Nuclear overhauser effect spectroscopy structure determination

Benzoylation of D-g/ycero-D-gw/o-heptono-1,4-lactone with an excess of benzoyl chloride and pyridine afforded the hept-2-enono-1,4-lactone as the main product (198). The di- and triunsaturated compounds were isolated in very low yield from the mother liquors (199). Higher yields of the di- and triunsaturated derivatives 153 and 154 were obtained when the /5-elimination reaction was performed with triethylamine on the previously synthesized per-O-benzoyl D-g/ycero-D-gw/o-heptono-1,4-lactone. Employing 10% triethylamine in chloroform, the lactone 153 was obtained as an E, Z dias-tereomeric mixture in 9 11 ratio as determined by H n.m.r. When 20% triethylamine was used, the furanone 154 was obtained in 59% yield (200). Its structure was assigned, on the basis of H and 13C n.m.r. spectra, as 3 -benzoyloxy - (5Z)-[(Z)-3 - benzoyloxy - 2 - propenyliden] -2(5 H)- furanone. The stereochemistry of the exocyclic double bonds was established (201) by nuclear Overhauser effect spectroscopy (NOESY). 

Sowinski and coworkers40 reported a structure of vacidin A (63), an aromatic hep-taene macrolide antibiotic. The constitution of vacidin A, a representative of the aromatic heptaene macrolide antibiotics, was established on the basis of 13C and H- H double quantum filtered correlated spectroscopy, rotating frame nuclear Overhauser effect spectroscopy, 7-resolved 11 as well as H-13C correlation NMR spectra. The geometry of the polyene chromophore was determined as 22E, 24E, 26E, 28Z, 30Z, 32E, 34E. 

Two-Dimensional NMR—Basically, the two-dimensional NMR techniques of nuclear Overhauser effect spectroscopy (NOESY) and correlation spectroscopy (COSY) depend on the observation that spins on different protons interact with one another. Protons that are attached to adjacent atoms can be directly spin-coupled and thus can be studied using the COSY method. This technique allows assignment of certain NMR frequencies by tracking from one atom to another. The NOESY approach is based on the observation that two protons closer than about 0.5 nm perturb one another s spins even if they are not closely coupled in the primary structure. This allows spacial geometry to be determined for certain molecules. 

Due to the great complexity of this class of molecules, nuclear magnetic resonance (NMR) and mass spectroscopy (MS) are the tools most widely used to identify cucurbitacins. Both one- and two-dimensional NMR techniques have been employed for the structural elucidation of new compounds 2D NMR, 1H-NMR, 13C-NMR, correlated spectroscopy (COSY), heteronuclear chemical shift correlation (HETCOR), attached proton test (APT), distortionless enhancement by polarization transfer (DEPT), and nuclear Overhauser effect spectroscopy (NOESY) are common techniques for determining the proton and carbon chemical shifts, constants, connectivity, stereochemistry, and chirality of these compounds [1,38,45-47]. 

The elucidation of the scalar coupling network by the correlation experiments is, apart from small molecules, not sufficient for the unambiguous, sequential and stereo-specific assignment. The complementary information of spatially adjacent protons is obtained via cross-relaxation experiments, the laboratory-frame nuclear Overhauser enhancement spectroscopy (NOESY) and the rotating-frame nuclear Overhauser effect spectroscopy (ROESY). These experiments provide also the distance restraints for the structure determination and help to recognize exchange processes. 

In principle, 2D NMR structural determination of proteins in solution involves the linking of information derived from a combination of three complementary experiments - TOCSY (Total Correlated Spectroscopy), DQF-COSY (Double-Quantum Filtered Correlated Spectroscopy) and NOESY (Nuclear Overhauser Effect Spectroscopy). The TOCSY (58) spectra are used to identify spin systems of the amino acids in the protein. COSY (59,60) spectra yield complementary information to the TOCSY, which can be used to obtain the direct scalar connectives. The NOESY (61) spectra yield information on the through space relationship of protons in the protein. It should be noted that because of the vast abundance of hydrogen atoms in a protein molecule proton NMR is the preferred technique to determine the solution structure of a protein. 

In de novo three-dimensional structure determinations of proteins in solution by NMR spectroscopy, the key conformational data are upper distance limits derived from nuclear Overhauser effects (NOEs) [11, 14]. In order to extract distance constraints from a NOESY spectrum, its cross peaks have to be assigned, i.e. the pairs of hydrogen atoms that give rise to cross peaks have to be identified. The basis for the NOESY assignment... 

The isotope N, with a natural abundance of 99.9%, has nuclear spin 7 = 1 and gives broad signals which are of little use for structural determinations. The N nucleus, with I = 1/2, is therefore preferred. However, the low natural abundance of about 0.4% and the extremely low relative sensitivity (Table 1) make measurements so difficult that N NMR spectroscopy was slow to become an accepted analytical tool. A further peculiarity is the negative magnetogyric ratio since, in proton decoupled spectra, the nuclear Overhauser effect can strongly reduce the signal intensity. DEPT and INEPT pulse techniques are therefore particularly important for N NMR spectroscopy. 

Chemical shift correlated NMR experiments are the most valuable amongst the variety of high resolution NMR techniques designed to date. In the family of homonuclear techniques, four basic experiments are applied routinely to the structure elucidation of molecules of all sizes. The first two, COSY [1, 2] and TOCSY [3, 4], provide through bond connectivity information based on the coherent (J-couplings) transfer of polarization between spins. The other two, NOESY [5] and ROESY [6] reveal proximity of spins in space by making use of the incoherent polarization transfer (nuclear Overhauser effect, NOE). These two different polarization transfer mechanisms can be looked at as two complementary vehicles which allow us to move from one proton atom of a molecule to another proton atom this is the essence of a structure determination by the H NMR spectroscopy. 

The above data clearly suggest a dibenzazonine structure with two methoxyl and two hydroxyl groups as substituents, their locations being determined by nuclear Overhauser effect difference spectroscopy experiments (Fig. 1). Several derivatives of crassifolazonine (2) were prepared and characterized (2a-2c). Final proof for the proposed structure of crassifolazonine (2) was obtained by its total synthesis (15). 

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