AstraZeneca, Pharmaceutical Sciences, Silk Road Business Park, Macclesfield, SK10 2NA, UK

Supporting Information The Development and Scale-Up of an ADC Tubulysin Payload Jeremy S Parker, *, Marc McCormick, David W Anderson, Beatrice A Maltman, Lakshmaiah Gingipalli and Dorin Toader AstraZeneca, Pharmaceutical Sciences, Silk Road Business Park, Macclesfield, SK10 2NA, UK Almac Sciences, 5 The Fleming Building, Edinburgh Technopole, Milton Bridge, Midlothian, EH26 0BE, UK AstraZeneca, R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA Page 2. Large scale preparation of Fmoc-Ile-F 3. Synthesis of (2R,4R)-1,4-dimethylpiperidine-2-carboxylic acid (7) 5. Initial Synthesis of N-Boc N-Et Tuv (8c) 24. Improved Synthesis of N-Et Tuv (8a) 34. Improved Synthesis of N-Fmoc N -Alloc Tup (9b) 53. Improved Synthesis of mc-lys-mmeta (6) 62. Analysis of Impurities

Large scale preparation of Fmoc-Ile-F A solution of Fmoc-Ile-OH (3.50kg, 9.85mol) and pyridine (780g, 9.86mol) in dichloromethane (30L) was cooled to 0 C and a solution of DAST (1975g, 11.88mol) in dichloromethane (5.25L) added over 10min. The reaction mixture was then warmed to 25 C and stirred for 3.5h. The reaction mixture was then transferred to reactor containing water (53L) at 0 C. The biphasic mixture was stirred then the layers separated. The organic phase was washed with water (53L), dried over magnesium sulphate and the solvent removed in vacuo affording Fmoc-Ile-F (3.40kg, 96%) as an off-white solid which was used without further purification.

Synthesis of (2R,4R)-1,4-dimethylpiperidine-2-carboxylic acid (7) Paraformaldehyde (204g, 6.79mol) was added to a solution of (2R,4R)-4-methylpiperidine-2- carboxylic acid (84.5g, 590mmol) in water (792mL) and MeOH (792mL). The mixture was stirred at 60 C for 48h. The reaction mixture was then added to a hydrogenation vessel and wet Pd/C (5%w/w, 56.5g, 26.5mmol) was added. The reaction mixture was stirred at 20-25 C under H 2 (3bar) for 65h. The paraformaldehyde and Pd catalyst were removed by filtration through a pad of celite which was washed with MeOH (250mL). The filtrate was concentrated in vacuo and stripped with toluene (3x264mL). The resulting solid was washed with water (211mL), stripped with toluene (3x264mL) and dissolved in DCM (110mL) and MeOH (110mL). Filtration through alumina and concentration in vacuo afforded a white solid. This was dried at 40ºC for 3h to afford the product (7, 55.5g, 60% yield) as a white solid. 1 H NMR (500 MHz, D 2 O/DMSO) 1.02 (d, J = 6.2 Hz, 3H), 1.53 (s, 3H), 1.66 1.94 (m, 3H), 2.03 (s, 1H), 2.84 (s, 3H), 3.03 3.20 (m, 1H), 3.42 (s, 1H), 3.61 (s, 1H).

Initial Synthesis of N-Boc N-Et Tuv (8c) Preparation of 2-((1R,3R)-1-acetoxy-3-(tert-butoxycarbonyl(ethyl)amino)-4- methylpentyl)thiazole-4-carboxylic acid (8c) HPLC Analysis: Column: ZORBAX SB-CN, 4.6x150mm, 3.5μm Mobile phase A: 0.025% HCOOH in water Mobile phase B: 0.025% HCOOH in acetonitrile Gradient: Time %A %B 0 55 45 20 5 95 Flow rate: 1.0 ml/min Detection: UV @ 210 nm Temperature: 30 C

Preparation of (R)-3-(tert-Butoxycarbonylamino)-4-methylpentanoic acid H 2 N CO 2 H Boc 2 O BocHN CO 2 H Boc 2 O (21.5kg, 98.8mol) was added dropwise to a suspension of (R)-3-amino-4-methyl pentanoic acid (10.0kg, 76mol) and Na 2 CO 3 (16.1kg, 152mol) in acetone (50L) and water (100L) with stirring at room temperature. The reaction mixture was stirred overnight and the organic solvent was removed under reduced pressure. The residue was diluted with water (100L) and the resulting solution was washed with EtOAc/hexane (1:5, 50L 3). The aqueous phase was acidified with 2N aqueous HCl (to ph=3) and the resulting mixture was extracted with EtOAc (80L 3). The combined extracts were washed with brine (80L 1), dried over anhydrous Na 2 SO 4 and concentrated to afford compound the product (18.3kg, 100% yield) as an oil, which was used in the next step without further purification. Preparation of (R)-tert-Butyl 1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-3-ylcarbamate BocHN CO 2 H HNMe(OMe) EDCI BocHN O N O Triethylamine (7.7kg, 10.5L, 76mol) was added to a suspension of (R)-3-(tertbutoxycarbonylamino)-4-methylpentanoic acid (18.3kg, 76mol) and N,Odimethylhydroxylamine hydrochloride (4.78kg, 76mol) in CH 2 Cl 2 (100L) with stirring at 0 C. The suspension was stirred for 0.5h and EDCI (14.6kg, 76mol) was added in portions at 0 C. The reaction mixture was stirred for 8h at 0 C and water (80L) was added. The organic phase was separated and washed with 5% aqueous KHSO 4 (80L 3), saturated aqueous NaHCO 3 (80L 3) and brine (80L 1). The organic solution was dried over anhydrous Na 2 SO 4 and concentrated to dryness. The residue was purified by flash column chromatography on silica gel (EtOAc:Hexane, 1:10 to 1:5) to afford the product (15.1kg, 90% area HPLC purity, 72% yield) as an oil. HPLC analysis of (R)-tert-butyl 1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-3- ylcarbamate: Identity: (R)-tert-butyl 1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-3-ylcarbamate 8.753 minutes

Preparation of (R)-tert-Butyl ethyl(1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-3-yl) carbamate BocHN O N O EtI, NaH N Boc O N O Iodoethane (4.4kg, 28mol) was added to a solution of (R)-tert-butyl 1- (methoxy(methyl)amino)-4-methyl-1-oxopentan-3-ylcarbamate (960g, 3.5mol) in DMF (10L) with stirring at 0 C. Then NaH (60% suspension, 280g, 7.0mol) was added in portions at 0 C and the reaction mixture was allowed to warm to room temperature and stirred for 12 h. The reaction was quenched carefully with water (2L). Another fourteen batches (960g of (R)-tert-butyl 1-(methoxy(methyl)amino)-4-methyl-1- oxopentan-3-ylcarbamate for each batch) of this reaction were run following the same procedure and all fifteen batches were combined for a single work-up. The solvent was removed under reduced pressure and the residue was partitioned between EtOAc (200L) and water (200L). The organic phase was separated and washed with 5% aqueous KHSO 4 (100L 3), saturated aqueous NaHCO 3 (100L 3) and brine (100mL 1). The organic solution was dried over anhydrous Na 2 SO 4 and concentrated to dryness. The residue was purified by flash column chromatography on silica gel (EtOAc:Hexane, 1:20 to 1:10) to afford the product (5.5kg, >95% area HPLC purity, 34% yield) as an oil. 1 H NMR (400 MHz, CDCl 3 ): δ 3.69 (s, 3H), 3.62 (m, 1H), 3.10-3.30 (m, 5H), 2.60-2.95 (m, 2H), 1.95-2.15 (m, 1H), 1.40-1.47 (m, 9H), 1.10 (m, 3H), 0.92 (m, 6H). HPLC analysis of (R)-tert-butyl ethyl(1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-3-yl) carbamate: Identity: (R)-tert-butyl ethyl(1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-3-yl) carbamate 9.606 minutes

Current Data Parameters NAME AZ13669353-Int-3-XKS-1723P57 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20140804 Time 10.37 INSTRUM spect PROBHD 5 mm PABBO BB/ PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 16 DS 2 SWH 8012.820 Hz FIDRES 0.122266 Hz AQ 4.0894465 sec RG 32.27 DW 62.400 usec DE 6.50 usec TE 298.0 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== SFO1 400.1324710 MHz NUC1 1H P1 10.00 usec PLW1 16.00000000 W F2 - Processing parameters SI 65536 SF 400.1299983 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 7.288 5.305 3.690 3.620 3.242 3.222 3.205 3.171 3.125 3.107 3.089 3.072 2.957 2.921 2.896 2.662 2.124 2.045 1.969 1.474 1.440 1.277 1.260 1.242 1.127 1.110 1.092 1.052 0.937 0.920 0.914 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 3.03 0.91 4.48 1.08 1.05 1.36 9.00 3.15 6.11

Preparation of Ethyl 2-bromothiazole-4-carboxylate H 2 N S N OEt O NaNO 2, CuBr 2 Br S N OEt O tert-butyl nitrite (628g, 730mL, 6.1mol) was added dropwise to a suspension of CuBr 2 (2.6kg, 11.6mol) and ethyl 2-aminothiazole-4- carboxylate (1.0kg, 5.8mol) in acetonitrile (8L) at 0 C over 3h. The reaction mixture was allowed to warm to room temperature and stirred for 12h. The reaction was quenched with EtOAc (8L) and 10% aqueous ammonia (8L). Another nine batches (1.0kg of ethyl 2-aminothiazole-4- carboxylate for each batch) of this reaction were run following the same procedure and all ten batches were combined for a single work-up. The resulting mixture was filtered and the two phases were separated. The aqueous phase was extracted with EtOAc (80L 2). The combined organic extracts were washed with 10% aqueous ammonia (50L 2), 5% aqueous KHSO 4 (50L 3), saturated aqueous NaHCO 3 (50L 3) and brine (50L 1). The organic solution was dried over anhydrous Na 2 SO 4 and concentrated to a volume of ~30L under vacuum. The solution was cooled to room temperature and hexane (200L) was added dropwise. The mixture was stirred for 3h and the resulting precipitate was collected by filtration, washed with hexane (10L 3) and dried at 50 C to afford the product (6.8 kg, 50% yield) which was used in the next step without further purification.

Preparation of 2-Bromo-4-((tert-butyldimethylsilyloxy)methyl)thiazole Br S N OEt O 1. NaBH4 2. TBSCl Br S N OTBS NaBH 4 (1.65kg, 57.6mol) was added in portions to a solution of ethyl 2-bromothiazole-4- carboxylate (6.8kg, 28.8mol) in ethanol (50L) at 50 C over 0.5h with stirring. The suspension was heated under reflux for 5h. The mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was dissolved in CH 2 Cl 2 (100L) and the resulting solution was washed with saturated aqueous NaHCO 3 (30L 3) and brine (30L 1), dried over anhydrous Na 2 SO 4 and concentrated to dryness to afford the corresponding alcohol (ca 10kg). The alcohol was dissolved in DMF (30L) and imidazole (2.83kg, 41.6mol) was added. Then a solution of TBSCl (6.3kg, 41.6mol) in THF (10L) was added dropwise at room temperature. The reaction mixture was stirred for 12h and water (80L) was added. The resulting mixture was extracted with EtOAc (80L 3). The combined organic extracts were washed with 5% aqueous KHSO 4 (100L 3), saturated aqueous NaHCO 3 (100L 3) and brine (50L 1). The organic solution was dried over anhydrous Na 2 SO 4 and concentrated to dryness. The residue was purified distillation under reduced pressure (bpt 130-140 C/13.3pa) to afford the product (4.5kg, >95% area HPLC purity, 51% yield) as an oil. 1 H NMR (400 MHz, CDCl 3 ): δ 7.02 (s, 1H), 4.71 (s, 2H), 0.83 (s, 9H), 0.00 (s, 6H). HPLC analysis of 2-bromo-4-((tert-butyldimethylsilyloxy)methyl)thiazole: Identity: 2-bromo-4-((tert-butyldimethylsilyloxy)methyl)thiazole 7.987 minutes

Current Data Parameters NAME PTC1448-ZYT-C EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20140721 Time 19.54 INSTRUM spect PROBHD 5 mm PABBO BB/ PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 16 DS 2 SWH 8012.820 Hz FIDRES 0.122266 Hz AQ 4.0894465 sec RG 32.27 DW 62.400 usec DE 6.50 usec TE 298.0 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== SFO1 400.1324710 MHz NUC1 1H P1 10.00 usec PLW1 16.00000000 W F2 - Processing parameters SI 65536 SF 400.1300501 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 7.159 7.024 4.706 0.828-0.000 7 6 5 4 3 2 1 0 ppm 1.00 2.12 9.56 6.81

Preparation of (R)-tert-Butyl ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-4- methyl-1 -oxo pentan-3-yl)carbamate Br N OTBS nbuli S N Boc O N O N Boc O S N OTBS n-buli (1714mL, 2.5N in hexanes, 4.3mol) was added dropwise to a solution of 2-bromo-4- ((tert-butyldimethylsilyloxy)methyl)thiazole (1.06kg, 2.5mol) in THF (5L) at -78 C under N 2 with stirring over 5h. The suspension was stirred for 30min and then a solution of (R)-tertbutyl ethyl(1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-3-yl) carbamate (1.2kg, 3.9mol) in THF (2L) was added dropwise over 30min at -78 C. The reaction mixture was stirred for 1h at -78 C and then allowed to warm to room temperature and stirred for 12h. Another three batches (1.2kg of (R)-tert-butyl ethyl(1-(methoxy(methyl)amino)-4-methyl-1- oxopentan-3-yl) carbamate for each batch) of this reaction were run following the same procedure and all four batches were combined for a single work-up. The combined reactions were quenched with 20% aqueous ammonium chloride (4L) and the organic solvent was removed under reduced pressure. The remaining mixture was extracted with EtOAc (5L 3). The combined organic phases were washed with 5% aqueous KHSO 4 (8L 3), saturated aqueous NaHCO 3 (8L 3) and brine (8L 1). The organic solution was dried over anhydrous Na 2 SO 4 and concentrated to dryness. The residue was purified by flash column chromatography on silica gel (EtOAc:Hexane, 1:30 to 1:10) to afford the product (4.2kg, >95% area HPLC purity, 73% yield) as an oil. 1 H NMR (400 MHz, CDCl 3 ): δ 7.39 (m, 1H), 4.76 (s, 2H), 3.90 (m, 1H), 3.17-3.37 (m, 2H), 3.00 (m, 2H), 1.91 (m, 1H), 1.26 (s, 9H), 0.80-1.00 (m, 21H), 0.00 (s, 6H). HPLC analysis of (R)-tert-butyl ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-4- methyl-1 -oxo pentan-3-yl)carbamate: Identity: (R)-tert-butyl ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-4-methyl-1 - oxo pentan-3-yl)carbamate 11.100 minutes

Current Data Parameters NAME AZ13669353-Int-4-XKS-1723P59 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20140807 Time 10.08 INSTRUM spect PROBHD 5 mm PABBO BB/ PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 16 DS 2 SWH 8012.820 Hz FIDRES 0.122266 Hz AQ 4.0894465 sec RG 16.03 DW 62.400 usec DE 6.50 usec TE 298.0 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== SFO1 400.1324710 MHz NUC1 1H P1 10.00 usec PLW1 16.00000000 W F2 - Processing parameters SI 65536 SF 400.1300559 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 7.408 7.387 7.144 7.116 7.114 7.111 4.792 4.790 4.762 4.699 4.692 4.685 3.902 3.378 3.338 3.234 3.172 3.037 3.022 3.004 2.984 2.969 1.919 1.912 1.906 1.883 1.833 1.778 1.308 1.260 0.974 0.957 0.946 0.930 0.848 0.831 0.819 8 7 6 5 4 3 2 1 0 ppm 0.83 2.10 0.85 1.86 2.15 1.09 9.00 3.05 3.04 9.05 3.27 6.10

Preparation of tert-butyl (1R,3R)-ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)- 1- hydroxy- 4-methylpentan-3-yl)carbamate LiBH 4 (76.0g, 3.45mol) was added in portions to a solution of (R)-tert-butyl ethyl-(1-(4-((tertbutyldimethylsilyloxy)methyl)thiazol-2-yl)-4-methyl-1 -oxo pentan-3-yl)carbamate (1.62kg, 3.45mol) in methanol (5L) at room temperature over 0.5h with stirring. The suspension was stirred for 1h at 0 C and the reaction was analysed by HPLC analysis giving a ratio of (R)- tert-butyl ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-4-methyl-1 -oxo pentan- 3-yl)carbamate:tert-butyl (1R,3R)-ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)- 1- hydroxy- 4-methylpentan-3-yl)carbamate:tert-butyl (1S,3R)-ethyl-(1-(4-((tertbutyldimethylsilyloxy)methyl)thiazol-2-yl)-1- hydroxy- 4-methylpentan-3-yl)carbamate (diastereomer) =0:35:65). HPLC analysis of reaction mixture: Identity: (R)-tert-butyl ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-4-methyl-1 - oxo pentan-3-yl)carbamate 11.180 minutes tert-butyl (1R,3R)-ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-1- hydroxy- 4-methylpentan-3-yl)carbamate 10.000 minutes tert-butyl (1S,3R)-ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-1- hydroxy- 4-methylpentan-3-yl)carbamate (diastereomer) 9.112 Water (5L) was added carefully to quench the reaction mixture at 0 C, then the organic solvent was removed under reduced pressure. The resulting aqueous was extracted with EtOAc (4L 3). The combined organic phases were washed with brine (5L 2), dried over anhydrous Na 2 SO 4 and concentrated to dryness. Another batch (2.54kg of (R)-tert-butyl ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol- 2-yl)-4-methyl-1 -oxo pentan-3-yl)carbamate) of this reaction was run following the same procedure and the two batches were combined for purification. The residue was purified by flash column chromatography on silica gel (EtOAc:Hexane, 1:30 to 1:10) to afford the product (1.4kg, >98% area HPLC purity, diastereomer content <2% area, 33% yield) and the diastereomer (2.4kg, 57% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.01 (m, 1H), 4.81 (m, 1H), 4.70 (s, 2H), 4.64 (m, 1H), 2.95-3.13 (m, 2H), 1.80-1.95 (m, 2H), 1.66 (s, 1H), 1.38 (s, 9H), 1.15 (t, J = 7.2 Hz, 3H), 0.80-1.10 (m, 21H), 0.00 (s, 6H). HPLC analysis of tert-butyl (1R,3R)-ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2- yl)-1- hydroxy- 4-methylpentan-3-yl)carbamate:

Identity: tert-butyl (1R,3R)-ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-1- hydroxy- 4-methylpentan-3-yl)carbamate 10.000 minutes Recycle of (3R)-ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-1- hydroxy- 4- methylpentan-3-yl)carbamate Dess-Martin periodinane (2.03kg, 4.8mol) was added to a solution of (3R)-ethyl-(1-(4-((tertbutyldimethylsilyloxy)methyl)thiazol-2-yl)-1- hydroxy- 4-methylpentan-3-yl)carbamate (1.9kg, 4.0mol) in dichloromethane (10L) at 0 C over 0.5h. The reaction mixture was stirred at room temperature for 2h. Water (3L) was added to quench the reaction and the resulting mixture was stirred for 0.5 h and filtered. The filtration cake wash was washed with dichloromethane (2L 4). The filtrate and washing were combined and washed with 5% aqueous KHSO 4 (5L 3), aqueous sodium thiosulfate (1N, 3L 3), saturated aqueous NaHCO 3 (5L 3) and brine (3L 1). The organic layer was dried over anhydrous Na 2 SO 4 and concentrated to dryness to afford (R)-tert-butyl ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-4- methyl-1 -oxo pentan-3-yl)carbamate (1.88kg, ~100% yield). Reduction of recovered material (1.88kg, 4.0mol) with lithium borohydride was carried out following the same procedure to afford (1R,3R)-ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-1- hydroxy- 4-methylpentan-3-yl)carbamate (603g, >98% area HPLC purity, diastereomer content <2%, 32% yield) and the diastereomer (1065g, 56% yield).

Current Data Parameters NAME AZ13669353-Int-5-XKS-1723P66 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20140829 Time 9.48 INSTRUM spect PROBHD 5 mm PABBO BB/ PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 16 DS 2 SWH 8012.820 Hz FIDRES 0.122266 Hz AQ 4.0894465 sec RG 32.27 DW 62.400 usec DE 6.50 usec TE 298.0 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== SFO1 400.1324710 MHz NUC1 1H P1 10.00 usec PLW1 16.00000000 W F2 - Processing parameters SI 65536 SF 400.1300471 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 7.166 7.008 5.193 4.812 4.805 4.723 4.659 4.632 3.872 3.126 3.108 3.090 2.992 2.974 2.956 1.958 1.928 1.875 1.867 1.847 1.840 1.663 1.385 1.322 1.154 1.142 1.124 1.107 0.865 0.849 0.835 0.819 0.796 0.779 0.771 0.759 0.754 0.738 0.703 0.689 0.684 0.677-0.000-0.031-0.105 7 6 5 4 3 2 1 0 ppm 1.07 1.04 2.10 1.06 1.00 1.02 1.05 1.02 1.09 1.03 9.15 3.04 19.51 6.50

Current Data Parameters NAME AZ13669353-XKS-1723P67-ISOMER EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20140916 Time 14.50 INSTRUM spect PROBHD 5 mm PABBO BB/ PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 16 DS 2 SWH 8012.820 Hz FIDRES 0.122266 Hz AQ 4.0894465 sec RG 32.27 DW 62.400 usec DE 6.50 usec TE 293.3 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== SFO1 400.1324710 MHz NUC1 1H P1 10.00 usec PLW1 16.00000000 W F2 - Processing parameters SI 65536 SF 400.1300495 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 7.160 6.980 5.193 4.867 4.709 4.431 3.618 2.945 2.927 2.910 2.892 2.633 2.616 2.272 2.238 2.009 1.993 1.980 1.958 1.716 1.379 1.335 1.117 1.078 0.991 0.893 0.879 0.832 0.786 0.772 0.675 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 0.90 1.00 2.06 0.86 0.80 1.65 0.82 1.01 1.09 1.10 9.09 2.19 2.26 9.34 4.16 6.04

Preparation of (1R,3R)-3-(tert-Butoxycarbonyl(ethyl)amino)-1-(4-((tertbutyldimethylsilyloxy)methyl) thiazol-2-yl)-4-methylpentyl acetate N Boc OH S N OTBS AcCl, Py N Boc OAc S N OTBS Acetyl chloride (1268g, 1.15L, 16.2mol) was added dropwise to a solution of tert-butyl (1R,3R)-ethyl-(1-(4-((tert-butyldimethylsilyloxy)methyl)thiazol-2-yl)-1- hydroxy- 4- methylpentan-3-yl)carbamate (953g, 72mol) in pyridine (500mL) at 0 C with stirring over 10min. The reaction mixture was allowed to warm to room temperature and stirred for 12h. The resulting precipitate was removed by filtration and the solid washed with EtOAc (2L 4). The filtrate and washings were combined and concentrated to dryness. The residue was purified by flash column chromatography on silica gel (EtOAc:Hexane, 1:30 to 1:10) to afford the product (830g, 95% area HPLC purity, 80% yield) as an oil. Another two batches (250g and 946g of tert-butyl (1R,3R)-ethyl-(1-(4-((tertbutyldimethylsilyloxy)methyl)thiazol-2-yl)-1- hydroxy- 4-methylpentan-3-yl)carbamate) of this acylation were run following the same procedure to afford (1R,3R)-3-(tertbutoxycarbonyl(ethyl)amino)-1-(4-((tert-butyldimethylsilyloxy)methyl) thiazol-2-yl)-4- methylpentyl acetate (216g in 80% yield and 820g in 80% yield). All three batches were combined for the next step 1 H NMR (400 MHz, CDCl 3 ): δ 7.01 (m, 1H), 5.78 (m, 1H), 4.73 (s, 2H), 3.95 (m, 1H), 2.98 (m, 2H), 2.22 (m, 1H), 2.04 (s, 3H), 1.94 (m, 1H), 1.59 (m, 1H), 1.33 (s, 9H), 1.15 (m, 3H), 0.75-0.90 (m, 21H), 0.00 (s, 6H). HPLC analysis of (1R,3R)-3-(tert-butoxycarbonyl(ethyl)amino)-1-(4-((tertbutyldimethylsilyloxy)methyl) thiazol-2-yl)-4-methylpentyl acetate: Identity: (1R,3R)-3-(tert-butoxycarbonyl(ethyl)amino)-1-(4-((tert-butyldimethylsilyloxy)methyl) thiazol-2-yl)-4-methylpentyl acetate 11.014 minutes

Current Data Parameters NAME AZ13669353-Int-6-XKS-1723P68 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20140829 Time 9.52 INSTRUM spect PROBHD 5 mm PABBO BB/ PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 16 DS 2 SWH 8012.820 Hz FIDRES 0.122266 Hz AQ 4.0894465 sec RG 32.27 DW 62.400 usec DE 6.50 usec TE 298.0 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== SFO1 400.1324710 MHz NUC1 1H P1 10.00 usec PLW1 16.00000000 W F2 - Processing parameters SI 65536 SF 400.1300481 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 7.164 7.025 7.010 5.805 5.799 5.775 5.770 5.194 4.727 3.012 2.998 2.995 2.981 2.978 2.963 2.235 2.228 2.206 2.198 2.036 1.953 1.939 1.921 1.889 1.608 1.593 1.583 1.350 1.333 1.189 1.152 1.124 1.107 1.090 0.877 0.870 0.860 0.832 0.803 0.786 0.762 0.674-0.000-0.034-0.106 7 6 5 4 3 2 1 0 ppm 0.92 0.97 2.00 0.74 1.72 1.01 1.15 1.03 9.03 3.00 15.76 5.86

Preparation of (1R,3R)-3-(tert-Butoxycarbonyl(ethyl)amino)-1-(4-(hydroxymethyl)thiazol-2- yl)-4- methylpentyl acetate N Boc OAc S N OTBS TBAF N Boc OAc S N OH Tetrabutylammonium fluoride (219g, 0.84mol) was added in portions to a solution of (1R,3R)-3-(tert-butoxycarbonyl(ethyl)amino)-1-(4-((tert-butyldimethylsilyloxy)methyl) thiazol- 2-yl)-4-methylpentyl acetate (216.0g, 0.42mmol) in THF (1L) at room temperature, then the reaction mixture was stirred for 15min. Water (3L) was added to quench the reaction. Another two batches (817g of (1R,3R)-3-(tert-butoxycarbonyl(ethyl)amino)-1-(4-((tertbutyldimethylsilyloxy)methyl) thiazol-2-yl)-4-methylpentyl acetate for each batch) of this reaction were run following the same procedure and all three batches were combined for a single work-up. The organic solvent was removed under reduced pressure and the remaining mixture was extracted with EtOAc (5L 3). The combined organic phases were washed with water (10L 3) and brine (10L 1). The organic solution was dried over anhydrous Na 2 SO 4 and concentrated to dryness. The residue was purified by flash column chromatography on silica gel (EtOAc:Hexane, 1:10 to 1:1) to afford the product (1.44kg, >95% area HPLC purity, 100% yield) as an oil. 1 H NMR (300 MHz, CDCl 3 ): δ 7.15 (m, 1H), 5.91 (m, 1H), 4.75 (s, 2H), 4.05 (m, 1H), 2.90-3.10 (m, 2H), 2.34 (m, 1H), 2.16 (s, 3H), 2.07 (m, 1H), 1.70 (m, 1H), 1.45 (s, 9H), 1.22 (m, 3H), 0.97 (m, 3H), 0.87 (m, 3H). HPLC analysis of (1R,3R)-3-(tert-butoxycarbonyl(ethyl)amino)-1-(4-(hydroxymethyl)thiazol-2- yl)-4- methylpentyl acetate: Identity: (1R,3R)-3-(tert-butoxycarbonyl(ethyl)amino)-1-(4-(hydroxymethyl)thiazol-2-yl)-4- methylpentyl acetate 4.366 minutes

Current Data Parameters NAME AZ13669353-INT-7-XKS-172 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20141120 Time 16.32 INSTRUM spect PROBHD 5 mm PABBO BB/ PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 16 DS 2 SWH 8012.820 Hz FIDRES 0.122266 Hz AQ 4.0894465 sec RG 50.71 DW 62.400 usec DE 6.50 usec TE 291.6 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== SFO1 400.1324710 MHz NUC1 1H P1 10.00 usec PLW1 16.00000000 W F2 - Processing parameters SI 65536 SF 400.1300053 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 7.272 7.163 7.153 5.931 5.924 5.901 5.895 5.305 4.750 3.134 3.117 3.099 3.082 2.384 2.375 2.355 2.347 2.338 2.317 2.309 2.156 2.095 2.090 2.059 2.049 1.722 1.712 1.706 1.696 1.680 1.455 1.445 1.261 1.254 1.238 1.220 1.203 0.985 0.969 0.911 0.895 0.873 0.866 0.863-0.000 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 0.90 0.99 2.00 0.72 1.61 1.46 1.15 2.89 1.05 1.07 9.40 3.35 3.27 3.36

Preparation of 2-((1R,3R)-1-acetoxy-3-(tert-butoxycarbonyl(ethyl)amino)-4- methylpentyl)thiazole-4-carboxylic acid (8c) N Boc OAc S N OH 1. Dess-Martin N 2. NaClO 2 Boc OAc S N OH O Dess-Martin periodinane (916g, 2.16mol) was added to a solution of (1R,3R)-3-(tertbutoxycarbonyl(ethyl)amino)-1-(4-(hydroxymethyl)thiazol-2-yl)-4- methylpentyl acetate (720g, 1.8mol) in dichloromethane (3.5L) and the reaction mixture was stirred at room temperature for 3h. Water (3L) was added and the resulting mixture was stirred for 0.5 h. The mixture was filtered and the filtration cake was washed with dichloromethane (2L 4). The filtrate and washings were combined and then washed with 5% aqueous KHSO 4 (5L 3), saturated aqueous sodium thiosulfate (1N, 3L 3), saturated aqueous NaHCO 3 (5L 3) and brine (3L 1). The organic layer was dried over anhydrous Na 2 SO 4 and concentrated to dryness to afford the intermediate aldehyde (720g). Another batch (720 g of (1R,3R)-3-(tert-butoxycarbonyl(ethyl)amino)-1-(4- (hydroxymethyl)thiazol-2-yl)-4- methylpentyl acetate) of this reaction was run following the same procedure and the two batches were combined for the next step. This crude aldehyde (approxiately 1.4kg, 3.5mol) was dissolved in tert-butyl alcohol (5L) and a solution of sodium chlorite (80%, 2.0kg, 17.6mol) and sodium dihydrogenphosphate monohydrate (7.14kg, 52.2mol) in water (7L) was added dropwise over 1h at 0-5 C. The reaction mixture was stirred for 0.5h then extracted with EtOAc (5L 3) and the combined organic layers washed with 5% aqueous KHSO 4 (5L 3) and brine (5L 1), dried over anhydrous Na 2 SO 4 and concentrated to dryness. The residue was purified by flash column chromatography on silica gel (CH 2 Cl 2 :MeOH 100:5) to afford the product (8c, 760g, >98% area HPLC purity, 51% yield over two steps) as a white solid. 1 H NMR (400 MHz, CDCl 3 ): δ 8.25 (s, 1H), 5.95 (m, 1H), 4.07 (m, 1H), 3.11 (m, 2H), 2.35 (m, 1H), 2.16 (s, 3H), 2.13 (m, 1H), 1.72 (m, 1H), 1.46 (s, 9H), 1.22 (m, 3H), 0.98 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H). HPLC analysis of 8c: Identity: 8c 6.292 minutes

Current Data Parameters NAME AZ13669353-XKS-1723P75 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20140911 Time 13.06 INSTRUM spect PROBHD 5 mm PABBO BB/ PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 16 DS 2 SWH 8012.820 Hz FIDRES 0.122266 Hz AQ 4.0894465 sec RG 56.05 DW 62.400 usec DE 6.50 usec TE 298.0 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== SFO1 400.1324710 MHz NUC1 1H P1 10.00 usec PLW1 16.00000000 W F2 - Processing parameters SI 65536 SF 400.1300057 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 8.256 8.243 7.270 5.999 5.954 5.931 5.302 4.753 4.071 3.138 3.122 3.104 3.088 2.388 2.379 2.351 2.322 2.314 2.178 2.156 2.130 1.734 1.719 1.709 1.616 1.460 1.300 1.234 1.217 1.200 0.988 0.972 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 1.02 1.06 1.05 2.22 1.08 1.09 9.00 3.10 6.02

Improved Synthesis of N-Et Tuv (8a) Preparation of (R)-tert-butoxy(2-((R)-3-(tert-butoxycarbonyl(ethyl)amino)-4-methylpentanoyl) thiazol-4-yl)methyl acetate (18) HPLC Analysis: Column: ZORBAX Eclipse Plus C18, 4.6x100mm, 3.5μm Mobile phase A: 0.1% H 3 PO 4 in water Mobile phase B: Acetonitrile Gradient: Time %A %B 0 90 10 9 5 95 12 0 100 12.1 90 10 15 90 10 Flow rate: 1.5 ml/min Detection: UV @ 210 nm Temperature: 40 C

Preparation of tert-butyl 2-bromothiazole-4-carboxylate (20) A solution of 2-bromothiazole-4-carboxylic acid (3.20kg, 15.38mol), CDI (3.12kg, 19.23mol, 1.25eq) and tbuoh (4.41L, 46.14mol, 3.00eq) in DMF (16.0L, 5vol) was cooled to 0 C under nitrogen and DBU (2.65L, 17.69mmol, 1.15eq) added dropwise. The mixture was stirred at 20 C for 4-6h. The reaction mixture was added slowly to water (48.0L, 15vol) at 0 C resulting the precipitation of an off-white solid. The suspension was stired at 0 C for 2h then the solid isolated by filtration. The solid was washed with water (16.0L, 5vol) then dried to afford the product (20, 3.49kg, 98.56% area HPLC purity, 85%) as a white solid. 1 H NMR (500 MHz, DMSO): 1.54 (s, 9H), 8.43 (s, 1H). HPLC analysis of 20: Identity: 20 6.420 minutes A second batch of 2-bromothiazole-4-carboxylic acid (3.00kg) afforded a second batch of product (20, 3.34kg, 99.14% area HPLC purity, 87%) as a white solid.

Current Data Parameters NAME 170517071 EXPNO 10 PROCNO 1 F2 - Acquisition Parameters Date_ 20170517 Time 16.34 h INSTRUM A500 PROBHD Z118636_0004 ( PULPROG zg TD 262144 SOLVENT DMSO NS 2 DS 0 SWH 10302.198 Hz FIDRES 0.078600 Hz AQ 12.7227221 sec RG 14.2 DW 48.533 usec DE 15.84 usec TE 300.0 K D1 77.27728271 sec TD0 1 SFO1 499.9030871 MHz NUC1 1H P1 10.95 usec PLW1 5.30000019 W F2 - Processing parameters SI 262144 SF 499.8999937 MHz WDW EM SSB 0 LB 0.19 Hz GB 0 PC 1.00 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0-1 -2 ppm 9.34 2.04 4.13 84.49

Preparation of (R)-tert-butyl 2-(3-(tert-butoxycarbonyl(ethyl)amino)-4-methylpentanoyl) thiazole-4-carboxylate (21) A solution of (R)-tert-butyl ethyl(1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-3- yl)carbamate (1.90kg, 6.28mol) and tert-butyl 2-bromothiazole-4-carboxylate (20, 2.07kg, 7.85mol, 1.25eq) in 2-methyl tetrahydrofuran (19L, 10vol) was cooled to 0 C and isopropyl magnesium bromide solution (2M in 2-methyl tetrahydrofuran, 7.85L, 15.70mol, 2.5eq) was added maintaining the temperature between 0 and 10 C. The reaction mixture was then stirred at 0 C for 2h. The reaction was quenched by the addition of aqueous ammonium chloride solution (20%w/w, 19L) and the layers separated. The organic phase was evaporated to dryness. The procedure was repeated for a second batch of (R)-tert-butyl ethyl(1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-3-yl)carbamate (1.90kg). The combined residues were purified by flash column chromatography on silica gel (EtOAc:Hexane:Triethylamine, 1:150:0.3 to 1:50:0.3) to afford the product (21, 2.20kg, 90.6% area HPLC purity, 35% yield) as an oil. 1 H NMR (400 MHz, DMSO, 90 C): 0.86 (d, J = 6.6 Hz, 3H), 0.99 (d, J = 6.6 Hz, 3H), 1.05 (t, J = 7.0 Hz, 3H), 1.28 (s, 9H), 1.57 (s, 9H), 1.93 2.04 (m, 1H), 3.10 (q, J = 7.0 Hz, 2H), 3.25 (dd, J = 9.7, 14.9 Hz, 1H), 3.47 (dd, J = 3.9, 14.9 Hz, 1H), 4.04 (s, 1H), 8.69 (s, 1H). 13 C NMR (126 MHz, DMSO, 90 C) 191.55, 165.98, 158.98, 154.15, 148.60, 133.53, 81.27, 77.81, 60.24, 40.86, 40.85, 30.54, 27.49, 27.37, 19.65, 19.35, 13.79. HPLC analysis of 21: Identity: 21 8.861 minutes

Current Data Parameters NAME 170504017 EXPNO 10 PROCNO 1 F2 - Acquisition Parameters Date_ 20170505 Time 13.50 h INSTRUM A500 PROBHD Z118636_0004 ( PULPROG zg TD 262144 SOLVENT DMSO NS 4 DS 0 SWH 10302.198 Hz FIDRES 0.078600 Hz AQ 12.7227221 sec RG 9 DW 48.533 usec DE 10.02 usec TE 363.0 K D1 77.27728271 sec TD0 1 SFO1 499.9030871 MHz NUC1 1H P1 10.57 usec PLW1 5.30000019 W F2 - Processing parameters SI 262144 SF 499.9000081 MHz WDW EM SSB 0 LB 0.19 Hz GB 0 PC 1.00 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0-1 -2 ppm 2.70 97.30

Current Data Parameters NAME 170504017 EXPNO 11 PROCNO 1 F2 - Acquisition Parameters Date_ 20170505 Time 14.05 h INSTRUM A500 PROBHD Z118636_0004 ( PULPROG zgpg30 TD 65340 SOLVENT DMSO NS 256 DS 0 SWH 29761.904 Hz FIDRES 0.910986 Hz AQ 1.0977120 sec RG 1290 DW 16.800 usec DE 52.37 usec TE 363.0 K D1 2.00000000 sec D11 0.03000000 sec TD0 1 SFO1 125.7125244 MHz NUC1 13C P1 12.00 usec PLW1 136.00000000 W SFO2 499.9019996 MHz NUC2 1H CPDPRG[2 waltz16 PCPD2 80.00 usec PLW2 5.30000019 W PLW12 0.10020000 W PLW13 0.05032100 W F2 - Processing parameters SI 65536 SF 125.7000731 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40 191.55 165.98 158.98 154.14 148.60 133.53 81.27 77.81 60.24 39.99 39.91 39.82 39.74 39.65 39.57 39.49 39.32 39.15 38.98 30.54 27.49 27.37 27.21 27.15 27.09 19.65 19.35 13.79 200 180 160 140 120 100 80 60 40 20 0 ppm

Preparation of tert-butyl (R)-1-(4-((S)-tert-butoxy(hydroxy)methyl)thiazol-2-yl)-4-methyl-1- oxopentan-3-yl(ethyl)carbamate (22) To a solution of (R)-tert-butyl 2-(3-(tert-butoxycarbonyl(ethyl)amino)-4-methylpentanoyl) thiazole-4-carboxylate (21, 2.20kg, 5.16mol) in THF (11L, 5vol) and water (110ml, 0.05vol) was added formic acid:triethylamine complex (5:2, 4.33L, 51.6mol, 2.00eq) and (Mes)RuCl(R,R-MsDPEN) (28.2g, 10.32mmol, 0.01eq). The mixture was degassed with nitrogen for 10min then stirred at 20 C for 4h followed by 40 C for 12h. The reaction mixture was partitioned between aqueous sodium hydrogen carbonate solution (9%w/w, 11L, 5vol) and MTBE (11L, 5vol). The layers were separated and the organic phase washed with aqueous sodium chloride solution (10%w/w, 11L, 5vol) then evaporated to dryness to afford the product (22, 85.70% area HPLC purity) as an oil. HPLC analysis of 22: Identity: 22 8.757 minutes

Preparation of (R)-tert-butoxy(2-((R)-3-(tert-butoxycarbonyl(ethyl)amino)-4-methylpentanoyl) thiazol-4-yl)methyl acetate (18) A solution of tert-butyl (R)-1-(4-((S)-tert-butoxy(hydroxy)methyl)thiazol-2-yl)-4-methyl-1- oxopentan-3-yl(ethyl)carbamate (22, 5.16mol) in pyridine (22L, 10vol) was cooled to 0 C and acetyl chloride (366mL, 11.54mol, 2.25eq) added maintaining the temperature below 5 C. The reaction mixture was warmed to 20 C and stirred for 4h. The resulting slurry was filtered and the solid washed with MTBE (44L, 20vol). The combined organic solutions were washed with aqueous sodium hydrogen sulfate solution (20%w/w, 28.6L, 13vol then 22L, 10vol then 19.8L, 9vol). Active carbon (220g, 10%w/w) was added to the organic solution, the mixture stirred for 2h then filtered and the solid washed with MTBE (2x2.2L, 2x1vol). The combined organic solutions were evaporated to dryness and heptane (11L, 5vol) added to the residue. The mixture was evaporated to dryness and heptane (11L, 5vol) added to the residue. The resulting solution was stirred at 20 C and seed material added resulting in a white solid precipitating. The slurry was stirred at 20 C for 3h then the solid isolated by filtration and dried affording the product (18, 2.00kg, 96.56% area HPLC purity, 82% yield over 2 steps) as a white solid. 1 H NMR (500 MHz, DMSO, 90 C): 0.83 (d, J = 6.7 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H), 1.15 (t, J = 6.9 Hz, 3H), 1.39 (s, 9H), 1.53 (s, 9H), 1.75 1.86 (m, 1H), 2.10 (s, 3H), 2.20 (dd, J = 4.8, 9.1 Hz, 2H), 3.03 (q, J = 7.5, 7.9 Hz, 1H), 3.08 3.24 (m, 1H), 5.82 (d, J = 7.1 Hz, 1H), 8.30 (s, 1H). 13 C NMR (126 MHz, DMSO, 90 C) 169.49, 168.93, 159.21, 154.75, 147.12, 127.60, 80.73, 77.99, 69.38, 57.76, 37.46, 34.86, 29.99, 27.62, 27.40, 20.04, 19.58, 19.33, 14.22. HPLC analysis of 18: Identity: 18 9.286 minutes

Current Data Parameters NAME 170517069 EXPNO 10 PROCNO 1 F2 - Acquisition Parameters Date_ 20170517 Time 16.05 h INSTRUM A500 PROBHD Z118636_0004 ( PULPROG zg30 TD 65536 SOLVENT DMSO NS 1 DS 0 SWH 10302.198 Hz FIDRES 0.314398 Hz AQ 3.1806805 sec RG 22.6 DW 48.533 usec DE 14.43 usec TE 363.1 K D1 0.81931949 sec TD0 1 SFO1 499.9030871 MHz NUC1 1H P1 11.00 usec PLW1 5.30000019 W F2 - Processing parameters SI 65536 SF 499.9000099 MHz WDW EM SSB 0 LB 0.19 Hz GB 0 PC 1.00 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0-1 -2 ppm 2.34 2.57 8.54 86.55

Current Data Parameters NAME 170517069 EXPNO 14 PROCNO 1 F2 - Acquisition Parameters Date_ 20170518 Time 1.21 h INSTRUM A500 PROBHD Z118636_0004 ( PULPROG zgpg30 TD 65340 SOLVENT DMSO NS 2048 DS 0 SWH 29761.904 Hz FIDRES 0.910986 Hz AQ 1.0977120 sec RG 1150 DW 16.800 usec DE 52.37 usec TE 300.0 K D1 2.00000000 sec D11 0.03000000 sec TD0 1 SFO1 125.7125244 MHz NUC1 13C P1 12.00 usec PLW1 136.00000000 W SFO2 499.9019996 MHz NUC2 1H CPDPRG[2 waltz16 PCPD2 80.00 usec PLW2 5.30000019 W PLW12 0.10020000 W PLW13 0.05032100 W F2 - Processing parameters SI 65536 SF 125.7000739 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40 169.49 168.92 159.21 154.75 147.12 127.60 80.73 77.99 69.38 40.07 39.99 39.91 39.82 39.74 39.65 39.57 39.48 39.41 39.32 39.15 38.98 37.46 34.86 29.98 27.62 27.40 27.23 20.04 19.57 19.33 14.22 200 180 160 140 120 100 80 60 40 20 0 ppm

Improved Synthesis of N-Fmoc N -Alloc Tup (9b) Preparation of (2S,4R)-4-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-(4- (allyloxycarbonylamino)phenyl)-2-methylpentanoic acid (9b) HPLC Analysis Method 1: Column: Xbridge C18N, 4.6x250mm, 5μm Mobile phase A: 0.05% TFA in water Mobile phase B: 0.05% TFA in acetonitrile Time %A %B 0 90 10 20 5 95 23 5 95 30 90 5 Flow rate: 1.0mL/min Detection: UV@254 nm Temperature: 35 C HPLC Analysis Method 2: Column: Xbridge C18N, 4.6x250mm, 5μm Mobile phase A: 0.05% TFA in water Mobile phase B: 0.05% TFA in acetonitrile Time %A %B 0 60 40 20 30 70 22 5 95 26 5 95 30 60 40 Flow rate: 1.0mL/min Detection: UV@254 nm Temperature: 35 C

HPLC Analysis Method 3: Column: Xselect HSS T3, 3.0x150mm, 3.5μm Mobile phase A: 20mM NH 4 Ac in water Mobile phase B: acetonitrile Time %A %B 0 90 10 10 55 45 15 55 45 17 5 95 21 5 95 30 90 10 Flow rate: 0.8mL/min Detection: UV@254 nm Temperature: 35 C HPLC Analysis Method 4: Column: Xselect HSS T3, 3.0x150mm, 3.5μm Mobile phase A: 20mM NH 4 Ac in water Mobile phase B: acetonitrile Time %A %B 0 90 10 15 5 95 21 5 95 30 90 10 Flow rate: 0.8mL/min Detection: UV@254 nm Temperature: 35 C

Chiral HPLC Analysis Method 1: Column: AD-RH, 4.6x150mm Mobile phase A: water Mobile phase B: acetonitrile Time %A %B 0 70 30 20 5 95 25 5 95 37 70 30 Flow rate: 0.6mL/min Detection: UV@280nm Temperature: 35 C Chiral HPLC Analysis Method 2: Column: AD-RH, 4.6x150mm Mobile phase A: 0.05% TFA in water Mobile phase B: 0.05% TFA in acetonitrile Time %A %B 0 70 30 20 5 95 25 5 95 30 70 30 Flow rate: 1.0mL/min Detection: UV@275 nm Temperature: 35 C Chiral HPLC Analysis Method 3: Column: AS-RH, 4.6x150mm Mobile phase A: 20mM NH 4 Ac in water Mobile phase B: acetonitrile Time %A %B 0 70 30 20 5 95 25 5 95 30 70 30 Flow rate: 1.0mL/min Detection: UV@254 nm Temperature: 35 C

Preparation of (S)-tert-butyl 1-hydroxy-3-(4-nitrophenyl)propan-2-ylcarbamate (10) Dimethyl sulfate (768g, 6.09mol, 1.05eq) was added dropwise to a suspension of (S)-2-(tertbutoxycarbonylamino)-3-(4-nitrophenyl)propanoic acid (1.80kg, 5.80mol) and Na 2 CO 3 (882g, 6.38mol, 1.1eq) in THF (14.4L) at 0 C. The reaction mixture was warmed to 20 C and stirred for 18h. Diethylamine (63.6g, 0.87mol, 0.15eq) was added (to consume residual dimethyl sulfate) and the mixture stirred for 2h. The mixture was filtered and the filter cake washed with THF (3.6L). The combined organic portions were combined and concentrated to remove the majority of the THF. The remaining material was dissolved in EtOAc (18L), washed with 10% brine (10.8L 3) and the sovent removed to afford the crude intermediate ester. The ester was dissolved in methanol (10L) and THF (10L) and cooled to 0 C. NaBH 4 (659g, 17.42mol, 3.0eq) was added slowly in portions, ensuring that the reaction temperature remained below 2 C. On completion of addition the reaction mixture was allowed to warm to 20 C and stirred for 16h. The reaction was then cooled to 0 C and quenched with acetic acid (2L) and water (10L). The resultant solution was concentrated to remove the majority of the solvent and the remaining material dissolved in EtOAc (20L). The organic solution was washed with 5% brine (12L 3) then the solvent removed to obtain the crude product. This material was reslurried in MTBE, filtered and then dried under vacuum at 40 C to afford the product (10, 1.5kg, 99.2% area HPLC purity, 99.9% area Chiral HPLC purity, 87%) as an offwhite solid. 1 H NMR (500 MHz, CDCl 3 ) 1.39 (s, 9H), 2.54 (s, 1H), 2.99 (m, 2H), 3.58 (dd, J = 4.2, 10.6 Hz, 1H), 3.68 (dd, J = 3.9, 10.9 Hz, 1H), 3.91 (s, 1H), 4.89 (s, 1H), 7.41 (d, J = 8.6 Hz, 2H), 8.09 8.22 (d, J = 8.7 Hz, 2H).

HPLC analysis of 10 (Method 1): Identity: 10 13.234 minutes Chiral HPLC analysis of 10 (Method 1): Identity: 10 14.095 minutes 10-Enantiomer 15.251 minutes

Current Data Parameters NAME 170613026 EXPNO 10 PROCNO 1 F2 - Acquisition Parameters Date_ 20170613 Time 14.09 h INSTRUM A500 PROBHD Z118636_0004 ( PULPROG zg TD 262144 SOLVENT CDCl3 NS 2 DS 0 SWH 10302.198 Hz FIDRES 0.078600 Hz AQ 12.7227221 sec RG 28.5 DW 48.533 usec DE 15.92 usec TE 300.0 K D1 77.27728271 sec TD0 1 SFO1 499.9030871 MHz NUC1 1H P1 10.83 usec PLW1 5.30000019 W F2 - Processing parameters SI 262144 SF 499.9000057 MHz WDW EM SSB 0 LB 0.19 Hz GB 0 PC 1.00 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0-1 -2 ppm 9.68 9.65 0.78 4.82 30.43 43.73 0.90

Preparation of tert-butyl (4S)-4-(4-nitrobenzyl)-1,2,3-oxathiazolidine-3-carboxylate 2-oxide (11) A solution of (S)-tert-butyl 1-hydroxy-3-(4-nitrophenyl)propan-2-ylcarbamate (10, 1.00kg, 3.37mol) in DCM (12L) was added over 4h to a solution of thionyl chloride (1.20kg, 3.0eq) and pyridine (1.87kg, 7.0eq) in DCM (8L) at -25 C. The reaction mixutre was then warmed to 20 C and stirred for 48h. The reaction was then cooled to -10 C and quenched by the slow addition of 10% brine (7L). The temperature was maintained at -10 C, the phases separated and the organic layer washed with 10% brine (7L 2). The organic layer was then concentrated (batch temperature below 40 C), diluted with acetonitrile (5L) and reconcentrated (batch temperature below 40 C) then diluted with acetonitrile (5L) affording the product (11) as a solution in acetonitrile (containing less than 2% DCM). Preparation of tert-butyl (4S)-4-(4-nitrobenzyl)-1,2,3-oxathiazolidine-3-carboxylate 2,2- dioxide (12) To a solution of tert-butyl (4S)-4-(4-nitrobenzyl)-1,2,3-oxathiazolidine-3-carboxylate 2-oxide (11, 3.37mol) in acetonitrile (5L) was added acetonitrile (12L), water (3L) and ruthenium (III) chloride trihydrate (8.8g, 0.034mol, 0.01eq). The reaction mixture was cooled to 0 C and sodium periodate (1.08kg, 5.06mol, 1.5eq) added portionwise. The reaction mixture was stirred at 0 C for 6h then EtOAc (15L) added and the mixture warmed to 10 C. The mixture was filtered and the phases separated. The organic layer was maintained at 10 C and washed with 20% Na 2 SO 3 (5L 2) and 10% brine (5L). The organic layer was then concentrated (batch temperature below 50 C), diluted with MTBE (5L)and reconcentrated (batch temperature below 50 C) then diluted with MTBE (5L). The final slurry was cooled to 10 C, the solid collected by filtration, washed with MTBE (1L) and dried under vacuum at 30 C to afford the product (12, 0.9kg, 94.2% area HPLC purity, 98.8% area Chiral HPLC purity, 74% over 2 steps) as a white solid. 1 H NMR (500 MHz, CDCl 3 ): 1.53 (s, 9H) 3.12 (dd, J=8.7, 13.5Hz, 1H) 3.41 (dd, J=5.2, 13.5Hz, 1H) 4.31 (d, J=9.2Hz, 1H) 4.51-4.60 (m, 2H) 7.44 (d, J=8.7Hz, 2H) 8.21 (d, J=8.7Hz, 2H). 13 C NMR (126 MHz, CDCl 3 ) 148.04, 147.11, 142.37, 130.17, 123.86, 85.73, 68.58, 57.59, 37.58, 27.55.

HPLC analysis of 12 (Method 2): Identity: 12 15.299 minutes Chiral HPLC analysis of 12 (Method 2): Identity: 12 12.110 minutes 12-Enantiomer 13.082 minutes

Current Data Parameters NAME 170613027 EXPNO 10 PROCNO 1 F2 - Acquisition Parameters Date_ 20170613 Time 14.25 h INSTRUM A500 PROBHD Z118636_0004 ( PULPROG zg TD 262144 SOLVENT CDCl3 NS 2 DS 0 SWH 10302.198 Hz FIDRES 0.078600 Hz AQ 12.7227221 sec RG 28.5 DW 48.533 usec DE 15.91 usec TE 300.0 K D1 77.27728271 sec TD0 1 SFO1 499.9030871 MHz NUC1 1H P1 10.84 usec PLW1 5.30000019 W F2 - Processing parameters SI 262144 SF 499.9000070 MHz WDW EM SSB 0 LB 0.19 Hz GB 0 PC 1.00 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0-1 -2 ppm 10.80 0.81 10.76 16.20 10.81 49.74 0.87

Current Data Parameters NAME 170613027 EXPNO 11 PROCNO 1 F2 - Acquisition Parameters Date_ 20170613 Time 14.40 h INSTRUM A500 PROBHD Z118636_0004 ( PULPROG zgpg30 TD 65340 SOLVENT CDCl3 NS 256 DS 0 SWH 29761.904 Hz FIDRES 0.910986 Hz AQ 1.0977120 sec RG 1030 DW 16.800 usec DE 52.37 usec TE 300.0 K D1 2.00000000 sec D11 0.03000000 sec TD0 1 SFO1 125.7125244 MHz NUC1 13C P1 12.00 usec PLW1 136.00000000 W SFO2 499.9019996 MHz NUC2 1H CPDPRG[2 waltz16 PCPD2 80.00 usec PLW2 5.30000019 W PLW12 0.10020000 W PLW13 0.05032100 W F2 - Processing parameters SI 65536 SF 125.6999956 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40 148.04 147.11 142.37 130.17 123.86 85.73 76.99 76.74 76.48 68.57 57.59 37.58 27.70 27.55 27.38-0.31 200 180 160 140 120 100 80 60 40 20 0 ppm

Preparation of (S)-diethyl 2-(2-(tert-butoxycarbonylamino)-3-(4-nitrophenyl)propyl)-2- methylmalonate (13) A solution of diethyl methylmalonate (885g, 5.08mol, 1.3eq) in THF (11.2L) was cooled to - 5 C and NaH (188g, 4.69mol, 1.2eq, 60% w/w) was added portionwise. The reaction mixture was stirred at -5 C for 1h then tert-butyl (4S)-4-(4-nitrobenzyl)-1,2,3-oxathiazolidine- 3-carboxylate 2,2-dioxide (12, 1.40kg, 3.91mol) was added portionwise. The mixture was stirred at -5 C for 3h then quenched by the addition of 5% citric acid (2.8L). The mixture was concentrated to remove the THF then diluted with EtOAc (14L). The organic solution was washed with 5% brine (7L 2) then concentrated to afford the crude product (13). This material was used in the following step without further purification. Preparation of (R)-4-amino-2-methyl-5-(4-nitrophenyl)pentanoic acid (14) A solution of (S)-diethyl 2-(2-(tert-butoxycarbonylamino)-3-(4-nitrophenyl)propyl)-2- methylmalonate (13, 3.91mol) in 8N aqueous hydrochloric acid (7L) was stirred at 95 C for 72h. The solution was cooled to 20 C, washed with DCM (7L 4), then concentrated (batch temperature below 65 C), to afford the crude product (14). This material was used in the following step without further purification. Preparation of (R)-4-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-5-(4- nitrophenyl)pentanoic acid (rac-9a) (R)-4-amino-2-methyl-5-(4-nitrophenyl)pentanoic acid (14, 3.91mol) was dissolved in water (10L) and stirred at 20 C. NaHCO 3 (3.50kg, 41.7mol, 10.7eq) was added portionwise followed by acetone (10L). Fmoc-OSu (1.10kg, 3.26mol, 0.83eq) was added portionwise at 20 C then the mixture was stirred for 18h. The ph was adjusted to 2 with 1N HCl then the reaction mixture was concentrated to remove the acetone. EtOAc (10L) was added, the layers separated and the aqueous layer further extracted with EtOAc (5L 2). The combined organic extracts were washed with 10% brine (7L 2) then evaporated to remove the EtOAc. The crude product was dissolved in methanol (8L) and stirred at 20 C then water (2.5L) was added dropwise over 16h. The resultant solid was collected by filtration and dried under vacuum at 40 C to afford the product (rac-9a, 1.36kg, 89.5% area HPLC purity, 72% over 3 steps) as a white solid.

1 H NMR (400 MHz, d 6 -DMSO): δ 1.05 (br t, J=8.03Hz, 3H) 1.31-1.54 (m, 1H) 1.69-1.93 (m, 1H) 2.23-2.43 (m, 1H) 2.70-2.99 (m, 2H) 3.63-3.84 (m, 1H) 4.07-4.17 (m, 1H) 4.18-4.27 (m, 2H) 7.19-7.35 (m, 3H) 7.36-7.49 (m, 4H) 7.53-7.69 (m, 2H) 7.88 (dd, J=7.28, 3.26Hz, 2H) 8.08 (d, J=8.53Hz, 2H) 12.09 (br. s, 1H). HPLC analysis of rac-9a (Method 3): Identity: rac-9a 19.412 and 19.863 minutes

Preparation of (2S,4R)-4-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-5-(4- nitrophenyl)pentanoic acid (9a) The two diastereomers of (R)-4-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-5-(4- nitrophenyl)pentanoic acid (rac-9a, 950g, mol) were separated using preparative Supercritical Fluid Chromatography: Column: AD, 250x50mm Mobile phase A: water Mobile phase B: 0.1% AcOH in MeOH Mobile phase ratio A:B: 1:1 Flow rate: 260g/min Detection: UV@254nm Back Presure: 100bar The resultant methanol solution was diluted with water (5-10% of total volume), then evaporated to remove the MeOH affording a suspension. The product was isolated by filtration and purified by reslurrying in MeOH (1.5ml per g of solid) at 20 C. The purified product was isolated by filtration and dried under vacuum at 50 C to afford the product (9a, 384g, 93.6% area HPLC purity, 40%) as a white solid. 1 H NMR (400 MHz, DMSO) 1.08 (d, J = 7.1 Hz, 3H), 1.42 (ddd, J = 4.5, 10.0, 14.0 Hz, 1H), 1.80 (ddd, J = 4.1, 9.4, 13.5 Hz, 1H), 2.38 2.49 (m, 1H), 2.72 2.93 (m, 2H), 3.68 3.82 (m, 1H), 4.15 (t, J = 6.7 Hz, 1H), 4.19 4.31 (m, 2H), 7.24 (d, J = 8.8 Hz, 1H), 7.32 (t, J = 7.4 Hz, 2H), 7.36 7.47 (m, 4H), 7.62 (dd, J = 7.5, 13.9 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H), 8.09 (d, J = 8.7 Hz, 2H), 12.07 (br s, 1H). 13 C NMR (101 MHz, DMSO) 155.60, 147.25, 145.95, 143.88, 143.78, 140.72, 130.48, 127.54, 126.96, 126.93, 125.05, 123.06, 120.07, 64.99, 50.43, 46.75, 40.60, 38.06, 35.82, 18.04. HPLC analysis of 9a (Method 3): Identity: 9a 19.468

Current Data Parameters NAME 170706002 EXPNO 10 PROCNO 1 F2 - Acquisition Parameters Date_ 20170706 Time 8.35 h INSTRUM A400 PROBHD Z104450_0265 ( PULPROG sth.z TD 65536 SOLVENT DMSO NS 32 DS 0 SWH 7062.147 Hz FIDRES 0.215520 Hz AQ 4.6399488 sec RG 189.19 DW 70.800 usec DE 11.13 usec TE 300.0 K D1 5.36000109 sec TD0 1 SFO1 400.1324710 MHz NUC1 1H P1 10.10 usec PLW1 16.70000076 W F2 - Processing parameters SI 65536 SF 400.1299984 MHz WDW EM SSB 0 LB 0.19 Hz GB 0 PC 1.00 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0-1 ppm 2.91 42.29 54.80

Current Data Parameters NAME 170706002 EXPNO 13 PROCNO 1 F2 - Acquisition Parameters Date_ 20170706 Time 9.25 h INSTRUM A400 PROBHD Z104450_0265 ( PULPROG stc.z TD 65536 SOLVENT DMSO NS 800 DS 2 SWH 24671.053 Hz FIDRES 0.752901 Hz AQ 1.3281963 sec RG 2050 DW 20.267 usec DE 6.50 usec TE 300.0 K D1 0.69999999 sec D11 0.03000000 sec TD0 1 SFO1 100.6228303 MHz NUC1 13C P1 8.40 usec PLW1 52.00000000 W SFO2 400.1316005 MHz NUC2 1H CPDPRG[2 waltz16 PCPD2 90.00 usec PLW2 16.70000076 W PLW12 0.20616999 W PLW13 0.10370000 W F2 - Processing parameters SI 65536 SF 100.6128180 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40 176.99 155.60 147.25 145.94 144.02 143.88 143.78 140.72 130.48 127.54 126.96 126.92 125.05 124.76 123.06 120.07 65.33 64.99 51.18 50.43 46.75 40.60 40.12 39.92 39.71 39.50 39.29 39.08 38.87 38.06 35.82 18.04 200 180 160 140 120 100 80 60 40 20 0 ppm