Solid-Phase Synthesis and Combinatorial Technologies

564 BIOSYNTHETIC COMBINATORIAL LIBRARIES

OH

OH

10.102

L30 = 1222 discretes

Figure 10.51 Combinatorial biocatalysis: synthesis of the BOD-focused biocatalytic discrete library L30.

introduced new functional groups on BOD (halohydration) or selectively functionalized one of the existing alcoholic functions (glycosylation, acylation). The reactions were carried out either in organic or in aqueous solvents, and the experimental conditions were adjusted to take account of the stability of the enzyme and the solubility of the substrate. Each biotransformation was monitored using chromatographic methods, and the final products were characterized by MS and NMR. The different sequence of enzymes used and the specificity of each of them produced a large, diverse library of BOD analogues. Some selected structures of this biotransformation library L30 (1222 derivatives in total) are shown in Fig. 10.52 to illustrate the variety of functional groups, products, and physicochemical properties that can be obtained by such an approach. It is noteworthy that all the isolated compounds are also optically active.

Khmelnitsky also reported using adenosine and 2,3-(methylene dioxy)benzaldehyde as substrates to create libraries of 92 and 457 compounds, respectively. Taxol has been acylated to give a library of 200 compounds, among which two showed a significant improvement in water solubility compared to the parent compound.

10.4.3 An Example: Synthesis of a Bergenin-Derived Library By Combinatorial Biocatalysis

Mozhaev et al. (310) have reported the synthesis of an 167-member, focused library L31 obtained by biocatalytic manipulation of bergenin, a polyhydroxylated flavonoid

HO

Glycosylation

Acylation I Acylation I

Acylation II

Figure 10.52 Combinatorial biocatalysis: structures of several individuals from the biocatalytic focused discrete library L30.

(10.103, Fig. 10.53). A thorough search among commercially available acylating enzymes was carried out to select those able to regioselectively acylate any of the five OH groups. However, although many 11-regioselective enzymes able to discriminate the primary OH from the others were found, only one 4,11-diacylating enzyme, subtilisin Carlsberg, was found that was able to acylate any of the other hydroxyls. The enzymes were purchased and immobilized (311) or lyophilized in the presence of KCl (312) to facilitate handling and to increase their catalytic activity. Various chemical conditions for acylation of 10.103 were also attempted, but invariably only mixtures of mono-, di-, and triacylated bergenins were obtained.

The library synthetic scheme is reported in Fig. 10.53. The synthesis was performed in 96-well plates using an automated liquid dispensing unit for sampling liquid aliquots. Bergenin was first submitted to 11-regioselective acylation with a mixture of four immobilized lipases (step a) and the acyl donor monomer set M1 (12 representatives, Fig. 10.54) in organic solvents, using 168 reaction wells. Purification of the

566 BIOSYNTHETIC COMBINATORIAL LIBRARIES

L31 = 10.104+10.105+10.106 = 167 discretes (one non-confirmed diacylated library member)

a:lipase catalytic mixture (PS30, FAP-15, Chirazyme L-2, Chirazyme L-9), acetonitrile, 45ºC;

b:separation of the immobilized enzyme mixture by filtration; c: Subtilisin Carlsberg/95% KCl, acetonitrile, toluene, DMSO, 45ºC; d: separation of liophilized subtilisin by centrifugation; e: lipase mixture

as in step a, acetonitrile/water 98/2, 45ºC, 96 hrs.

Figure 10.53 Synthesis of the bergenin-focused discrete library L31 using combinatorial biocatalysis.

products (step b) included removal of the immobilized enzymes by filtration of the solutions through the filter of the plates, evaporation of solvents and extraction of the excess of the acyl donor with n-hexane. Twelve wells containing twelve 11-acyl bergenins 10.104 were analyzed and archived, while the monoacylated residues in the other wells were treated with lyophilized subtilisin Carlsberg and the same acyl donor set M1 (step c) followed by centrifugation to remove the enzyme (step d). One hundred and forty-four wells containing all the possible combinations of homoand hetero- 4,11-diacylated bergenins 10.105 were characterized and archived, while the remaining 12 were treated with the same enzyme cocktail used in step a but in presence of water (step e). In this case, the 11-acyl group was regioselectively hydrolyzed and twelve 4-acylated bergenins 10.106 were obtained after the usual purification procedure (step b, Fig. 10.53).

Only one diacylated derivative out of the 168 reaction products in L31 was not confirmed after HPLC/MS quality control, probably due to the steric hindrance of the acyl donor, whereas all the other library individuals were obtained in 60–90% yields

N

Figure 10.54 Monomers M1 used to prepare the bergenin-focused discrete library L31.

and good purities. The synthesis of L31 by conventional organic synthesis would have required a complex multistep protection–deprotection strategy, and the final products would undoubtedly have been obtained with lower yields.

A larger 600-member bergenin-based library using acylations, oxidations, halogenations, and glycosylations and a 24-member 3,6-dihydroxytropane library have been prepared by the same group (313); a library of acylated taxol derivatives was also reported (314). A survey of theoretical applications for combinatorial biocatalysis has recently appeared (315).

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