RELAY-NOESY

Both homonuclear and heteronuclear versions of relayed nOe experiments are known. The homonuclear relayed NOESY experiment involves both an incoherent transfer of magnetization between two spins H and H/ that are not coupled but close in space, and a coherent transfer of magnetization between two spins H(and H that are /-coupled together. The magnetization pathway may be depicted as... 

Two-dimensional relayed NOESY experiments (Wagner, 1984 Kessler et al., 1988) give cross-peaks that could be a superposition of nOe s resulting from different relay nuclei. This complicates the extracting of crossrelaxation rates. The 1D NOESY experiment (Kessler et al., 1989a), however, allows the path of the magnetization transfer to be clarified. 

Figure 7.5 Pulses sequences for ID NOESY and ID relayed NOESY experiments, (a) A ID NOESY sequence with full Gaussian pulses is inferior to the ID NOESY sequence (b) with half-Gaussian excitation, (c) A 90° Gaussian pulse in the pulse sequence of 1D relayed NOESYis appropriate for excitation, since antiphase magnetization is required for the first mixing step. (Reprinted from Mag. Reson. Chem. 29, H. Kessler el al, 527, copyright (1991), with permission from John Wiley and Sons Limited, Baffins Lane, Chichester, Sussex P019 lUD, England.)...

An interesting possibility for assignment of NOEs from two or more overlapping anomeric protons is provided by a 1D RELAY-NOESY experiment [38] (fig. 13(d)). In this experiment the magnetization is transferred from H-1 protons to H-2 protons first and after a chemical-shift-selective... 

Fig. 17. ID RELAY-NOESY spectra of the oligosaccharide 5 acquired using the pulse sequence of fig. 13(d) with the initial polarization transfer from overlapping anomeric protons of two terminal glucoses in 5. (a) Partial H spectrum of 7 at 600 MHz after 8 scans. The following parameters were used to acquire the two original ID RELAY-NOESY spectra (not shown) rsei = 50 ms, tq = 39 ms, A = 9.09 ms, ti = 120 ms, Tr = 64 ms, r, = 400 ms.

For example, ambiguities in the sequential assignment of peptide or protein spectra can often be eliminated through use of relayed NOESY-type experiments (Wagner, 1984), where the mixing process consists of... 

The pulse sequence used for relayed NOESY (Figure 5.75A) is identical to the normal NOESY pulse sequence until the third 90° pulse. During the preceding period, magnetization is transferred from the spatially remote spin (Hr) to the neighbor spin (H ) via NOE. The third 90° pulse converts the... 

All these experiments involve at least three distinct time periods preparation (tp), evolution (tl), and detection (t2) these periods are usually separate by rf pulses. Some experiments (e.g., NOESY, RELAY) further contain an additional "Mixing period, tm, between the evolution and detection periods. 

Figure 1. Pulse sequences of some typical 2D-NMR experiments. COSY = correlation SpectroscopY, DQFCOSY = Double Quantum Filtered COSY, RELAY = RELAYed Magnetization Spectroscopy, and NOESY = Nuclear Overhauser Effect SpectroscopY.

It was straightforward to identify the spin systems of four valines, five threonines, four alanines, three glycines, two glutamates, and AMX type residues (Asp, Cys, Phe, Tyr, Trp, and Ser) of this protein from the COSY, RELAY, and NOESY spectra in D O solution. The RELAY spectrum was particularly useful in identification of Val, Ala, Thr, and Glu spin systems. 

Figure 1.45 Coherence transfer pathways in 2D NMR experiments. (A) Pathways in homonuclear 2D correlation spectroscopy. The first 90° pulse excites singlequantum coherence of order p= . The second mixing pulse of angle /3 converts the coherence into detectable magnetization (p= —1). (Bra) Coherence transfer pathways in NOESY/2D exchange spectroscopy (B b) relayed COSY (B c) doublequantum spectroscopy (B d) 2D COSY with double-quantum filter (t = 0). The pathways shown in (B a,b, and d) involve a fixed mixing interval (t ). (Reprinted from G. Bodenhausen et al, J. Magn. Resonance, 58, 370, copyright 1984, Rights and Permission Department, Academic Press Inc., 6277 Sea Harbor Drive, Orlando, Florida 32887.)...

Oil and 0)2, and (b) 2D shift-correlation spectra, involving either coherent transfer of magnetization [e.g., COSY (Aue et al, 1976), hetero-COSY (Maudsley and Ernst, 1977), relayed COSY (Eich et al, 1982), TOCSY (Braunschweiler and Ernst, 1983), 2D multiple-quantum spectra (Braun-schweiler et al, 1983), etc.] or incoherent transfer of magnedzation (Kumar et al, 1980 Machura and Ernst, 1980 Bothner-By et al, 1984) [e.g., 2D crossrelaxation experiments, such as NOESY, ROESY, 2D chemical-exchange spectroscopy (EXSY) (Jeener et al, 1979 Meier and Ernst, 1979), and 2D spin-diffusion spectroscopy (Caravatti et al, 1985) ]. 

The pioneering work in this field, a two-dimensional relayed-NOE experiment proposed by Wagner [7], was quickly followed by the appearance of several related NMR techniques [8-17]. Application of isotropic mixing during the J-transfer period yielded the 2D TOCSY-NOESY [11, 15] and NOESY-TOCSY [12, 14] experiments. When spin-lock sequences were applied to both J and NOE-transfers, the 2D TOCSY-ROESY and ROESY-TOCSY experiments [10, 16, 17] emerged. 

As an alternative to selective pulses, chemical-shift-selective filters (CSSF) were successfully used in ID COSY, ID RELAY and ID NOESY experiments when signals partially overlapping, but different in their chemical... 

The pulse methods rely on selective irradiation of a particular resonance line with a radio frequency (rf) and observation of the resulting effects in the rest of the spectrum. Among commonly employed methods are 2D correlated spectroscopy (COSY), 2D spin-echo correlated spectroscopy (SECSY), 2D nuclear Overhauser and exchange spectroscopy (NOESY), 2D J-resolved spectroscopy (2D-J), and relayed coherence-transfer spectroscopy (RELAYED-COSY) (Wutrich, 1986). 

Fig. 8. 2D C/ H shift correlation spectrum of the xyloglucan polysaccharide shown in Fig. 7. The anomeric region is not shown. A partial assignment of the H spectrum was first made from COSY, RELAY and NOESY experiments and was completed using the spectrum shown above. (I.J. Colquhoun, P. Ryden and R.R. Selvendran, unpublished.)...

AM-500 spectrometer operating at 500.13 MHz for proton magnetic resonance. Samples were prepared in d -DMSO and 1-D and 2-D spectra obtained. Several experiments were run for the 2-D spectra COSY, Relay COSY, HOHAHA, and NOESY which were zero-filled in the FI dimension only. D2O exchange spectra were also obtained. 

Further modifications (the addition of a spin-echo) allow the direct and relayed peaks to be differentiated through inversion of the direct correlations [38], and gradient-selected versions of these sequences without [39, 40] and with [10,41] such editing have also been proposed. Although the addition of TOCSY transfer is probably the most useful extension, any homonuclear mixing scheme can, in principle, be added, including a COSY, NOESY (NOE spectroscopy) or ROESY (ROE spectroscopy) step. 

The inverse-detected 2D NMR experiments that have been discussed to this point have all been discrete, single-purpose experiments, e.g. correlating protons with their directly bound heteronucHde (typically or N). There are another class of inverse-detected 2D NMR experiments that are generally referred to as hyphenated 2D experiments. These are experiments that first establish one type of correlation, followed by an additional experiment segment that then pursues a further spectroscopic task. Predecessors of the inverse-detected variants of these experiments were the HC-RELAY (proton—carbon heteronuclear relayed coherence transfer) experiments pioneered by Bolton [151—155]. Examples of these include, but are by no means hmited to HXQC-COSY and -TOCSY [156—158], -NOESY [159], -ROESY [160], and more recent gradient variants [161] etc., where X = S (single) or M (multiple) quantum variants of the experiments. 

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