D NMR Experiments

Fig. 5. A 2-D nmr experiment of 2-methyl-5-bromopentane [626-88-0] where and correspond to evolution and acquisition time, respectively.

Table F1.4.1 Parameters for 2-D NMR Experiments Performed on a Typical Anthocyanin" ...

Today, it is possible to make complete assignments of all proton and carbon atoms in the NMR spectra of most isolated anthocyanins. These assignments are normally based on chemical shifts (8) and coupling constants (J) observed in 1-D H and l3C NMR spectra (Fig. FI.4.2), combined with correlations observed as cross-peaks in various homo- and heteronu-clear 2-D NMR experiments (see below for details on COSY, TOCSY, HSQC, HMBC, NOESY, and ROESY). 

Introduction to 2-D NMR experiments The purpose of the standard 1-D H NMR experiment is to achieve structure-related information about sample protons (i.e., chemical shifts, spin-spin couplings, and integration data) describing the relative number of protons. Applied to anthocyanins, this information may help to identify the aglycone (anthocyanidin), number of monosaccharides present, and anomeric configuration of the monosaccharides. However, for most anthocyanins, the information gained by a standard 1 -D H NMR experiment is insufficient for complete structure elucidation. In recent years, various 2-D NMR experiments have evolved as the most powerful tools for complete structure elucidation of anthocyanins. 

The partial long range C-H chemical shift correlation spectrum presented in figure 3 shows signals from the aliphatic side chains of the trimeric compound. This 2-D NMR experiment provides information about the H-C connection 2 to 3 bonds away from the carbon (coupling constants less than 20 Hz). For example, Ha shows... 

We now consider multiple-pulse experiments and two-dimensional (2-D) NMR. Exactly what does the term dimension in NMR mean The familiar proton spectrum is a plot of frequency (in S units) versus intensity (arbitrary units)—obviously 2-D but called a 1-D NMR experiment, the one-dimension referring to the frequency axis. It is important to remember that the frequency axis, with which we are comfortable, is derived from the time axis (the acquisition time) of the FID through the mathematical process of Fourier transformation. Thus, experimentally, the variable of the abscissa of a 1-D experiment is in time units. 

Many readers will already be aware that acronyms for 2-D NMR experiments have proliferated along with available experiments. This chapter attempts neither an encyclopedic approach to describing these acronyms nor their experimental counterparts. This chapter does, however, cover enough important experiments to enable the reader to interpret nearly any 2-D experiment that one is likely to encounter. Acronyms are listed in the index. 

All the spectroscopic approaches applied for structural characterization of mixtures derive from methods originally developed for screening libraries for their biological activities. They include diffusion-ordered spectroscopy [15-18], relaxation-edited spectroscopy [19], isotope-filtered affinity NMR [20] and SAR-by-NMR [21]. These applications will be discussed in the last part of this chapter. As usually most of the components show very similar molecular weight, their spectroscopic parameters, such as relaxation rates or selfdiffusion coefficients, are not very different and application of these methodologies for chemical characterization is not straightforward. An exception is diffusion-edited spectroscopy, which can be a feasible way to analyze the structure of compounds within a mixture without the need of prior separation. This was the case for the analysis of a mixture of five esters (propyl acetate, butyl acetate, ethyl butyrate, isopropyl butyrate and butyl levulinate) [18]. By the combined use of diffusion-edited NMR and 2-D NMR methods such as Total Correlation Spectroscopy (TOCSY), it was possible to elucidate the structure of the components of this mixture. This strategy was called diffusion encoded spectroscopy DECODES. Another example of combination between diffusion-edited spectroscopy and traditional 2-D NMR experiment is the DOSY-NOESY experiment [22]. The use of these experiments have proven to be useful in the identification of compounds from small split and mix synthetic pools. 

In a one-dimensional (1-D) NMR experiment, t, is kept constant, t2 varies and the FT is taken with respect to t2 only in two-dimensional (2-D) NMR experiments, both t, and t2 are varied and the FT is taken with respect to both time variables. 

Commonly, the sample produced for heteionuclear triple-resonance 3-D NMR experiments must be ImM or greater in macromolecule concentration in approximately 400-600uL of solution in a SmM NMR tube. However, it should be noted that there are efforts underway in several laboratories aimed at developing probes which can accommodate larger sample volumes. The solution used is routinely 90% H2O with 10% D2O added for field locking. The sample should be free of impurities, especially other proteins which may copurify and may also be labeled with and N isotopes. 

Stop-flow requires the calibration of the delay time, which is the time required for the sample to travel from the UV detector to the NMR flow cell, which depends, in turn, on the flow rate and the length of the tubing connecting HPLC with NMR. Because the chromatographic run is automatically stopped when the chromatographic peak of interest is in the flow cell, the amount of sample required for the analysis can be reduced and 2-D NMR experiments, such as correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and others, can be obtained because the sample can remain inside the flow cell for days. It is possible to obtain NMR... 

The so-called 2-D NMR spectrum is actually a three-dimensional (3-D) plot the omitted dimension in all NMR experiments (1-D, 2-D, 3-D, etc.) is always the intensity in arbitrary units. The two dimensions referred to in a 2-D NMR experiment are both frequency axes. It requires two Fourier transformations at right angles to each other on two independent time axes to arrive at two orthogonal frequency axes. 

FIGURE 6.2. Prototype pulse sequence for a 2-D NMR experiment. The incremental delay, f, and the acquisition time, t2, are Fourier transformed into frequencies, Vo and /, respectively. (7t/2)x represents a 90° pulse along the x axis. The interval r, is of the order of microseconds t is of the order of seconds. 

Natural product extracts are generally complex and comprise mixtures of neutral, acidic, basic, lipophilic, hydrophilic, and amphiphilic (e.g., amino acids) compounds and, as a consequence, there is rarely one method that will serve for all eventualities. It is sometimes worthwhile to carry out H or NMR spectroscopy of the extract or fraction to determine the class of compound(s) to be separated (1)—deuterated NMR solvents are cheap ( 1.00 for CDCI3) and 1 D NMR experiments are quicker to run than the extensive... 

Many NMR probes now available are designed so that probe tuning response is relatively insensitive to sample changes. Consequently, we do not need to tune our probe or calibrate our RF pulses before carrying out various 1-D and 2-D NMR experiments. A probe of this nature is an essential feature of a high-throughput instrument. 

For conducting 2-D NMR experiments such as the HSQC, HMBC, and NOESY experiments, we may elect to tune and calibrate the 90° pulse for each sample. 

The DEPT experiment is another 1-D NMR experiment that can he used to distinguish the number of attached H s on the various resonances we observe. The DEPT experiment is used more commonly than the APT experiment however, the DEPT experiment employs a strategy similar to that used in the APT experiment. A DEPT experiment is often used when molecules are relatively simple or when the resonances found in the spectmm have already been mostly assigned. Figure 6.10 shows the DEPT spectmm of longifolene in CDCI3 (longifolene also appears as Problem 12.1). 

The 1-coupling allows us to obtain extremely useful information about solute molecules. The list of 2-D NMR experiments that employ 1-couplings for correlating chemical shifts continues to expand only a few key experiments will be discussed here. The major 2-D NMR... 

The homonudear 2-D NMR experiments that use I-coupling indude the correlation spectroscopy (COSY, and variants induding gradient-selected COSY or gCOSY, double-quantum filtered COSY or DQF-COSY) experiment, the total correlation spectroscopy (TOCSY) experiment, and the incredible natural abimdance double quantum transfer experiment (INADEQUATE) [3]. 

There are two basic 2-D NMR experiments that make use of the NOE the NOESY and the ROESY [1] experiments. NOESY stands for nuclear Overhauser effect spectroscopy and ROESY stands for rotational Overhauser effect spectroscopy. The ROESY experiment is also referred to in some of the literature as the CAMELSPIN experiment. The principal difference between the NOESY and ROESY experiments lies in the time scale associated with the dipolar relaxation mechanism. 

Rotational Overhauser effect spectroscopy, ROESY. Syn. CAMELSPIN experiment A 2-D NMR experiment similar to the 2-D NOESY experiment except that the ROESY experiment employs a spin-lock using the B, field of the applied RE, thus skirting the problem of the cancellation of the NOE cross peak when correlation times become long enough to reduce the rate constant for the dipolar doubl quantum spin flip. 

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