Separating an overlapped ¹H peak and identifying its ¹H-¹H correlations with the use of single-channel ¹H solid-state NMR at fast MAS

Highlights

• A MIxed Selective Excitation/Recoupling And Broadband recoupLing Experiment (MISERABLE) is designed where the selective recoupling is SERP and broadband recoupling is BABA;

• MISERABLE can separate the overlapped ¹H peaks and map out spatially proximal protons to the selectively recoupled spins;

• By varying only the cycle time of SERP, MISERABLE can monitor different spin pairs to identify and assign proton correlations in ¹H DQ/¹H SQ spectra;

• MISERABLE simplifies the 2D spectra with two sets of correlations depicted in opposite signs.

Abstract

Fast magic-angle spinning (≥60 ​kHz) technique has enabled the acquisition of high-resolution ¹H NMR spectra of solid materials. However, the spectral interpretation is still difficult because the ¹H peaks are overlapped due to the narrow chemical shift range and broad linewidths. An additional ¹³C or ¹⁴N or ¹H dimension possibly addresses the issues of overlapped proton resonances, but it leads to the elongated experimental time. Herein, we introduce a single-channel ¹H experiment to separate the overlapped ¹H peak and identify its spatially proximal ¹H–¹H correlations. This sequence combines selective excitation, selective ¹H–¹H polarization transfer by selective recoupling of protons (SERP), and broadband ¹H recoupling by back-to-back (BABA) recoupling sequences. The concept for ¹H separation is based on (i) the selective excitation of a well-resolved ¹H peak and (ii) the selective dipolar polarization transfer from this isolated ¹H peak to one of the ¹H peaks in the overlapped/poor resolution region by SERP and (iii) the detection of ¹H–¹H correlations from these two ¹H peaks to other neighboring ¹Hs by BABA. We demonstrated the applicability of this approach to identify overlapped peaks on two molecules, β-L-aspartyl-l-alanine and Pioglitazone.HCl. The sequence allows the clear observation of ¹H–¹H correlations from an overlapped ¹H peak without an additional heteronuclear dimension and ensures efficient polarization transfers that leads to twelve fold reduction in experimental time compared to ¹⁴N edited experiments. The limitation and the conditions of applicability for this approach are discussed in detail.

Introduction

Proton (¹H) is the most favorable nucleus for nuclear magnetic resonance (NMR) spectroscopy in terms of high natural abundance (99.9%) and gyromagnetic ratio, together with its ubiquitous nature. On the one hand, the gyromagnetic ratio offers high sensitivity; on the other hand, it causes strong ¹H–¹H homonuclear dipolar interactions. Since the random tumbling motions are absent in rigid solids, these ¹H–¹H interactions severely broaden ¹H NMR linewidths, making it difficult to extract structural information. Therefore, the popular ¹H solid-state NMR has a limited range of applications, particularly in samples with overlapped ¹H resonances.

The fast magic-angle spinning (MAS) technique is the easiest and most efficient approach for removing the broadening induced by ¹H–¹H interactions [[1], [2], [3], [4], [5]]. Consequently, fast MAS improves the ¹H resolution and sensitivity that allows the ¹H-detection NMR experiments. Two-dimensional ¹H double-quantum/¹H single-quantum (2D ¹H DQ/¹H SQ) experiments at fast MAS are widely used to extract the homonuclear correlations [[6], [7], [8], [9], [10], [11], [12], [13]]. However, partially or fully overlapped ¹H peaks are still observed due to the narrow ¹H chemical shift range (0–20 ​ppm). This causes the ambiguous ¹H peak assignments.

An additional heteronuclear (¹³C or ¹⁴N) or homonuclear dimension is used to improve the resolution in ¹H DQ/¹H SQ experiments [[14], [15], [16]]. For ¹³C, the chemical shift range (in ppm) is ten times larger than that of ¹H (200 ​ppm versus 20 ​ppm, respectively) and the linewidth is narrower than that of ¹H because of negligible homonuclear ¹³C–¹³C dipolar couplings. Therefore, in ¹H–¹³C heteronuclear correlation experiments, the overlapped ¹H peaks are possibly separated [15,[17], [18], [19]]. Nevertheless, due to the low natural abundance of ¹³C (1.1%), this approach suffers from low sensitivity, thus a long experimental time. Otherwise, dynamic nuclear polarization or isotopic labeling is required. ¹⁴N is an attractive nucleus due to its high natural abundance (99.6%). However, a 3D¹⁴N SQ/¹H DQ/¹H SQ experiment is still time-intensive, not to mention the ¹⁴N resolution is rather limited due to the second-order quadrupolar broadening [16]. More importantly, this approach is limited to the case where overlapped ¹H peaks bond to nitrogen. A selective pulse on ¹³C or ¹⁴N overtone [[20], [21], [22]] can be used to reduce the dimensionality, thus experimental time; however, the low natural abundance of ¹³C or the poor efficiency of ¹⁴N overtone excitation again compromises the sensitivity. Nevertheless, for the heteronuclear case, the selection of one of the overlapped ¹H peaks is feasible owing to the selective polarization transfer from a¹³C or ¹⁴N nucleus. For the homonuclear case that is ¹H, 3D ¹H SQ/¹H DQ/¹H SQ [23], ¹H DQ/¹H DQ/¹H SQ [24], and ¹H TQ/¹H DQ/¹H SQ [25] experiments were demonstrated to improve the resolution in ¹H DQ/¹H SQ, but they require long experiment time. Moreover, the simultaneous transfers from a ¹H nucleus to multiple ¹Hs partially hamper the resolution enhancement due to the broadband nature of the ¹H–¹H polarization transfer. A selective pulse on ¹H might also be used to reduce the dimensionality and experiment time. However, this approach is only applicable to well-resolved ¹H peaks and is not suitable to separate the overlapped ¹H resonances.

In order to separate the overlapped ¹H peaks in a reasonable experimental time, we propose a novel single-channel sequence called MIxed Selective Excitation/Recoupling And Broadband recoupLing Experiment (MISERABLE). Herein, the selective ¹H recoupling sequence is achieved using SERP [[26], [27], [28]]) whereas the broadband recoupling sequence employed is BABA [[29], [30], [31], [32]]. Different from the conventional approaches that are based on chemical shifts, our proposed method is based on the distances between a well-resolved ¹H peak to the overlapped ¹H peaks together with the isotropic chemical shifts. This novel approach is feasible since SERP can selectively polarize the spatially closest ¹H nucleus among the overlapped ¹H peaks, provided that the distance is within 3.5 ​Å [26,27]. Fortunately, the presence of such a well-resolved peak is frequently observed, such as OH and NH moieties. The concept of this novel method includes three steps. Firstly, a well-resolved peak is selected by a conventional selective pulse. Secondly, this isolated peak selectively transfers its polarization to a specific ¹H peak among the overlapped ¹H peaks by the SERP sequence. The ¹H carrier frequency during the SERP transfer is set between the isolated peak and overlapped peaks, ensuring the selective polarization transfer to the nearest ¹H nucleus among overlapped resonances and thus, the separation can be achieved. Thirdly, the spatial proximities of the two specific ¹Hs to neighboring ¹Hs are studied by the BABA sequence. The feasibility and applicability of MISERABLE to resolve the overlapped ¹H peaks and to distinguish their ¹H–¹H correlations is demonstrated on β-L-aspartyl-l-alanine (AspAla) and Pioglitazone.HCl (PioHCl).