Reaction monitoring using online vs tube NMR spectroscopy: Seriously different results

Article abstract of DOI:10.1002/mrc.4259

We report findings from the qualitative evaluation of nuclear magnetic resonance (NMR) reaction monitoring techniques of how each relates to the kinetic profile of a reaction process. The study highlights key reaction rate differences observed between the various NMR reaction monitoring methods investigated: online NMR, static NMR tubes, and periodic inversion of NMR tubes. The analysis of three reaction processes reveals that rates derived from NMR analysis are highly dependent on monitoring method. These findings indicate that users must be aware of the effect of their monitoring method upon the kinetic rate data derived from NMR analysis.

Full text of DOI:10.1002/mrc.4259

Special issue research article
Received: 19 August 2014
Revised: 11 March 2015
Accepted: 15 April 2015
Published online in Wiley Online Library: 6 August 2015
(wileyonlinelibrary.com) DOI 10.1002/mrc.4259
Reaction monitoring using online vs tube NMR
spectroscopy: seriously different results
David A. Foley,a* Anna L. Dunna,b and Mark T. Zella
We report findings from the qualitative evaluation of nuclear magnetic resonance (NMR) reaction monitoring techniques of how
each relates to the kinetic profile of a reaction process. The study highlights key reaction rate differences observed between the
various NMR reaction monitoring methods investigated: online NMR, static NMR tubes, and periodic inversion of NMR tubes. The
analysis of three reaction processes reveals that rates derived from NMR analysis are highly dependent on monitoring method.
These findings indicate that users must be aware of the effect of their monitoring method upon the kinetic rate data derived from
NMR analysis. Copyright © 2015 John Wiley & Sons, Ltd.
Keywords: NMR reaction monitoring; online NMR; reaction mechanism
vessel allows replication of reaction conditions while allowing
for analysis in an essentially unperturbed state. There are exam-
Introduction
Nuclear magnetic resonance (NMR) is an extremely powerful tool
for the analysis of reaction mixtures. Not only can NMR be used
as a quantitative method of monitoring reaction processes, it also
has the advantage of providing detailed structural information
about reaction components in solution. These two characteristics
give NMR a distinct advantage over other analytical techniques
typically used for reaction monitoring purposes. Some of these
techniques (i.e. infrared or Raman) can identify functional groups
of the molecules being analyzed; however, they lack the compre-
hensive characterization that NMR can provide.
There are four common approaches to conducting NMR reaction
monitoring: (i) static, in standard NMR tubes, (ii) online monitoring,
(iii) stopped-flow, and (iv) rapid injection NMR. Static NMR tube
monitoring (i) is the simplest and therefore most extensively used
of these techniques, and involves placing reagents into a standard
NMR tube, then monitoring reaction progress of the static solution
within the spectrometer.[1] This approach requires no specialized
equipment beyond that normally used to run NMR samples and
can be practically conducted in deuterated solvent because of the
small volume required. This has been shown to be an effective
method for probing mechanistic details of reactions.[2] An alterna-
tive method is to remove aliquots from a reaction vessel (some-
times followed by a quench step) and transfer the sample to an
NMR tube for offline analysis.[3] This could equally be conducted
in protonated solvents using proton gradient shimming for efficient
shimming without the presence of a deuterium source.[4]
ples of online NMR reaction monitoring being applied at both
high and low fields.[6]
The third method, stopped-flow NMR (iii), is typically used for the
detection of rapid kinetics (within 2.5–100ms of reagents mixing), a
feature that both NMR tube and online reaction monitoring cannot
provide because of the time required to mix and get reagents to
the detection region.[7] Stopped-flow NMR typically employs
custom probes designed to flow reagents first into a mixing region
and then into the detection region of the spectrometer with ex-
tremely high flow rates to minimize dead time. Prior to acquisition,
the flow must be stopped because of the short residence times of
magnetized sample at the high flow rates. The flow system allows
for multiple time points to be easily taken by flowing more
unreacted starting material into the mixing, then detection regions
with different delay values in the pulse sequence. The stopped-flow
NMR technique has allowed detection of short-lived intermediates,
study of complex protein-folding mechanisms,[8] and insights into
the effects of fast kinetics on NMR lineshape.[9]
The fourth approach, rapid injection NMR (iv), allows acquisition
typically 40 ms–1 s after reagents have mixed. Rapid-injection NMR
often utilizes custom probes or probe inserts that allow for quick in-
sertion of reagents into the NMR tube and sometimes include a me-
chanical mixing period along with injection.[10] Rapid-injection NMR
techniques have allowed direct observation of multiple reactive in-
termediate species, providing valuable mechanistic information.[11]
However, the lack of a flow system means that subsequent
The second approach, online NMR (ii), transfers a flowing stream
of the reaction mixture from a reaction vessel to the NMR probe
where the analysis is conducted. The speed of flow is optimized
to allow enough residence time of the magnetized sample for
acquisition.[5] The reaction mixture can then be returned to the
reaction vessel or directed to a waste container, depending on
the requirements of the experiment. Online NMR requires special
probes or custom NMR tubes, however it does facilitate online
sampling, obviating the need for operator input once the experi-
ment is running. Monitoring an online sample from a reaction
*
Correspondence to: David Foley, Analytical Research and Development, Pfizer
Worldwide Research and Development, 445 Eastern Point Road, Groton, CT
06340, USA. E-mail: DavidAnthony.Foley@pfizer.com
a Analytical Research and Development, Pfizer Worldwide Research and
Development, 445 Eastern Point Road, Groton, CT, 06340, USA
b Department of Chemistry, University of Wisconsin, 1101 University Avenue,
Madison, WI, 53706, USA
Magn. Reson. Chem. 2016, 54, 451–456
Copyright © 2015 John Wiley & Sons, Ltd.

D. A. Foley, A. L. Dunn and M. T. Zell
measurements may only be taken after replacement or cleaning of
parts of the system.
4-fluorobenzaldehyde was also analyzed using the same three reac-
tion monitoring methods (online, tube, and periodic inversion).
Finally, the acid-catalyzed transesterification of isopropanol and
acetic anhydride was also studied to show that the results obtained
are not limited to only very slow reactions.
In the last few decades, there have been numerous reports on
the analysis of reaction mixtures using NMR. The majority use stan-
dard NMR tube reaction monitoring.[12] While there is little doubt
that monitoring a static NMR tube gives excellent mechanistic infor-
mation, there is a lack of proof of how kinetic information gathered
from this type of experiment is representative of how a reaction
process proceeds under standard laboratory-scale synthetic chem-
istry conditions. We evaluated the validity of the following NMR
reaction monitoring techniques from a kinetic standpoint: online
NMR, standard NMR tubes, and NMR tubes with periodic inversion.
The results that we present here demonstrate that reaction moni-
toring techniques have a significant influence on the kinetic under-
standing of a reaction.[13]
Heterogeneous reaction
The L-proline-catalyzed self-condensation of propionaldehyde 1
was chosen for the kinetic comparison study because of the well-
established mechanism and the ability to observe many intermedi-
ate species via NMR spectroscopy.[16] In 2011, Zeitler and Gschwind
reported an elegant mechanistic study of the L-proline-catalyzed
self-condensation of propionaldehyde 1. The conclusions of their
study, which involved observing reaction progression in an NMR
tube, indicated that the aldol addition pathway to 2a/2b is compet-
itive with aldol condensation to 3 (Scheme 1A), and the mechanism
is a double-activation Mannich-type mechanism involving two cat-
alyst molecules (Scheme 1B). According to their results, the mecha-
nism involves simultaneous activation with proline (Pro) of the
donor aldehyde to form 4a and the acceptor aldehyde to form
4d. The interconversion between 4a and 4d goes through 4b/4c.
The C–C bond formation step was indicated to be the rate deter-
mining step and could be monitored by observing the species 4b
– the maximum concentration of 4b was determined to correspond
with the fastest formation rate of 3.
Results and discussion
This study was undertaken to evaluate how basic NMR tube moni-
toring techniques compare with following the progress of a reac-
tion using an online NMR system such as the one developed in
our laboratory.[14] While numerous reports of kinetic and mechanis-
tic studies using both standard NMR tube and online approaches
have appeared in the literature, none have undertaken a systematic
comparison of NMR reaction monitoring techniques. This investiga-
tion was designed to look at both homogeneous and heteroge-
neous reactions, to provide evidence that the NMR monitoring
technique can influence the kinetic results for a wide variety of
reactions.
Three modes of NMR spectroscopy reaction monitoring were
investigated; online NMR spectroscopy, NMR tube reaction moni-
toring with and without periodic agitation of the reaction mixture.
The online NMR method, using our custom reaction monitoring
system, was directly compared with the basic method of reaction
monitoring in an NMR tube.[15] A third method was also used
involving periodic inversion by inverting a standard NMR tube
three times during the interval between each NMR spectrum
acquisition. Periodic inversion gave insight into an intermediate
mixing regime between constant mixing (online NMR) and a static
sample (NMR tube).
Mechanistic observations from this study were consistent with
those reported by Zeitler and Gschwind, so all chemical assign-
ments used in this study are in agreement with their published
chemical shifts.
The progress of the L-proline-catalyzed aldol condensation was
monitored by 1H NMR by three methods; (i) online NMR, (ii) periodic
inversion, and (iii) a static NMR tube. This heterogeneous reaction
clearly displays the effect of efficient mixing on reaction progres-
sion. All three experiments were conducted at the same concentra-
tion (50 mM). The results of these experiments are displayed in
Fig. 1. Characteristic resonances outlined by Gschwind et al. were
also tracked as reaction monitoring handles.[16a] Because of overlap
of the aldehyde protons related to 1 and 2a at δH 9.65 ppm, these
two components were tracked together. Intermediate 4b was
monitored using the resonance at δH 5.06ppm and the aldol
condensation product 3 at δH 9.34 ppm. It can be clearly seen from
the reaction profiles that the time to reaction completion is very
different when adequate mixing is applied. Intermediate 4b, which
was correlated with the rate determining step in Zeitler and
Gschwind’s study, forms at a much faster rate when agitation is
Three reaction types were evaluated in this study. The L-proline-
catalyzed self-condensation of propionaldehyde was examined to
compare different NMR methods of observing heterogeneous reac-
tion progression. The homogenous coupling reaction of aniline and
Scheme 1. (A) Aldol addition to form 2a/2b indicated a competitive process with the desired aldol condensation to 3. (B) Double-activation Mannich-type
pathway indicated in the mechanistic study by Zeitler and Gschwind. Species in brackets could not be directly detected by NMR.
wileyonlinelibrary.com/journal/mrc
Copyright © 2015 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2016, 54, 451–456

Online vs tube NMR: a kinetic evaluation of NMR reaction monitoring approaches
Figure 1. Study of the L-proline-catalyzed self-condensation of propionaldehyde 1 in dimethyl sulfoxide at 27 °C as observed by (A) online NMR, symbol ×;
(B) NMR tube with periodic inversion, symbol □; (C) static NMR tube, symbol ●. All methods have initial 1 and Pro concentrations of 50.0 mM.
applied (Fig. 1A and B). When the reaction is diffusion controlled, as
for the static NMR tube (Fig. 1C), there is much less 4b observed,
corresponding to the much slower rate of product 3 formation.
Mol% was calculated by setting all propionaldehyde methyl signals
and the methylene signal from the one intermediate without a dis-
tinct methyl (4d) from the first spectrum to 100%.
Figure 2 displays an overlay of the rate of product formation from
all three methods (online NMR, static NMR tube, and periodic inver-
sion) on a single plot, demonstrating clearly that rate of formation
of product is dependent on amount of mixing. For clarity, minor
components involved in the reaction mechanism (i.e. 2b, 4b, 4c,
and 4d) are not shown; however, these were included in the mass
balance calculation and conversion to mol%. The method with con-
tinuous mixing, online NMR, has the fastest growth of product. The
method with an intermediate amount of mixing, periodic inversion
– where there are some periods of mixing and some periods of no
mixing – shows an intermediate rate of product growth. The NMR
tube reaction, which is entirely under diffusion control, displays
by far the slowest rate of product growth, clearly demonstrating
that any kinetics derived from the reaction conditions are highly
dependent upon the amount of mixing in the system and do not
necessarily display the true kinetics of the reaction under investiga-
tion. In addition, variability in the rates derived from the NMR tube
monitoring was observed, likely because of the inability to control
how much catalyst stuck to the walls of the NMR tube upon the ini-
tial mixing of reagents.
Homogenous reaction
The second reaction investigated was the homogeneous coupling
reaction of aniline 6 and 4-fluorobenzaldehyde 7 to produce the
corresponding imine at 25°C (Scheme 2). This reaction was chosen
for the investigation as a comparative study to the heterogeneous
case, outlined in the previous section. The reaction kinetics were
found to be relatively slow, and 19 F NMR spectroscopy could be
used to monitor the progress of the reaction.
The results from this experiment are outlined in Fig. 3. There is a
less pronounced difference between the kinetic results obtained
(online vs tube) when monitoring the homogeneous reaction as
compared with the heterogeneous reaction. Mol% was calculated
by setting the total amount of 4-fluorobenzaldehyde 7 and imine
8 detected by 19 F NMR at each time point to 100% (at -105.2 and
À110.2 ppm, respectively). The method with the most mixing
(online NMR) has decidedly the quickest conversion to product.
Periodic inversion of the NMR tube does not increase the rate of re-
action as compared with the static NMR tube, showing that for this
Figure 2. Overlay of product formation profiles for all methods used to
study the L-proline-catalyzed self-condensation of propionaldehyde 1 to
form 3 in dimethyl sulfoxide at 27 °C.
Scheme 2. The coupling reaction of aniline 6 and 4-fluorobenzaldehyde 7 to form imine 8 in 1 : 1 methanol: acetonitrile at 25 °C.
Magn. Reson. Chem. 2016, 54, 451–456
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

D. A. Foley, A. L. Dunn and M. T. Zell
efficient stirring is applied in the reaction vessel in the case of the
experiment monitored by online NMR spectroscopy, while
diffusion-controlled kinetics have a greater effect on the result
obtained from the static NMR tube experiment.
There are positive and negative aspects to each reaction
monitoring technique that must be evaluated when designing an
experimental procedure. Table 1 summarizes some of these consid-
erations. Online NMR monitoring allows the closest replication to a
non-monitored experiment, achieving control over parameters
such as temperature, rate of mixing, and rate of addition of re-
agents, which is key to developing a chemical reaction process.
There are some limitations associated with monitoring a reaction
online because of the distance between the reaction vessel and
the detection cell, which can influence sample transfer time and
temperature gradients. Details of the online NMR spectroscopy sys-
tem used in this study have been published previously,[14] and a
number of reports of the characteristics of online NMR systems
have provided in-depth analysis of the considerations when
conducting this type of analysis.[5,6b,17] Online NMR also allows for
the introduction of other analytical instruments in-line, but requires
specialized equipment that may not be available to all users.
Conducting a reaction in an NMR tube requires only basic equip-
ment, but at the cost of sacrificing efficient mixing during the reac-
tion. Temperature gradients are likely between the top and bottom
of the NMR tube, especially at elevated temperatures, also evapora-
tion and condensation of volatile solvents could be experienced,
which could result in concentration gradients or changes because
of solvent loss. These effects will influence the kinetic results obtained.
The periodic inversion method also requires only basic equip-
ment; however, it has the major drawback of the operator having
to physically invert the NMR tube between each spectrum for the
entirety of the reaction. Acquiring data points in this way can also
lead to brief loss of temperature control of the sample, which can
affect the results obtained. Taking aliquots from a stirred reaction
vessel should give similar results to a non-monitored experiment,
but again requires the operator to be present at each time point. Al-
iquots of the L-proline-catalyzed aldol condensation were also
taken, but upon diluting the reaction mixture with deuterated sol-
vent to lock, the sample was far too dilute to get meaningful results.
Also, taking aliquots causes brief loss of temperature control as well
as loss of material.
Figure 3. A summary of all methods used to study the aniline 6 and 4-
fluorobenzaldehyde 7 coupling reaction to form the imine 8 in 1 : 1
methanol: acetonitrile at 25 °C. Online nuclear magnetic resonance (NMR)
(×) initial concentrations were 64 mM each of 6 and 7. Periodic inversion
(□) and NMR tube (●) reactions’ initial concentrations were 69 mM each of
6 and 7.
homogenous reaction, inversion every 6–7 min is not enough to
show improvement upon diffusion-limited mixing. Regardless, it is
clear that any kinetic rates derived from the static NMR tube would
reflect more upon being diffusion controlled than the actual rates
of reaction.
Homogeneous reaction, fast kinetics
In order to test the effect of mixing on reactions with shorter half-
lives (i.e. on the order of a few minutes), the transesterification of
acetic anhydride 9 and isopropanol 10 to produce acetic acid 11
and isopropyl acetate 12 at 25 °C in acetonitrile was studied
(Scheme 3).
Figure 4 shows that even for reactions with half-lives on the order
of minutes, mixing is still crucial to obtaining accurate rate values.
The periodic inversion results for the transesterification reaction
are not presented because of the reaction completion being too
fast to obtain an appropriate number of data points. Mol% was cal-
culated by setting the total integration of isopropanol 10 methine
(3.9 ppm) and isopropyl acetate 12 methine (4.9 ppm) detected at
each time point to 100%. Because of the residence time character-
istics of the online system used, the early portion of the online data
(<3.2min) cannot be used for kinetic calculations; however, there is
still a clear difference in the reaction completion time between the
two experiments. This difference is again due to the mixing effects,
One must also consider the larger purpose of the NMR monitor-
ing study when deciding upon a monitoring method. If the data
from the monitoring study are to be used in the development of
a chemical process for scale-up, it would be wise to use a monitor-
ing method that facilitates similar reaction conditions to the large-
scale reaction, such as that provided by online NMR.[18] Online NMR
is particularly amenable to tracking flow chemistry processes,
Scheme 3. The sulfuric acid-catalyzed transesterification reaction of acetic
anhydride 9 and isopropanol 10 in acetonitrile at 25 °C.
Table 1. Summary of some important considerations when choosing
an NMR monitoring method
Conditions
Online NMR NMR tube Periodic inversion
Automated sampling
Temperature control
Efficient mixing
✔
✔
✔
✘
✔
✔
✘
✘
✘
✘
Standard equipment
Deuterated solvent
Scalability
✔
✔
✘
✔
✔
✘
Figure 4. A summary of the methods used to study the transesterification
reaction of acetic anhydride 9 and isopropanol 10 in acetonitrile at 25 °C.
Online NMR initial concentrations were 2.5 M each of 9 and 10 and 44 mM
H2SO4. Tube initial concentrations were 2.3 M each of 9 and 10 and 46 mM
H2SO4.
✘
✔
NMR, nuclear magnetic resonance.
wileyonlinelibrary.com/journal/mrc
Copyright © 2015 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2016, 54, 451–456

Online vs tube NMR: a kinetic evaluation of NMR reaction monitoring approaches
L-proline-catalyzed aldol self-condensation of propionaldehyde at 27 °C
where the output stream can be readily monitored in real-time,
allowing quick optimization of experimental conditions. Offline
sampling will not produce accurate results for a flow chemistry pro-
cess as a result of averaged response because of pooling of the
sample stream in the detection cell before acquisition. Accurate
results would require the use of a flow-through NMR cell, rather
than the online NMR tube system used to monitor the reactions
described in the current study, because of similar pooling effects.
Online NMR. L-proline (Pro), (Amresco) (208.9mg, 1.8mmol) was
suspended in 36 ml protio dimethyl sulfoxide (DMSO) in a 50 ml re-
actor, and propionaldehyde 1 (Sigma–Aldrich) (0.13ml, 1.8 mmol)
1
was added in one portion via syringe. H NMR spectra were ac-
quired at intervals of 5 until 200 min, then at 10min intervals with
a 90° pulse angle and a 15 s relaxation delay. The DMSO singlet
was suppressed using the WET (water suppression enhanced
through T1 effects) solvent suppression pulse sequence.[19]
Conclusion
Static NMR tube reaction. L-proline (Pro) (3.7 mg, 0.03 mmol) was
suspended in 0.6 ml DMSO-d6 in an NMR tube. Propionaldehyde
1 (2.2μl, 0.03 mmol) was added in one portion via micropipette,
and the tube was inverted three times. H NMR spectra were ini-
tially acquired at 5 min intervals, then at 10 min intervals. All spectra
were acquired with eight scans and a 15 s relaxation delay.
In conclusion, we present evidence that mixing has a large effect on
the rate of reaction determined by NMR spectroscopy for three
different types of reactions [heterogeneous, homogeneous with
long (>1 h) reaction times, and homogeneous with short (<1 h)
reaction times]. All three reactions studied show conclusive evi-
dence that the NMR monitoring technique can have a significant
effect on reaction rates, providing support for the application of
continuous flow online NMR methods for kinetic studies when
the most accurate results are required. Studies in static NMR tubes
can provide good mechanistic and structural information particu-
larly for labile or reactive intermediates. However, as the results
from this work demonstrate, caution should be applied when rely-
ing on kinetic data acquired from systems lacking adequate mixing.
1
NMR tube with periodic inversion. L-proline (Pro) (3.7mg, 0.03 mmol)
was suspended in 0.6ml DMSO-d6 in an NMR tube.
Propionaldehyde 1 (2.2 μl, 0.03 mmol) was added in one portion
via micropipette. 1H NMR spectra were acquired with eight scans.
Aniline and 4-fluorobenzaldehyde coupling at 25 °C
Online NMR. 4-Fluorobenzaldehyde 7 (Sigma–Aldrich) (0.28 ml,
2.6 mmol) was added to 20 ml protio acetonitrile and 20 ml protio
methanol in a 50 ml reaction vessel. Aniline 6 (Sigma–Aldrich)
(0.24 ml, 2.6 mmol) was added in one portion via micropipette.
19 F{1H}, and 1H NMR spectra were acquired at 5 min intervals.
Experimental
General procedure for online NMR
Static NMR tube reaction. 4-Fluorobenzaldehyde 7 (7μl, 0.07 mmol)
was added to an NMR tube with 0.5ml methanol-d4 and 0.5ml
acetonitrile-d3 via micropipette. Aniline 6 (6μl, 0.07mmol) was
added in one portion via micropipette, and the NMR tube was
inverted three times. 19 F{1H} spectra were acquired at 5 min inter-
vals. 1H NMR spectra were also acquired but not used for analysis.
The online NMR setup was used as previously described with re-
moval of the needle splitting valve.[15] Temperature control was
achieved by setting reaction vessel, sample loop, and spectrometer
to the specified temperature. The flow rate was set to 4 ml/min.[14]
Spectra were acquired on the flowing sample on a Bruker 400 MHz
Avance III NMR (Billerica, MA, USA) equipped with broad band fluo-
rine observation (BBFO) probe. 1H NMR spectra were acquired with
four scans, a 30° pulse angle, and a 10 s relaxation delay, unless oth-
erwise noted. If applicable, 19 F{1H} spectra were acquired with eight
scans, 90° pulse angle, and 30 s relaxation delay.
NMR tube with periodic inversion. 4-Fluorobenzaldehyde 7 (7μl,
0.07 mmol) was added to an NMR tube with 0.5ml methanol-d4
and 0.5ml acetonitrile-d3 via micropipette. Aniline 6 (6μl,
0.07 mmol) was added in one portion via micropipette. 19 F{1H}
spectra were acquired for analysis.
General procedure for static NMR tube reactions
Isopropanol and acetic anhydride transesterification at 25 °C
Reagents were added to a 5 mm NMR tube, inverted three times, and
inserted into spectrometer that began taking spectra immediately.
Spectra were acquired on a Bruker 400 MHz Avance III NMR equipped
with BBFO probe. The temperature of the sample was controlled
using the variable temperature heater of the probe. 1H NMR spectra
were acquired with four scans, a 30° pulse angle, and a 10 s relaxation
delay, unless otherwise noted. If applicable, 19 F{1H} spectra were ac-
quired with eight scans, a 90° pulse angle, and a 30 s relaxation delay.
Online NMR. Acetic anhydride 9 (Sigma–Aldrich) (10 ml, 106 mmol)
and isopropanol 10 (Sigma–Aldrich) (8.1ml, 106 mmol) were dis-
solved in 25ml protio acetonitrile in a 50ml reaction vessel. Con-
centrated sulfuric acid (J.T. Baker) (100 μl, 1.9mmol) was added in
one portion via syringe. 1H NMR spectra were acquired in 60 s
intervals.
Static NMR tube reaction. Acetic anhydride 9 (0.2ml, 2 mmol),
isopropanol 10 (0.16 ml, 2 mmol), and acetonitrile-d3 (0.5 ml) were
added to an NMR tube. Concentrated sulfuric acid (2μl, 0.04 mmol)
was added in one portion via syringe. 1H NMR spectra were
acquired at 60 s intervals.
General procedure for NMR tube reactions with periodic inversion
Reagents were added to a 5 mm NMR tube, inverted three times,
and inserted into spectrometer where initial spectrum was
acquired. Prior to each subsequent spectrum, the NMR tube was
removed from the spectrometer and inverted three times before
inserting back into the spectrometer. Spectra were acquired on a
Bruker 400 MHz Avance III with BBFO probe. The temperature of
the sample was controlled by the variable temperature heater of
the probe. 1H NMR spectra were acquired with four scans, 30° pulse
angle, and 10s relaxation delay, unless otherwise noted. If applica-
ble, 19 F{1H} spectra were acquired with eight scans, 90° pulse angle,
and 30 s relaxation delay.
References
[1] F. Susanne, D. S. Smith, A. Codina. Org. Process Res. Dev. 2012, 16,
61–64.
[2] Y. Yokoyama, M. Nakakoshi, H. Okuno, Y. Sakamoto, S. Sakurai. Magn.
Reson. Chem. 2010, 48, 811–817.
[3] T. Bartik, B. Bartik, B. E. Hanson, T. Glass, W. Bebout. Inorg. Chem. 1992,
31, 2667–2670.
Magn. Reson. Chem. 2016, 54, 451–456
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

D. A. Foley, A. L. Dunn and M. T. Zell
[4] T. R. Hoye, B. M. Eklov, T. D. Ryba, M. Voloshin, L. J. Yao. Org. Lett. 2004,
6, 953–956.
[5] M. Maiwald, H. H. Fischer, Y.-K. Kim, K. Albert, H. Hasse. J. Magn. Reson.
2004, 166, 135–146.
3492–3493; (c) J. F. McGarrity, J. Prodolliet. J. Org. Chem. 1984, 49,
4465–4470; (d) J. F. McGarrity, J. Prodolliet, T. Smyth. Org. Magn.
Reson. 1981, 17, 59–65.
[12] (a) N. M. Do, M. A. Olivier, J. J. Salisbury, C. B. Wager. Anal. Chem. 2011,
83, 8766–8771; (b) D. H. Pieper, K. Pollmann, P. Nikodem, B. Gonzalez,
V. Wray. J. Bacteriol. 2002, 184, 1466–1470; (c) J. Bures, A. Armstrong,
D. G. Blackmond. J. Am. Chem. Soc. 2011, 133, 8822–8825; (d)
I. M. Clegg, C. M. Gordon, D. S. Smith, R. Alzaga, A. Codina. Anal.
Methods 2012, 4, 1498–1506; (e) C. J. Morten, J. A. Byers,
T. F. Jamison. J. Am. Chem. Soc. 2011, 133, 1902–1908.
[13] D. G. Blackmond. Angew. Chem. Int. Ed. 2005, 44, 4302–4320.
[14] D. A. Foley, E. Bez, A. Codina, K. L. Colson, M. Fey, R. Krull, D. Piroli,
M. T. Zell, B. L. Marquez. Anal. Chem. 2014, 86, 12008–12013.
[15] D. A. Foley, J. Wang, B. Maranzano, M. T. Zell, B. L. Marquez, Y. Xiang,
G. L. Reid. Anal. Chem. 2013, 85, 8928–8932.
[16] (a) M. B. Schmid, K. Zeitler, R. M. Gschwind. J. Org. Chem. 2011, 76,
3005–3015; (b) M. B. Schmid, K. Zeitler, R. M. Gschwind. Angew. Chem.
Int. Ed. 2010, 49, 4997–5003.
[17] F. Dalitz, M. Maiwald, G. Guthausen. Chem. Eng. Sci. 2012, 75,
318–326.
[6] (a) M. Maiwald, H. H. Fischer, Y.-K. Kim, H. Hasse. Anal. Bioanal. Chem.
2003, 375, 1111–1115; (b) F. Dalitz, M. Cudaj, M. Maiwald,
G. Guthausen. Prog. Nucl. Magn. Reson. Spectrosc. 2012, 60, 52–70; (c)
F. Dalitz, L. Kreckel, M. Maiwald, G. Guthausen. Appl. Magn. Reson.
2014, 45, 411–425; (d) A. C. Barrios Sosa, R. T. Williamson, R. Conway,
A. Shankar, R. Sumpter, T. Cleary. Org. Process Res. Dev. 2011, 15, 449–454;
(e) D. A. Foley, C. W. Doecke, J. Y. Buser, J. M. Merritt, L. Murphy,
M. Kissane, S. G. Collins, A. R. Maguire, A. Kaerner. J. Org. Chem..
2011, 76, 9630–9640; (f) M. A. Bernstein, M. Stefinovic, C. J. Sleigh.
Magn. Reson. Chem. 2007, 45, 564–571.
[7] (a) P. V. Yushmanov, I. Furo. J. Magn. Reson. 2005, 175, 264–270; (b)
M. D. Christianson, E. H. P. Tan, C. R. Landis. J. Am. Chem. Soc. 2010,
132, 11461–11463.
[8] C. Frieden, S. D. Hoeltzli, I. J. Ropson. Protein Sci. 1993, 2, 2007–2014.
[9] (a) M. D. Christianson, C. R. Landis. Concepts Magn. Reson., Part A 2007,
30A, 165–183; (b) W. A. McGee, L. J. Parkhurst. Anal. Biochem. 1990, 189,
267–273; (c) R. O. Kuehne, T. Schaffhauser, A. Wokaun, R. R. Ernst.
J. Magn. Reson. 1979, 35, 39–67.
[18] (a) J. M. Merritt, J. Y. Buser, A. N. Campbell, J. W. Fennell, N. J. Kallman,
T. M. Koenig, H. Moursy, M. A. Pietz, N. Scully, U. K. Singh. Org. Process
Res. Dev. 2014, 18, 246–256; (b) A. C. Barrios Sosa, R. Conway,
R. T. Williamson, J. P. Suchy, W. Edwards, T. Cleary. Org. Process Res.
Dev. 2011, 15, 1458–1463.
[10] S. E. Denmark, B. J. Williams, B. M. Eklov, S. M. Pham, G. L. Beutner. J. Org.
Chem. 2010, 75, 5558–5572.
[11] (a) A. C. Jones, A. W. Sanders, W. H. Sikorski, K. L. Jansen, H. J. Reich.
J. Am. Chem. Soc. 2008, 130, 6060–6061; (b) A. C. Jones,
A. W. Sanders, M. J. Bevan, H. J. Reich. J. Am. Chem. Soc. 2007, 129,
[19] S. H. Smallcombe, S. L. Patt, P. A. Keifer. J. Magn. Reson., Ser. A 1995, 117,
295–303.
wileyonlinelibrary.com/journal/mrc
Copyright © 2015 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2016, 54, 451–456