Subtilisin-CLEC

A beneficial feature of subtilisin, and in particular subtilisin-CLECs, is their high catalytic activity in polar and non-polar organic solvents, allowing for transesterifications of alcohols in the presence of small amounts of water. Transesterifications catalyzed by subtilisin were mostly done with vinyl acetate. Apparently, the acetaldehyde formed during transesterification is not harmful to the enzyme as it is in the case of some lipases and pig liver esterase. Although resolution of such alcohols either through hydrolysis of the corresponding esters or transesterification is the domain of lipase, in some cases useful selectivities were achieved with subtilisin (1-9) (Table 11.1-26). 

In addition, the (S)-seleetive DKR of aleohols with subtilisin-CLEC (cross-linked ciystal) was also possible in ionie liquids at room temperature." In this case, a cymene ruthenium eomplex was used as the catalyst, and the optical purities of the final (S)-esters were lower than those of the (R)-esters obtained by using lipases, as shown in Seheme 8.34. 

Regioselective reactions in systems which contain multiple functional groups are an area ideally suited for biocatalysis. Linhardt and co-workers at the University of Iowa s Division of Medicinal and Natural Products Chemistry recently published the synthesis of a series of 1 -O-acyl sucrose derivatives [31], Using Chiro-CLEC -BL (the CLC of subtilisin) and vinyl esters of the acylating agent in pyridine as solvent, the authors prepared l -O-lauryl sucrose, l -O-myristyl sucrose, and l -O-stearyl sucrose in 80-90% yield (Fig. 9). Their method represents a green alternative to the tin chemistry previously used [32],... 

Y-F Wang, K Yakovlevsky, AL Margolin. An efficient synthesis of chiral amino acid and peptide alkylamides via CLEC-subtilisin catalyzed coupling and in situ resolution. Tetrahedron Lett 37 5317-5320, 1996. 

Much of the current interest in making simple derivatives of (+ )-castanospermine (239) can be traced to a seminal publication in 1989, which showed that the alkaloid s anti-HIV activity could be increased by as much as twenty times upon esterification (216). Positionally selective acylation procedures usually involve sequential protection, acylation, and deprotection steps e.g. the preparation of esters at the C-6 and C-7 (217) or the C-8 hydroxy groups (218). Also of interest are procedures that take advantage of enzyme-catalyzed transesterification with activated esters, e.g. the use of subtilisin for ester formation at C-1, pancreatic porcine lipase for preferential reaction at C-6 and C-7 (219-221), and cross-linked enzyme crystals (CLECs) of subtilisin for making the potentially valuable antitumor agent 1-0-butanoylcastanospermine (222). A cautionary note was sounded, however, when it was observed that 6-0-acyl castanospermine esters could equilibrate to a mixture of... 

Currently, Candida rugosa and Pseudomonas cepacia lipases, thermolysin, subtilisin, and penicillin acylase are commercially available from Altus Biologies Inc. and have proved to be very effective catalysts for the synthesis of peptides and esters, and the resolution of acids, alcohols, amines, and amino acids in a variety of media [370-372,374-377,379-381]. In addition, proteinase K, lactate dehydrogenase, horse liver alcohol dehydrogenase, and rabbit muscle aldolase have also been crossUnked and shown to operate efficiently in synthetic reactions [305,374,378,382,383]. The unique features, and the advantages and drawbacks of using CLECs, are outlined in Table 9. 

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