Additional Conformational Restraints

One remaining puzzle is the decreasing ApA), per D from methylamine to dimethylamine to trimethylamine in solution. Such behavior was ascribed to an inductive effect,51,53 but inductive effects ought to be linear in the number of deuteriums. It may be that conformational restraints due to additional methyls increase the negative hyperconjugation. Computations might be informative. 

A remarkable case of RCM assisted by conformational restraint is that shown in equation 39. This involves a substrate in which the two double bonds are separated by 13 single bonds only one of which forms part of a ring, but in which an additional constraint is imposed by the hydrogen bond. When a mixture of the four stereoisomers denoted by 57 is treated with catalyst 19 under the conditions indicated, only one of the isomers undergoes RCM to give (S,S,S)-5H, showing that in this case the configuration... 

Two conformations of EpoA in complex with tubulin have been proposed on the basis of EC [26] and NMR [76, 96] data, respectively (Fig. 11). The tubulin-bound conformation of EpoA was determined by solution NMR spectroscopy [96] before the EC structure of EpoA bound to tubulin was available. The observation that, in a 100 1 mixture with tubulin, NOE cross-peaks of EpoA have negative sign, indicated that there is a fast exchange equilibrium in solution. This offered the opportunity to measure transferred NMR experiments, that report on the bound conformation of the ligand. A total of 46 interproton distances were derived from cross-peak volumes in tr-NOE spectra. However, these distance restraints did not suffice to define a unique conformation, as several distinct structures were consistent with them. Transferred cross-correlated relaxation (Sect. 2.2.1.3) provided the additional dihedral restraints that were crucial to define the bound conformation [96, 97], One requirement to measure CH-CH dipolar and CH-CO dipolar-CSA CCR rates is that the carbon atoms involved in the interaction are labeled with 13C. The availability of a 13C-labeled sample of EpoA offered the opportunity to derive seven of these dihedral angle restraints from tr-CCR measurements (Fig. 12). 

The final step in protein structure determination using NMR is to use a computer program that combines the NMR-derived conformational restraints with additional restraints resulting from the covalent structure of the protein (i.e., bond lengths and bond angles) in order to calculate a 3D structure that is consistent with all of these... 

The addition of the growing radical to the double bond is analogous to the first step of 1-5 addition, but apparently conformational restraints caused by the cyclobutane ring prevents normal cyclopropyl ring opening. Instead a hydrogen shift resulting in a resonance-stabilized radical takes place. The possibility of initial thermal isomerization followed by polymerization was excluded because monomer 13 when heated in the presence of DPPH did not isomerize or lymerize. 

High-resolution H n.m.r. spectroscopy has been used to probe the conformations of a number of o-ribofuranosylamine derivatives and such rigid molecules as 2,2 -cyclonucleosides and nucleoside 3, 5 -phosphates in aqueous solution. H N.m.r. spectroscopy has also been used to study details of the intramolecular association and conformations of a- and j8-linked pyridine ribo-nucleosides and their 5 -phosphates. The results were analysed in terms of base-D-ribose, o-ribose-side-chain, and base-side-chain interactions and the conformational restraints imposed by the cis HO-2-HO-3 interaction in jS-nucleo-tides and the additional cis HO-2 -base interaction in a-nucleotides. H N.m.r. measurements - including measurements of nuclear Overhauser effects and paramagnetic relaxations effected by Mn + cations - have been used to investigate the preferred conformation about the jV-glycosidic bond of 8-amino-, 8-methyl-amino-, and 8-dimethylamino-adenylic acid, all of which competitively inhibit the coenzyme NADH in the reaction with chicken-muscle lactate dehydrogenase. The primary and secondary amines were shown to prefer anti conformations, whereas the tertiary amine prefers a syn conformation. 

The proposed concept that the conformational restraint imposed on the pyrrolidine framework in the form of bicycUc framework could lead to improved stereoselectivity was experimentally verified by Armstrong and coworkers [33]. Additional experimental verification, particularly on highly selective bicycUc catalysts, would indeed be of interest... 

As it was mentioned in Section 9.4.1, 3D structures generated by DG have to be optimized. For this purpose, MD is a well-suited tool. In addition, MD structure calculations can also be performed if no coarse structural model exists. In both cases, pairwise atom distances obtained from NMR measurements are directly used in the MD computations in order to restrain the degrees of motional freedom of defined atoms (rMD Section 9.4.2.4). To make sure that a calculated molecular conformation is rehable, the time-averaged 3D structure must be stable in a free MD run (fMD Sechon 9.4.2.5J where the distance restraints are removed and the molecule is surrounded by expMcit solvent which was also used in the NMR measurement Before both procedures are described in detail the general preparation of an MD run (Section 9.4.2.1), simulations in vacuo (Section 9.4.2.2) and the handling of distance restraints in a MD calculation (Section 9.4.2.3) are treated. Finally, a short overview of the SA technique as a special M D method is given in Sechon 9.4.2.6. 

At this point it is essential to compare the calculated structure with both the experimental data and the results of the rMD run (6). On the one hand, the interatomic distances of the final model must match the NMR restraints additionally, the fMD-averaged structure should correspond with the refined conformation obtained by the rMD. Only if the rMD and the fMD simulations result in the same conformational model and no experimental restraints are severely violated the calculated structure can be presented as a 3D image (7). 

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