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1 Supporting Information Visible-Light-Induced Specific Desulfurization of Cysteinyl Peptide and Glycopeptide in Aqueous Solution Xiao-Fei Gao, Jing-Jing Du, Zheng Liu and Jun Guo* Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, CCNU-uOttawa Joint Research Centre, College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan, Hubei (P.R. China). Contents 1. General materials and methods... S2 2. General procedures for peptide synthesis... S3 3. General procedure for photoredox desulfurization... S4 4. Desulfurization of cysteine... S5 5. Desulfurization of glutathione (GSH)... S6 6. Synthesis of peptides 10a, 11a, 12a, 13a, 14a, 15a... S7 7. HPLC and ESI-MS spectra of peptides... S10 8. NMR spectra of alanine and 1b... S38 9. Reference... S40 S1

2 1. General Materials and Methods Materials: Rink Amide AM resin, 2-chlorotrityl chloride resin, all Fmoc-amino acids and Boc-amino acids were obtained from GL Biochem (Shanghai), with different side chain protecting group: Arg(Pbf), Asn(Trt), Asp(OtBu), Cys(Acm), Cys(Boc), Cys(Trt), Glu(OtBu), Gln(Trt), His(Boc), Lys(Boc), Ser(tBu), Thr(tBu) Trp(Boc), Tyr(tBu) (Fmoc = 9-Fluorenylmethoxycarbonyl, Boc = tert-butoxycarbonxyl, Pbf = 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl, Trt = Trityl, tbu = tert-butyl, Acm = Acetamidomethyl). Beznotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), N-Methylmorpholine (NMM), 2-(1H-7-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyl-amonium hexafluorophosphate (HATU), and N-Hydroxy-7-azabenzotriazole (HOAt), 1-Hydroxybenzotriazole (HOBt) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), trifluoroacetic acid (TFA), trifluoroethanol (TFE) and triisopropylsilane (TIPS) were obtained from InnoChem Science &Technology Co., Ltd. Unless stated otherwise, all other reagents and solvents were purchased from commercial sources and used without further purification. NMR spectrometry: NMR spectra were recorded on a Varian Mercury Plus 400 MHz. Chemical shifts (δ) are reported in ppm. Mass spectrometry: electrospray ionization mass (ESI-MS) was performed on TSQ Quantum Access MAX (ThermoFisher Scientific). High performance liquid chromatography (HPLC): analytical reversed-phase HPLC was run on an Agilent (1100 series HPLC system) instrument using an analytical column (Agilent mm, 5 μm). Semi-preparative reversed-phase HPLC was run on an Agilent (1100 series HPLC system) instrument using a semi-preparative column (Agilent mm, 5 μm). Linear gradients of acetonitrile B (0.1% TFA) in water A (0.1% TFA) were used for all systems to elute peptides. The flow rates were 1.0 ml/min (analytical) and 4.0 ml/min (semi-preparative). S2

3 2. General Procedures for Peptide Synthesis Proloading Rink amide AM resin: Rink Amide AM resin (0.65 mmol/g) was initially swollen in DCM for 50 min, then washed with DCM (3 3 ml) and DMF (3 3 ml), followed by removal of the Fmoc group with 20% piperidine/dmf (v/v) (3 ml, 3 5 min). Subsequently, the resin was submitted to iterative peptide assembly. 1 Preloading 2-chlorotrityl chloride resin: 2-chlorotrityl chloride resin (1.15 mmol/g) was swollen in dry DCM for 50 min, then washed with DCM (3 3 ml) and DMF (3 3 ml). A solution of Fmoc-AA-OH (3.0 equiv) and N,N-diisopropylethlamine (DIPEA) (6.0 equiv) in DMF was added to the resin and the mixture was bubbling with N 2 at room temperature overnight. The resin was washed with DMF (3 3 ml), DCM (3 3 ml) and DMF (3 3 ml) and subsequently submitted to iterative peptide assembly. 1 Manual solid-phase peptide synthesis: peptides were synthesized manually according to the general procedure for iterative peptide assembly. The general procedures are as follows Removal of Fmoc group: the resin was treated with 20% piperidine/dmf (3 ml, 3 5 min) washed with DMF (3 3 ml), DCM (3 3 ml) and DMF (3 3 ml). 2. Resin tests: a few resin beads were washed with ethanol and transferred to a small glass tube. 2 drops of Kasier test solution were added to the tube and heated to 120 for 5 min. A positive test is indicated by blue or brown resin beads (indicating the removal of N-terminal protecting group). 3. Amino acid coupling: a solution of PyBOP (3.0 equiv), N-methylmorpholine (NMM) (6.0 equiv) and Fmoc-AA-OH (3.0 equiv) in DMF were added to the resin (final concentration 0.1 M) and was bubbling with N 2. After 2 h, the resin was washed with DMF (3 3 ml), DCM (3 3 ml) and DMF (3 3 ml). 4. Cleavage: the cleavage reagent A (TFA/TIPS/H 2 O/EtSH, 90/5/2.5/2.5, v/v/v/v); the cleavage reagent B (DCM/TFE/AcOH, 3/1/1, v/v/v). 5. Work-up procedure: TFA was removed azeotropically with toluene under vaccum. The residue was precipitated with Et 2 O. The resulting solid was ready for purificaiton by semi-preparative HPLC and analysis by ESI-MS spectrometry. S3

4 Table S1 Screening Experiments of Transformation of Cysteinyl Peptide to Alanyl Peptide a entry photocatalyst TPPTS (equiv) solvent sulfur additive yield (%) b Water Water Water - <10 4 c PB DTT PB ME 64 6 d PB TBM 83 7 e PB TBM 82 8 f PB TBM 85 9 g PB TBM 87 Reaction conditions: 1a (20.0 mm), TPPTS, photocatalyst (5 mol %), sulfur additive (80.0 mm), 16 h. b Yield was determined by 1 H NMR. c DTT is 40.0 mm. d Gn HCl (200 mm). e 1a (10.0 mm). f 1a (5.0 mm), 8 h. g 1a (1.0 mm), 5 h. PB = phosphate buffer (ph = 7.4, 200 mm), DTT = dithiothreitol, ME = 2-mercptoethanol, TBM = t-butyl mercaptan, N.D. = not detected. Table S2 Comparison between visible-light-induced and heat-induced radical reaction radical reaction conditions visible-light-induced heat-induced ref substrate concentration (mm) 1-20 < reaction temperature ( ) rt , 5-8 radical initiator (equiv) phosphine (mm) Ru(bpy) 3 Cl VA-044 or V-50 - >5 TPPTS 50 - TCEP - > , 9 4, 5, 9 allowed additives (TBM, DTT, ME) Yes Yes 3, 4, 8 3. General Procedure for Photoredox Desulfurization To a solution of cysteinyl peptide in phosphate buffer (or binary system of phosphate buffer and organic solvent), phosphine, photocatalyst, sulfur additive and guanidine were added. The resulting clear solution was degased with an argon balloon for 15 min and sealed. Then, it was stirred and irradiated by 36 W household at room temperature. The desulfurization product was purified by semi-preparative HPLC and was analyzed by ESI-MS spectrometry. S4

5 4. Desulfurization of Cysteine (A) in D 2 O: to a solution of cysteine (20.0 mm) in D 2 O (2 ml), TPPTS (50.0 mm) and Ru(bpy) 3 Cl 2 (5 mol %) were added. The resulting clear solution was degased with an argon balloon for 15 min and sealed. After the reaction solution was stirred and irradiated with 36 W household bulb for 16 h (Figure S1), it was purified by column chromatography on silylated silica gel to give withe solid (3.0 mg, 84%). 1 H NMR (400 MHz, D 2 O): δ H 3.87 (t, J = 7.7 Hz, 1 H, CH (b 2 )), 1.53 (dt, J = 7.4, 1.8 Hz, 2 H, CH 2 (b 1 )). ESI-MS calcd for C 3 H 7 DNO 2 [M + H] + m/z = 91.06, found: Figure S1. 1 H NMR spectrum of desulfurization product of cysteine in D 2 O (Inset: ESI-MS of the desulfurization product). (B) in H 2 O: to a solution of cysteine (20.0 mm) in H 2 O (2 ml), TPPTS (50.0 mm) and Ru(bpy) 3 Cl 2 (5 mol %) were added. The resulting clear solution was degased with an argon balloon for 15 min and sealed. After the reaction solution was stirred and irradiated with 36 W household bulb for 16 h (Figure S2), it was purified by column chromatography on silylated silica gel to give withe solid (3.0 mg, 84%). 1 H NMR (400 MHz, D 2 O): δ H 3.83 (q, J = 7.3 Hz, 1 H), 1.53 (d, J = 7.3 Hz, 3 H); 13 C NMR (100 MHz, D 2 O): δ C 175.7, 50.3, ESI-MS calcd for alanine C 3 H 8 NO 2 [M + H] + m/z = 90.10, found: S5

6 Figure S2. 1 H NMR spectrum of crude reaction mixtures (top) and purified product of desulfurization (bottom). 5. Desulfurization of Glutathione (GSH) To a solution of GSH (20.0 mm) in H 2 O (2 ml), TPPTS (50.0 mm) and Ru(bpy) 3 Cl 2 (5 mol %) were added. The resulting clear solution was degased with an argon balloon for 15 min and sealed. After the reaction solution was stirred and irradiated with 36 W household bulb for 16 h (Figure S3), it was purified by column chromatography on silylated silica gel to give withe solid (9.0 mg, 85%). 1 H NMR (400 MHz, D 2 O): δ H 4.47 (q, J = 7.3 Hz, 1 H), 4.00 (t, J = 6.6 Hz, 1 H), 3.93 (d, J = 3.7 Hz, 2 H), (m, 2 H), (m, 2 H), 1.53 (d, J = 7.3 Hz, 3 H); 13 C NMR (100 MHz, D 2 O): δ C ESI-MS calcd for 2b C 10 H 17 N 3 O 6 [M - H] + m/z = , found: Figure S3. 1 H NMR spectrum of crude product after crude reaction mixtures (top) and purified product of desulfurization (bottom). S6

7 6. Synthesis of Peptides 10a, 11a, 12a, 13a, 14a, 15a Peptide 10a: the carboxylic acid precursor of 10a was synthesized manually on 2-chlorotrityl chloride resin by the general SPPS procedure. The resin was subjected to a cleavage reagent B for 2 h, and this procedure was repeated twice. The 2-chlorotrityl chloride resin was removed by filtration and the resulting solution was concentrated in vacuo and precipitated by Et 2 O to get crude peptide. To a solution of the crude peptide (16.0 mg, 15.0 μmol) in dried DMF (0.3 ml), benzyl mercaptan (37.0 μl, 0.3 mmol), EDCI (28.0 mg, 0.15 mmol) and HOBt (20.0 mg, 0.15 mmol) were added. The reaction mixture was stirred at room temperature for 2 h and the solvent was removed in vacuo. The residue was purified by silica gel chromatography (MeOH/DCM, 1/20, v/v) to give the side-chain protected thioester peptide (15.6 mg). The side-chain protected thioester peptide was treated with cleavage reagent A for 2 h. The majority of the solvents were removed azeotropically with toluene under vaccum and the remaining residue was precipitated with Et 2 O. The resulting white solid was purified by semi-preparative HPLC and analyzed by ESI-MS spectrometry (see Figure S22). Peptide 11a: all amino acids in glycopeptide 11a was condensted one by one on Rink Amide AM resin acoording to the general procedure for iterative peptide assembly except the building block glycosylamino acid Fmoc-Thr(AcO 3 -α-d-galnac)-oh (17). The (17) (1.5 equiv), which was prepared according to the reported procedure, 9 was condensted to the resin using HATU (3.0 equiv), HOAt (3.0 equiv) and DIPEA (6.0 equiv). The mixture in DMF (final concentration 0.1 M) was preactived for 5 min before added to the resin and was bubbling with N 2 for 4 h. The resin was washed thoroughly with DMF (3 3 ml), DCM (3 3 ml) and MeOH (3 3 ml) and then was swollen in DCM (3 ml) and washed with DMF to be ready for the condenstion of next amino acid. After the completion of the peptide, the resin was washed with DMF (3 3 ml), DCM (3 3 ml) and was treated with cleavage reagent A for 2 h. The resin was filtered and washed with TFA (2 2 ml). The TFA was removed azeotropiccaly with toluene, and the residue was precipitated using Et 2 O. The crude peptide was purified by semi-preparative HPLC and analyzed by ESI-MS spectrometry (see Figure S24). S7

8 Peptide 12a: all amino acids in glycopeptide 12a was condensted one by one acoording to the general procedure like 11a. Before the last Fmoc-Thr(tBu)-OH was coupled to the resin, the glycopeptide was deacetylation on the resin with 60% hydrazine in MeOH for 2 h. After the completion of the peptide, the resin was washed with DMF (3 3 ml), DCM (3 3 ml) and was treated with cleavage reagent A for 2 h. The resin was filtered and washed with TFA (2 2 ml). The TFA was removed azeotropiccaly with toluene, and the residue was precipitated using Et 2 O. The crude peptide was purified by semi-preparative HPLC and analyzed by ESI-MS spectrometry (see Figure S26). Glycopeptide 13a: the carboxylic acid precursor of 13a was synthesized manually on 2-chlorotrityl chloride resin as the same procedure of 11a. The resin was subjected to a cleavage reagent B for 2 h, and the procedure was repeated twice. The 2-chlorotrityl chloride resin was removed by filtration and the resulting solution was concentrated in vacuo and precipitated by Et 2 O to get crude peptide. To a solution of the crude peptide (28.0 mg, 15.0 μmol) in dried DMF (0.3 ml), benzyl mercaptan (37.0 μl, 0.3 mmol), EDCI (28.0 mg, 0.15 mmol) and HOBt (20.0 mg, 0.15 mmol) were added. The reaction mixture was stirred at room temperature for 2 h and the solvent was removed in vacuo. The residue was purified by silica gel chromatography (MeOH/DCM 1/20, v/v) to give the side-chain protected peptide (26.0 mg). The side-chain protected peptide was treated with cleavage reagent A for 2 h. The majority of the solvents were removed azeotropically with toluene under vaccum and the remaining residue was precipitated with Et 2 O. The resulting white solid was purified by semi-preparative HPLC and analyzed by ESI-MS spectrometry (see Figure S28). Peptide 14a: peptide 14a 1 was synthesized manually on 2-chlorotrityl chloride resin as the general procedure. The crude peptide was purified by semi-preparative HPLC. Ligation S8

9 conditions: 14a 1 (37.0 mm), 14a 2 (33.0 mm), TCEP (20.0 equiv), sodium 2-mercaptoethanesulfonate (MESNa) (10.0 equiv), Gn HCl (200 mm), phosphate buffer (200 mm, ph = 7.4), 5 h. The crude peptide was purified by semi-preparative HPLC and analyzed by ESI-MS spectrometry (see Figure S30). Figure S4. HPLC profiles of the ligation reaction: (a) t = 0 h, (b) t = 2.5 h, (c) t = 5 h, (d) purified product. Peptide 15a: peptide 15a 1 was synthesized manually on 2-chlorotrityl chloride resin as the procedure of thioester peptide synthesis. Ligation conditions: 15a 1 (30.0 mm), TCEP (20 equiv), sodium 2-mercaptoethanesulfonate (10.0 equiv), H 2 O/DMF/Et 3 N (150/100/1, v/v/v), 30 h, 81%. The crude peptide was purified by semi-preparative HPLC and analyzed by ESI-MS spectrometry (see Figure S32). Figure S5. HPLC profiles of the ligation reaction: (a) t = 0 h, (b) t = 12 h, (c) t = 30 h, (d) purified product. S9

10 7. HPLC and ESI-MS spectra of peptides Figure S6. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 2a. Analytical HPLC: 5-10% B in A over 40 min, λ = 254 nm, ESI-MS calcd for 2a C 35 H 53 N 11 O 13 S [M + Na] + m/z = , [M + H] + m/z = , [M + K + H] 2+ m/z = , [M + 2 H] + m/z = , found: , , , S10

11 Figure S7. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 2b. Analytical HPLC: 5-10% B in A over 40 min, λ = 254 nm, ESI-MS calcd for 2b C 35 H 53 N 11 O 13 [M + Na] + m/z = , [M + H] + m/z = , [M + K + H] 2+ m/z = , found: , , S11

12 Figure S8. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 3a. Analytical HPLC: 5-10% B in A over 50 min, λ = 254 nm, ESI-MS calcd for 3a C 37 H 61 N 13 O 11 S [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , S12

13 Figure S9. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 3b. Analytical HPLC: 5-10% B in A over 50 min, λ = 254 nm, ESI-MS calcd for 3b C 37 H 61 N 13 O 11 [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , S13

14 Figure S10. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 4a. Analytical HPLC: 5-30% B in A over 30 min, λ = 254 nm, ESI-MS calcd for 4a C 59 H 85 N 15 O 20 S [M + Na] + m/z = , [M + H] + m/z = , [M + Na + H] 2+ m/z = , found: , , S14

15 Figure S11. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 4b. Analytical HPLC: 5-30% B in A over 30 min, λ = 254 nm, ESI-MS calcd for 4b C 59 H 85 N 15 O 20 [M + Na] + m/z = , [M + H] + m/z = , [M + Na + H] 2+ m/z = , found: , , S15

16 Figure S12. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 5a. Analytical HPLC: 5-10% B in A over 50 min, λ = 254 nm, ESI-MS calcd for 5a C 42 H 68 N 14 O 14 [M + Na] + m/z = , [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , , S16

17 Figure S13. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 5b. Analytical HPLC: 5-10% B in A over 50 min, λ = 254 nm, ESI-MS calcd for 5b C 42 H 68 N 14 O 14 [M + Na] + m/z = , [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , , S17

18 Figure S14. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 6a. Analytical HPLC: 5-30% B in A over 30 min, λ = 254 nm, ESI-MS calcd for 6a C 48 H 79 N 21 O 13 S [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , S18

19 Figure S15. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 6b. Analytical HPLC: 5-30% B in A over 30 min, λ = 254 nm, ESI-MS calcd for 6b C 48 H 79 N 21 O 13 [M + Na] + m/z = , [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , , S19

20 Figure S16. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 7a. Analytical HPLC: 5-10% B in A over 50 min, λ = 280 nm, ESI-MS calcd for 7a C 40 H 64 N 12 O 12 S 2 [M + Na] + m/z = , [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , , S20

21 Figure S17. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 7b. Analytical HPLC: 5-10% B in A over 50 min, λ = 280 nm, ESI-MS calcd for 7b C 40 H 64 N 12 O 12 S [M + Na] + m/z = , [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , , S21

22 Figure S18. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 8a. Analytical HPLC: 5-30% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 8a C 56 H 88 N 16 O 12 S 2 [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , S22

23 Figure S19. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 8b. Analytical HPLC: 5-30% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 8b C 56 H 88 N 16 O 12 S [M + Na] + m/z = , [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , , S23

24 Figure S20. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 9a. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 9a C 61 H 87 N 13 O 21 S 2 [M + Na] + m/z = , [M + H] + m/z = , [M + 2 H] 2+ m/z =, found: , S24

25 Figure S21. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 9b. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 9b C 61 H 87 N 13 O 21 S [M + Na] + m/z = , [M + H] + m/z = , found: , S25

26 Figure S22. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 10a. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 10a C 53 H 76 N 12 O 13 S 2 [M + K] + m/z = , [M + Na] + m/z = , [M + H] + m/z = , found: , , S26

27 Figure S23. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 10b. Analytical HPLC: 5-90% B in A over 30min, λ = 280 nm, ESI-MS calcd for 10b C 53 H 76 N 16 O 13 S [M + Na] + m/z = , [M + H] + m/z = , found: , S27

28 Figure S24. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 11a. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 11a C 71 H 99 N 15 O 25 S [M + K] + m/z = , [M + Na] + m/z = , [M + H] + m/z = , found: , , S28

29 Figure S25. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 11b. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 11b C 71 H 99 N 15 O 25 [M + K] + m/z = , [M + Na] + m/z = , [M + H] + m/z = , found: , , S29

30 Figure S26. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 12a. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 12a C 65 H 93 N 15 O 22 S [M + H] + m/z = , [M + K + H] 2+ m/z = , found: , S30

31 Figure S27. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 12b. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 12b C 65 H 93 N 15 O 22 [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , S31

32 Figure S28. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 13a. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 13a C 79 H 107 N 17 O 27 S 5 [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , S32

33 Figure S29. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 13b. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 13b C 79 H 107 N 17 O 27 S 4 [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , S33

34 Figure S30. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 14a. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 14a C 63 H 95 N 19 O 23 S [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , S34

35 Figure S31. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 14a. Analytical HPLC: 5-90% B in A over 30 min, λ = 280 nm, ESI-MS calcd for 14a C 63 H 95 N 19 O 23 [M + H] + m/z = , [M + 2 H] 2+ m/z = , found: , S35

36 Figure S32. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 15a. Analytical HPLC: 5-90% B in A over 30 min, λ = 254 nm, ESI-MS calcd for 15a C 37 H 61 N 13 O 11 S [M + Na] + m/z = , [M + H] + m/z = , found:520.48, S36

37 Figure S33. Analytical HPLC (left) and ESI-MS (right) analysis of the purified 15b. Analytical HPLC: 5-90% B in A over 30 min, λ = 254 nm, ESI-MS calcd for 15b C 37 H 61 N 13 O 11 [M + Na] + m/z = , [M + H] + m/z = ,found: , S37

38 S38

39 S39

40 9. Reference (1) L. R. Malins, A. M. Giltrap, L. J. Dowman, R. J. Payne, Org. Lett. 2015, 17, (2) P. Siman, S. V. Karthikeyan, A. Brik, Org. Lett. 2012, 14, (3) Wan, Q.; Danishefsky, S. J. Angew. Chem. Int. Ed. 2007, 46, (4) Chen, J.; Wan, Q.; Yuan, Y.; Zhu, J.; Danishefsky, S. J. Angew. Chem. Int. Ed. 2008, 47, (5) Haase, C.; Rohde, H.; Seitz, O. Angew. Chem. Int. Ed. 2008, 47, (6) El Oualid, F.; Merkx, R.; Ekkebus, R.; Hameed, D. S.; Smit, J. J.; de Jong, A.; Hilkmann, H.; Sixma, T. K.; Ovaa, H. Angew. Chem. Int. Ed. 2010, 49, (7) Ding, H.; Shigenaga, A.; Sato, K.; Morishita, K.; Otaka, A. Org. Lett. 2011, 13, (8) Thompson, R. E.; Chan, B.; Radom, L.; Jolliffe, K. A.; Payne, R. J. Angew. Chem. Int. Ed. 2013, 52, (9) Cergol, K. M.; Thompson, R. E.; Malins, L. R.; Turner, P.; Payne, R. J. Org. Lett. 2014, 16, (10) Z. Wu, X. Guo, Z. Guo, Chem. Commun. 2010, 46, S40