Short Total Synthesis of Ajoene, (E,Z)‐4,5,9‐Trithiadodeca‐1,6,11‐triene 9‐oxide, in Batch and (E,Z)‐4,5,9‐Trithiadodeca‐1,7,11‐triene in Continuous Flow

Abstract A short total synthesis of ajoene, (E,Z)‐4,5,9‐trithiadodeca‐1,6,11‐triene 9‐oxide, has been achieved over six steps. In addition, a continuous flow synthesis under mild reaction conditions to (E,Z)‐4,5,9‐trithiadodeca‐1,7,11‐triene is described starting from simple and easily accessible starting materials. Over four steps including propargylation, radical addition of thioacetate, deprotection, and disulfide formation/ allylation, the target product can be obtained at a rate of 0.26 g h−1 in an overall yield of 12 %.


S3
Biological Assay Techniques 1. MBIC Assay [1] In short, compounds were assayed for their ability to inhibit S. aureus or P. aeruginosa biofilm formation as follows. All compounds were prepared as a 30 mM stock in DMSO and diluted in TSB to 144 µM.
The compounds were further serially diluted two-fold from 144 µM to 2.25 µM and added to a 96-well plate at 100 µL per well. Overnight cultures of bacteria were diluted 1:2000 in fresh TSB and 100 µl of the diluted bacterial culture was added to the compound containing wells. The bacteria were incubated in the presence of the compounds overnight at 37 ºC. After incubation, the planktonic bacteria were removed by three washes using H2O and the remaining biofilm was stained using crystal violet and quantified photometrically at A570. Absorbances corresponding to biofilm mass were plotted versus compound concentration and IC50 values were calculated using Graphpad Prism 8.0. [2] To summarise, the P. aeruginosa lasB-gfp strain was cultured overnight in ABT with gentamicin (selective antibiotic; 120µg/mL) at 37 ºC, 150 rpm. Test compounds were solubilized to a stock concentration of 30 mM in DMSO (100%) and diluted 1:100 in ABT media. The compounds were further diluted 1:1 in the wells of a black 96 well plate to produce a range of test concentrations; the final concentrations tested were 150 µM, 75 µM, 37.5 µM and 18.75 µM. ABT medium without compound was added to rows one and twelve of the 96 well plate to provide untreated controls and blank medium controls respectively. The overnight culture was diluted to an OD600 of 0.08 in ABT medium (the values were blanked against ABT medium) and added 1:1 to each well with the exception of the blank medium control. The plate was placed into the BMG Fluostar Omega whereby the plate was read for absorbance (OD600) and fluorescence (excitation 485 nm, emission 520 nm) every 15 minutes for 16 hours at 37 ºC. The resulting readings were blank corrected, replicates were averaged, and data were normalized (Relative Fluorescence Units (RFU)/OD). The maximum fluorescence of each sample was compared to the mean maximum fluorescence of the untreated controls; the ratio was expressed as percent inhibition. Each compound was tested 3 times, except for ajoene which was tested 6 times. The data were reported as the percent inhibition of fluorescence when treated with 75 µM compound, a measure S4 mM). The plates were incubated at 37 ºC for 16 h. Data were measured as the diameter (mm) of the zone of blue colouration around the disc.

Flow Procedure:
Potassium carbonate (8.4 g, 0.06 mol) and sand (11.6 g) were shaken until a homogenous mixture was achieved by eye. The K2CO3/sand (20 g) was packed into a glass OmniSep® (150 x 15 mm). The packed column was weighed, and dry tetrahydrofuran (THF) was flowed through at 0.4 mL/min for 1 hour, after which, the column was reweighed. The mass difference equated to the calculated column volume (CV, 7.1 mL, residence time: 18 minutes).

S,S'-(Prop-1-ene-1,3-diyl) diethanethioate (6)
Batch Procedure: S-(Prop-2-yn-1-yl) ethanethioate 5 (398 mg, 3.5 mmol) was dissolved in dry toluene (5 mL) and the solution was heated to 85 °C under argon atmosphere. Azobisisobutyronitrile (AIBN) (57 mg, 0.35 mmol, 0.1 equiv.) was added to the solution directly, followed by the dropwise addition of thioacetic acid (0.28 mL) in toluene (5 mL) over 40 minutes using a syringe pump. The mixture was left to stir at 85 °C for a further 1 hour. The reaction was then quenched with aqueous saturated solution of sodium carbonate S6 (5 mL) and the toluene was removed in vacuo. The remaining residue was dissolved diethyl ether (20 mL) and the organic layer was washed with brine (2 x 10 mL) and dried over MgSO4. The solvent was evaporated in vacuo and the resulting residue was purified by column chromatography using Biotage Isolera Four [gradient: 100% hexane for 3 column volumes (CV), then increased to 80:20 hexane:diethyl ether over 15 CV, then increased to 100% diethyl ether over 3 CV] to afford compound 6 as a colourless oil (0.4 g, 60%, E:Z 1.2:1.0).

Combined Flow Procedure for steps (i) and (ii):
Potassium carbonate (8.4 g, 0.06 mol) and sand (11.6 g) were shaken until a homogenous mixture was achieved by eye. The K2CO3/sand (20 g) was packed into a glass OmniSep® (150 x 15 mm). The

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The thioacetate, S-(3-(allyldisulfaneyl)allyl) ethanethioate 7 (100 mg, 0.45 mmol) was dissolved in methanol (4.5 mL) and cooled to -40 °C. Potassium hydroxide (31 mg, 0.48 mmol, 1.05 equiv.) in methanol (4.5 mL) was added to the solution, followed by the direct addition of allyl bromide (54 mg, 0.44 mmol, 1.0 equiv.). The solution was left at -40 °C for 1 hour and warmed to room temperature until thin layer chromatography (TLC) indicated full consumption of starting material. The reaction was quenched with a saturated solution of ammonium chloride (5 mL), and solvent was removed in vacuo.

Combined Flow Procedure:
Potassium carbonate (9.7 g, 0.07 mol) and sand (14.3 g) were shaken until a homogenous mixture was achieved by eye. The K2CO3/sand (24 g, 42 wt.%) was packed into a 10 g Biotage® Isolera column (C1). The packed column was weighed, and dry tetrahydrofuran (THF) was flowed through at 0.4 mL/min for 1 hour, after which, the column was reweighed. The mass difference equated to the The outlet of which delivered the reaction mixture to C4, a PTFE coil with volume 24 mL and a resulting residence time of 11 minutes. C4 was submerged in an ice bath to allow the reaction to occur at 0 °C.
The outlet of the continuous system was collected for 17 minutes (41 mL) and quenched with ammonium chloride. The organic layer was extracted using diethyl ether and washed with water and brine. The organic layer was dried over magnesium sulfate and concentrated in vacuo. The resulting residue was purified by column chromatography using Biotage Isolera Four [gradient: 100% hexane for 10 column volumes (CV), then increased to 90:10 hexane:diethyl ether over 10 CV, then increased to 100% diethyl ether over 3 CV] to afford compound 8 as a pale yellow oil (73 mg, 12%).

S-(3-((4-Methoxybenzyl)thio)allyl) ethanethioate (14)
S-(Prop-2-yn-1-yl) ethanethioate 5 (3.64 g, 32 mmol) was dissolved in dry toluene (20 mL) and the solution was heated to 85 °C under argon atmosphere. Azobisisobutyronitrile (AIBN) (1.05 g, 6.4 mmol, 0.2 equiv.) was added to the solution directly, followed by the dropwise addition of p-methoxy benzyl mercaptan (7.4 mL) in toluene (20 mL) over 45 minutes using a syringe pump. The mixture was left to stir at 85 °C for a further 1 hour. The reaction was then quenched with aqueous saturated solution of sodium carbonate (20 mL) and the toluene was removed in vacuo. The remaining residue was dissolved S11 diethyl ether (20 mL) and the organic layer was washed with brine (2 x 10 mL) and dried over MgSO4.
The solvent was evaporated in vacuo and the resulting residue was purified by column chromatography using Biotage Isolera Four [gradient: 100% hexane for 3 column volumes (CV), then increased to 80:20 hexane:diethyl ether over 15 CV, then increased to 100% diethyl ether over 3 CV] to afford compound 14 as a pale yellow oil (6.13 g, 71%).
As a mixture of E/Z isomers: 1
Potassium hydroxide (1.7 g, 26.3 mmol, 1.5 equiv.) was added to the solution, followed by the direct addition of allyl bromide (3 mL, 35 mmol, 2 equiv.). The solution was left at -40 °C for 1 hour and warmed to room temperature until thin layer chromatography (TLC) indicated full consumption of starting material. The reaction was quenched with a saturated solution of ammonium chloride (10 mL), and solvent was removed in vacuo. The resulting residue was dissolved in diethyl ether (20 mL), and washed with water (20 mL), brine (2 x 20 mL), dried over MgSO4, and concentrated in vacuo. The resulting residue was purified using Biotage Isolera Four [gradient: 100% hexane for 10 column volumes (CV), then increased to 90:10 hexane:diethyl ether over 10 CV, then increased to 100% diethyl ether over 3 CV] to afford compound 15 as a pale yellow oil (3.5 g, 88%).   -1-en-1-yl)
Potassium hydroxide (20 mg, 0.31 mmol, 1.1 equiv.) in methanol (2 mL) was added to the solution, followed by the addition of allyl thiotosylate (76.7 mg, 0.34 mmol, 1.2 equiv.). The solution was left at -40 °C for 1 hour and warmed to room temperature until thin layer chromatography (TLC) indicated full consumption of starting material. The reaction was quenched with a saturated solution of ammonium chloride (5 mL), and solvent was removed in vacuo. The resulting residue was dissolved in diethyl ether S13 (20 mL), and washed with water (20 mL), brine (2 x 20 mL), dried over MgSO4, and concentrated in vacuo. The resulting residue was purified using Biotage Isolera Four [gradient: 100% hexane for 10 column volumes (CV), then increased to 90:10 hexane:diethyl ether over 10 CV, then increased to 100% diethyl ether over 3 CV] to afford compound 12 as a pale yellow oil (43 mg, 70%).

Design of Experiment for Step (iii) and (iv)
Umetrics MODDE Pro 12.1 was used for the design of experiment software. The 'Design Wizard' was used to create an optimised design that investigated two quantitative factors with two levels (time and temperature) and two quantitative factors with three levels (equivalence of electrophile and base).
Umetric MODDE Pro 12.1 produced a randomised design of 21 experiments, including 3 repeats. The equivalence of electrophile and base were controlled by the concentration whilst keeping the flow rate constant. The first one and a half column volumes of each run was discarded and then approximately 750 µL of solution was collected into a 1 mL HPLC vial containing 3 drops of saturated solution of ammonium chloride. From the quenched reaction mixture, a 50 µL aliquot was diluted into 1 mL acetonitrile and analysed by Reverse Phase HPLC. The solvent system for the starting material was chosen as a mixture of THF, dichloromethane and methanol to best represent the final system. Modde software based on the results analysed by HPLC predicted that at 2.0 equiv. of base and 3.0 equiv. of allyl thiotosylate at 0 °C and 5 minutes residence time, 7 could be obtained as in 54%, with 41% of 9 and unreacted 3% of 6 by integrated peak area at 254 nm. Modde software based on the results analysed by HPLC predicted that at 3.0 equiv. of base and 3.0 equiv. of allyl bromide at 0 °C and 10 minute residence time, 7 could be obtained in <60% as a percentage of peak area at 254 nm.