Chiroptical techniques, absolute configuration

On the basis of these results, it is apparent that the derivation of the absolute configuration of a COOH- or NH2-terminal amino acid in a dipeptide from its chiroptical properties is hazardous, and a comparison of spectral data with those of proper model peptides is needed. Thus, a more reliable technique is to react the NH2-terminal (or COOH-terminal) amino acid of a small peptide with a proper chromophoric reagent, as discussed above (via Edman degradation or other techniques). Cyclic peptides have to undergo at least partial acid hydrolysis before they can be reacted with methyl isothiocyanate, fluorescamine, etc. 

In this chapter, we have attempted to assess the usefulness of chiroptical techniques for determining the absolute configuration of amino acids and small peptides, either directly or via their chromophoric derivatives. Several empirical and semiempirical rules have been described. The importance of conformational equilibria has been stressed repeatedly, and solvent effects have been discussed. It should be emphasized once again that the widest possible variety of standards should be studied when an absolute configuration is to be assigned to an unknown on the basis of empirical chiroptical rules. 

In spite of the fact that nowadays well established and widely applicable X-ray techniques to determine the absolute configuration are available, these methods cannot displace chiroptical analyses, not only because the X-ray analyses needs good crystalline material. With BCD, VCD, and ROA, besides configurational chirality the conformational chirality of the diverse conformations of nonrigid molecules can also be analyzed. 

The relatively new chiroptical technique, vibrational circular dichroism spectroscopy, is coming of age. Instrumentation and theoretical models are sufficiently good to establish VCD as the only spectroscopic method for the determination of absolute configurations of molecules in solution and potentially in the gas phase. 

From the examples discussed in this chapter it results clearly that chiroptical techniques supply a powerfull method for investigating the molecular conformation of macromolecules in solution. The evident limitation of the method is that optically active polymers only display chiroptical properties. While this occurs in the most naturally occurring polymers, it is not true for the most common stereoregular synthetic polymers. In these last the asymmetric carbon atomsliave either one or the other absolute configuration with the same probability and inter-and intra-molecular compensations cancel any optical rotation. 

Spectroscopic methods can work with the chiral selector associated with the ligand either in solid state or in solution. The chiroptical spectroscopies, circular dichroism, and optical rotatory dispersion, represent an important means for evaluating structural properties of selector-ligand adducts [14]. NMR can specifically investigate proton or carbon atom positions and differentiate one from the other. X-ray crystallography is a powerful technique to investigate the absolute configuration of diastereoisomeric complexes but in the solid state only. Fluorescence anisotropy is a polarization-based technique that is a measure, in solution, of the rotational motion of a fluorescent molecule or a molecule + selector complex [15]. 

The correct structure of humulone, in particular the enolization pattern and the absolute configuration of the chiral centre, has been established by a combination of chemical, spectrometrical and chiroptical techniques (11). Humulone has structure 3 (Fig.1) and is (-) (6R)-2-(3-methylbutanoyl)-4,6-bis(3-methyl-2-butenyl)-3,5,6-trihydroxycyclohexa-2,4-dienone (empirical formula C21H3QO0) (20). 

The humulate anion, obtained in alkaline solution, has a positive specific optical rotation. The salts of the D-2-hydroxycarboxylic acids also show a more positive value compared to the free acids. From this analogy the D-configuration has been assigned to (-) humulone (29,30). As shown later, this conclusion, which was only a judicious guess, is wrong. The exact absolute configuration of (-) humulone has been proved independently by two techniques, which will now be discussed. a) From chiroptical studies. 

The configurational assignments are based on the Horeau method and chiroptical techniques, applied to the dihydrodeoxohumulinic acids (see 8.4.3.1.3.). This procedure has been explained in detail when the absolute configuration of the isohumulones was determined (see 5.1.3.2.). It follows that (+) ds humulinic acid (121) has the (4R,5S)-configuration and (-) trans humulinic acid (122) has the (4S.5S) configuration (33). 

Careful analysis by other experts in the chiroptical techniques, however (S. F. Mason, Chem. Comm., 1973, 239 A. M. F. Hezemans and M. P. Groenewege, Tetrahedron, 1973, 29, 1223) and by X-ray crystallographers, now indicates that the error lay in the method used for the calculation of the absolute configurations from the spectroscopic data, and that the Bijvoet method is indeed correct. This interlude did show, however, how slender is the thread of argument, albeit correct, on which the Bijvoet assignments are made, and also how few chemists are truly competent to pass judgement on these matters. 

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