Selective Nuclear Magnetic Resonance Experiments for Sign-Sensitive Determination of Heteronuclear Couplings: Expanding the Analysis of Crude Reaction Mixtures

Article abstract of DOI:10.1021/acs.analchem.0c02976

State-of-the-art nuclear magnetic resonance (NMR) selective experiments are capable of directly analyzing crude reaction mixtures. A new experiment named HD-HAPPY-FESTA yields ultrahigh-resolution total correlation subspectra, which are suitable for sign-sensitive determination of heteronuclear couplings, as demonstrated here by measuring the sign and magnitude for proton-fluorine couplings (JHF) from major and minor isomer products of a two-step reaction without any purification. Proton-fluorine couplings ranging from 51.5 to -2.6 Hz could be measured using HD-HAPPY-FESTA, with the smallest measured magnitude of 0.8 Hz. Experimental JHF values were used to identify the two fluoroketone intermediates and the four fluoroalcohol products. Results were rationalized and compared with the density functional theory (DFT) calculations. Experimental data were further compared with the couplings reported in the literature, where pure samples were analyzed.

Full text of DOI:10.1021/acs.analchem.0c02976

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Article
Selective NMR experiments for sign-sensitive determination of
heteronuclear couplings: expanding the analysis of crude reaction mixtures
Guilherme Dal Poggetto, João Vitor Soares, and Cláudio Francisco Tormena
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.0c02976 • Publication Date (Web): 14 Sep 2020
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Selective NMR experiments for sign-sensitive determination of
heteronuclear couplings: expanding the analysis of crude reaction
mixtures
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Guilherme Dal Poggetto,* João Vitor Soares and Cláudio F. Tormena
Institute of Chemistry, University of Campinas (UNICAMP), PO BOX 6154, Campinas (SP), CEP 13083-970, Brazil.
*Email: dalpgt@unicamp.br
ABSTRACT: State-of-the-art nuclear magnetic resonance (NMR) selective experiments are capable of direct analysis of crude
reaction mixtures. A new experiment named HD-HAPPY-FESTA yields ultrahigh-resolution total correlation subspectra, which is
suitable for the sign-sensitive determination of heteronuclear couplings,
as demonstrated here by measuring sign and magnitude for proton-
fluorine couplings (JHF) from major and minor isomer products of a two-
step reaction without any purification. Proton-fluorine couplings ranging
from 51.5 to -2.6 Hz could be measured using HD-HAPPY-FESTA, with
the smallest measured magnitude of 0.8 Hz. Experimental JHF values
were used to identify the two fluoroketone intermediates and the four
fluoroalcohol products. Results were rationalized and compared with
density functional theory (DFT) calculation. Experimental data were
further compared with the couplings reported in the literature, where
pure samples were analysed.
overlap.16 Although 19F NMR is used successfully for mixture
analysis,17-20 Fluorine spectra are information poor, unlike H
spectra, and therefore are not normally used for characterisation
but to probe various interactions,2,20-23 commonly by observing
changes in signal chemical shift and relaxation. One way to take
advantage of the spectral sparsity of 19F to unambiguously
assign the atomic connectivity, while still observing the more
informational 1H spectra, is by using 1D Fluorine Edited
Selective TOCSY Acquisition (FESTA).24,25 In FESTA, only
1H signals that are in a spin system coupled to a selected fluorine
nucleus are observed through magnetization transfer, i.e. TOtal
Correlation SpectroscopY (TOCSY)26. Various 2D NMR
experiments, such as hetero-COSY,27 HSQC-TOCSY,28,29 and
more,30,31 also give useful molecular knowledge for fluorinated
species, but these usually have long experiment duration and
are limited for high-concentration-dynamic-range mixtures,14
as opposed to 1D selective NMR experiments,32-34 which can be
used to observe signals coming from low concentration
components of complex matrixes. For the FESTA family of
1
INTRODUCTION
The incorporation of even a single fluorine atom in a
molecule can affect its physical and chemical properties.1,2 In
drugs, fluorination has shown to improve metabolic stability
and membrane permeation, and to increase binding affinity. The
several examples of fluorinated compound synthesis in the
literature 3-6 reflect the ever-growing interest in the discovery of
new fluorinated drug candidates by the pharmaceutical
industry.7,8 Solution-state nuclear magnetic resonance (NMR)
is arguably the most useful non-destructive spectroscopic
technique for the analysis and characterisation of chemical
reaction products. Typically, these products are purified by
physical separation methods (e.g. chromatography,
recrystallization) in many manual steps previous to NMR
analysis.9 These time-consuming, resource-intensive, and often
tedious practices can sometimes be avoided by analysing the
intact reaction mixture – either to bypass the necessity of
difficult component separation or by having an extra source of
information previous to isolation. Mixture analysis by NMR has
1
experiments, all H signals observed belong to the same spin
system connected to a selected 19F nucleus. It is thus possible to
obtain all 1H chemical shifts (H), homonuclear 1H-1H (JHH) and
heteronuclear 1H-19F (JHF) couplings, given that signals are well
resolved. Fluorine decoupling can declutter the partial spectra
even further, however, there are still difficulties for using
FESTA to measure JHF, which are abundant in structural and
conformational information.35 These couplings are frequently
used as a diagnostic tool for full characterisation.29,36 In contrast
with 2JHH, germinal 2JHF are large and positive. Vicinal 3JHF can
be both positive or negative, with a larger span of magnitudes
been used successfully in the past for the analysis of natural
15
products,10,11 beverages,12,13 pharmaceutical formulations,14
among others, yet it is still a cumbersome task.
,
Chemical and structural insight is typically extracted from
1H NMR spectra, in the form of chemical shifts () and scalar
couplings (J), but for complex mixtures, this information is
severely obscured by signal overlap. It is also possible to obtain
19F NMR spectra (when this is available in a molecule), which
has a much larger frequency dispersion (~500 ppm) in
1
comparison with H spectra (~10 ppm) and far rarer signal
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n
compared to JHH. Longer-range JHF are also quite common.
Frequently, the magnitude of JHH and JHF are similar within the
multiplet structure, making them hard to be distinguished from
one another. Suppressing the effects of homonuclear couplings
can be a very effective way of measuring the magnitude of
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heteronuclear couplings.37-39 This can be achieved with
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“ultrahigh-resolution”
pure shift NMR methods,43-45 in
which heteronuclear couplings are left unaffected.
Unfortunately, pure shift methods alone are hopeless to analyse
but the simplest mixtures, as most complex mixtures have wall-
to-wall peaks. Recently, an experiment combining Selective
Spin Eco (SSE)-TOCSY with PSYCHE (Pure Shift Yielded by
CHirp Excitation)40 was proposed,10 allowing the analysis of
high-dynamic-range reaction mixtures. This method simplifies
1H spectra by only observing subspectra from 1H spin systems
of interest (as in FESTA), and uses pure shift NMR to remove
the effects of JHH. As useful these may be, neither FESTA nor
SSE-TOCSY-PSYCHE is sensitive to the sign of couplings.
Only when both magnitude and sign are available this property
can be used to determine relative configuration above any
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doubt.36 For JHF, sign and magnitude are also crucial to
understanding electronic interactions behind scalar coupling
transmission mechanisms.46,47
Here we propose a combination of homonuclear decoupling
with a modified MODulated echO (MODO)-FESTA 25 for the
Figure 1. 500 MHz (a) conventional, (b) PSYCHE and (c-f) HD-
n
sign-sensitive determination of heteronuclear JHF in complex
HAPPY-FESTA (isotropic mixing time of 150 ms) 1H spectra of a
mixtures, adding a powerful tool to the FESTA family of
experiments. This new method, named HD-HAPPY-FESTA
(Homonuclear Decoupled Heteronuclear Anti-Phase Permuted
modulated echo Yielding Fluorine Edited Selective TOCSY
Acquisition), was used to obtain all relevant JHF, with ease and
high precision, for the two-step reaction (Scheme 1) of the
formation of fluorinated alcohol isomers with no purification
previous to NMR analysis. While conventional (Figure 1a) and
pure shift (Figure 1b) 1H spectra completely fail for molecular
identification, very clean spectra are obtained for each species
using HD-HAPPY-FESTA (Figure 1c-g). Experimental
coupling values were compared with the widely used quantum
mechanics density functional theory (DFT) coupling constant
calculations.48-51 Calculated NMR parameters (J and ) are
routinely applied to support synthetic and natural products
structure assignments.52-55 In our study, DFT was used to
support the experimental data by calculating the sign and
magnitude of all possible nJHF.
crude reaction mixture from the reduction of ketones 2a and 2b, in
CDCl3.
Ultrahigh-resolution
sign-sensitive
anti-phase
heteronuclear total correlation spectra of alcohols: (c) 3a; (d) 3b;
(e) 3c; and (f) 3d, respectively. Molecular structures are shown in
Scheme 1. (g) is the expansion of (f) between 1.2-2.1 ppm.
Lightning bolts in (c-f) indicate chemical shifts of band selective
1H pulses, selected using 24.4 ms REBURP pulses (1H bandwidth
of 200 Hz). A flip angle (β) of 24° and 80 data chunks of 12.5 ms
duration were used in (b-g). 32 transients were acquired in (a-e),
and 128 in (f-g), with maximum receiver gain in each experiment.
Complete experimental parameters can be found in the SI.
commercially available from Aldrich and were used without
further purification. Scheme 1 describes the two-step reaction
used to prepare all fluorinated molecules, following the
synthetic procedure described by Anizelli et. al.46
Commercially available 4-tert-butyl-cyclohexanone (1) was
fluorinated in the α-ketone position using Selectfluor, which
formed the 4-tert-butyl-2-fluoro-cyclohexanone isomers (2a
and 2b). 92 mg of the crude reaction product of the α-
fluorination was dissolved in 600μL of Acetone-d6 and analysed
by NMR without further purification. The remaining of the
crude reaction was used as a starting material for the second
EXPERIMENTAL SECTION
Sample preparation. All reactants and solvents were
Scheme 1. Reactions described in this paper. The first step describes the experimental conditions for the α-fluorination reaction of 4-tert-
butyl-cyclohexanone (1), generating both fluorinated ketones isomers, 2a and 2b, at a ratio of 4.2 /1.0, respectively. The second step describes
the condition for the ketone reduction from the crude reaction of the first step, generating simultaneously all four fluorinated alcohols isomers,
3a, 3b, 3c, and 3d, at a ratio of 9.1/4.8/2.8/1.0, respectively. The relative proportion of the products in each reaction step were measured by
integration of the signals in the 1H decoupled 19F spectra (Fig. 3).
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Figure 2. Pulse sequence for the HD-HAPPY-FESTA (Homonuclear Decoupled Heteronuclear Anti-Phase Permuted modulated echo
Yielding Fluorine Edited Selective TOCSY acquisition). The orange rectangle highlights the selective pMODO (permuted MODulated echO)
block, purple rectangle highlights the zTOCSY block and the green rectangle the PSYCHE (Pure Shift Yielded by CHirp Excitation) block.
Black narrow and white wide rectangles represent hard 90° and 180° pulses, respectively. Trapezoids with cross-diagonal arrows are low
power chirp pulses of small flip angle (β).40 Trapezoids on either side of the DIPSI-2 isotropic mixing element 56 are low power 180° chirp
pulses used to suppress zero-quantum coherences. 57 Selective (soft) 180° pulses, represented by shaped pulses, are applied at the resonance
frequency of coupled 1H and 19F. Typically, RSNOB or REBURP shapes are used for 1H refocus, and IBURP2 for 19F inversion.58,59 Δ is set
as 1/(4 × nF × JHF), where for JHF is the coupling between the 1H and 19F selected by the 180° soft pulses and nF is the number of equivalent
19F selected. A detailed description of the pulse sequence can be found in the SI. Removing the PSYCHE block reduces the dimensionality
and increases the sensitivity of the experiment, and homonuclear coupling is observed (i.e. HAPPY-FESTA).
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step. NaBH4 was used for ketone reduction generating all four
4-tert-butyl-2-fluoro-cyclohexanol isomers (3a, 3b, 3c, and 3d)
at once. 209 mg of the crude reaction of the ketone reduction
was dissolved in 600μL of CDCl3 and analysed by NMR
without further purification. § Detailed reaction conditions are
fully described in the Support Information (SI).
The NMR pulse sequence for HD-HAPPY-FESTA (Figure
2) is a modification of the conventional MODO-FESTA.25
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Selective pulses on both coupled H and 19F are used in a
selectively modulated echo, which generates heteronuclear
anti-phase signals only for the selected spins while refocussing
the evolution of all other scalar couplings (see SI Fig. S3b).
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Following the initial preparation period, a hard 90°y H pulse
Data acquisition and processing. All spectra were
recorded at 298K on an 11.4 T Bruker Avance III spectrometer
equipped with a 5mm BBFO smart probe, a QNP switch and a
z-gradient coil with a maximum nominal gradient strength of 53
aided by a
zero-quantum filter (ZQF)57,66 suppression,
eliminates all signals except the anti-phase component for the
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selected H-19F pair. Using DIPSI-2 as isotropic mixing (i.e.
TOCSY),56 the selected anti-phase magnetization is transferred
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G cm-1, operating at 499.87 MHz and 470.35 MHz for H and
to all other H spins that are part of the same spin system
19F, respectively. All data were processed using the software
TOPSPIN (version 3.5 pl7, Bruker Biospin). HAPPY-FESTA
experiment duration was of approximately 5 min, for 2 seconds
of relaxation delay (d1) and 32 transients. HD-HAPPY-FESTA
experiment duration was of approximately 2h 15 min, for 2
seconds of relaxation delay (d1), 32 transients and 80 chunks.
To avoid FID truncation, the number of pure shift chunks
collected were beyond what is needed for a resolution to
measure couplings with a precision better than 0.1 Hz. Peak
linewidth will ultimately determine the lower limit in J
measurement. The spectra showed here (and in the SI) were
processed with a Lorentz-to-Gauss window function and 128k
points, resulting in a spectral resolution of 0.03 Hz per point
after FT. All raw data, AU macros and pulse sequence programs
network (i.e. an unbroken chain of couplings), generating
subspectra with heteronuclear anti-phase to all H signals in
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respect to that selected fluorine (see SI Fig. S3c). As the sense
of heteronuclear anti-phase is preserved for each signal, the
positive/negative sign of each JHF is directly observable and are
extracted from their relative slope. Lastly, homonuclear
decoupling is achieved using PSYCHE (see SI Fig. S3d).40 As
the resulting spectra are composed by (normally) heteronuclear
anti-phase doublets, it is less adequate for these spectra to be
described as “pure (chemical) shift”, hence these will be
referred to as “homonuclear decoupled” spectra. In principle,
other pure shift methods could be used for broadband
homonuclear decoupling,67-69 however PSYCHE generally
gives the best sensitivity, spectral purity, and it is the simplest
pure shift method for set-up and automation.
used
in
this
paper
are
available
at
https://doi.org/10.25824/redu/LNVQT9 free of charge.
Detailed acquisition and processing parameters are described
extensively in the SI (see Fig. S1).
The modified MODO, described here as “permuted
MODO” (pMODO), differs from the conventional MODO in
three aspects: i) the coupling evolution periods (Δ, see Fig. 2)
used here are optimized to maximize anti-phase, as opposed to
in-phase, contribution; ii) pMODO uses phase cycling of the
hard 90°y 1H pulse to reinforce the selected coherence transfer
pathways (CTP). In conventional MODO, this selection is
achieved with differential experiments,70 which does not apply
to anti-phase signal selection; iii) While in MODO only a single
19F pulse is used, in pMODO two 19F selective inversion pulses
are used: a standard and a time-reversed pulse (see Fig. 2).
Partial JHF evolution during the first inversion pulse is
refocussed during its time-reversed pair,71 which allows the use
of more selective (and longer) 19F pulses. In conventional
DFT calculations. All calculations were performed using
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the software Gaussian 16
applying B3LYP hybrid
functional.61-63 The basis set used for geometry optimizations
was the aug-cc-pVTZ,64 and for the spin-spin couplings
constants, the aug-cc-pVTZ-J.65 Computational details and
Cartesian geometries are available in the SI.
RESULTS AND DISCUSSION
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MODO the duration of selective 19F pulses is restricted to the
duration of Δ. The narrower a pulse bandwidth the longer its
duration, which causes extra loss of magnetization due to
transverse relaxation (T2), and yet it allows the analysis of
cognate structures, such as position isomers, where 19F signals
may not be much dispersed.
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The products of the α-fluorination of ketone 1 were
analysed to test the new methodology. The 19F spectrum for this
mixture (Figure 3a) gives signals which can be used to measure
the relative quantities of each formed isomer (ketones 2a or 2b,
the structural motif is shown in Scheme 2), but it is not very
informative about their identity. The many superimposed
signals in the conventional and pure shift 1H spectra (Figure 4a
and 4b, respectively) prevent characterization – even for this
relatively simple mixture, containing mostly products signals.
Two deshielded 1H signals, at chemical shifts of 5.12 and 4.68
ppm, each with a JHF of approximately 50 Hz (as typical for
2JHF), can be attributed as the proton germinal to the fluorine of
each ketone, making these signals ideal for HD-HAPPY-
FESTA. As for best set-up practice, the less sensitive Selective
Reverse INEPT (SRI)24,70 can be first used to find exactly the
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Scheme 2. Numbered structural motifs of fluoroketones (left) and
fluoroalcohols (right).
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chemical shift of a JHF proton signal. This was not necessary
here. In the first homonuclear decoupled heteronuclear anti-
phase 1H subspectrum (Figure 4c) three relatively large
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couplings were measured in the aliphatic H spectral region:
9.6, 6.7 and 5.4 Hz. Similarly, in the second subspectrum
(Figure 4d), the three couplings measured were: 41.9, 13.9 and
5.1 Hz. All signals were determined to be positive, as all of them
had the same phase as the 2JHF proton signal, which is typically
largely positive (+48.5 and +50.7 Hz for Fig. 4c and 4d,
respectively). The coupling of 41.9 Hz was attributed as the
vicinal diaxial 3JHF of H3ax in ketone 2b, identifying ketone 2a
from the subspectrum of Fig. 4c, and therefore ketone 2b from
Fig. 4b. Vicinal 3JHF values follow similar rules as observed for
vicinal 3JHH in six-membered ring systems.72,73 It is not unusual
though to observe larger magnitudes for couplings involving
fluorine since it is a very electron-rich atom, favouring coupling
transmission mechanisms.47,74,75 Experimental and theoretical
coupling constants are in good agreement (see SI section C) for
both signs and magnitudes. The comparison between
experimental and theoretical data could have been used to
obtain complementary structural information, easily
differentiating between 2a and 2b, for example. While in ketone
Figure 3. 470 MHz 19F spectra of the crude reaction mixture from:
a) the α-fluorination of ketone 1, in Acetone-d6; and b) the
reduction of ketones 2a and 2b, in CDCl3.
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2a the JHF between the axial H4 and equatorial F is
approximately -1.8 Hz, in ketone 2b, this coupling (with the
axial F) is of +1.0 Hz, agreeing with the DFT calculated
couplings: -1.8 and +1.4 Hz, respectively. This demonstrates
how useful for structural characterization the analysis of JHF can
be, even in positions distant to the probed fluorine, and
highlights the importance to determine the sign of coupling
constants.
In Figure 5, HAPPY-FESTA and its homonuclear
decoupled version are compared for the spectra of ketone 2b.
One important aspect to be noted is that the homonuclear
multiplet structure is the main source of signal overlap in the
heteronuclear anti-phase subspectrum, as can be seen for the
signals between 2.30 and 2.45 ppm in Fig. 5b and 5c. It is also
obvious that the Signal-to-Noise Ratio (SNR) is severely
reduced by the suppression of JHH (about 100 times smaller per
time unit), as it would for any broadband pure shift NMR
method. Despite that, the SNR penalty does not constrict signal
identification. Signal amplitude is limited by short T2s and very
Figure 4. 500 MHz (a) conventional, (b) PSYCHE and (c-d) HD-
HAPPY-FESTA (isotropic mixing time of 150 ms) 1H spectra of a
crude reaction mixture from the α-fluorination of 1, in Acetone-d6.
Ultrahigh-resolution sign-sensitive anti-phase heteronuclear total
correlation spectra of ketones: (c) 2a; and (d) 2b, respectively.
Molecular structures are shown in Scheme 1. Lightning bolts in (c-
1
d) indicate chemical shifts of band selective H pulses, selected
using 9.25 ms RSNOB pulses (1H bandwidth of 200 Hz). A flip
angle (β) of 22° and 80 data chunks of 12.5 ms duration were used
in (b-g). 32 transients were acquired in (a-d), with maximum
receiver gain in each experiment. Complete experimental
parameters can be found in the SI.
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3d, leaving two isomers unidentified. Here is where DFT results
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can fully demonstrate support for spectra interpretation.
Comparison between experimental and theoretical results led to
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the attribution of the JHF of 12.1 and 7.7 Hz to the species 3a
and 3b respectively. The calculated values by DFT were of 14.0
and 8.3 Hz. As described for the fluoroketones, the sign of 4JHF
with H4 could be used here to differentiate between axial (3a-
b) from equatorial (3c-d) fluorine cyclohexanols. DFT
calculations showed that this coupling is negative when the 19
F
is at the equatorial position and positive when 19F is at the axial
position, for the compounds studied here. Traditional 2D NMR
experiments were not used here as correlations from minor
dilute components are overshadowed by strong signals of major
components, either because of truncation and/or sideband
artefacts or due to overlap, and are not adequate for these sort
of sample complexity.
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Experimental data were further compared with the literature
(see SI Tables S9-S14),46,76 where purified compounds were
studied. Most JHF are omitted in these accounts because they are
hard to identify even in pure samples, particularly the long-
range couplings with small magnitude. On top of that, most 1H
chemical shifts are reported as ranges due to signal
superposition, which is circumvented here with the use of
homonuclear decoupling. As discussed before, 1H signals with
no (or relatively small) JHF that are part of the selected spin
system are not observable by HAPPY-FESTA, therefore their
chemical shifts must be extracted from in-phase FESTA
experiments. SSE-TOCSY-PSYCHE could also have been used
Figure 5. Expansion of 500 MHz (a) conventional (expansion of
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Fig. 4a), (b) HAPPY-FESTA and (c) HD-HAPPY-FESTA
(expansion of Fig 4d) 1H spectra of a crude reaction mixture from
the α-fluorination of 4-tert-butyl-cyclohexanone, in Acetone-d6.
Ketone 2b is been observed selectively in (b-c).
1
to complement the analysis, but H signals not coupled to the
small JHF (i.e. if signal linewidth is large compared to the
magnitude of the coupling, positive and negative edges of the
anti-phase signal get cancelled, reducing signal intensity), while
the furthest heteronuclear coupling measurable is limited by the
proton- proton magnetization transfer during the TOCSY block.
selected 19F will potentially cause extra complications if signals
are still superimposed after homonuclear decoupling (see SI
Fig. S6).
Other approaches, on the top of the one described here, can
be used for recording FESTA. Selective 19F pulses can be
replaced by hard 180° 19F pulses, at the cost of spectral purity,
when selected 1H signals are isolated and low SNR is an issue
due to transverse relaxation. Another approach for FESTA is
replacing MODO by a Selective Reverse INEPT (SRI)24,70
block to provide anti-phase coherence selection. SNR is
reduced up to two-fold in SRI in comparison to MODO (see SI
Fig. S4), although spectral purity is often improved. For
inspecting very dilute components, the extra sensitivity is
welcomed to be sacrificed by the homonuclear decoupling.
There are other alternatives described in the literature for the
sign-sensitive measurement of heteronuclear couplings. These
include the use of 1D-selective-HSQC-TOCSY,77 2D-selective-
HSQC-TOCSY,78 2D-HSQC-TOCSY,79 and 1D HSQCMBC-
CPMG,69,80 which are normally used in pure compounds and not
mixtures. Different nuclei will have different advantages and
compromises by using either HSQC-based or modulated echo-
based experiments. Linear combination of in-phase (IP) and
anti-phase (AP) signals, with IPAP methods,81 can also be
employed as an alternative to pure shift NMR methods for
disentangling heteronuclear from homonuclear coupling
contributions, however, differential experiments are prone to
subtraction artefacts (i.e. cross-talk signals), relying on very
stable magnetic fields. In practice, artefacts are common in both
IPAP and pure shift NMR spectra.82 While the effects of
homonuclear couplings, such as J-modulation to name one,
lessen the spectral quality of IPAP, in pure shift NMR, strong
coupling introduces characteristic extra signals in the spectrum,
which are shown in the SI Fig. S9.
5
Heteronuclear couplings as far as JHF could be measured for
the set of molecules studied here. In some cases, where signal
overlap (due to homonuclear multiplet structure) is not a
challenge, HAPPY-FESTA alone should be enough for
obtaining sign sensitive coupling information much quicker
than its HD version.
To fully demonstrate the power of HD-HAPPY-FESTA,
the crude of the fluorination reaction was used as the starting
material for a ketone reduction. 19F spectrum (Fig. 3b) or 1H
spectra (conventional Fig. 1a and pure shift Fig. 1b) of such
sample are ineffective in observing the signals belonging to the
four alcohol structures. The sundry number of close 19F signals,
with various intensities due to high-dynamic-range, makes the
use of very selective 19F inversion pulses crucial for the
application of FESTA, which is achieved with no difficulties
employing the pMODO block. Here, each alcohol was analysed
3
1
first by their JHF from the carbinolic H signals, with typical
and easy to identify chemical shifts (~3.5 to 4.0 ppm). These
measured couplings were: 12.1, 7.7, 29.1 and 5.5 Hz for spectra
of Fig. 1c, 1d, 1e and 1f, respectively (structural motif is shown
in Scheme 2). This leads to the identification of alcohol 3c for
the subspectrum of Figure 1e, as the largest coupling constant
(29.1 Hz) is attributed to the diaxial H1-F coupling (26.7 Hz by
DFT). Using only this set of couplings for the identification of
the remaining molecules is less obvious, those being different
arrangements of axial-equatorial and equatorial-equatorial
couplings. The subspectra of Figure 1f also shows a very large
coupling of 48.0 Hz at 1.49 ppm (i.e. aliphatic region),
identifying that as the diaxial 3JHF of the axial H3 for the alcohol
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Page 6 of 16
facility. The authors also gratefully acknowledge Dr. Luís G. T. A.
Duarte for valuable corrections in this manuscript.
The long experimental duration for acquiring pure shift
experiments (~ 2 hours) can be avoided by combining MODO
and HAPPY-FESTA to produce IPAP spectra (under 15
minutes) but, typically, pure shift spectra will require no further
manipulation and are easily interpretable by non-NMR experts.
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CONCLUSIONS
We have demonstrated, for two samples with different
complexities, that the novel HD-HAPPY-FESTA method can
be used for the direct NMR analysis of fluorinated molecules in
crude reaction products. The 1H subspectra of spins coupled to
a selected 19F nucleus, extracted with HAPPY-FESTA, unlocks
the ability to characterise challenging unpurified reaction
products, acting as a magnifying glass for dilute fluorine-
containing components in a mixture. Heteronuclear couplings
ranging from 51.4 to -2.3 Hz could be measured using HD-
HAPPY-FESTA, with the smallest measured magnitude of 0.8
Hz. This is the first time (to the best of our knowledge) that
selective NMR experiments are used to analyse a completely
unfractionated complex reaction mixture, as shown here for the
analysis of fluorinated isomers. FESTA is not limited to 19F and,
in principle, it applies to any NMR active nuclei, such as 31P or
77Se. We expect that the new approach finds a wide application
not just in synthetic chemistry, but also in the fields of
medicinal chemistry and biochemistry. Besides, we expect that
it may be used by the industry and Academy complementarily
to physical separation methods.
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ASSOCIATED CONTENT
Supporting Information
All raw data, AU macros and pulse sequence programs used in this
paper are available at https://doi.org/10.25824/redu/LNVQT9 free
of charge.
The Supporting Information (SI) is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal.chemxxxxxxx.
Full NMR pulse sequence programs, experimental
parameters, synthetic procedure, computational details,
additional NMR spectra, and comparison between
experimental, theoretical and literature data (PDF)
AUTHOR INFORMATION
Corresponding Author
* Dr Guilherme Dal Poggetto: dalpgt@unicamp.br
Author Contributions
All authors have approved the final version of the manuscript.
Notes
§ The sample was left in the fridge for 6 weeks before analysis, as
the University was closed due to the COVID-19 crisis.
ACKNOWLEDGMENT
The authors thank the Fundação de Amparo à Pesquisa do Estado
de São Paulo (FAPESP) for providing financial support for this
research, thought grants No. 2015/08541-6 and for a scholarship to
G.D.P. (Process No. 2018/19253-0), to Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for a
scholarship to J.V.S. (Process No. 88887.339268/2019-00), and the
Institute of Chemistry of UNICAMP for the multi-user NMR
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K.; Lindon, J. C. "Impurity profiling in bulk pharmaceutical batches
using 19F NMR spectroscopy and distinction between monomeric and
dimeric impurities by NMR-based diffusion measurements". J. Pharm.
Biomed. Anal. 1999, 19, 511-517.
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HSQMBC methods". Magn. Reson. Chem. 2018, 56, 910-917.
(70) Freeman, R.; Mareci, T. H.; Morris, G. A. "Weak Satellite
Signals in High-Resolution NMR Spectra: Separating the Wheat from
the Chaff". J. Magn. Reson. 1981, 42, 341-345.
(71) Kupče, Ē.; Freeman, R. "Compensation for Spin–Spin Coupling
Effects during Adiabatic Pulses". J. Magn. Reson. 1997, 127, 36-48.
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Figure 1. 500 MHz (a) conventional, (b) PSYCHE and (c-f) HD-HAPPY-FESTA (isotropic mixing time of 150
1
ms) H spectra of a crude reaction mixture from the reduction of ketones 2a and 2b, in CDCl . Ultrahigh-
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resolution sign-sensitive anti-phase heteronuclear total correlation spectra of alcohols: (c) 3a; (d) 3b; (e)
3c; and (f) 3d, respectively. Molecular structures are shown in Scheme 1. (g) is the expansion of (f)
1
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ms duration were used in (b-g). 32 transients were acquired in (a-e), and 128 in (f-g), with maximum
receiver gain in each experiment. Complete experimental parameters can be found in the ESI.
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Scheme 1. Reactions described in this paper. The first step describes the experimental conditions for the α-
fluorination reaction of 4-tert-butyl-cyclohexanone (1), generating both fluorinated ketones isomers, 2a and
2b, at a ratio of 4.2 /1.0, respectively. The second step describes the condition for the ketone reduction
from the crude reaction of the first step, generating simultaneously all four fluorinated alcohols isomers, 3a,
3b, 3c, and 3d, at a ratio of 9.1/4.8/2.8/1.0, respectively. The relative proportion of the products in each
1
19
reaction step were measured by integration of the signals in the H decoupled F spectra (Fig. 3).
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Figure 2. Pulse sequence for the HD-HAPPY-FESTA (Homonuclear Decoupled Heteronuclear Anti-Phase
Permuted modulated echo Yielding Fluorine Edited Selective TOCSY acquisition). The orange rectangle
highlights the selective pMODO (permuted MODulated echO) block, purple rectangle highlights the zTOCSY
block and the green rectangle the PSYCHE (Pure Shift Yielded by CHirp Excitation) block. Black narrow and
white wide rectangles represent hard 90° and 180° pulses, respectively. Trapezoids with cross-diagonal
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arrows are low power chirp pulses of small flip angle (β). Trapezoids on either side of the DIPSI-2 isotropic
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mixing element
are low power 180° chirp pulses used to suppress zero-quantum coherences. Selective
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(soft) 180° pulses, represented by shaped pulses, are applied at the resonance frequency of coupled H and
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58,59
F. Typically, RSNOB or REBURP shapes are used for H refocus, and IBURP2 for F inversion.
Δ is set
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as 1/(4 × n × J ), where for J is the coupling between the H and F selected by the 180° soft pulses
F
HF
HF
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and n is the number of equivalent F selected. A detailed description of the pulse sequence can be found in
F
the ESI. Removing the PSYCHE block reduces the dimensionality and increases the sensitivity of the
experiment, and homonuclear coupling is observed (i.e. HAPPY-FESTA).
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Scheme 2. Numbered structural motifs of fluoroketones (left) and fluoroalcohols (right).
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Figure 3. 470 MHz F spectra of the crude reaction mixture from: a) the α-fluorination of ketone 1, in
Acetone-d ; and b) the reduction of ketones 2a and 2b, in CDCl .
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Figure 4. 500 MHz (a) conventional, (b) PSYCHE and (c-d) HD-HAPPY-FESTA (isotropic mixing time of 150
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ms) H spectra of a crude reaction mixture from the α-fluorination of 1, in Acetone-d . Ultrahigh-resolution
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sign-sensitive anti-phase heteronuclear total correlation spectra of ketones: (c) 2a; and (d) 2b,
respectively. Molecular structures are shown in Scheme 1. Lightning bolts in (c-d) indicate chemical shifts
1
1
of band selective H pulses, selected using 9.25 ms RSNOB pulses ( H bandwidth of 200 Hz). A flip angle (β)
of 22° and 80 data chunks of 12.5 ms duration were used in (b-g). 32 transients were acquired in (a-d),
with maximum receiver gain in each experiment. Complete experimental parameters can be found in the
ESI.
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Figure 5. Expansion of 500 MHz (a) conventional (expansion of Fig. 4a), (b) HAPPY-FESTA and (c) HD-
HAPPY-FESTA (expansion of Fig. 4d) H spectra of a crude reaction mixture from the α-fluorination of 4-
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tert-butyl-cyclohexanone, in Acetone-d . Ketone 2b is been observed selectively in (b-c).
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91x94mm (300 x 300 DPI)
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