Sulfoxidation mutants

Sulfoxide Mutant Amino acid modifications" sulfoxide ee sulfone... 

The original library of 10 000 clones used in the Baeyer-Villiger reaction [89] was screened for performance as potential catalysts in the sulfoxidation [32]. This led to the discovery of at least 20 mutants having enantiomeric excess values in the range of 85-99%, some being (R) selective and others being (S) selective. Five mutants resulting in enantiomeric excess values of >95% were sequenced (Table 2.2) [32]. 

Sulfoxidations are not restricted to MOs but can also be carried out by dioxygenases. For example. Pseudomonas mutant strain UV4 producing a toluene dioxygenase (TOO) and Pseudomonas NCIMB 8859 expressing a naphthalene dioxygenase (NDO) were used to oxidize aryl sulfides to antipodal chiral sulfoxides [203]. 

Parallel to the modification of the catalytic performance in Baeyer-Villiger oxidations, random mutagenesis was successfully applied to improve the stereoselectivity of CHMOAcineto hi cascs of essentially racemic sulfoxide formation. In addition, enantiodivergent clones with >98% ee for both antipodal products were identified (Table 9.5) [205]. However, improvement in stereoselectivity of mutant enzymes was often accompanied by increased formation of sulfone. This effect can also be utilized to resolve racemic sulfoxides. 

Enzymes, in particular peroxidases, catalyze efficiently the enantioselective oxidation of alkyl aryl sulfides and also dialkyl sulfides, provided that the alkyl substituents are sterically differentiable by the enzyme. The peroxidases HRP, CPO, MP-11, and the mutants of HRP, e. g. F41L and F4IT, were successfully used as biocatalysts for the asymmetric sulfoxidation (Eq. 14). A selection of sulfides. 

In the sulfoxidation, small to appreciable amounts of over oxidation with formation of undesired sulfone were observed, a result that implies that kinetic resolution may be involved in influencing the overall stereochemical result 105). This was shown to be the case. Indeed, some of the mutants are also excellent catalysts in the kinetic resolution of racemic sulfoxides such as 25 105). Directed evolution was then applied successfully to eliminate undesired sulfone formation, specifically, by going through a second cycle of epPCR 105). This is significant because it shows for the first time that an undesired side reaction can be eliminated by directed evolution. 

Although a systematic study of the mutants as catalysts in the sulfoxidation of a wide variety by thioethers has not been completed, initial results are promising 105). For example, when thioether 56 was used as the substrate, previously identified mutant 1-C5-H3 induced complete reversal of enantioselectivity. The WT CHMO resulted in 47% ee in favor of (R)-57, whereas 1-C5-H3 led to 98.9% ee in favor of (5)-57 105). An interpretation of the results on a molecular basis remains to be offered. Since a homology model of the CHMO has been generated 183), this may in fact be possible. 

The oxidative stability of subtilisin has been extensively studied and improved stability has been engineered. In subtilisin BPN two methionines, MET " and MET are especially susceptible to oxidation. To prevent the negative influenee eaused by the formation of methioiune sulfoxide the MET can be substituted with ALA, SER or LEU, without loosing more than 12-53% of the activity. One such mutant MET222. ALA is currently in use as a commercial detergent enzyme Durazyme (Riisgard, 1990). 

Some mutant mice have extended lifespans. The Ames dwarf mouse has a mutation in p66shc, a cell-surface protein that contains both Src-homology and collagen-homology domains. It lives almost one-third longer than do wild-type mice.538 Mice deficient in methionine sulfoxide reductase have a reduced lifespan539 but fruit flies with overexpressed activity of the enzyme are more resistant than wild-type flies to oxidative damage.540... 

Asymmetric sulfoxidation by Mb mutants are also applicable to other sulfides bearing simple substituents. Table III shows examples of asymmetric sulfoxidation catalyzed by wild-type and L29H/H64L Mb (48). For a wide range of substrates, L29H/H64L Mb exhibits high activity and selectivity. 

Thioanisole and styrene share a similar structure, thus the configuration of the sulfoxide and epoxide are expected to be the same, since both substrates could be accommodated in a similar manner in the active site of Mb mutants. 

However, the (M)-phenyl methyl sulfoxide has the opposite configuration to (Mi-styrene oxide (Fig. 8, inset) (49). A possible structural difference between styrene and thioanisole is the fact that the ethylene group in styrene is in the plane of the phenyl ring while the S-methyl group in thioanisole is perpendicular to the phenyl group. If the Mb mutants discriminate this steric difference, one could expect (S)-sulfoxide formation if cyclic sulfides are employed as substrates, since the cyclic sulfides should have planar structures (Fig. 8c) similar to styrene. Table III lists representative results of cyclic and acyclic sulfide... 

Ozaki S-I, Ortiz de Montellano PR (1995) Molecular engineering of horseradish peroxidase thioether sulfoxidation and styrene epoxidation by Phe-41 leucine and threonine mutants. J Am Chem Soc 117 7056-7064... 

Ozaki S, Matsui T, Watanabe Y (1997) Conversion of myoglobin into a peroxygenase a catalytic intermediate of sulfoxidation and epoxidation by the F43FI/H64L mutant. J Am Chem Soc 119 6666-6667... 

In a related study, we also evolved CHMO-mutants which catalyse the sulfoxidation of thio-ethers such as 6 (Reetz et al. 2004b) (Scheme 3). 

A question of particular interest concerns the catalytic advantage that a selenol in the active site of an enzyme has in comparison to a thiol group. It has been approached by replacing the selenocysteine in the formate dehydrogenase Ft by a cysteine residue and by determining the catalytic parameters of the Se-wild-type species with the mutant S-species. Table 1 presents the data obtained. They show that koit of the Se enzyme is more than 300-fold higher than that of the S variant and that the affinity to the substrate is only marginally affected. Similar replacements of selenocysteine by cysteine in other enzymes like the deiodinase type 1 or methionine sulfoxide reductase B corroborated these results. 

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