Anisotropy methods

Calibration method. The measured fluorescence parameter should be independent of indicator concentration, geometry of sample, and sensitivity of detection system. Thus, an intensity-based method requires wavelength-ratiometric probes. Lifetime and anisotropy methods do not require wavelength-ratiometric probes, but the lifetime or anisotropy must be sensitive to analyte. 

The theory of high order corrections to the Lamb shift described above for H and D may also be applied to other light hydrogenlike ions. The simplest such ion is He+. Originally the classic Lamb shift in 17e+ was measured in [50] by the quenching-anisotropy method with the result L 2Si — 2Pi,17e+) = 14 042.52 (16) MHz. Later the authors of [50] discovered a previously unsuspected source of systematic error in their experiment. Their new measurement of the classic Lamb shift in 17e+ by the anisotropy method resulted in the value L 2Si — 2Pi,He ) = 14 041.13 (17) MHz [51]. Besides the experimental data this result depends also on the theoretical value of the hne structure interval. In [51] the value AE 2Pz —2Pi) = 175 593.50 (2) MHz was used. We recalculated this interval using tire latest theoretical results discussed above and obtained AE 2P3 — 2Pi) = 175 593.33 (1) MHz. Then the value of the... 

Brownbridge, G. G., Lowe, P. N., Moore, K. J., Skinner, R. H., and Webb, M. R. (1993). Interaction of GTPase activating proteins (GAPs) with p21ras measured by a novel fluorescence anisotropy method. Essential role of ARG-903 of GAP in activation of GTP hydrolysis on p21ras. J. Biol. Chem. 268, 10914-10919. 

Various fluorinated diphenylmethanols 36-39 were also enantioresolved as CSDP esters (entries 23-26). In the case of alcohols 36, 37, and 39, their absolute configurations were determined by X-ray crystallography. To those fluorinated alcohols, the method of MaNP acid 3 could be applied for enantioresolution and also for determination of their absolute configurations by the H NMR anisotropy method, as discussed below. Meto-substituted diphenylmethanols 40 and 41 were enantioresolved by the CSDP acid method yielding enantiopure alcohols, the abso-... 

We have discovered that this novel carboxylic acid, MaNP acid 3, is also effective for enantioresolution and simultaneous determination of the absolute configuration of various secondary alcohols by the H NMR anisotropy method [41-52, 56-58]. The results obtained by the H NMR anisotropy mefhod are, of course, consistent wifh those by the X-ray method. Therefore, the methods of CSDP and MaNP acids are useful as complementary molecular tools, as discussed in fhis chapter. 

Determination of Their Absolute Configurations by the NMR Anisotropy Method... 

The NMR Anisotropy Method for Determining the Absolute Configuration of Secondary Alcohols the Sector Rule and Applications [42, 50]... 

Fig. 9.16 Enantioresolution of racemic alcohol as (S)-MaNP esters, and determination of the absolute configuration of the first-eluted fraction by the NMR anisotropy method [38, 45, 50],...

Fig. 9.17 Determination of the absolute configurations of the alcoholic part of the first-eluted esters by the NMR anisotropy method using (S)-(+)-MaN P acid, 3 and the observed Ad values [44],...

Table 9.3 HPLC > separation of diastereomeric esters formed from alcohols with MaNP acid (S)-(+)-3, determination of their absolute configurations by the H NMR anisotropy method, and absolute configurations of recovered chiral alcohols.

Fig. 9.22 The S absolute configuration of alcohol (+)-40 as determined by the H NMR anisotropy method using MaN P acid (a) DCC, DMAP, CSA/CH2CI2, r.t. [39].

As described above, the methods of CSDP acid and MaNP acid are very powerful for the preparation of enantiopure alcohols and also for simultaneous determination of their absolute configurations. If single crystals of CSDP esters are not available, the NMR anisotropy method using MaNP acid is powerful as the complementary tool. 

We next apphed the method of MaNP acid 3, which is very effective for enantior-esolving racemic alcohols and also for determining the absolute configuration of chiral alcohols by the H NMR anisotropy method as described above. We explain here the apphcation of the MaNP acid method to the chiral thyroid hormone analogue KAT-2003 (-i-)-9 to determine its absolute configuration. 

Fig. 9.27 The H N M R chemical shift data (500 M Hz, CDCI3) of esters (+)-81aand (-)-81band the absolute configuration ofthe first-eluted ester (4-)-81 a as determined by the H NMR anisotropy method using (S)-MaN P acid [52].

As described above, the absolute configuration of the chiral thyroid hormone analog KAT-2003 (-t-)-9 was unambiguously determined as S by the H NMR anisotropy method using novel MaNP acid (S)-(-i-)-3. This result indicates that MaNP method is very powerful for determining the absolute configurations of chiral synthetic drugs. 

Table 8.7. Comparison of the anisotropies of the two classes of Trp residues of ai-acid glycoprotein and Lens culinaris agglutinin. Measurements were performed at 20°C with the Quenching Resolved Emission Anisotropy method.

Time-resolved fluorescence methods are discussed elsewhere in this volume, and we shall not enter into lengthy descriptions of the technique but rather will point out some interesting aspects of time-resolved anisotropy methods as they apply to binding studies. The two principal time-resolved methodologies presently utilized are the impulse-response... 

R 624 Y. Kasai, J. Naito, S. Kuwahara, M. Watanabe, A. Ichikawa and N. Harada, Novel Chiral Molecular Tools for Preparation of Enantio-pure Alcohols by Resolution and Simultaneous Determination of Their Absolute Configurations by the H NMR Anisotropy Method , Yuki Gosei Kagaku Kyokaishi, 2004,62,1114... 

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