SSNMR

Solid-state nuclear magnetic resonance spectroscopy (SSNMR) [13]... 

Tensor correlation methods refer to the selection of two anisotropic interactions so that the pattern of the 2D correlation spectrum can reveal the magnitudes and relative orientation of the two tensorial interactions. Below we will discuss many different SSNMR techniques suggested for the determination of peptide backbone... 

Fig. 2 Mechanically oriented bilayer samples as a membrane model for ssNMR. (a) Illustration of the hydrated lipid bilayers with MAPs embedded, the glass supports, and the insulating wrapping, (b) A real sample consists of 15 stacked glass slides, (c) Schematic solid-state 19F-NMR lineshapes from an oriented CF3-labelled peptide (red), and the corresponding powder lineshape from a non-oriented sample (grey), (d) Illustration of typical orientational defects in real samples - the sources of powder contribution in the spectra...

For the orientation-based structure analysis of MAPs, uniformly oriented lipid bilayers are typically prepared on solid supports as illustrated in Fig. 2 [23, 47, 55]. These mechanically oriented membranes are advantageous for static ssNMR experiments, as they provide a robust way to orient a sample with any desired lipid composition, peptide concentration, and at any desired temperature. The lipids... 

Fig. 3 Important 19F-labelled amino acids, (a) Compounds that are wo-steric to native amino acids can be incorporated into proteins biosynthetically, but they possess too many degrees of torsional freedom to be useful for ssNMR structure analysis, (b) In these artificial amino acids the 19F-reporter group is rigidly attached to the peptide backbone. They can be incorporated by solid-phase peptide synthesis, but some problems can arise due to racemisation (4F-Phg, 4CF3-Phg), steric hindrance of coupling (F3-Aib) or HF elimination (fluoro-Ala, F3-Ala). 4F-Phg is additionally problematic due to an ambiguity of the side-chain rotamer. The preferred 19F-labels for ssNMR structure analysis are CF3-Bpg and CF3-Phg (as suitable substitutes for Leu, lie, Met, Val and Ala), as well as F3-Aib and CF3-MePro...

Fig. 17. Models of A/ (l-40) fibrils, viewed down the fibril axis. (A, B) Cartoon representations of the hairpin model proposed by Petkova et al. (2002). (A) The proposed /1-strands span residues 9-24 and 30-40, with a main-chain bend spanning residues 25-29. Aspartate 23 and lysine 28 are proposed to form a salt bridge (dotted line) based on distance constraints provided by ssNMR. The hairpins stack in-register to form two parallel //-sheets. (B) Two hairpin stacks pack together to form the smallest observed fibrils, or protofilaments, burying the hydrophobic residues of the C-terminal strand. Two or more protofilaments may pack together to form thicker fibrils. (C) Cartoon representation of the parallel /i-helix-like model proposed byj. T. Guo and Y. Xu (unpublished model shown in Fig. 1 of Shivaprasad and Wetzel, 2004). The gray oval highlights residues 17 and 34, proposed to sit in close proximity.

In general, liquid-state NMR is preferable as a starting point over SSNMR for several compelling reasons. The primary reason is due to the ability of liquid-state NMR to yield much narrower lines. In practice, liquid-state NMR line shapes are typically an order of the magnitude or more narrower than the solid state. This provides much greater spectral resolution and often an apparently greater signal-to-noise ratio for liquids spectra as compared with solids spectra. NMR... 

As is the case for NMR of liquids, another important consideration for SSNMR spectroscopy is relaxation. There are different types of relaxation present during a SSNMR experiment. The spin-lattice relaxation, Ti, dictates how fast one can repeat scans. This time between experiments must be set greater than five times T1 in order for complete relaxation to occur. Otherwise, the full signal intensity will not be... 

There are several practical aspects to consider before launching a SSNMR experiment. As was mentioned at the beginning of this section, SSNMR experiments are usually much more demanding on the instrument hardware than NMR of liquids experiments. Because of the type of high power capacitors and other electronics necessary to perform... 

Representative commercial CPMAS SSNMR probe specifications... 

Fig. 10.24. 13C CPMAS SSNMR spectrum of drug formulation (bottom) together with the spectrum of the solvated form A of the API (second from the bottom), formulation excipients (second from the top) and the solvated form B of API (top).

Fig. 10.25. 13C CPMAS and 19F MAS SSNMR spectra of the drug formulation (top) and the physical mixture of API with excipients (bottom). Shown above each peak are the relaxation times calculated from the 13C detected...

A comparison of the carbon SSNMR spectra of the manufactured formulation to that of the equivalent physical mixture, both shown on the left of Fig. 10.25, shows no significant differences. The chemical shift and line shape differences between the top and bottom carbon spectra in the figure are minor and thus do not themselves prove an interaction between the API and excipients. Small spectral differences such as these may arise from minor fluctuations in sample temperature, for instance. One may, albeit incorrectly, conclude at this point that no drug-excipi-ent interactions exist. However, as we shall soon see, it is risky to make such conclusions based on the lack of an observed change. 

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