PAUL VALERIE J (US)
MATTHEW SUSAN (US)
CHEN QI-YIN (US)
RATNAYAKE RANJALA (US)
SMITHSONIAN INST (US)
US20200121607A1 | 2020-04-23 | |||
US20190194166A1 | 2019-06-27 | |||
US20140200319A1 | 2014-07-17 |
CLAIMS What is claimed: 1. A compound according to Formula I: 1 GB1 R = OH 2 GB2 R = H wherein, each R is independently H or OH; and pharmaceutically acceptable salts, thereof. 2. The compound of claim 1, wherein R is independently H. 3. The compound of claim 2 wherein R is OH. 4. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier. 5. The pharmaceutical composition of claim 4 further comprising an additional therapeutic agent. 6. The pharmaceutical composition of claim 5 wherein the additional therapeutic agent is an anti-cancer agent, chemotherapeutic agent, an anti-angiogenesis agent, cytotoxic agent, or an anti-proliferation agent. 7. A kit comprising an effective amount of a compound of claim 1, in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to a cell proliferation disorder. 8. A method of modulating the activity of cell proliferation in a subject, comprising contacting the subject with a compound of formula I in claim 1, in an amount and under conditions sufficient to modulate cell proliferation. 9. The method of claim 8, wherein the cell is a cancer cell. 10. The method of claim 8, wherein the cell is a tumor cell. 11. The method of claim 8, wherein the modulation is inhibition. 12. A method of treating a subject suffering from or susceptible to a cell proliferation related disorder or disease, wherein the subject has been identified as in need of treatment for a cell proliferation related disorder or disease, comprising administering to said subject in need thereof, an effective amount of a compound or pharmaceutical composition of formula I in claim 1, such that said subject is treated for said disorder. 13. The method of claim 12, wherein the compound of formula I is one of Compounds 1 or 2 in Table A. 14. The method of claim 13, wherein the disorder is cancer, solid tumor, colon cancer, breast cancer, bone, brain and others (e.g., osteosarcoma, neuroblastoma, colon adenocarcinoma the like). 15. The method of claim 12, wherein the subject is a mammal. 16. The method of claim 12 wherein the subject is a primate or human. 17. The method of claim 12, wherein the effective amount of the compound of formula I ranges from about 0.005 µg/kg to about 200 mg/kg. 18. The method of claim 12, wherein the effective amount of the compound of formula I ranges from about 0.1 mg/kg to about 200 mg/kg. 19. The method of claim 12, wherein the effective amount of compound of formula I ranges from about 10 mg/kg to 100 mg/kg. 20. The method of claim 12, wherein the effective amount of the compound of formula I ranges from about 1.0 pM to about 500 µM 21. The method of claim 13, wherein the compound of formula I is administered intravenously, intramuscularly, subcutaneously, intracerebroventricularly, orally or topically. 22. The method of claim 13, wherein the compound of formula I is administered alone or in combination with one or more other therapeutics. 23. The method of claim 13, wherein the additional therapeutic agent is an anti-cancer agent, chemotherapeutic agent, an anti-angiogenesis agent, cytotoxic agent, or an anti- proliferation agent. 24. A method of treating cancer or tumors, comprising administering to said subject in need thereof, an effective amount of any of Compounds 1-2, and pharmaceutically acceptable salts thereof. 25. A compound that is Gatorbulin-1 (compound 1a), or pharmaceutically acceptable salt thereof. 26. A method of treating cancer or tumors, comprising administering to said subject in need thereof, an effective amount of Gatorbulin-1 (compound 1a), or pharmaceutically acceptable salt thereof. 27. The method of claim 26, further comprising further comprising administering to said subject an additional therapeutic agent. 28. The method of claim 27, wherein the additional therapeutic agent is an anti-cancer agent, chemotherapeutic agent, an anti-angiogenesis agent, cytotoxic agent, or an anti- proliferation agent. 29. The method of claim 27, wherein the additional therapeutic agent is a tubulin- interactive anticancer agent. |
Total synthesis of gatorbulin-1 (1a) Scheme S2. Synthesis of building block 4. (2S,3R)-3-Hydroxy-N-methylaspartic acid 2 (11). Methylamine water solution (41%) (100 mL) was added to solid of (2R,3R)-epoxysuccinic acid (10) (6.0 g, 45.43 mmol) at 0 ºC. The mixture was stirred for 4.5 h under refluxing (75-80 ºC), then cooled down to room temperature and concentrated under reduced pressure. Water (3 × 15 mL) was added to the concentrated residue and evaporated again for three times to remove unreacted methylamine. The crude product was purified by column of Dowex (H + ) resin, eluted by deionized water and then 4 M aqueous ammonia to provide product 11 (6.7 g, 90%). 1 H NMR (400 MHz, D 2 O): δ 4.64 (d, J = 2.8 Hz, 1H), 4.10 (d, J = 3.2 Hz, 1H), 2.78 (s, 3H) ppm. HRMS (ESI) m/z calcd for C 5 H 9 NO 5 (M+H) + 164.0559, found 164.0550. (2S,3R)-3-Hydroxy-N-methylaspartic acid β-methyl ester (12). Concentrated chloric acid (1.7 mL, 12 M) was added to the solution of compound 11 (1.649 g) in MeOH (50 mL) and the reaction mixture was refluxed for 3 h. The resulting mixture was concentrated under reduced pressure. MeOH (3 × 20 mL) was added to the concentrated residue and evaporated again for three times to remove excess chloric acid. The crude product was dried by oil pump to provide product 12 (1.73 g, 100%) as white foam. 1 H NMR (400 MHz, D 2 O): δ 4.87 (d, J = 2.4 Hz, 1H), 4.44 (d, J = 2.8 Hz, 1H), 3.80 (s, 3H), 2.83 (s, 3H) ppm. 13 C NMR (100 MHz, CDC1 3 ): δ 171.9, 168.0, 67.4, 63.0, 53.2, 31.6 ppm. HRMS (ESI) m/z calcd for C6H11NO5 (M+H) + 178.0715, found 178.0712. (2S,3R)-3-hydroxy-Nα-methyl-asparagine (13). Compound 12 (1.7 g, 10.49 mmol) was dissolved in anhydrous MeOH (40 mL). The resulting solution was saturated (bubbled) with ammonia (gas) for 5–7 min each day (total 3 days). After stirring for three days, the reaction solution was evaporated to dryness in vacuo. The resulting solid was washed with cold methanol, then diethyl ether and to give the compound 13 as a white solid (1.60 g, 95%). [α] 20 D: +27 (c 0.09, H 2 O). 1 H NMR (600 MHz, D2O, mixtures of rotamers): δ 4.61 (br m, 1H), 4.00 (br m, 1H), 2.82 (s, 1H) ppm. 13 C NMR (150 MHz, D 2 O, mixtures of rotamers): δ 175.4, 169.2, 68.7, 65.1, 32.0 ppm. HRMS (ESI) m/z calcd for C 5 H 10 N 2 O 4 (M+H) + 163.0719, found 163.0710. (2S,3R)-3-Hydroxy-Nα-methyl-Nα-Fmoc-asparagine (14).9-Fluorenylmethyl chloroformate (Fmoc-Cl) (2.945g, 11.386 mmol) and Na2CO3 (2.413 g, 22.767 mmol) were dissolved in the solution of the mixture of 1,4-dioxane and water (45 mL-45 mL). Compound 13 (1.23 g, 7.589 mmol) was added to the above solution at 0 ºC ant stirred 10 min at this temperature. After the reaction mixture was moved to room temperature and stirred at this temperature overnight, it was diluted with water (80 mL) and concentrated under reduced pressure to move most of 1,4-dioxane. The concentrated mixture was extracted with diethyl ether (20 mL × 4). The water layer was acidified with 2M HCl (aq.) to pH 2 and extracted with EtOAc (150 mL × 3). The combined organic phase was dried with anhydrous MgSO 4 and evaporated in vacuo to give product 14 (2.393 g, 82%). [α] 20 D : +1.5 (c 0.32, MeOH). 1 H NMR (400 MHz, CDCl 3, mixtures of rotamers): δ 7.68 (d, J = 7.7 Hz, 2H), 7.50 (t, J = 8.0 Hz, 2H), 7.36-7.30 (br m, 3H), 7.26-7.21 (br m, 3H), 4.86 (s, 1H), 4.49 (s, 1H), 4.36-4.25 (br m, 2H), 4.18-4.12 (br m, 1H), 3.01 (s, 3H) ppm. 13 C NMR (125 MHz, CDCl 3 ): δ 176.2, 171.1, 158.4, 143.8, 143.5, 141.3, 141.3, 72.9, 68.5, 64.6, 47.0, 36.1 ppm. HRMS (ESI) m/z calcd for C 20 H 20 N 2 O 6 (M+H) + 385.1400, found 385.1385. Usually, compound 14 was used in the next step without further purification and characterization. (2S,3R)-3-Hydroxy-N α -methyl-N α -Fmoc-asparagine benzyl ester (15). BnBr (2.94 mL, 24.735 mmol) was added to the solution of compound 14 (2.351 g, 6.121 mmol) and NaHCO 3 (1.55 g, 18.45 mmol) in anhydrous DMF (45 mL) at 0 ºC and was stirred at this temperature for 1 h, room temperature for 20 h. The resulting mixture was quenched with water (100 mL) and was extracted with EtOAc (150 mL × 4). The combined organic phase was washed with water (100 mL × 4), dried with anhydrous MgSO4, evaporated in vacuo and purified by flash chromatography column on silica gel (eluted by 40-80% ethyl acetate in hexane) to give product 15 (2.14 g, 74%). [α] 20 D: +2.8 (c 0.12, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixtures of rotamers): δ 7.78 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.33-7.27 (br m, 7H), 6.83 (s, 1H), 5.96-5.86 (br m, 2H), 5.23-5.15 (m, 2H), 4.76 (br s, 1H), 4.50-4.45 (m, 2H), 4.39-4.35 (m, 1H), 4.24 (t, J = 7.0 Hz, 1H), 3.08 (s, 3H) ppm. 13 C NMR (125 MHz, CDC1 3 ): δ 173.7, 167.7, 158.6, 143.9, 143.5, 141.4, 141.3, 135.2, 128.5, 128.4, 128.2, 127.9, 127.9, 127.2, 125.1, 125.0, 120.1, 73.7, 68.5, 67.5, 66.4, 47.1, 37.5 ppm. HRMS (ESI) m/z calcd for C 27 H 26 N 2 O 6 (M+H) + 475.1869, found 475.1854. (2S,3R)-3-Hydroxy-Nα-methyl-Nα-Fmoc-Nγ-trityl-asparagine benzyl ester (16). Compound 15 (2.443 g, 5.153 mmol) and trityl alcohol (13.414 g, 51.528 mmol) was dissolved in AcOH (17.5 mL). The solution was heated to 50 °C and was treated successively with concentrated sulfuric acid (168 μL, 3.092 mmol) and acetic anhydride (1.22 mL, 12.882 mmol). The reaction mixture was cooled to room temperature after stirred at 50 °C for 2.5 h, then it was diluted with EtOAc (100 mL) and quenched with saturated aqueous NaHCO 3 solution (70 mL). Excessive NaHCO 3 powder was added slowly to the quenched solution to that there was no bubble (CO 2 ) produced any more. EtOAc layer was separated and the aqueous layer was further extracted with EtOAc, and the combined organic layers were dried (MgSO4), filtered, concentrated in vacuo and purified by flash chromatography column (SiO 2 , 33% EtOAc in hexane) to provide 16 as a white solid (3.5 g, 95%). [α] 20 D : +28.4 (c 0.0.48, MeOH). 1 H NMR (400 MHz, CDCl 3 ): δ 8.23 (s, 1H), 7.79 (d, J = 7.6 Hz, 2H), 7.59 (dd, J = 7.6, 3.2 Hz, 2H), 7.43 (dd, J = 7.6, 7.6 Hz, 2H), 7.35-7.16 (m, 22H), 6.03 (s, 1H), 5.42 (d, J = 12.4 Hz, 1H), 5.11 (d, J = 12.4 Hz, 1H), 4.72 (br s, 1H), 4.50-4.46 (m, 2H), 4.41-4.36 (m, 1H), 4.26 (dd, J = 7.2, 7.2 Hz, 1H), 3.03 (s, 3H) ppm. 13 C NMR (100 MHz, CDCl 3 ): δ 169.1, 167.7, 158.8, 144.8, 144.0, 143.5, 141.5, 141.4, 135.5, 128.8, 128.6, 128.5, 128.4, 128.0, 128.0, 127.2, 127.1, 125.1, 125.1, 120.1, 75.1, 70.2, 68.5, 67.4, 67.2, 47.1, 38.1 ppm. HRMS (ESI) m/z calcd for C 46 H 40 N 2 O 6 (M+Na) + 739.2748, found 739.2761. (2S,3R)-3-OTBS-N α -methyl-N α -Fmoc-N γ -trityl-asparagine benzyl ester (17). To the solution of 16 (1.48 g, 2.066 mmol) in anhydrous CH 2 C1 2 (20 mL) were added 2,6-lutidine (2.4 mL, 20.661 mmol) and tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf) (2.37 mL, 10.33 mmol) at 0 ºC under argon. After stirring at the same temperature for 20 min, the reaction mixture was moved to room temperature and stirred for another 1 h, then it was quenched with MeOH (10 mL) and saturated aq. NH 4 Cl (30 mL), and extracted with EtOAc (50 mL × 3). The combined organic layer was washed with 0.5 M HCl (20 mL × 3), saturate aq. NaHCO 3 (20 mL × 2) and brine (20 mL), dried with anhydrous MgSO 4 and evaporated in vacuo. The resulting crude mixture was purified by flash chromatography column (SiO 2 , eluted by 10-12.5% EtOAc in hexane) to give product 17 (1.71 g, 93%) as a white solid. [α] 20 D +11.6 (c 0.22, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixture of rotamers, major and minor (7/3)): δ 7.82 (s, 1H), 7.77 (d, J = 7.6 Hz, 1.7H), 7.73-7.70 (m, 1H), 7.57-7.53 (m, 2H), 7.49 (d, J = 7.6 Hz, 0.3H), 7.40 (dd, J = 7.6, 7.6 Hz, 2H), 7.36-7.11 (m, 21H), 5.30 (d, J = 6.8 Hz, 0.7H), 5.19-5.07 (m, 2.3H) 4.73 (d, J = 6.8 Hz, 0.7H), 4.63 (d, J = 6.0 Hz, 0.3H), 4.58- 4.52 (m, 0.3H), 4.37 (dd, J = 10.0, 7.2 Hz, 0.7H), 4.29 (dd, J = 7.2, 7.2 Hz, 0.7H), 4.23 (dd, J = 7.2, 7.2 Hz, 0.7H), 4.11-4.05 (m, 0.6 H), 2.83 (s, 3H), 0.81 (s, 9H), 0.15 (s, 2.1H), 0.06 (s, 2.1H), -0.01 (s, 0.9H), -0.02 (s, 0.9H) ppm. 13 C NMR (100 MHz, CDC1 3 , mixture of rotamers, major and minor (7/3)): δ 169.0, 168.5, 168.1, 157.0, 155.8, 144.4, 144.3, 144.0, 143.9, 141.4, 135.7, 135.5, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.1, 128.0, 127.8, 127.7, 127.3, 127.2, 127.2, 127.1, 125.3, 125.0, 120.1, 120.0, 73.2, 72.3, 70.6, 70.5, 68.2, 68.0, 67.3, 67.1, 61.9, 61.2, 34.8, 34.6, 32.2, 31.9, 31.7, 25.7, 25.7, 25.4, 22.8, 17.9, 17.9, 14.3, -4.6, -4.7, -5.2, -5.3 ppm. HRMS (ESI) m/z calcd for C52H54N2O6Si (M+H) + 831.3829, found 831.3802. (2S,3R)-3-OTBS-Nα-methyl-Nα-Fmoc-Nγ-trityl-asparagine (4). MeOH (50 mL) was added cautiously to the mixture of compound 17 (1.6 g, 1.927 mmol) and Pd/C (10% wt) (160 mg). The suspension was degassed with argon (balloon) and hydrogen (balloon) successively, then it was stirred under hydrogen gas (balloon) at room temperature for 30 min. The catalyst was removed by filtration through Celite and the filtrate cake was washed by MeOH. The combined filtrate was concentrated in vacuo and purified by flash chromatography column (SiO 2 , eluted by 2-7% MeOH in CH 2 C1 2 ) to provide product 4 as a white solid (1.21 g, 85%). [α] 20 D +32.4 (c 0.18, MeOH). 1 H NMR (400 MHz, CDC1 3, mixture of rotamers, major and minor (3/1)): δ 9.03 (br s, 1H), 7.93 (br s, 0.75H), 7.78 (d, J = 7.2 Hz, 1.75H), 7.72 (dd, J = 7.6, 7.6 Hz, 0.5H), 7.59 (dd, J = 6.4, 6.4 Hz, 1.75H), 7.53 (d, J = 7.6 Hz, 0.25H), 7.43-7.38 (m, 1.75H), 7.36-7.20 (m, 17.25H), 5.17 (d, J = 6.4 Hz, 0.75H), 4.99 (d, J = 6.0 Hz, 0.25H), 4.84 (d, J = 6.4 Hz, 0.75H), 4.71-4.66 (m, 0.5H), 4.42-4.32 (m, 1.5H), 4.26 (dd, J = 7.2, 7.2 Hz, 1H), 4.18-4.10 (m, 0.5H), 2.75 (s, 0.75H), 2.74 (s, 2.25H), 0.87 (s, 6.75H), 0.85 (s, 2.25H), 0.20 (s, 2.25H), 0.16 (s, 2.25H), 0.04 (s, 0.75H), 0.00 (s, 0.75H) ppm. 13 C NMR (100 MHz, CDCl 3, mixture of rotamers, major and minor (3/1)): δ 177.2, 172.5, 172.0, 169.3, 169.0, 157.3, 155.9, 144.1, 144.1, 144.0, 143.9, 141.4, 141.3, 128.8, 128.1, 127.8, 127.3, 127.2, 127.1, 125.3, 125.2, 120.0, 72.7, 72.0, 70.5, 68.3, 67.9, 61.8, 61.5, 47.2, 36.2, 34.8, 34.6, 32.5, 32.1, 29.2, 27.0, 25.8, 25.7, 25.4, 20.8, 18.9, 17.9, 14.2, 11.6, -4.7, -5.2 ppm. HRMS (ESI) m/z calcd for C 45 H 48 N 2 O 6 Si (M+H) + 741.3360, found 741.3341. Scheme S3. Synthesis of building block 5 2,3 Allyloxamine (19) 3,4 . Alloxyamine.HCl (6.0 g) was mixed with KOH pellets (15.0 g) in a distillation flask with distillation assembly. The mixture was subjected to distillation by heating at 1 atm under the atmosphere of nitrogen. The product was collected at 86-90 ºC as colorless liquid (3.5 g, 88%). 1 H NMR (400 MHz, CDC1 3 ): δ 5.93-5.83 (m, 1H), 5.37 (br s, 2H), 5.29-5.19 (m, 2H), 1.06 (d, J = 6.5 Hz, 2H) ppm. 13 C NMR (100 MHz, CDC1 3 ): δ 134.0, 118.5, 76.8 ppm. (S)-N-Allyloxyalanine t-butyl ester (5) 3,4 . Triflic anhydride (4.2 mL, 24.981 mmol) was added dropwise to the solution of tert-butyl (R)-lactate (3.044 g, 20.817 mmol) in anhydrous CH 2 C1 2 (90 mL) at -70 ºC. The mixture was stirred 5 min, then 2,6-lutidine (3.044 mL, 26.022 mmol) was added dropwise at the same temperature. After the reaction mixture was stirred at -70 ºC for 1.5 h, alloxyamine (19) (3.35 mL, 41.635 mmol) was added dropwise at the same temperature. When the resulting mixture was stirred at -70 ºC for another 15 min, it was warmed to room temperature and stirred overnight (about 19 h). The reaction was diluted with CH 2 C1 2 (40 mL) and quenched with water (40 mL). The organic layer was washed successively with water (50 mL × 2), 2% citric acid (50 mL × 2), 5% NaHCO 3 (50 mL × 2), dried with anhydrous MgSO 4, filtered and concentrated in vacuo. The residue was purified by flash chromatography column (SiO 2 , eluted by 10% EtOAc in hexanes) to provide product 5 (3.77 g, 90%). [α] 20 D: -8.3 (c 0.12, MeOH). 1 H NMR (400 MHz, CDC1 3 ): δ 5.90 (dddd, J = 17.3, 10.4, 5.6, 5.6 Hz, 1H), 5.20 (m, 2H), 5.86-5.76 (m, 1H), 5.70 (br, 1H), 4.17 (d, J = 5.6 Hz, 2H), 3.59 (q, J = 6.8 Hz, 1H), 1.46 (s, 9H), 1.17 (d, J = 7.2 Hz, 3H) ppm. 13 C NMR (100 MHz, CDCl 3 ): δ 173.7, 134.6, 117.5, 81.4, 75.1, 59.7, 28.1, 15.0 ppm. HRMS (ESI) m/z calcd for C 10 H 19 NO 3 (M+H) + 202.1443, found 202.1449. Scheme S4. Synthesis of building block 6. N-Boc-4-MePro-Lac-OBn (20). To the suspension of 4-MePro-OH (9) (3.247 g, 14.160 mmol) in toluene (5.0 ml) was added N, N-diisopropylethylamine (DIEA) (3.69 ml, 21.24 mmol), 2,4,6-trichlorobenzoyl chloride (3.32 ml, 21.24 mmol) at room temperature under argon, and stirred at the same temperature for 40 min. Then the benzyl lactate (8) (2.5 ml, 15.575 mmol) and DMAP (2.941 g, 24.07 mmol) were added to the above mixture at 0 ºC. The reaction was stirred at 0 ºC for 20 min and at room temperature for another 4 h, then it was quenched with water (50 ml) and extracted with diethyl ether (50 ml × 4). The combined organic layer was washed with saturated NH 4 C1 (50 ml × 2), saturated NaHCO 3 (50 ml × 2), brine (50 ml), dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel flash chromatography column (eluted by 10% ethyl acetate in hexane) to give ester 20 (4.738 g, 86%). [α] 20 D: -86 (c 0.43, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixture of rotamers, major and minor (1/1)): δ 7.39-7.26 (m, 5H), 5.25-5.09 (m, 3H), 4.65 (d, J = 10.8 Hz, 1H), 4.31 (t, J = 8.4 Hz, 0.5H), 4.22 (t, J = 8.4 Hz, 0.5H), 4.22 (t, J = 8.4 Hz, 0.5H), 3.73 (dd, J = 10.0, 6.8 Hz, 0.5H), 3.65 (dd, J = 10.4, 7.2 Hz, 0.5H), 3.65 (dd, J = 10.4, 7.2 Hz, 0.5H), 2.95 (dd+dd, J = 10.0, 10.0 Hz, 1H), 2.41-2.32 (m, 1H), 2.26-2.13 (m, 1H), 1.58-1.49 (m, 4H), 1.45 (s, 4.5H), 1.39 (s, 4.5H), 1.00 (d, J = 6.4 Hz, 1.5H), 0.98 (d, J = 6.0 Hz, 1.5H) ppm. 13 C NMR (100 MHz, CDC1 3 , ,mixture of rotamers, major and minor (1/1)): δ 172.8, 172.4, 170.7, 170.3, 154.3, 153.5, 135.3, 135.2, 128.6, 128.6, 128.5, 128.5, 128.4, 128.3, ,128.3, 80.0, 79.9, 68.8, 68.7, 67.2, 67.0, 59.1, 58.7, 53.9, 53.3, 38.7, 37.7, 33.3, 32.6, 28.5, 28.2, 17.0, 16.9, 16.9, 16.8 ppm. HRMS (ESI) m/z calcd for C 21 H 29 NO 6 (M+Na) + 414.1893, found 414.1878. N-Fmoc-4-MePro-Lac-OBn (21). Trifluoroacetic acid (TFA) (30 ml) was added to the solution of compound 20 (4.738 g, 12.108 mmol) in CH 2 C1 2 (30 ml) at 0 ºC. The reaction mixture was stirred at 0 ºC for 20 min, then moved room temperature and stirred for 1.5 h. Toluene (30 ml) was added in and the solvent was evaporated. Another toluene (5 ml × 3) was added to the concentrated residue and evaporated again for three times to move excess TFA. The residue was dried under vacuum and used in the next step without purification. The dried residue was dissolved in 1,4-dioxane (30 ml) and water (30 ml). NaHCO 3 (2.543 g, 30.271 mmol), Fmoc-C1 (3.759 g, 14.53 mmol) were added the above solution successively at 0 ºC. After the reaction mixture was stirred at 0 ºC for 20 min, at room temperature for 4.5 h, it was diluted water (30 ml) and extracted with EtOAc (50 mL × 3). The combined organic layer was washed water (30 mL × 3), dried with anhydrous MgSO 4 , filtered and concentrated in vacuo. The residue was purified by flash chromatography column (SiO 2 , eluted by 10-25% EtOAc in hexane) to provide product 21 (5.874 g, 95%). [α] 20 D : -81 (c 0.21, MeOH). 1 H NMR (500 MHz, CDCl 3, mixture of rotamers, major and minor (5.5/4.5)): δ 7.77 (dd, J = 7.0, 7.0 Hz, 2H), 7.64 (d, J = 7.5 Hz, 0.55H), 7.61 (dd, J = 6.5, 6.5 Hz, 1H), 7.56 (d, J = 7.5 Hz, 0.45H), 7.42-7.30 (m, 9H), 5.27 (q, J = 7.0 Hz, 0.55H), 5.23-5.07 (m, 2.45H), 4.51 (dd, J = 10.5, 6.0 Hz, 0.45H), 4.45 (dd, J = 10.0, 6.5 Hz, 0.55H), 4.41 (t, J = 8.5 Hz, 0.55H), 4.35 (t, J = 8.0 Hz, 0.45H), 4.33-4.26 (m, 1.55H), 4.16 (dd, J = 6.5, 6.5 Hz, 0.45H), 3.85 (dd, J = 10.5, 7.0 Hz, 0.45H), 3.80 (dd, J = 10.0, 7.0 Hz, 0.55H), 3.13 (dd, J = 10.0, 10.0 Hz, 0.55H), 3.05 (dd, J = 10.0, 10.0 Hz, 0.45H), 2.46 (dq, J = 7.5, 7.5 Hz, 1H), 2.34-2.26 (m, 0.55H), 2.26- 2.18 (m, 0.45H), 1.69-1.62 (m, 1H), 1.56 (d, J = 7.0 Hz, 1.65H), 1.47 (d, J = 7.0 Hz, 1.35H), 1.07 (d, J = 6.5 Hz, 1.65H), 1.03 (d, J = 6.5 Hz, 1.35H) ppm. 13 C NMR (125 MHz, CDC1 3 , mixture of rotamers, major and minor (5.5/4.5)): δ 172.2, 172.1, 170.6, 170.3, 154.8, 154.3, 144.5, 144.2, 144.0, 143.7, 141.4, 141.4, 141.3, 135.3, 135.2, 128.7, 128.7, 128.6, 128.5, 128.3, 128.3, 127.8, 127.7, 127.7, 127.1, 127.1, 127.0, 125.3, 125.2, 125.2, 125.0, 120.0, 120.0, 120.0, 69.0, 69.0, 67.6, 67.4, 67.2, 67.1, 59.3, 58.8, 54.2, 53.7, 47.4, 47.3, 38.8, 37.6, 33.5, 32.6, 17.1, 17.0, 16.9, 16.9 ppm. HRMS (ESI) m/z calcd for C 31 H 31 NO 6 (M+Na) + 536.2049, found 536.2029. N-Boc-4-MePro-Lac-OH (6). MeOH (250 ml) was added cautiously to the mixture of compound 21 (6.6 g, 12.86 mmol) and Pd/C (10% wt) (660 mg). The suspending mixture was degassed with argon (balloon) and hydrogen (balloon) successively, then it was stirred under hydrogen gas (balloon) at room temperature for 30 min. The catalyst was removed by filtration through Celite and the filtrate cake was washed with MeOH. The combined filtrate was concentrated in vacuo and purified by flash chromatography column (SiO 2 , eluted by 10- 15% MeOH in CH 2 C1 2 ) to provide product 6 as a white solid (5.0 g, 92%). [α] 20 D: -82.5 (c 0.11, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixture of rotamers, major and minor (5.5/4.5)): δ 8.73 (br, 1H), 7.77-7.74 (m, 2H), 7.62-7.58 (m, 1.55H), 7.55 (d, J = 7.6 Hz, 0.45H), 7.39 (d, J = 7.6,7.6 Hz, 2H), 7.33-7.28 (m, 2H), 5.20-5.13 (m, 0.55H), 5.00 (q, J = 7.2 Hz, 0.45H), 4.52- 4.39 (m, 1.55H), 4.36-4.24 (m, 2H), 4.15 (dd, J = 6.8, 6.8 Hz, 0.45H), 3.83 (dd, J = 10.8, 7.6 Hz, 0.45H), 3.78-3.72 (m, 1H), 3.12-3.01 (m, 1H), 2.54-2.45 (m, 1H), 2.35-2.19 (m, 1H), 1.78-1.68 (m, 1H), 1.51 (d, J = 7.2 Hz, 1.65H), 1.44 (d, J = 7.2 Hz, 1.35H), 1.08 (d, J = 6.4 Hz, 1.65H), 1.04 (d, J = 6.4 Hz, 1.35H) ppm. 13 C NMR (100 MHz, CDCl 3, mixture of rotamers, major and minor (5.5/4.5)): δ 175.2, 175.0, 172.2, 172.0, 155.1, 154.5, 144.3, 144.1, 143.8, 143.6, 141.3, 141.3, 141.2, 127.8, 127.8, 127.7, 127.1, 127.1, 127.0, 125.2, 125.2, 125.2, 125.0, 120.0, 120.0, 120.0, 119.9, 69.4, 69.1, 67.8, 67.6, 59.4, 58.8, 54.2, 53.7, 53.5, 47.3, 47.2, 38.6, 37.6, 33.3, 33.3, 32.6, 17.3, 17.1, 17.0, 16.9 ppm. HRMS (ESI) m/z calcd for C 24 H 25 NO 6 (M+Na) + 446.1580, found 446.1563. Scheme S4. Alternative synthesis of building block 6. N-Boc-4-MePro-Lac-OBn (20). To the suspension of 4-MePro-OH (9) (3.247 g, 14.160 mmol) in toluene (5.0 mL) was added N, N-diisopropylethylamine (DIEA) (3.69 mL, 21.24 mmol), 2,4,6-trichlorobenzoyl chloride (3.32 mL, 21.24 mmol) at room temperature under argon, and stirred at the same temperature for 40 min. Then the benzyl lactate (8) (2.5 mL, 15.575 mmol) and DMAP (2.941 g, 24.07 mmol) were added to the above mixture at 0 ºC. The reaction was stirred at 0 ºC for 20 min and at room temperature for another 4 h, then it was quenched with water (50 mL) and extracted with diethyl ether (50 mL × 4). The combined organic layer was washed with saturated NH4Cl (50 mL × 2), saturated NaHCO 3 (50 mL × 2), brine (50 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel flash chromatography column (eluted by 10% ethyl acetate in hexane) to give ester 20 (4.738 g, 86%). [α] 20 D: -86 (c 0.43, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixture of rotamers, major and minor (1/1)): δ 7.39-7.26 (m, 5H), 5.25-5.09 (m, 3H), 4.65 (d, J = 10.8 Hz, 1H), 4.31 (t, J = 8.4 Hz, 0.5H), 4.22 (t, J = 8.4 Hz, 0.5H), 4.22 (t, J = 8.4 Hz, 0.5H), 3.73 (dd, J = 10.0, 6.8 Hz, 0.5H), 3.65 (dd, J = 10.4, 7.2 Hz, 0.5H), 3.65 (dd, J = 10.4, 7.2 Hz, 0.5H), 2.95 (dd+dd, J = 10.0, 10.0 Hz, 1H), 2.41-2.32 (m, 1H), 2.26-2.13 (m, 1H), 1.58-1.49 (m, 4H), 1.45 (s, 4.5H), 1.39 (s, 4.5H), 1.00 (d, J = 6.4 Hz, 1.5H), 0.98 (d, J = 6.0 Hz, 1.5H) ppm. 13 C NMR (100 MHz, CDCl 3, , mixture of rotamers, major and minor (1/1)): δ 172.8, 172.4, 170.7, 170.3, 154.3, 153.5, 135.3, 135.2, 128.6, 128.6, 128.5, 128.5, 128.4, 128.3, ,128.3, 80.0, 79.9, 68.8, 68.7, 67.2, 67.0, 59.1, 58.7, 53.9, 53.3, 38.7, 37.7, 33.3, 32.6, 28.5, 28.2, 17.0, 16.9, 16.9, 16.8 ppm. HRMS (ESI) m/z calcd for C 21 H 29 NO 6 (M+Na) + 414.1893, found 414.1878. N-Fmoc-4-MePro-Lac-OBn (21). Trifluoroacetic acid (TFA) (30 mL) was added to the solution of compound 20 (4.738 g, 12.108 mmol) in CH 2 C1 2 (30 mL) at 0 ºC. The reaction mixture was stirred at 0 ºC for 20 min, then moved room temperature and stirred for 1.5 h. Toluene (30 mL) was added in and the solvent was evaporated. Another toluene (5 mL × 3) was added to the concentrated residue and evaporated again for three times to move excess TFA. The residue was dried under vacuum and used in the next step without purification. The dried residue was dissolved in 1,4-dioxane (30 mL) and water (30 mL). NaHCO 3 (2.543 g, 30.271 mmol), Fmoc-Cl (3.759 g, 14.53 mmol) were added the above solution successively at 0 ºC. After the reaction mixture was stirred at 0 ºC for 20 min, at room temperature for 4.5 h, it was diluted water (30 mL) and extracted with EtOAc (50 mL × 3). The combined organic layer was washed water (30 mL × 3), dried with anhydrous MgSO 4 , filtered and concentrated in vacuo. The residue was purified by flash chromatography column (SiO 2 , eluted by 10-25% EtOAc in hexane) to provide product 21 (5.874 g, 95%). [α] 20 D: -81 (c 0.21, MeOH). 1 H NMR (500 MHz, CDC1 3 , mixture of rotamers, major and minor (5.5/4.5)): δ 7.77 (dd, J = 7.0, 7.0 Hz, 2H), 7.64 (d, J = 7.5 Hz, 0.55H), 7.61 (dd, J = 6.5, 6.5 Hz, 1H), 7.56 (d, J = 7.5 Hz, 0.45H), 7.42-7.30 (m, 9H), 5.27 (q, J = 7.0 Hz, 0.55H), 5.23-5.07 (m, 2.45H), 4.51 (dd, J = 10.5, 6.0 Hz, 0.45H), 4.45 (dd, J = 10.0, 6.5 Hz, 0.55H), 4.41 (t, J = 8.5 Hz, 0.55H), 4.35 (t, J = 8.0 Hz, 0.45H), 4.33-4.26 (m, 1.55H), 4.16 (dd, J = 6.5, 6.5 Hz, 0.45H), 3.85 (dd, J = 10.5, 7.0 Hz, 0.45H), 3.80 (dd, J = 10.0, 7.0 Hz, 0.55H), 3.13 (dd, J = 10.0, 10.0 Hz, 0.55H), 3.05 (dd, J = 10.0, 10.0 Hz, 0.45H), 2.46 (dq, J = 7.5, 7.5 Hz, 1H), 2.34-2.26 (m, 0.55H), 2.26- 2.18 (m, 0.45H), 1.69-1.62 (m, 1H), 1.56 (d, J = 7.0 Hz, 1.65H), 1.47 (d, J = 7.0 Hz, 1.35H), 1.07 (d, J = 6.5 Hz, 1.65H), 1.03 (d, J = 6.5 Hz, 1.35H) ppm. 13 C NMR (125 MHz, CDC1 3 , mixture of rotamers, major and minor (5.5/4.5)): δ 172.2, 172.1, 170.6, 170.3, 154.8, 154.3, 144.5, 144.2, 144.0, 143.7, 141.4, 141.4, 141.3, 135.3, 135.2, 128.7, 128.7, 128.6, 128.5, 128.3, 128.3, 127.8, 127.7, 127.7, 127.1, 127.1, 127.0, 125.3, 125.2, 125.2, 125.0, 120.0, 120.0, 120.0, 69.0, 69.0, 67.6, 67.4, 67.2, 67.1, 59.3, 58.8, 54.2, 53.7, 47.4, 47.3, 38.8, 37.6, 33.5, 32.6, 17.1, 17.0, 16.9, 16.9 ppm. HRMS (ESI) m/z calcd for C 31 H 31 NO 6 (M+Na) + 536.2049, found 536.2029. N-Boc-4-MePro-Lac-OH (6). MeOH (250 mL) was added cautiously to the mixture of compound 21 (6.6 g, 12.86 mmol) and Pd/C (10% wt) (660 mg). The suspending mixture was degassed with argon (balloon) and hydrogen (balloon) successively, then it was stirred under hydrogen gas (balloon) at room temperature for 30 min. The catalyst was removed by filtration through Celite and the filtrate cake was washed with MeOH. The combined filtrate was concentrated in vacuo and purified by flash chromatography column (SiO 2 , eluted by 10- 15% MeOH in CH 2 C1 2 ) to provide product 6 as a white solid (5.0 g, 92%). [α] 20 D : -82.5 (c 0.11, MeOH). 1 H NMR (400 MHz, CDC1 3, mixture of rotamers, major and minor (5.5/4.5)): δ 8.73 (br, 1H), 7.77-7.74 (m, 2H), 7.62-7.58 (m, 1.55H), 7.55 (d, J = 7.6 Hz, 0.45H), 7.39 (d, J = 7.6,7.6 Hz, 2H), 7.33-7.28 (m, 2H), 5.20-5.13 (m, 0.55H), 5.00 (q, J = 7.2 Hz, 0.45H), 4.52- 4.39 (m, 1.55H), 4.36-4.24 (m, 2H), 4.15 (dd, J = 6.8, 6.8 Hz, 0.45H), 3.83 (dd, J = 10.8, 7.6 Hz, 0.45H), 3.78-3.72 (m, 1H), 3.12-3.01 (m, 1H), 2.54-2.45 (m, 1H), 2.35-2.19 (m, 1H), 1.78-1.68 (m, 1H), 1.51 (d, J = 7.2 Hz, 1.65H), 1.44 (d, J = 7.2 Hz, 1.35H), 1.08 (d, J = 6.4 Hz, 1.65H), 1.04 (d, J = 6.4 Hz, 1.35H) ppm. 13 C NMR (100 MHz, CDCl 3, mixture of rotamers, major and minor (5.5/4.5)): δ 175.2, 175.0, 172.2, 172.0, 155.1, 154.5, 144.3, 144.1, 143.8, 143.6, 141.3, 141.3, 141.2, 127.8, 127.8, 127.7, 127.1, 127.1, 127.0, 125.2, 125.2, 125.2, 125.0, 120.0, 120.0, 120.0, 119.9, 69.4, 69.1, 67.8, 67.6, 59.4, 58.8, 54.2, 53.7, 53.5, 47.3, 47.2, 38.6, 37.6, 33.3, 33.3, 32.6, 17.3, 17.1, 17.0, 16.9 ppm. HRMS (ESI) m/z calcd for C 24 H 25 NO 6 (M+Na) + 446.1580, found 446.1563.
Scheme S5. Synthesis of building block 7. N-Boc-L-serine β-lactone (23) 5,6 . Diethyl azodicarboxylate (DEAD) (14.0 mL, 30.73 mmol) was added to the solution of triphenylphosphine (Ph 3 P) (8.06 g, 30.73 mmol) in THF (250 mL) at -78 °C. The resulting solution was stirred at -78 °C for 15 min, then warmed to room temperature and stirred for another 15 min. Then it was cooled to -78 °C. A solution of N- Boc-Ser-OH (6.31 g, 30.73 mmol) in THF (30 mL) was added dropwise to the above solution at -78 °C. The reaction mixture was stirred at -78 °C for 30 minutes, room temperature for another 2 h, then concentrated in vacuo. The residue was triturated in EtOAc/hexane (200 mL, 1:1) and filtered. The filtrate was concentrated in vacuo and purified by flash column chromatography (SiO 2 , EtOAc/hexane, 1:3) to provide β-lactone 23 (3.01 g, 52%) as white solid, which was confirmed by MS (ESI) and used in next step directly without characterization by NMR. MS (ESI) m/z calcd for C8H13NO4 (M+Na) + 210.08, found 210.1; C8H13NO4 (M-H)- 186.08, found 186.0. (S)-N-Boc-L-(Se)-phenylselenocysteine (24) 5,6 . Sodium trimethoxyborohydride (NaBH(OMe) 3 ) (2.47 g, 19.316 mmol) was added to the solution of diphenyl diselenide ((PhSe)2) (3.014 g, 9.659 mmol) in absolute ethanol (150 mL) at room temperature under argon. The resulting mixture was stirred for 30 min at room temperature, then β-lactone (23) (2.583 g, 13.797 mmol) was added. The reaction mixture was stirred for 3 h at room temperature under argon and concentrated in vacuo. Saturated aqueous NaHCO 3 (100 mL) was added to the residue and stirred 20 min at room temperature. The above mixture was washed with Et 2 O (30 mL × 3). The water layer was acidified with 3 M aqueous HCl to pH 2 and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried with anhydrous MgSO 4 and concentrated to give a yellowish oil, which was added hexanes (20 mL) and kept -20 ºC freezer overnight. The precipitate was filtrated to give product 24 (4.52 g, 95%) as a white solid. 1 H NMR (400 MHz, CDC1 3 ): δ 7.56-7.54 (m, 2H), 7.27-7.25 (m, 3H), 5.29 (d, J = 6.8 Hz, 1H), 4.65-4.61 (m, 1H), 3.39-3.29 (m, 2H), 1.41 (s, 9H) ppm. LRMS (ESI) m/z calcd for C 14 H 19 NO 4 78 Se (M+Na) + 368.05, found 368.0; C14H19NO4 78 Se (M-H)- 344.05, found 344.1. Fm (S)-N-Boc-L-(Se)-phenylselenocysteine ester (7) 7 . DMAP (150 mg, 1.228 mmol), DCC (2.767 g, 13.409 mmol) were added to a solution of 24 (4.205 g, 12.19 mmol) and 9- fluorenylmethanol (FmOH) (2.632 g, 13.415 mmol) in anhydrous CH 2 C1 2 (70 mL) at 0 ºC under argon. After stirring at room temperature for 4 h, the reaction mixture was evaporated in vacuo. EtOAc (200 mL) was added to the residue and the suspension was filtered. The filtrate was evaporated, and purified by flash chromatography column on silica gel (eluted by 6-12% AcOEt in hexane) to provide 7 (4.791 g, 67%). 1 H NMR (400 MHz, CDC1 3 ): δ 7.78- 7.75 (m, 2H), 7.55-7.51 (m, 4H), 7.44-7.39 (m, 2H), 7.35-7.29 (m, 2H), 7.25-7.19 (m, 3H), 5.38 (d, J = 8.4 Hz, 1H), 4.76-4.71 (m, 1H), 4.25 (dd, J = 10.4, 7.2 Hz, 1H), 4.14-4.04 (m, 2H), 3.29 (br d, J = 5.2 Hz, 2H), 1.45 (s, 9H) ppm. 13 C NMR (100 MHz, CDC1 3 ): δ 170.7, 155.1, 143.5, 143.4, 141.4, 141.3, 133.8, 129.2, 128.9, 128.0, 127.6, 127.3, 127.2, 125.2, 125.1, 120.1, 120.1, 80.2, 67.3, 53.4, 46.6, 30.6, 28.4 ppm. HRMS (ESI) m/z calcd for C 28 H 29 NO 4 Se (M+H) + 524.1340, found 524.1319. ((2S,3R)-3-OTBS-Nα-methyl-Nα-Fmoc-Nγ-trityl)-Asn-(N-OAlly l)-Ala-Ot-Bu (25). (COC1) 2 (2.0 mL, 22.89 mol) was added to the solution of acid 4 (1.107 g, 1.496 mmol) in benzene (22 ml) under nitrogen. After the reaction mixture was stirred at room temperature for 1 h, it was concentrated in vacuo. The residue was added dry benzene (5 mL× 3) and evaporated again for another three times to remove traces of (COC1) 2 , and dried under high vacuum. The residue was dissolved in dry benzene (24 ml) and the solution of compound 5 (352 mg, 1.75 mmol) in dry benzene (2 ml) was added under N2. The reaction flask was equipped with a reflux condenser and covered with Al foil. AgCN (260 mg, 1.942 mmol) was added in a single portion. The reaction mixture was stirred at room temperature for 10 min and then at 80 °C to for 20 min (pre-heated oil bath). The reaction mixture was cooled to room temperature and diluted with CH 2 C1 2 (40 ml) and filtered through Celite. The filtrate was then concentrated in vacuo and purified by flash chromatography column (SiO 2 , eluted by 7-15% EtOAc in hexanes) to afford 25 (690 mg, 50%) as a white solid. [α] 20 D: +20.3 (c 0.1, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixture of rotamers, major and minor (3/1)): δ 7.88 (br s, 0.25H), 7.78 (d, J = 7.6 Hz, 2H), 7.75 (br s, 0.75H), 7.72 (d, J = 7.6 Hz, 0.75H), 7.64-7.60 (m, 2H), 7.49 (d, J = 6.8 Hz, 0.25H), 7.41 (dd, J = 7.6, 7.6 Hz, 2H), 7.36-7.19 (m, 16H), 6.12-5.92 (m, 0.75H), 5.61 (d, J = 8.4 Hz, 0.75H), 5.47 (d, J = 6.0 Hz, 0.25H), 5.38-5.19 (m, 2H), 4.73-4.41 (m, 5H), 4.30-4.20 (m, 2H), 3.06 (s, 2.25H), 2.96 (s, 0.75H), 1.53-1.45 (m, 12H), 0.84 (s, 2.25H), 0.81 (s, 6.75H), 0.09 (s, 2.25H), 0.03 (s, 0.75H), -0.01 (s, 0.75H), -0.07 (s, 2.25H) ppm. 13 C NMR (100 MHz, CDC1 3 , mixture of rotamers, major and minor (3/1)): δ 169.5, 169.4, 168.7, 456.4, 155.7, 144.8, 144.6, 144.5, 144.2, 144.1, 143.4, 141.5, 141.3, 141.3, 141.1, 131.7, 131.0, 129.0, 128.9, 128.1, 128.0, 127.9, 127.8, 127.7, 127.2, 127.1, 127.1, 127.0, 125.5, 125.4, 120.6, 120.2, 120.1, 120.0, 119.1, 82.0, 81.9, 78.0, 77.4, 77.3, 73.4, 71.9, 70.8, 70.3, 68.2, 60.1, 58.8, 58.2, 58.0, 47.2, 46.9, 34.8, 34.6, 32.0, 31.9, 29.8, 29.8, 28.0, 26.0, 25.9, 22.8, 22.8, 17.9, 17.8, 14.8, 14.5, 14.2, -4.7, -4.8, -4.9 ppm. HRMS (ESI) m/z calcd for C 55 H 65 N 3 O 8 Si (M+H) + 924.4619, found 924.4594. (N-Fmoc-4-Me)-Pro-Lac-((2S,3R)-3-OTBS-N α -methyl-N α -Fmoc-N γ -trityl)-Asn-(N- OAllyl)-Ala-Ot-Bu (26). Dipeptide 25 (283.0 mg, 0.306 mmol) was treated with diethylamine (Et 2 NH) (4.0 ml) in MeCN (8.0 ml) at room temperature for 1 h. The reaction mixture was concentrated in vacuo and the residue was co-evaporated with toluene (5 ml × 3) for three times to remove trace of Et 2 NH, and dried with high vacuum for 1 h. DIEA (160 μl, 0.919 mmol) was added to the solution of the above residue and BEP (125.9 mg, 0.46 mmol) in dry CH 2 C1 2 (18 ml) at 0 ºC. The reaction mixture was stirred at room temperature overnight under argon, then concentrated in vacuo and purified by flash chromatography column (SiO 2 , eluted by EtOAc/hexane (1:3, v/v) to afford product 26 (359 mg, 78%). [α] 20 D: -42.0 (c 0.2, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixture of rotamers, major and minor (6/4)): δ 7.82-7.76 (m, 3H), 7.68 (d, J = 6.8 Hz, 0.4H), 7.63-7.58 (m, 1.6H), 7.41-7.38 (m, 2.4H), 7.35-7.10 (m, 16.6H), 6.00 (br, 1H), 5.81 (br m, 0.6H), 5.72 (br d, J = 5.6 Hz, 0.6H), 5.65 (br m, 0.4H), 4.62 (br m, 1H), 4.58-4.48 (m, 2.4H), 4.44-4.20 (m, 4H), 3.79 (br m, 0.4H), 3.72 (br m, 0.6H), 3.10-2.95 (m, 4H), 2.16 (br m, 2H), 1.77-1.69 (m, 0.4H), 1.66-1.57 (m, 0.6H), 1.50-1.45 (m, 13.8H), 1.34 (d, J = 6.4 Hz, 1.2H), 1.00-0.92 (m, 3H), 0.92-0.83 (m, 2.4H), 0.79 (s, 3.6H), 0.78 (s, 5.4H), 0.07 (s, 1.8H), -0.12 (s, 1.8H) ppm. 13 C NMR (100 MHz, CDC1 3 , , mixture of rotamers, major and minor (6/4)): δ 172.1, 172.0, 169.6, 169.5, 169.3, 169.0, 168.9, 154.8, 154.4, 144.6, 144.5, 144.3, 144.0, 143.7, 141.1, 141.3, 141.3, 131.8, 131.7, 129.0, 128.0, 127.8, 127.7, 127.7, 127.2, 127.1, 127.1, 127.0, 125.5, 125.3, 125.2, 125.1, 121.0, 120.0, 82.0, 78.1, 77.4, 72.1, 72.1, 70.4, 67.8, 67.5, 67.0, 59.8, 59.3, 58.7, 58.6, 57.1, 54.3, 53.8, 47.4, 47.3, 38.7, 37.6, 34.6, 34.8, 33.5, 32.9, 32.6, 29.8, 28.1, 26.0, 17.9, 17.1, 17.0, 15.0, -4.7, -4.9 ppm. HRMS (ESI) m/z calcd for C 64 H 78 N 4 O 11 Si (M+Na) + 1129.5334, found 1129.5320. (N-Fmoc-4-Me)-Pro-Lac-((2S,3R)-3-OTBS-Nα-methyl-Nα-Fmoc-N -trityl)-Asn-(N- OAllyl)-Ala-OH (27). To the solution of 26 (355 mg, 0.321 mmol) in anhydrous CH 2 C1 2 (20 mL) were added 2,6-lutidine (1.86 mL, 16.04 mmol) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) (1.74 mL, 9.62 mmol) at 0 ºC under argon. After stirred at the same temperature for 20 min, the reaction mixture was moved to room temperature and stirred for 2.5 h, then it was quenched with saturated aqueous NaHCO 3 (30 mL) and extracted with EtOAc (50 mL × 3). The combined organic layer was washed with 5% KHSO 4 (30 mL × 5), water (30 mL × 2) and brine (30 mL), dried with anhydrous MgSO 4 and evaporated in vacuo. The resulting crude mixture was purified by flash chromatography column (SiO 2 , eluted by 5% MeOH in CH 2 C1 2 ) to provide acid 27 (285 mg, 85%) as a white solid. [α] 20 D : - 55.0 (c 0.09, MeOH). 1 H NMR (400 MHz, CDCl 3, , mixture of rotamers, major and minor (5.5/4.5)): δ 8.15 (s, 0.45H), 8.11 (s, 0.55H), 7.76 (dd, J = 6.4, 6.4 Hz, 2H), 7.63-7.55 (m, 2H), 7.41-7.36 (m, 2H), 7.32-7.25 (m, 11H), 7.16-7.14 (m, 6H), 6.03-5.91 (m, 1H), 5.87 (dd, J = 7.6 Hz, 1H), 5.57 (br d, J = 6.0 Hz, 0.55H), 5.41-5.27 (m, 2.45H), 4.90-4.80 (m, 2H), 4.60-4.50 (m, 2H), 4.48-4.41 (m, 1H), 4.38-4.26 (m, 2.55H), 4.17 (dd, J = 6.4 Hz, 0.45H), 3.85-3.75 (m, 1H), 3.09-3.03 (m, 0.55H), 2.98 (br s, 3.45 H), 2.50-2.38 (br m, 1H), 2.31-2.17 (br m, 1H), 1.70-1.56 (m, 1H), 1.50-1.45 (m, 4.65H), 1.37 (d, J = 6.4 Hz, 1.35H), 1.05 (d, J = 6.4 Hz, 1.65H), 1.01 (d, J = 6.4 Hz, 1..35H), 0.80 (s, 4.95H), 0.79 (s, 4.05H), 0.09 (s, 1.65H), 0.07 (s, 1.35H), 0.07 (s, 3H) ppm. 13 C NMR (100 MHz, CDCl 3, mixture of rotamers, major and minor (5.5/4.5)): δ 171.9, 171.8, 171.5, 171.3, 170.8, 170.9, 170.8, 169.6, 169.6, 154.8, 154.4, 144.4, 144.3, 143.9, 143.7, 141.4, 141.3, 141.3, 131.0, 130.9, 128.7, 128.2, 127.8, 127.8, 127.7, 127.5, 127.2, 127.1, 127.0, 125.4, 125.3, 125.2, 125.0, 121.7, 120.0, 120.0, 79.4, 77.3, 71.2, 70.3, 70.1, 67.6, 67.5, 59.3, 58.8, 58.1, 57.2, 54.2, 53.8, 47.4, 47.3, 38.7, 37.5, 33.4, 33.0, 32.5, 31.7, 25.9, 22.8, 17.9, 17.2, 16.6, 16.4, 14.2, -4.7, -5.1 ppm. HRMS (ESI) m/z calcd for C60H70N4O11Si (M+Na) + 1073.4708, found 1073.4691. (N-Fmoc-4-Me)-Pro-Lac-((2S,3R)-3-OTBS-N α -methyl-N α -Fmoc-N γ -trityl)-Asn-(N- OAllyl)-Ala- ((S)-N-Boc-L-(Se)-phenylseleno)-Cys-OFm (3). Compound 7 (244.7 mg, 0.468 mmol) was treated with 4 M HCl in dry EtOAc (15 ml) at 0 ºC for 10 min and then room temperature for 1 h. The reaction mixture was concentrated in vacuo, and the residue was co-evaporated with toluene (5 ml × 3) for three times to move trace of HCl. The residue was dried under vacuum and used in next step without purification. Compound 27 (277 mg, 0.264 mmol), BOP (256.4 mg, 0.58 mmol), DIEA (230 μl, 1.321 mmol) were added successively to the solution of the above residue from 7 in dry THF (20 ml) at room temperature. After the reaction mixture was stirred at the same temperature for 3 h, it was evaporated in vacuo and purified by flash chromatography column (SiO 2 , eluted by 15-20% EtOAc in hexane) to provide product 3 (330 mg, 86%). [α] 20 D: -54.4 (c 0.09, MeOH). 1 H NMR (400 MHz, CDC1 3 , , mixture of rotamers, major and minor (5.5/4.5)): δ 8.21 (s, 0.45H), 8.19 (s, 0.55H), 8.09 (d, J = 7.6 Hz, 1H), 7.79-7.76 (m, 4H), 7.65-7.57 (m, 2H), 7.53 (d, J = 7.6 Hz, 1H), 7.48-7.12 (m, 28H), 6.07-5.90 (m, 2H), 5.59 (q, J = 5.6 Hz, 0.55H), 5.50-5.38 (m, 1.45H), 5.30 (dd, J = 10.0, 1H), 5.25-5.19 (br m, 1H), 4.98 (d, J = 7.6 Hz, 1H), 4.78-4.71 (m, 1H), 4.66-4.60 (m, 1H), 4.50-4.26 (m, 6H), 4.18 (dd, J = 6.8, 6.8 Hz, 0.55H), 4.09 (t, J = 8.8 Hz, 1H), 4.02 (dd, J = 6.8, 6.8 Hz, 1H), 3.89-3.79 (m, 1H), 3.12-3.00 (m, 4.45H), 2.89- 2.84 (br m, 1H), 2.58-2.47 (m, 1H), 2.37-2.27 (m, 1H), 2.25-2.14 (m, 1.55H), 1.74-1.62 (m, 1.45H), 1.50 (br d, J = 6.8 Hz, 4.65H), 1.40 (d, J = 6.8 Hz, 1.35H), 1.09 (d, J = 6.4 Hz, 1.65H), 1.05 (d, J = 6.4 Hz, 1.35H), 0.82 (s, 4.05H), 0.81 (s, 4.95H), 0.15-0.12 (m, 6H) ppm. 13 C NMR (100 MHz, CDC1 3 ): δ 172.0, 171.9, 171.8, 171.8, 170.5, 170.1, 169.3, 168.8, 168.7, 154.8, 154.4, 144.4, 144.3, 144.1, 144.0, 143.8, 143.3, 141.4, 141.4, 141.4, 141.4, 141.2, 133.7, 133.7, 131.1, 131.0, 129.3, 129.0, 128.8, 128.2, 127.9, 127.8, 127.8, 127.8, 127.8, 127.7, 127.7, 127.5, 127.5, 127.3, 127.2, 127.1, 127.1, 127.0, 125.7, 125.3, 125.3, 125.3, 125.2, 125.0, 120.7, 120.1, 120.0, 120.0, 79.0, 79.0, 77.4, 71.0, 70.9, 70.4, 70.3, 67.6, 67.6, 67.5, 67.2, 59.3, 58.7, 57.0, 57.0, 55.4, 54.2, 53.8, 52.9, 47.5, 47.3, 46.7, 38.7, 37.6, 33.5, 33.3, 33.3, 32.6, 31.7, 29.8, 28.5, 25.9, 22.8, 17.9, 17.3, 17.2, 16.6, 15.0, 14.2, -4.7, -5.0 ppm. HRMS (ESI) m/z calcd for C 83 H 89 N 5 O 12 SeSi (M+Na) + 1478.5340, found 1478.5337. Macrocycle 2. Linear compound 3 (222.5 mg, 0.153 mmol) was treated with diethylamine (Et 2 NH) (10 ml) in MeCN (20 ml) under nitrogen atmosphere at room temperature for 2.5 h. The reaction mixture was concentrated in vacuo and the residue was co-evaporated with the mixture of toluene/CH 2 C1 2 /DIEA (10:10:1, 5 ml × 3) for three times and CH 2 C1 2 (5 ml × 2) two times to remove traces of Et 2 NH, and dried with high vacuum for 0.5 h. The reaction mixture was purified by a preparative column (Alltech, 1 gram, silica). The column was eluted sequentially by CH 2 C1 2 , EtOAc/hexane (1:1) and CH 2 C1 2 /MeOH (4:1). The fraction eluted by CH 2 C1 2 /MeOH (4:1) was collected, evaporated, dried and used in next step directly. DIEA (239.2 μl, 1.376 mmol) was added to the solution of the above residue, PyBOP (238.6 mg, 0.459 mmol) and HOAt (64.5 mg, 0.474 mmol) in dry CH 2 C1 2 (250 ml) at room temperature. The reaction mixture was stirred at the same temperature for 24 h under argon, then concentrated in vacuo and purified by flash chromatography column (SiO2, eluted by EtOAc/hexane (1:3 to 1:1, v/v) to afford product 2 (95.2 mg, 60.3%) and 28 (5.1 mg, 3.8%). [α] 20 D: -42.3 (c 0.11, MeOH). 1 H NMR of 2 (400 MHz, CDC1 3 ): δ 7.75 (s, 1H), 7.46-7.43 (m, 2H), 7.29-7.20 (m,12H), 7.15-7.12 (m, 6H), 6.82 (d, J = 9.2 Hz, 1H), 6.01 (dddd, J = 17.0, 10.7, 6.4, 6.0 Hz, 1H), 5.52-5.46 (m, 2H), 5.40-5.30 (m, 2H), 4.89 (ddt, J = 11.2, 6.0, 1.2 Hz, 1H), 4.79 (td, J = 9.6, 4.8 Hz, 1H), 4.69 (d, J = 9.2 Hz, 1H), 4.66 (q, J = 6.8 Hz, 1H), 4.56 (dd, J = 8.8, 5.2 Hz, 1H), 4.19 (ddt, J = 10.8, 6.4, 1.6 Hz, 1H), 3.57 (dd, J = 12.0, 7.2 Hz, 1H), 3.15 (dd, J = 12.8, 10.4, 1H), 3.06 (dd, J = 12.8, 5.2 Hz, 1H), 2.92 (s, 3H), 2.78 (dd, J = 12.0, 6.4 Hz, 1H), 2.47-2.40 (m, 1H), 2.15-2.07 (m, 1H), 1.69-1.63 (m, 1H), 1.60 (d, J = 6.8 Hz, 3H), 1.11 (d, J = 7.2 Hz, 3H), 0.90 (d, J = 7.2 Hz, 3H), 0.82 (s, 9H), 0.21 (s, 3H), -0.05 (s, 3H) ppm. 13 C NMR (100 MHz, CDC1 3 ): δ 169.9, 169.7, 169.4, 168.5, 167.8, 165.4, 144.3, 131.3, 131.1, 129.6, 129.1, 128.0, 127.2, 127.2, 120.1, 78.7, 77.4, 74.8, 70.5, 69.7, 59.8, 59.2, 56.5, 53.7, 49.9, 39.2, 32.3, 30.9, 29.8, 28.4, 26.0, 18.6, 18.0, 16.6, 16.1, -3.6, -5.0 ppm. HRMS (ESI) m/z calcd for C54H67N5O9SeSi (M+Na) + 1060.3771, found 1060.3753. Scheme S6. Alternative fusing of all fragments and macrocyclization. ((2S,3R)-3-OTBS-N α -methyl-N α -Fmoc-N γ -trityl)-Asn-(N-OAllyl)-Ala-Ot-Bu (25). (COCl) 2 (2.0 mL, 22.89 mol) was added to the solution of acid 4 (1.107 g, 1.496 mmol) in benzene (22 ml) under nitrogen. After the reaction mixture was stirred at room temperature for 1 h, it was concentrated in vacuo. The residue was added dry benzene (5 mL× 3) and evaporated again for another three times to remove traces of (COC1) 2 , and dried under high vacuum. The residue was dissolved in dry benzene (24 mL) and the solution of compound 5 (352 mg, 1.75 mmol) in dry benzene (2 mL) was added under N2. The reaction flask was equipped with a reflux condenser and covered with Al foil. AgCN (260 mg, 1.942 mmol) was added in a single portion. The reaction mixture was stirred at room temperature for 10 min and then at 80 °C to for 20 min (pre-heated oil bath). The reaction mixture was cooled to room temperature and diluted with CH 2 C1 2 (40 mL) and filtered through Celite. The filtrate was then concentrated in vacuo and purified by flash chromatography column (SiO 2 , eluted by 7-15% EtOAc in hexanes) to afford 25 (690 mg, 50%, 85% BRSM) as a white solid. [α] 20 D: +20.3 (c 0.1, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixture of rotamers, major and minor (3/1)): δ 7.88 (br s, 0.25H), 7.78 (d, J = 7.6 Hz, 2H), 7.75 (br s, 0.75H), 7.72 (d, J = 7.6 Hz, 0.75H), 7.64- 7.60 (m, 2H), 7.49 (d, J = 6.8 Hz, 0.25H), 7.41 (dd, J = 7.6, 7.6 Hz, 2H), 7.36-7.19 (m, 16H), 6.12-5.92 (m, 0.75H), 5.61 (d, J = 8.4 Hz, 0.75H), 5.47 (d, J = 6.0 Hz, 0.25H), 5.38-5.19 (m, 2H), 4.73-4.41 (m, 5H), 4.30-4.20 (m, 2H), 3.06 (s, 2.25H), 2.96 (s, 0.75H), 1.53-1.45 (m, 12H), 0.84 (s, 2.25H), 0.81 (s, 6.75H), 0.09 (s, 2.25H), 0.03 (s, 0.75H), -0.01 (s, 0.75H), -0.07 (s, 2.25H) ppm. 13 C NMR (100 MHz, CDC1 3 , mixture of rotamers, major and minor (3/1)): δ 169.5, 169.4, 168.7, 456.4, 155.7, 144.8, 144.6, 144.5, 144.2, 144.1, 143.4, 141.5, 141.3, 141.3, 141.1, 131.7, 131.0, 129.0, 128.9, 128.1, 128.0, 127.9, 127.8, 127.7, 127.2, 127.1, 127.1, 127.0, 125.5, 125.4, 120.6, 120.2, 120.1, 120.0, 119.1, 82.0, 81.9, 78.0, 77.4, 77.3, 73.4, 71.9, 70.8, 70.3, 68.2, 60.1, 58.8, 58.2, 58.0, 47.2, 46.9, 34.8, 34.6, 32.0, 31.9, 29.8, 29.8, 28.0, 26.0, 25.9, 22.8, 22.8, 17.9, 17.8, 14.8, 14.5, 14.2, -4.7, -4.8, -4.9 ppm. HRMS (ESI) m/z calcd for C 55 H 65 N 3 O 8 Si (M+H) + 924.4619, found 924.4594. (N-Fmoc-4-Me)-Pro-Lac-((2S,3R)-3-OTBS-Nα-methyl-Nα-Fmoc-N -trityl)-Asn-(N- OAllyl)-Ala-Ot-Bu (26). Dipeptide 25 (283.0 mg, 0.306 mmol) was treated with diethylamine (Et 2 NH) (4.0 mL) in MeCN (8.0 mL) at room temperature for 1 h. The reaction mixture was concentrated in vacuo and the residue was co-evaporated with toluene (5 mL × 3) for three times to remove traces of Et 2 NH, and dried with high vacuum for 1 h. DIEA (160 μL, 0.919 mmol) was added to the solution of the above residue and BEP (125.9 mg, 0.46 mmol) in dry CH 2 C1 2 (18 mL) at 0 ºC. The reaction mixture was stirred at room temperature overnight under argon, then concentrated in vacuo and purified by flash chromatography column (SiO 2 , eluted by EtOAc/hexane (1:3, v/v) to afford product 26 (359 mg, 78%). [α] 20 D : -42.0 (c 0.2, MeOH). 1 H NMR (400 MHz, CDCl 3, mixture of rotamers, major and minor (6/4)): δ 7.82-7.76 (m, 3H), 7.68 (d, J = 6.8 Hz, 0.4H), 7.63-7.58 (m, 1.6H), 7.41-7.38 (m, 2.4H), 7.35-7.10 (m, 16.6H), 6.00 (br, 1H), 5.81 (br m, 0.6H), 5.72 (br d, J = 5.6 Hz, 0.6H), 5.65 (br m, 0.4H), 4.62 (br m, 1H), 4.58-4.48 (m, 2.4H), 4.44-4.20 (m, 4H), 3.79 (br m, 0.4H), 3.72 (br m, 0.6H), 3.10-2.95 (m, 4H), 2.16 (br m, 2H), 1.77-1.69 (m, 0.4H), 1.66-1.57 (m, 0.6H), 1.50-1.45 (m, 13.8H), 1.34 (d, J = 6.4 Hz, 1.2H), 1.00-0.92 (m, 3H), 0.92-0.83 (m, 2.4H), 0.79 (s, 3.6H), 0.78 (s, 5.4H), 0.07 (s, 1.8H), -0.12 (s, 1.8H) ppm. 13 C NMR (100 MHz, CDC1 3 , , mixture of rotamers, major and minor (6/4)): δ 172.1, 172.0, 169.6, 169.5, 169.3, 169.0, 168.9, 154.8, 154.4, 144.6, 144.5, 144.3, 144.0, 143.7, 141.1, 141.3, 141.3, 131.8, 131.7, 129.0, 128.0, 127.8, 127.7, 127.7, 127.2, 127.1, 127.1, 127.0, 125.5, 125.3, 125.2, 125.1, 121.0, 120.0, 82.0, 78.1, 77.4, 72.1, 72.1, 70.4, 67.8, 67.5, 67.0, 59.8, 59.3, 58.7, 58.6, 57.1, 54.3, 53.8, 47.4, 47.3, 38.7, 37.6, 34.6, 34.8, 33.5, 32.9, 32.6, 29.8, 28.1, 26.0, 17.9, 17.1, 17.0, 15.0, -4.7, -4.9 ppm. HRMS (ESI) m/z calcd for C64H78N4O11Si (M+Na) + 1129.5334, found 1129.5320. (N-Fmoc-4-Me)-Pro-Lac-((2S,3R)-3-OTBS-Nα-methyl-Nα-Fmoc-N -trityl)-Asn-(N- OAllyl)-Ala-OH (27). To the solution of 26 (355 mg, 0.321 mmol) in anhydrous CH 2 C1 2 (20 mL) were added 2,6-lutidine (1.86 mL, 16.04 mmol) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) (1.74 mL, 9.62 mmol) at 0 ºC under argon. After stirred at the same temperature for 20 min, the reaction mixture was moved to room temperature and stirred for 2.5 h, then it was quenched with saturated aqueous NaHCO 3 (30 mL) and extracted with EtOAc (50 mL × 3). The combined organic layer was washed with 5% KHSO4 (30 mL × 5), water (30 mL × 2) and brine (30 mL), dried with anhydrous MgSO4 and evaporated in vacuo. The resulting crude mixture was purified by flash chromatography column (SiO 2 , eluted by 5% MeOH in CH 2 C1 2 ) to provide acid 27 (285 mg, 85%) as a white solid. [α] 20 D : - 55.0 (c 0.09, MeOH). 1 H NMR (400 MHz, CDC1 3 , , mixture of rotamers, major and minor (5.5/4.5)): δ 8.15 (s, 0.45H), 8.11 (s, 0.55H), 7.76 (dd, J = 6.4, 6.4 Hz, 2H), 7.63-7.55 (m, 2H), 7.41-7.36 (m, 2H), 7.32-7.25 (m, 11H), 7.16-7.14 (m, 6H), 6.03-5.91 (m, 1H), 5.87 (dd, J = 7.6 Hz, 1H), 5.57 (br d, J = 6.0 Hz, 0.55H), 5.41-5.27 (m, 2.45H), 4.90-4.80 (m, 2H), 4.60-4.50 (m, 2H), 4.48-4.41 (m, 1H), 4.38-4.26 (m, 2.55H), 4.17 (dd, J = 6.4 Hz, 0.45H), 3.85-3.75 (m, 1H), 3.09-3.03 (m, 0.55H), 2.98 (br s, 3.45 H), 2.50-2.38 (br m, 1H), 2.31-2.17 (br m, 1H), 1.70-1.56 (m, 1H), 1.50-1.45 (m, 4.65H), 1.37 (d, J = 6.4 Hz, 1.35H), 1.05 (d, J = 6.4 Hz, 1.65H), 1.01 (d, J = 6.4 Hz, 1..35H), 0.80 (s, 4.95H), 0.79 (s, 4.05H), 0.09 (s, 1.65H), 0.07 (s, 1.35H), 0.07 (s, 3H) ppm. 13 C NMR (100 MHz, CDCl 3, mixture of rotamers, major and minor (5.5/4.5)): δ 171.9, 171.8, 171.5, 171.3, 170.8, 170.9, 170.8, 169.6, 169.6, 154.8, 154.4, 144.4, 144.3, 143.9, 143.7, 141.4, 141.3, 141.3, 131.0, 130.9, 128.7, 128.2, 127.8, 127.8, 127.7, 127.5, 127.2, 127.1, 127.0, 125.4, 125.3, 125.2, 125.0, 121.7, 120.0, 120.0, 79.4, 77.3, 71.2, 70.3, 70.1, 67.6, 67.5, 59.3, 58.8, 58.1, 57.2, 54.2, 53.8, 47.4, 47.3, 38.7, 37.5, 33.4, 33.0, 32.5, 31.7, 25.9, 22.8, 17.9, 17.2, 16.6, 16.4, 14.2, -4.7, -5.1 ppm. HRMS (ESI) m/z calcd for C 60 H 70 N 4 O 11 Si (M+Na) + 1073.4708, found 1073.4691. (N-Fmoc-4-Me)-Pro-Lac-((2S,3R)-3-OTBS-N α -methyl-N α -Fmoc-N γ -trityl)-Asn-(N- OAllyl)-Ala- ((S)-N-Boc-L-(Se)-phenylseleno)-Cys-OFm (3). Compound 7 (244.7 mg, 0.468 mmol) was treated with TFA (2.5 mL) in CH 2 C1 2 (5 mL) at room temperature for 30 min. The reaction mixture was concentrated in vacuo, and the residue was co-evaporated with toluene (5 mL × 3) for three times to remove traces of TFA. The residue was dried under vacuum and used in next step without purification. Compound 27 (277 mg, 0.264 mmol), BOP (256.4 mg, 0.58 mmol), DIEA (230 μL, 1.321 mmol) were added successively to the solution of the above residue from 7 in dry THF (20 mL) at room temperature. After the reaction mixture was stirred at the same temperature for 3 h, it was evaporated in vacuo and purified by flash chromatography column (SiO 2 , eluted by 15-20% EtOAc in hexane) to provide product 3 (330 mg, 86%). [α] 20 D: -54.4 (c 0.09, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixture of rotamers, major and minor (5.5/4.5)): δ 8.21 (s, 0.45H), 8.19 (s, 0.55H), 8.09 (d, J = 7.6 Hz, 1H), 7.79-7.76 (m, 4H), 7.65-7.57 (m, 2H), 7.53 (d, J = 7.6 Hz, 1H), 7.48-7.12 (m, 28H), 6.07-5.90 (m, 2H), 5.59 (q, J = 5.6 Hz, 0.55H), 5.50-5.38 (m, 1.45H), 5.30 (dd, J = 10.0, 1H), 5.25-5.19 (br m, 1H), 4.98 (d, J = 7.6 Hz, 1H), 4.78-4.71 (m, 1H), 4.66-4.60 (m, 1H), 4.50-4.26 (m, 6H), 4.18 (dd, J = 6.8, 6.8 Hz, 0.55H), 4.09 (t, J = 8.8 Hz, 1H), 4.02 (dd, J = 6.8, 6.8 Hz, 1H), 3.89-3.79 (m, 1H), 3.12-3.00 (m, 4.45H), 2.89-2.84 (br m, 1H), 2.58-2.47 (m, 1H), 2.37-2.27 (m, 1H), 2.25-2.14 (m, 1.55H), 1.74-1.62 (m, 1.45H), 1.50 (br d, J = 6.8 Hz, 4.65H), 1.40 (d, J = 6.8 Hz, 1.35H), 1.09 (d, J = 6.4 Hz, 1.65H), 1.05 (d, J = 6.4 Hz, 1.35H), 0.82 (s, 4.05H), 0.81 (s, 4.95H), 0.15-0.12 (m, 6H) ppm. 13 C NMR (100 MHz, CDCl 3 ): δ 172.0, 171.9, 171.8, 171.8, 170.5, 170.1, 169.3, 168.8, 168.7, 154.8, 154.4, 144.4, 144.3, 144.1, 144.0, 143.8, 143.3, 141.4, 141.4, 141.4, 141.4, 141.2, 133.7, 133.7, 131.1, 131.0, 129.3, 129.0, 128.8, 128.2, 127.9, 127.8, 127.8, 127.8, 127.8, 127.7, 127.7, 127.5, 127.5, 127.3, 127.2, 127.1, 127.1, 127.0, 125.7, 125.3, 125.3, 125.3, 125.2, 125.0, 120.7, 120.1, 120.0, 120.0, 79.0, 79.0, 77.4, 71.0, 70.9, 70.4, 70.3, 67.6, 67.6, 67.5, 67.2, 59.3, 58.7, 57.0, 57.0, 55.4, 54.2, 53.8, 52.9, 47.5, 47.3, 46.7, 38.7, 37.6, 33.5, 33.3, 33.3, 32.6, 31.7, 29.8, 28.5, 25.9, 22.8, 17.9, 17.3, 17.2, 16.6, 15.0, 14.2, -4.7, -5.0 ppm. HRMS (ESI) m/z calcd for C 83 H 89 N 5 O 12 SeSi (M+Na) + 1478.5340, found 1478.5337. Macrocycle 2. Linear compound 3 (222.5 mg, 0.153 mmol) was treated with diethylamine (Et 2 NH) (10 mL) in MeCN (20 mL) under nitrogen atmosphere at room temperature for 2.5 h. The reaction mixture was concentrated in vacuo and the residue was co-evaporated with the mixture of toluene/CH 2 C1 2 /DIEA (10:10:1, 5 mL × 3) for three times and CH 2 C1 2 (5 mL × 2) two times to remove traces of Et 2 NH, and dried with high vacuum for 0.5 h. The reaction mixture was purified by a preparative column (Alltech, 1 gram, silica). The column was eluted sequentially by CH 2 C1 2 , EtOAc/hexane (1:1) and CH 2 C1 2 /MeOH (4:1). The fraction eluted by CH 2 C1 2 /MeOH (4:1) was collected, evaporated, dried and used in next step directly. DIEA (239.2 μL, 1.376 mmol) was added to the solution of the above residue, PyBOP (238.6 mg, 0.459 mmol) and HOAt (64.5 mg, 0.474 mmol) in dry CH 2 C1 2 (250 mL) at room temperature. The reaction mixture was stirred at the same temperature for 24 h under argon, then concentrated in vacuo and purified by flash chromatography column (SiO 2 , eluted by EtOAc/hexane (1:3 to 1:1, v/v) to afford product 2 (95.2 mg, 60%). [α] 20 D: -42.3 (c 0.11, MeOH). 1 H NMR of 2 (400 MHz, CDC1 3 ): δ 7.75 (s, 1H), 7.46-7.43 (m, 2H), 7.29-7.20 (m,12H), 7.15-7.12 (m, 6H), 6.82 (d, J = 9.2 Hz, 1H), 6.01 (dddd, J = 17.0, 10.7, 6.4, 6.0 Hz, 1H), 5.52-5.46 (m, 2H), 5.40-5.30 (m, 2H), 4.89 (ddt, J = 11.2, 6.0, 1.2 Hz, 1H), 4.79 (td, J = 9.6, 4.8 Hz, 1H), 4.69 (d, J = 9.2 Hz, 1H), 4.66 (q, J = 6.8 Hz, 1H), 4.56 (dd, J = 8.8, 5.2 Hz, 1H), 4.19 (ddt, J = 10.8, 6.4, 1.6 Hz, 1H), 3.57 (dd, J = 12.0, 7.2 Hz, 1H), 3.15 (dd, J = 12.8, 10.4, 1H), 3.06 (dd, J = 12.8, 5.2 Hz, 1H), 2.92 (s, 3H), 2.78 (dd, J = 12.0, 6.4 Hz, 1H), 2.47- 2.40 (m, 1H), 2.15-2.07 (m, 1H), 1.69-1.63 (m, 1H), 1.60 (d, J = 6.8 Hz, 3H), 1.11 (d, J = 7.2 Hz, 3H), 0.90 (d, J = 7.2 Hz, 3H), 0.82 (s, 9H), 0.21 (s, 3H), -0.05 (s, 3H) ppm. 13 C NMR (100 MHz, CDC1 3 ): δ 169.9, 169.7, 169.4, 168.5, 167.8, 165.4, 144.3, 131.3, 131.1, 129.6, 129.1, 128.0, 127.2, 127.2, 120.1, 78.7, 77.4, 74.8, 70.5, 69.7, 59.8, 59.2, 56.5, 53.7, 49.9, 39.2, 32.3, 30.9, 29.8, 28.4, 26.0, 18.6, 18.0, 16.6, 16.1, -3.6, -5.0 ppm. HRMS (ESI) m/z calcd for C 54 H 67 N 5 O 9 SeSi (M+Na) + 1060.3771, found 1060.3753. Macrocycle 28. The pre-mixed buffer of TBAF (1.35 mL, 0.5 M in THF, 0.675 mmol) and AcOH (1.62 mL, 0.5 M in THF, 0.81 mmol) was added to the solution of macrocycle 2 (70 mg, 0.0675 mmol) in dry THF (14 mL). After the reaction mixture was stirred at room temperature under argon for 5 h, it was diluted with EtOAc and quenched with saturate NaHCO 3 (10 mL) extracted with EtOAc (15 mL × 3). The combined organic layers were dried over anhydrous MgSO4 and purified by preparative TLC plate (SiO 2 , developed by acetone/hexanes 1:2, Rf = 0.4) to provide product 28 (59.2 mg, 95%). [α] 20 D: -91.5 (c 0.09, MeOH). 1 H NMR (400 MHz, CDC1 3 ): δ 8.10 (s, 1H), 7.47-7.44 (m, 2H), 7.30-7.18 (m,18H), 6.76 (d, J = 9.2 Hz, 2H), 6.09-5.99 (m, 1H), 5.49 (q, J = 6.8 Hz, 1H), 5.43-5.33 (m, 2H), 4.91 (dd, J = 10.8, 5.6 Hz, 1H), 4.85-4.79 (m, 1H), 4.53 (dd, J = 8.8, 6.0 Hz, 1H), 4.46 (br s, 2H), 4.30 (dd+dd, J = 11.2, 6.8 Hz, 2H), 3.64 (dd, J = 11.6, 7.2 Hz, 1H), 3.18 (dd, J = 12.4, 10.0 Hz, 1H), 3.02 (dd, J = 12.4, 5.6 Hz, 1H), 3.03 (s, 3H), 2.88-2.83 (m, 1H), 2.51-2.44 (m, 1H), 2.17-2.09 (m, 1H), 1.65 (ddd, J = 13.2, 6.4, 6.4 Hz, 1H), 1.57 (d, J = 6.8 Hz, 3H), 1.26 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H) ppm. 13 C NMR (100 MHz, CDC1 3 ): δ 169.6, 169.5, 169.5, 169.3, 169.0, 166.8, 144.5, 131.6, 131.2, 129.5, 129.2, 128.8, 128.0, 127.3, 127.1, 120.8, 79.2, 77.4, 71.7, 70.1, 60.0, 59.5, 53.7, 49.7, 39.3, 31.1, 28.9, 18.3, 16.7, 16.5 ppm. HRMS (ESI) m/z calcd for C48H53N5O9Se (M+Na) + 946.2906, found 946.2878. Macrocycle 29. NaIO 4 (67.8 mg, 0.314 mmol) was added to the solution of compound 28 (73.2 mg, 0.0793 mmol) in the mixture of MeCN-H 2 O (15 mL–11.2 mL) at the room temperature. After the reaction mixture was stirred at the same temperature under argon for 2 h, CH 2 C1 2 (25 mL) and aqueous saturate NaHCO 3 (15 mL) were added and the resulting mixture was extracted with CH 2 C1 2 (20 mL × 4). The combined organic layers were dried with anhydrous MgSO4, evaporated in vacuo and purified by preparative TLC plate (SiO 2 , developed by acetone/hexanes 2:3, Rf = 0.5) to afford product 29 (57.2 mg, 94%). [α] 20 D: - 48.4 (c 0.07, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixture of three conformers, major, medium and minor (0.65:0.25:0.1)): δ 8.38 (s, 0.1H), 8.30 (s, 0.1H), 8.28 (s, 0.65H), 8.24 (s, 0.25H), 8.19 (s, 0.65H), 7.99 (s, 0.25H), 7.28-7.20 (m, 15H), 6.48 (s, 0.65H), 6.25 (s, 0.25H), 6.23 (s, 0.1H), 6.14-6.04 (m, 0.25H), 5.95-5.86 (m, 0.65H), 5.86-5.77 (m, 0.1H), 5.63 (q, J = 6.8 Hz, 0.75H), 5.48-5.27 (m, 3H), 5.13 (s, 0.65H), 5.05 (br m, 0.25H), 5.01 (s, 0.25H), 4.91 (s, 0.1H), 4.72 (dd, J = 8.4, 6.0 Hz, 0.35H), 4.69 (d, J = 9.2 Hz, 1H), 4.56 (dd, J = 9.6, 7.2 Hz, 0.65H), 4.48 (dd, J = 10.8, 6.8 Hz, 0.65H), 4.42-4.39 (m, 2.65H), 4.34-4.31 (m, 1H), 4.30- 4.24 (m, 0.35H), 4.18 (q, J = 6.8 Hz, 1H), 4.07 (dd, J = 10.4, 6.8 Hz, 0.9H), 4.02 (dd, J = 11.6, 7.2 Hz, 0.1H), 3.75 (dd, J = 11.6, 7.2 Hz, 0.35H), 3.40 (dd, J = 10.0, 8.4 Hz, 0.65H), 3.24 (t, J = 11.2, 0.25H), 3.14 (t, J = 11.2 Hz, 0.1H), 3.05 (s, 1.95H), 3.03 (s, 0.3H), 2.97 (s, 0.75H), 2.63-2.42 (m, 2.25H), 2.37-2.27 (m, 0.65H), 2.00 (d, J = 7.2 Hz, 0.3H), 1.81 (d, J = 7.6 Hz, 1.95H), 1.62-1.55 (m, 1.75H), 1.48 (d, J = 7.2 Hz, 0.3H), 1.40 (d, J = 6.8 Hz, 1.95H), 1.33 (d, J = 6.4 Hz, 0.75H), 1.15-1.11 (m, 3H) ppm. 13 C NMR (100 MHz, CDCl 3, mixture of three conformers, major, medium and minor): δ 172.3, 171.4, 171.0, 170.4, 170.2, 170.0, 169.6, 169.4, 169.0, 168.7, 167.9, 166.5, 166.0, 165.8, 144.5, 144.5, 144.5, 144.4, 135.8, 134.3, 134.3, 131.5, 131.3, 130.6, 130.5, 129.5, 128.7, 128.7, 128.4, 128.2, 128.0, 127.8, 127.1, 121.7, 120.6, 120.3, 104.8, 104.2, 103.1, 78.8, 77.4, 76.3, 74.7, 73.5, 73.4, 70.1, 70.0, 67.9, 67.6, 63.9, 63.6, 63.4, 62.0, 61.7, 60.7, 56.1, 54.7, 53.4, 40.0, 37.4, 36.9, 36.0, 35.9, 34.0, 31.7, 29.8, 17.7, 17.6, 17.3, 17.0, 16.9, 16.7, 16.0, 14.6, 14.2 ppm. HRMS (ESI) m/z calcd for C42H47N5O9 (M+Na) + 788.3271, found 788.3244. Macrocycle 30. TFA (2.0 mL) was added to the solution of 29 (26.3 mg, 0.0344 mmol) in dry CH 2 C1 2 (10 mL) at 0 ºC. The reaction was stirred room temperature for 1 h, then it was diluted with toluene (10 mL). The solvent was evaporated in vacuo and the residue was purified by preparative TLC plate (SiO 2 , developed by MeOH/CH 2 C1 2 5:95, Rf = 0.2) to afford product 30 (11.8 mg, 66%). [α] 20 D: -54.5 (c 0.08, MeOH). 1 H NMR (400 MHz, CDC1 3 , mixture of conformers, major/minor (2:1)): δ 8.24 (s, 0.67H), 7.95 (s, 0.33H), 6.49 (s, 0.67H), 6.27 (s, 0.33H), 6.06-5.96 (m, 1H), 5.80 (br m, 1H), 5.67 (q, J = 6.8 Hz, 0.67H), 5.29 (d, J = 10.0 Hz, 0.33H), 5.14 (s, 0.67H), 5.02 (br s, 1H), 4.96-4.92 (m, 0.33H), 4.60-4.36 (m, 5H), 4.20-4.07 (m, 2H), 3.74 (dd, J = 12.0, 7.2 Hz, 0.33H), 3.35 (dd, J = 10.4, 7.6 Hz, 0.67H), 3.23 (t, J = 11.6, 0.33H), 3.14-3.12 (m, 1.32H), 3.09-3.06 (m, 2.68H), 2.55-2.43 (m, 2H), 2.38-2.27 (m, 0.67H), 1.67-1.64 (m, 3.33H), 1.47-1.43 (m, 3H), 1.14-1.11 (m, 3H), 7-5.03 (m, 2H), 4.65 (d, J = 10.8 Hz, 1H), 4.45 (d, J = 10.8 Hz, 1H), 3.86-3.80 (m, 1H), 3.80 (s, 3H), 3.08 (dd, J = 7.2, 3.6 Hz, 1H), 2.31-2.20 (m, 2H), 1.87 (br m, 1H), 1.66 (ddd, J = 13.2, 9.2, 2.8 Hz, 1H),1.46-1.34 (m, 2H), 1.06 (d, J = 6.8 Hz, 3H),0.91 (s, 9H), 0.86 (s, 9H) ppm. 13 C NMR (100 MHz, CDC1 3 , mixture of conformers, major/minor (2:1)): δ 173.8, 173.6, 170.7, 170.5, 170.0, 169.4, 168.6, 168.4, 166.9, 166.3, 165.3, 134.3, 134.2, 131.2, 130.6, 122.4, 120.3, 104.8, 104.1, 78.2, 77.4, 70.7, 70.4, 63.4, 62.0, 61.8, 60.9, 56.2, 54.7, 40.0, 36.1, 35.3, 33.9, 32.0, 31.8, 29.8, 29.8, 17.9, 17.3, 17.1, 16.7, 15.8, 14.3 ppm. HRMS (ESI) m/z calcd for C 23 H 33 N 5 O 9 (M+H) + 524.2357, found 524.2341. Gatorbulin-1 (1a). PhSiH 3 (10 μL, 0.081 mmol) and Pd(PPh 3 ) 4 (2.43 mg, 0.0021 mmol, in degassed CH 2 C1 2 (0.5 mL) were added to the solution of 30 (11.0 mg, 0.021 mmol) in degassed dry CH 2 C1 2 (4.0 mL) sequentially at room temperature. The reaction flask was protected by aluminum foil and the reaction mixture was stirred at the room temperature for 1.5 h under argon. Then the reaction was concentrated under reduced pressure and the residue was purified by preparative reverse TLC plate (C18, developed by MeOH/H 2 O (1:1), Rf = 0.5). The product band was scraped and washed down by MeOH/CH 2 C1 2 (3:7). The washed down product fraction was concentrated to dryness under reduced pressure and rinsed with hexanes. The residue was purified again by reverse preparative cartridge (C18, 100 mg, Alltech, eluted by MeOH). The product fractions were collected, concentrated and dried to provide product 1a (6.6 mg, 65%) as off-white solid. The 1 H NMR and 13 C NMR were identical to natural product 1a (mixture of two conformers (1:1)). [α] 20 D : –119.2 (c 0.17, MeOH) (natural 1, [α] 20 D: – 84.0 (c 0.10, MeOH)). (Integrated two conformers separately) Conformer 1: 1 H NMR (600 MHz, DMF-d 7 ), δ 11.38 (br s, 1H), 8.28 (s, 1H), 7.28 (s, 1H), 7.09 (s, 1H), 6.46 (s, 1H), 6.07 (br, 1H), 5.51 (br d, J = 9.6 Hz, 1H), 5.47 (q, J = 7.2 Hz, 1H), 5.23 (s, 1H), 4.51 (br d, 1H), 4.41 (t, J = 7.8 Hz, 1H), 4.31 (m, 1H), 3.25 (dd, J = 10.2, 7.8 Hz, 1H), 3.09 (s, 3H), 2.58 (m, 1H), 2.53 (m, 1H), 1.58 (m, 1H), 1.53 (d, J = 6.6 Hz, 3H), 1.42 (d, J = 7.2 Hz, 3H), 1.12 (d, J = 6.6 Hz, 3H) ppm; 13 C NMR (150 MHz, DMF-d7), δ 175.1, 170.8, 170.5, 169.9, 165.9, 136.2, 101.9, 72.9, 68.8, 64.6, 62.6, 57.9, 56.9, 37.0, 34.7, 34.0, 18.0, 17.6, 15.6, 13.8 ppm. Conformer 2: 1 H NMR (400 MHz, d7-DMF): δ 10.61 (br s, 1H), 7.91 (s, 1H), 7.41 (s, 1H), 7.21 (s, 1H), 6.22 (s, 1H), 5.90 (br, 1H), 5.38 (q, J = 7.2 Hz, 1H), 5.22 (s, 1H), 5.07 (br, 1H), 4.93 (dd, J = 9.6, 7.2 Hz, 1H), 4.71 (br q, J = 7.2 Hz, 1H), 4.64 (br m, 1H), 4.29 (q, J = 6.6 Hz, 1H), 3.71 (dd, J = 11.4, 7.2 Hz, 1H), 3.13 (s, 3H), 3.11 (dd, J = 11.4, 11.4 Hz, 1H), 2.38 (m, 1H), 1.66 (m, 1H), 1.52 (d, J = 6.0 Hz, 3H), 1.40 (d, J = 6.6 Hz, 3H), 1.10 (d, J = 6.6 Hz, 3H) ppm; 13 C NMR (100 MHz, CDC1 3 ): δ 174.9, 170.4, 170.2, 169.7, 165.4, 166.8, 136.2, 72.2, 69.7, 63.8, 60.0, 55.8, 55.6, 40.8, 34.5, 32.4, 17.3, 16.9 ppm. HRMS (ESI) m/z calcd for C20H29N5O9 (M+H) + 484.2044, found 484.2028. (Integrated two conformers together): 1 H NMR (600 MHz, DMF-d7, mixture of conformers (1:1)): δ 11.38 (br s, 0.5H), 10.61 (br s, 0.5H), 8.28 (s, 0.5H), 7.91 (s, 0.5H), 7.41 (s, 0.5H), 7.28 (s, 0.5H), 7.21 (s, 0.5H),7.09 (s, 0.5H), 6.46 (s, 0.5H), 6.22 (s, 0.5H), 6.07 (br, 0.5H), 5.90 (br, 0.5H), 5.51 (br d, J = 9.6 Hz, 0.5H), 5.47 (q, J = 7.2 Hz, 0.5H), 5.38 (q, J = 7.2 Hz, 0.5H), 5.23 (s, 0.5H), 5.22 (s, 0.5H), 5.07 (br, 0.5H), 4.93 (dd, J = 9.6, 7.2 Hz, 0.5H), 4.71 (br q, J = 7.2 Hz, 0.5H), 4.64 (br m, 0.5H), 4.51 (br d, 0.5H), 4.41 (t, J = 7.8 Hz, 0.5H), 4.33-4.28 (m, 1H), 3.71 (dd, J = 11.4, 7.2 Hz, 0.5H), 3.25 (dd, J = 10.2, 7.8 Hz, 0.5H), 3.15- 3.11 (m, 2H), 3.09 (s, 1.5H), 2.60-2.50 (m, 1.5H), 2.41-2.34 (m, 0.5H), 1.69-1.63 (m, 1H), 1.60-1.56 (m, 1H), 1.53 (d, J = 6.6 Hz, 1.5H), 1.52 (d, J = 6.0 Hz, 1.5H), 1.42 (d, J = 7.2 Hz, 1.5H), 1.40 (d, J = 6.6 Hz, 1.5H), 1.12 (d, J = 6.6 Hz, 1.5H), 1.10 (d, J = 6.6 Hz, 1.5H) ppm; 13 C NMR (150 MHz, DMF-d7, mixture of conformers (1:1)): δ 175.1, 174.9, 170.8, 170.5, 170.4, 170.2, 169.9, 169.7, 169.4, 166.8, 165.9, 165.4, 136.2, 136.2, 101.9, 72.9, 72.2, 69.7, 68.8, 64.6, 63.8, 62.6, 60.0, 57.9, 56.9, 55.8, 55.6, 40.8, 37.0, 34.7, 34.5, 34.0, 32.4, 18.0, 17.6, 17.3, 16.9, 15.6, 13.8 ppm. HRMS (ESI) m/z calcd for C 20 H 29 N 5 O 9 (M+H) + 484.2044, found 484.2028. Qualitative test for Fe(III) chelating ability of GB1 (1a) We applied a solution of GB1 (1a) in CH 2 C1 2 to reverse TLC (C18) (MeOH/H 2 O 1:1, v/v), then dipped the TLC into FeCl 3 solution in ethanol (5% wt). The compound quickly caused an orange spot (positive reaction). References for Synthesis and Characterization 1. Sendai, M., Hashiguchi, S., Tomimoto, M., Kishimoto, S., Matsuo, T., Ochiai, M. Synthesis of carumonam (AMA-1080) and a related compound starting from (2R, 3R)- epoxysuccinic acid. Chem. Pharm. Bull.33, 3798-3810 (1985). 2. Koyama, J., Sugita, T., Suzuta, Y., Irie, H. Thermolysis of oxime O-allyl ethers: a new method for pyridine synthesis. Chem. Pharm. Bull.31, 2601-2606 (1985). 3. Shustov, G. V., Chandler, M. K., Wolfe, S. Stereoselective synthesis of multiply substituted [1,2] oxazinan-3-ones via ring-closing metathesis Can. J. Chem.83, 93–103 (2005). 4. Ley, S. V., Priour, A. Total synthesis of the cyclic peptide argyrin B, Eur. J. Org. Chem. 3995-4004 (2002). 5. Okeley, N. M., Zhu, Y., van der Donk, W. A. Facile chemoselective synthesis of dehydroalanine-containing peptides, Org. Lett.2, 3603-3606 (2000). 6. Mori, T., Higashibayashi, S., Goto, T., Kohno, M., Satouchi, Y., Shinko, K., Suzuki, K., Suzuki, S., Tohmiya, H., Hashimoto, K., Nakata, M. Total synthesis of siomycin A: completion of the total synthesis. Chem. Asian J.3, 984-1012 (2008). B. Biological Methods Cell Lines Isogenic HCT116 cell lines were generated and authenticated as described (S.Y. Chun, et al., Oncogenic KRAS modulates mitochondrial metabolism in human colon cancer cells by inducing HIF-1α and HIF-2α target genes. Mol. Cancer 9, 293 (2010)), and normal colon CCD841-CoN cells obtained from ATTC. HeLa cell lines were purchased from the American Type Culture Collection (ATCC) and authenticated by Genetica. MTT Cell Viability Assay Parental HCT116, HCT116 HIF-1α-/-HIF-2α-/- , HCT116 HIF-1α-/- , HCT116 HIF-2α-/- , HCT116 WT KRAS along with normal colon cell line CCD841-CoN were cultured in Dulbecco’s modified Eagle medium (DMEM, Life Technologies, USA) supplemented with 10% Fetal Bovine Serum (FBS, Sigma, USA) and maintained in 5% CO 2 at 37 ° C. HCT116 cells (8,000 cells/well) and normal cells (3,000 cells/well) were seeded in 96-well plates, allowed to attach overnight and then treated with different concentrations of GB1 (1a) or solvent control (0.5% DMSO). Cell viability was measured 48 h following treatment with MTT dye using manufacturer’s protocol (Promega). IC 50 was determined by non-linear regression analysis using GraphPad Prism 8. Data are represented as average ± SD (n = 3). HCT116 Cell Cycle Analysis HCT116 cells were seeded in 6-well plates (400,000 cells/well, DMEM/10% FBS and allowed to attach overnight prior to treatment with GB1. Media was replaced by fresh DMEM/10% FBS after 24 h of incubation and treated with compound and solvent control (0.25% DMSO) for 24 h. The medium in each well was collected separately, and the cells washed with 500 μL PBS and collected into the corresponding tubes. Cells were detached using trypsin (Invitrogen) and collected into the corresponding tubes and centrifuged at 400g for 10 min at 4 °C. The supernatant was discarded and the cell pellets were resuspended in 500 μL ice-cold PBS, centrifuged at 400g for 10 min at 4 °C and supernatant was discarded. The cell pellets were resuspended in 300 μL ice-cold PBS and 700 μL ice-cold EtOH was added slowly to each cell suspension with gentle pipetting. Cells were incubated at –20 °C overnight and centrifuged at 400g for 10 min at 4 °C next day. The EtOH/PBS was discarded and cells resuspended in 300 μL PBS containing 1 mM EDTA and 100 μg/mL RNase A (Invitrogen). The cells were incubated at 37 °C for 30 min, with shaking at 800 rpm, followed by addition of 1 μL propidium iodide (1 mg/mL, Invitrogen). Fluorescence from propidium iodide–DNA complexes were quantified using FACScan (BD Biosciences LSRFortessa) and data were analyzed using ModFit LT (Verity Software House). RNA Isolation, Reverse Transcription and Quantitative Polymerase Chain Reaction HCT116 cells were seeded in 6-well plates at a density of 4 × 10 5 per well and incubated overnight for cells to attach. Cells were treated with 3.2 µM GB1 or 0.25 % DMSO (vehicle). RNA was isolated 16 h later using the RNeasy mini kit (QIAGEN, Valencia, CA). Total RNA was quantified using NanoDrop 2000. cDNA synthesis was carried out using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) and oligo (dT) (Invitrogen) from 2 μg of total RNA. The qPCR after reverse transcription (RT-qPCR) was performed on a 25 μL reaction solution containing a 0.3 μL aliquot of cDNA, 12.5 μL of TaqMan gene expression master mix, 1.25 μL of probes, and 11 μL RNase-free water. qPCR was carried out on an ABI 7300 sequence detection system using the thermocycler program: 2 min at 50 °C, 10 min at 95 °C, and 15 s at 95 °C (40 cycles) and 1 min at 60 °C. Experiments were performed in triplicate. Probes for target gene: VEGFA (Hs00900055_m1) and for endogenous control: β-actin (Hs99999903_m1). Statistical analysis for comparison between treatment and vehicle group was done using a student t test ( * p < 0.05). Tubulin polymerization assay: Tubulin polymerization was performed using fluorescence based in vitro polymerization kit (Cytoskeleton, #BK011P) per the manufacturer recommendation. Briefly, the provided tubulin was reconstituted to 10 mg/mL stock, as recommended, and used at final concentration of 2 mg/mL in 80 mM PIPES pH 6.9, 2.0 mM MgC1 2 , 0.5 mM EGTA, 1.0 mM GTP and 15% glycerol for the assays. The compound was prepared in 3-fold dilutions and kept on ice until the assay was started. Buffer components and tubulin stocks were kept on ice until use. The assay plate was pre-warmed at 37 ºC for 10minutes and the compounds were added to the plate wells at 5uL (<10% DMSO) for 1 minute to warm. Fifty microliters of tubulin reaction mix (2 mg/mL in 80 mM PIPES pH 6.9, 2.0 mM MgC1 2 , 0.5 mM EGTA, 1.0 mM GTP and 15% glycerol) was added to each well and mixed gently with the inhibitor to avoid bubble formation. The plate was read on FlexStation3 reader (Molecular Devices) at 360 nm excitation/450 nm emission, every minute for 1 hours at 37 °C. Decrease in fluorescent signal indicated inhibition of polymerization. C. Description of the Examples There is continued demand for new microtubule-targeting agents, ideally against novel binding sites, since anticancer efficacy and the pharmacological effects vary depending on the chemical scaffold and binding site. Current approved drugs and other known tubulin agents bind to six distinct sites at the α/β-tubulin heterodimer. We have discovered a new microtubule-destabilizing cyclodepsipeptide with unique chemotype from a marine cyanobacterium, termed gatorbulin-1. The chemical structure was elucidated through multidimensional characterization. The structure was determined by 1 H, 13 C and 15 N NMR and mass spectrometry, revealing the modified pentapeptide possessing a functionally-critical hydroxamate group, and developed a total synthesis to solve the supply issue. We probed the pharmacology using isogenic cancer cell screening, extensive cellular profiling, and complementary phenotypic assays. The unique chemical structure and observed pharmacology suggested that GB1 might interact with tubulin in a distinct way and different from other agents. Results Isolation and structure determination. Various collections of the cyanobacterium Lyngbya cf. confervoides during blooms off the coast near Ft. Lauderdale 11 (Broward County) were extracted with EtOAc–MeOH (1:1). The extracts, previously proven to be rich in secondary metabolites and possessing antifeedant activity 12,13 , w were either solvent-solvent partitioned first or directly applied onto a Diaion HP-20 column and then fractions subjected to reversed- phase HPLC to afford gatorbulin-1 (GB1) as an optically active white solid ([α] 20 D –84.0 (c 0.10, MeOH)) and as the most antiproliferative extract component by bioassay-guided isolation using colon cancer cells, along with a minor analogue, gatorbulin-2 ( FIG.19). Doubling of virtually every signal in the 1 H NMR spectrum of gatorbulin-1 recorded in DMF- d 7 suggested the presence of an asymmetric dimer or the presence of conformers in a ratio of 1:1. This observation coupled with the [M + H] + ion peak at m/z 484.2043 obtained by HRESI/APCIMS and 13 C NMR data suggested a molecular formula of C 20 H 29 N 5 O 9 (calcd for C 20 H 30 N 5 O 9 , 484.2044) and consequently the presence of conformers in the NMR solvent. NMR analysis using 1 H NMR, 13 C NMR, COSY, HMQC, and 1 H– 13 C HMBC data was carried out for both conformers, revealing two sets of five spin-coupled systems as part of a pentapeptide structure; one signal set appeared slightly broader (FIG.18 FIG.20). For both signal sets, one putative NH singlet each (δH 8.28 and 8.60) showed COSY correlations to sp 2 -methylene protons (δH 6.46/5.22 for conformer 1, 6.22/5.11 for conformer 2) which also appeared as singlets in the 1 H NMR spectrum. Correlations from the NH to the corresponding olefinic methylene carbon (δC 101.8 and 103.0), to a quaternary sp 2 hybridized carbon (δC 136.3 and 136.2) and to two carbonyl carbons (δC 165.9/170.5 for conformer 1 and 166.8/170.2 for conformer 2) defined the first unit as a dehydro-alanine (DhAla) residue (FIGs.18 and 19) as a dehydro-alanine (DhAla) residue (FIG.18, FIG.20). The second multi-proton spin system consisted of two methylenes (δH 4.31/3.25, 2.58/1.58 for conformer 1), two methines (δH 4.42, 2.53) and one methyl group (δH 1.12 d). COSY analysis established their arrangement supported by HMBC data (FIG.18, FIG.20). The terminal methine and methylene carbons of this spin system appeared to be nitrogenated (δC 62.6, 56.9) and the HMBC correlation of one of the methylene protons (δH 4.31) to one of the methine carbons (δC 62.6) clarified that the carbons were joined in a 3-methyl pyrrolidine structure (FIG.20), which upon further analysis of HMBC correlations to a carbonyl carbon (δH 170.8) identified the second residue as a 4-methylproline unit (4-MePro). In a similar fashion, a signal set corresponding to the second conformer for this unit was unambiguously identified (FIG.18). Analysis of the third spin system was straightforward and this unit consisted of only one methine and a methyl group and NMR data (FIG.18 and FIG.20), consistent with an acylated lactic acid (Lac) moiety (FIG.19). Two singlets of another signal set for heteroatom-bound protons (δH 7.26/7.09 for conformer 1 and δ H 7.39/7.21 for conformer 2) showed cross-peaks in the COSY spectrum, suggesting a primary amide. Another set of singlets at δ H 3.09 and 3.14 was indicative of an N-methyl tertiary amide group; expectedly these signals exhibited HMBC correlations with a carbonyl carbon of the adjacent residue (δ C 169.9 and 169.7) and for conformer 1 also to the α-carbon of the N-methylated amino acid (δ C 58.0). Rigorous 2D NMR analysis established the second unit as an N(α)-methyl-β-hydroxy-asparagine (N(α)-Me-β-OH-Asn). Even though the significant broadening of all signals for this unit for the second conformer resulted in fewer HMBC correlations, all 1 H and 13 C NMR resonances could be assigned except for the α-carbon since its NMR signal was too broad to be observed (FIG.18). The last unit exhibited similarity to a lactic acid or alanine moiety, yet the α-carbon resonated at higher field than the corresponding carbon for lactic acid (δ C 64.7 and 60.0) and thus more likely bore a nitrogen atom, which then in turn had to bear a substituent that was not accounted for yet. This NMR analysis so far led to the assignment of all atoms except one oxygen and hydrogen based on HRMS analysis. In the 1 H NMR spectrum, the only unassigned signal at this point was a signal for an exchangeable proton at δH 11.35 (br s) for conformer 1 and at δH 10.58 (br) putatively for conformer 2, which could not be rationalized by a secondary amide since it did not show a COSY correlation to the nitrogen-bearing methine while also resonating too far downfield. The chemical shifts were consistent with carboxylic acid protons which could exist in a linear structure; however, it would not leave a substituent for the nitrogen atom in the alanine-like moiety. Therefore, a bond between two heteroatoms, nitrogen and oxygen, had to be invoked which led us to propose a N-hydroxy group in a cyclic hydroxamate (N-OH-Ala); its hydroxy proton was also expected to resonate between δH 10–12 as observed. The doubling and overlap of signals for several carbonyl carbons for different conformers slightly complicated the sequencing of the individual units. To ultimately prove the existence of the hydroxamate and to validate the nature of the nitrogen atoms we carried out a 1 H– 15 N HMBC analysis (FIG.18). Correlations of the H-3a/b methylene protons of the DhAla unit to a nitrogen atom resonating at δN –258.2 (relative to external MeNO2, δN 0.0) supported the earlier assignment of a secondary amide (FIGs.20-21). The N-Me protons of the N-Me-3-OH-Asn showed HMBC correlations to a nitrogen possessing a chemical shift of δN –273.8, an expected value for a tertiary amide (FIGs.20-21). Most importantly and confirmatory for the hydroxamate moiety were two- and three-bond correlations from the α- methine and the β-methyl protons to a signal at δN –202.6 (FIGs.20-21) which is in agreement with literature values for hydroxamate nitrogens (δN –199.2 for polyoxypeptin A 14 ), further validating the proposed structure for GB1 (1a). The 1 H– 15 N HSQC spectrum, in addition to the secondary amide proton for DhAla (δ N –258.2), also showed one-bond correlations for both protons of the primary amide functionality for both conformers (δN – 280.5 and –279.6, FIG.21). The presence of the hydroxamate functionality was supported analytically by ferric hydroxamate complex formation 15 . Further support for the proposed structure was found with the isolation of its N- deoxy-derivative, termed gatorbulin-2 (GB2), or N-deoxy-gatorbulin-1 (FIG.19). The 1 H NMR spectrum was strikingly similar to the one of GB1, including the presence of conformers in a 1:1 ratio in DMF-d 7 . The most significant difference appeared to be the lack of the N-OH protons at δ H 10–12; instead a new set of doublets appeared in the range for amide protons (δ H 8.21 and 8.38 for conformers 1 and 2). To establish the absolute configuration, we performed acid hydrolysis to liberate the individual units and synthesized all isomers of the amino acid standards for comparative chiral HPLC analysis and advanced Marfey’s analysis. The 4-MePro standards were prepared as described previously 16 and the N(α)-Me-β-OH-aspartic acid stereoisomers as described as provided herein. We detected L-erythro-N(α)-Me-β-OH-Asp, (2S,4S)-4-Me-Pro, and L-Lac in the hydrolyzate of 1a. The structures and identical absolute configurations were confirmed by conversion of 1a into 1b via TiCl3-mediated reduction (FIG.19) 17 . Upon acid hydrolysis, 1b yielded L-Ala as detected by chiral HPLC analysis, establishing the remaining stereogenic center. Synthesis of gatorbulin-1. To prove the structure and overcome the supply issue, we embarked on the total synthesis. The retrosynthetic analysis of total synthesis of gatorbulin-1 (GB1) is shown in FIG.22. The final product GB1 could be obtained from the fully masked cyclized precursor 2 by sequential deprotection. The site between 4-MePro and (Se)- phenylselenocysteine (Sec(Ph)) was chosen for macrolactamization. In linear precursor 3, Fmoc-Fm pair was designed as the protection groups of amino and carboxy termini, respectively, which could be cleaved simultaneously with base to provide the precursor of macrocyclization 20 . Sec(Ph) was proposed as the pro-unit of DhAla 21 . Linear compound 3 was disconnected into four building blocks 4–7, which could be constructed from commercially available reagents (e.g., 6 from 8 and 9) using established or modified protocols. FIG.23 depicts the synthetic route to gatorbulin-1 (GB1). The synthesis of acid 4 was adopted from published proedures 22 . (2R,3R)-epoxysuccinic acid (10) was converted to erythro-N(α)-methyl-3-hydroxy-L-aspartic acid (11) by treatment with methylamine-water under reflux. Then 11 was selectively esterified with acidic methanol under refluxing 23 to provide monoester 12. Without purification, aminolysis of 12 with ammonia (gas) in MeOH provided N(α)-methyl-β-hydroxy-asparagine (13) 22,23 , which had poor solubility in MeOH, so that pure product could be obtained by simple filtration. Sequential protections of the groups of 13 using standard methods 24 provided full masked compound 17. Finally, acid 4 was obtained from 17 by hydrogenation with palladium catalyst. The synthesis of building block of allyloxamine 5 adopted the triflate method 25 . Allyl was chosen as NOH protecting group as it could be selectively removed by Pd(Ph 3 P) 4 in the presence of dehydropeptide. Acid 6 was synthesized from (4S)-N-Boc-4-methyl-Pro (9) and benzyl-L-lactate (8) by standard protocols of esterification, protection and deprotection. Following established procedures 21 , N-Boc-serine (22) was converted to BocSec(Ph) (24) via β-lactone 23, which was esterified with FmOH to provide building block 7. Dehydroalanine could be obtained from phenylselenocysteine by oxidative β-elimination. The fusion of building blocks was initiated by coupling of acid 4 with allyloxamine 5. Acid 4 was activated to the acid chloride, which was then coupled with 5 in presence of AgCN 26,27 to provide 25 in 50% yield and 85% yield based on recovered starting materials (BRSM). This acylation was not successful when other common coupling reagents were used because of poor nucleophilicity of the nitrogen and high steric hindrance 28 . Compound 26 was obtained in 78% yield by BEP-mediated coupling 29 of acid 6 with the free methyl amine generated by selective deprotection of 25. Selective removal of t-butyl group of 26 by TMSOTf/2,6-lutidine 30 afforded acid 27, which was coupled with free amine from 7 using BOP as coupling reagent to yield the linear compound 3 in 86% yield. The use of the t-butyl protecting group prevented diketopiperazine 31 formation upon coupling of 6 and 25, and ensured that trityl and TBS groups were intact for the generation of acid 27. Both Fmoc and Fm protection groups were removed simultaneously when compound 3 was exposed to Et2NH in MeCN. The macrocyclization was mediated by PyBOP/HOAt to give macrocycle 2 in 60% yield for two steps. Removal of TBS of 2 with the TBAF/HOAc buffer 24,32 provided 28, and subsequent oxidation of SePh with NaIO 4 21 yielded dehydropeptide 29. Trityl was removed with TFA in CH 2 C1 2 33 to yield primary amide 30. The removal of allyl group by Pd(PPh3)4/PhSiH3 32,34 provided final product 1a. The removal sequence for trityl and allyl groups are interchangeable; however, the yield would drop from 66% to 35%. GB1 (1a) was purified by reverse TLC plate (C18) with acceptable purity. The synthetic sample was identical to the isolated natural product, which was verified by NMR, HRMS and optical rotation (natural 1a: [α] 20 D –84.0 (c 0.10, MeOH); synthetic 1a: [α] 20 D –119.2 (c 0.17, MeOH). HIF-selectivity and cellular profiling identifies the mechanism of action. GB1 (1a) was identified as the extract’s active component against colon cancer cells and showed an IC50 of 0.80 µM (MTT assay) against HCT116 colorectal cancer cells (FIG.24, while GB2 (1b) was inactive at the highest concentration tested (IC50 > 10 µM), indicating that the hydroxamate moiety is indispensable to the antiproliferative activity. Isogenic cell line selectivity screening indicated preferential activity against parental HCT116 compared with the oncogenic KRAS knockout or double knockout of both HIF-1α and HIF-2α transcription factors (FIG.24 7 . Deconvolution using single knockouts of HIF-1α and HIF-2α clearly demonstrated that only cells depleted in HIF-1α had reduced susceptibility to GB1 (1a), which is consistent with HIF-1α being in the same pathway and activated by oncogenic KRAS. Furthermore, normal epithelial colon cells (CCD841CoN), were less inhibited indicating an additional promising level of selectivity (FIG.24). The preference for HIF-1α expressing cells parallels the selectivity profiles for microtubule agents we previously discovered (dolastatins 10 and 15) 4,7. DNA content analysis revealed the concentration-dependent G2/M cell cycle accumulation characteristic for antimitotic agents (FIG.25). A concentration of GB1 (4 x IC50) that results in a complete antiproliferative response, initiated a concomitant downregulation of the HIF-1α target gene VEGFA in parental HCT116 cells (FIG.26), even more pronounced than for dolastatin 15 4 , which also elevated its potential as antigangiogenic agent. Taken together, this reveals an antiangiogenic role of the compound in inhibiting the formation and stabilization of a three-dimensional vascular network. The additional VEGF downregulation in growth factor secreting cells is expected to more pronounced angiogenic action. The NCI-60 screen data (FIG.27), analyzed by the COMPARE algorithm 19,34b , were indicative of a cytotoxicity profile most related to antimitotic/tubulin agents, including paclitaxel, eribulin, colchicine and vinca bis-indole alkaloid derivatives (P 0.75–0.85), suggesting that the biochemical mechanisms of action are related. While GB1 displayed an IC50 > 10 µM against normal mucosal colon cells, it had submicromolar activity against HCT116 cells (GI50306 nM) and was even more potent against COLO205 cells (GI5092 nM) based on the NCI-60 data (sulforhodamine B assay). Other susceptible cell types included certain melanoma (SK-MEL-5), ovarian (OVCAR-3) and prostate (DU-145) cancer cells, which correspond to certain cancers where microtubule agents have been successful. Furthermore, cervical and breast cancers are relevant indications, prompting additional studies in these cell types 35 . Target identification and inhibition of tubulin polymerization by gatorbulin-1. First, using a biochemical assay, we demonstrated that gatorbulin-1 (1a) directly inhibits tubulin polymerization in vitro (FIG.28). We then measured the caspase-3/7 activity in a concentration- and time-dependent manner, validating that treatment with GB1 induces apoptosis at concentrations that affect tubulin dynamics in cells, especially after 24 h and beyond, similar to CA-4 (FIG.29). Discussion We took an integrated approach towards natural products drug discovery by targeting minor, highly bioactive compounds from a chemically prolific cyanobacterium, combining innovative screening and rigorous bioassay-guided isolation and structure determination with chemical synthesis to overcome the supply problem, identified tubulin as direct target and performing achieving in depth-mechanistic studies as well as direct target and binding site identification. We advocated for such an approach to fully exploit the proven potential of natural products and increase the value of bioactive natural products 42 . The ultimate key for a successful natural product drug discovery campaign is the choice of the source organism. We have been focusing on marine cyanobacteria, which are prolific yet underexplored marine prokaryotes with a tremendous biosynthetic potential. The gatorbulin-yielding sample was derived from a blooming “superproducer” of secondary (specialized) metabolites (natural products) that previously yielded lyngbyastatins 4-6, pompanopeptins A and B, tiglicamides, largamides/largamide D oxazolidine, most of which are noncytotoxic serine protease inhibitors 12,13,43-45 . Beyond showcasing the biosynthetic capacity of marine cyanobacteria, our discovery of gatorbulin-1 exemplifies that marine cyanobacterial natural products occupy therapeutically relevant chemical space that could lead to the discovery of new biology, chemical tools or even drug leads. Gatorbulin-1 is a small (MW < 500 g/mol) cyclodepsipeptide, unique from most cyanobacterial modified peptides or peptide-polyketide hybrids, which dominate the landscape of bioactive natural products produced by marine cyanobacteria 49 . GB1 is densely functionalized with all amino acids being modified and the presence of one hydroxy acid. Natural produces possessing all of these unusual structural features of GB1(i.e., the hydroxamate, C-hydroxylated and dehydro-amino acids, modified proline) have not been reported. The 4-methylproline residue is a rare feature but has been previously found in cyanobacterial natural products 16 . Interestingly, the hydroxamate group that is typical for metal chelators (especially ion siderophores) and present in other antiproliferative compounds 46 , plays an iron-dependent functional role in GB1’s binding to tubulin as the major mechanism of antiproliferative action. The additional metal binding ability potentially increases the pharmacological complexity of GB1 and remains to be investigated. However, the iron complexing ability due to the hydroxamate functionality was proven but appeared not to play a predominant role with respect to the antiproliferative activity, based on the NCI-60 profile and cell cycle arrest characteristic for tubulin targeting agents. We neither found the metal was necessary for GB1 binding to tubulin and the inhibition of tubulin assembly into microtubules in our in vitro assays. We previously reported the discovery of the microtubule-destabilizers dolastatins 10 and 15 2-4 , which bind to the vinca domain, as indirect HIF inhibitors in our cell-based phenotypic screens. Dolastatin 10 originated from a new genus of marine cyanobacteria 2,3,47 , further validating the unexplored genetic diversity of cyanobacteria that translates into chemical diversity. HIF inhibition likely contributes to the overall anticancer activity of most microtubule binding drugs, including GB1 48 . Gatorbulin-1 is a cyclic depsipeptide that represents a new chemotype that differs from other peptides targeting tubulin such as dolastatin 10 and possesses low toxicity and molecular weight, adding to its promising small-molecule, drug-like properties and translational potential. References for Description of the Examples 1. Risinger, A. L. & Du, L. Targeting and extending the eukaryotic druggable genome with natural products: cytoskeletal targets of natural products. Nat. Prod. Rep.37, 634–652 (2020). 2. Luesch, H., Moore, R. E., Paul, V. J., Mooberry, S. L. & Corbett, T. H. Isolation of dolastatin 10 from the marine cyanobacterium Symploca species VP642 and total stereochemistry and biological evaluation of its analogue symplostatin 1. J. Nat. Prod.64, 907–910 (2001). 3. Salvador-Reyes, L. A., Engene, N., Paul, V. J. & Luesch, H. Targeted natural products discovery from marine cyanobacteria using combined phylogenetic and mass spectrometric evaluation. J. Nat. 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EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended with be encompassed by the following claims.