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

Article abstract of DOI:10.3762/bjoc.9.215

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; synthesi

Full text of DOI:10.3762/bjoc.9.215

Raman spectroscopy as a tool for monitoring
mesoscale continuous-flow organic synthesis:
Equipment interface and assessment in four
medicinally-relevant reactions
Trevor A. Hamlin and Nicholas E. Leadbeater*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 1843–1852.
Department of Chemistry, University of Connecticut, 55 North
Eagleville Road, Storrs, CT 06269, USA
doi:10.3762/bjoc.9.215
Received: 24 May 2013
Email:
Accepted: 15 August 2013
Published: 11 September 2013
Nicholas E. Leadbeater* - nicholas.leadbeater@uconn.edu
* Corresponding author
This article is part of the Thematic Series "Chemistry in flow systems III".
Guest Editor: A. Kirschning
Keywords:
flow processing; Raman spectroscopy; reaction monitoring;
α,β-unsaturated carbonyl
© 2013 Hamlin and Leadbeater; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
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.
Introduction
Continuous-flow processing is used in the chemical industry on of reactions performed using flow chemistry and optimizing
production scales. In a research and development setting, there reaction conditions, one option is to use inline product analysis.
has been increasing interest in using flow chemistry on smaller This opens the avenue for fast, reliable assay in comparison
scales. To this end, a wide range of companies now produce with the traditional approach in which performance is evalu-
equipment for both micro- and mesofluidic flow chemistry ated based on offline product analysis. When interfaced with
[1,2]. Some of the advantages of these devices are increased microreactors, inline analysis has taken significant strides in
experimental safety, easy scale-up and thorough mixing of recent years [7,10]. Spectroscopic tools such as infrared [11-
reagents [3-7]. It is not surprising, therefore, that a wide range 15], UV–visible [16-18], NMR [19,20], Raman [21-25], and
of synthetic chemistry transformations have been reported using mass spectrometry [26,27] have all been interfaced with
this equipment [8,9]. When it comes to evaluating the outcome success. There have been less reports when it comes to
1843

Beilstein J. Org. Chem. 2013, 9, 1843–1852.
mesoflow systems. Perhaps most developed is the area of
infrared monitoring. The now ubiquitous ReactIR equipment
has been interfaced with commercially available flow equip-
ment to allow for real-time analysis of reactions and on-the-fly
optimization of conditions [28-30].
In our laboratory we have had success interfacing a Raman
spectrometer with a scientific microwave unit [31]. This has
allowed us to monitor reactions from both a qualitative [32-35]
and quantitative [36,37] perspective. A recent report of the use
of Raman spectroscopy for monitoring a continuous-flow palla-
dium-catalyzed cross-coupling reaction [38] sparked our
interest in interfacing our Raman spectrometer with one of our
continuous-flow units and employing it for inline reaction
monitoring of a number of key medicinally-relevant organic
transformations. Our results are presented here.
Results and Discussion
Interfacing the spectrometer to the flow unit
In interfacing our Raman spectrometer with a continuous-flow
reactor, our objective was to use a similar approach to that
which proved successful when using microwave heating.
Borosilicate glass is essentially “Raman transparent”. There-
fore reactions could be monitored by placing a Raman probe
near the reaction vessel, without requirement to place any parts
of the spectrometer inside the reaction vessel. The exposure of
metallic components to the microwave field was avoided using
a quartz light-pipe extending both the excitation laser and the
acquisition fiber optic components of the spectrometer almost
without any loss of light. The optimum distance of the light-
Figure 1: (a) Flow cell and (b) Raman interface used in the present
study.
pipe to the outside wall of the reaction vessel was found to be experience of monitoring this reaction both qualitatively [32]
approximately 0.5 mm. Moving to our continuous-flow reactor, and quantitatively [36] when using microwave heating so
we decided to place the spectroscopic interface just after the believed it would be a good starting point for our present study.
back-pressure regulator assembly. This meant that we did not The reaction works well when using ethyl acetate as the solvent.
need to engineer a flow cell capable of holding significant pres- However, 1 is not completely soluble at room temperature. To
sure. Instead we used an off-the-shelf flow cell traditionally overcome potential clogging of the back-pressure regulator as
used in conjunction with other spectroscopic monitoring tools. well as mitigating the risk of having solid particles in the flow
The cell had screw-threaded inlet and outlet tubes of the same cell (which would perturb signal acquisition), we leveraged a
diameter as the tubing of the flow unit (i.d. 1 mm). The sample technique we developed for this and other reactions previously
chamber had a width of 6.5 mm, height of 20 mm and a path [39]. Once the reaction stream has exited the heated zone, it is
length of 5 mm giving the cell a nominal internal volume of intercepted with a flow of a suitable organic solvent. This solu-
0.210 mL (Figure 1a). We built an assembly to allow us to hold bilizes the product and allows it to pass through the back-pres-
the cell in a fixed location and vary the distance of the quartz
light-pipe so as to optimize the Raman signal intensity. The
apparatus is shown in Figure 1b.
Testing the interface: The synthesis of
3-acetylcoumarin
As our first reaction for study, we selected the piperidine-
Scheme 1: The reaction between salicylaldehyde and ethyl aceto-
acetate to form 3-acetyl coumarin (1).
catalyzed synthesis of 3-acetylcoumarin (1) from salicylalde-
hyde with ethyl acetoacetate (Scheme 1). We had extensive
1844

Beilstein J. Org. Chem. 2013, 9, 1843–1852.
sure regulator unimpeded. In the case of 1, we intercept the
product stream with a flow of acetone.
Our first objective was to determine whether we could observe
spectroscopically a slug of the coumarin passing through the
flow cell. The Raman spectrum of 1 (Figure 2) exhibits strong
Raman-active stretching modes at 1608 cm−1 and 1563 cm−1
while the salicylaldehyde and ethyl acetoacetate starting ma-
terials exhibit minimal Raman activity in this area. As a result,
we chose to monitor the 1608 cm−1 signal. To mimic a product
mixture, we pumped a solution of 1 in acetone through our flow
reactor, intercepted it with an equal volume of ethyl acetate and
passed this mixture through the flow cell. We recorded a Raman
spectrum every 15 s in an automated fashion as the coumarin
passed through the cell by using the “continuous-scan” func-
tion on our spectrometer. By subtracting the spectrum of the
solvent mixture (1:1 ethyl acetate:acetone) from the spectra
recorded, we were able to clearly see the growth of the signal
due to 1 followed by a plateau as it passed through the cell and
then a drop back to the baseline as the final aliquot exited
(Figure 3).
Figure 3: Monitoring an aliquot of 3-acetyl coumarin (1) as it passes
through the flow cell (scan time = 15 s, integration = 10 s).
Figure 4: Monitoring the conversion of salicylaldehyde and ethyl
acetoacetate to 3-acetylcoumarin (1) across a range of reaction condi-
tions (scan time = 15 s, integration = 10 s).
In an attempt to quantify product conversion, we needed next to
obtain a calibration curve to allow us to convert units of Raman
intensity to units of concentration in standard terms. To achieve
this, we passed solutions of various concentrations of 3-acetyl-
coumarin (3) in ethyl acetate/acetone through the flow cell and
collected the Raman spectrum. When the signal intensity at
1608 cm−1 is plotted against concentration, after subtraction of
signals due to the solvent, the result is a straight line (Figure 5).
Figure 2: The Raman spectrum of 3-acetylcoumarin (1) generated
using Gaussian 09 [40] at the B3LYP/6-31g(d) level of theory. The
inset molecule illustrates the stretching mode responsible for the signal
calculated at 1602 cm−1 (actual: 1608 cm−1).
Knowing we could observe the product as it passed through the The Stokes shift (which is being monitored) is inversely propor-
flow cell, we next performed the complete reaction. As a tional to the temperature. Since the flow cell is situated after the
starting point, we chose as conditions a flow rate of 1 mL/min product mixture exits the heated zone and because of the very
through a 10 mL PFA coil at room temperature. We were efficient heat transfer observed using narrow-gauge tubing, the
indeed able to monitor the reaction as shown in Figure 4. In an product mixture was essentially at room temperature by the
effort to optimize the reaction conditions, we varied both the time it passed through the flow cell. As a result, it was not
temperature of the reactor coil and also the flow rate, moni- deemed necessary to involve a scaling factor to account for
toring each run and then compiling the data (Figure 4). While temperature effects.
increasing the reaction temperature to 130 °C led to a marked
increase in product conversion, reducing the flow rate from With the appropriate calibration curve in hand, we were able to
1 mL/min to 0.5 mL/min at this temperature did not have a obtain product conversion values for each set of reaction condi-
significant impact on the outcome of the reaction.
tions screened, taking into account the fact that the product
1845

Beilstein J. Org. Chem. 2013, 9, 1843–1852.
Scheme 2: The Knoevenagel condensation of benzaldehyde and ethyl
acetoacetate to yield (Z)-ethyl 2-benzylidene-3-oxobutanoate (2a).
Performing the reaction across a range of conditions, flowing
the reaction mixture at 1 mL/min through the 10 mL coil heated
to 130 °C proved to be optimal (Figure 6). A 67% conversion to
2a was obtained, as determined by GC analysis. Purification of
the product mixture gave a 60% isolated yield of the Z-isomer
of 2a. Using these optimized reaction conditions, we screened
Figure 5: Plot of Raman intensity of the peak arising at 1608 cm-1 vs
concentration of 3-acetyl coumarin (1), yielding a straight line, y = mx +
b; m = Raman intensity·M−1 of 1.
concentration is halved by the interception with acetone. To
determine their accuracy, we also determined product conver-
sion using NMR spectroscopy. Comparison of the values shows
a good correlation (Table 1).
Expanding the technique to other reactions
The Knovenagel condensation
We turned our attention next to the Knoevenagel condensation
of ethyl acetoacetate with a range of aromatic aldehydes
(Scheme 2). Our objective was to optimize conditions using one
aldehyde substrate spectroscopically from a qualitative stand-
point and then screen other examples. We chose benzaldehyde
as our initial substrate, ethyl acetate as the solvent and piperi-
dine as a base catalyst. In order to determine the optimal spec-
tral frequency at which to monitor we wanted to find a quick
way to derive the Raman spectrum of the product 2a. As was
the case with 1, this could be achieved computationally using
Gaussian 09 at the B3LYP/6-31g(d) level of theory [40], and a
signal at 1598 cm−1 selected for monitoring.
Figure 6: Monitoring the conversion of benzaldehyde and ethyl aceto-
acetate to (Z)-ethyl 2-benzylidene-3-oxobutanoate (2a) across a range
of reaction conditions (scan time = 15 s, integration = 10 s).
Table 1: Comparison of product conversion values obtained from Raman spectra with those obtained using NMR spectroscopy for the conversion of
salicylaldehyde and ethyl acetoacetate to 3-acetylcoumarin (1).
Conditions
Raman monitoring
NMR
Concentration of 1 when Concentration of 1 after
diluted with acetone
(mol L–1)
normalizing for dilution
by acetone (mol L−1)
Conv. (%)
Conv. (%)
25 °C, 1 mL/min
65 °C, 1 mL/min
130 °C, 1 mL/min
130 °C, 0.5 mL/min
0.125
0.27
0.37
0.39
0.25
0.55
0.74
0.78
25
55
74
78
22
58
79
80
1846

Beilstein J. Org. Chem. 2013, 9, 1843–1852.
Table 2: Product conversion obtained for four aldehyde substrates in the Knoevenagel reaction with ethyl acetoacetate.
R
Product
Conv. (%)
H
OMe
Me
F
2a
2b
2c
2d
67 (60)a
53
66
63
aIsolated yield.
three para-substituted aldehyde substrates (Table 2). As be readily detected. It does however highlight the fact that there
expected, placing an electron-donating methoxy group on the may be both significant dispersion along the length of the
aromatic ring led to lower product conversion as compared to reactor and the product is slow in clearing the flow cell. Disper-
benzaldehyde (Table 2, entry 2). A methyl- or fluoro-substituent sion is the consequence of laminar flow and some of the ma-
has little effect on the outcome of the reaction (Table 2, entries terial takes longer to travel through the reactor than the rest.
3 and 4).
Thus, when a flow reactor is used to process a finite volume of
reagents, the leading and trailing ends of the product emerging
from the end of the reactor will have mixed to some extent with
The Claisen–Schmidt condensation
We moved next to study the Claisen–Schmidt condensation of the solvent that preceded or followed it. This means that there
benzaldehyde with acetophenone to yield chalcone (Scheme 3). are zones at the leading and trailing ends of the product stream
Chalcones display interesting biological properties such as anti- in which the concentration of product is variable. Our optimal
oxidant, cytotoxic, anticancer, antimicrobial, antiprotozoal, conditions for the reaction were heating at 65 °C with a flow
antiulcer, antihistaminic, and anti-inflammatory activity [41].
They are also intermediates on the way to highly fluorescent
cyanopyridine and deazalumazine dyes [42]. The calculated
Raman spectrum of the product 3a shows a very strong signal at
1604 cm−1 which was selected for monitoring. Using sodium
hydroxide as the catalyst, the reaction was monitored under a
range of reaction conditions (Figure 7). We fast discovered that
at temperatures in excess of 65 °C we observed decomposition
or else formation of a highly fluorescent byproduct, as evi-
denced by collapse of the Raman spectrum. We also observed a
significant “tail” on the plot of signal intensity at 1604 cm−1 vs
time. We attribute this to the fact that the chalcone product is
very highly Raman active and even a trace in the flow cell can
Figure 7: Monitoring the conversion of benzaldehyde with acetophe-
Scheme 3: Claisen-Schmidt condensation of benzaldehyde with
acetophenone to yield chalcone, 3a.
none to chalcone, 3a, across a range of reaction conditions (scan time
= 15 s, integration = 10 s).
1847

Beilstein J. Org. Chem. 2013, 9, 1843–1852.
Table 3: Product conversion obtained for four aldehyde substrates in the Claisen-Schmidt reaction with acetophenone.
R
Product
Conv. (%)
H
OMe
Me
F
3a
3b
3c
3d
90
66
84
98 (90)a
aIsolated yield.
rate of 1 mL/min, this corresponding to a product conversion of reaction of benzaldehyde, ethyl acetoacetate and urea catalyzed
90%, as determined by GC analysis. Performing the reaction by sulfuric acid (Figure 8).
under these conditions using three substituted benzaldehydes as
substrates, we obtained product conversions of 66−98% The calculated Raman spectrum of the product, 4a, shows a
depending on how electron rich or deficient the aromatic ring of strong signal at 1598 cm−1 which was selected for monitoring.
the aldehyde was (Table 3).
Using a catalyst loading of 10 mol % and a flow rate of
1 mL/min, we monitored the reaction over a temperature range
from 25–120 °C. Seeing that the reaction did not reach comple-
The Biginelli reaction
As our final reaction for study, we turned to the Biginelli reac- tion within the 10 min in the heated zone, we then repeated the
tion (Scheme 4) [43-48]. This acid-catalyzed cyclocondensa- process at lower flow rates; first to 0.5 mL/min and then
tion of urea, β-ketoesters and aromatic aldehydes to yield dihy- 0.25 mL/min. Our optimal conditions as determined by Raman
dropyrimidines has received significant attention, these prod- monitoring were heating at 120 °C with a flow rate of
ucts having pharmacological activity including calcium channel 0.25 mL/min, this corresponding to a product conversion of
modulation, mitotic kinesin Eg5 inhibition, and antiviral and
antibacterial activity [49,50]. The Biginelli reaction has been
performed in flow previously as a route to densely functional-
ized heterocycles using HBr generated in a prior step as the
catalyst for the reaction [51]. Copper catalysis has also been
used in flow mode for preparing PEG-immobilized dihydropy-
rimidines [52]. We decided to screen a set of conditions for the
Figure 8: Monitoring the conversion of benzaldehyde, ethyl aceto-
Scheme 4: The Biginelli cyclocondensation of benzaldehyde, ethyl
acetoacetate, and urea to yield 5-ethoxycarbonyl-6-methyl-4-phenyl-
3,4-dihydropyrimidin-2(1H)-one (4a).
acetate, and urea to 5-ethoxycarbonyl-6-methyl-4-phenyl-3,4-dihydro-
pyrimidin-2(1H)-one (4a) across a range of reaction conditions (scan
time = 15 s, integration = 10 s).
1848

Beilstein J. Org. Chem. 2013, 9, 1843–1852.
89%, as determined by GC analysis, and a product yield of 78% 19F NMR spectra were referenced to hexafluorobenzene
after purification. Performing the reaction using three other (−164.9 ppm) [53]. Reactions were monitored by an Agilent
aldehyde substrates resulted in similar product conversions Technologies 7820A Gas Chromatograph attached to a 5975
(Table 4).
Mass Spectrometer or 1H NMR. Flash chromatography and
silica plugs utilized Dynamic Adsorbants Inc. Flash Silica Gel
(60Å porosity, 32-63 µm).
Table 4: Product conversion obtained for four aldehyde substrates in
the Biginelli reaction with ethyl acetoacetate and urea.
Apparatus configuration
The Raman system used was an Enwave Optronics Spectrom-
eter, Model EZRaman-L [32]. 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 1 mm i.d.
PFA tubing (the void volume between the flow reactor and the
flow cell was 4.79 mL). The flow cell was secured in place in a
custom-made box and the fiber-optic probe from the spectrom-
eter inserted so it touched the wall of the flow cell. During a
reaction, spectral data was recorded at pre-determined time
intervals using the EZ Raman software provided with the instru-
ment. The data was then exported to Excel for processing.
R
Product
Conv. (%)
H
OMe
Me
F
4a
4b
4c
4d
88 (78)a
85
Typical procedure for monitoring the formation of
3-acetylcoumarin (1)
87
Performing the reaction: 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
91
aIsolated yield.
Conclusion
In conclusion, we describe here an apparatus for real-time transferred to a 20 mL vial equipped with a Teflon-coated stir
Raman monitoring of reactions performed using continuous- bar. The flow reactor was readied using the equipment manu-
flow processing. We assess its capability by studying four reac- facturer’s suggested start-up sequence. Ethyl acetate was
tions. We find that it is possible to monitor reactions and also, pumped at 1 mL/min to fill the reactor coil. The back-pressure
by means of a calibration curve, determine product conversion regulator was adjusted to 7 bar and the reactor coil heated to
from Raman spectral data as corroborated by data obtained 65 °C. After the heating coil, the product stream was inter-
using NMR spectroscopy. Work is now underway to expand the cepted with a stream of acetone (1 mL/min) by means of a
scope of the method to other classes of useful reactions.
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
Experimental
General experimental
All reagents are used as received from the various vendors then automatically subtracted from all subsequent scans,
without purification. Sodium sulfate, MeOH, EtOH, EtOAc, thereby removing any signals from the solvent. The Raman
DMF and Et2O (ACS Grade and reagent grade), were purchased spectrometer was set to acquire a spectrum every 15 s through-
from Sigma-Aldrich and used without further purification. out the run, with 10 s integration time, boxcar = 3, and average
Deuterated NMR solvents (CDCl3) were purchased from = 1. When the flow unit was ready, piperidine (0.099 mL,
Cambridge Isotope Laboratories. CDCl3 stored over 4Å molec- 0.1 mmol, 0.1 equiv.) was injected all at once into the vial
ular sieves and K2CO3. NMR Spectra (1H, 13C, 19F) were containing the reagents and, after mixing for 15 s, the reaction
performed at 298 K on either a Bruker DRX-400 MHz NMR, or mixture was loaded into the reactor at a flow rate of 1 mL/min.
Bruker Avance 500 MHz NMR. 1H NMR Spectra obtained in After the reaction mixture had been completely loaded into the
CDCl3 were referenced to residual non-deuterated chloroform reactor, ethyl acetate was again pumped through the coil at
(7.26 ppm) in the deuterated solvent. 13C NMR Spectra 1 mL/min. After the product had been fully discharged from the
obtained in CDCl3 were referenced to chloroform (77.3 ppm). flow cell, the scans were halted. While the product mixture was
1849

Beilstein J. Org. Chem. 2013, 9, 1843–1852.
passing through the flow cell, a drop of the exit stream was sequence. Ethanol was pumped at 0.5 mL/min to fill the reactor
removed and an NMR spectrum recorded to obtain product coil. The back-pressure regulator was adjusted to 7 bar and the
conversion for comparison with data obtained by Raman spec- reactor coil heated to 65 °C. After the heating coil, the product
troscopy. NMR conversions were determined by comparing stream was intercepted with a stream of acetone (0.5 mL/min)
signals from the starting salicylaldehyde (9.84 ppm) and the by means of a T-piece to ensure complete solubility of the prod-
coumarin product (8.45 ppm) [36].
uct. The Raman spectrometer was configured as in the case of
monitoring formation of 1. When the flow unit was ready, 2 M
Obtaining a relationship between signal strength and NaOH (0.125 mL, 0.25 mmol) was injected all at once and after
concentration: To obtain a calibration curve, spectra of mixing for 15 s the reaction mixture was loaded into the reactor
3-acetylcoumarin in 1:1 ethyl acetate/acetone were recorded at a coil at a flow rate of 0.5 mL/min. After the reaction mixture had
range of concentrations by passing the solutions through the been completely loaded into the reactor, ethanol was again
flow cell. A plot of signal strength due to the peak at 1608 cm−1 pumped through the coil at 0.5 mL/min. After the product had
versus concentration of 1 was constructed (Figure 5). From this, been fully discharged from the flow cell, the scans were halted.
units of Raman intensity could be converted to units of concen- The yellow product solution was poured into a beaker
tration in standard terms and hence product conversion deter- containing ice (100 g) causing an immediate precipitation of the
mined.
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
in air to yield (E)-3-(4-fluorophenyl)-1-phenylprop-2-en-1-one,
Typical procedure for monitoring the Knoevenagel
reaction
An analogous approach was used to prepare the Knoevenagel (3d, 0.5421 g, 91%) as a pale yellow solid. 1H NMR (CDCl3,
product as for the case of 1, benzaldehyde (5.306 g, 50 mmol, 1 400 MHz) δ ppm 7.11 (t, J = 8.68 Hz, 2H), 7.46 (d, J = 15.89
equiv) being used in place of salicylaldehyde and there being no Hz, 1H), 7.49–7.55 (m, 2H), 7.56–7.69 (m, 3H), 7.78 (d, J =
need for acetone interception of the product mixture. The 15.65 Hz, 1H), 8.02 (d, J = 7.34 Hz, 2H); 13C NMR (CDCl3,
Raman spectrometer was programmed to take continuous scans 100 MHz) δ ppm 116.42 (d, JC-C-F = 22.01 Hz, CH), 122.09 (d,
using the same parameters as in the case of 1. After the product JC-C-C-C-F = 2.20 Hz, C), 128.76 (s, 10C), 128.94 (s, 9C),
had been fully discharged from the flow cell, the scans were 130.62 (d, JC-C-C-F = 8.80 Hz, CH), 131.45 (d, JC-C-C-C-C-F =
halted. The resulting clear yellow solution was poured over 3.67 Hz, CH), 133.12 (C), 138.43 (C), 143.78 (CH), 164.35 (d,
aqueous 2 M HCl and extracted with ethyl acetate. The JC-F = 250.89 Hz, C), 190.59 (C); 19F NMR (CDCl3, 377 MHz)
combined organic layers were washed with brine, dried over δ ppm −113.59, −111.32 (m) [55,56].
sodium sulfate, and the solvent was removed in vacuo by rotary
evaporation affording the crude product. The crude product was Typical procedure for monitoring the Biginelli reac-
loaded on a 15-cm silica gel column (55 g silica gel) and a tion
gradient eluting system (99:1, 95:5, 90:10; Hex:EtOAc) was In a 50 mL volumetric flask was dissolved urea (3.003 g, 50
used to obtain (Z)-ethyl 2-benzylidene-3-oxobutanoate (2a, mmol, 1 equiv.) in methanol (~30 mL). Into the flask was then
1.3095 g, 60%) as a clear yellow oil. 1H NMR (CDCl3, 400 added benzaldehyde (1.306 g, 50 mmol, 1 equiv) and ethyl
MHz) δ ppm 1.26 (t, J = 7.21 Hz, 3H), 2.41 (s, 3H), 4.32 (q, J = acetoacetate (6.507 g, 50 mmol, 1 equiv). Methanol was added
7.09 Hz, 2H), 7.33–7.41 (m, 3H), 7.42–7.47 (m, 2H), 7.56 (s, to bring the total volume to 50 (1 M) and the reagents were
1H); 13C NMR (CDCl3, 100 MHz) δ ppm 14.08 (CH3), 26.74 thoroughly mixed. An aliquot of this solution (10 mL) was
(CH3), 61.92 (CH2), 129.06 (CH), 129.74 (CH), 130.93 (CH), transferred to a 20 mL vial equipped with a Teflon-coated stir
133.17 (C), 134.88 (C), 141.48 (CH), 168.00 (C), 194.87 (C) bar. The flow reactor was readied using the equipment manu-
[54].
facturer’s suggested start-up sequence. Methanol was pumped
at 0.25 mL/min to fill the reactor coil. The back-pressure regu-
lator was adjusted to 7 bar and the reactor coil heated to 120 °C.
After the heating coil, the product stream was intercepted with a
Typical procedure for monitoring the
Claisen–Schmidt reaction
Into a 50 mL volumetric flask was added 4-fluorobenzaldehyde stream of N,N-dimethylformamide (0.25 mL/min) by means of
(1.551 g, 12.5 mmol, 1 equiv) and acetophenone (1.637 g, 12.5 a T-piece to ensure complete solubility of the product. The
mmol, 1 equiv). Ethanol was added to bring the total volume to Raman spectrometer was set to acquire a spectrum every 25 s,
50 mL (0.25 M) and the reagents were thoroughly mixed. An with 20 s integration time, boxcar = 3, and average = 1. When
aliquot of this solution (10 mL) was transferred to a 20 mL vial the flow unit was ready, 6 M H2SO4 (0.2 mL, 0.1 equiv) was
equipped with a Teflon-coated stir bar. The flow reactor was injected all at once and after mixing for 15 s the reaction mix-
readied using the equipment manufacturer’s suggested start-up ture was loaded into the reactor coil at a flow rate of
1850

Beilstein J. Org. Chem. 2013, 9, 1843–1852.
6. Mark, D.; Haeberle, S.; Roth, G.; von Stetten, F.; Zengerle, R.
Chem. Soc. Rev. 2010, 39, 1153–1182. doi:10.1039/b820557b
7. van den Broek, S. A. M. W.; Leliveld, J. R.; Delville, M. M. E.;
Nieuwland, P. J.; Koch, K.; Rutjes, F. L. J. T. Org. Process Res. Dev.
2012, 16, 934–938. doi:10.1021/op2003437
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. After the product had been fully
discharged from the flow cell, the scans were halted. The reac-
tion mixture was transferred to a separatory funnel, diluted with
diethyl ether and quenched with satd. sodium bicarbonate
(100 mL) and deionized water (100 mL). The layers were sep-
arated and the aqueous layer was extracted with diethyl ether (3
× 100 mL). The combined organic layers were washed with
brine (2 × 100 mL) and dried over sodium sulfate. The solvent
was removed in vacuo by rotary evaporation affording the crude
product. The resulting solid was transferred to a filter funnel
and was washed with cold methanol. The solid was isolated and
air dried to afford 5-ethoxycarbonyl-6-methyl-4-phenyl-3,4-di-
hydropyrimidin-2(1H)-one, (4a, 2.030 g, 78%) as a fluffy white
solid. 1H NMR (DMSO-d6, 500 MHz) δ ppm 1.18 (s., 3H), 2.34
(s., 3H), 3.67–4.60 (m, 2H), 5.24 (s., 1H), 7.34 (s., 5H), 7.80 (s.,
1H), 7.74 (s, 1H), 9.26 (s, 1H); 13C NMR (DMSO-d6, 125
MHz) δ ppm 14.5 (CH3), 18.2 (CH3), 54.4 (CH), 59.6 (CH2),
99.7 (C), 126.7 (CH), 127.7 (CH), 128.8 (CH), 145.3 (C), 148.8
(C), 152.6 (C), 165.8 (C) [57].
8. Baxendale, I. R. J. Chem. Technol. Biotechnol. 2013, 88, 519–552.
doi:10.1002/jctb.4012
9. Malet-Sanz, L.; Susanne, F. J. Med. Chem. 2012, 55, 4062–4098.
doi:10.1021/jm2006029
10.McMullen, J. P.; Jensen, K. F. Annu. Rev. Anal. Chem. 2010, 3, 19–42.
doi:10.1146/annurev.anchem.111808.073718
11.Moore, J. S.; Jensen, K. F. Org. Process Res. Dev. 2012, 16,
1409–1415. doi:10.1021/op300099x
12.Prim, D.; Crelier, S.; Segura, J.-M. Chimia 2011, 65, 815–816.
doi:10.2533/chimia.2011.815
13.Greener, J.; Abbasi, B.; Kumacheva, E. Lab Chip 2010, 10,
1561–1566. doi:10.1039/c001889a
14.Steinfeldt, N.; Bentrup, U.; Jähnisch, K. Ind. Eng. Chem. Res. 2010,
49, 72–80. doi:10.1021/ie900726s
15.Floyd, T. M.; Schmidt, M. A.; Jensen, K. F. Ind. Eng. Chem. Res. 2005,
44, 2351–2358. doi:10.1021/ie049348j
16.Ferstl, W.; Klahn, T.; Schweikert, W.; Billeb, G.; Schwarzer, M.;
Loebbecke, S. Chem. Eng. Technol. 2007, 30, 370–378.
doi:10.1002/ceat.200600404
17.Benito-Lopez, F.; Verboom, W.; Kakuta, M.; Gardeniers, J. G. E.;
Egberink, R. J. M.; Oosterbroek, E. R.; van den Berg, A.;
Reinhoudt, D. N. Chem. Commun. 2005, 2857–2859.
doi:10.1039/b500429b
Supporting Information
18.Lu, H.; Schmidt, M. A.; Jensen, K. F. Lab Chip 2001, 1, 22–28.
doi:10.1039/b104037p
Supporting Information File 1
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.
19.Gökay, O.; Albert, K. Anal. Bioanal. Chem. 2012, 402, 647–669.
doi:10.1007/s00216-011-5419-z
20.Jones, C. J.; Larive, C. K. Anal. Bioanal. Chem. 2012, 402, 61–68.
doi:10.1007/s00216-011-5330-7
21.Mozharov, S.; Nordon, A.; Littlejohn, D.; Wiles, C.; Watts, P.; Dallin, P.;
Girkin, J. M. J. Am. Chem. Soc. 2011, 133, 3601–3608.
doi:10.1021/ja1102234
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-215-S1.pdf]
22.Rinke, G.; Ewinger, A.; Kerschbaum, S.; Rinke, M.
Microfluid. Nanofluid. 2011, 10, 145–153.
doi:10.1007/s10404-010-0654-8
Acknowledgements
23.Leung, S. A.; Winkle, R. F.; Wootton, R. C. R.; deMello, A. J. Analyst
2005, 130, 46–51. doi:10.1039/b412069h
Funding from the National Science Foundation (CAREER
award CHE-0847262) is acknowledged. Vapourtec Ltd and
Enwave Optronics are thanked for equipment support. Daniel
Daleb of the University of Connecticut is thanked for his
support in the construction of the flow cell apparatus.
24.Lee, M.; Lee, J.-P.; Rhee, H.; Choo, J.; Chai, Y. G.; Lee, E. K.
J. Raman Spectrosc. 2003, 34, 737–742. doi:10.1002/jrs.1038
25.Fletcher, P. D. I.; Haswell, S. J.; Zhang, X. Electrophoresis 2003, 24,
3239–3245. doi:10.1002/elps.200305532
26.Browne, D. L.; Wright, S.; Deadman, B. J.; Dunnage, S.;
Baxendale, I. R.; Turner, R. M.; Ley, S. V.
References
Rapid Comm. Mass. Spectrosc. 2012, 26, 1999–2010.
doi:10.1002/rcm.6312
1. Wiles, C.; Watts, P. Micro Reaction Technology in Organic Synthesis;
CRC Press: Boca Raton, FL, USA, 2011.
27.Koster, S.; Verpoorte, E. Lab Chip 2007, 7, 1394–1412.
doi:10.1039/b709706a
2. Luis, S. V.; Garcia-Verdugo, E., Eds. Chemical Reactions and
Processes under Flow Conditions; Royal Society of Chemistry:
Cambridge, U.K, 2010.
28.Carter, C. F.; Lange, H.; Ley, S. V.; Baxendale, I. R.; Wittkamp, B.;
Goode, J. G.; Gaunt, N. L. Org. Process Res. Dev. 2010, 14, 393–404.
doi:10.1021/op900305v
3. Wiles, C.; Watts, P. Chem. Commun. 2011, 47, 6512–6535.
doi:10.1039/c1cc00089f
29.Brodmann, T.; Koos, P.; Metzger, A.; Knochel, P.; Ley, S. V.
Org. Process Res. Dev. 2012, 16, 1102–1113. doi:10.1021/op200275d
30.Rueping, M.; Bootwicha, T.; Sugiono, E. Beilstein J. Org. Chem. 2012,
8, 300–307. doi:10.3762/bjoc.8.32
4. Wegner, J.; Ceylan, S.; Kirschning, A. Chem. Commun. 2011, 47,
4583–4592. doi:10.1039/c0cc05060a
5. Razzaq, T.; Kappe, C. O. Chem.–Asian J. 2010, 5, 1274–1289.
doi:10.1002/asia.201000010
1851

Beilstein J. Org. Chem. 2013, 9, 1843–1852.
31.Leadbeater, N. E.; Schmink, J. R.; Hamlin, T. A. In Microwaves in
Organic Synthesis, 3rd ed.; de la Hoz, A.; Loupy, A., Eds.; Wiley-VCH:
Weinheim, Germany, 2012.
License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
32.Leadbeater, N. E.; Schmink, J. R. Nat. Protoc. 2008, 3, 1–7.
doi:10.1038/nprot.2007.453
33.Leadbeater, N. E.; Smith, R. J.; Barnard, T. M. Org. Biomol. Chem.
2007, 5, 822–825. doi:10.1039/b615597a
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
34.Leadbeater, N. E.; Smith, R. J. Org. Lett. 2006, 8, 4589–4591.
doi:10.1021/ol061803f
35.Leadbeater, N. E.; Smith, R. J. Org. Biomol. Chem. 2007, 5,
2770–2774. doi:10.1039/b707692d
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
36.Schmink, J. R.; Holcomb, J. L.; Leadbeater, N. E. Chem.–Eur. J. 2008,
14, 9943–9950. doi:10.1002/chem.200801158
(http://www.beilstein-journals.org/bjoc)
37.Schmink, J. R.; Holcomb, J. L.; Leadbeater, N. E. Org. Lett. 2009, 11,
365–368. doi:10.1021/ol802594s
The definitive version of this article is the electronic one
which can be found at:
38.Chaplain, G.; Haswell, S. J.; Fletcher, P. D. I.; Kelly, S. M.;
Mansfield, A. Aust. J. Chem. 2013, 66, 208–212. doi:10.1071/CH12379
39.Kelly, C. B.; Lee, C.; Leadbeater, N. E. Tetrahedron Lett. 2011, 52,
263–265. doi:10.1016/j.tetlet.2010.11.027
doi:10.3762/bjoc.9.215
40.Gaussian 09, Revision A.02; Gaussian, Inc.: Wallingford CT, , 2009.
41.Batovska, D. I.; Todorova, I. T. Curr. Clin. Pharmacol. 2010, 5, 1–29.
doi:10.2174/157488410790410579
42.Bowman, M. D.; Jacobson, M. M.; Blackwell, H. E. Org. Lett. 2006, 8,
1645–1648. doi:10.1021/ol0602708
43.Singh, K.; Singh, K. Adv. Heterocycl. Chem. 2012, 105, 223–308.
doi:10.1016/B978-0-12-396530-1.00003-6
44.Panda, S. S.; Khanna, P.; Khanna, L. Curr. Org. Chem. 2012, 16,
507–520. doi:10.2174/138527212799499859
45.Kappe, C. O. QSAR Comb. Sci. 2003, 22, 630–645.
doi:10.1002/qsar.200320001
46.Mukhopadhyay, C.; Datta, A.; Banik, B. K. J. Heterocycl. Chem. 2011,
44, 979–981. doi:10.1002/jhet.5570440439
47.Fang, Z.; Lam, Y. Tetrahedron 2011, 67, 1294–1297.
doi:10.1016/j.tet.2010.11.075
48.Dallinger, D.; Kappe, C. O. Nat. Protoc. 2007, 2, 317–321.
doi:10.1038/nprot.2006.436
49.Singh, K.; Arora, D.; Singh, K.; Singh, S. Mini-Rev. Med. Chem. 2009,
9, 95–106. doi:10.2174/138955709787001686
50.Kappe, C. O. Eur. J. Med. Chem. 2000, 35, 1043–1052.
doi:10.1016/S0223-5234(00)01189-2
51.Pagano, N.; Herath, A.; Cosford, N. D. P. J. Flow Chem. 2011, 1,
28–31. doi:10.1556/jfchem.2011.00001
52.Prosa, N.; Turgis, R.; Piccardi, R.; Scherrmann, M.-C.
Eur. J. Org. Chem. 2012, 2188–2200. doi:10.1002/ejoc.201101726
53.Ravikumar, I.; Saha, S.; Ghosh, P. Chem. Commun. 2011, 47,
4721–4723. doi:10.1039/c0cc03469j
54.Li, Z.-N.; Chen, X.-L.; Fu, Y.-J.; Wang, W.; Luo, M.
Res. Chem. Intermed. 2012, 38, 25–35.
doi:10.1007/s11164-011-0322-y
55.Stroba, A.; Schaeffer, F.; Hindie, V.; Lopez-Garcia, L.; Adrian, I.;
Froehner, W.; Hartmann, R. W.; Biondi, R. M.; Engel, M.
J. Med. Chem. 2009, 52, 4683–4693. doi:10.1021/jm9001499
56.Wijnberg, H.; Vries, T. R.; Pouwer, K.; Nieuwenhuijzen, J.
3-Oxopropane-1-sulphonic acids and sulphonates. U.S. Patent
6,639,103, Oct 28, 2003.
57.Xu, D.-Z.; Li, H.; Wang, Y. Tetrahedron 2012, 68, 7867–7872.
doi:10.1016/j.tet.2012.07.027
1852