Direct regioisomer analysis of crude reaction mixtures: Via molecular rotational resonance (MRR) spectroscopy

Article abstract of DOI:10.1039/d0sc01853h

Direct analyses of crude reaction mixtures have been carried out using molecular rotational resonance (MRR) spectroscopy. Two examples are presented, a demonstration application in photocatalytic CH-arylation as well as generation of an intermediate in a natural product synthesis. In both cases, the reaction can proceed at more than one site, leading to a mixture of regioisomers that can be challenging to distinguish. MRR structural parameters were calculated for the low lying conformers for the desired compounds, and then compared to the experimental spectra of the crude mixtures to confirm the presence of these species. Next, quantitation was performed by comparing experimentally measured line intensities with simulations based on computed values for the magnitude and direction of the molecular dipole moment of each species. This identification and quantification was performed without sample purification and without isolated standards of the compounds of interest. The values obtained for MRR quantitation were in good agreement with the chromatographic values. Finally, previously unknown impurities were discovered within the photocatalytic CH-arylation work. This paper demonstrates the utility of MRR as a reaction characterization tool to simplify analytical workflows. This journal is

Full text of DOI:10.1039/d0sc01853h

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Direct regioisomer analysis of crude reaction
mixtures via molecular rotational resonance (MRR)
Cite this: DOI: 10.1039/d0sc01853h
spectroscopy†
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Leo A. Joyce, ‡a Danielle M. Schultz,
a Edward C. Sherer, b Justin L. Neill,
c
*
*
Reilly E. Sonstromd and Brooks H. Pated
Direct analyses of crude reaction mixtures have been carried out using molecular rotational resonance
(MRR) spectroscopy. Two examples are presented, a demonstration application in photocatalytic CH-
arylation as well as generation of an intermediate in a natural product synthesis. In both cases, the
reaction can proceed at more than one site, leading to a mixture of regioisomers that can be challenging
to distinguish. MRR structural parameters were calculated for the low lying conformers for the desired
compounds, and then compared to the experimental spectra of the crude mixtures to confirm the
presence of these species. Next, quantitation was performed by comparing experimentally measured line
intensities with simulations based on computed values for the magnitude and direction of the molecular
dipole moment of each species. This identification and quantification was performed without sample
purification and without isolated standards of the compounds of interest. The values obtained for MRR
quantitation were in good agreement with the chromatographic values. Finally, previously unknown
impurities were discovered within the photocatalytic CH-arylation work. This paper demonstrates the
utility of MRR as a reaction characterization tool to simplify analytical workflows.
Received 31st March 2020
Accepted 30th May 2020
DOI: 10.1039/d0sc01853h
rsc.li/chemical-science
The characterization of novel synthetic chemical reactions – in conditions and pooling them together. Next, the crude reaction
particular, the identication and quantication of the products mixture is puried to isolate the individual components (typi-
arising from a synthesis – is a critical, but oen difficult task. cally using chromatography such as TLC, HPLC, SFC etc.),
Reactions oen yield complex mixtures with structurally similar however multiple separations are oen required to generate
analytes, such as diastereomers, regioisomers, and positional sufficiently pure compounds.9 Once the pure components have
isomers.1–3 The last few decades have seen the development of been isolated, the next challenge is to elucidate their structure.
many synthetic platforms that give unique and exciting chem- This generally requires either high-resolution mass spec
ical transformations,4–8 so effective and standardized methods (HRMS) or elemental analysis (EA) to understand the elemental
for reaction characterization are highly useful.
composition, followed by NMR analysis to unambiguously
In order to fully understand the products of a reaction of assign the atomic connectivity.10–13
interest, researchers oen follow a procedure similar to the
While this approach is amenable to a variety of synthetic
following. First, the reaction conditions are scaled up to platforms, there are several drawbacks regarding structural
generate a sufficient quantity of material for analysis. This may characterization. First, this is a very laborious process that
require running several concurrent reactions with identical requires many manual steps to obtain pure products for
subsequent analyses. If the separation is challenging, this can
oen require additional purications to be carried out, costing
not only time but losing precious amounts of material to
aDepartment of Process Research & Development, Merck & Co., Inc., Rahway, NJ,
07065, USA. E-mail: danielle.schultz@merck.com
multiple separation passes. Additionally, it may be challenging
bDepartment of Computational and Structural Chemistry, Merck & Co., Inc., Rahway,
to detect and isolate a sufficient quantity of small components
NJ, 07065, USA
cBrightSpec, Inc., 770 Harris St., Suite 104b, Charlottesville, VA, 22904, USA. E-mail: of larger mixtures. Identifying these small abundance
justin.neill@brightspec.com
dDepartment of Chemistry, University of Virginia, McCormick Road, Charlottesville,
compounds may be crucial to understanding the reaction
mechanism and can even lead to the development of new
reaction methodologies. Finally, accurately quantifying prod-
ucts and reactants from a crude mixture can be challenging due
VA, 22904, USA
† Electronic supplementary information (ESI) available: MRR spectra,
calculations, comparison between experimental and theoretical, separation
to different ionization properties or UV responses of the various
methods. See DOI: 10.1039/d0sc01853h
components. For all of these reasons, it is necessary to develop
‡ Current address: Arrowhead pharmaceuticals, 502 S. Rosa Rd., Madison, WI
52719, USA, ljoyce@arrowheadpharma.com
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techniques that allow reaction yield and byproduct content to
be determined directly on crude materials.
Molecular rotational resonance (MRR), also widely known in
the literature as microwave or rotational spectroscopy, is a eld
with a long history within chemistry.14,15 This technique has
historically been used in chemical physics because of its ability
to provide insights into the electronic structure of gas phase
molecules and weakly bound complexes, particularly in the
study of intermolecular and intramolecular interactions.16–19
Recent advances in instrumentation have facilitated the
measurement of broad-bandwidth spectra, enabling rapid
characterization of mixtures, in a reasonable amount of time
Scheme 1 (A) Tetrabutylammonium decatungstate (TBADT) mediated
C–H arylation, and (B) ketone methylation reactions analyzed directly
(minutes to hours),20–22 including for increasingly large, con- with MRR.
formationally complex, and chiral compounds.23–27 Several
recent studies have also demonstrated the capability for rota-
tional spectroscopy to resolve numerous mixture components
The second reaction was part of an investigation into the
without purication.24,28–30 As a result, MRR has become suit-
able for characterization of compounds of industrial and
pharmaceutical relevance.31
absolute conguration of a natural product, frondosin B.35 As
part of this study, previously reported syntheses of frondosin B
were re-examined to determine the cause of a reversal in the
sign of the optical rotation. While a stereochemical inversion
was initially expected, it was discovered that near the end of the
synthesis a two-step gem-dimethylation of ketone 4 produced
a signicant amount of regioisomeric impurity 6 (Scheme 1b).
This impurity, as opposed to stereochemical inversion,
accounted for the previously noted discrepancy.
MRR identies molecules through characterization of their
pure rotational transitions in the gas phase, the energies of
which are governed by their molecular rotational constants.
These constants (A, B, and C) have an inverse proportional
relationship with the molecule's three-dimensional moments of
inertia and give rise to spectra with a multitude of highly
resolved lines, as long as the compound has a permanent dipole
moment. The permanent dipole moment magnitude and
orientation directly affects the transition intensities, and
quadrupolar nuclei (for example, 14N) also add hyperne
structure to the spectrum, which can be compared to calcula-
tions to provide additional structural information. Subtle
differences in structure (including between regioisomers, dia-
stereomers, and isotopologues) produce signicant and calcu-
lable effects on the rotational constants, allowing for
unambiguous conrmation of molecular identity. These
parameters can be accurately calculated from molecular
geometry, and have been used to benchmark computational
methods for molecular structure.32 Additionally, techniques to
elicit enantiomerically selective MRR spectra have recently been
Both of these reactions were characterized in previous
studies by separation of the distinct products followed by
structure elucidation, as described above. The purpose of the
present study is to demonstrate the use of MRR spectroscopy to
directly characterize the crude reaction mixtures and quantify
their components with a single measurement. We successfully
identied a number of components in both reactions, including
both previously known and new impurities. All analyses were
performed without the need for pure reference standards of the
different products.
Methods
reported.24,26,33 The close relationship between spectra and MRR spectra of the two crude reaction mixtures were measured
calculations, along with the intrinsically high resolution of the using a broadband chirped-pulse Fourier transform microwave
technique, enables the identication of compounds directly spectrometer based at the University of Virginia, operating over
within a mixture, without the need for pure reference standards. the 2–8 GHz frequency range. The design of this spectrometer
Hence, MRR is an ideal tool for the characterization of complex has been described elsewhere.21 In order to isolate the compo-
synthetic organic reactions.
nents in the gas phase and rotationally cool the samples for
In this paper, we demonstrate the ability of MRR to identify analysis, pulsed solenoid valves coupled to 0.9 mm pinhole
and quantify impurities in two different previously reported nozzles are employed, which create a supersonic expansion of
synthetic reactions. The rst was presented in a recent study volatilized analytes in a carrier gas stream within a chamber
that reported the use of decatungstate photocatalysis to facili- maintained at high vacuum. This typically results in samples
tate direct arylation of aliphatic C–H bonds to access complex with rotational temperatures of approximately 1 K, cools out
and pharmaceutically relevant molecules.34 However, due to the most vibrationally excited states, and also results in some
variety and abundance of C–H bonds present in a given mole- conformational cooling (see further discussion below). The
cule, C–H arylation isomers oen resulted. Herein, we report nozzles also include a heated reservoir for holding solid or
the application of MRR for the direct analysis of the photo- liquid samples. The pulsed sample injection is congured to
catalytic arylation of cyclohexanone
(triuoromethyl)pyridine 2 (Scheme 1a).
1
with 5-bromo-2- synchronize to a series of short (1–4 ms), high-power broadband
chirped pulses. Between each pulse, the broadband microwave
spectrum is recorded as a time-domain free induction decay
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(FID) signal, and Fourier transformed to yield the spectra pre- correction has been reported to improve the accuracy of the
sented below. three-dimensional geometry, to which the MRR spectrum is
In these experiments, the samples (ꢀ50 mg of unpuried extremely sensitive. In addition to observing substantial
oil) are dissolved in approximately 300 mL of dichloromethane, differences in the rotational constants between regioisomers,
and divided between three identical reservoirs, which are different conformational isomers of the same compound have
congured to inject sample simultaneously to increase the distinct rotational spectra, and so it is important to include all
measurement sensitivity. Neon (>99.999%) was used as the low-energy conformers in the search. As an example, the
carrier gas. The sample reservoirs were rst heated to 40 ꢁC conformers of 3a (the major product of the arylation reaction)
and pulsed to remove the dichloromethane solvent. Once this are presented in Fig. 1. In two of these conformers (conf. 1 and
was completed, the reservoir temperatures were increased in conf. 3), the pyridine is in an equatorial position on the
steps of approximately 15 ꢁC at a time, and the MRR spectrum cyclohexanone ring, differing only by 180ꢁ rotation about the
was measured for approximately 5 minutes at each tempera- carbon-to-carbon bond between the two rings. Fig. 1 shows
ture step so that the presence of any impurities that are volatile that despite this subtle difference in structure, the rotational
at that temperature can be noted. The nozzles are operated at constants are substantially different. The direction of the
a 3 Hz repetition rate, with eight spectra measured on each molecular dipole moment, presented through its projections
injection pulse, and accumulated in the time domain to along the three principal axes (ma, mb, and mc), and the orien-
improve the sensitivity through signal averaging. The nal tation of the 14N quadrupolar coupling constant (not shown
measurement temperature, where the signals of the target here, but available in the ESI†), are also distinct between the
compounds were optimized, was 165 ꢁC for the cyclohexanone two conformers, enabling their unambiguous identication in
reaction, and 195 ꢁC for the frondosin B mixture. In the the sample. Likewise, the two axial conformers (conf. 2 and
cyclohexanone sample, volatile impurities were also observed conf. 4), can also be differentiated through their MRR
at low temperature, which will be described in more detail parameters. The same computational method was carried out
below.
for the other species under consideration in both reactions,
The MRR parameters of each compound of interest were with full parameters available in the ESI.†
also predicted computationally to facilitate component iden-
tication and quantication. First, a conformational search
was performed on each compound to generate the possible
minima, which is carried out using previously described
methodology.36 These structures are then optimized using
B3LYP calculations with the D3BJ dispersion correction as
implemented in Gaussian 09.37–39 The D3BJ dispersion
Results and discussion
Cyclohexanone arylation reaction
The 165 ꢁC broadband MRR spectrum of the crude arylation
reaction mixture was measured over the course of 2.3 h (200 000
total spectral acquisitions). The spectrum is presented in the
top panel of Fig. 2. The primary components in the sample (3a
and 3b) can be observed within a matter of seconds, while signal
averaging enables the detection of minor components (1% or
less). A minor regioisomer not indicated in the previous study,
where arylation occurs at the 2-position, is identied. Each
species is assigned by a t to a rotational Hamiltonian with
Fig. 1 Structures of the four lowest energy conformers of the primary
product 3a (3-substituted) of the arylation reaction, and their calcu-
lated MRR parameters (rotational constants and dipole moment
projections). Energies and rotational constants are calculated at
a B3LYP-D3BJ/def2TZVP level of theory.
Fig. 2 The top spectrum (in black) shows the full MRR spectrum of the
cyclohexanone reaction mixture at a temperature of 165 ꢁC. Beneath
this are isolated MRR spectra of the three detected conformers of the
major (3-substituted) product 3 in the arylation reaction.
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standard methods and soware, using the theoretical rotational has been explored in a number of previous studies.40–42 In
constants described above as a guide. Identications are based order to quantify the total abundance of each isomer in the
on satisfactory agreement between experimental and computed mixture, we sum the contributions of all of the identied
rotational constants. Full t results and procedures are avail- conformers. The relative concentration of each conformer in
able in the ESI.†
the sample vapor was determined by simulating the MRR
A total of three conformers of the primary 3-substituted spectral pattern with the calculated molecular dipole moment
product 3a were identied in the MRR spectrum, along with components to predict the expected relative line intensities
two conformers of the 4-substituted isomer 3b, and one of the for each transition in the spectrum, using a rotational
2-substituted isomer. Each conformer is t separately in the temperature of 1.5 K as this temperature best describes the
spectrum. In Fig. 2, the full spectrum of the product mixture spectral intensity pattern. The ratio between the experimental
is shown in black, and the three observed conformers of 3a and simulated intensity therefore provides a relative measure
are shown in different shades of blue in order to highlight of that component's abundance in the mixture. Averaging
their different contributions to the spectra. In the colored across all of the detected transitions of each conformer
spectra, the spectral lines attributed to the indicated improves the quantitative accuracy. Once a scale factor is
conformer have been kept in the spectrum while all of the determined for each conformer of each species, the total
other spectral lines have been cut for visual clarity. In Fig. 3, abundance of each component can be calculated. Further
the full spectrum is again shown in black in the top panel. In information on the quantication procedure used here is
the middle and lower panels, the transitions are color-coded available in the ESI.† This analysis did not account for any
based on which of the three regioisomers they have been possible difference in vapor pressure between the
assigned to. A portion of the spectrum of the minor 2- regioisomers, which are expected to be small.
substituted isomer can be seen in the inset of the lower right
Table 1 shows the combined results of the analysis on the
corner of this gure.
crude arylation sample. The ratio between isomers 3a and 3b
Notably, the conformational population in pulsed super- were previously determined by HPLC is in excellent agreement
sonic expansion measurements does not reect thermody- with the MRR results. Aer the MRR results were obtained, the
namic equilibrium at the sample vaporization temperature, low-level presence of the 2-substituted isomer in this reaction
as conformational cooling is typically observed. This effect mixture was conrmed by HPLC-MS. The estimated relative
error for the quantitative results presented in this paper is about
10%, with the primary source being the calculation of the dipole
moment of each species.
As noted above, this sample also contained additional
impurities that were more volatile than the arylation products.
While the current MRR measurement procedure does not
reliably quantify these components (since they are measured
at lower temperature), it is still notable that MRR can identify
additional non-isomeric byproducts in mixtures as well as
isomeric ones. Fig. 4 shows a spectrum that was measured
ꢁ
across the temperature range 120–130 C, where the vapor is
dominated by lower molecular-weight impurities in the
sample. Three primary components were identied in this
spectrum. Two of these, cyclohexanone and acetic acid, had
previously characterized MRR spectra43,44 and so were imme-
diately identied using a spectral library. Cyclohexanone, in
particular, is a starting material in the reaction, and was
charged in excess, accounting for its presence in the mixture.
The third byproduct identied was arylated acetonitrile
(reaction solvent) with 2. The structure was determined by rst
Table 1 Comparison of amounts of each substituted product of 3
determined by MRR and UPLC (note that the amount by UPLC
represents area percent at 210 nm)
Fig. 3 (Top) Broadband MRR spectrum of the arylation product
mixture at 165 ꢁC (see the text for full measurement description). The
Amount
lower panels show the same spectrum, with the assigned contribu-
tions from each of the three product isomers colored to show their
presence in the spectrum. (Different conformers of the same isomer
are colored the same in this figure). The bottom row shows two
expanded views to show that there are no spectral overlaps between
the features of different species.
Compound
(MRR)
Amount (UPLC)
3a (3-substituted)
3b (4-substituted)
2-substituted
57.0%
42.7%
0.3%
58.0%
41.6%
0.4%
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Fig. 5 (Top) MRR spectrum of the OMe-frondosin B 5 reaction
mixture (displayed from 4–8 GHz for clarity). The blue spectrum
consists of the isolated spectra of four conformers of OMe-frondosin
B (5), which make up all the strong lines observed. (Bottom) Expanded
views of different regions of the spectrum to indicate some of the
identified transitions of the regioisomeric impurity and starting mate-
rial in the reaction. Because of the low concentration of these impu-
rities, fewer lines are visible, but still can be matched unambiguously to
a set of rotational constants that agrees with the calculations for these
species.
Fig. 4 Lower-temperature (120–130 ꢁC) spectrum of the arylation
product mixture. The strongest line is due to the cyclohexanone
reagent. Weaker lines are due to the solvent-derived byproduct. The
arylation reaction products are also present at lower levels.
noting hyperne structure in the impurity consistent with two
14N nuclei, and observing that acetonitrile is also a viable
substrate for the arylation reaction. Electronic structure
calculations were then performed on the proposed structure to
conrm the molecular identication.
this to correct for the populations expected for the low-dipole
components. Under this model, only 17% of the OMe-
frondosin B population is calculated to be in the low-polarity
conformers, and so the precise value of the equilibrium
temperature does not signicantly affect the quantitative
results presented below. This procedure is described in more
detail in the ESI.†
As the regioisomeric byproduct 6 contains two chiral
centers, in the previous study of this reaction35 the relative
stereochemistry was determined on the basis of 1H ROESY 2-D
NMR experiments on an isolated sample with comparison to
chemical shis predicted using DFT. Just as for regioisomers,
MRR is extremely precise in distinguishing between diaste-
reomers on the basis of their differences in moments of
inertia. This is illustrated in Fig. 6, where the calculated
minimum-energy conformers of compound 6 (R,R) and its
diastereomer (R,S) are presented in comparison to the exper-
imentally determined rotational constants for the impurity.
The signicant difference between the rotational constants of
the two diastereomers, and the excellent agreement between
the experimental constants and the calculation for the (R,R)
diastereomer, demonstrates that MRR is able to unambigu-
ously determine the stereochemistry of the impurity directly in
the reaction mixture.
Frondosin B intermediate reaction
The gem-dimethylation of the ketone 4 to form OMe-frondosin B
(5) was measured for 295 000 signal averages (3.4 h). The
resulting spectrum was then analyzed following the same
procedure as for the rst study. Fig. 5 presents the MRR spec-
trum of the crude reaction mixture. The spectrum is again
dominated by several conformers of the primary product, with
lower-abundance species appearing as weaker patterns in the
spectrum. As was observed for the arylation reaction, the low-
abundance species do not spectrally overlap with the primary
mixture component.
The compounds in this sample did present additional
conformational exibility, due to the fused cyclohexene and
cycloheptadiene rings that can obtain several energetically
favorable conformations; these were previously described in
greater detail.35 Additionally, the methyl ether group can adopt
two congurations that are planar with the phenyl ring it is
connected to. Crucially for the MRR analysis, one of these two
congurations of OMe-frondosin B 5 results in a strong
molecular dipole moment (>2 Debye), while the other has very
low dipole moment (<0.3 Debye). The MRR signal response in
the spectrometer used in this study is proportional to the
square of the dipole moment, so the measurement sensitivity
to half of the conformations is reduced by a factor of approx-
imately 50 and are not detected. We assumed a conforma-
tional equilibrium at a temperature of 200 K (a best-t based
on the populations of the four observed conformers) and use
The nal quantitative results for the OMe-frondosin B
sample are presented in Table 2. Again we note that the agree-
ment of MRR results with the previously reported chromato-
graphic analysis (in this case, SFC) are in excellent agreement
considering the measurement uncertainty of the two
techniques.
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Conflicts of interest
Authors J. L. N and B. H. P. have a nancial interest in Bright-
Spec, Inc.
Acknowledgements
The authors thank Rina Dukor for many helpful discussions.
Work at the University of Virginia was supported by the National
Science Foundation (CHE-153193). Work at BrightSpec and at
the University of Virginia was also supported by the Virginia
Biosciences Health Research Corporation (the Catalyst).
Fig. 6 Calculated structures of the lowest-energy conformers of the Notes and references
regioisomeric byproduct in the frondosin B reaction (6) and its dia-
stereomer. The table shows the experimental rotational constants
assigned for this compound in the MRR crude reaction mixture anal-
ysis, and the percent differences between the experiment and each of
the two calculations. The identified impurity is unambiguously
assigned as the (R,R) diastereomer, consistent with the previous study.
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synthesis of OMe-frondosin B 5 determined by MRR and SFC (note that
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(MRR)
Amount
(SFC)
Compound
OMe frondosin B (5)
Regioisomer (6)
Starting material (4)
91.2%
6.8%
2.0%
92.9%
7.1%
n.d.a
a
The presence of the starting material was not noted on the SFC
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Conclusions
We have demonstrated that MRR can be used for the direct
characterization of crude reaction mixtures without reference
standards. This technique was applied to two systems, both
involving the identication and quantication of regioiso-
meric impurities in crude reaction mixtures: the photo-
catalytic C–H arylation of cyclohexanone, and the
quantication of an isomeric impurity in an intermediate step
in the synthesis of frondosin B. MRR streamlines the typical
workow of extensive purications and structure elucidation
techniques used for separation and conrmation of chemical
structures. The quantitation results that were determined for
the composition of both reaction mixtures are in good agree-
ment with analytical results. Additionally, MRR can readily
identify low-level impurities that would be challenging to
separate and identify by alternative techniques. The ability to
identify additional sites of activation in small quantities can
be very important in a discovery chemistry setting where it
could enable more rapid exploration of structure–activity
relationships.
14 E. B. Wilson, Science, 1968, 162, 59.
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