Correlation time COSY-NOESY

LC-NMR can be operated in two different modes on-flow and stopped-flow. In the onflow mode, LC-NMR spectra are acquired continuously during the separation. The data are processed as a two-dimensional (2D) NMR experiment. The main drawback is the inherent low sensitivity. The detection limit with a 60 p.1 cell in a 500 MHz instrument for a compound with a molecular weight around 400 amu is 20 pig. Thus, on-flow LC-NMR runs are mainly restricted to the direct measurement of the main constituents of a crude extract and this is often under overloaded HPLC conditions. Typically, 1 to 5 mg of crude plant extract will have to be injected on-column.In the stopped-flow mode, the flow of solvent after HPLC separation is stopped for a certain length of time when the required peak reaches the NMR flow cell. This makes it possible to acquire a large number of transients for a given LC peak and improves the detection limit. In this mode, various 2D correlation experiments (COSY, NOESY, HSQC, HMBC) are possible. 

At this point NOE and NOESY experiments are needed. In this way we learn which proton is close to which other proton, and the picture becomes sharper. Sometimes, when the correlation times governing cross relaxation are unfavorable, ROE or ROESY experiments can be a valid alternative. Both NOE and ROE types of experiment also provide information on the presence of chemical equilibria when the interconversion rate is of the order of T. It is also clear that NOE and ROE types of experiment may not always provide a unique picture, because they ignore chemical bonds. COSY and TOCSY experiments provide the information on which proton is chemically bound to which other proton. 

Laurie was one of the first to apply two-dimensional (2D) NMR to carbohydrates. With students Subramaniam Sukumar and Michael Bernstein, and visiting scientist Gareth Morris, he demonstrated and extended the application of many of the directly observed 2D NMR techniques of the time. These included the homo- and hetero-nuclear 2D /-resolved techniques, delayed proton /-resolved NMR that allowed broad resonances to be suppressed, for example, those of dextran in the presence of methyl /Lxvlopyranoside. proton-proton chemical shift correlation spectroscopy (COSY), nuclear Overhauser enhancement spectroscopy (NOESY), proton-carbon chemical shift correlation (known later as HETCOR), and spin-echo correlated spectroscopy (SECSY). Trideuteriomethyl 2,3,4,6-tetrakis-<9-trideuterioacetyl-a-D-glucopyranoside served as a commonly used model compound for these studies. 

All proton NMR spectra were recorded on a Varian Unity 600 at 25 C. 6 to 10 mg of the disulfide linked c-Myc-Max heterodimeric LZ were dissolved in 0.5 mL of potassium phosphate buffer (50 mM, 10% DiO / 90% H2O and pH 4.7) containing 100 mM KCl and ImM 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) to yield solutions ranging from 0.75 to 1.25 mM. Proton resonances were assigned from two-dimensional double quantum filtered correlation spectroscopy (DQF-COSY (21)), two-dimensional total correlation spectrocopy (TOCSY mixing time = 50 ms (22)) and two-dimensional nuclear Overhauser enhancement spectrocopy (NOESY mixing times = 150 and 200 ms (23)) experiments. Sequential assignment of the proton resonances was performed as described by Wuthrich (24). 

Multidimensional correlation experiments can be arduous to analyse at the best of times. Therefore, any practitioner ofbiomolecularNMR spectroscopy will want to do the minimum number of experiments to achieve unique and unambiguous resonance assignment of as many amino-acid residue nuclei as necessary in order to enable the critical NOESY experiments. Two of the simplest 3D correlation experiments that have been used are 3D //- NTOCSY-HSQC and 3D HCCH COSY/TOCSY. Such 3D correlation experiments are known as doubleresonance experiments in that they generate intensity data /(Fi, Fj, F3) emanating from the double resonance of two entirely different populations of nuclei, either Hand Nnuclei, or H and C nuclei respectively. 

For natural abundance H, correlation experiments, the 2D HMQC pulse sequence as described by Bax et al. 3 s used. A proton spectral width of 5(X)0 Hz and a carbon-13 spectral width of 25000 Hz were used, with 2K X 512 data points in the t2 and tl dimensions respectively. H, P correlation experiments were obtained using a hetero-TOCSY sequence, with an isotropic mixing time of 67 ms. The spectral width was set to 4000 Hz for protons and 607 Hz for phosphorus, with typically 2K X 256 data points in the two dimensions respectively. The proton 2D DQF-COSY and TOCSY experiments were recorded with standard pulse sequences the data size was 2K X 512, with spectral width of 5500 Hz in both dimensions. The NOESY sequence with a jump-return excitation pulse was used for optimal imino proton detection . Mixing times from 100 to 300 ms were used. The spectral widths were 11000 Hz in both dimensions. 

A new 2D C- C correlation experiment in solids, in which C- C /-correlation (COSY) and dipolar correlation (DARR) are recorded at the same time, is described. The sequence is similar to COCONOSY in the liquid-state NMR, in which H- H COSY and NOESY spectra are obtained in a single experiment. The combined COSY and DARR experiment facilitates assignment of C signals in solids. 

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