Enzymatic synthesis subtilisin

Finally, a recent example on the biocatalyzed synthesis of new cytotoxic derivatives of the anthracycline doxurobicin (8) deserves to be mentioned. In the presence of Mucorjavanicus lipase or subtilisin Carlsberg ion-paired with Aerosol , the primary C-14 OH was chemoselectively acylated to give the corresponding derivatives, such as the valerate 8a [19]. However, optimization of this enzymatic synthesis did not provide a synthetic approach suitable for the preparative synthesis of gram quantities of Valrubicin (N-trifluoroacetyl doxurobicin-14-valerate, 8b)... 

Klein AE, Freiberg J, Same S et al. (1989) Rapid colorimetric determination of activity of subtilisin enzymes in cleaning products. Assoc Off Anal Chem 72(6) 881-882 Klibanov AM (1977) A new approach to preparative enzymatic synthesis. Biotechnol Bioeng 28 417 21... 

Enzymatic acylation reactions offer considerable promise in the synthesis of specific ester derivatives of sucrose. For example, reaction of sucrose with an activated alkyl ester in /V, /V- dim ethyl form am i de in the presence of subtilisin gave 1 -0-butyrylsucrose, which on further treatment with an activated fatty acid ester in acetone in the presence of Hpase C. viscosum produced the 1, 6-diester derivative (71,72). 

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

It was known from the work of Richards that RNase A could be cleaved between residues 20 and 21 by digestion with subtilisin. 29 The N-terminal 20-residue peptide (S-peptide) could be separated from the 104-residue 21-124 protein (S-protein), and each was enzymatically inactive however, when they were mixed in a 1 1 ratio, full RNase activity was regenerated even though the covalent bond between residues 20 and 21 was not re-formed. This finding was the basis for a great many structure/function studies by the synthesis of analogues of the S-peptide and their noncovalent recombination with natural S-protein.13"31 ... 

The presence of crown ethers in aqueous medium before lyophilization can greatly enhance the activity of proteases (e.g., chymotrypsin, subtilisin, trypsin) for peptide synthesis in organic solvents. - The crown ether must also be rinsed away from the lyophilized enzyme before use. The activity enhancement is solvent dependent. It is speculated that crown ethers accelerate enzymatic rates either by preventing a salt bridge from forming in the enzyme s secondary structure or inducing microscopic changes in the structure of the solid phase. ... 

It has been demonstrated that the combination of metal-catalysed racemisation and enzymatic kinetic resolution is a powerful method for the synthesis of optically active compounds from racemic alcohols and amines. There are many metal complexes active for racemisation, but the conditions for enzymatic acylation often limit the application of the metal complexes to DKR. In the case of DKR of alcohols, complementary catalyst systems are now available for the synthesis of both (R)- and (5)-esters. Thus, (R)-esters can be obtained by the combination of an R-selective lipase, such as CAL-B or LPS, and a racemisation catalyst, whereas the use of an A-selective protease, such as subtilisin, at room temperature provides (5)-esters. The DKR of alcohols can be achieved not only for simple alcohols but also for those bearing various additional functional groups. The DKR of alcohols has also been applied to the synthesis of chiral polymers and coupled to tandem reactions, producing various polycyclic compounds. 

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