Facile kinetic profiling of chemical reactions using MISER chromatographic analysis

Article abstract of DOI:10.1016/j.tet.2017.05.048

Direct MISER (Multiple Injections in a Single Experimental Run) chromatographic analysis can afford easy to interpret kinetic profiles of chemical reactions without the need for peak integration, data input or graphing. Several examples are presented illustrating how this approach can provide researchers with straightforward access to information-rich kinetic reaction profiles.

Full text of DOI:10.1016/j.tet.2017.05.048

Tetrahedron xxx (2017) 1e6
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Facile kinetic profiling of chemical reactions using MISER
chromatographic analysis
Kerstin Zawatzky*, Shane Grosser, Christopher J. Welch*
Department of Process Research & Development, Merck & Co., Inc., Rahway, NJ, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Direct MISER (Multiple Injections in a Single Experimental Run) chromatographic analysis can afford
easy to interpret kinetic profiles of chemical reactions without the need for peak integration, data input
or graphing. Several examples are presented illustrating how this approach can provide researchers with
straightforward access to information-rich kinetic reaction profiles.
Received 31 March 2017
Received in revised form
10 May 2017
Accepted 12 May 2017
Available online xxx
© 2017 Elsevier Ltd. All rights reserved.
Dedicated to longtime friend and colleague,
Ben Feringa, with heartfelt congratulations
on his receipt of the 2016 Nobel Prize in
Chemistry.
Keywords:
Online analysis
High throughput analysis
Kinetic profiling
Reaction monitoring
MISER chromatography
EasySampler
1. Introduction
throughput analysis that facilitate the direct and easy to interpret
visualization of experimental results. MISER chromatographic
Studies of the kinetics of organic reactions have become
increasingly popular in recent years, with a growing number of
synthetic chemists embracing physical organic chemistry tools and
strategies that can lead to better understanding of organic reaction
mechanisms.1 In part inspired by the elegant kinetic isomerization
studies underlying molecular switches, rotors and machines,2 this
renaissance has also been motivated by the practical benefits of
gaining improved performance and control of organic reactions
through better understanding of reaction mechanism. A number of
simplifying innovations have helped to bring what was once a
somewhat esoteric subject within range of most practicing chem-
ists.3 In addition, new experimental approaches and analytical tools
have become available, simplifying kinetic profiling and providing
new windows on a heretofore hidden world of chemical reactivity.4
In an effort to provide more user-friendly tools for organic
chemists we have developed analytical techniques for high
analysis (Multiple Injections in a Single Experimental Run) utilizes
sequential rapidly executed (typically 10s-1min) injections of
related samples using minimal chromatographic separation of
analytes of interest from interfering substances, with detection by
UVeVis, CD, MS or ICP-MS.5 Typically, MISER analysis is used to
compare related samples containing the same compound of inter-
est that have been prepared using different conditions, however,
the technique can also be used for reconstructing kinetic reaction
profiles from reaction timecourse aliquots.6 In the present study we
investigate kinetic profiling using the direct and real time MISER
analysis of organic reactions carried out within HPLC autosampler
vials or via online sampling of reactions carried out in typical lab-
oratory reactors.
2. Results and discussion
Reaction monitoring by MISER LC generally works well when
compound specific detection is possible, but even simple UV
absorbance can afford clear kinetic profiles when reaction
* Corresponding authors.
E-mail address: whelko@comcast.net (C.J. Welch).
http://dx.doi.org/10.1016/j.tet.2017.05.048
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K. Zawatzky et al. / Tetrahedron xxx (2017) 1e6
components are well resolved by chromatography. Fig. 1 shows
MISER kinetic profile monitoring of the basic hydrolysis of a classic
set of three different substituted methyl benzoates originally
studied by Hammett to investigate substituents effects on reaction
rates.7 In each of these examples, the starting ester and product
acid can be baseline resolved in <20s using isocratic elution on a
3 cm reversed phase C18 column. MISER kinetic profiling affords a
convenient direct readout of substrate consumption and product
formation that allows easy comparison of different substrates
without the need for integration, data analysis or graphing. For
instance, in this study it is clear that the relative rates of hydrolysis
for the three reactions can be readily discerned, with the p-nitro-
benzoate 1 being fastest (t1/2 ~3e4 min), the p-acetoxybenzoate 2
being considerably slower (t1/2 ~14 min) and the p-methox-
ybenzoate 3 being slowest, with virtually no hydrolysis being
observed throughout the entire experiment.
For the sake of clarity, the first injection in the sequence con-
tains a solution of starting material at the initial concentration.
Subsequent injections are from a second vial, following addition of
the hydrolysis reagents (NaOH) and using a sampling interval of
14 s.
MISER kinetic profiling is particularly powerful when used in
conjunction with MS analysis, as this can often allow the signals for
starting materials, intermediates and products to be placed into
separate channels for analysis. Fig. 2 shows an example where
MISER kinetic profiling was used to obtain information about a
fairly complex maleimide-thiol coupling reaction where multiple
products and intermediates are observed. Bis maleimide cross-
linkers are commonly used at pH 6.5e7.5 to form protein conju-
gates and to characterize protein structures and interactions by
providing information on distances between cross-linked sulfhy-
dryl residues.8 We investigated the coupling reaction of a single bis
maleimide reagent, 4, with two different thiol-containing com-
pounds, cysteine 5 and N-acetyl cysteine 6. As illustrated in Fig. 2b,
two different mono adduct intermediates are possible, whereas
three possible bis adduct final products can be formed. As all 5 of
these components have unique masses that also differ from the
starting materials used in the study, MISER HPLC-MS analysis
provides a useful window into the reaction. This very fast reaction
can be accurately monitored by using high speed autosampler
technology that allows for an injection cycle time of 14 s.
Quantitative interpretation can be somewhat challenging, as MS
detection can exhibit significant nonlinear response and can also be
prone to interference by ion suppression and enhancement.
Despite these concerns, trends in reactivity can clearly be observed.
Very fast consumption of the starting material in less than 5 min
can be noted, but also the rapid formation and subsequent con-
sumption of intermediate 1 (7) which exhibits a faster reaction rate
Fig. 1. MISER kinetic profiling of the base catalyzed ester hydrolysis of a series of substituted methyl benzoates using a 30s injection period. The chromatogram serves as a graph,
providing immediate information about progress of the reaction and relative reaction rates. MISER Conditions: Waters CORTECS UPLC C18þ (30 ꢀ 2.1 mm, 1.6 m); 0.8 mL/min;
m
mobile phase ¼ 1:1 A:B with A ¼ 90% ACN in H2O (2 mM HCOONH4) and B ¼ H2O (2 mM HCOONH4). The first injection contains a starting material standard at the initial
concentration. Following the first injection, 100 ml of a 4 mM NaOH solution was added to a second unstirred vial at room temperature containing 1 mL of 0.01 mM starting material,
with online sampling every 14 s.
Please cite this article in press as: Zawatzky K, et al., Facile kinetic profiling of chemical reactions using MISER chromatographic analysis,
Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.05.048

K. Zawatzky et al. / Tetrahedron xxx (2017) 1e6
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K. Zawatzky et al. / Tetrahedron xxx (2017) 1e6
than formation and consumption of intermediate 2 (8). At the same
time appearance of the 3 different bis adduct products can be
simultaneously monitored, allowing for an easy visual comparison
of relative product formation rates, with product 1 (9) > product 2
(10) > product 3 (11).
intermediate that is subsequently hydrolyzed to the carboxylic acid.
Selected ion monitoring at the target mass for the intermediate and
the carboxylic acid product provides a clear kinetic profile for their
formation.
One of the distinct benefits of the EasySampler probe technol-
ogy is the ability to perform reaction quenching locally within the
reactor, as well as suitably preparing the probe for future sampling
activities to minimize sample carryover, and to mitigate the risk of
quench solution entering the reaction solution. A drawback of these
features is a comparatively longer sampling frequency than other
MISER approaches.
While this sampling interval of 5 min provides a useful kinetic
profile, a higher sampling rate with a fast chromatography method
would provide additional insight into the initial rate portion of the
reaction. Similarly, although this home built modification of the
EasySampler allows proof of principle for direct online MISER ki-
netic profiling, a more versatile system combining fast sampling
rates with convenient software control could easily be imagined.
Finally, conventional HPLC kinetic profiling approaches with inte-
gration of peaks, data input, graphing and curve fitting are certainly
appropriate in instances where exact measurements are critical for
understanding reaction mechanisms. Nevertheless, MISER kinetic
profiling can provide a generally accessible approach for initial
studies of reaction kinetics and for rapid range finding of the effect
of reagents or conditions on reactivity.
While the preceding examples serve to illustrate the value of
MISER kinetic profiling, the study of homogeneous reactions carried
out in an HPLC vial at room temperature without stirring, inertion or
ongoing reagent addition is clearly not representative of the ma-
jority of organic chemical reactions. Great strides have been made in
recent years in the use of online HPLC analysis for kinetic reaction
profiling, where samples can be withdrawn from organic reactions,
quenched, diluted and then analyzed by HPLC.9 Such studies typi-
cally utilize conventional individual chromatogram data files, from
whichdata isextractedand displayed aseither waterfall plots orarea
% vs. time curves, but we reasoned that MISER kinetic profiling could
provide a more straightforward visualization of the reaction time
course. We investigated the hydrolysis of a pharmaceutical inter-
mediate, using the Mettler-Toledo EasySampler for sample collec-
tion. In comparison to other commercially available online reaction
monitoring systems, the EasySampler combines a robust probe-
based reaction sampling device with the ability to perform reac-
tion quenching locally within the reactor, at reaction temperature
and pressure. This sampling technology has been demonstrated
under demanding reaction conditions to routinely and reliably
deliver a quenched sampled at an accurate dilution from both ho-
mogeneous and heterogeneous reaction mixtures. The EasySampler
probe normally collects reaction aliquots, preparing and storing
samples in a 12 vial carrousel for subsequent offline analysis. We
explored a modification of this setup in which the transfer of the
quenched sample tothe storage vial is intercepted, instead filling the
injection loop of an HPLC instrument, thereby allowing direct online
analysis of reaction aliquots in real time. The tubing from the Easy-
Sampler was directly connected to an external 10 port/2 position
valve integrated in an Agilent 1290 UHPLC system as illustrated in
Fig. 3. The dilution solvent of the EasySampler is used to push the
quenched sample through the tubing from the sampling system, to
the LC system. Dilution factorand tubing length canbe adjustedsuch
3. Conclusion
MISER kinetic profiling provides a simplified approach for
studying chemical reactions without the need for peak integration,
data input, graphing or curve fitting. When used in combination
with fast chromatography and fast autosampler technology, sam-
pling intervals on the order of ~10s are possible, enabling kinetic
investigations into relatively fast reactions. The technique is also
useful for online HPLC-MS analysis of reaction aliquots collected by
sampling devices such as the Mettler-Toledo EasySampler.
4. Experimental section
that an aliquot of the sample is stored in the 5 ml loop when the flow
from the EasySampler ceases. Switching the valve at this point in-
jects the sample onto the LC system.
4.1. Instrumentation
Preliminary robustness/accuracy testing of this configuration
revealed an RSD of injection of up to 3%. This variance is not due to
inaccuracies of the sample removed from the reaction solution, but
rather due to slight inaccuracies of the EasySampler pump, which
results in minor inconsistencies in the sampling of the quenched
aliquot's concentration profile by the injection loop. Presumably,
integrating a more precise pump into the sampling system would
overcome this issue and allow for improved repeatability in the LC
injection.
The MISER chromatogram in Fig. 4 shows the kinetic profile of
the reaction over the first 2 h with sampling every 5 min. The
consumption of the t-Bu ester starting material (12) and the for-
mation of the carboxylic acid product (14) can easily be visualized
by UV detection. In addition, the formation of the methyl ester (13)
can be also observed, which grows to a maximum concentration
around 40 min reaction time and which is completely consumed at
the end of the reaction (4 h). Methanol is a critically important
component for this hydrolysis reaction,10 which is believed to
proceed through initial transesterification to form a methyl ester
High performance liquid chromatography (HPLC) experiments
were performed on an Agilent 1290 Infinity II system, equipped
with a G7104A binary pump, a G7167B multisampler, a G7117B
diode array detector and a 6120B Quadrupole LC/MS detector with
electrospray ionization controlled by the Agilent ChemStation
software.
Online reaction monitoring experiments were performed by
integrating a Mettler-Toledo EasySampler 1210 with an EasyMax
102 Advanced Synthesis workstation and an Agilent 1290 Infinity
HPLC system, equipped with a G4204A quaternary pump, a G4226A
HiP autosampler, a G1316c column compartment, a G1157A 2 po-
sition/10 port valve, a G4212A HDR detector and a 6120 Quadrupole
LC/MS detector with electrospray ionization controlled by the
Agilent ChemStation software.
4.2. Chemicals, reagents and stationary phases
Acetonitrile (HPLC-MS grade), water (HPLC-MS grade) and
methanol (HPLC grade) were purchased from Fisher Scientific. Formic
Fig. 2. MISER kinetic profiling of the reaction of a bis maleimide with two different thiols a) Reaction scheme for the addition of a 1:1 mixture of cysteine/acetylcysteine to
bismaleimidohexane including all possible intermediates and products. b) LC-MS reaction monitoring by extracting MS signals for starting material, intermediates and products.
Reaction conditions: 1.5 mM BMH in DMSO (2eq. N-acetylcysteine/cysteine in aq. NH4COO pH 6.6). MISER Conditions: Sampling interval ¼ 14s; Waters CORTECS UPLC C18þ
(30 ꢀ 2.1 mm, 1.6 m); 0.8 mL/min; mobile phase ¼ 1:1 A:B where A ¼ 90% ACN in H2O (2 mM HCOONH4) and B ¼ H2O (2 mM HCOONH4).
m
Please cite this article in press as: Zawatzky K, et al., Facile kinetic profiling of chemical reactions using MISER chromatographic analysis,
Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.05.048

K. Zawatzky et al. / Tetrahedron xxx (2017) 1e6
5
Fig. 3. Instrumental setup for online sampling and LC analysis.
Fig. 4. MISER kinetic profiling for online HPLC analysis of the hydrolysis of a pharmaceutical intermediate using a modification of the Mettler-Toledo EasySampler system for
reaction sampling, quenching and dilution. The presence of methanol is critically important for this reaction, which proceeds through initial transesterification and formation of a
methyl ester intermediate (13). Reaction conditions: 0.13 M (substrate 12) in MeOH/THF, NaOH (5eq.), 40 ꢁC. EasySampler conditions: 20
ml sample, dilution factor 1:180, quench
and diluent 72% ACN in H2O (2 mM NH4COO) MISER conditions: Zorbax SB-Phenyl 150 ꢀ 4.6 mm, 3.5
mm, mobile phase 72% ACN in H2O, 2 mM NH4COO. Samples were taken and
injected every 5 min. The first injection corresponds to the sample taken after 5 min and injected on the LC system after 9 min.
Please cite this article in press as: Zawatzky K, et al., Facile kinetic profiling of chemical reactions using MISER chromatographic analysis,
Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.05.048

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K. Zawatzky et al. / Tetrahedron xxx (2017) 1e6
acid (HCOOH), ammonium formate (HCOONH4), methyl 4-
nitrobenzoate, methyl 4-methoxybenzoate, methyl 4-
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400
and 200
mixed with 500
m
l BMH (5 mM in DMSO) added to 200
l cysteine (10 mM in aq. NH4COOH pH 6.6; 2 eq.) pre-
l DMSO and cooled to 10 ꢁC. Final concentration in
ml N-acetylcysteine
m
m
mix 1.5 mM in BMH, 3 mM in N-acetylcysteine and cysteine. After
mixing the sample was immediately analyzed by MISER-LC-MS.
In a reactor controlled by an EasyMax 102 workstation, 5.63 g of
t-butyl ester (12) were dissolved in 65.5 g 45% w/w MeOH in THF
and heated to 40 ꢁC with stirring under nitrogen. 3.92 g NaOH (5
eq., 50% in H2O) were added to the mixture and samples were taken
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Acknowledgements
We are grateful to the MRL Postdoctoral Research Fellows pro-
gram for financial support provided by a fellowship (K.Z.).
Please cite this article in press as: Zawatzky K, et al., Facile kinetic profiling of chemical reactions using MISER chromatographic analysis,
Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.05.048