Communication: Ultrafast homonuclear correlation spectroscopy with diagonal suppression

Abstract

A novel ultrafast 2D NMR experiment is introduced for homonuclear correlation spectroscopy in solution state, with diagonal peak suppression in each scan of a two scan procedure. This experiment permits clear visualization of cross peaks between spins whose chemical shifts are very close, which could otherwise be masked by diagonal peaks. The present report describes the principles of its design and illustrates actual performance. Nuclear magnetic resonance (NMR) has emerged as a powerful technique in physics, chemistry, materials science, biochemistry, and biology owing to its high resolution, which results in the ability to study molecular structure and dynamics in great detail. In particular, spin connectivity in molecules may be visualized handily by multi-dimensional NMR1,2 not only in small molecules, but also in the case of biomolecules or mixtures of metabolites. 2D correlation spectroscopy (COSY), which allows visualization of coupled networks of homonuclear spin systems by driving coherent magnetization transfer among coupled spins, is among the most popular multi-dimensional (nD) NMR experiments.2 nD NMR experiments are, however, generally time consuming when standard data acquisition strategies are employed, requiring multiple repetitions of the experiment with evolution time incrementation for the indirect dimensions. Several methods have been proposed in the last decade to speed up nD experiments significantly.3–6 Frydman and co-workers proposed single scan or ultrafast (UF) nD NMR, which replaces parametric evolution time incrementation with spatial encoding and Echo Planar Imaging (EPI) type of acquisition.7–10 Practical applications of the UF method have been demonstrated in several recent studies11–13 including magnetic resonance imaging.14 The UF implementation of 2D COSY has been employed to study dynamic processes,15 as also for quantification of metabolites.16 Spin echo correlation spectroscopy (SECSY), which may be viewed as a delayed COSY experiment, generates information similar to that from COSY, but gives rise to mixed phase (or phase twisted) lineshapes. It is based on coherence transfer echo pathway selection, and acquisition of the signal from the echo top. SECSY has some advantages over COSY, and comes into its own especially in inhomogeneous media, and in the study of biological macromolecules,17 as well as in in vivo applications.18 An ultrafast version of SECSY has also been reported recently.19,20 One of the interesting features of SECSY is that it requires a reduced spectral width in the indirect dimension compared to COSY, and hence smaller acquisition gradients9 (Ga) may be employed in its UF version. While mapping spin connectivity by way of coherence transfer is the focus of correlation spectroscopy, a major concern with COSY, SECSY, and their ultrafast variants however is that these experiments also give rise in general to peaks from magnetization components that have not been involved in any coherence transfer. Such spectral multiplets are centred at the same frequency in both dimensions in COSY, and at zero frequency in the virtual frequency dimension F1 in SECSY. In a generalized sense we may call these peaks as “diagonal” peaks in both experiments. They are to be distinguished from peaks that could arise from longitudinal magnetization that is brought into the transverse plane by the second pulse, which are routinely eliminated however by standard procedures. These latter multiplets are centred at F1 = 0 in COSY, but are centred in SECSY at an F1 frequency that equals half the individual chemical shift, i.e., half the frequency in F2. These peaks may in a generalized sense be called “axial” peaks in both experiments. Here we propose and demonstrate a simple strategy that basically suppresses diagonal peaks in each scan in the ultrafast SECSY environment, and leads ultimately to a two scan procedure. This approach is in fact valid under any general conditions of limited resolution and short acquisition time in the directly detected dimension; such conditions are commonly encountered especially in volume localized Magnetic Resonance Spectroscopy (MRS), UF NMR, and Overhauser dynamic nuclear polarization (ODNP). To put this in perspective, we recall that while axial peaks may be easily suppressed by suitable phase cycling (for example, by phase alternation of the first pulse together with the receiver phase), suppression of diagonal peaks, which could arise both from coupled spins, as well as from “isolated” spins (i.e., spins that are not coupled to others), has thus far required more elaborate strategies. Diagonal peaks could often obscure the more informative cross peaks, and indeed overlap between cross and diagonal peaks could be especially severe in UF experiments due to their typical linewidths, which are larger in both dimensions – and especially so in the indirect dimension – than in standard COSY or SECSY. Several approaches have been reported in the literature to suppress diagonal peaks in COSY spectra. Double quantum filtered COSY21 is a popular approach to get a correlation spectrum with reduced diagonal peaks. This method, which has half the sensitivity of COSY, effectively suppresses diagonal peaks arising from isolated spins, but not those from coupled spins. Some other methods have been proposed for diagonal suppression, based on the subtraction of two spectra.22,23 Typically, one spectrum is acquired with both cross and diagonal peaks, while a second is the spectrum with only diagonal peaks, acquired with a modified pulse sequence. Neglecting relaxation losses during the mixing time, the efficiency of diagonal suppression in such experiments depends on the reproducibility of the two different experiments, as well as the efficiency of the relevant additional modules, e.g., refocusing pulses22 or the z-filter.23 Diagonal peak suppression was also recently investigated with the help of spatially selective and frequency selective pulses:24 the sequence uses a selective pulse combined with a weak field gradient to excite the sample. The magnetization that does not get transferred during the mixing time (and thus generates diagonal peaks) is suppressed with an excitation sculpting block before signal acquisition. The disadvantages of such an approach include the considerable loss of sensitivity owing to slice selective excitation, the dependence of the efficiency of diagonal suppression on the selectivity of the 180° pulse in the excitation sculpting block, and the suppression of cross peaks in the close vicinity of diagonal peaks as well. In contrast, our present approach is a simple single scan strategy for diagonal suppression in the ultrafast SECSY environment, that leads ultimately to a two step phase cycle. We term our experiment UF-DISSECT (UltraFast DIagonal Suppressed Spin-Echo Correlation specTroscopy). The basic sequence is very similar to UF-SECSY except for an additional 90° pulse with specified phase just before the start of data acquisition (the “DISSECT pulse”). In UF-DISSECT, as shown in Fig. 1, this additional 90° pulse is used at the top of the coherence transfer echo.