Cross-linked enzyme crystals CLECS

RA Persichetti, N St. Clair, JP Griffith, MA Navia, AL Margolin. Cross-linked enzyme crystals (CLECs) of thermolysin in the synthesis of peptides. J Am Chem Soc... 

N St. Clair, Y-F Wang, AL Margolin. Cofactor bound cross-linked enzyme crystals (CLEC) of alcohol dehydrogenase. Angew Chem Int Ed 39 380-383, 2000. 

The use of enzymes to stereospecifically form amide bonds has been described in many texts (115) however, the commercial availability cf cross-linked enzyme crystals (CLECs), for example, PeptiCLEC-TR, which is an immobilized form of Thermolysin protease, has been used in the synthesis of D2163 (68), a novel matrix metalloproteinase inhibitor (116). In vitro enzyme screening identified the all-natural SSS-isomer as the active product. The elegant CLEC (117) technology used in this example makes the enzyme stable to typical organic reaction conditions and enables facile removal of the enzyme at the end of the reaction by simple filtration. On this basis, it is... 

Cross-linked enzyme crystals (CLEC, [63]) can have greatly improved catalytic activity over native enzyme. Their practical utility has recently been demonstrated for the hydrolytic kinetic resolution of the acetates of 1-phenylethanol (Figure 3.28) [64]. The catalyst was recovered and recycled eight times, on a 100-liter scale. The future will show increased use of enzymes. 

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... 

The most common methods for the immobilization are entrapment in matrices such as alginate beads, cross-linking, and covalent or adsorptive binding to a carrier. A very recent method is the development of cross-linked enzyme crystals (CLECS) (see also Chapter 6)[23 . A. survey of different immobilization methods can be found in in[24, 25>. 

T. Zelinski, H. Waldmann, Cross-linked enzyme crystals (CLECs) Efficient and stable biocatalysts for preparative organic chemistry, Angew. Chem. Int. Ed. Engl. 1996, 7, 722-725. 

One interesting technology uses lipases in the form of cross-linked enzyme crystals (CLECs) (Margolin 1996). This immobilization method does not use any solid support and the lipase specific activity (units of activity/g of immobilized catalyst) of the immobilized lipase derivative can be enhanced by 10-fold because there is no inert support, that usually represent more than 90% of the catalyst weight in the case of carrier-bound enzymes. These cross-linked crystals have been used for the chiral resolution of commercially important organic compounds, such as ibuprofen. 

Enzymes do have their disadvantages. Their solvent incompatibility and instability restrict their industrial use. However, researchers strive to find means to overcome such drawbacks to make use of the catalytic efficiency of enzymes. Aims Biologies (1997) has developed cross-linked enzyme crystals (CLECs) to increase the versatility of enzymes in organic reactions. CLECs exhibit a high level of stability in extreme conditions of temperature and pH and in exposure to both aqueous and organic solvents. The synthesis of the antibiotic cephalexin was carried out using CLECs (see Fig. 3.21). The N-protection step of methyl phenyl glycinate in the classical synthesis was eliminated. 

---