19F-centred NMR analysis of mono-fluorinated compounds

Addressing limitations of the existing NMR techniques for the structure determination of mono-fluorinated compounds, we have developed methodology that uses 19F as the focal point of this process. The proposed 19F-centred NMR analysis consists of a complementary set of broadband, phase-sensitive NMR experiments that utilise the substantial sensitivity of 19F and its far reaching couplings with 1H and 13C to obtain a large number of NMR parameters. The assembled 1H, 13C and 19F chemical shifts, values of JHF, JHH, and JFC coupling constants and the size of 13C induced 19F isotopic shifts constitute a rich source of information that enables structure elucidation of fluorinated moieties and even complete structures of molecules. Here we introduce the methodology, provide a detailed description of each NMR experiment and illustrate their interpretation using 3-fluoro-3-deoxy-d-glucose. This novel approach performs particularly well in the structure elucidation of fluorinated compounds embedded in complex mixtures, eliminating the need for compound separation or use of standards to confirm the structures. It represents a major contribution towards the analysis of fluorinated agrochemicals and (radio)pharmaceuticals at any point during their lifetime, including preparation, use, biotransformation and biodegradation in the environment. The developed methodology can also assist with the investigations of the stability of fluoroorganics and their pharmacokinetics. Studies of reaction mechanisms using fluorinated molecules as convenient reporters of these processes, will also benefit.


Introduction
Fluorine's unique properties, such as high electronegativity, strength of a single uorine-carbon bond and small atomic radius, impart signicant benets to uorinated organic molecules. 1 Fluorination has been shown to enhance potency and/or specicity of molecular interactions, increase membrane permeability, modulate metabolism, moderate the pK a of proximal functionalities, inuence conformation, stabilise inherently reactive functionalities and produce viable bioisosteres. 2,3 Currently, about 20% of the commercial pharmaceuticals contain uorine and the proportion of newly approved uoro-pharmaceuticals is rising steadily. 4,5 The proportion of uoro-agrochemicals is even larger; 53% of all active agrochemicals registered during 1998-2020 belong to this category. 6 Similarly, 18 F is the most frequently used radioisotope in positron emission tomography radiopharmaceuticals. 7 Fluorination also has the potential to become a useful tool for improving properties of fragrance and semiochemical molecules. 8 To capitalise on the ability of uorine to improve molecular properties, there is a drive to design efficient and environmentally-safe chemical, 9,10 enzymatic 11 and chemoenzymatic 12-14 uorination methods. To assist these efforts, efficient analytical methods for the characterisation of uorinated molecules are required. 19 F NMR spectroscopy plays a prominent role in this area due to the favourable properties of 19 F, such as its high sensitivity, 100% natural abundance, large chemical shi dispersion, large and far-reaching spin-spin interactions and 13 C induced 19 F isotopic shis.
The lack of background 19 F signals, due to the scarcity of uorinated endogenous compounds, makes 19 F NMR perfect for the analysis of mixtures produced by chemical or chemoenzymatic reactions with minimum clean-up steps or compound separation required. 1D 19 F NMR spectroscopy has been widely used in studies of biodegradation and biotransformation of uorinated compounds 15-21 mostly relying on the use of known standards 15 or tabulated 19 F chemical shis. In a similar manner, 19 F NMR has also been used for probing the mechanism and kinetics of chemical reactions, were uorine is a convenient reporter of the processes taking place. 22,23 In support of such wide ranging activities, we have developed a 19 F-centred NMR approach for the analysis of mono-uorinated compounds, taking 19 F NMR beyond recording simple 1D NMR spectra. Put together, the information obtained allows the structure elucidation of uorine-containing molecular moieties and complete structure determination of small uorine-containing molecules. It is well suited for the studies of complex mixtures.
The 19 F-centred NMR shares similarities to the "NMR spy" approach developed for the analysis of complex mixtures of soil organic matter, where -O 13 CH 3 tags are introduced to a subset of molecules. [24][25][26] Nevertheless, there are signicant differences between the two approaches. Firstly, uorinated molecules already contain 19 F and therefore do not require additional chemical modications. Secondly, the uorine atom is typically closer to the protons and carbons of an organic molecule than are the nuclei of the -O 13 CH 3 group which, when combined with far reaching 19 F couplings, allows to inspect parts of the molecule that are more remote from the 19 19 F-centered approach is illustrated using 3-uoro-3-deoxy-D-glucose, 1, which can be characterized as a simple mixture of two 19 F-containing molecules. Application of this methodology to a very complex mixture of compounds produced by chloramination of a single uorinated molecule is presented elsewhere. 30

Experimental
The sample of 3-uoro-3-deoxy-D-glucose (30 mg), 1, was dissolved in 600 mL of D 2 O (Merck, 99.9 atom% D) and placed into a 5 mm NMR tube. Spectra involving 19 F were acquired at 300 K on a 400 MHz Bruker Avance III NMR spectrometers equipped with a TBO BB-H/F-D probe. A 1D 1 H spectrum was acquired on an 800 MHz Bruker Avance III NMR spectrometer equipped with a TCI 5 mm probe. Parameters of the performed NMR experiments are presented in Table S1 † and Bruker pulse sequences compatible with TopSpin 3 can be found in the ESI † (pp. 1-6).
The following symbols are used to depict the pulse sequences in Fig. 1-6: the thin and thick lled rectangles represent high power 90 ( 1 H, p1 or 19 F, p3) and 180 ( 1 H, p2) pulses, respectively. 1 ms adiabatic CHIRP pulses with a peak power of 10.3 kHz (p44, shaded trapezoid with an inclined arrow) were applied to 19 F. A 20 ms 60 kHz CHIRP 1 H pulse with a peak power of 2294 Hz (p32, trapezoid with inclined arrow) was used as part of the z-lter. A 500 ms CHIRP pulse (p14) and 2 ms composite CHIRP pulse (p24) were applied to 13 C with a peak power of 9800 Hz. Unless stated otherwise, the r.f. pulses were applied from the x-axis. The 100% pulsed eld gradient strength corresponds to 53.5 G cm À1 .   19 F HETCOR. In a non z-filtered experiment, the part within the dashed rectangle is not included. For explanation of symbols used for pulses see Experimental. The NMR parameters used are given in Table S1. † The delays used were as follows: D 1 ¼ p44; D 2 ¼ one half of the J HF evolution; t 1 (0), the initial t 1 evolution delay time ¼ 0.5 Â in0, where in0 is the t 1 increment. The gradient strengths were as follows: The following phase cycling was used: 4 1 ¼ x, Àx; 4 2 ¼ 4x, 4(Àx); 4 3 ¼ 2y, 2(Ày); J ¼ x, 2(Àx), x. States-TPPI protocol was used for sign discrimination in F 1 with the phase 4 1 incremented by 90 . Purging of 19 F magnetisation at the beginning of the pulse sequence by a composite 90 19 F pulse and pulsed field gradients (PFGs) minimises the cancellation artefacts. (b) An overlay of the 19 F-detected 2D 1 H, 19 F HETCOR spectra with (blue/turquoise) and without the z-filter (red/magenta). For clarity, the spectrum acquired without a z-filter was offset horizontally to the right. Insets show 1D F 1 traces taken at positions indicated by arrows. 1D 1 H and 19 F spectra are shown along the left and top, respectively.

Results and discussion
1D 1 H and 19 F spectra of 3-uoro-3-deoxy-D-glucose, 1 A 400 MHz 1D 1 H spectrum of 1 with the suppression of the HOD signal shows considerable overlap of 1 H resonances (Fig. 1a). A 1 H-coupled 1D 19 F spectrum 1 (Fig. 1b) 32,33 Acquisition of pure-phase absorption signals in a phase-sensitive manner is much preferred, as it provides higher sensitivity and allows for accurate determination of coupling constants, including identication of the active coupling constants. Some existing phase-sensitive experiments yield complicated cross peak structures that lower their sensitivity. 34,35 The optimal performance of experiments constituting the 19 F-centred NMR approach across a range of 19 F frequencies, is ensured by the use of adiabatic inversion pulses. 36,37 The experiments provide pure phase multiplets with simple structure afforded by 1 H or 19 F decoupling and were designed to minimise the effect of passive spins; they do not use refocusing intervals, which maximises their sensitivity. NMR hardware capable of pulsing simultaneously on 1 H and 19 F frequencies is  Table S1. †The delays were as follows: The gradient strengths were are follows: The following phase cycling was used: States-TPPI protocol was used for sign discrimination in F 1 with the phase 4 1 incremented by 90 . Purging of 19 F magnetisation after the z-filter by a composite 90 19 F pulse followed by the G 2 PFG minimises the cancellation artefacts. (b) An overlay of the 19 F-detected 2D 1 H, 19 F TOCSY-HETCOR spectrum (blue/turquoise) and a z-filtered VT 19 F-detected 2D 1 H, 19 F HETCOR spectrum (red/magenta, horizontally offset to the right) of 1 acquired with the pulse sequence shown in (a) and   Table S1. †The dashed line indicates signal acquisition before an optional 1 H-1 H spin-lock. For description of pulses see Experimental. The delays were as follows: is the t 1 increment. The gradient strengths were as follows: The following phase cycling was used: 4 1 ¼ y, Ày; 4 2 ¼ 4x, 4(Àx); 4 3 ¼ 2y, 2(Ày); J ¼ x, 2(Àx), x. The states-TPPI protocol was used for sign discrimination in F 1 with the phase 4 1 incremented by 90 . Purging of 19 F magnetisation at the beginning of the pulse sequence by a composite 90 19 F pulse and PFGs minimises the cancellation artefacts. (b) An overlay of two 2D 19 F, 1 H CP-DIPSI3-DIPSI2 spectra acquired with 20 ms 19 F / 1 H cross-polarisation (CP) only (red) and an additional 50 ms 1 H / 1 H spin-lock (blue) using the pulse sequence shown in (a). The red spectrum was offset vertically to facilitate visualisation of the cross peaks. The two insets show overlaid 1D F2 traces through 19 F resonances of aand b-D-glucose from both spectra. Twice as many scans were acquired for the blue spectrum as for the red spectrum. 1D 19 F and 1 H projections of the blue spectrum are shown along the left and top, respectively. required; fortunately, such systems are more widespread now. To access the rich information provided by 13 C-19 F interactions, a three-channel NMR spectrometer is necessary. Maximum benets are realised on systems equipped with highly sensitive low temperature probes. These have also become more widely available, mainly due to their use in binding studies of biomacromolecules with uorinated ligands.

Fluorine-proton and proton-proton correlation
Following the acquisition of 1 H-decoupled and 1 H-coupled 1D 19 F spectra, mapping of the 1 H-19 F correlations is the natural next step in investigating the structure of uorinated compounds. For this task a choice of three types of experiment exist: hetero-COSY, HETCOR or HMBC. 31,32 Most of these can be implemented using 19 F or 1 H as the directly detected nucleus. The NMR parameters used are given in Table S1. † The delays were as follows: The gradient strengths were are follows: ) and sfo1 and sfo2 are 19 F and 13 C frequencies, respectively. The following phase cycling was used: . The echoantiecho protocol was used with PFGs changing sign between real and imaginary increments. Phases 4 2 and J were incremented by 180 together with the PFG sign change, (b) A 2D 19 F, 13 C HMBC spectrum of 1 optimised for n J FC of 20 Hz acquired using the pulse sequence of shown in (a). The two insets show 1D F 2 traces for individual 13 C resonances of the aand b-forms of 1. 1D 1 H-decoupled 19 F NMR spectrum and the 13 C projection are shown on the top and along the left of the spectrum, respectively.  Table S1. †The delays were as follows: where p16 and d16 are the PFG length and the recovery time, respectively. The gradient strengths were as follows: The following phase cycling was used: 4 1 ¼ y, Ày; 4 2 ¼ 4x, 4(Àx); 4 3 ¼ 2x, 2(Àx); 4 4 ¼ 2y, 2(Ày); J ¼ x, 2(Àx), x, Àx, 2x, Àx. The echo-antiecho protocol was used with G 1 changing sign between real and imaginary increments. Phases 4 1 and J were incremented by 180 together with the sign change. Two interleaved experiments were acquired applying either the 4 3 or 4 4 phase to the last 90 13 C pulse, (b) an F 1 antiphase (3, 2)D H 1 C n F spectra of 1 acquired using the pulse sequence shown in (a) showing the cross peaks of the b-anomer of 1. Positive and negative cross peaks are shown in blue and turquoise, respectively. The insets contain vertical and horizontal traces through the H1, F cross peaks. The 1 H chemical shift of protons directly attached to 13 C atoms and the associated kU 13C frequencies are indicated. Antiphase doublets in F 2 show n J FC coupling constants. Horizontal and vertical internal projections are shown on the top and along the left side of all spectra, respectively. The editing process that simplifies this spectrum is explained in the text and shown in Fig. S5. † Using 19 F as the directly detected nucleus, the 2D 1 H, 19 F HMBC has the highest sensitivity, but yields mixed-phased multiplets. 2D 1 H, 19 F hetero-COSY can be implemented with either nucleus being sampled in the directly detected (F 2 ) dimension. Nevertheless, sampling 19 F in the F 2 dimension has a distinct advantage of acquiring spectra with the high digital resolution required for the identication of active and passive J HF coupling constants and potentially also for their measurements. A disadvantage of COSY type spectra is the antiphase nature of their cross peaks (particularly in F 1 ) and their large footprint.
Choosing to obtain the 1 H-19 F correlations using a phasesensitive 19 F-detected 2D 1 H, 19 F HETCOR experiment (Fig. 2a) retains the advantages of 19 F detection. Its uniform performance across a large 19 F chemical shi range is guaranteed by the use of broadband inversion CHIRP pulses 38 arranged in a double inversion adiabatic sweep (Fig. S1 †), a feature applied in several experiments presented here to eliminate phase evolution of the transverse magnetisation during pulses. [39][40][41] This allows the use of such pulses not only for spin inversion but also refocusing.
The structure of cross peaks in HETCOR spectra is simplied by the application of a 180 19 F pulse in the middle of the t 1 interval, reducing the probability of signal overlap in spectra of complex mixtures. A drawback of this experiment is the evolution of 1 H-1 H couplings during the defocusing interval 2D 2 , which competes with the evolution of 1 H-19 F couplings, decreasing its sensitivity. This decrease can oen be tolerated because of the 100% natural abundance of both nuclei.
Due to diverse sizes of J HF coupling constants, no attempt was made to refocus 19 F magnetisation prior to detection and 1 H decoupling was not applied during t 2 . Preserving the antiphase character of cross peaks is important, as it allows the identication of active couplings. Nevertheless, if a 1 H-coupled 19 F 1D spectrum is overlap free, it is advised to read the coupling constants from this spectrum, where accurate values are readily obtained (see Fig. 1b).
In a basic HETCOR experiment, 32 the evolution of 1 H-1 H couplings during the 1 H-19 F defocusing interval, 2D 2 , leads to the appearance of mixed phase proton multiplets in F 1a feature that is masked by the magnitude mode presentation of spectra. This issue was resolved in the proposed phase-sensitive experiment by inserting a z-lter 42 aer the t 1 period, which separates the evolution of 1 H-1 H couplings during the t 1 and the 2D 2 defocusing interval. Providing the t 1max is kept short (<30 ms), the cross peaks appear as singlets in F 1 . The described features of the experiment are illustrated on a 2D 1 H, 19 F HETCOR spectrum of 1 (Fig. 2b), where correlations with many 19 F coupled protons are observed.
Protons not coupled by a sizable (>1.0 Hz) coupling constant to a 19 F, but which are part of a spin system containing at least one 1 H coupled to a 19 F, are detected in a 2D 1 H, 19 F TOCSY-HETCOR experiment (Fig. 3a). Here, the 1 H chemical shis are labelled before their magnetisation is spread through the network of J HH coupled spins by a DIPSI-2 spin-lock. 43 Part of the magnetisation that has reached the 19 F-coupled protons is then transferred to 19 F for detection in a subsequent HETCOR step. An overlay of the 2D 1 H, 19 F HETCOR and 2D 1 H, 19 F TOCSY-HETCOR spectra (Fig. 3b) revealed several protons with a J HF close to zero, which were not detected by the HETCOR experiment. Other protons of both anomeric forms of 1 coupled with small coupling constants to 19 F showed increased intensities.
In addition to J HF coupling constants, J HH coupling constants provide important structural information that for complex mixtures is inaccessible by standard 2D experiments, but can be retrieved when some form of 19 F editing is used. In principle, 1 H-1 H couplings modulate cross peaks in the F 1 dimension of the 2D (TOCSY-)HETCOR experiments discussed above but in practice, typical t 1 acquisition times used to record such spectra are too short to resolve them. The 1 H-1 H couplings are more likely to be resolved in the F 2 dimension of 1 Hdetected experiments considering a non-refocused 2D 1 Hdetected 19 F, 1 H HETCOR, this experiment shows F 2 multiplets with J HF and J HH coupling constants as anti-phase and inphase splitting, respectively, complicating access to J HH coupling constants (data not shown).
The J HH coupling constants can be measured more effectively from inphase proton multiplets acquired in the presence of 19 F decoupling. Developed for simple mixtures of uorinated compounds, this reasoning has led to the design of FESTA experiments. [27][28][29] These 1D selective experiments require that both 19 F and 1 H multiplets are amenable to selective inversion, which is rarely the case for complex mixtures; experiments that do not rely on selective manipulations of spins are more robust.
A suitable alternative involving the use of 19 F / 1 H crosspolarisation (CP) that produces inphase 1 H multiplets was already proposed in the form of a 3D CP 19 F, 1 H heteronuclear TOCSY experiment. 44 We did not nd it necessary to label the 1 H chemical shis aer the initial 19 F / 1 H magnetisation transfer and present here a 2D version of this experiment in the form of a 2D 19 F, 1 H CP-DIPSI3-DIPSI2 (Fig. 4a). Here, the signal acquisition can start immediately aer the z-lter 42 that follows the CP step. Note that signals of protons not coupled to 19 F can appear in the spectrum even at this point due to the 1 H-1 H TOCSY transfer that takes place simultaneously with the heteronuclear CP step.
This pulse sequence can be extended by a dedicated 1 H-1 H DIPSI-2 spin-lock propagating the magnetisation transfer to more remote parts of the spin system. Application of two z-lters and 19 F decoupling ensures that pure inphase 1 H multiplets are eventually acquired. DIPSI-3, 45 using 40 ms 19 F/ 1 H pulses, was applied for the CP step covering a AE4 kHz frequency range with >75% efficiency. A slight improvement was achieved with the FLOPSY-16 mixing scheme 46 covering AE4.7 kHz, i.e. 25 ppm of 19 F resonances on a 400 MHz NMR spectrometer with >65% efficiency relative to the on-resonance signal (Fig. S2 †). Further improvements, not explored here, can be achieved by using broadband pulses during the CP step. 47 An overlay of two 400 MHz 2D 19 F, 1 H CP-DIPSI3-DIPSI2 spectra acquired with a 20 ms 19 F / 1 H cross-polarisation (red) and an additional 50 ms 1 H / 1 H spin-lock (blue) using the pulse sequence of Fig. 4a is presented in Fig. 4b. Both spectra are suitable for the determination of the J HH coupling constants. The former spectrum contains pure in phase multiplets of protons H2, 3 and 4 of 1, while the latter spectrum also shows all their other protons. Note the dominance of the H3 signals in the red spectra caused by an effective CP via large J H3F3 ($50 Hz).

Fluorine-carbon correlation
Structure determination of sparsely protonated uorinated molecules, such as heavily substituted aromatic rings, based only on 1 H and 19 F chemical shis and coupling constants could be problematic. Thanks to the far-reaching 19 F-13 C couplings ( n J FC , n ¼ 1-5), many 19 F-coupled 13 C atoms can be identied by 2D 19 F, 13 C correlated experiments such as HMBC or HSQC, making structure determination of such molecules possible. A 2D 19 F, 13 C HSQC experiment 33,41 was not considered in this study mainly because of a larger complexity of the double INEPT transfer. For small molecules, the slower relaxation of single-quantum (HSQC) relative to multiple-quantum (HMBC) coherences does not make a substantial difference to their sensitivity and for mono-uorinated compounds F 1 singlets are produced by both experiments.
As the 1 J FC coupling constants are large ($150-250 Hz), while the n>1 J FC typically range from 0 to 50 Hz, 48 the one-bond (Fig. S3 †) and long-range correlation (Fig. 5a) experiments are best performed separately. A single long-range optimised experiment can also yield one-bond correlations if multiple rotations of the 19 F magnetisation vectors during the evolution interval fall outside of even multiples of 0.5/ 1 J FC . This approach can only be used when values of 1 J FC coupling constants are known, and if dealing with mixtures, their spread is narrow. Values of 1 J FC coupling constants required for such optimisation can be obtained from 1D 1 H-decoupled 19 F spectra acquired with a sufficient S/N ratio. Alternatively, accordion optimisation 49 can be used to obtain simultaneously both types of correlations. Both experiments perform best when 1 H decoupling is applied during most of the pulse sequence. Such decoupling removes splitting of cross peaks by 1 H-13 C couplings in F 1 and by J HF in F 2 . Resulting F 1 singlets and F 2 anti-phase doublets split by 19 F-13 C interactions (Fig. 5b) allow accurate measurement of J FC coupling constants that provide valuable structural information.
A comparison of 19 F chemical shis of 13 C isotopomers obtained from 2D 19 F, 13 C HMBC spectra with the 19 F signal in a 1D 1 H-decoupled 19 F spectrum yields 13 C induced 19 F isotopic shis (see a large isotopic shi of C3 resonances in Fig. 5b). In aliphatic systems these decrease with the number of bonds separating the two atoms and are generally measurable to up to four bonds separating 13 C and 19 F. A careful alignment of the one-bond correlation trace from the pure phase HMBC spectrum and the satellites from the 1D 19 F spectrum is required to obtain accurate values of these isotopic shis.
Proton-carbon-uorine correlation 1 H-1 H and 1 H-13 C interactions are the cornerstone of NMR structure determination of small molecules. For uorinated compounds, the existence of 1 H-19 F and 19 F-13 C couplings makes this process even more robust. However, for complex mixtures, mapping of these interactions separately, can compromise iden-tication of the nuclei belonging to individual molecules. This ambiguity can be avoided by correlating all three spin types in a dedicated HCF experiment. There are numerous possibilities for how such an experiment can be designed. Inspired by the 3D HNCA, a pulse sequence for assigning protein backbone resonances, 50 a 1 H-detected 3D tripleresonance 1 H, 13 C, 19 F experiment has been proposed previously. 51,52 This out-and-back 3D experiment contains J FC defocusing and refocusing intervals, samples 19 F and 13 C chemical shis indirectly and applies simultaneous 13 C and 19 F decoupling during the direct detection of 1 H. We prefer to use a unidirectional polarisation transfer pathway and direct detection of 19 F; both of these features are well suited for molecules with a large spread of coupling constants, as is typical for 19 F-13 C interactions. The pulse sequence of such an experiment starts with a one-bond 1 H-13 C correlation step followed by a 13 C, 19 F long-range transfer step. It incorporates a reduced dimensionality approach 53-55 and samples 13 C chemical shis simultaneously with the indirect labelling of 1 H resonances. The resulting 2D experiment is referred to as (3, 2)D H 1 C n F, where the superscripts indicate the type of 13 C and 19 F interactions (1-one-bond, n-long-range) mediating the polarisation transfer (Fig. 6a).
In the (3, 2)D H 1 C n F experiment, the 1 H chemical shis are recorded rst, while suppressing the evolution of 1 H-1 H and 1 H-19 F couplings by a BIRD r,X pulse 56,57 and a 180 19 F pulse applied in the middle of the t 1 period, respectively.
The magnetisation is then transferred in an INEPT step to 13 C via one-bond 1 H-13 C couplings, where it is refocused before starting 1 H decoupling. During the subsequent evolution interval, the 19 F-13 C anti-phase magnetisation is developed while the central 180 13 C and 19 F pulses move simultaneously with the t 1 incrementation. This causes modulation of 1 H chemical shis by 13 C offsets, U 13C (¼d( 13 C) À 13 C r.f. carrier frequency) of their directly bonded 13 C, splitting the signals into doublets centred at the 1 H chemical shi. The size of 13 C doublets can be scaled down relative to the t 1 evolution (k factor), keeping the F 1 spectral width small and without any limitations for setting the length of the constant-time 19 F-13 C coupling evolution interval, 2D 5 .
The signal is nally transferred to 19 F, where it is detected during t 2 under 1 H decoupling as a pure phase doublet in antiphase with regard to J FC (Fig. 6b).
Interleaved acquisition of two spectra, differing by 90 in the phase of the last 90 13 C pulse of the pulse sequence, generates inphase and anti-phase F 1 doublets, respectively, allowing spectra to be simplied by spectral editing 58,59 as illustrated in Fig. S5. † A pulsed eld gradient assisted echo-antiecho protocol is used to obtain pure phase signals in F 1 .
Overall, the reduced dimensionality experiment retains the full information content of 3D spectra with substantially increased digital resolution. Due to the use of a single n J FC evolution interval, sensitivity is also improved relative to the original 3D HCF experiment. 51 Detecting 19 F under 1 H decoupling during t 2 further increases sensitivity of this experiment, while providing values of J FC coupling constants. The (3, 2)D H 1 C n F experiment thus complements the 2D 19 F, 13 C HMBC technique discussed above and for protonated carbons correlates unambiguously three atom types, HCF, instead of aiming to achieve the same through a combined interpretation of 2D 1 H, 13 C HSQC and 2D 19 F, 13 C HMBC spectra, which for complex mixtures, is problematic.

Structure determination process in 19 F-centred NMR
This process is briey summarised with the help of a graphical representation in Fig. 7, using the b-anomeric form of 1 as an example. The 19 F-1 H correlations experiments, 2D 1 H, 19 F HETCOR and 2D 1 H, 19 F TOCSY-HETCOR spectra, together with 1D 1 H-coupled/decoupled 19 F spectra provided the parameters summarised in Fig. 7a, while 2D 19 F, 1 H CP-DIPSI3-DIPSI2 experiments extended the identied spin system to protons not directly coupled to uorine (Fig. 7b).
These experiments thus provide 19 F and 1 H chemical shi correlations together with n J HF (n ¼ 2-4) 60 and n J HH (n ¼ 2-3) coupling constants, enabling the start of a structure determination process.
Experiments involving 19 F-13 C correlations are very informative. Central to these is the 2D 19 F, 13 C HMBC experiment, which provides long-range 19 F-13 C correlations and n J FC coupling constants and in conjunction with a 1D 1 H decoupled 19 F spectrum also the 13 C induced 19 F isotopic chemical shis (Fig. 7c). The subsequent (3, 2)D H 1 C n F experiment provides correlations of HC pairs, in which the carbon is coupled to 19 F, and if present, a distinction between non-protonated and protonated carbons (Fig. 7d).
Occasionally, a 2D 1 H, 19 F HOESY experiment 31,61 can be used to identify protons not accessible by exploring J coupled networks of spins. In general, at this point, the chemical shi assignment and of numerous 1 H, 13 C and 19 F resonances, values of J HF , J HH and J FC coupling constants and 13 C induced 19 F isotopic shis are known and the structure determination of uorine containing moieties can be completed.
For larger molecules, which contain spin systems isolated from those containing 19 F, the 19 F-centered approach provides a starting point by identifying protons and carbons that appear in both the 19 F-centered and the standard 1 H-1 H and 1 H-13 C 2D chemical shi correlated spectra. These resonances can then be used to extend the structures and connect the uorinated and non-uorinated parts of molecules, e.g. via 1 H-1 H NOESY experiments or 1 H-13 C HMBC experiments, which can bridge such spin-systems. This approach is particularly benecial for analyses of mixtures, where the identity of cross peaks belonging to the non-uorinated parts of the molecule could be difficult to establish.
Although the discussed NMR experiments were developed for mono-uorinated compounds, they can also be applied to compounds bearing more than one uorine atom. Nevertheless, the presence of multiple 19 F atoms should be taken into account when setting up some of the experiments, as the existence of passive 1 H-19 F (or 19 F-13 C) couplings need to be re-ected in the parameters used as outlined in Table S3. † It should be emphasised, that the 19 F-centered approach takes full advantage of the high sensitivity of 19 F to its environment and minute differences in the 19 F chemical shi of the order of few Hz are sufficient to obtain the kind of information illustrated here on a very simple mixture provided by 1. Application of the 19 F-centered approach to a very complex mixture of chloramination by-products is presented elsewhere. 30

Conclusions
The described methodology is based on a concerted use of several NMR experiments, nevertheless, these can also be used in their own right. Collectively, these experiments represent the most effective NMR approach for the structure determination of mono-uorinated compounds, particularly those contained in mixtures.
The 19 F-centred approach developed here is applicable at any point during the lifetime of uorinated compounds, e.g. in analysing reaction mixtures during their production, performing mechanistic studies to understand reaction mechanisms and to optimise chemical reactions, investigating their stability, pharmacokinetics, biodegradation and biotransformation and ultimately to follow their fate in the environment. 62

Data availability
The spectra obtained in this study are available here: https:// doi.org/10.7488/ds/3422.

Conflicts of interest
There are no conicts to declare.