 CH (1H, t, 9.7) 72.6 CH (1H, m) 75.1 CH 6a 3.80 (1H, m) 63.1 CH 2 6b 3.89 (1H, m) 1' 3.62 (1H, dd, 2.7, 10.0) 81.8 CH 2' 4.22 (1H, t, 2.7) 74.3 CH 3' 3.55 (1H, dd, 2.7, 9.9) 73.6 CH 4' 3.65 (1H, t, 9.7) 74.7 CH 5' 3.32 (1H, t, 9.4) 76.9 CH 6' 3.80 (1H, t, 9.5) 74.7 CH 1'' C 2'' 4.58 (1H, dd, 5.2, 8.9) 56.9 CH 3''a 3.00 (1H, dd, 9.3, 14.3) 35.3 CH 2 3''b 3.16 (1H, dd, 5.1, 14.3) 4'' C 5'' 2.10 (3H, s) 24.4 CH 3 a In D 2 O, 500MHz for 1 H and 125MHz for 13 C NMR; Chemical shifts are reported in ppm. All signals are determined by 1 H - 1 H COSY, HSQC, HMBC and NOESY correlation. 46

47 Supplementary Table 7. NMR spectroscopic data for PPL a. position δ H (mult., J in Hz) δ C mutl (1H, dd, 3.8, 9.4) 63.5 CH 2-C=O C 3a 2.29 (1H, m) 37.4 CH 2 3b 1.97 (1H, m) (1H, m) a 3.59 (1H, dd, 7.6, 11.4) 53.3 CH 2 5b 2.92 (1H, dd, 8.9, 11.4) (2H, m) 36.2 CH (2H, m) 23.2 CH (3H, t, 7.3) 15.8 CH 3 a In D 2 O, 500MHz for 1 H and 125MHz for 13 C NMR; Chemical shifts are reported in ppm. All signals are determined by 1 H - 1 H COSY, HSQC, HMBC and NOESY correlation. 47

48 5. Supplementary References 1. Lee, S. & Rossaza, J. P. First total synthesis of mycothiol and mycothiol disulfide. Org. Lett. 6, (2004). 2. Ajayi, K., Thakur, V. V., Lapo, R. C. & Knapp, S. Intramolecular α-glucosaminidation: synthesis of mycothiol. Org. Lett. 12, (2010). 3. Moloney, G. P., Craik, D. J., Iskander, M. N. & Munro, S. L. A. 1 H N.M.R. and theoretical studies of the conformation of the antibiotic lincomycin. Aust. J. Chem.43, (1990). 4. Verdier, L., Bertho, G., Gharbi-Benarous, J. & Girault, J-P. Lincomycin and clindamycin conformations. A fragment shared by macrolides, ketolides and lincosamides determined from TRNOE ribosome-bound conformations. Bioorg. Med. Chem. 8, (2000). 5. Davisson, J., Davis, D. R., Dixit, V. M. & Poulter, C. D. Synthesis of nucleotide 5'-diphosphates from 5'-O-tosyl nucleosides. J. Org. Chem. 52, (1987). 6. Xu, J.-Z. & Yadan, J. C. Synthesis of L-(+)-ergothioneine. J. Org. Chem. 60, (1995). 7. Koskinen, Ari M. P. & Rapoport, H. Synthesis of 4-substituted prolines as conformationally constrained amino acid analogues. J. Org. Chem. 54, (1989). 48