Relaxation Editing Using Long-Lived States and Coherences for

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Relaxation Editing using Long-Lived States and Coherences for analysis of mixtures Maninder Singh, Vineet Kumar Soni, Rituraj Mishra, and Narayanan Damodar Kurur Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00050 • Publication Date (Web): 16 Feb 2016 Downloaded from http://pubs.acs.org on February 18, 2016

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Analytical Chemistry

Relaxation editing using Long-Lived States and Coherences for analysis of Mixtures Maninder Singh*,†, Vineet Kumar Soni‡, Rituraj Mishra†, Narayanan D Kurur*,† †

Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.

‡

Department of Chemistry, Indian Institute of Technology Jodhpur, Ratanada, Rajasthan 342011, India.

ABSTRACT: Nuclear Magnetic Resonance (NMR) is a powerful tool for structural and dynamical studies of molecules. Although widely applicable, the search for novel spectral editing methods that facilitate spectral assignment of peaks in high resolution NMR is highly desirable. Earlier, the sensitivity of lifetime of spin states (spin-lattice relaxation time, ) and coherences (spin-spin relaxation time, ) to the immediate environment was utilized for spectral editing in solution NMR. Long-Lived States (LLS) and Coherences (LLCs) were recently uncovered to have longer and more domain sensitive lifetime than other type of states and coherences. Herein, this longevity and increased sensitivity of LLS and LLC lifetime is utilized for more enhanced dispersion in relaxation editing in NMR. The generality of the method as a powerful tool in spectral editing is confirmed with molecules containing a mixture of strongly and weakly coupled spin systems and finally with metabolomic mixture. Extension to INEPT, COSY and HSQC are also demonstrated.

Determination of the high resolution solution structure of molecules, especially macromolecules and metabolomic mixtures using NMR spectroscopy requires that resonances observed in the NMR spectra to be unequivocally assigned to individual nuclei.1 Therefore, the search for new spectral resolution techniques continues. The 1H NMR spectra of many important macromolecules or biological samples are information rich, but unravelling it is complex owing to the superposition of resonances from the presence of different chemical species. This makes resonance assignments difficult. In order to find a solution, various spectral-editing NMR experiments were introduced.2-7 Spectral editing is one of the major building blocks for spectral resolution in high resolution NMR. One approach combines spin-relaxation filters (based on lifetimes and ) with both one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy.2-7 These methods separate NMR resonances on the basis of their relaxation times which results in simplification of the complex spectra. Relaxation/Spectral editing experiments are based on the principle that the NMR peaks, especially in macromolecule/complex mixture, often have different lifetimes (either or ). This difference is exploited for the selective suppression of NMR signals.8-11 In this way, the signal of larger species may be zeroed enhancing the detection of signals from smaller molecules. Often in macromolecules or samples containing a mixture of molecules, the spread in the longitudinal or transverse relaxation time between different 1H nuclei is not sufficient to differentiate their attenuation in spectrum. Extending this range of timescales in order to efficiently

filter the spectrum is welcome. Recently some methods have been proposed that create long-lived states (LLS)12 and coherences (LLC)13 in coupled spin systems with nuclear spin memory beyond the longitudinal and transverse relaxation time. For example, the lifetime of LLS, , of 37 is reported in a partially deuterated saccharide14 and in N labelled N O molecules, it has been observed to be several tens of minutes.15 The LLC lifetime ( ) has also been observed to vary 3 − 9 in different motional regimes.13,16 Long-Lived (Singlet) States and Coherences offer an exciting opportunity to use the enhanced lifetime of states and coherences in NMR for applications to various structural and dynamical studies.17-22 The characteristic long lifetime, , of LLS is due to the difference in the symmetry properties of the intramolecular dipole-dipole relaxation operator and singlet state. Unlike , the LLC lifetime, , is not attributable to fundamental symmetry properties. More importantly, the LLCs are immune to the spatial inhomogeneity of the main magnetic field as well as to its time fluctuations.23,24 Earlier, metabolic profiling of cells, tissues and biological fluids was successfully performed using and filtering strategies.25 Here we propose and demonstrate the use of and for spectral editing in NMR. In this work, we describe an approach for stepwise filtering of peaks in the NMR spectra using LLS and LLC. The usual method for generating LLS and LLC requires region specific excitation of two coupled spins which restricts their usefulness for various studies. Hence, the ability to excite LLS or LLC in a broadband manner is required to apply relaxation editing strategy similarly for or as for or . The methods used for broad-

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band LLS and LLC excitation are discussed in detail in the supporting information (ESI). We have first tested the and filtering strategy containing two and three coupled spin-1/2 pairs. Eventually these methods are demonstrated for a sample containing metabolomic mixture and for several one and two-dimensional (INEPT, COSY and HSQC) experiments. The LLC measurements were performed on a 300 MHz Bruker Avance III spectrometer equipped with a 5mm multi-nuclear probe, whereas the LLS measurements were done on a 400 and 500 MHz Bruker Avance III spectrometer. All the experiments were done at room temperature (298 K). The generality of the LLS and LLC methods has been tested for relaxation editing, which requires stepwise filtering of NMR spectra by varying the length of filter. In the sequence shown Figure S1 and Figure S2(b), the length of spin lock (LLS or LLC sustaining) interval corresponds to the length of filter for spectral editing. The feasibility of the strategy is first demonstrated in two molecules 2,3,6trichloro--nitrostyrene and 1,3-di-p-tolyl-propenone, containing two and three pairs of coupled protons. Figure 1 and 2 depicts the structure, the , , and edited spectra of these molecules, which shows well resolved signals for different proton pairs. 1

Figure 2. H relaxation edited spectra of 1,3-di-p-tolylpropenone having three pairs of coupled protons (shown as Roman numerals). The relaxation times are shown in the table. The spectra were acquired in solvent CDCl3 (298 K) with the sequence given in Figure S1, S2, inversion recovery, and CPMG sequence.

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Figure 1. H relaxation edited spectra of 2,3,6-trichloro-nitrostyrene containing two pairs of coupled protons (A and B). The relaxation times are shown in the table. The spectra were acquired in solvent CDCl3 (298 K) with the sequence given in Figure S1, S2, inversion recovery, and CPMG sequence.

The potential of , , and filters for selective suppression of the 1H signals (having different , , and values) for different pairs was assessed by plotting the 1H signal intensities as a function of the incremented time. As shown in Figure 1 and 2, the signal intensity for different pairs decay ( , and ) or recover ( ) with times that are dependent on their chemical environment. In Figure 1, the signals due to the two protons pairs (A and B) in 2,3,6-trichloro--nitrostyrene are clearly visible for a filter length of 2.5 s. At the filter length of 12.5 s however, the signals from the B pair have almost vanished while that from A are still present. Similarly, for filter length of 20 s, the peaks from the A pair is visible while that for B pair is missing. Likewise, in Figure 2, the signals for the three proton pairs (I, II, III) in 1,3-di-p-tolyl-propenone are all present upto 14 s and 10 s of and filter lengths respectively. Nevertheless, the use of a and filter length of 14.5 s and 11 s results in the decay of peaks from (II) pair only while decay of peaks from (III) pair are observed at and filter length of 15 s and 19 s respectively. This discrimination is not possible with any length of filter due to the small variation in the of the various protons. Signal discrimination is better with and is optimal when the filter length corresponds to the null point in the recovery curve.


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Figure 3. 300 MHz H LLC spectra of metabolomic mixture (at neutral pH) edited on the basis of differences in lifetimes using the pulse sequence of Figure S2(b). Top: LLC spectrum with spin-lock time or filter length of 0 s. Peaks arising from the different components of metabolomic mixture are: Histidine ‘A’, Uracil ‘B’, Threonine ‘C’, Valine ‘D’ and Citric acid ‘E’. The large and broad peak present is of water as impurity in D2O. Middle to bottom: All spectra are obtained at different filter lengths to filter each component of metabolomic mixture.

Indeed, as indicated by an arrows in filtered spectra, sequential suppression of each component was possible by implementation of increasing filters from 2.2 s (Citric acid) to 7.8 s (Histidine) to 9.4 s (Valine) to 13.8 s (Threonine) and finally affording a spectra showing the most isolated protons of Uracil. The occurrence of signals upto length 14 allows one to effectively and easily set the length of filter which is restricted earlier to a maximum 9 and 8 in and filtering strategies (see Figure 4). While examining Figure 3 and 4 carefully, we found that our goal of selective suppression of peaks could be efficiently achieved by using LLC filtering strategy owing to increase in time difference between adjacent filters lengths ∆ 2 − 5 in contrast to ∆ 2 − 3 or ∆ 0.3 − 2 . The same is true for filtering strategy using broadband LLS excitation. Nevertheless, it is important to point out that the 1D method for broadband LLS excitation (Figure S1) depends upon the coupling information of the spin system, resulting in some phase distortions (see Figure 2) in spectrum. However, this problem have been taken care of in its 2D variant.14 Finally, the application of this LLS filtering strategy is explored for several important 1D and 2D-NMR experiments such as INEPT, COSY and HSQC. In these LLS filtering experiments, the first excitation pulse in original experiments is replaced with broadband LLS pulse sequence. Typically, these methods are similar to the several 2D-NMR spectral filtering techniques already reported by Pinto et.al.,26 and by Liu et. al.11 The unique feature of this LLS filtering is that signal originating from single spins is eliminated in much the same way as it happens in

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Figure 4. H and edited spectra of metobolomic mixture. These spectra were acquired by using standard Inversion recovery (I.R) and CPMG sequence in solvent D2O (298 K). The window (the time difference between adjacent filters) ∆ 0.3 − 2 and ∆ 2 − 3 is found to be less than ∆ 2 − 5 (see Figure 3).

The fidelity of the strategy in relaxation editing is further tested by analyzing a sample of metabolomic mixture (at pH = 7) containing several biologically important metabolites – Histidine, Uracil, Threonine, Citric acid and Valine. Figure 3 shows the 1H NMR LLC spectra acquired by using sequence given in Figure S2(b). The topmost LLC spectrum (Figure 3) is obtained without any spin locking. The peaks pertinent to each metabolomic component in Figure 3 are labelled as ‘A to E’. As shown in Figure 3, the intensity for each component decreases with the filter length that ensures their stepwise selective suppression.

Figure 5. Top: Structure of Uridine 5’-monophosphate 1 13 (UMP). Middle to bottom: LLS filtered H to C transfer of 13 magnetization is shown through a) normal C spectra b) 13 original INEPT spectra and c) C filtered INEPT spectra of UMP. The LLS is excited in a coupled proton pair (labelled as red) as shown in the structure above. The magnetization due to all other protons is effectively filtered through the LLS filter. Only the remaining magnetization due to the proton 13 pair (labelled as red) gets transferred to their adjacent C 13 nuclei as shown in C filtered INEPT spectra. These spectra were recorded on 400 MHz Bruker AVANCE III spectrometer containing 5mm BBO multinuclear probe with 512 transients each. The filter length used for recording spectra (c) is 300 ms.



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mixture and several 1D and 2D NMR experiments (INEPT, COSY and HSQC). A comparison is also made with earlier methods of relaxation editing using relaxation time and . The enhanced lifetime of LLC/LLS for each spin pair compared to or results in a wider spread of time scale and facilitates selective suppression of peaks. This extension of lifespan eases the process of setting/calibrating the filter length to be used for spectral editing. Application of the above strategies is expected to aid complex metabolomic analysis and LLS enhanced ligand-protein binding studies.22 In the latter, instead of monitoring intensities from the complex 1H NMR spectra, one can follow the carbon intensities by using these LLS filtered 13C-INEPT experiment. The method may also assist in desired synthesis or post functionalization of large macromolecules.

ASSOCIATED CONTENT Supporting Information 1

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Figure 6. Original and LLS filtered COSY and H- C HSQC spectra of UMP. The spectral peaks are circled in red to distinguish them from background noise. These spectra were recorded on 400 MHz Bruker AVANCE III spectrometer containing 5mm BBO multinuclear probe with 512 transients in dimension and 1024 transients in dimension. The LLS was excited in a pair of coupled protons present in the uridine moiety of UMP (as shown in structure in Figure 5). The filter length used for recording these LLS filtered HSQC and COSY spectra is 300 ms.

Methods for broadband LLS and LLC excitation were explained in details with GAMMA simulations. Also synthetic procedure for 2,3,6-trichloro--nitrostyrene and 1,3-di-ptolyl-propenone along with NMR and IR data is provided. This material is available free of charge via the Internet at http://pubs.acs.org.

double quantum filtered (DQF) experiments.27 The relaxation editing strategy can then be further used for filtering these surviving LLS signals. The applicability of these -filtered 1D and 2D methods has been ascertained for a nucleotide uridine 5’- monophosphate (UMP). The 1DLLS filtered INEPT and 2D-LLS filtered COSY and HSQC spectra are illustrated in Figure 5 and 6. In LLS filtered 13 C-INEPT spectra (see Figure 5), the peaks originating from 13C nuclei that are directly attached with LLS proton pair persist while all other peaks are suppressed. Similar behaviour is seen for LLS filtered COSY and HSQC spectra (see Figure 6). These spectra confirm the validity of these LLS filtered 1D and 2D NMR methodologies.

Notes

In summary, we have demonstrated and to be good candidates for relaxation editing in NMR. The characteristic range of or values within the sample containing a mixture of components or components within a large macromolecule can be exploited in spectral edited NMR experiments for the stepwise filtering of complicated spectra. The resulting spectra benefits from reduced signal overlap unravelling signal assignment and characterization. The LLS and LLC filtering strategies are demonstrated in weakly and strongly coupled spin systems separately (shown in ESI) which is extended to molecules containing two or three pairs of weakly and strongly coupled spins. The potential of these strategies was further substantiated by an application to metabolomic

AUTHOR INFORMATION Corresponding Author * Email for M. Singh: [email protected]. * Email for N. D. Kurur: [email protected]. The authors declare no competing financial interest.

ACKNOWLEDGMENT MS thanks the University Grants Commission (UGC), India for financial support. We thank Dr. Neel Bhavesh, Staff scientist at International Center for Genetic Engineering and Biotechnology (ICGEB) India, for providing access to the high field NMR spectrometers at ICGEB and National Institute of Immunology (NII), New Delhi. The NMR spectrometers at the ICGEB and NII were procured through support from Department of Biotechnology (DBT), Government of India.

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Graphical Abstract


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Relaxation Editing Using Long-Lived States and Coherences for

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