Cross‐Peaks in Simple Two‐Dimensional NMR Experiments from Chemical Exchange of Transverse Magnetisation

Two‐dimensional correlation measurements such as COSY, NOESY, HMQC, and HSQC experiments are central to small‐molecule and biomolecular NMR spectroscopy, and commonly form the basis of more complex experiments designed to study chemical exchange occurring during additional mixing periods. However, exchange occurring during chemical shift evolution periods can also influence the appearance of such spectra. While this is often exploited through one‐dimensional lineshape analysis (“dynamic NMR”), the analysis of exchange across multiple chemical shift evolution periods has received less attention. Here we report that chemical exchange‐induced cross‐peaks can arise in even the simplest two‐dimensional NMR experiments. These cross‐peaks can have highly distorted phases that contain rich information about the underlying exchange process. The quantitative analysis of such peaks, from a single 2D spectrum, can provide a highly accurate characterisation of underlying exchange processes.     

Chemical shift assignment and conformational analysis of monoalkylated acylguanidines

Monoalkylated acylguanidines are important functional groups in many biologically active compounds and additionally applied in coordination chemistry. Yet a straightforward assignment of the individual NH chemical shifts and the acylguanidine conformations is still missing. Therefore, in this study, NMR spectroscopic approaches for the chemical and especially the conformational assignment of protonated monoalkylated acylguanidines are presented. While NOESY and ³J_{H, H} scalar couplings cannot be applied successfully for the assignment of acylguanidines, ⁴J_{H, H} scalar couplings in ¹H,¹H COSY spectra allow for an unambiguous chemical shift and conformational assignment. It is shown that these ⁴J_{H, H} long‐range couplings between individual acylguanidinium NH resonances are observed solely across all‐trans (w) pathways. Already one cis orientation in the magnetisation transfer pathway leads to signal intensities below the actual detection limit and significantly lower than cross‐peaks from ²J_{NH, NH} couplings or chemical exchange. However, it should be noted that also in the case of conformational exchange being fast on the NMR time scale, averaged cross‐peaks from all‐trans ⁴J_{H, H} scalar couplings are detected, which may lead at first glance to an incomplete or even wrong conformational analysis. Copyright © 2010 John Wiley & Sons, Ltd.     

Stacking Structure of Confined 1‐Butanol in SBA‐15 Investigated by Solid‐State NMR Spectroscopy

Understanding the complex thermodynamic behavior of confined amphiphilic molecules in biological or mesoporous hosts requires detailed knowledge of the stacking structures. Here, we present detailed solid‐state NMR spectroscopic investigations on 1‐butanol molecules confined in the hydrophilic mesoporous SBA‐15 host. A range of NMR spectroscopic measurements comprising of ¹H spin–lattice (T₁), spin–spin (T₂) relaxation, ¹³C cross‐polarization (CP), and ¹H,¹H two‐dimensional nuclear Overhauser enhancement spectroscopy (¹H,¹H 2D NOESY) with the magic angle spinning (MAS) technique as well as static wide‐line ²H NMR spectra have been used to investigate the dynamics and to observe the stacking structure of confined 1‐butanol in SBA‐15. The results suggest that not only the molecular reorientation but also the exchange motions of confined molecules of 1‐butanol are extremely restricted in the confined space of the SBA‐15 pores. The dynamics of the confined molecules of 1‐butanol imply that the ¹H,¹H 2D NOESY should be an appropriate technique to observe the stacking structure of confined amphiphilc molecules. This study is the first to observe that a significant part of confined 1‐butanol molecules are orientated as tilted bilayered structures on the surface of the host SBA‐15 pores in a time‐average state by solid‐state NMR spectroscopy with the ¹H,¹H 2D NOESY technique.     

An NMR investigation of the interaction of polyethylene oxide with water‐soluble poly(vinyl phenol‐co‐potassium styrene sulfonate)

The water‐soluble complex of polyethylene oxide (PEO) with poly (vinyl phenol‐co‐potassium styrene sulfonate) (PVPh‐co‐KSS) was studied by liquid‐state NMR. PEO showed two peaks in the ¹H spectra, which corresponded to the free and complexed PEO. The ratio of the free PEO/complexed PEO was decreased with the increase in the mixing ratio of PVPh‐co‐KSS/PEO. Some of the complex formation disappeared when the pH was raised from 6.4 to 12.0. It had been thought that at high pH, the phenolic groups dissociate and thus cannot form hydrogen bonds. The fact that NMR indicates some interaction at pH 12.0 implies there are some other interactions, such as hydrophobic interactions between the aromatic rings and the polyether methylene groups, contributing to PEO and PVPh‐co‐KSS complex formation. Nuclear Overhauser effect (NOE) cross peaks were observed between PEO and the aromatic protons of PVPh‐co‐KSS in nuclear Overhauser effect spectra (NOESY) suggesting that the distance between PEO and the aromatic protons of PVPh‐co‐KSS was less than 5 Å. The exchange between the complexed PEO and the free PEO was slow on the NMR time scale. The ratio of the integral of the complexed PEO to the free PEO increased with temperature, indicating that the number of PEO segments interacting with the aromatic ring increases with temperature. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 1276–1284, 2000     

Real‐time homonuclear broadband and band‐selective decoupled pure‐shift ROESY

Unambiguous spectral assignments in ¹H solution‐state NMR are central, for accurate structural elucidation of complex molecules, which is often hampered by signal overlap, primarily because of scalar coupling multiplets, even at typical high magnetic fields. The recent advances in homodecoupling methods have shown powerful means of achieving high resolution pure‐shift ¹H spectra in 1D and also in 2D J‐correlated experiments, by effectively collapsing the multiplet structures. The present work extends these decoupling strategies to through‐space correlation experiments as well and describes two new pure‐shift ROESY pulse schemes with homodecoupling during acquisition, viz., homodecoupled broadband (HOBB)‐ROESY and homodecoupled band‐selective (HOBS)‐ROESY. Furthermore, the ROESY blocks suppress the undesired interferences of TOCSY cross peaks and other offsets. Despite the reduced signal sensitivity and prolonged experimental times, the HOBB‐ROESY is particularly useful for molecules that exhibit an extensive scalar coupling network spread over the entire ¹H chemical shift range, such as natural/synthetic organic molecules. On the other hand, the HOBS‐ROESY is useful for molecules that exhibit well‐separated chemical shift regions such as peptides (NH, Hα and side‐chain protons). The HOBS‐ROESY sensitivities are comparable with the conventional ROESY, thereby saves the experimental time significantly. The power of these pure‐shift ROESY sequences is demonstrated for two different organic molecules, wherein complex conventional ROE cross peaks are greatly simplified with high resolution and sensitivity. The enhanced resolution allows deriving possibly more numbers of ROEs with better accuracy, thereby facilitating superior means of structural characterization of medium‐size molecules. Copyright © 2014 John Wiley & Sons, Ltd.     

Recovering Invisible Signals by Two‐Field NMR Spectroscopy

Nuclear magnetic resonance (NMR) studies have benefited tremendously from the steady increase in the strength of magnetic fields. Spectacular improvements in both sensitivity and resolution have enabled the investigation of molecular systems of rising complexity. At very high fields, this progress may be jeopardized by line broadening, which is due to chemical exchange or relaxation by chemical shift anisotropy. In this work, we introduce a two‐field NMR spectrometer designed for both excitation and observation of nuclear spins in two distinct magnetic fields in a single experiment. NMR spectra of several small molecules as well as a protein were obtained, with two dimensions acquired at vastly different magnetic fields. Resonances of exchanging groups that are broadened beyond recognition at high field can be sharpened to narrow peaks in the low‐field dimension. Two‐field NMR spectroscopy enables the measurement of chemical shifts at optimal fields and the study of molecular systems that suffer from internal dynamics, and opens new avenues for NMR spectroscopy at very high magnetic fields.     

NMR Backbone Assignment of Large Proteins by Using 13Cα‐Only Triple‐Resonance Experiments

Nuclear magnetic resonance (NMR) is a powerful tool to interrogate protein structure and dynamics residue by residue. However, the prerequisite chemical‐shift assignment remains a bottleneck for large proteins due to the fast relaxation and the frequency degeneracy of the ¹³C_α nuclei. Herein, we present a covariance NMR strategy to assign the backbone chemical shifts by using only HN(CO)CA and HNCA spectra that has a high sensitivity even for large proteins. By using the peak linear correlation coefficient (LCC), which is a sensitive probe even for tiny chemical‐shift displacements, we correctly identify the fidelity of approximately 92 % cross‐peaks in the covariance spectrum, which is thus a significant improvement on the approach developed by Snyder and Brüschweiler (66 %) and the use of spectral derivatives (50 %). Thus, we calculate the 4D covariance spectrum from HN(CO)CA and HNCA experiments, in which cross‐peaks with LCCs above a universal threshold are considered as true correlations. This 4D covariance spectrum enables the sequential assignment of a 42 kDa maltose binding protein (MBP), in which about 95 % residues are successfully assigned with a high accuracy of 98 %. Our LCC approach, therefore, paves the way for a residue‐by‐residue study of the backbone structure and dynamics of large proteins.     

Influence of NMR Data Completeness on Structure Determinations of Homodimeric Proteins

The structure determination of homodimeric proteins by NMR using conventional NOESY experiments is still challenging due to the degeneracy of the chemical shifts in the identical monomers, which causes ambiguity in the NOE assignments. Residues involved in the interface between two monomers provide essential intermolecular NOEs for the structure determinations of homodimeric proteins. Hence NMR data, such as NOE peak lists and chemical shift assignments of these interface residues, play a crucial role for the successful structure determination of homodimeric proteins. This paper extends our previous report (Lin, Y.‐J.; Kirchner, D. K.; Güntert, P. J. Magn. Reson.­ 2012 , 222, 96) and investigates the influence of incomplete NOESY peak lists combined with incomplete ¹H chemical shift assignments of the interface residues on the structure determination of homodimeric proteins using the program CYANA. Data incompleteness was simulated by random omission of both NOESY cross peaks and interface ¹H chemical shifts. Our results for three proteins with different percentages of interface residues reveal that the algorithm can tolerate about 40–50% NOESY peak omission with complete interface chemical shift assignments, which indicates that partial NOESY peak omission does not cause severe problems when the interface chemical shifts are completely assigned. Combining NOESY peak omission with incomplete interface chemical shift assignments, the tolerance for interface chemical shift omission decreases with the extent of omitted NOESY peaks. The tolerance for unassigned interface side chain, methyl and aromatic chemical shifts is affected more strongly by NOESY peak omission than that for the omission of general interface ¹H chemical shifts including the backbone. In general about 10–30% peaks omission is tolerated in conjunction with missing chemical shift assignments. If more NOESY peaks are omitted calculations gradually become unstable and tend not to tolerate any missing interface chemical shifts. A large amount of omitted NOESY peaks, for instance 30% omission in our calculations, could decrease the tolerance for missing aromatic or methyl interface ¹H chemical shifts to as few as 2–4 missing chemical shifts, suggesting that complete aromatic and methyl ¹H chemical shift assignments are important when the NOESY peak data is significantly incomplete. Finally, for homodimeric proteins with a low percentage of interface residues, our results reveal that the omission of NOESY peaks, even at an extent of only 10%, can result in no tolerance against the omission of interface ¹H chemical shifts, suggesting that the completeness of both interface ¹H chemical shift assignments and NOESY peaks are important for the successful structure determination of proteins with a small homodimer interface.     

Simplifying LR‐HSQC spectra using a triple‐quantum filter: The LR‐HTQC experiment

Long‐range heteronuclear single quantum correlation (LR‐HSQC) experiments may be applied for detecting long‐range correlations but suffer from two disadvantages, common to all heteronuclear long‐range correlation experiments: (i) The information density in LR‐HSQC spectra may be too high to be used directly without “filtering out” shorter range correlations, and (ii) often, substantial differences in intensity among cross peaks exist, potentially hampering the visualization of weak, often crucial cross peaks. In this contribution, we propose a modified LR‐HSQC experiment, the LR‐HTQC experiment (Long‐Range Heteronuclear Triple Quantum Correlation) that partially solves the problems aforementioned. We show theoretically and experimentally that the LR‐HTQC experiment removes the intense cross peaks of CH spin pairs, substantially reduces the medium intensity of cross peaks originating from CHH' spin systems, whereas the typically weak intensity of cross peaks of CHH'H″ and C(H)_n, _{n > 3} spin systems is less affected. Consequently, the LR‐HTQC experiment affords simplified long‐range heteronuclear shift correlation spectra and scales down large intensity differences among different types of cross peaks, although a certain general reduction of signal intensities has to be accepted.     

NMR studies on 1,2‐dihydro‐2‐(4‐aminophenyl)‐4‐[4‐(4‐aminophenoxy)‐4‐phenyl]‐(2H)phthalazin‐1‐one

An unsymmetrical heterocyclic diamine, 1,2‐dihydro‐2‐(4‐aminophenyl)‐4‐[4‐(4‐aminophenoxy)‐4‐phenyl]‐(2H)phthalazin‐1‐one, was synthesized. Its ¹H and ¹³C NMR spectra were completely assigned by utilizing the two‐dimensional heteronuclear ¹³C–¹H multiple‐bond coherence (HMBC) spectroscopy, and heteronuclear ¹³C–¹H one‐bond correlation spectroscopy, homonuclear shift correlation spectroscopy (H,H‐COSY) and rotating frame Overhauser enhancement spectroscopy (ROESY). The structure of the compound was shown to be the phthalazinone rather than the phthalazine ether from cross peaks and chemical shifts of the protons. Copyright © 2002 John Wiley & Sons, Ltd.     

NMR Spectroscopic Study of the Self‐Aggregation of 3‐Hexen‐1,5‐diyne Derivatives

The self‐assembly of polycatenar molecules derived from 1,6‐diphenyl‐3,4‐dipropyl‐3‐hexen‐1,5‐diyne has been studied in detail by solution NMR spectroscopy. The analysis of the concentration‐ and temperature‐dependent evolution of the chemical shifts and the diffusion coefficients in [D₁₂]cyclohexane agrees well with an isodesmic model of association in this solvent. The association constants for the stacking and entropy and enthalpy of the process have been obtained. The driving force for the aggregation process is provided by a negative enthalpy (ΔH), which is partially compensated by a negative entropy (ΔS). A structural study of the self‐assembly in solution has been carried out with the help of NOESY NMR spectroscopic experiments.     

Complete NMR assignment of 3, 4‐seco‐lup‐20(29)‐en‐3‐oic acid from Decatropis bicolor

Complete ¹H and ¹³C NMR chemical shift assignments for 3,4‐seco‐lup‐20(29)‐en‐3‐oic acid ( 1 ) have been established by means of two‐dimensional COSY, HSQC, HMBC and NOESY spectroscopic experiments as well as by analysis of MS data. Compound 1 was isolated from Decatropis bicolor (Zucc.) Radlk. (Rutaceae) in addition to six coumarins and one alkaloid of known structure. Copyright © 2012 John Wiley & Sons, Ltd.     

Solid‐State Structures and Solution Analyses of a Phenylpropylpyridine N‐Oxide and an N‐Methyl Phenylpropylpyridine

The crystal structures of phenylpropylpyridine‐N‐oxide and N‐methyl‐phenylpropylpyridinium iodide are compared, revealing that hydrogen bonding with the solvent molecule plays an important role in the N‐oxide compound, whilst electrostatic interactions are predominant in controlling the solid‐state orientation of the N‐methylated compound. Fluorescence spectroscopy and NOESY indicate that in contrast to the previously reported pyridinium iodide, the N‐oxide is not subject to intramolecular π‐stacking, as judged by excimer emission and a lack of corresponding cross peaks, respectively.     

Hydroxo‐Bridged Dimers of Oxo‐Centered Ruthenium(III) Triangle: Synthesis and Spectroscopic and Theoretical Investigations

The homometallic hexameric ruthenium cluster of the formula [Ru^III₆(μ₃‐O)₂(μ‐OH)₂((CH₃)₃CCO₂)₁₂(py)₂] ( 1 ) (py=pyridine) is solved by single‐crystal X‐ray diffraction. Magnetic susceptibility measurements performed on 1 suggest that the antiferromagnetic interaction between the Ru^III centers is dominant, and this is supported by theoretical studies. Theoretical calculations based on density functional methods yield eight different exchange interaction values for 1 : J₁=?737.6, J₂=+63.4, J₃=?187.6, J₄=+124.4, J₅=?376.4, J₆=?601.2, J₇=?657.0, and J₈=?800.6 cm^?1. Among all the computed J values, six are found to be antiferromagnetic. Four exchange values (J₁, J₆, J₇ and J₈) are computed to be extremely strong, with J₈, mediated through one μ‐hydroxo and a carboxylate bridge, being by far the largest exchange obtained for any transition‐metal cluster. The origin of these strong interactions is the orientation of the magnetic orbitals in the Ru^III centers, and the computed J values are rationalized by using molecular orbital and natural bond order analysis. Detailed NMR studies (¹H, ¹³C, HSQC, NOESY, and TOCSY) of 1 (in CDCl₃) confirm the existence of the solid‐state structure in solution. The observation of sharp NMR peaks and spin‐lattice time relaxation (T1 relaxation) experiments support the existence of strong intramolecular antiferromagnetic exchange interactions between the metal centers. A broad absorption peak around 600–1000 nm in the visible to near‐IR region is a characteristic signature of an intracluster charge‐transfer transition. Cyclic voltammetry experiments show that there are three reversible one‐electron redox couples at ?0.865, +0.186, and +1.159 V with respect to the Ag/AgCl reference electrode, which corresponds to two metal‐based one‐electron oxidations and one reduction process.     

Gradient‐based solvent suppression methods on a benchtop spectrometer

Benchtop NMR emerges as an appealing alternative to widely extend the scope of NMR spectroscopy in harsh environments and for on‐line monitoring. Obviously, the use of low‐field magnets induces a dramatic reduction of the spectral resolution leading to frequent peak overlaps. This issue is even more serious because applications such as chemical process monitoring involve the use of non‐deuterated solvents, leading to intense and broad peaks overlapping with the signals of interest. In this article, we highlight the need for efficient suppression methods compatible with flowing samples, which is not the case of the common pre‐saturation approaches. Thanks to a gradient coil included in our benchtop spectrometer, we were able to implement modern and efficient solvent suppression blocks such as WET or excitation sculpting to deliver quantitative spectra in the conditions of the on‐line monitoring. While these methods are commonly used at high field, this is the first time that they are investigated on a benchtop setting. Their analytical performance is evaluated and compared under static and on‐flow conditions. The results demonstrate the superiority of gradient‐based methods, thus highlighting the relevance of implementing this device on benchtop spectrometers. The comparison of major solvent suppression methods reveals an optimum performance for the WET‐180‐NOESY experiment, both under static and on‐flow conditions. Copyright © 2016 John Wiley & Sons, Ltd.     

Solid‐state 13C NMR study on thermal dehydration process of poly(methacrylic acid)(PMAA)

Thermal dehydration process of PMAA was investigated by solid‐state ¹³C NMR. For heat‐treated PMAA at 150°C, at which the dehydration goes very slowly, we observed three ¹³C peaks at 172, 178, and 187 ppm in the carboxyl group region. The peak at 172 ppm is due to the intramolecular cyclic anhydrides by comparing the reported value of ¹³C chemical shift. The peaks at 178 and 187 ppm were assigned to regularly aligned free carboxylic acids and intermolecular acid dimers, respectively, from the 2D‐exchange ¹³C NMR spectra, ¹³C chemical shift values and IR spectra. We concluded that by heat‐treatment the rearrangement of intermolecular hydrogen bonding of the carboxylic acids in PMAA occurs firstly to form the regularly aligned acid dimers, and the dimers dissociated to be the regularly aligned free carboxylic acids at high temperatures. The adjacent free carboxyl acids dehydrate with each other, resulting in the formation of intramolecular anhydrides. © 1999 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 37: 2007–2012, 1999     

Multidimensional solid‐state NMR studies of the structure and dynamics of pectic polysaccharides in uniformly 13C‐labeled Arabidopsis primary cell walls

Plant cell wall (CW) polysaccharides are responsible for the mechanical strength and growth of plant cells; however, the high‐resolution structure and dynamics of the CW polysaccharides are still poorly understood because of the insoluble nature of these molecules. Here, we use 2D and 3D magic‐angle‐spinning (MAS) solid‐state NMR (SSNMR) to investigate the structural role of pectins in the plant CW. Intact and partially depectinated primary CWs of Arabidopsis thaliana were uniformly labeled with ¹³C and their NMR spectra were compared. Recent ¹³C resonance assignment of the major polysaccharides in Arabidopsis thaliana CWs allowed us to determine the effects of depectination on the intermolecular packing and dynamics of the remaining wall polysaccharides. 2D and 3D correlation spectra show the suppression of pectin signals, confirming partial pectin removal by chelating agents and sodium carbonate. Importantly, higher cross peaks are observed in 2D and 3D ¹³C spectra of the depectinated CW, suggesting higher rigidity and denser packing of the remaining wall polysaccharides compared with the intact CW. ¹³C spin–lattice relaxation times and ¹H rotating‐frame spin–lattice relaxation times indicate that the polysaccharides are more rigid on both the nanosecond and microsecond timescales in the depectinated CW. Taken together, these results indicate that pectic polysaccharides are highly dynamic and endow the polysaccharide network of the primary CW with mobility and flexibility, which may be important for pectin functions. This study demonstrates the capability of multidimensional SSNMR to determine the intermolecular interactions and dynamic structures of complex plant materials under near‐native conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

Offset‐compensated and zero‐quantum suppressed ROESY provides accurate 1H–1H distances in small to medium‐sized molecules

A robust version of the off‐resonance ROESY pulse scheme is suggested for the measurement of proton–proton distances or slow chemical exchange in small to medium‐sized molecules. The method implements adiabatic ramps to establish a pair of opposite frequency off‐resonance spin lock fields – with optionally randomized duration – and adiabatic inversion pulses with simultaneous gradients for efficient zero‐quantum suppression. The amended pulse sequence yields pure absorption cross‐peaks and works safely for small to medium‐sized molecules. The applicability of the method has been demonstrated using small, rigid molecules (strychnine and codeine) and was also applied for a cyclic peptide and a small protein. We found that the pure phase cross‐peaks of the new ROESY version are beneficial for distance measurements. The one‐dimensional (selective) version of the new method is also powerful for measuring selected pair‐wise interactions and distance determination. Copyright © 2016 John Wiley & Sons, Ltd.