Raman spectroscopy as a tool for monitoring mesoscale continuous-flow organic synthesis: Equipment interface and assessment in four medicinally-relevant reactions

An apparatus is reported for real-time Raman monitoring of reactions performed using continuous-flow processing. Its capability is assessed by studying four reactions, all involving formation of products bearing α,β-unsaturated carbonyl moieties; synthesis of 3-acetylcoumarin, Knoevenagel and Claisen–Schmidt condensations, and a Biginelli reaction. In each case it is possible to monitor the reactions and also in one case, by means of a calibration curve, determine product conversion from Raman spectral data as corroborated by data obtained using NMR spectroscopy.

NMR spectra of the isolated products (1, 2a, 3d, 4a), further experimental information, and pictures of the Raman interface and Cartesian coordinates of the stationary points Specifics of the Raman spectrometer / flow chemistry interface S2 Photographs of the Raman spectrometer / flow chemistry interface S3 Experimental details for the four reactions studied S4-S11 Catesian Coordinates of All Stationary Points S12-S15 1 H NMR spectra of isolated compounds (1a, 2a, 3d, 4a) S16-S19 13 C NMR spectra of isolated compounds (1a, 2a, 3d, 4a) S20-S23 19 Specifics of the Raman spectrometer / flow chemistry interface The Raman system used was an Enwave Optronics Spectrometer, Model EZRaman-L (www.enwaveopt.com). The continuous-flow unit used was a Vapourtec E-series. A Starna 583.65.65-Q-5/Z20 flow-cell (width: 6.5 mm, height: 20 mm, path length: 5 mm) was placed inline after the back-pressure regulator using 1mm i.d. PFA tubing. The flow cell was secured in place in a custom-made box and the fiber-optic probe from the spectrometer inserted so it touched the wall of the flow cell. SYSTEM SOFTWARE: EZ Raman 3.5.4MAS. Data files exported into Microsoft Excel.
The flow system used in the study was a Vapourtec E-series (www.vapourtec.co.uk) equipped with a 10 mL PFA reactor coil.

3-Acetylcoumarin (1a), compound 1 of the main article:
Into a 50 mL volumetric flask was added salicylaldehyde (6.106 g, 50 mmol, 1 equiv) and ethyl acetoacetate (6.507 g, 50 mmol, 1 equiv). Ethyl acetate was added to bring the total volume to 50 mL (1 M) and the reagents were thoroughly mixed. An aliquot of this solution (10 mL) was transferred to a 20 mL vial equipped with a Teflon-coated stir bar. The flow reactor was readied using the equipment manufacturer's suggested start-up sequence. Ethyl acetate was pumped at 1 mL/min to fill the reactor coil. The back-pressure regulator was adjusted to 7 bar and the reactor coil heated to 65 °C. After the heating coil, the product stream was intercepted with a stream of acetone (1 mL/min) by means of a T-piece to ensure complete solubility of the product. The Raman probe was inserted into the box containing the flow cell and was properly focused. A background scan of the ethyl acetate/acetone solvent system was taken. This background was then automatically subtracted from all subsequent scans, thereby removing any signals from the solvent. The Raman spectrometer was set to acquire a spectrum every 15 s throughout the run, with 10 s integration time, boxcar = 3, and average = 1. When the flow unit was ready, piperidine (0.099 mL, 0.1 mmol, 0.1 equiv) was injected all at once into the vial containing the reagents and, after mixing for 15 s, the reaction mixture was loaded into the reactor at a flow rate of 1 mL/min. After the reaction mixture had been completely loaded into the reactor, ethyl acetate was again pumped through the coil at 1 mL/ min. After the product had been fully discharged from the flow cell, the scans were halted. After the product had been fully discharged from the flow cell, the scans were halted and the resulting clear yellow solution was then concentrated in vacuo by rotary evaporation. The crude was transferred to a filter funnel and washed with cold hexanes. The product was recrystallized from ethanol to afford 3acetylcoumarin 1a (1.56 g, 85%) as a pale yellow solid.

Knoevenagel reaction (Z)-Ethyl 2-benzylidene-3-oxobutanoate (2a):
Into a 50 mL volumetric flask was added benzaldehyde (5.306 g, 50 mmol, 1 equiv) and ethyl acetoacetate (6.507 g, 50 mmol, 1 equiv). EtOAc was added to bring the total volume to 50 mL (1 M) and the reagents were thoroughly mixed. An aliquot of this solution (10 mL) was transferred to a 20 mL vial equipped with a Teflon-coated stir bar. The flow reactor was readied using the equipment manufacturer's suggested start-up sequence. Ethyl acetate was pumped at 1 mL/min to prime the system as the coil was heated to 130 °C. The back-pressure regulator was adjusted to 7 bar to prevent the solvent from boiling in the coil. The Raman probe was inserted into the box containing the flow cell and was properly focused. The background scan of the solvent system was taken that would be subtracted from all subsequent scans. Piperidine (0.099 mL, 0.1 mmol, 0.1 equiv) was injected all at once and, after mixing for 15 s, the reaction mixture was loaded into the reactor at a flow rate of 1 mL/min. Product collection was commenced immediately after this switch. After the reaction mixture had been completely loaded into the reactor, the reactor pump was set back to pumping ethyl acetate. The resulting clear yellow solution was poured over aqueous 2 M HCl and extracted with ethyl acetate. The combined organic layers washed with brine, dried over Na 2 SO 4 , and the solvent was removed in vacuo by rotary evaporation affording the crude product. The crude product was loaded on a 15cm silica gel column (55 g silica gel) and a gradient eluting system (99:1, 95:5, 90:10; Hex:EtOAc) was used to obtain (Z)-ethyl 2-benzylidene-3-oxobutanoate, 2a (1.3095 g, 60%) as a clear yellow oil.
The Raman spectrum of 2a generated using Gaussian 09 at the B3LYP/6-31g(d) level of theory. The inset molecule illustrates the complex stretching mode responsible for the signal calculated at 1600 cm − 1 (actual: 1598 cm −1 ).

Claisen-Schmidt reaction (E)-3-(4-Fluorophenyl)-3-phenylprop-2-en-1-one (3d):
Into a 50 mL volumetric flask was added 4-fluorobenzaldehyde (1.551 g, 12.5 mmol, 1 equiv) and ethyl acetoacetate (1.637 g, 12.5 mmol, 1 equiv). Ethanol was added to bring the total volume to 50 mL (0.25 M) and the reagents were thoroughly mixed. An aliquot of this solution (10 mL) was transferred to a 20 mL vial equipped with a Teflon-coated stir bar. The flow reactor was readied using the equipment manufacturer's suggested start-up sequence. Ethanol was pumped at 0.5 mL/min to prime the system as the coil was heated to 65 °C. The back-pressure regulator was adjusted to 7 bar to prevent the solvent from boiling in the coil. After the heating coil, the product stream was intercepted by pumping acetone at 0.5 mL/min by means of a Tpiece to ensure complete solubility of the product. The Raman probe was inserted into the box containing the flow cell and was properly focused. The background scan of the solvent system was taken that would be subtracted from all subsequent scans. 2 M NaOH (0.125 mL, 0.25 mmol) was injected all at once and after mixing for 15 s the reaction mixture was loaded into the reactor coil at a flow rate of 0.5 mL/min. Product collection was commenced immediately after this switch. After the reaction mixture had been completely loaded into the reactor, the reactor pump was set back to pumping ethanol. The Raman spectrometer was programmed to take continuous scans using the same parameters as the coumarin synthesis, above. After the product had been fully discharged from the flow cell, the scans were halted. The yellow solution was poured into a beaker containing ice (100 g) causing an immediate precipitation of the product. To ensure complete precipitation, the solution was stirred at 0 °C. The solid product was collected via vacuum filtration and washed with cold ethanol. The material was dried on top of the oven for 6 hours to yield (E)-3-(4-fluorophenyl)-3-phenylprop-2-en-1-one, 3d (0.5421 g, 91%) as a pale yellow solid.   S9 Chalcone (3a) (90% GC conversion) was prepared according to the representative procedure for the synthesis of 3d from benzaldehyde (0.2653 g, 2.5 mmol).

Ethyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4a):
In a 50 mL volumetric flask was dissolved urea (3.003 g, 50 mmol, 1 equiv) in methanol (~30 mL). Into the flask was then added benzaldehyde (1.306 g, 50 mmol, 1 equiv), ethyl acetoacetate (6.507 g, 50 mmol, 1 equiv). Methanol was added to bring the total volume to 50 mL (1 M) and the reagents were thoroughly mixed. An aliquot of this solution (10 mL) was transferred to a 20 mL vial equipped with a Teflon-coated stir bar. The flow reactor was readied using the equipment manufacturer's suggested start-up sequence. Methanol was pumped at 0.25 mL min -1 to prime the system as the coil was heated to 120 °C. The back-pressure regulator was adjusted to 7 bar to prevent the solvent from boiling in the coil. After the heating coil, the product stream was intercepted by pumping DMF (0.25 mL/min) by means of a T-piece to ensure complete solubility of the product. The Raman spectrometer was set to acquire a spectrum every 25 s, with 20 s integration time, boxcar = 3, and average = 1. When the flow unit was ready, 6 M H 2 SO 4 (0.2 mL, 0.1 equiv) was injected all at once and after mixing for 15 s the reaction mixture was loaded into the reactor coil at a flow rate of 0.25 mL/min. After the reaction mixture had been completely loaded into the reactor, methanol was again pumped through the coil at 0.25 mL/ min. Product collection was commenced immediately after this switch. After the reaction mixture had been completely loaded into the reactor, the reactor pump was set back to pumping methanol. After the product had been fully discharged from the flow cell, the scans were halted. The reaction mixture was transferred to a separatory funnel, diluted with diethyl ether and sat. NaHCO 3 (100 mL) and water (100 mL) added. The layers were separated and the aqueous layer extracted with ether (3 × 100 mL). The combined organics were washed with brine (2 × 100 mL) and dried over Na 2 SO 4 . The solvent was removed in vacuo by rotary evaporation affording the crude product. The resulting solid was transferred to a filter funnel, washed with cold methanol and air dried to afford 4a (2.03 g, 78%) as a fluffy white solid.