One-dimensional NMR experiment

In the one-dimensional NMR experiments discussed earlier, the FID was recorded immediately after the pulse, and the only time domain involved (ij) was the one in which the FID was obtained. If, however, the signal is not recorded immediately after the pulse but a certain time interval (time interval (the evolution period) the nuclei can be made to interact with each other in various ways, depending on the pulse sequences applied. Introduction of this second dimension in NMR spectroscopy, triggered byjeener s original experiment, has resulted in tremendous advances in NMR spectroscopy and in the development of a multitude of powerful NMR techniques for structure elucidation of complex organic molecules. 

NMR is a remarkably flexible technique that can be effectively used to address many analytical issues in the development of biopharmaceutical products. Although it is already more than 50 years old, NMR is still underutilized in the biopharmaceutical industry for solving process-related analytical problems. In this chapter, we have described many simple and useful NMR applications for biopharmaceutical process development and validation. In particular, quantitative NMR analysis is perhaps the most important application. It is suitable for quantitating small organic molecules with a detection limit of 1 to 10 p.g/ml. In general, only simple one-dimensional NMR experiments are required for quantitative analysis. The other important application of NMR in biopharmaceutical development is the structural characterization of molecules that are product related (e.g., carbohydrates and peptide fragments) or process related (e.g., impurities and buffer components). However, structural studies typically require sophisticated multidimensional NMR experiments. 

Distinguish clearly between a one-dimensional NMR experiment that uses a time increment, such as the inversion-recovery technique (Section 2.9 and Fig. 8.8a), and a two-dimensional NMR experiment, such as NOESY. 

Steady-state, or dummy, scans are used to allow a sample to come to equilibrium before data collection begins. As in a regular experiment, a number of scans are taken, but data are not collected during what would be the normal acquisition time. Steady-state scans are usually performed before the start of an experiment, but, for certain experiments on older instruments, may be acquired before the start of each incremented time value. This technique is not necessary in typical one-dimensional NMR experiments, but is employed in onedimensional methods that involve spectral subtraction (e.g., DEPT Section 7-2b) and virtually all two-dimensional experiments. 

The one-dimensional NMR experiment is derived from measuring the FID as a function of time. If the pulse program also contains a second time period which is incremented, then a second frequency axis can be derived from a second Fourier transform. This is the basis for two-dimensional NMR and its extension to three or even four dimensions. For example, a simple sequence such as... 

In a one-dimensional NMR experiment, data are taken as a function of a single time parameter, and the relation between these data and the frequency spectrum is the previously discussed Fourier transform relation. Over the past few years, a number of experiments have been developed in which the time intervals in the NMR experiments are divided into regions, a region ti, followed by another region, f2. The time domain signal, then, is a function of both of these times S(t) = S t, t2). An immediate result of this statement is that the frequency domain signal, 5(ft>i, C02), now becomes a three-dimensional contour plot, as shown in Fig. 8. 

The amount of computation necessary to try many conformers can be greatly reduced if a portion of the structure is known. One way to determine a portion of the structure experimentally is to obtain some of the internuclear distances from two-dimensional NMR experiments, as predicted by the nuclear Over-hauser effect (NOE). Once a set of distances are determined, they can be used as constraints within a conformation search. This has been particularly effective for predicting protein structure since it is very difficult to obtain crystallographic structures of proteins. It is also possible to define distance constraints based on the average bond lengths and angles, if we assume these are fairly rigid while all conformations are accessible. 

NOESY NMR spectroscopy is a homonuclear two-dimensional experiment that identifies proton nuclei that are close to each other in space. If one has already identified proton resonances in one-dimensional NMR spectroscopy or by other methods, it is then possible to determine three dimensional structure through NOESY. For instance, it is possible to determine how large molecules such as proteins fold themselves in three-dimensional space using the NOESY technique. The solution structures thus determined can be compared with solid-state information on the same protein obtained from X-ray crystallographic studies. The pulse sequence for a simple NOESY experiment is shown in Figure 3.23 as adapted from Figure 8.12 of reference 19. 

Finally, the last step of the procedure for optimizing experimental conditions is to identify the denaturation temperature of the protein. This step is important because the rotational tumbling rate of a protein increases with temperature, and faster tumbling results in sharper resonance lines. Therefore, the temperature during the NMR experiments should be as high as possible without denaturating the protein. The denaturation temperature can best be determined by either CD-spectroscopy or one-dimensional NMR. 

GC analyses of the pupal secretion of E. borealis have indicated the presence of vitamin E acetate and other tocopherol derivatives [49,50]. However, in tests with ants, these compounds proved to be essentially inactive, whereas the secretion itself was potently deterrent. To find and identify the active components in the pupal Epilachna borealis secretion, NMR spectroscopic studies on the fresh secretion were carried out. One and two-dimensional NMR experiments revealed that the tocopheryl acetates account for only a relatively small percentage of the beetles5 total secretion (20%), whereas the major components represented a group of previously undetected compounds. By analysis of the COSY, HSQC and HMBC spectra of the mixture, these components were shown to be esters and amides derived from three (co-l)-(2-hydroxyethylamino)alka-noic acids 44-46. HPLC analyses coupled to a mass spectrometric detector revealed that the secretion contain a highly diverse mixture of macrocyclic polyamines, the polyazamacrolides (PAMLs) 47-52 (Fig. 8). 

Standard onedimensional NMR One-dimensional NMR — simple one-pulse experiment, typically with presaturation of solvent during the recycle delay with a weak RF field To quantify small molecules To identify some simple small molecules... 

In many cases, the analytical tasks are simply to detect and quantify a specific known analyte. Examples include the detection and quantification of commonly used buffer components (e.g., Tris, acetate, citrate, MES, propylene glycol, etc.). These simple tasks can readily be accomplished by using a standard one-dimensional NMR method. In other situations, the analytical tasks may involve identifying unknown compounds. This type of task usually requires homonuclear and heteronuclear two-dimensional NMR experiments, such as COSY, TOCSY, NOESY, HSQC, HMBC, etc. The identification of unknown molecules may also require additional information from other analytical methods, such as mass spectrometry, UV-Vis spectroscopy, and IR spectroscopy.14... 

If substantial conformational differences between the in vivo and the in vitro states of the protein exist the in vitro assignments of the chemical shifts might not be transferable to the in-cell NMR spectra, making a new chemical shift assignment necessary. One of the main difficulties of measuring multi-dimensional NMR spectra of proteins inside hving cells is that the life time of the cells or of the protein inside the cells is often smaller than the measurement time for the average multi-dimensional NMR experiment. 

With current commercially available equipment the ideal set-up for online analysis would be an HPLC-SPE system, a cryogenic flow probe (30 p,l active volume) that is in permanent use within an actively shielded magnet operating at 500 MHz or higher. The system would offer the optimum LC-NMR sensitivity (no dependency on LC peak volumes), and complex impurities as low as 0.1% could be identified by one- and two-dimensional NMR experiments, provided that the impurities are sufficiently stable to permit isolation on the SPE cartridges,... 

The idea of back transformation of a three-dimensional NMR experiment involving heteronuclear 3H/X/Y out-and-back coherence transfer can in principle be carried to the extreme by fixing the mixing time in both indirect domains. Even if one-dimensional experiments of this kind fall short of providing any information on heteronuclear chemical shifts, they may still serve to obtain isotope-filtered 3H NMR spectra. A potential application of this technique is the detection of appropriately labelled metabolites in metabolism studies, and a one dimensional variant of the double INEPT 111/X/Y sequence has in fact been applied to pharmacokinetics studies of doubly 13C, 15N labelled metabolites.46 Even if the pulse scheme relied exclusively on phase-cycling for coherence selection, a suppression of matrix signals by a factor of 104 proved feasible, and it is easily conceivable that the performance can still be improved by the application of pulsed field gradients. 

H is particularly important in NMR experiments because of its high sensitivity and natural abundance. For macromolecules, 1H NMR spectra can become quite complicated. Even a small protein has hundreds of 1H atoms, typically resulting in a one-dimensional NMR spectrum too complex for analysis. Structural analysis of proteins became possible with the advent of two-dimensional NMR techniques (Fig. 3). These methods allow measurement of distance-dependent coupling of nuclear spins in nearby atoms through space (the nuclear Overhauser effect (NOE), in a method dubbed NOESY) or the coupling of nuclear spins in atoms connected by covalent bonds (total correlation spectroscopy, or TOCSY). 

The development of carbon-13 NMR during the last eight years has been characterized by a continual increase in the sensitivity and quality of spectra. A reduction in measuring time - equivalent to an enhancement in sensitivity has been achieved mainly by cryomagnet technology. The efficiency with which NMR information can be obtained has been substantially improved by new computer-controllable pulse sequences for one-and two-dimensional NMR experiments. A selection of these new methods, in particular, those used for multiplicity analysis and homo- or heteronuclear shift correlations, is presented in chapter 2 of this edition. 

In Chapters 7 and 8, one-dimensional NOE experiments and a few two-dimensional experiments are presented. Strategies to minimize adverse paramagnetic effects are discussed, as well as ways to exploit such effects to extract structural and dynamic properties. Partial orientation and cross correlation between the Curie magnetic moment relaxation and nuclear dipolar relaxation are also discussed. Chapter 9 deals with the experimental strategies necessary to achieve the highest level of performance in NMR of paramagnetic compounds in solution. 

NMR spectroscopy has proven to be an invaluable tool in structure determination of hydrogenated fullerenes ever since the first synthesis of C60H36 (Haufler et al. 1990) and the first structure determination by NMR was reported for C60H2 by Henderson and Cahill in 1993 (Henderson and Cahill 1993). Today, a multitude of both one- and two-dimensional NMR experiments are available for this purpose. Recent developments in NMR technology with higher magnetic field strengths and cryoprobes have dramatically increased the sensitivity and improved the usefulness of NMR in this field even further. 

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