Subtilisin Carlsberg selectivity

Early reports on the effects of the choice of solvent on enzymatic enantioselectivity showed that substantial changes may be observed. For the transesterification reaction of sec-phenethyl alcohol with vinyl butyrate catalyzed by subtilisin Carlsberg, a 20-fold increase in the E-value was reported when the medium was changed from acetonitrile to dioxane [59]. Similar changes were recorded for the prochiral selectivity of Pseudomonas sp. lipase in the hydrolysis of 2-substituted... 

Naturally occurring Upases are (R)-selective for alcohols according to Kazlauskas rule [58, 59]. Thus, DKR of alcohols employing lipases can only be used to transform the racemic alcohol into the (R)-acetate. Serine proteases, a sub-class of hydrolases, are known to catalyze transesterifications similar to those catalyzed by lipases, but, interestingly, often with reversed enantioselectivity. Proteases are less thermostable enzymes, and for this reason only metal complexes that racemize secondary alcohols at ambient temperature can be employed for efficient (S)-selective DKR of sec-alcohols. Ruthenium complexes 2 and 3 have been combined with subtilisin Carlsberg, affording a method for the synthesis of... 

For the two proteases trypsin and subtilisin Carlsberg (Table 2, Entries 6 and 7), only the latter showed some activity under these conditions, also with some selectivity for the longer acyl chains similar to HLE. (3-Galactosidase ((3-Gal, Table 2, Entry 5), did not, however, show any activity, and control experiments with bovine serum albumin (BSA, Table 2, Entry 8) resulted in no hydrolysis products. 

The resolution of ( )-Z/77 7 o-mcthylphcnidate (10) free base by enantioselective enzymatic hydrolysis was first reported by us (Novartis) (Scheme 9).[ ] a-Chy-motrypsin and subtilisin carlsberg exhibited selectivity towards the hydrolysis of the (2/ ,2 / )-enantiomer. 

Enantioselective esterification can be pressure dependent in supercritical fluoroform. For example, the selectivity of the transesterification of A -acetyl-phenylalanine ethyl ester with methanol catalyzed by Subtilisin Carlsberg was shown by Kamat et al. (26) to be greater at higher pressures than at lower pressures [Eq. (9)] ... 

In the original process [2, 3] the crystalline substrate had been applied as a DMSO or ethanol solution. Thus, the substrate concentration could not be increased significantly without increasing the co-solvent concentration, which in turn would affect the enzyme activity. Elevating the temperature to 36 °C or above could solve the problem. At this temperature the suspended substrate starts melting while Subtilisin Carlsberg retained its activity and selectivity. 

A very straightforward approach in route C (Fig. 2) would have been the direct enzyme-catalyzed peptide formation (cf. Chen et al. [18]) by enantioselective aminolysis of diester 9 with (S)-tert-leucine methylamide 13 or even racemic 13. This would combine three synthetic objectives the resolution of (rac)-9, the resolution of (roc)-13 and the coupling step. In orientating experiments monoester 10 was tested as a model substrate. It was contacted with an equal amount of (S)-amine 13 in the presence and absence of an organic solvent. Solid or liquid subtilisin Carlsberg preparations (Alcalase 2.0 T or Alcalase 2.5 L, respectively) were used as the catalyst. Only with the liquid enzyme preparation was the formation of minor amounts of one of two possible diastereoisomeric peptides observed [19], whereas most of the ester was hydrolyzed to the acid. Likewise, a few selected lipases also provided negative results. 

The starting point of the parameter screening was the relatively plain standard conditions used in our initial experiments described in note [21]. Since Subtilisin Carlsberg is known to tolerate high substrate concentrations [22], besides the 0.4% concentration, 7.2% was also tested. The initial experiments were already revealing excellent selectivity (>98% ee at 49% conversion (E>100 [23]). Therefore, no further additives such as co-solvents or salts were tested. Instead, the optimization was aimed at a simpler reaction system and technically more attractive conditions. 

A further possibility for the enzymatic removal of C-terminal blocking groups is opened up by the application of enzymes which generally display a high esterase/ protease ratio. Such a biocatalyst is the alkaline protease from Bacillus subtilis DY which shows similarities to Subtilisin Carlsberg. For this enzyme the ratio of esterase to protease activity is >105. It selectively removes methyl, ethyl and benzyl esters from a variety of Trt-, Z- and Boc-protected di- and tripeptides and a pentapeptide at pH 8 and 37 °C (Fig. 18-9) (91l... 

Products from the ( -selective DKR of secondary alcohols with subtilisin Carlsberg and 5. 

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