More than ADEQUATE: doubling the sensitivity of 13 CH– 13 CH correlations in double-quantum NMR experiments

We present modifications of the ADEQUATE experiment which more than double the sensitivity of the carbon-carbon correlations of 13 CH– 13 CH moieties. Additionally, these improvements can be applied without a sensitivity penalty to obtain spectra with 13 C chemical shift axis in the indirectly detected dimension, instead of a double-quantum frequency, allowing simpler interpretation of spectra. The modified experiments, which use refocussing of 1 J CH couplings and 1 H decoupling during J CC evolution intervals, were tested on several molecules, including a pentasaccharide (20 mg, 19 mM), where on average a 2.6-fold signal-to-noise improvement was achieved and the number of observable correlations increased. Doubling sensitivity results in a 4-fold reduction of the experimental time, allowing ADEQUATE spectra to be recorded overnight instead of over multiple days


Justinas Sakas a and Dušan Uhrín* a
We present modifications of the ADEQUATE experiment which more than double the sensitivity of the carbon-carbon correlations of 13 CH-13 CH moieties. Additionally, these improvements can be applied without a sensitivity penalty to obtain spectra with 13 C chemical shift axis in the indirectly detected dimension, instead of a double-quantum frequency, allowing simpler interpretation of spectra. The modified experiments, which use refocussing of 1 JCH couplings and 1 H decoupling during JCC evolution intervals, were tested on several molecules, including a pentasaccharide (20 mg, 19 mM), where on average a 2.6-fold signal-to-noise improvement was achieved and the number of observable correlations increased. Doubling sensitivity results in a 4-fold reduction of the experimental time, allowing ADEQUATE spectra to be recorded overnight instead of over multiple days.
The INADEQUATE (incredible natural abundance doublequantum transfer experiment) NMR experiment 1,2 is of substantial interest to chemists as it allows to trace out the carbon skeleton of a molecule. 3 It relies on the detection of double-quantum (DQ) coherences between two coupled 13 C spins and therefore has inherently inadequate sensitivity, as the probability of a molecule containing a pair of 13 C atoms is approximately 1-in-8,300. Historically, this has limited its use, as long experimental times and/or high sample concentrations were typically required. However, the development of cryogenically cooled NMR probes and the associated sensitivity gains enabled INADEQUATE and its variants to become routine experiments, which have been reviewed extensively. [4][5][6][7][8][9] For protonated 13 C atoms, sensitivity of INADEQUATE can be increased by using 1 H detection as demonstrated by the INEPT-INADEQUATE experiment. 10 The addition of the INEPT (insensitive nuclei enhancement by polarisation transfer) step offers increased sensitivity due to the higher gyromagnetic ratio (γ) of 1 H compared to 13 C. Considering proton singlets and neglecting the sensitivity enhancement of 13 C-detected INADEQUATE through heteronuclear NOE, the maximum theoretical signal-to-noise ratio (SNR) increase associated with 1 H detection is (γH/γC) 5/2 ≈ 32. 11 However, such a SNR increase is not achieved for various reasons. 12 A factor of √2 is lost due to pulsed-field gradient (PFG) selection, 13 and a loss of a further factor of 2 occurs because in the 1 H-detected experiment the DQ coherences start and end on the same proton, whereas in 13 C-detected INADEQUATE the coherences are generated from two nuclei. Additional sensitivity loses occur due to relaxation effects: the INEPT transfer of the 1 H-detected experiments generates mixed proton-carbon coherences that in medium size molecules relax faster than pure carbon coherences. While sharp 1 H-decoupled antiphase 13 C-13 C doublets are acquired in 13 C-detected INADEQUATE, complex 1 H multiplets degrade sensitivity of 1 H-detected methods.
As a result, approaches have been developed to improve the sensitivity of 1 H detection. It has been demonstrated that removing the proton term from mixed CH coherences prolongs their relaxation times 12 and increases the overall sensitivity of experiments. This approach is especially beneficial for longrange ( n JCC, n ≥ 2) optimised 1 H-detected experiments containing longer n JCC evolution delays. The sensitivity of 1 Hdetected INADEQUATE was substantially increased by 1 Hdetected ADEQUATE (adequate sensitivity double-quantum spectroscopy). 14,15 This method employs a 120° 13 C pulse to optimise the 13 C multiple-quantum to single-quantum coherence transfer and incorporates the preservation of equivalent pathways (PEP) element that offers sensitivity improvements of up to √2-fold. 16 Sensitivity can be further improved by homonuclear decoupling that at least partially simplifies the structure of 1 H multiplets. 17,18 In order to avoid complications associated with the setup and interpretation of experiments that produce DQ frequencies in the indirectly detected dimension (F1), a version of the ADEQUATE experiment was reported that samples 13 C singlequantum (SQ) coherences in F1. 14,15 This modification, originally termed ω1-refocussed ADEQUATE, is referred to as SQ ADEQUATE herein. It works by incorporating an additional t1 period into the pulse sequence, during which the 13 C-13 C DQ coherences are modulated by a single-quantum 13 C frequency with an opposite sign to the DQ 13 C-13 C frequency. For an Hn-Cn-Cm-Hm spin system this amounts to Hn being modulated by the Cm frequency (and Hm by Cn), and thus the 1 JCC-optimised SQ ADEQUATE experiment produces a spectrum containing pseudo-2-bond C-H correlations at the (F2, F1) chemical shifts of (Hn, Cm) and (Hm, Cn). A comparison of SQ ADEQUATE and 2D 1 H, 13 C HSQC spectra (the latter acquired in a fraction of time) This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins allows for a straightforward interpretation of 13 C-13 C correlations observed in SQ ADEQUATE experiments.
In this work, we present modifications of the ADEQUATE experiments that significantly increase the sensitivity of 13 C-13 C correlations for molecular moieties containing CH-CH fragments. These modifications can be applied to experiments that sample either DQ or SQ frequencies in F1 and are herein referred to collectively as 1 JCH-refocussed ADEQUATE (Fig. 1). The full pulse sequence parameters and experimental details are presented in Fig. S1 (ESI †).
The first minor modification of the original experiments is the addition of a 90° 13 C pulse and a PFG purge element at the beginning of the pulse sequence, which defocusses any 13 C magnetisation, thus eliminating polarisation transfer pathways starting on 13 C. Although the ADEQUATE experiment employs phase cycling and PFG selection to remove the strong signals coming from 12 C-bonded protons, isolated 13 C-bonded protons or 13 C atoms, imperfect defocussing of these signals can obscure correlations of interest. The addition of this purge element reduced cancellation artefacts and slightly increased the SNR as shown in Fig. S2 (ESI †).
A major sensitivity increase was achieved by refocussing the 1 JCH coupling at the beginning of the 1/(2 1,n JCC) evolution interval following the initial 1 H  13 C INEPT transfer. This conversion of carbon-proton antiphase coherences into pure 13 C coherences prior to excitation of DQ coherence removes the leakage of signal into undetectable zero-quantum coherences and generates a 2-fold sensitivity increase. 19 Application of 1 H decoupling while SQ or DQ 13 C coherences are present, reduces relaxation and further increases the sensitivity of experiments. 12 To regenerate the antiphase proton-carbon coherences required for the reverse 13 C  1 H INEPT step, the decoupling is turned off for a period of 1/(2 1 JCH) at the end of the 1/(2 1,n JCC) refocussing interval.
The increased SNR of the modified ADEQUATE experiments was evaluated using a concentrated sample of methyl β-Dxylopyranoside (I, 1.1 M, Scheme 1). This model compound was chosen because it contains CH, CH2 and CH3 carbons; its high concentration enabled fast and accurate comparison of related spectra. Relative SNR changes for CH groups are presented in Table 1 and Fig. 2; full spectra are shown in Fig. S3 (ESI †). SNR improvement for selected correlations is highlighted in Fig. S4 (ESI †). Not reported previously in the literature, these results indicated that the original 1 JCC-optimised SQ (ω1-refocussed) ADEQUATE experiment shows an up to 32% sensitivity loss compared to the DQ ADEQUATE experiment. This loss, which  Please do not adjust margins Please do not adjust margins can be tolerated in exchange for a more straightforward analysis of spectra when sample concentration is not limiting, is associated with the appearance of HSQC-like artefacts at frequencies of (Hn, Cn) as shown in Fig. S3c (ESI †). These extra correlations, which can be much more efficiently obtained from 1 H, 13  Overall, the data summarised in Table 1 indicate that 1 JCHrefocussed SQ ADEQUATE experiment performs best for both one-bond and long-range 13 CH-13 CH correlations. Therefore, simpler spectra, displaying SQ 13 C chemical shift along F1 can now be obtained without sensitivity penalty and a 1.8-2.0-fold enhancement compared to the original DQ ADEQUATE. As the 1 JCH-refocussed ADEQUATE experiments are designed to refocus couplings of CH groups only, the sensitivity of CH3detected correlations are reduced and CH2-detected correlations are absent. The CH-detected CH-CHx (x = 0, 2, 3) connectivities are present with improved sensitivity (1.3-2.0×) compared to the original ADEQUATE spectra as evaluated on the spectra of isoleucine (Fig. S5, ESI †).
In order to investigate the SNR improvements on a compound with a more diverse structure, one-bond and longrange optimised SQ ADEQUATE spectra were acquired for strychnine (II, 32 mM). Despite the wide range of coupling constants reported for II ( 1 JCH = 124-168 Hz, 1 JCC = 32-71 Hz, 3 JCC = 3-7 Hz), 20,21 experiments optimised for 1 JCH (150 Hz) and 1,n Jcc (50 Hz or 6 Hz) yielded sensitivity improvements similar to those observed for I. A SNR gain of (1.9 ± 0.4) was observed for CH-CH correlations in 1 JCC-optimised ADEQUATE (Fig. S6, Table S1, ESI †). A more significant increase in sensitivity was observed in the long-range spectra (Fig. S7, Table S2, ESI †), with an average SNR increase of 2.3 for CH-CH correlations (Fig. 3). The sensitivity is also improved for CH-CHx (x = 0, 2) moieties, although by not as much, especially for quaternary carbons. The lower increase can be attributed to quaternary carbons of II having relaxation times of ≥ 20 s, 22 therefore not benefiting much from reducing relaxation losses.
To illustrate the benefits of the improved ADEQUATE experiment on a weaker sample of a larger molecule, one-bond and long-range 13 CH-13 CH correlations of a sulfated pentasaccharide, fondaparinux Na (III, Scheme 1, Mw = 1,728 g/mol, 19 mM), were obtained on an 800 MHz spectrometer equipped with a TCI cryoprobe in 7 and 21 hours, respectively (Figs. 4, S8−S10, ESI †). SNR improvements in the range of 1.4-to 4.4-fold were obtained for the 1 JCH refocussed  (Table S3, ESI †). Low SNR of the spectra is responsible for such large variations, yielding an average (2.6  0.7)-fold sensitivity increase. Increased SNRs were also seen in the n JCC-optimised 1 JCH-refocussed SQ ADEQUATE spectra (2.2  0.5-fold, Table S4, ESI †). The increase in sensitivity is larger than for I or II, which illustrates that larger molecules benefit more from reduced relaxation losses due to their shorter 1 H relaxation times. The sensitivity increase in both one-bond and long-range optimised 1 JCH-refocussed experiments is significant, however the values only refer to SNR increases for signals present in both the original and improved spectra. In fact, 1 JCH-refocussing revealed extra correlations not present in the spectra of the original experiments, therefore the sensitivity gains are even larger than the calculated factors. This is particularly true for the n JCC-optimised experiments.
Correlations between adjacent monomer units in the pentasaccharide, providing valuable ring connectivity information, were observed. These experiments demonstrate that with < 4 mg per monosaccharide residue, 1 JCH-refocussed SQ ADEQUATE is an efficient experiment for structure determination of oligosaccharides.
It is worth noting that due to fast evolution of 1 JCC compared to n JCC coupling constants, one-bond correlations can appear in long-range optimised spectra. In this work, these correlations were identified by comparison to 1 JCC-optimised spectra, however methods exist to allow discrimination between 1 JCC and n JCC correlations in n JCC-optimised experiments. 23 In conclusion, the pulse sequences presented in this work provide significant improvements for the detection of 13 CH-13 CH correlation by double-quantum NMR experiments. Larger than 2-fold Increases have been achieved, which translate to a 4-times reduction in spectrometer time. Double quantum 13 C− 13 C experiments are regularly recorded over multiple days, yet the sensitivity-improved experiments will be able to provide the same information overnight. The new method is compatible with other schemes, such as homonuclear decoupling and nonuniform sampling, that have already been successfully applied to ADEQUATE experiments. 17,18,24,25 Additionally, the proposed modifications allow observation of SQ 13 C frequency in the F1 dimension without a sensitivity penalty. These sensitivity enhancements increase the potential of using 13 C-13 C correlations for structure elucidation and will benefit areas such as carbohydrates, natural products or mixture analysis, as well as general NMR applications when sample quantities are limited.
This research was supported by EPSRC grants EP/T517884/1 and EP/R030065/1. The authors thank Mr Juraj Bella and Dr Lorna Murray for the maintenance of the NMR spectrometer. Raw data for the spectra presented in this communication can be found at DOI….

Conflicts of interest
There are no conflicts to declare.