Swartz Analytical Techniques in Combinatorial Chemistry

Introduction

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is that the kinetics for reaction of the substrate with each of the diverse building blocks may not be identical. When reaction kinetics are not identical, the resulting product mixture will not be equimolar, and it is possible that some of the presumed products will not be present in the product mixture at all. Furthermore, each individual polymer bead in the mixture will contain a mixture of products rather than a single product, thus complicating analysis and deconvolution (as will be discussed later).

A better way to prepare mixtures by solid phase synthesis is by using the ‘‘split-and-pool’’ method. With this method a single substrate is bound to the resin; then the resin is split into a number of equal size pools, where the number of pools represents the number of diverse building blocks to be used in the first step of the synthesis. Each pool is then reacted with its designated building block in a separate reaction vessel under conditions where the reactions are driven to completion (e.g., by using excess reagents). Following reaction, the pools are filtered to remove excess reagents and washed with fresh solvent. All of the polymer from all of the pools is then recombined and mixed thoroughly. The combined resin is then split again into a new set of pools representing the number of diverse building blocks to be used in the second step of the synthesis. At this point, each resin pool should contain an equimolar amount of each of the products created in the first synthesis step. The process is then repeated through as many cycles as needed to complete synthesis of the entire library.

The split-and-pool method is illustrated in Fig. 2 for a two-step synthesis involving three building blocks at each step resulting in three equimolar mixtures of three compounds each. The starting functionalized resin is divided into three equal pools, and each pool is reacted with a specific reagent [R1(a), R1(b), or R1(c)] in a separate container. At the end of this step, each reaction container contains a single specific resin–bound product. Reagents are then removed from the reactions and the solid resins are washed with fresh solvent. All three resin batches are then combined and mixed thoroughly to provide an equimolar mixture of the three possible products. Note that, in principle, one could obtain the same mixture by simply reacting all of the starting resin with a mixture of the three reagents in a single vessel. In practice, though, this process will not provide an equimolar mixture of products due to differential reaction kinetics. It is the desire to obtain equimolar mixtures of products that necessitates the split-and-pool method.

Returning to the hypothetical split and pool synthesis (Fig. 2), the chemist would next split the equimolar mixture of three products into another three identical pools. Reaction of each of these pools separately with three different

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Figure 2 Split-and-pool synthesis.

Introduction

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reagents [R2(a), R2(b), and R2(c)] results in three equimolar mixtures of three products each, for a total of nine new products. The products can be cleaved from the solid support to provide three mixtures of three compounds each.

The split-and-pool method has several interesting ramifications. First, since it does not depend on identical reaction kinetics, it should produce an equimolar mixture of products assuming that all reactions are driven to completion. Second, since any individual polymer particle follows only one synthetic path, all of the product molecules bound to that particle should be the same. That is, one compound is prepared per bead. Third, since one compound is prepared per bead, there must be at least as many polymer beads in the total synthesis pool as there are theoretical products being formed during the synthesis. Imagine, for example, if the split-and-pool synthesis outlined in Fig 2 was begun with only six resin particles! In practice a statistical sampling of the total resin pool is made with each ‘‘split’’ and statistical analysis can be done to determine the required number of particles to ensure adequate sampling of a given pool (30,31). Finally, split-and-pool methodology results in significant reduction in the total number of reactions needed to produce the library. Imagine, for example, a library where building blocks are added in three sequential chemical steps and where each building block is represented by 10 different variations. The total library size will be 10 10 10 1000 compounds. To prepare the compounds conventionally would require 1000 individual reactions at each step or 3000 reactions in total. Using split-and- pool methods, only 10 reactions are required at each step or 30 reactions total to prepare the library. Counterbalancing the synthesis efficiency is the effort required to split and pool the resin particles.

At the end of a split-and-pool synthesis, the chemist has the option to recombine all resin particles into a single pool and then cleave products, or to keep the final split mixtures separate and cleave each individually. Using the example in Fig. 2, the chemist could recombine all polymer resin prior to cleavage to obtain an equimolar mixture of all nine compounds in the library. Alternately, the chemist could keep the three pools used in the final step separate and cleave each individually, resulting in three separate mixtures of three compounds each as illustrated in the figure. The choice of which path to take is dictated largely by the target mixture size. If there are too few compounds in the final mixture the synthetic advantages of mixtures are lost, whereas if there are too many compounds in the mixture there is significant risk that a single active compound will be missed during bioassay due to the dilution effect of other mixture members. In the early days of combinatorial synthesis, very large mixture libraries were common, with mixtures containing tens of thousands or even hundreds of thousands of compounds commonly reported.

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For example, a library containing 19 105 (nearly 2 million) pentapeptides was prepared from 19 of the 20 naturally occurring amino acids in 1991 (32), as was a library of over 34 million hexapeptides consisting of 324 mixtures of 104,976 compounds each (33).

Because of concerns about not detecting low concentrations of active compounds in such large mixtures, the current trend is toward much smaller mixtures containing only hundreds or even dozens of compounds. For example, the Affymax group prepared a library containing mixtures of only 540 compounds and identified potent cyclooxygenase-1 inhibitors after deconvolution (34). The issue of optimum mixture size is not yet settled and discussion will no doubt continue for some time.

When bioassay of a mixture produces apparent activity, one must then ‘‘deconvolute’’ the mixture to identify the active component (or components). The most straightforward deconvolution method is independent synthesis of each individual member of the original mixture. For large mixtures, though, such an approach is not practical. Instead, iterative resynthesis of increasingly smaller mixtures is used to ultimately identify the active component. For example, a mixture of 104,976 hexapeptides was deconvoluted by four iterative syntheses of 20 compounds or mixtures each (35). In the first iteration, 20 mixtures of 6859 peptides were synthesized. The most active mixture was then used as the starting point for 20 mixtures of 361 compounds. Next, 20 mixtures of 19 compounds each were synthesized and, finally, the 19 individual compounds of the most active mixture were synthesized to identify the single most active compound in the original mixture.

Iterative deconvolution works best when a single compound of the mixture is much more active than all others. Conversely, deconvolution is difficult when all members of a library have similar activity—as is often the case in the later stages of a drug discovery process. Because of the difficulties associated with mixture deconvolution, split-and-pool synthesis is best suited to the early lead discovery phase of the drug discovery process. Later stages, requiring direct comparison of one compound against another, are best served by other methods described below.

B. Chemically Encoded Libraries

The synthetic advantages of preparing mixtures instead of individual compounds are offset by the need to deconvolute mixture libraries following bioassay. Deconvolution shortcuts, such as synthesis of multiple libraries containing orthogonal pools (36), are sometimes useful but can only be relied on when the activity of the mixture results from a single highly active compound. Keep-

Introduction

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ing in mind that split-and-pool methodology results in only one compound per polymer bead, the difficulties associated with deconvolution and the uncertainties of screening mixtures can be eliminated by the screening of individual compounds from single beads. Methods have been developed for screening products while still attached to the beads. Alternately, following split-and- pool synthesis, the individual beads are separated and the products cleaved individually from the beads. The individual products are then screened in the bioassay. Active compounds discovered in the bioassay are individual compounds rather than components of mixtures; thus, no deconvolution is necessary. How, though, can one identify the structure of the active compounds from the small (subnanomole) amount prepared on a single bead? Direct analysis of the products by sensitive analytical methods has been reported. For example, peptides and oligonucleotides have been analyzed in the 5-pmol range by Edman degradation or DNA sequencing (37). Molecular weights have been obtained for small organic molecules from single-bead synthesis (38), but large libraries may contain more than a single member with the same mass. The solution to the problem of product identification from single bead synthesis lies mainly with encoded libraries.

Chemically encoded libraries are synthesized from bifunctionalized polymers. During each step of the split-and-pool synthesis, two separate chemical reactions take place. The first adds a building block to the polymer-bound substrate and leads to the target product. The second adds a building block to a unique chemical tag that will be used to later identify the product associated with that bead. The method is shown schematically in Fig. 3 using an example of a two-step synthesis with three variations at each step. Following synthesis, products from individual beads are cleaved and screened. When active compounds are found, the original bead is subjected to an orthogonal cleavage step that releases the chemical tag. The chemical tag is subjected to a sensitive and precise analytical method that reveals the structure of the tag and therefore the structure of the product molecule.

Success of an encoded library strategy depends on the availability of a method for screening products from individual beads, and of a suitably precise and sensitive analytical method for decoding the chemical tag. Four primary tagging methods have been reported. As mentioned above, sensitive methods exist for sequencing of peptides and oligonucleotides; thus, peptides and oligonucleotides make suitable chemical tags for encoded library synthesis. For example, Needles and coworkers (39) demonstrated the suitability of oligonucleotide tags for the synthesis of a peptide library using an orthogonal protecting group strategy. The growing peptide chain was protected by the baselabile 9-Fluorenylmethoxycarbonyl (FMOC) protecting group, while the

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Figure 3 Chemically encoded split-and-pool synthesis.

Introduction

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growing oligonucleotide chain was protected by the acid-labile dimethoxytrityl (DMT) group. A similar strategy should work well for synthesis of small organic molecules instead of peptides, providing the chemical sequence needed for product synthesis is compatible with the oligonucleotide tag. Similar strategies have been reported using peptides as the chemical tag (40). A binary encoding strategy has been reported using substituted haloaromatic tags that can be detected at very low levels by electron capture gas chromatography (41). The tags are attached via a photolabile (42) or oxidatively labile (43) linker, thus allowing most types of chemistry to be used in building the desired product molecules. This method has been reported for synthesis of both peptides (44) and nonpeptide molecules (45). A similar coding method utilizing secondary amines attached via amide linkages has also been reported (46,47). In this case, the tags are hydrolyzed using 6 N HCl, the resulting secondary amines derivatized, separated by HPLC, and detected by fluorescence spectroscopy.

The power of the encoded library strategy is illustrated by an example from Borchardt and Still (48). A 50,000-compound encoded acyl tripeptide amide library was prepared using the split-and-pool method. Beads from the library were mixed with a dye-linked synthetic receptor. After 24 hours, beads containing compounds that bind tightly to the synthetic receptor were stained deep red. Stained beads were mechanically selected, their binary coded tags were cleaved by photolysis, and the codes were read by electron capture gas chromatography to determine the structures of the tight-binding substrates. In this way, 50 compounds with high receptor affinity were selected from the library of over 50,000 compounds.

Chemically encoded libraries are a very powerful method for preparing large numbers of individual compounds whose structures can be determined. Successful application of a chemically encoded library requires access to a suitably sensitive high-throughput analytical method for reading the chemical code. Since code reading itself can become time consuming if many members of a library are found to be active, and the amount of each compound produced is limited to the amount that can be synthesized on a single bead, encoded libraries are well suited to lead discovery and early phase lead optimization but may be less well suited as the drug discovery process approaches candidate selection.

C. Mechanically Encoded Libraries

The main limitations of chemically encoded libraries are the necessity to perform an extra chemical reaction at each step to introduce the coding tag, the

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limitations introduced by the presence of the tag on the chemistries that may be performed on the target substrate, and the limited amount of material that can be obtained from a single polymer bead. These limitations can all be overcome using mechanically encoded libraries instead of chemically encoded libraries. With mechanically encoded libraries, polymer resin is held in a porous container that allows soluble reagents to pass through but not resin beads. The container is labeled with a mechanical tag (perhaps as simple as a printed label) and the containers of resin are treated in the same way that individual beads are treated in the split-and-pool method. While the split-and-pool method provides one compound per bead, the mechanically labeled split-and- pool method provides one compound per container of beads. If the synthetic path of each container is recorded at each synthetic step, then simply reading the container label will identify the product contained within. In practice, a database is generally created prior to execution of the synthesis outlining the precise synthetic pathway intended for each resin container. After each synthetic step the containers are retrieved, their labels read, and they are pooled for the next synthetic step according to the predetermined sequence from the database. This method is referred to as ‘‘deterministic’’ split-and-pool synthesis as opposed to ‘‘statistical’’ split-and-pool synthesis. Because the fate of each container is determined ahead of time, statistics play no role and the number of containers required is exactly equal to the number of products being synthesized.

The first example of a mechanically encoded library was reported by Houghten in 1985 (49). Resin was contained in a polypropylene mesh packet resembling a tea bag. The label was mechanically inscribed on the packet and was read visually with manual sorting of the packets. In this way, 247 analogs of a 13-amino-acid peptide were individually prepared. Synthesis of a 500,000-compound mixture library was recently reported using this method (50). The packets used were porous polyethylene tubes with printed labels that could be automatically read by optical character recognition (OCR) software. Each packet contained a mixture of 21 different functionalized resins, and the effort resulted in synthesis of approximately 26,000 individual containers each theoretically containing a mixture of 21 compounds for a total library of 551,070 compounds. The reader will recall, however, that in syntheses of this type each packet will contain an equimolar mixture of 21 products only if the reaction kinetics at each step are identical and that this was the reason for moving away from direct mixture synthesis and toward split-and-pool mixture synthesis in the first place. Two groups have recently reported use of radiofrequency tags in place of visually readable tags (51,52). These machine-readable tags offer the possibility of inexpensive automated reading and sorting. All of the above methods require the use of a nonreactive porous resin container.

Introduction

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Radiofrequency tags encapsulated in grafted functionalized polymers have recently been reported (53), as have laser optical–encoded ceramics with grafted polystyrene supports (54). These devices have the properties of ‘‘really big beads’’ that contain the identifying tag and thus combine the features of chemically encoded libraries with the advantages of mechanically encoded libraries.

Mechanically encoded libraries are best suited to synthesis of individual compounds rather than mixtures. The amount of each compound produced can be small or large and is limited only by the size of the packets and related physical limitations. Since reactions can be carried out in conventional glassware, mechanically encoded libraries offer an inexpensive entry into largelibrary synthesis. The method is applicable throughout most of the drug discovery process with the possible exception of the very earliest discovery phase and the very latest candidate selection phase.

Mechanically encoded libraries offer their greatest advantage over individual compound synthesis when the library consists of a dense symmetrical array. Consider, for example, synthesis of a 100-compound library with 10 structural variations at each of two positions (a 10 10 array). Individual compound synthesis would require that all 100 compounds undergo two chemical synthesis steps (not counting cleavage from the resin) or a total of 200 chemical reactions. Synthesis of the same library using mechanical tags would require only 20 reactions (10 reaction vessels each containing 10 packets for the first step, and the same for the second step). For a 100-compound library consisting of a 2 50 array, though, individual synthesis would still require 200 reactions whereas the mechanically encoded library would require 52 reactions (two reactions each with 50 packets in the first step, and 50 reactions each with two packets at the second step). Thus the advantage over individual synthesis is diminished. The advantage is also reduced for synthesis of ‘‘sparse’’ arrays. Consider again the 10 10 array, but suppose that sophisticated library design software has excluded some of the possible combinations so that there are ‘‘holes’’ in the array and a total of only 50 targets. Individual synthesis would require 50 reactions at each of 2 steps or 100 reactions total, while the encoded library would still require all 10 reactions at each step (though with less than 10 packets at each step) for a total of 20 reactions. For these reasons, encoded library synthesis will probably take its place alongside individual compound synthesis and split-and-pool mixture synthesis (rather than replace them) in an overall drug discovery program.

D. Automated Parallel Synthesis

The simplest conceptual manifestation of combinatorial chemistry is that of parallel synthesis, which is simply synthesis of several compounds in distinct

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reaction vessels at once (in parallel) rather than sequentially (in series). Any chemist who has had several reaction flasks stirring at once has practiced a crude form of parallel synthesis. Parallel synthesis results in individual compounds that can be treated in downstream handling steps identically to compounds synthesized conventionally. Thus it is a conceptually easy first entry into combinatorial chemistry. The tedium of handling many reaction flasks at a time has quickly led to a wide variety of apparatus for automating, or partially automating, parallel synthesis. The first equipment available was modeled after the venerable peptide synthesizer and offered complete automation of a multistep reaction sequence. Such equipment is now available commercially from vendors such as Advanced Chemtech (55) and Arognaut Technologies (56). The advantages of such systems (total automation) are offset by relatively complex software and relatively low synthesis capacity. The latter is a result of the fact that the actual synthesis reaction vessels must remain mated to apparatus for reagent addition throughout the process in order to meet the needs of total automation. A number of modular workstations have now been reported, and some are now available commercially from vendors such as Diversomer Technologies (57), Bohdan Automation (58), and Tecan (59). One of the first workstations reported was the Diversomer apparatus from ParkeDavis (60,61). Solid phase resin is contained in glass gas dispersion tubes that are arrayed in solvent containment wells. Reagents are added via robotic liquid handler through a septum that seals the top of the apparatus. A number of other reaction blocks have been reported. For example, an enclosed reaction block that can be heated and cooled and to which reagents are added robotically was described by the Ontogen group (62).

Regardless of the source, reactors for automated parallel synthesis have certain common characteristics. They all offer a group of reaction vessels that can be handled as a unit. All offer a means of liquid addition either via robotic liquid handler or via a closed pneumatic or pumping system. All offer a means of separating soluble reagents from insoluble polymer-supported products, and all offer a means of collecting soluble products after cleavage from the polymer resin. Systems that offer total automation of a multistep chemical sequence are generally referred to as ‘‘synthesizers.’’ With synthesizers, the reactions generally take place entirely within the confines of the synthesizer and proceed completely unattended. Throughput per run is limited to the capacity of the synthesizer. Systems that require manual intervention and movement of the reactions from place to place in assembly line fashion are referred to as ‘‘workstations.’’ In this case, a group of reaction vessels, perhaps contained in a single ‘‘reaction block,’’ is manually moved from station to station as the synthesis progresses. For example, the reaction block may be loaded