
Solid-Phase Synthesis and Combinatorial Technologies
.pdf10.3 COMBINATORIAL BIOSYNTHESIS OF NATURAL PRODUCTS 553
determine the exact structure of an active compound. Whereas in combinatorial libraries the members of the libraries are effectively present in roughly equimolar amounts and their complexity can be resolved with relative ease, natural extracts contain many chemical entities including macromolecules, inorganic materials, and small organic molecules in different amounts, such that an active component may well be present at a very low concentration and the work-up procedures to purify, separate, and structurally characterize any individual from the extract can be long and laborious. Finally, the large majority of extracts (>99%) showing an interesting biological activity are known and therefore unexploitable compounds, while combinatorial libraries may be designed to contain only novel, druglike individuals. Over time a number of efficient methods have been introduced to reduce the time required to purify and isolate a natural product and determine the novelty of its structure. Nevertheless, the diversity embedded in NPs is mostly applied in specific therapeutic areas or where combinatorial methods have failed to produce active compounds.
The biosynthesis of a natural product is an extremely complex event that is carried out by multienzyme systems showing a high specificity for the sequential elaboration of simple precursors into complex end products. Often these systems perform iterative processes to synthesize the final NP as an assembly of units, with each cycle being performed by a multifunctional protein containing several active sites responsible for each transformation in a given cycle (e.g., polyketides, vide infra). The cluster of active sites in a multifunctional protein is referred to as a module (240). Genetic manipulation of these modules has allowed the full characterization of several of these pathways in order to understand the features of the many enzymes involved and to test their structural specificity. Combinatorial alteration of naturally occurring modules, usually referred to as combinatorial biosynthesis (241), involves either modification of the order of individual enzymes in the module complex or deletion or duplication of an enzyme activity through deletion or addition of a component to the module. This process can be completely controlled by genetic manipulation and results in the production of modified analogues of the parent NP. Libraries of modified NPs obtained by combinatorial biosynthesis are structurally defined, in that the design of several module modifications determines the expected structure of any member of the library in any specific library well (Fig. 10.42). The final compounds are novel, because only modifications leading to unprecedented compounds are considered and can be detected, purified, and isolated from the biological mixture using well-known protocols. These modified NP libraries ensure access to biologically relevant NP diversity that is constantly increased by the elucidation and characterization of novel biosynthetic pathways. The following sections will focus on the most studied biosynthetic pathways and on some specific examples of their combinatorial modification.
10.3.2 Combinatorial Biosynthesis of Polyketides
Polyketides (PKs) are a typical example of a large and diverse class of NPs that derive from several related biosynthetic pathways. Their structures contain repeating units iteratively assembled into a range of diverse chemical structures (Fig. 10.43). PKs can be taken as an example of the application of combinatorial biosynthesis as both the

10.3 COMBINATORIAL BIOSYNTHESIS OF NATURAL PRODUCTS 555
chemistry and the biology of their biosynthesis are well known (242–245) and they are suitable for extensive manipulation to produce libraries of modified PKs. Standard protocols for the genetic manipulation of PK producers and for the expression of engineered, novel PKs (246, 247) are now routinely used.
Two major classes of multifunctional polyketide synthase (PKS) enzymes can be considered. Iterative PKS enzymes are made up of a single module that iteratively processes biosynthetic units for a synthetic cycle, adding a new monomeric unit and repeating the whole cycle until the assembly of the final PK is finished. Modular PKS enzymes are made up of several modules embedded into multifunctional proteins that are specifically responsible for one of the several cycles necessary to produce the final PK. The biosynthetic intermediates remain anchored to an individual module until the cycle is successfully terminated; then they are shunted to the next module in order to continue the PK biosynthesis. Among iterative PKS enzymes, actinorhodin synthase produces actinorhodin (10.69, Fig. 10.44) by the iterative action of a minimal PKS enzyme composed of a ketosynthase (KS), which carries the growing PK chain and couples it with a carboxylate extender unit 10.67 loaded onto an acyl carrier protein (ACP) and transported by an acyltransferase (AT). Iteration of this cycle is sometimes punctuated by other activities such as a ketoreductase (KR), an aromatase (ARO), and a cyclase (CYC). The advanced intermediate 10.68 is converted into 10.69 by several tailoring enzymes (Fig. 10.44). The combinatorial potential of these pathways is high, including different starter and extender units, manipulation of ARO and CYC activities, the use of KR activities from several different producing organisms, and the use of tailoring enzymes from other sources. Several reviews (248–252) and papers (253–264) have extensively covered the subject including an example of iterative PKS from plants (265).
Modular PKS enzymes are responsible for the synthesis of a wide diversity of structures and seem to have more relaxed specificities in several of the enzymatic steps. Their enormous appeal for combinatorial purposes, though, derives from the presence of multiple modules that can be manipulated independently, allowing the production of rings of different sizes and with potential stereochemical variation at each PK carbon. The higher complexity of these pathways has somewhat hindered their exploitation, but recently, several have been fully characterized. Among them, by far the most studied modular multienzyme complex is 6-deoxyerythronolide B synthase (DEBS; 240, 266, 267), which produces the 14-member macrolide 6-deoxyerythronolide B (10.70, Fig. 10.45). DEBS contains three large subunits each of which contains two PKS enzyme modules. Each module contains the minimal PKS enzyme (vide supra) and either none (M3), one (ketoreductase KR; M1, M2, M5, and M6), or three (dehydratase DH–enoyl reductase ER–ketoreductase KR, M4) catalytic activities that produce a keto (M3), an hydroxy (M1, M2, M5 and M6), or an unsubstituted methylene (M4) on the last monomeric unit of the growing chain (Fig. 10.45). A final thioesterase (TE) activity catalyzes lactone formation with concomitant release of 10.70 from the multienzyme complex. Introduction of TE activity after an upstream module allows various reduced-size macrolides (10.71–10.73, Fig. 10.45) to be obtained.


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6-deoxy erythronolide B
Figure 10.45 Modular PKS enzymes: biosynthesis of 6-deoxy erythronolide B (10.70) and modified biosynthetic products 10.71–10.73.
execute novel combinatorial biosynthetic approaches. The same is true for modular biosynthetic pathways other than PKS enzymes, such as nonribosomal peptide synthetases (276, 289, 290), which have also been reviewed recently (241, 291, 292), and deoxy sugar biosynthetic pathways (293, 294).
Jacobsen et al. (295) reported the biosynthesis of a 16-membered macrolide from a modified DEBS multienzyme in which KS in M1 was inactivated and the unnatural di- (10.74) and triketides (10.75 and 10.76, Fig. 10.46) were used to feed M1 or M2 (Fig. 10.46) (295). The products 10.70, 10.77, and 10.78 show how not only decreased ring sizes but also larger ones (path 3, Fig. 10.46) are available by simple genetic manipulations, and they also demonstrate the selectivity of the modules even for the chirality of a single atom (compare paths 2 and 3, Fig. 10.46). Jacobsen also reported

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Figure 10.46 Combinatorial biosynthesis: manipulation of the ring size and the stereochemical transformations of DEBS modules to produce analogues 10.77 and 10.78.
the first successful shuffling of entire modules, rather than single enzymatic activities, to increase the diversity of PKs obtained. The small macrolide 10.79 was produced by hybrid bimodular subunits M1–M3 or M1–M6, where a polypeptide linker connected the two modules and allowed the processing of the substrates, as for the natural bimodular M1–M2 (Fig. 10.47). More significantly, though, the replacement of M2 in DEBS with a module from rifamycin PKS (rapM5) containing the same activities of M2 gave the natural PK 10.70 with reasonable yield (Fig. 10.47) (296).



10.3 COMBINATORIAL BIOSYNTHESIS OF NATURAL PRODUCTS 561
L29
>100-member macrolide library multiple modifications:
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Figure 10.49 Combinatorial biosynthesis of the PK library L29 from modified DEBS modules: doubleand triple-domain substitution products.
562 BIOSYNTHETIC COMBINATORIAL LIBRARIES
rifampicin M2, rapAT2, gave various desmethyl erythronolide B derivatives 10.80– 10.84, whereas the replacement of their KR domains with a synthetic amino acidic linker to suppress ketone reduction gave keto erythronolides 10.85–10.86. Anhydro erythronolides 10.87–10.89 were obtained by introducing the rapDH/KR4 domains of rifampicin M4, and inclusion of the rapDH/ER/KR4 M4 domains led to deoxy erythronolides 10.90–10.91. The epimeric erythronolide 10.92 was formed when the rapKR2 was incorporated. Double or triple mutations were also inserted into DEBS (Fig. 10.49), in the same module (10.93–10.95), in two modules (10.96–10.99), or even in three modules (10.100–10.101), to generate a total of >100 novel PKs, including several by-products obtained from each of the manipulated multienzyme proteins, to give the library L29.
Another report by Xue et al. (298) presented a multiple-plasmid strategy employed to increase exponentially the number of polyketides obtainable from a limited number of experiments using DEBS as a test PKS. A library of 43 fully characterized polyketides (6dEB, 11 single mutations, 26 double mutations, 5 triple mutations) was obtained.
The flexibility of each DEBS module was proven by this work, which will almost certainly be followed in the near future by further work aimed at the mutation of this and other macrolide pathways. The major obstacle encountered during this work was the marked reduction in productivity (ranging from 1 to 70% of the wild-type 10.70), especially when several modules were modified. The use of higher yielding replacements and alterations along with careful optimization of productivity by genetic means could overcome this problem.
10.4 COMBINATORIAL BIOCATALYSIS
10.4.1 General Considerations
Combinatorial chemistry has proven its usefulness for the synthesis of chemical libraries with different degrees of complexity embedded in the scaffolds and the building blocks used. The synthesis of polyfunctionalized molecules in a combinatorial format, though, often requires the careful adjustment of experimental conditions and the selection of orthogonal protecting groups to prevent side reactions, degradation, or problems with regioselectivity. The synthesis of libraries of complex, chiral compounds has mostly been an unattainable target for combinatorial chemists.
Enzymes have often been used as reagents in organic reactions (299, 300). However, several new directions in the development of biocatalysts such as the utilization of enzymes from extremophiles (301, 302), nonaqueous enzyme technology (303, 304), and directed evolution (305, 306) now ensure the wider applicability of purified enzymes, and even whole cells, to organic biotransformations (307). Many of these enzymes are commercially available, inexpensive, and able to perform a wide range of chemical transformations, including the introduction of new functional groups on a scaffold, the modification of existing functionalities, and addition onto functional groups. Their most appealing features as reagents in combinatorial chemistry are the