2'3JCH (long-range couplings)


Homonuclear experiments are characterized by a diagonal defined by F1 = F2 and by pairs of crosspeaks at symmetrical positions across the diagonal: F1 = F2 = and F1 = ^b, F2 = This is because both magnetization transfer pathways, 1Ha ^ 1Hb and 1Hb ^ 1Ha, can be observed. Heteronuclear experiments lack a diagonal and diagonal symmetry. The range of J values can be selected in mixing schemes: for 1H-13C couplings, 1JCH (the one-bond or direct coupling) is very large (125-180 Hz) whereas the long-range 2'3 JCH (two and three-bond coupling) is small (2-12 Hz). Some mixing sequences can allow multiple "jumps" of magnetization: the TOCSY experiment allows for many jumps based on J coupling—the first from the F1 nucleus (A) to an intermediate (undetected) nucleus (C). Other jumps may transfer the magnetization to other undetected nuclei (e.g., D and E) and a final jump carries it to the F2 nucleus (B), thus spreading the magnetization out over an entire "spin system" or group of protons interconnected by J couplings.

From these basic experiments have grown many variants. The COSY has been extended to DQF-COSY (reduced diagonal intensity and improved phase properties) and COSY-35 (simplified crosspeak structure for J-value determination). A common variant of NOESY is the ROESY, which gives better results for molecules in the size range of peptides, oligosac-charides and large natural products. HSQC gives the same results as HMQC but has better relaxation properties for large molecules such as proteins. All 2D experiments and even 3D and higher dimensional experiments are based on the above list of basic 2D experiments.

In all this complexity of acronyms it is easy to forget that all 2D experiments do the same thing: they allow you to correlate two atoms (nuclei) in a molecule based on an interaction that is either through bond (J coupling) or through space (NOE). The two nuclei are identified by their chemical shifts, and the correlation appears in the 2D spectrum as a crosspeak at the F1 chemical shift ("y coordinate") of the nucleus where magnetization starts and the F2 chemical shift ("x coordinate") of the nucleus to which the magnetization is transferred. Thus the basis of all 2D experiments is the "jump" or transfer of magnetization. The information (J value or NOE intensity) can be used to define structural relationships (dihedral angle or distance) but is only useful if we can unambiguously assign the two chemical shifts (Fi and F2) to specific positions within the molecule.


COSY is the first and the simplest 2D experiment. It correlates one proton (Ha) to another (Hb) via a single J coupling that may be 2-bond (geminal), 3-bond (vicinal) or in rare cases 4-bond or 5-bond (long range). The pulse sequence is simply 90o - t1 - 90o - FID (Fig. 9.16). Consider the interaction of two J-coupled protons, Ha and Hb (Fig. 9.17). The preparation pulse rotates the Ha magnetization from the z axis into the x-y plane. During the evolution (t1) period, Ha magnetization precesses in the rotating frame at a rate dependent upon its chemical-shift offset, Qa. At the same time, J-coupling evolution occurs to produce Ha magnetization that is antiphase with respect to its J coupling with Hb. As with the INEPT transfer in the HETCOR experiment, simultaneous 90o pulses applied to Ha and Hb transfer antiphase Ha magnetization to antiphase Hb magnetization (this is actually accomplished with a single nonselective 90o 1H pulse). During the detection period (FID), Hb magnetization precesses at its characteristic rate (^b) in the rotating frame, inducing a voltage in the probe coil, which is digitized as the FID. Fourier transformation in F2 and then in F1 leads to a 2D data matrix with a crosspeak at F1 = Qa, F2 = Qb (Fig. 9.18). This basic COSY sequence is not used much any more—everyone uses the double-quantum filtered COSY or DQF-COSY sequence. Until we get to the actual DQF-COSY pulse sequence and understand how it works, however, we will treat these two experiments as equivalent.

The appearance of a homonuclear 2D spectrum is different from what you have seen for HETCOR, the 1H-13C correlation. Because both frequency scales, F2 and F1, are 1H chemical-shift scales, we can observe the transfer of coherence from Ha to Hb as a crosspeak at F1 = Qa, F2 = (Fig. 9.18, lower right), as well as the opposite sense of transfer from Hb to Ha, a symmetrically disposed crosspeak at F1 = Qb, F2 = Qa (upper left crosspeak). In HETCOR we observe 13C coherence (in F2) that was transferred from the attached proton, whose chemical shift appears in F1. Transfer in the opposite sense (13Cto1H) is not possible

Figure 9.17

because the pulse sequence starts with XH excitation and the detector is set up to observe only signals at the radio frequency of13 C. Furthermore, if transfer of magnetization "fails" (i.e., if some coherence remains on the 1H after the mixing step), the 1H coherence is not detected and cannot be displayed in the 2D spectrum. But in a homonuclear experiment like COSY, "failed" coherence transfer from Ha means that we encode the frequency of Ha during t1 and then observe the same frequency in t2. The FID has the form:

which leads to a peak in the 2D spectrum at F1 = and F2 = We call this a diagonal peak because it falls on the diagonal line defined by F1 = F2, running from the lower left corner to the upper right corner of the 2D data matrix (Fig. 9.18). Because coherence transfer is never 100% complete at the end of the mixing sequence, we will always see a diagonal peak for each resonance in the 1H spectrum. That means that if we trace along the

'H Chemical shift

Figure 9.18

diagonal line from lower left to upper right we will trace out the 1D proton spectrum. Keep this in mind because you do not always have the luxury of a 1H spectrum displayed along the top and side of the 2D spectrum. After a while you can wean yourself away from this crutch and begin to view the diagonal as your 1D spectrum.

Figure 9.19 shows a COSY spectrum of a molecule with two simple spin systems separated by a quaternary carbon: -CHa-CHb-CHc-Cq-CHd-CHe-. One can "walk" through each spin system by moving from diagonal to cross-peak vertically, back to the diagonal horizontally, and repeating this process. Note that either of the two symmetrical crosspeaks can be used for this "walk". The Hb-Hc crosspeak (upper left) shows how the Hb peak in the 1D spectrum at the top is correlated to the Hc peak in the 1D spectrum on the left side. This crosspeak represents coherence on Hc that transferred to Hb in the mixing step. We will always label crosspeaks as shown, with the F2 assignment on the top or bottom and the F1 assignment to the left or right of the crosspeak. This process of "walking" (diagonal-crosspeak-diagonal) is especially useful when each carbon has only one proton, or group of equivalent protons, so that each crosspeak is a jump from a proton on one carbon to a proton on the next carbon (a vicinal coupling), a common occurrence in carbohydrates. The walk sometimes doubles back on itself, since the chemical shift changes can reverse direction as we move through a spin system (Fig. 9.20). In this case we walk from the crosspeak to the diagonal peak, passing over the crosspeak for the next jump. The biggest problem occurs when two adjacent (vicinal) protons have identical or nearly identical chemical shifts: then the crosspeak connecting them is on the diagonal or very near the diagonal. Consider the molecular fragment -CH1-CH2-CH3-Cq4-CH5-CH6-CH7- with two spin systems separated by the quaternary carbon C4. Suppose that the Ha resonance can be assigned to H1 and the Hf resonance can be assigned to H7 (Fig. 9.21) based on chemical shifts or coupling constants. Using the COSY data, we can connect both Ha (H1) and Hf (H7) to the overlapped two-proton peak Hd/e. So we know that this peak contains

Figure 9.20

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