COSY Principle

Differential relaxation of in-phase and anti-phase operators involving a spin C [10], which are due to additional Tj relaxation effects active only for the anti-phase components and which depend on the geometry of the spin system, can lead to systematic errors of the coupling constant derived from cross-peak multiplets observed in an E. COSY-type experiment [11]. Since these errors depend for a given differential relaxation rate Ap on the frequency difference of the coherences with C in the a or yS state, according to Eq. (1) a remedy to the problem is to maximize the relevant J such that the condition J 3 Ap/2n is fulfilled  

we present the example of the trans hydrogen bond coupling between the C of the acceptor and the N of the donor h/(N, C ) that is measured by excitation of double-quantum and zero-quantum coherence between the HN and the C nuclei [12] in a protein. Thus, the double-quantum coherence is split by h /(N, C )+ /(N, H) while the zero-quantum coher- 

HN(CO) experiment. In this experiment doublequantum and zero-quantum coherence between Hn and the C bound to the proton via a hydro- 

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Fig. 7.2 Explanation of the E. COSY principle. The E. COSY spectrum can be conceived as a superposition of spectra originating from two...

For the qualitative and quantitative determination of coupling constants, Hartmann-Hahn transfer can be of assistance and also provides a number of new approaches. These approaches are based on Hartmann-Hahn transfer functions or on the efficient transfer of coherence in one subset of the spin system while the polarization of a second subset of spins remains untouched (E.COSY principle). Furthermore, in combination with other experiments, the in-phase multiplets of Hartmann-Hahn experiments can be used as a reference in an iterative fitting of coupling constants in antiphase multiplets. 

Willker and Leibfritz (1992a) introduced an extension of the E.COSY principle that yields additional flexibility. In addition to coherence transfer between the active spins i and j, polarization of spin p, which is passive during is transferred to a spin q, which plays the role of the passive spin during t - Hence, in general, the E.COSY triad is opened up. The two- and three-dimensional JHH-TOCSY experiments for the determination of coupling constants of Willker and Leibfritz (1992a) use a combination of homonuclear TOCSY transfer and two BIRD (bilinear rotation decou-... 

These signals in the NOE spectra therefore in principle make it possible to determine which fingerprint in the COSY spectrum comes from a residue adjacent to the one previously identified. For example, in the case of the lac-repressor fragment the specific Ser residue that was identified from the COSY spectrum was shown in the NOE spectrum to interact with a His residue, which in turn interacted with a Val residue. Comparison with the known amino acid sequence revealed that the tripeptide Ser-His-Val occurred only once, for residues 28-30. 

In the absence of a large associated coupling J(A,C), which is a requirement for the successful application of E. COSY or DQ/ZQ methodology, the FIDS (fitting of doublets from singlets) procedure can be applied. The basic principle of this experiment is outlined in Fig. 7.8. 

As an example and to clarify the principle, the acquisition and the processing schemes for the modified heteronuclear inverse detected ID COSY experiment (pulse sequence IVa in fig. 1) are shown below (tables 1 and 2). 

The principle of multiple selective excitation has been incorporated into a few ID and 2D experiments, the schemes of which are shown below (fig. 1). Depending on the experiment, either a DANTE pulse train (ID TOCSY [2]), frequency selective 180° pulses (ID NOE [3], ID INADEQUATE [4], ID C/H COSY [5] and 2D TOCSY-COSY [6]) or frequency selective 90° pulses (2D HMBC [11]) are applied to selectively perturb and uniquely label selected spins. Besides the DANTE pulse , composed itself of a series of non-selective rectangular pulses, Gaussian-shaped 180° and... 

In principle, all the combinations of homonuclear 2D spectroscopies can be performed to originate a 3D spectrum (COSY-COSY, NOESY-COSY, NOESY-TOCSY, etc.). The considerations made in this chapter for the most basic experiments can be easily extended to their combinations. The general guideline should always be that the more complex the pulse sequence is, the more the experimental sensitivity will suffer from fast nuclear relaxation. 

The complete assignment of individual resonances for a protein can, in principle, be achieved by using multidimensional NMR spectroscopic techniques. For simplicity, the following three 2D NMR H techniques will be discussed. They are COSY (Section 13.3), NOESY (Section 13.4), and TOCSY. These techniques allow for the identification of resonances for nuclei that are connected through bonds, those that are in close proximity in space, and those that are within a given spin system, respectively. 

Selective excitation of a resolved resonance followed by homonuclear or heteronuclear Hartmann-Hahn transfer can also be advantageous in the preparation period of two-dimensional experiments. For example, two-dimensional COSY, NOESY, TOCSY, and two-dimensional /-resolved subspectra of individual spin systems can be acquired based on this principle (Homans, 1990 Sklenaf and Feigon, 1990 Nuzillard and Massiot, 1991 Gardner and Coleman, 1994). In selective two-dimensional experiments like soft COSY (Briischweiler et al., 1987 Cavanagh et al., 1987),... 

One way to partially alleviate this problem is to couple the DOSY filter with a 2-D-NMR sequence where overlapping signals are less of a problem. This results in 3-D sequences, referred to as 3-D-DOSY sequences. Since diffusion is a filter that can easily be coupled to nearly any 2-D-NMR sequence, many 3-D-DOSY sequences have been developed with relative ease. However, there are only a limited number of applications of these techniques in real chemical systems. The principle of a 3-D-DOSY in virtual separation is schematically outlined in Fig. 6.5b. Here, the result of a 3-D-DOSY-COSY sequence is presented, where COSY maps of each compound in the mixture are separated on the diffusion axis and appear on a separate plan. 

The principle of 2D-COSY imaging has been demonstrated on the hypocotyl of the castor bean, enabling the formation of images showing the distributions of sucrose. 

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