Proteases synthesis, using affinity

The constmction of synthetic selenocysteine-containing proteins or selenium-containing proteins attracts considerable interest at present, mainly for the reason that it can be used to solve the phase problem in X-ray crystallography. Selenomethionine incorporation has been used mostly uutil now for this purpose. There are also two reports ou uew synthetic selenocysteine-containing proteins. In one case, the active site serine of subtUisin has been converted into a selenocysteine residue by chemical means, with the result that the enzyme gains a predominant esterase instead of protease activity. In the second case, automated peptide synthesis was carried out to produce a peptide in which all seven-cysteine residues of the Neurospora crassa metallothioueiu (Cu) were replaced by selenocysteine. The replacement resulted iu au alteration of both the stoichiometry and the affinity of copper binding. ... 

Numerous biotranformation processes for fhe synthesis of amino acids have been described and for fhe purpose of this chapter, we have restricted the discussion to fhe unnatural amino acids fhat are not accessible by fermentation. For this class of amino acids, commercialized biotranformations are either based on asymmetric synthesis starting from a prochiral compound or on (dynamic) kinetic resolutions of a racemate. As an illustration the published processes for (R)- and (S)-tert-leucine are outlined in Scheme 4.4. Both stereoisomers of tert-leucine have been used for fhe synfhesis of peptides that serve as protease inhibitors acting against viral infections (e.g. Hepatitis C, HIV), bacterial infections, autoimmune diseases and cancer [27]. This particular amino acid is versatile in fhese applications since fhe tert-butyl moiety provides resistance against endogenous proteases and can enhance the binding affinity of fhe peptide to fhe target protease. 

In the second example, in which desolvation influences were considered, Reddy et al. used a computer-assisted ligand design method that combines molecular mechanics, molecular dynamics, FEP calculations, synthesis, and biochemical testing of peptidomimetic inhibitors, plus crystallographic structure determination of the protein-inhibitor complexes. This combination of techniques successfully led to the design of novel inhibitors for the HIV-1 protease enzyme. This study involved a large set of molecules whose relative binding affinities, predicted via the FEP method prior to synthesis, were later confirmed by experimental measurements. 

The principle of transition state affinity requires the development during enzyme catalysis of very powerful forces of attraction between the enzyme and the altered form of the substrate that is present in the transition state. > This principle has served as a basis for the synthesis of some very strong reversible enzyme inhibitors/" and may also in principle be useful in the design of affinity labeling reagents. The latter possibility remains to be explored, but seems already to be partly substantiated by a few serendipitous examples of inhibitors of glycosidases and proteases. 

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