Inline Reaction Monitoring of Amine-Catalyzed Acetylation of Benzyl Alcohol Using a Microfluidic Stripline Nuclear Magnetic Resonance Setup

Article abstract of DOI:10.1021/jacs.9b00039

We present an in-depth study of the acetylation of benzyl alcohol in the presence of N,N-diisopropylethylamine (DIPEA) by nuclear magnetic resonance (NMR) monitoring of the reaction from 1.5 s to several minutes. We have adapted the NMR setup to be compatible to microreactor technology, scaling down the typical sample volume of commercial NMR probes (500 μL) to a microfluidic stripline setup with 150 nL detection volume. Inline spectra are obtained to monitor the kinetics and unravel the reaction mechanism of this industrially relevant reaction. The experiments are combined with conventional 2D NMR measurements to identify the reaction products. In addition, we replace DIPEA with triethylamine and pyridine to validate the reaction mechanism for different amine catalysts. In all three acetylation reactions, we find that the acetyl ammonium ion is a key intermediate. The formation of ketene is observed during the first minutes of the reaction when tertiary amines were present. The pyridine-catalyzed reaction proceeds via a different mechanism.

Full text of DOI:10.1021/jacs.9b00039

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Article
Inline reaction monitoring of amine-catalyzed acetylation
of benzyl alcohol using a microfluidic stripline NMR setup
Anna Jo Oosthoek - de Vries, Pieter J. Nieuwland, Jacob Bart, Kaspar Koch, Johannes W.G. Janssen,
P. Jan M. van Bentum, Floris P. J. T. Rutjes, Han J.G.E. Gardeniers, and Arno P.M. Kentgens
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b00039 • Publication Date (Web): 13 Mar 2019
Downloaded from http://pubs.acs.org on March 13, 2019
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Inline reaction monitoring of amine-catalyzed
acetylation of benzyl alcohol using a microfluidic
stripline NMR setup
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Anna Jo Oosthoek - de Vries,† Pieter J. Nieuwland,‡,† Jacob Bart,†,§ Kaspar
Koch,‡,† Johannes W.G. Janssen,† P. Jan M. van Bentum,† Floris P.J.T.
Rutjes,† Han J.G.E. Gardeniers,¶ and Arno P.M. Kentgens∗,†
†Institute of Molecules and Materials, Radboud University Nijmegen, Nijmegen, The
Netherlands
‡FutureChemistry Holding B.V., Nijmegen, The Netherlands
¶Mesoscale Chemical Systems, Univerity of Twente, Enschede, The Netherlands
§current address: Nouryon, Expert Capability Group Process Technology, Research
Development & Innovation, Zutphenseweg 10, 7418 AJ Deventer, The Netherlands
E-mail: a.kentgens@nmr.ru.nl
Abstract
We present an in-depth study of the acetylation of benzyl alcohol in the presence of
N,N-diisopropylethylamine (DIPEA) by NMR monitoring the reaction from 1.5 seconds
to several minutes. We have adapted the NMR setup to be compatible to microreac-
tor technology, scaling down the typical sample volume of commercial NMR probes
(500 µL) to a microfluidic stripline setup with 150 nL detection volume. In line spec-
tra are obtained to monitor the kinetics and unravel the reaction mechanism of this
industrially relevant reaction. The experiments are combined with conventional 2D
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NMR measurements to identify the reaction products. In addition, we replace DIPEA
with triethylamine and pyridine to validate the reaction mechanism for different amine
catalysts. In all the three acetylation reactions, we find that the acetyl ammonium ion
is a key intermediate. The formation of ketene is observed during the first minutes
of the reaction in case tertiary amines were present. The pyridine catalyzed reaction
proceeds via a different mechanism.
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Introduction
Spectroscopic techniques are extensively used in organic chemistry for analysing molecu-
lar compounds and for monitoring chemical reactions, because they provide quantitative
chemical information at the molecular level. Gas/liquid chromatography-mass spectroscopy
(GC-MS, LC-MS), infrared (IR) spectroscopy and nuclear magnetic resonance (NMR) spec-
troscopy are methods that are frequently employed.1–4 Nuclear Magnetic Resonance (NMR)
spectroscopy is a particularly versatile technique and is the method of choice for elucida-
tion of molecular structure of organic compounds in a broad range of fields.5 NMR has also
proven to be very suitable for the study of organic reactions.6–8
Microscale chemical reactions are attracting increasing attention in chemical research.9–11
Compared to conventional batch reactors, microreactors have extremely high surface-to-
volume ratios, which allows for better heat exchange and mass transfer.12,13 The small vol-
umes, which are typically involved, enable potentially dangerous and/or fast reactions, such
as exothermic reactions or reactions with flammable, explosive, toxic or hazardous chemicals
to be performed under relatively safe conditions. Some recent examples employ the controlled
environment of the microreactor and the increased mass and heat transfer capabilities. The
autoxidation of olefins was performed in a microreactor, which improved safety and yield due
to increased mass transfer and increased temperature.14 Also, fluorine reactions, that are in
batch difficult to control and unstable, involving hazardous compounds that are difficult to
handle, were sucessfully performed using microreactor-based continuous flow chemistry, due
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to the fast mixing, high heat and mass transfer in a microreactor.15 Cantillo16 et al. devel-
oped a procedure for the synthesis of triaminophlorogluconol, an important compound for
industrial and medical use, involving a very unstable and explosive intermediate, which could
be safely performed in continuous flow in a microreactor using a thermostated ultrasound
bath for controlling the temperature of this exothermic reaction.
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Mixing of reactants in conventional reactions occurs by convection and turbulence. Mi-
crofluidic systems have low Reynold numbers, and therefore operate in the laminar flow
regime, mixing takes place mainly by mass transfer through diffusion. The diffusion dis-
tance may be decreased by using split-and-recombine mixing elements in the microreactor:
flows are split up, deformed and recombined, creating thin layers of laminar flows. Whereas
turbulent mixing can give rise to large concentration gradients, microfluidic diffusion-limited
mixing is more homogeneous and the reaction progress is more reproducible as a result. This
enhances chemical selectivity and significantly suppresses side product formation.17 The re-
producibility is also a great advantage in efficient screening or optimisation of reactions,
which is of considerable interest for a variety of pharmaceutical and industrial processes
and for research and development in organic chemistry. High throughput optimisation in
microfluidic setups is achieved at reduced costs, due to low material requirements and low
waste generation,18 even for dangerous and explosive compounds.19
With the developments in microreactor technology, comes a growing interest in online
spectroscopic analysis techniques.20,21 For accurate monitoring of fast reaction intermediates,
it is required that the applied spectroscopic technique operates at the same volumetric scale
as the microreactor,22 and in order to be able to follow the reaction in situ, this method
should be integrated with the reaction element, which calls for the scaling down of the NMR
volume. Miniaturization of the NMR detection coil increases mass sensititivy,23 i.e. scaling
down of the NMR coil increases the sensitivity per unit mass, but decreases the sensitivity
per unit concentration.24 For a sample with a certain, limited concentration, but sufficient
volume available, the sensitivity of a measurement is decreased for smaller coil. For samples
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with limited mass, however, sensitivity increases when the coil is better fitted to the size of the
sample. This is not only beneficial for mass-limited samples, but also for the limited volumes
that are present in the microfluidic reactions. Considerable effort has been devoted to the
development of microscale NMR techniques and several approaches for microcoil NMR have
been explored.25–28 Different types of microcoils can be distinguished: microsolenoids wound
around a capillary,29–31 planar spiral microcoils32–35 and transmission line type (stripline or
microslot) NMR detector.36–43 Our stripline based NMR chip38,44 consists of a planar copper
structure in which a central flat wire is defined that excites and detects the nuclear spins,
having a constriction where a high and homogeneous radio frequency (rf) field is generated,
with a fluidic channel running directly above the copper strip. Furthermore, microfluidic
connections can be straightforwardly applied so that a straightforward microfluidic setup for
the study of microscale reactions in flow is realised.
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Several groups have investigated the applicability of microscale NMR devices for inline
reaction monitoring;45 Ciobanu et al.46 studied the reaction of D-xylose and borate by mul-
tiple physically distinct solenoidal microcoils. Wensink et al.47 presented a microfluidic chip
with an integrated planar microcoil for the real-time monitoring of imine formation from
benzaldehyde and aniline. Kakuta et al.48 monitored ubiquitin protein conformation by
coupling a micromixer to a solenoidal NMR microcoil. More recently, Br¨acher et al.49 com-
bined a microreactor with a capillary NMR flow cell, where the flow path and the solenoid
NMR coil are thermostatted using FC-43 (perfluorotributylamine). In this setup as a test
system a catalytic esterification of methanol with acetic acid was studied under isothermal
conditions. Hyphenation of a continuous flow microreactor and a microfluidic NMR chip to
determine kinetic parameters of a reaction with a single on-flow experiment was employed
by Gomez et al.50
In an earlier study, we showed that the stripline based microfluidic NMR setup could be
conveniently used for reaction monitoring and analysing mass-limited biological samples sam-
ples.44 This setup is further optimized for monitoring the amine base-catalysed acetylation
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of benzyl alcohol in situ. Acetylation of hydroxyl groups is an important and fundamental
process in organic chemistry. The acetylation is mostly used to protect alcohol groups from
undesired side reactions, but also to turn hydroxyl substituents into better leaving groups.
It is frequently performed using an acid chloride in the presence of an amine to significantly
speed up the reaction. We performed the acetylation with acetyl chloride in the presence of
DIPEA, which gives a fast and exothermic reaction. The timescale of this reaction is several
minutes which can be perfectly monitored with our microfluidic NMR setup.
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Despite the abundance of esterification examples with acetyl chloride, there is still discus-
sion about the exact mechanism. The reaction mechanism for a base catalysed acetylation
in general can be thought to proceed via base-assisted nucleophilic attack of the alcohol with
acetyl chloride.51 However, it has also been suggested that the reaction proceeds via a tetra-
hedral intermediate (the quaternary acetyl ammonium ion)52 or via a highly reactive ketene
intermediate that is formed through base-assisted alpha-elimination of HCl from acetyl chlo-
ride.53 Our setup enables the observation of unstable intermediates such as ketene. Comple-
mented with conventional NMR measurements this allows us to fully unravel the reaction
mechanism. Furthermore, we applied different base catalysts to compare the mechanisms
when different amines are involved.
Experimental
Chemicals
All chemicals were used as received without further purification, and consisted of: acetyl chlo-
ride (Fluka Analytical from Sigma Aldrich), acetyl-2-13C chloride, 99 atom% 13C (Aldrich),
benzyl alchohol (reagent Plus, Sigma Aldrich), N,N-diisopropylethylamine (DIPEA) (Biotech
grade 99.5%, Sigma Aldrich), triethylamine (Sigma Aldrich), pyridine (Fluka analytical,
puriss.p.a.), and chloroform-d3 + 0.05% v/v TMS (Cambridge Isotope Laboratories, Inc) as
a solvent.
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Microfluidic stripline NMR setup
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The stripline NMR chip used in these experiments has been described before.54 The stripline
NMR chip is a microfabricated chip, where the rf coil consists of a copper stripline structure
sputtered and electroplated onto the glass substrate. The analyte flows through the detection
area via a microfluidic channel. The volume sensitive for detection is 150 nL. The chip is
coupled to a standard microfluidic setup. Syringe pumps are used for providing a continuous
flow of the reactants. Prior to entering the stripline NMR chip, the reactants are brought
together using a Y-junction and subsequently flow into the chip using a fused silica (FS)
capillary with 75 µm inner diameter (I.D.). A reaction volume of approximately 1 µL
results.
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From the point of the Y-junction mixing takes place by laminar diffusion. By a rough
approximation the mixing time from Fick’s law can be calculated to be approximately 1.4
s. More accurately, the Damko¨hler number can be estimated which compares diffusion to
reaction rate.55 For our reaction process the Damk¨ohler number is around 1, so the reaction
rate is possibly limited by the mixing process.
In a pressure-driven laminar flow through the capillary a parabolic Poiseuille flow profile
arises, instead of plug flow with a linear profile. Radial diffusion takes place which disrupts
the parabolic profile. Bodenstein numbers can be estimated, that gives an indication on the
validity of assuming plug flow for the reaction.55 We find that for our microfluidic system
the Bodenstein number is below 1000 for reaction times up to 30 s. So small deviations from
plug flow can be present up to reaction times of 30 s, where the differences between center
velocity and flow rate at the wall may cause a velocity distribution.
An effective reaction time can be calculated by dividing flow rate with the reaction
volume, so that depending on the applied flow rates, detection can take place at effective
reaction times ranging from 1.5 seconds and 5 minutes. Details regarding the chip, probe and
microfluidics setup can be found in the Supporting Information. Pictures and schematics of
the stripline NMR chip and probe are shown in Figure S1.
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In flow measurements
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For the reaction monitoring a continuous flow of the reaction mixture to the stripline was
established. After having set a new flow rate, a stabilisation time depending on the flow rate
was taken into account (varying from at least 1 minute for high flow rates up to 15 minutes
for low flow rates). Flow rates ranging from 20 µL/min down to 0.1 µL/min correspond
to effective reaction times of 1.5 s to 5 minutes at the NMR measurement. A steady state
spectrum is recorded and saved. The spectra were taken acquiring 4 or 16 scans, depending
on the concentration of the analyte. The acquisition delay between scans varied between 5
seconds for the lower flow rates to 1 second for the higher flow rates, when the detection
volume is refreshed faster. All spectra were recorded at room temperature on a VNMRS
600MHz Varian NMR spectrometer operated with VNMRJ software.
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The flow of analyte through the NMR detection area continuously replaces depolarised
spins with polarised spins that did not yet receive an rf pulse. As a result, it is not nec-
essary to wait for 5 times the relaxation time T1 for the spins to repolarise, therefore the
pulse repetition rate can be increased for an improved Signal to Noise ratio (SNR) per unit
time.24,56 On the other hand, the residence time will increase signal linewidth for any spin
not being in the detection volume of the NMR probe for a period long enough to record a full
Free Induction Decay (FID), as determined by the transverse relaxation T2.57 The resulting
increase in linewidth due to flow is inversely proportional to the residence time. Although
the expected decrease in resolution with increasing flow rate occurs, the effect is minor, so
that the intrinsic high resolution of the stripline chip still permits a spectral resolution of
approximately 2 Hz for flow rates up to 50 µL/min.
From the spectrum of 0.5 M acetyl chloride in flow a single scan SNR of 683 is estimated
for the methyl peak. For calculation of the concentration within 1% error at least a SNR of
150 is needed,59 which is valid for a concentration of more than 0.11 M. When accumulating
4 or 16 scans, the minimum concentration becomes 55 mM and 27 mM, respectively. A SNR
of 1:3 is necessary for detection of a peak, corresponding to a minimum concentration of 2
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mM in a single scan. The first measurement is acquired at an effective reaction time of 2 s.
Intermediate products that have a lifetime of substantially less than 2 s will therefore not be
observed. The time between the effective reaction times at the measurements varies between
a few seconds at the beginning of the reaction up to a minute at the end of the reaction
(selected spectra are shown in the figures). Intermediates that are not present at a time
of measurement will not be present in the spectra. Furthermore, the acquisition time is 1
second, intermediates appearing and/or disappearing during this period will give dispersive
and/or broadened lines.58
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Temperature changes can be present as the reaction generates heat or in hot spots.
According to guidelines provided by Westermann and Mleczko60 we operate in a regime that
does not have a high risk on hot spots, due to the small diameter of the reaction channel
(75 µm) temperature rise is expected to remain well below 1K.
Conventional NMR experiments
The reaction mixtures were prepared in the fumehood, mixed in a tube and allowed to
equilibrate. After 15 minutes the mixtures were relatively stable, and the conventional
NMR measurements were performed typically after a reaction time of around 2 hours. After
the desired reaction time, a sample was taken and put into a 5 mm (500 µL) NMR tube
and subsequently measured with a commercially available probe in a Bruker Avance III 600
MHz NMR spectrometer operated with Bruker TopSpin 3.0 software. For each sample, a 1H
spectrum, a 13C spectrum, a heteronuclear single quantum coherence (HSQC)61 spectrum
and a heteronuclear multiple-bond correlation (HMBC)62 spectrum was taken.
Data processing
The data were processed with VnmrJ and matNMR.63 Advanced Chemistry Development,
Inc. ACD/NMR Processor was used for plotting the conventional 2D experiments.64 The
concentration of the methyl products during the reaction were monitored from the spectra
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measured in the stripline probe by deconvolution fitting of the peaks with MatNMR.63
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Results and Discussion
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The acetylation of benzyl alcohol with DIPEA
The acetylation of benzyl alcohol without a base catalyst is a slow reaction. The reaction
proceeds via a tetrahedral intermediate and is completed in one day. In the Supporting In-
formation the reaction mechanism (Scheme S1) and a series of NMR spectra (Figure S2) that
are taken during the conversion are shown. However, the presence of an amine significantly
increases the reaction rate. Several mechanisms that can play a role have been suggested
in literature.52 First of all, HCl is formed in the nucleophilic addition-elimination reaction,
and a basic amine can absorb HCl to form the corresponding ammonium salt. This would
shift the equilibrium of the reaction to accelerate it. Therefore, we would expect to see
protonation of DIPEA, and possibly the tetrahedral intermediate. Second, the basic amine
can deprotonate the alcohol in trace amounts. However, this process is not expected to be
a significant factor, since acetyl chloride and DIPEA react vigorously. Third, it has been
suggested that an acetyl ammonium ion might be formed.52,53 Acetyl chloride and the amine
then react to give ketene and the protonated amine. Ketene is very reactive and reacts with
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the alcohol into an ester (reaction k5 in Scheme 1)
or with the protonated amine to give
an acetyl ammonium ion 9 (reaction k4 in Scheme 1).66,67 These various insights have been
brought together in Scheme 1 which we will validate by detailed NMR analyses as described
below.
In situ NMR spectra
To get detailed insights in the reaction mechanism of this fast amine catalyzed acetylation,
we performed the reactions in a microfluidic setup (Figure S1 in the Suporting Information).
The syringes were loaded with A: 0.5 M benzyl alcohol with 0.5 M DIPEA and B: 0.5 M acetyl
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Figure 1: Selected spectra for the reaction of benzyl alcohol 2 (0.5 M) with acetyl chloride 1
(0.5 M) in the presence of DIPEA 5 (0.5 M). Top: unreacted compounds: acetyl chloride (A),
DIPEA (D), benzyl alcohol (BnOH). The series of in situ spectra show the broadening and
shifting of DIPEA peaks, formation of product benzyl acetate 4 (BnOAc) and intermediate
peaks marked ’k’ (ketene 7) and ’AA4’ (acetyl group of acetyl ammonium ion 9). Bottom:
conventional NMR spectrum after 2 hours reaction time, the slightly broadened DIPEA
peaks are shifted to a position of protonated DIPEA 8 and/or acetyl ammonium ion 9
(’AA’).
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Scheme 1: Proposed reaction mechanism of benzyl alcohol 2 with acetyl chloride 1 and
DIPEA 5. Acetyl chloride and DIPEA form an unstable tetrahedral intermediate 6, which
gives ketene 7, protonated DIPEA 8 and acetyl-N,N-diisopropylethylammonium ion 9. Ben-
zyl alcohol 2 reacts with ketene 7 or acetyl ammonium ion 9 into benzyl acetate 4. Diketene
10 is formed as a side product in trace amounts.
chloride. Comparing the spectra of DIPEA and DIPEA with benzyl alcohol, protonation fo
DIPEA from benzyl alcohol is not observed. By keeping the flow rates constant at a certain
flow rate during the acquisition we obtained a steady state spectrum while the reaction
is in progress. By adjustment of the flow rates A and B, a series of steady state spectra
was obtained at effective reaction times ranging from 1.5 seconds up to 5 minutes. Selected
spectra and a conventional NMR spectrum are shown in Figure 1. Table S1 in the Supporting
Information gives an overview of the methyl peaks observed in the reactions discussed in this
paper.
In the ”conventional” NMR spectrum (bottom of Figure 1), taken after 2 hours reaction
time, we observe the DIPEA peaks at a position (marked ’AA’) shifted with respect to
the original position (marked ’D’ in the top spectrum of the unreacted compounds), which
indicates the protonation of the amine. There are two main methyl resonances present; the
benzyl acetate peak at 2.09 ppm (BnOAc1), and a smaller peak at 2.23 ppm which will be
discussed in more detail in the next section.
More information can be obtained from the analysis of the stripline NMR spectra of the
ongoing reaction shown in Figure 1. The conversion into benzyl acetate can be monitored
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nicely using the resonances of the alpha protons. The spectra show the shifting and broad-
ening of the DIPEA peaks (from ’D’ to ’D+’ and ’AA’), and four peaks in the methyl region
can be observed: acetyl chloride (2.69 ppm, labeled ’A’), two intermediates at 2.4 ppm (’k’)
and 2.23 ppm (’AA4’) and benzyl acetate at 2.09 ppm (’BnOAc1’).
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Figure 2: Selected spectra for the reaction of acetyl chloride (0.5 M) with DIPEA (0.5 M).
Top: unreacted compounds: acetyl chloride 1 (A) and DIPEA 5 (D). The series of in situ
spectra show the broadening and shifting of DIPEA peaks, intermediate peaks marked ’k’
(ketene 7 ) and ’AA4’ (acetyl group of acetyl ammonium ion 9). Bottom: two conventional
NMR spectra. After 2 hours reaction time, the DIPEA peaks are found at original position
(D) and at shifted position acetyl ammonium ion 9 (’AA1,2,3’), the main methyl product is
associated with the acetyl group of the acetyl ammonium ion 9 (AA4). After 2 days reaction
time, the DIPEA/acetyl ammonium ion peaks are broadened.
To simplify the identification of the different steps in the reaction, the interaction of
DIPEA 5 and acetyl chloride 1 was studied separately. Experiments are performed in a
similar way, with syringe A: acetyl chloride (0.5 M) and syringe B: DIPEA (0.5 M). Figure 2
shows selected spectra acquired in the stripline NMR chip, and two conventional NMR
spectra acquired after 2 hours and 2 days reaction time. Since all of these peaks are still
present in the conventional NMR spectrum after 2 hours reaction time, we were able to
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perform conventional 2D NMR experiments of these reaction products. In order to come
to a reaction mechanism we first need to assign the various resonances in the stripline and
conventional NMR spectra.
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Protonation of DIPEA
The spectra in Figure 2 clearly show that the DIPEA peaks first broaden, then split. Three
main resonances are present in the methyl region (2.69 ppm (AcCl), 2.4 ppm, 2.23 ppm),
see also Table S1 in the Supporting Information. A similar effect is seen in the spectra of
the full reaction obtained with the stripline probe (Figure 1) showing that the DIPEA peaks
broaden and shift as the reaction proceeds. This suggests (partial) protonation of DIPEA.
k1
D + H+ ꢀDH+
(1)
k
−1
If the resulting protonation/deprotonation process is a fast exchange process, the position
of the resulting (narrow) peak in the NMR spectrum is the weighted average of the shift
of the protonated and unprotonated resonances, whereas in the slow exchange limit these
separate resonances would both be present in the spectrum.68 Since we observe a broadened,
averaged signal, we conlude that this is an intermediate exchange process, meaning that the
proton is exchanged from one molecule to another on the NMR timescale. The observed
chemical shift δ is the population averaged shift, where the populations γi are the relative
concentrations:
n
X
δ(t) =
γi(t)δi
(2)
i=1
For intermediate exchange rates NMR peaks broaden as observed in the spectra, meaning
the life time of the species is shorter than the transverse relaxation time T2 and of similar
magnitude of the frequency difference of the individual resonances.68 As the reaction pro-
gresses, the position of the broadened DIPEA peak moves from the original chemical shift
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of the unprotonated DIPEA to a position similar to the protonated DIPEA chemical shift.
This shift reflects the gradually increasing protonation of DIPEA during the course of the
reaction.
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Reaction products of acetyl chloride
To unravel which resonances correspond to the reaction products of acetyl chloride, labeled
acetyl chloride-2-13C was used for the reaction with DIPEA. Figure 3 shows the conventional
1
13C and H NMR spectra after two hours reaction time for reactions using either natural
abundance or 13C labeled acetyl chloride. Since we observed protonated DIPEA 8, the
formation of ketene 7 as an intermediate in the reaction is a possible consequence in this
part of the reaction. The 13C chemical shifts of ketene are known from literature to be
2.5 ppm and 194 ppm.69 Both peaks are indeed observed in the 13C spectrum (Figure 3a),
confirming the presence of ketene 7 in the reaction mixture.
The peaks that belong to the reaction products of acetyl chloride can be identified by
their increased peak intensity in the spectrum of the reaction performed with labeled acetyl
chloride relative to the spectrum with natural abundance acetyl chloride. The methyl region
(below 50 ppm) shows that three of the main resonances have much higher intensity (marked
with dots in Figure 3a). Since ketene 7 is a reactive compound we assume that it will react
with protonated DIPEA 8 and form acetyl N,N-diisopropylethylammonium ion 9. With the
peaks corresponding to the resonances of acetyl chloride and ketene already identified, we
assign the third peak to the acetyl group of the acetyl ammonium ion (AA4). Interestingly,
we also observe diketene 10 as a side product of the reaction.
To verify that the peaks at 2.5 ppm and 194 ppm in the conventional 13C NMR spectra
in Figure 3a indeed belong to ketene 7, and to determine which peak in the proton spec-
trum corresponds to ketene, a heteronuclear multiple-bond correlation (HMBC) spectrum
is acquired (Figure 4b). It is clear from the connection between the 2.5 ppm and 194 ppm
1
13C peaks to the 2.4 ppm H peak, that the ketene protons resonate at 2.4 ppm peak in the
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Figure 3: DIPEA (0.5 M) and AcCl (0.5 M) after two hours reaction time, for natural
abundance and 13C-labeled AcCl, measured in a conventional 600 MHz NMR spectrometer.
In the 13C spectra a) the peaks of labeled products in the bottom spectrum are enlarged, the
bottom spectrum has been scaled down (1:6, relative to the CDCl3 peaks), to accomodate
for these intensity differences. The peaks of labeled products that are increased are mainly
acetyl chloride 1, the acetyl group of acetyl ammonium ion 9 (AA4) and ketene 7, but also
diketene 10 and some side products are found. In the natural abundance spectrum we see
also the carbons from the carbonyl groups for ketene 7 and acetyl ammonium ion 9 (AA).
1
In the H spectra b), the peaks belonging to DIPEA 5 are indicated with D1a-1b, D2 and
D3. The acetyl ammonium ion peaks are at a position shifted from the DIPEA, indicated
with AA1a-1b (partly overlapping), AA2 and AA3. Due to 13C-labelling of acetyl chloride,
splitting due to JCH coupling of the acetyl chloride 1 and product methyl peaks occurs:
ketene 7, and acetyl ammonium ion 9 (AA4), marked with dots. Due to hindered rotation
the acetyl ammonium ion peaks (AA1-3) are split, which is visible in this spectrum.
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Figure 4: Acetyl chloride with DIPEA: conventional 2D spectra after 2 hours reaction time,
a) HSQC, b) HMBC. The HSQC shows the separation of the DIPEA 5 and acetyl ammonium
ion 9 peaks in the 13C spectrum. In the HMBC, other than multiple peaks from DIPEA 5
and acetyl ammonium 9, we observe ketene 7 (k) at 2.4 ppm in 1H spectrum, and at 2.5 ppm
and 194 ppm in the 13C spectrum. Furthermore, acetyl chloride 1 (A), the acetyl group of
the acetyl ammonium ion 9 (AA), side products (SP) and diketene 10 are found.
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proton spectrum.
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Figure 3b shows the proton spectra for reactions of DIPEA with the natural abundance
and the 13C labeled acetyl chloride. For the 13C labeled acetyl chloride, the peaks in the
spectra that belong to the 13C labeled compound are split due to the JCH coupling. This
splitting can be observed for the resonances at 2.69 ppm, 2.4 ppm and 2.23 ppm, in agreement
with the previous findings. In addition, some low intensity peaks at 6 ppm, 2.30 ppm and
2.27 ppm (too small to be marked in the 13C labeled spectrum) exhibiting JCH couplings
are observed. For acetyl chloride a JCH coupling of 133 Hz is perceived, for ketene 177 Hz
and for the acetyl ammonium ion the JCH coupling is 131 Hz.
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The acetyl-N,N-diisopropylethyl ammonium ion 9 has a hindered rotation around the
N-CO bond, therefore the protons 1a are inequivalent and show a split resonance.70,71 When
the initial (fast) part of the reaction is completed, the acetyl ammonium ion peaks are split
as can be seen in Figure 3. This is also observed in the series of spectra in Figure 2 (at 5 min
reaction time and the first conventional NMR spectrum). The protons from the acetyl group
of the acetyl ammonium ion, marked 1a and 1b in Figure 3, are found as two doublets with
51 Hz splitting with a 1:1 ratio (1a and 1a’) and one triplet (1b) partly overlapping with
the doublet. The other acetyl ammonium peaks (2 and 3) exhibit a 4.2 Hz splitting. The
observed chemical shift difference between the cis-trans isomers, due to hindered rotation
around the N-CO bond, confirms the presence of acetyl ammonium ion as an intermediate.
As can be observed in the bottom spectrum in Figure 2, the DIPEA peaks broaden again
after 2 days. Since the acetyl ammonium ion 9 is not stable this is not unexpected. Upon
dissociation of the acetyl moiety, it may form side products and protonated DIPEA 8 which,
due to exchange with the acetyl ammonium ion 9, will broaden the peaks.
Since both acetyl ammonium ion 9 and ketene 7 are unstable compounds, side products
are formed during the reaction. Diketene 10 is identified by its resonances at 4.88, 4.53
and 3.93 ppm. Furthermore, a product with resonances at 2.27, 2.3 and 6 ppm is observed.
This may be a product of (instable) ketene and/or diketene, since both disappeared while
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this product appears. Some minor products at 1.97 ppm and 2.05 ppm that are present in
all of the reactions are observed as well. The peak at 2.05 ppm might be from acetic acid,
which can be formed from acetyl chloride and has approximately this chemical shift. Table
S1 in the Supporting Information gives an overview of the main peaks that were found in
the spectra.
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Considering the observed intermediates, acetyl ammonium ion and ketene, several reac-
tion steps can be envisioned. Acetyl chloride 1 and DIPEA 5 were shown to react, forming
either ketene 7 and protonated DIPEA 8 or acetyl ammonium 9. An explanation for this
could be that the reaction proceeds via an unstable tetrahedral intermediate 6, which re-
sults from addition of the amine to the carbonyl group. The proposed reaction mechanism
is shown in Scheme 2.
Scheme 2: Proposed reaction mechanism of acetyl chloride 1 and DIPEA 5. The reaction
proceeds via tetrahedral intermediate 6, which gives either ketene 7 and protonated DIPEA
8 or acetyl-N,N-diisopropylethylammonium ion 9. Diketene 10 is a side product.
Reaction mechanism
As ketene 7 and acetyl ammonium 9 are products in the reaction of acetyl chloride 1 with
DIPEA 5, it is very likely that they are also present in the first minutes of the DIPEA
catalyzed acetylation of benzyl alcohol. Small quantities of ketene 7 are indeed observed in
the stripline NMR spectra shown in Figure 1. During the reaction, the DIPEA peaks are
broadened and shifted to lower field. The acetyl group of the acetyl ammonium ion 9 appears
at the start of the reaction and remains present throughout the progressing reaction. At the
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Figure 5: Acetyl chloride 1 and benzyl alcohol 2 with DIPEA 5: conventional 2D spectra
after 2 hours reaction time, a) HSQC, b) HMBC. The HSQC shows the direct correlation be-
tween 1H and 13C peaks for benzyl acetate 4 (BnOAc), benzyl alcohol 2 (BnOH), protonated
DIPEA 8 or acetyl ammonium ion 9 (D+/AA) and its acetyl group (AA4). In the HMBC
we find the correlated peaks of the benzyl acetate 4 (BnOAc), benzyl alcohol 2 (BnOH) and
their peaks in the aromatic region, acetyl chloride 1 (A), and side products.
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end of the reaction, acetyl ammonium is identified by the slightly broadened and shifted
multiplets in the conventional NMR spectrum (bottom trace of Figure 1). As discussed
before, the broadening indicates an exchange process, suggesting the presence of protonated
9
1
DIPEA 8. The 2D NMR spectra in Figure 5 show the correlations between the H NMR
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and the 13C chemical shifts. The peaks of the acetyl group of acetyl ammonium ion are
found at the same chemical shift position as in the reaction with DIPEA, see Table S1 in
the Supporting Information, which confirms that the acetyl ammonium ion 9 is a reaction
product.
The observed reaction products and the protonation of DIPEA suggests that the increased
reaction rate of the acetylation in the presence of DIPEA is induced by the reaction of benzyl
alcohol with ketene and acetyl ammonium. Since these products were found in the reaction
of acetyl chloride and DIPEA as well, the proposed reaction mechanism for the acetylation
of benzyl alcohol in the presence of DIPEA is an extension of Scheme 2. Benzyl alcohol
may react with either ketene and/or the acetyl ammonium ion, forming benzyl acetate. This
gives credibility to the reaction mechanism as shown in Scheme 1.
Kinetics
The proposed reaction schemes are explored further using a fitting procedure to a kinetic
model described in the Supporting Information. Considering the large number of reaction
constants (seven for the full reaction) in relation to the limited number of experimental
points we do not claim that we can fully characterize the kinetics with this approach. Nev-
ertheless the analysis is useful to determine the relative importance of the various steps in
the reaction. Based on the integrated intensities of the resonances, the concentrations of
the reaction products during the reaction progress are calculated, as shown in Figure 6. A
set of differential equations representing the reaction scheme (Eq. (2) in the Supporting
Information) is solved, while varying the k values to minimize the difference between experi-
mental and fitted values via an object function F,72 for the concentrations of acetyl chloride
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1, ketene 7 and the acetyl group of the acetyl ammonium ion 9.
5
6
7
8
For the reaction of acetyl chloride and DIPEA, the reaction scheme shown in Scheme 2
gives the best fit to the data, compared to a similar reaction mechanism without an interme-
diate and/or different equilibria. Thus we postulate that the tetrahedral intermediate 6 is
formed in the first step of the reaction acetyl chloride and DIPEA. Comparing the backwards
and forwards reactions, k−1 is much smaller than k1 and so the backwards reaction is assumed
to be negligible. Since the concentration of the tetrahedral intermediate 6 is very small in
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the model, the backwards reaction can not be accurately fit and k is set to zero. The
−1
tetrahedral intermediate 6 disintegrates forming ketene 7 with protonated DIPEA 8 (k2) or
the acetyl ammonium ion 9 (k3). These are the fastest steps in the reaction mechanism; the
values of k2 and k3 are much larger than all other reaction constants so the corresponding
reactions can be considered instantaneous. For this reason, the relative ratio between k2 and
k3 (2.4 ± 0.5) is more relevant than their absolute values. Likewise the ratio of the (much
smaller) equilibrium constants of the reactions between ketene 7 and DIPEA 5 with acetyl
ammonium ion 9 (K = k4/k = 1.7 ± 0.5) can be determined more accurately than the
−4
absolute value of the individual rates.
After optimisation we find the k values: k1 = 0.16(±0.03)M−1s−1, k2 = 12s−1, k3 = 5s−1,
k4 = 0.0035(±0.002)M−1s−1 and k = 0.0020(±0.002)M−1s−1. The error margin given for
−4
the reaction constants reflects the influence on the accuracy of the model. One of the reaction
constants is changed while the other k-values remain at their optimal value. The relative
effect of such a change in k-values on the fit result is different for each reaction constant.
The given amount of deviation of the k-value will decrease the object function72 of the fit
(summed squared residuals) with 5 %. The values of k2 and k3 can be set much larger
which slightly improves the fit, as long as the ratio remains 2.4, however the calculation
slows down when these k values are set too high. The resulting k values suggest that the
tetrahedral intermediate 4 is indeed very unstable, and breaks down into ketene 7 and
protonated DIPEA 8 and acetyl ammonium ion 9, with a preference for the ketene route.
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Figure 6: Modelling of the kinetics of the acetylation of benzyl alcohol, a) reaction of acetyl
chloride with DIPEA, b) reaction of acetyl chloride with benzyl alcohol in the presence of
DIPEA. Experimentally derived values of concentrations in the mixture during the reaction
are marked with *, the solid line is the result of the fit. Starting product acetyl chloride 1
(AcCl), intermediates ketene 7 and acetyl ammonium ion 9 (AA), and end poduct benzyl
acetate 4 (BnOAc). The concentrations have been estimated by the relative deconvoluted
areas of the peaks.
Some exchange between ketene and protonated DIPEA with acetyl ammonium ion is possible,
favouring acetyl ammonium ion, which is the end product at the longest reaction times that
we used in the microfluidic stripline setup. We conclude from the broadening for very long
reaction times that the acetyl ammonium ion 9 partly dissociates, leaving protonated DIPEA
8 and some side products. This slower process has not been included in the reaction scheme
modeling.
The proposed reaction mechanism of the complete acetylation of benzyl alcohol (Scheme 1)
is modeled next. The reaction of DIPEA and acetyl chloride results in ketene 7, protonated
DIPEA 8 and acetyl ammonium ion 9. Benzyl alcohol 2 may react with ketene7 or with
acetyl ammonium ion 9, forming benzyl acetate 4. The direct reaction of benzyl alcohol
and acetyl chloride is not taken into account, because it much slower (hours) relative to the
reaction times we studied in-line (seconds to minutes).
Analogous to the reaction of acetyl chloride with DIPEA, the first reaction step is the
formation of the tetrahedral intermediate 6, in which the forward reaction rate is much
higher than the backwards reaction rate, so again k assumed to be negligible and set to
−1
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zero. Also as before, the ratio of k2 and k3, in which the tetrahedral intermediate 6 breaks
down into ketene 7 and protonated DIPEA 8 or acetyl ammonium ion 9, can be determined
more accurately than the actual values. For the ratio between k2 and k3, the best fit is found
for the value k2/k3 = 2.7(±0.3), thus favouring ketene and protonated DIPEA formation.
Since the concentration of ketene is very small, k4 does not critically influence the outcome
of the model, as long as it is very small and k4 is therefore set to zero.
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Seven k values as indicated in Scheme 1 are thus needed for representation of the re-
action with a set of differential equations (Eq. (3) in Supporting Information), which is
optimised with respect to the concentrations of acetyl chloride 1, ketene 7, the acetyl group
of acetyl ammonium ion 9 and benzyl acetate 4. The optimised k values that were found
are: k1 = 0.23(±0.05)M−1s−1, k2 = 3s−1, k3 = 1s−1, k4 = 0M−1s−1, k = 0.04(±0.2)s−1,
−4
k5 = 2.6(±1.3)M−1s−1 and k6 = 0.030(±0.003)M−1s−1. The given error margin, as before,
decreases the variance of the fit with approximately 5 %, reflecting the influence of the fit
parameter on the accuracy of the model.
Interestingly, k5 is found to be much larger than k6, which suggests that the formation of
benzyl acetate 4 via the reaction of benzyl alcohol 2 with ketene 7 is the fastest. After five
minutes reaction time the conversion of benzyl alcohol 2 into benzyl acetate 4 is approx. 56%.
The fast part of the reaction is finished by then, since there is no ketene 7 and acetyl chloride
1 left in the mixture. If left to stand for 2 days, the acetyl ammonium 9 peak diminishes and
benzyl acetate 4 increases slightly, which indicates that the reaction proceeds slowly via the
acetyl ammonium route (reaction k6 in Scheme 1). The high reactivity/instability of both
ketene 7 and acetyl ammonium 9 prevents the achievement of full conversion as witnessed
by the formation of diketene 10 and protonated DIPEA 8 leading to various side products.
Variation of amines as base catalyst
Having established the role of DIPEA in the acetylation of benzyl alcohol we set out to study
different types of amines as catalysts to examine whether the reaction proceeds via a similar
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mechanism. DIPEA, triethylamine 11 (TEA) and pyridine 15 differ in reactivity; pyridine
is a weakly aromatic base (acid dissociation constant pKa ≈ 5.2), TEA and DIPEA have,
due to increasing steric hindrance, a reduced nucleophilicity and a higher base reactivity
(pKa ≈ 10.6 and pKa ≈ 11.4, respectively).73 The proton affinity of DIPEA is highest
(984 kJ/mol), that of TEA slightly lower (972 kJ/mol) and that of pyridine lowest (924
kJ/mol).74 With the high proton affinity the DIPEA molecule was postulated to act as a
proton scavenger while not taking part in the reaction, due to steric hindrance, contrary to
the findings in the previous section. Pyridine, having lower proton affinity, but little steric
effects, would instead be acetylated rather than protonated.
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Triethylamine
Figure S3 in the SI shows a series of stripline NMR spectra, and Figure S4 shows the
conventional 2D NMR spectra for the reaction of acetyl chloride (0.5 M) and TEA (0.5 M).
Peaks of intermediate products, ketene 7 and the acetyl group of acetyl ammonium ion 14
are found at the same positions as in the reaction with DIPEA. This suggests that similar
products are involved with the proposed reaction mechanism shown in Scheme 3. However,
the reaction kinetics are markedly different. The first step of acetyl chloride reacting with
TEA is much faster as compared to DIPEA. Ketene 7 and deketene 10 form at a much higher
rate, being already visible within 1.5 seconds in the spectra taken with the stripline probe.
With the highly reactive ketene 7 and diketene 10 being formed at high rate we observe the
formation of more side products which can be seen in the conventional 2D spectra in Figure
S4.
In Figure S5, a series of in situ spectra for the acetylation of benzyl alcohol in the presence
of TEA are shown, and Figure S6 shows the conventional 2D NMR spectra at later stages in
this reaction. In these spectra benzyl acetate 4 and acetyl ammonium ion 14 are observed
with a small amount of acetic acid as a side product. Much less side products are observed,
compared to the spectra of acetyl chloride and TEA. With the rapid formation of ketene that
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Scheme 3: Proposed reaction mechanism of acetyl chloride 1 and triethylamine 11. A
tetrahedral intermediate 12 is formed, which breaks down into ketene 7 and protonated
TEA 13 or acetyl ammonium ion 14. Diketene 10 is a side product.
is available for reaction with benzyl alcohol to form benzyl acetate, the reaction is completed
within 30 seconds, the overall conversion is 57% after 5 minutes reaction time.
From the NMR analyses we conclude that the TEA catalyzed acetylation proceeds sim-
ilarly to the acetylation in the presence of DIPEA, as summarized in Scheme 4. Despite
TEA being a somewhat weaker base, the reaction rates are higher than in the reaction with
DIPEA. This further corroborates that the amine is not merely a proton scavenger but
takes part in the reaction, leading to the formation of ketene, where steric factors are more
important than basicity.
Scheme 4: Proposed reaction mechanism of acetyl chloride 1 and triethylamine 11 with
benzyl alchohol 2. First acetyl chloride and TEA form a tetrahedral intermediate 12, from
which an equilibrium between acetyl ammonium ion 14 and ketene 7 with protonated TEA
13. Benzyl alcohol 2 reacts with ketene 7 or acetyl ammonium ion 14 into benzyl acetate
4.
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Pyridine
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Despite the fact that pyridine 15 is regularly used as base catalyst in similar reactions, it is
a very different base. From literature,75,76 we expect an acetyl pyridinium ion 16 to play an
important role in this reaction, similar to the acetyl ammonium ion that we observed in the
preceding reactions.
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Figure S7 in the Supporting Information shows the results of the in situ experiments in
the stripline probe using pyridine as a base catalyst. Figure S8 shows the conventional 2D
NMR spectra. In these spectra, a methyl peak at 2.22 ppm is observed at similar position as
the acetyl ammonium ion 9. Furthermore, during the reaction, the pyridine peaks are found
to shift and broaden suggesting the formation of a complex. Based on this we conclude that
the predicted acetyl pyridinium ion 16 is indeed formed, in agreement with literature.75,76
Ketene 7 is not observed at any point of this reaction. We conclude that acetyl chloride 1
and pyridine 15 directly react to give acetyl pyridinium ion 16 as shown in Scheme 5.
Scheme 5: Proposed reaction mechanism of acetyl chloride 1 and pyridine 15, giving acetyl
pyridinium ion 16
The spectra for the pyridine (0.5 M) catalyzed reaction of benzyl alcohol (0.5 M) with
acetyl chloride (0.5 M) are shown in Figure S9, with the conventional 2D NMR spectra at
a later stage of the reaction displayed in Figure S10 of the SI. In these spectra, a methyl
peak at 2.22 ppm and a broadening and shift of the pyridine peaks is observed as before,
suggesting the presence of acetyl pyridinium 16. Already after 1.5 seconds (top spectrum in
Figure S9), acetyl chloride 1 has almost completely reacted into benzyl acetate 4 and acetyl
pyridinium 16. Furthermore, benzyl alchohol 2 and some side product formation, probably
acetic acid, is observed. This corroborates that the acetylation takes place via the reaction
of benzyl alcohol with the acetyl pyridinium 16 intermediate as depicted in Scheme 6. The
reaction rates are much faster compared to the reaction performed with DIPEA. The overall
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conversion of benzyl alcohol to benzyl acetate is similar, however, being 55% after 5 minutes.
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Scheme 6: Proposed reaction mechanism of acetyl chloride 1 and pyridine 15 with benzyl
alchohol 2, via acetyl pyridinium 16, forming benzyl acetate 4 and protonated pyridine 17
Conclusion
In the work presented here we show that a microfluidic stripline NMR setup offers the
possibility to study fast reactions in situ. Microreactor technology is advantageous for very
fast and/or exothermic reactions. The acetylation of benzyl alcohol in the presence of DIPEA
was studied in detail, intermediates of the reaction were identified as ketene and acetyl
ammonium ion. The kinetics of this reaction were monitored and modeled by solving the rate
equations for the proposed reaction scheme. Based on these results, a reaction mechanism
via a tetrahedral intermediate was proposed. It was found that the product is formed
most rapidly by the reaction of benzyl alcohol with ketene. Replacing DIPEA with TEA
accelerates the reaction, but the mechanism remains similar, as suggested by the observation
of the same intermediates, ketene and acetyl ammonium. Using pyridine as a base catalyst,
no evidence is found that this reaction also proceeds via ketene, but acetyl pyridinium ion
16 was observed.
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Acknowledgement
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Ruud Aspers is acknowledged for acquiring of the conventional NMR spectra. This work
was financially supported by the Netherlands Organisation for Scientific Research (NWO),
ACTS, Process on a Chip programme.
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Supporting Information Available
Technical details of the stripline NMR chips, probe desgin, microfluidics. Description of
the fitting procedure for the kinetics and PDEs. Spectra and reaction mechanism of the
acetylation of benzyl alcohol without the presence of a base catalyst. Stripline NMR spectra
and conventional 2D NMR spectra (HSQC and HMBC) of the reactions of acetyl chloride
with/without benzyl alcohol and base catalysts TEA and pyridine. Table giving an overview
of the observed peaks in stripline and conventional NMR experiments.
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