Synthesis of Natural and Unnatural Cyclooligomeric Depsipeptides Enabled by Flow Chemistry

Abstract Flow chemistry has been successfully integrated into the synthesis of a series of cyclooligomeric depsipeptides of three different ring sizes including the natural products beauvericin (1 a), bassianolide (2 b) and enniatin C (1 b). A reliable flow chemistry protocol was established for the coupling and macrocyclisation to form challenging N‐methylated amides. This flexible approach has allowed the rapid synthesis of both natural and unnatural depsipeptides in high yields, enabling further exploration of their promising biological activity.


4.5
Boc  Melting points (mp) were measured using a Stanford Research Systems OptiMelt automated melting point system using a gradient of 1 °C/min, and are uncorrected.
Specific Optical Rotation was recorded on a Perkin-Elmer Model 343 digital polarimeter, using a Na/Hal lamp set at 589 nm and with a path length of 100 mm. All [α D ] values were measured using spectroscopy grade solvent at the specified concentration (in gcm −3 ) and temperature, with units of 10 -1 cm 2 g -1 .
The compound numbering system used is based on IUPAC conventions. Each amino acid is numbered individually, starting from the carboxyl group and proceeding up the side chain. Position labels thus refer first to the individual amino acid, and then to the position on it, with the atom to be defined (C or H) underlined. Non-amino acid derived functionality is numbered based on the position from which it branches. The entire branch is thus given the label of the position from which it begins, appended with a prime, and each position within the chain is further denoted by a lowercase letter.

Method A (Optimised Batch)
A solution of Ghosez reagent (0.013 mL, 0.1 mmol) in dichloromethane (5 mL) was added to a solution of Boc-leucine 14 (0.012g, 0.05 mmol) in dichloromethane (5 mL) at 0 °C and the resulting mixture was stirred for 5 min at this temperature. A solution of phenylalanine methyl ester HCl salt 15 (0.011 g, 0.05 mmol) and N,N-diisopropylethylamine (0.032 mL, 0.18 mmol) in dichloromethane (5 mL) was added and the reaction mixture was allowed to warm to room temperature and stirred for 6 min 40 s before cooling to 0 °C and addition of aqueous HCl (10 mL, 1M). The reaction mixture was extracted with dichloromethane (3 x 10 mL) and the combined organic layers were washed with water (10 mL), saturated sodium bicarbonate (NaHCO 3 ) solution (10 mL) and saturated sodium chloride (NaCl) solution (10 mL) before drying over magnesium sulfate (MgSO 4 ) and removing the solvent in vacuo. The crude product was purified via preparative TLC using 40-60 petroleum ether:ethyl acetate (1:1) as eluent to afford the title compound 16 (0.009 g, 0.023 mmol, 46%) as a colourless solid. Plug flow: Boc-leucine 14 (0.012 g, 0.05 mmol) was taken up in dichloromethane (5 mL) and filled into loop 1. Ghosez reagent (0.013 mL, 0.1 mmol) was taken up in dichloromethane (5 mL) and filled into loop 2. Phenylalanine methyl ester HCl salt 15 (0.011 g, 0.05 mmol) and diisopropylethylamine (0.032 mL, 0.18 mmol) were taken up in dichloromethane (4.968 mL) and filled into loop 3. Pumps 1 -3 were run a 1 mL.min -1 with dichloromethane and injection loops 1 -3 injected at the appropriate time to ensure coordinated meeting of streams at the T-pieces (determined by runs using Sudan red dye) -loop 1 at t=0, loop 2 at t=23 s and loop 3 at t=4 min 53 s. From t = 15 min to 1 = 26 min the reaction outflow was collected in stirring aqueous HCl (10 mL, 1M) at 0 °C. The reaction mixture was extracted with dichloromethane (3 x 20 mL) and the combined organic extracts were washed with water (10 mL), saturated NaHCO 3 solution (10 mL) and saturated NaCl solution (10 mL) before drying over MgSO 4 and removing the solvent in vacuo. The resultant crude product was then purified via preparative TLC using 40-60 petroleum ether:ethyl acetate (1:1) as eluent to afford the title compound 16 (0.009 g, 0.023 mmol, 46%) as a colourless solid, which was spectroscopically identical to that reported using method A.

Continuous flow:
The flow coupling was also run continuously with pump 1 pumping a solution of Boc-Leucine 14 (0.01 M) in dichloromethane, pump 2 pumping a solution of Ghosez reagent (0.02 M) in dichloromethane and pump 3 pumping a solution of phenylalanine methyl ester HCl salt 15 (0.01 M) and diisopropylethylamine (0.036 M) in dichloromethane. The reactor was allowed to reach steady state (30 min) and then the outflow was collected for 60 min into stirring aqueous HCl at 0 °C (150 mL, 1M). The reaction mixture was extracted with dichloromethane (3 x 150 mL) and the combined organic extracts washed with water (150 mL), saturated NaHCO 3 (150 mL) and saturated NaCl (150 mL). The combined organic extracts were dried over magnesium sulphate and the solvent removed in vacuo. The resultant crude product was purified using preparative TLC using 40-60 petroleum ether:ethyl acetate (1:1) as eluent to afford the title compound 16 (0.110 g, 0.28 mmol, 47%) as a colourless solid, which was spectroscopically identical to that reported using method A.

Acid deprotection
Coupling partner A (0.05 mmol) was taken up in solvent and this solution was hydrogenated using an H-cube® Pro (ThalesNano) with a 10% Pd/C CatCart®. The pump was run at 1 mL.min -1 using solvent with the temperature set to 60 o C and the pressure to 6 bar. The solvent was then removed in vacuo and the crude acid used directly in the coupling.

Amine deprotection
Coupling partner B (0.05 mmol) was taken up in dioxane (1 mL) and anhydrous HCl (1 mL, 4M in dioxane) was added and the reaction mixture stirred at room temperature until the reaction was judged to be complete using TLC. The solvent was then removed in vacuo and the crude oil taken up in ethanol (2 x 2 mL) and methanol (2 mL) successively, removing the solvent in vacuo after each addition to afford the amine HCl salt which was used directly in the coupling.

Coupling
Scheme 2. Flow equipment set up for General Method 1.
Acid (0.05 mmol) was taken up in dichloromethane (5 mL) and filled into loop 1. Ghosez reagent (0.013 mL, 0.1 mmol) was taken up in dichloromethane (5 mL) and filled into loop 2. Amine HCl (0.05 mmol) and diisopropylethylamine (0.032 mL, 0.18 mmol) were taken up in dichloromethane (4.968 mL) and filled into loop 3. Pumps 1 -3 were run a 1 mL.min -1 with dichloromethane and injection loops 1 -3 injected at the appropriate time to ensure coordinated meeting of streams at the T-pieces (determined by runs using Sudan red dye) -loop 1 at t=0, loop 2 at t=23 s and loop 3 at t=4 min 53 s. From t = 15 min to 1 = 26 min the reaction outflow was collected in stirring aqueous HCl (10 mL, 1M) at 0 °C. The reaction mixture was extracted with dichloromethane (3 x 20 mL) and the combined organic extracts were washed with water (10 mL), saturated NaHCO 3 solution (10 mL) and saturated NaCl solution (10 mL) before drying over MgSO 4 and removing the solvent in vacuo. The resultant crude product was then purified via flash chromatography or preparative TLC.

Acid deprotection
Protected precursor (0.05 mmol) was taken up in solvent and this solution was hydrogenated using an H-cube® Pro (ThalesNano) with a 10% Pd/C CatCart®. The pump was run at 1 mL.min -1 using solvent with the temperature set to 60 o C and the pressure to 6 bar. The solvent was then removed in vacuo and the crude acid used directly in the amine deprotection.

Amine deprotection
Crude acid (0.05 mmol) was taken up in dioxane (1 mL) and anhydrous HCl (1 mL, 4M in dioxane) was added and the reaction mixture stirred at room temperature until the reaction was judged to be complete using TLC. The solvent was then removed in vacuo and the crude oil taken up in ethanol (2 x 2 mL) and methanol (2 mL) successively, removing the solvent in vacuo after each addition to afford the cyclisation precursor HCl salt which was used directly in the coupling.

Coupling
Scheme 3. Flow equipment set up for General Method 2 Cyclisation precursor HCl (0.05 mmol) was taken up in dichloromethane (5 mL) and filled into loop 1. Ghosez reagent (0.013 mL, 0.1 mmol) was taken up in dichloromethane (5 mL) and filled into loop 2. Pumps 1 and 2 were run at 1 mL.min -1 with dichloromethane and pump 3 at 1 mL.min -1 with a solution of diisopropylethylamine (0.036M) in dichloromethane. Injection loops 1 and 2 were injected at the appropriate time to ensure coordinated meeting of streams at the T-pieces (determined by runs using Sudan red dye) -loop 1 at t=0, loop 2 at t=23 s. From t = 15 min to 1 = 26 min the reaction outflow was collected in stirring aqueous HCl (20 mL, 1M) at 0 °C. The reaction mixture was extracted with dichloromethane (3 x 20 mL) and the combined organic extracts washed with water (10 mL), saturated NaHCO 3 solution (10 mL) and saturated NaCl solution (10 mL) and was dried over MgSO 4 and the solvent was removed in vacuo before purification via flash chromatography or preparative TLC. D-Hydroxyisovaleric acid 3 was synthesised using a modification of the procedure previously reported by the Ley group. [5] A solution of D-valine (3.41 g, 29.1 mmol) in sulfuric acid (58.2 mL, 0.5 M in water) at a flow rate of 0.167 mL.min -1 was mixed with a solution of sodium nitrite (4.02 g, 58.2 mmol) in water (58.2 mL) at a flowrate of 0.167 mL.min -1 at a T-piece (0.333 mL.min -1 total). The reaction mixture was passed through a 20 mL reactor coil at 60 °C and a 100 psi back pressure regulator before the output was collected in stirring ethyl acetate (150 mL). The aqueous layer was extracted with ethyl acetate (4 x 100 mL) and the combined organic layers were washed with saturated NaCl solution (250 mL) before drying over MgSO 4 and removing the solvent in vacuo. The crude title compound 3 (2.74 g, 23.2 mmol, 80%) was obtained as a yellow solid, which was used in the next step without further purification. The 1 H and 13 C NMR data was in agreement with that reported in the literature. [

D-Hiv-OBn 4
Caesium carbonate (4.45 g, 13.7 mmol) was added to a solution of D-hydroxyisovaleric acid 3 (3.22 g, 27.3 mmol) in N,N-dimethylformamide (20 mL) at 0 °C and the resulting mixture was stirred for 40 min. Benzyl bromide (3.6 mL, 30.0 mmol) was added and the reaction mixture was stirred at room temperature for 15 h. The reaction mixture was filtered and the filtrate diluted with 40-60 petroleum ether:ethyl acetate (4:1, 120 mL). The organic layer was washed with saturated ammonium chloride solution (100 mL), saturated NaHCO 3 solution (100 mL) and saturated NaCl solution (100 mL) before drying over MgSO 4 and removing the solvent in vacuo. The crude product was purified via flash column chromatography using 40-60 petroleum ether:ethyl acetate (10:1) as eluent to afford the title compound 4 (4.56 g, 21.9 mmol, 80%) as a colourless oil. The 1 H and 13 C data was in agreement with that reported in the literature [7] .  Anhydrous HCl (2.5 mL, 4M in dioxane) was added to a solution of Boc-Me-Phe-D-Hiv-OBn 6a (0.31 g, 0.66 mmol) in dioxane (2.5 mL) at room temperature and the resulting mixture was stirred for 3 h. The solvent was removed in vacuo and the residue was taken up sequentially in ethanol (2 x 5 mL) and methanol (5 mL) with the solvent removed in vacuo after each addition to afford the amine HCl salt which was used in the next step without further purification.
Hydrogen gas was bubbled through a solution of Boc-Me-Phe-D-Hiv-OBn 6a (0.31 g, 0.66 mmol) and palladium (70 mg, 10% on charcoal, 0.07 mmol) in tetrahydrofuran (5 mL) for 3 min at room temperature before the resulting mixture was stirred under a hydrogen atmosphere for 2.5 h. The reaction mixture was filtered through celite, eluting with ethyl acetate (20 mL). The solvent was removed in vacuo from the filtrate to afford the acid which was used in the next step without further purification.
Ghosez reagent (0.096 mL, 0.72 mmol) was added to a solution of the crude acid in dichloromethane (2.5 mL) at 0 °C and the resulting mixture was stirred for 20 min at this temperature. A solution of the crude amine HCl salt and N,N-diisopropylethylamine (0.40 mL, 2.36 mmol) in dichloromethane (2.5 mL) was added and the reaction mixture was allowed to warm to room temperature and stirred for 17 h. Aqueous HCl (20 mL, 1M) was added and the phases were separated. The aqueous layer was extracted with dichloromethane (3 x 20 mL) and the combined organic layers were washed with water (40 mL), saturated NaHCO 3 solution (40 mL) and saturated NaCl solution (40 mL) before drying over MgSO 4 and removing the solvent in vacuo. The crude product was purified via flash column chromatography using 40-60 petroleum ether:ethyl acetate (10:1) as eluent to afford the title compound 8a (0.33 g, 0.45 mmol, 68%) as a colourless solid.

Method B (Flow)
According to General Method 1, a solution of coupling partner A 6a (0.024 g, 0.05 mmol) in dichloromethane (5 mL) was hydrogenated to give the acid and the nitrogen of coupling partner B 6a (0.024 g, 0.05 mmol) was deprotected using acid (reaction time = 3 h) and the resultant crude products were coupled in flow. The crude product was purified using preparative TLC using 40-60 petroleum ether:ethyl acetate (2:1) as eluent to afford the title compound 8a (0.031 g, 0.042 mmol, 82%) as a colourless solid, which was spectroscopically identical to that reported using method A.

Method A (Batch)
Anhydrous HCl (2 mL, 4M in dioxane) was added to a solution of Boc-Me-Phe-D-Hiv-Me-Phe-D-Hiv-OBn 8a (0.17 g, 0.23 mmol) in dioxane (2 mL) at room temperature and the resulting mixture was stirred for 6 h. The solvent was removed in vacuo and the residue was taken up sequentially in ethanol (2 x 5 mL) and methanol (5 mL) with the solvent removed in vacuo after each addition to afford the crude amine HCl salt which was used in the next step without further purification.
Hydrogen gas was bubbled through a solution of Boc-Me-Phe-D-Hiv-OBn 6a (0.11 g, 0.23 mmol) and palladium (24 mg, 10% on charcoal, 0.02 mmol,) in tetrahydrofuran (5 mL) for 3 min at room temperature before the resulting mixture was stirred under hydrogen for 2.5 h. The reaction mixture was filtered through celite, which was washed with ethyl acetate (20 mL). The solvent was removed in vacuo from the filtrate to afford the crude acid which was used in the next step without further purification.
Ghosez reagent (31 µL, 0.23 mmol) was added to a solution of the crude acid in dichloromethane (3 mL) at 0 °C and the resulting mixture was stirred for 20 min. A solution of the crude amine and N,N-diisopropylethylamine (0.14 mL, 0.84 mmol) in dichloromethane (3 mL) was added and the reaction mixture was stirred at room temperature for 18 h. Aqueous HCl (5 mL, 1M) was added, the reaction mixture was diluted with diethyl ether (10 mL) and the phases were separated. The aqueous layer was extracted with diethyl ether (3 x 10 mL) and the combined organic layers were washed with water (30 mL), saturated NaHCO 3 solution (30 mL) and saturated NaCl solution (30 mL) before drying over MgSO 4 and removing the solvent in vacuo. The crude product was purified via flash column chromatography using 40-60 petroleum ether:ethyl acetate (10:1  5:1) as eluent to afford the title compound 10a (0.18 g, 0.18 mmol, 75%) as a colourless solid.

Method B (Flow)
According to General Method 1, a solution of coupling partner A 6a (0.024 g, 0.05 mmol) in dichloromethane (5 mL) was hydrogenated to give the acid and the nitrogen of coupling partner B 8a (0.037 g, 0.05 mmol) was deprotected using HCl (reaction time = 6 h) and the resultant crude products were coupled in flow. The crude product was purified using preparative TLC using 40-60 petroleum ether:ethyl acetate (2:1) as eluent to afford the title compound 10a (0.039 g, 0.039 mmol, 78%) as a colourless solid, which was spectroscopically identical to that reported using method A.

Method A (Batch)
Anhydrous HCl (3 mL, 4M in dioxane) was added to a solution of 8a (0.10 g, 0.14 mmol) in dioxane (3 mL) and the reaction mixture was stirred at room temperature for 6 h before the solvent was removed in vacuo. Ethanol (2 x 3 mL) and methanol (3 mL) were added successively, removing the solvent in vacuo after each addition to afford the crude amine HCl salt which was used in the next step without further purification.
Hydrogen gas was bubbled through a solution of 8a (0.10 g, 0.14 mmol) and palladium (0.015 g, 10% on charcoal, 0.014 mmol) in tetrahydrofuran (4 mL) for 5 min before stirring under a hydrogen atmosphere for 6 h. The reaction mixture was filtered through cotton wool and a syringe filter and the solvent was removed in vacuo to afford crude acid which was used in the next step without further purification.
Ghosez reagent (0.02 mL, 0.15 mmol) was added to a solution of the crude acid in dichloromethane (5 mL) at 0 °C and the reaction mixture was stirred at this temperature for 20 min before a solution of the crude amine HCl salt and diisopropylethylamine (0.088 mL, 0.5 mmol) in dichloromethane (5 mL) was added and the reaction mixture was warmed to room temperature and stirred for 20.5 h.
The solvent was removed in vacuo and the crude taken up in diethyl ether (5 mL) and aqueous HCl (5 mL, 1M). The reaction mixture was extracted with diethyl ether (3 x 5 mL) and the combined organic extracts were washed with water (5 mL), saturated NaHCO 3 (5 mL) and saturated NaCl (5 mL) and were dried over MgSO 4 before the solvent was removed in vacuo to afford a crude oil. This was purified using preparative TLC using 40-60 petroleum ether:ethyl acetate (2:1) as eluent to afford the title compound 11a (0.10 g, 0.08 mmol, 57%) as a colourless solid.

Method B (Flow)
According to General Method 1, a solution of coupling partner A 8a (0.037 g, 0.05 mmol) in dichloromethane (5 mL) was hydrogenated to give the acid and coupling partner B 8a (0.037 g, 0.05 mmol) was acid deprotected (reaction time = 6 h) and the resultant crude products were coupled in flow. The crude product was purified using preparative TLC using 40-60 petroleum ether:ethyl acetate (2:1) as eluent to afford the title compound 11a (0.042 g, 0.034 mmol, 67%) as a colourless solid, which was spectroscopically identical to that reported using method A. 4.7 -[Me-Phe-D-Hiv-Me-Phe-D-Hiv]-9a 4.7.1 Method A (Batch) Palladium (0.02 g, 10% on charcoal, 0.01 mmol) was added to a solution of Boc-Me-Phe-D-Hiv-Me-Phe-D-Hiv-OBn 6a (0.10 g, 0.14 mmol) and hydrogen was bubbled through the reaction mixture for 5 min before stirring under a hydrogen atmosphere for 6 h. The reaction mixture was filtered through a plug of cotton wool and a syringe filter and the solvent was removed in vacuo from the filtrate to afford the crude acid. This was taken up in dioxane (3 mL) and anhydrous HCl (3 mL, 4M in dioxane) added. The reaction mixture was stirred at room temperature for 6 h before the solvent was removed in vacuo. Ethanol (2 x 3 mL) and methanol (3 mL) were added successively and the solvent removed in vacuo after each addition to afford the doubly deprotected depsipeptide HCl salt. This was taken up in dichloromethane (14 mL) and was cooled to 0 °C before Ghosez reagent (0.02 mL, 0.15 mmol) was added. The reaction mixture was stirred at 0 °C for 20 min before diisopropylethylamine (0.09 mL, 0.50 mmol) was added and the reaction mixture warmed to room temperature and stirred for 21 h. The solvent was removed in vacuo and the crude taken up in diethyl ether (5 mL) and aqueous HCl (5 mL, 1M). The reaction mixture was extracted with diethyl ether (3 x 5 mL) and the combined organic extracts were washed with water (5 mL), saturated NaHCO 3 (5 mL) and saturated NaCl (5 mL). The combined aqueous extracts were further extracted with ethyl acetate (3 x 5 mL) and the combined organics were dried over MgSO 4 before the solvent was removed in vacuo. The resulting crude oil was purified using preparative TLC using 40-60 petroleum ether:ethyl acetate (1:1) as eluent to afford the title compound 9a (0.012 g, 0.024 mmol, 17%) as a colourless solid and cyclic tetramer 2a (0.030 g, 0.029 mmol, 41%) as a colourless solid which was spectroscopically identical to that reported for this compound below. According to General Method 2, a solution of protected precursor 8a (0.037 g, 0.05 mmol) in dichloromethane (5 mL) was hydrogenated to give the acid and then the nitrogen acid deprotected (reaction time = 6 h) and the resultant crude product macrocyclised in flow. The crude product was purified using preparative TLC using toluene:ethyl acetate (2:1) as eluent to afford the title compound 9a (0.018 g, 0.034 mmol, 68%) as a colourless solid, which was spectroscopically identical to that reported using method A. and palladium (9 mg, 10% on charcoal, 8.4 µmol) in tetrahydrofuran (2 mL) for 3 min at room temperature before the mixture was stirred under hydrogen for 6 h. The reaction mixture was filtered through celite, washing with ethyl acetate (30 mL). The solvent was removed in vacuo from the filtrate to afford the crude acid, which was then taken up in dioxane (2 mL). Anhydrous HCl (2 mL, 4M in dioxane) was added and the resulting mixture was stirred for 6 h. The solvent was removed in vacuo and the residue was taken up sequentially in ethanol (2 x 5 mL) and methanol (5 mL) removing the solvent in vacuo after each addition to afford the deprotected linear precursor. This was taken up in dichloromethane (20 mL) and cooled to 0 °C before Ghosez reagent (12 µL, 92 µmol) was added at and the resulting mixture was stirred for 30 min at this temperature. N,N-diisopropylethylamine (51 µL, 301 µmol) was added and the reaction mixture was warmed to room temperature and stirred for 18.5 h. The solvent was removed in vacuo and the residue was taken up in diethyl ether (60 mL) and aqueous HCl (60 mL, 1M). The aqueous layer was extracted with diethyl ether (3 x 30 mL) and the combined organic layers were washed with water (100 mL), saturated NaHCO 3 solution (100 mL) and saturated NaCl solution (100 mL) before drying over MgSO 4 and removing the solvent in vacuo. The crude product was purified via flash column chromatography using 40-60 petroleum ether:ethyl acetate (2:1  1:1) as eluent to afford the title compound 1a (46 mg, 59 µmol, 70%) as a colourless solid. The 1 H and 13 C NMR spectrum were in agreement with that published in the literature. [9] R f = 0.15 [40-60 petroleum ether:diethyl ether = 1:1]; mp = 92 -93 °C (literature = 92 -93 °C [9] ); α D 24.7 = +75 (c 0.1 MeOH) literature [10]

Method B (Flow)
According to General Method 2, a solution of protected precursor 11a (0.063 g, 0.05 mmol) in methanol (5 mL) and dichloromethane (2 mL) and was hydrogenated to give the acid and then the nitrogen acid deprotected (reaction time = 6 h 25 min) and the resultant crude product macrocyclised in flow. The crude product was purified using preparative TLC using toluene:ethyl acetate (1:1) as eluent to afford the title compound 2a (0.041 g, 0.040 mmol, 80%) as a colourless solid, which was spectroscopically identical to that reported using method A.
5 Synthesis of N-Me-Leu containing depsipeptides

Method A (Batch)
Anhydrous HCl (2.5 mL, 4M in dioxane) was added to a solution of Boc-Me-Leu-D-Hiv-OBn 6b (0.12 g, 0.26 mmol) in dioxane (2.5 mL) at room temperature and the resulting mixture was stirred for 3 h. The solvent was removed in vacuo and the residue was taken up sequentially in ethanol (2 x 5 mL) and methanol (5 mL) with the solvent removed in vacuo after each addition to afford the amine HCl salt which was used directly in the next step.
Hydrogen gas was bubbled through a solution of Boc-Me-Leu-D-Hiv-OBn 6b (0.12 g, 0.26 mmol) and palladium (26 mg, 10% on charcoal, 26.4 µmol) in tetrahydrofuran (5 mL) for 3 min at room temperature before the resulting mixture was stirred under a hydrogen atmosphere for 3 h. The reaction mixture was filtered through celite, eluting with ethyl acetate (20 mL). The solvent was removed in vacuo from the filtrate to afford the acid which was used directly in the next step.
Ghosez reagent (38 µL, 0.29 mmol) was added to a solution of the acid in dichloromethane (5 mL) at 0 °C and the resulting mixture was stirred for 25 min. A solution of amine HCl salt and N,N-diisopropylethylamine (0.17 mL, 0.95 mmol) in dichloromethane (5 mL) was added, the reaction mixture was allowed to warm to room temperature and stirred for 22 h. Aqueous HCl (20 mL, 1M) was added, the reaction mixture was diluted with diethyl ether (40 mL) and the phases were separated. The aqueous layer was extracted with diethyl ether (3 x 20 mL) and the combined organic layers were washed with water (100 mL), saturated NaHCO 3 solution (100 mL) and saturated NaCl solution (100 mL) before drying over MgSO 4 and removing the solvent in vacuo. The crude product was purified via flash column chromatography using 40-60 petroleum ether:ethyl acetate (10:1  5:1) as eluent to afford the title compound 8b (0.11 g, 0.17 mmol, 64%) as a colourless solid.

Method B (Flow)
According to General Method 1, a solution of coupling partner A 6b (0.022 g, 0.05 mmol) in methanol (5 mL) was hydrogenated to give the acid and coupling partner B 6b (0.022 g, 0.05 mmol) was deprotected using HCl (reaction time = 5 h) and the resultant crude products were coupled in flow. The product was purified using flash column chromatography using 40-60 petroleum ether:ethyl acetate (10:1) as eluent to afford the title compound 8b (0.030 g, 0.045 mmol, 90%) as a colourless solid, which was spectroscopically identical to that reported using method A. 5.3 Boc-Me-Leu-D-Hiv-Me-Leu-D-Hiv-Me-Leu-D-Hiv-OBn 10b 5.3.1 Method A (Batch) Anhydrous HCl (3 mL, 4M in dioxane) was added to a solution of Boc-Me-Leu-D-Hiv-OBn 6b (0.10 g, 0.15 mmol) in dioxane (3 mL) at room temperature and the resulting mixture was stirred for 6 h. The solvent was removed in vacuo and the residue was taken up sequentially in ethanol (2 x 4 mL) and methanol (4 mL) with the solvent removed in vacuo after each addition to afford the amine HCl salt which was used directly in the next step.
Hydrogen gas was bubbled through a solution of Boc-Me-Leu-D-Hiv-OBn 6b (0.07 g, 0.15 mmol) and palladium (16 mg, 10% on charcoal, 0.015 mmol) in tetrahydrofuran (4 mL) for 5 min at room temperature before the resulting mixture was stirred under a hydrogen atmosphere for 3 h 15 min. The reaction mixture was filtered through cotton wool and syringe filter (0.2 μm) and the solvent was removed in vacuo from the filtrate to afford the acid which was used directly in the next step.
Ghosez reagent (0.022 mL, 0.17 mmol) was added to a solution of the acid in dichloromethane (5 mL) at 0 °C and the resulting mixture was stirred for 20 min. A solution of amine hydrochloride salt and N,N-diisopropylethylamine (0.094 mL, 0.54 mmol) in dichloromethane (5 mL) was added, the reaction mixture was allowed to warm to room temperature and stirred for 18 h. The solvent was removed in vacuo and aqueous HCl (5 mL, 1M) was added before the reaction mixture was extracted with ethyl acetate (3 x 15 mL) and the combined organic layers were washed with water (5 mL), saturated NaHCO 3 solution (5 mL) and saturated NaCl solution (5 mL) before drying over MgSO 4 and removing the solvent in vacuo. The crude product was purified via preparative TLC using 40-60 petroleum ether:ethyl acetate (2:1) as eluent to afford the title compound 10b (0.10 g, 0.12 mmol, 78%) as a colourless foam.