Combinatorial Chemistry and solid phase synthesis seminar and laboratory course Topic 1: Principles of combinatorial chemistry 1. Introduction: Why Combinatorial Chemistry? Until recently, a common drug discovery process took 6.5 years (average) before a suitable drug candidate was found for further clinical evaluation. It was necessary to apply the individual synthesis and biological evaluation of many organic compounds in an attempt to enhance their biological activity, selectivity and bioavailability, while, at the same time, decreasing its toxicity. Combinatorial synthesis as a novel high-throughput method enables the rapid production of thousand times more compounds than conventional serial organic synthesis allows. The increasing number of new biological targets is also expected to accelerate the process of discovering new and improved drug candidates in conjunction with high-throughput screening. Thus pharmaceutical industry will be able to cut the time and high costs associated with the serial drug discovery process. Scheme of drug discovery process to new or improved drug candidates in the R & D laboratories of pharmaceutical and agrochemical companies. 2. History Origin of the principles used in combinatorial chemistry is the field of peptide chemistry. Geysen s multipin apparatus (1984) and Houghten s tea-bag system (1985) are methods for multiple (parallel) synthesis of peptides, based on the techniques of solid-phase peptide synthesis according to Merrifield (presented 1963) Birth of Comb. Chem. in 1988 by the work of Furka (split-pool method for the synthesis of peptides). Since the early 1990s, combinatorial chemistry has attracted the attention of many companies.
3. Principles of Combinatorial Chemistry 3.1 Characteristic of combinatorial synthesis: - large number of different compounds - identical reaction conditions - systematic manner of synthesis products of all possible combinations of a given set of starting materials (building blocks) are obtained combinatorial library screening 3.2 Library The combinatorial libraries can be structurally related by a central core structure (termed scaffold, figure(a) below) or by a common backbone (B). In both cases, the accessible dissimilarity of the compounds within the library depends on the building blocks used for the construction. Size of combinatorial library: few tens to many hundreds of thousands Number of compounds determined by 2 factors: number of building blocks used per reaction number of reaction steps with new building blocks 2
Example: (A): In general, in a conventional synthesis, one starting material A reacts with one reagent B resulting in one product AB. (B): In a combinatorial synthesis, different building blocks of type A (A1-An) are treated simultaneously with different building blocks of type B (B1-Bn) according to combinatorial principles, i.e. each starting material A reacts separately with all reagents B resulting in a combinatorial library A1-nB1-n. Libraries can comprise compound mixtures, or separate, single substances, depending on the synthetic strategy, which are described as combinatorial. 3
4. Methods and Techniques of Combinatorial Synthesis 4.1 Synthetic Strategies towards Combinatorial Libraries 4.1.1 Split-Pool Synthesis towards Combinatorial Libraries In the first step, the resin beads, each of which is coupled with a single building block, are split into multiple, equally sized portions in separate reaction vessels. After the reaction, the resin-bound compounds from all vessels are pooled together in one vessel where they are washed and deprotected in one batch. Then the still resin-bound compounds are reapportioned into the necessary number of separate reaction vessels. The following second reaction provides compounds that incorporate all of the possible combinations of the two sets of building blocks. These split and pool operations are repeated until the desired combinatorial library has been assembled. Advantages of this method: - few reaction vessels needed - only one single compound is bound to each resin bead in a library Currently, split-pool synthesis is the most popular method for the synthesis of large combinatorial libraries of compound mixtures. 4
4.1.2 Parallel Synthesis towards Combinatorial Libraries Compounds are synthesized in parallel using ordered arrays (collections of separate single substances) of spatially separated reaction vessels. Advantages: - one vessel - one compound philosophy: compound is pure in its local area - location of the compound in the array provides the structure of the compound - synthesis easily automated - unlike split-pool synthesis, which requires a solid support, parallel synthesis can be done either on solid phase or in solution 4.1.3 Reagent Mixture Synthesis towards Combinatorial Libraries Each reaction step of this combinatorial synthesis is carried out with a building block mixture in one reaction vessel. Advantages: - reaction on solid support or in solution Problems: - The use of reagent mixtures requires good knowledge of the mechanism and kinetics involved in the specific reaction being carried out. - Therefore it is important that the relative reaction rates of the incoming reagents are approximately equal and relatively independent of the resin-bound compounds final compound mixture can comprise compounds of different yields 5
4.2 Synthetic Methodology for Organic Library Construction In principle, combinatorial synthesis can be performed both in solution and on solid phase. The majority of the compound libraries have been synthesized on solid phases such as resin beads, pins or chips. 4.2.1 Solid-Phase Organic Synthesis Throughout the solid-phase synthesis the compound under construction is covalently attached to a swollen insoluble solid support (usually a resin bead) by a linker that can be cleaved under specific conditions with an appropriate reagent to give the target compound in solution later on. Conditions: Reactions must be very selective and efficient, achieved by functionalized solid supports and specialized methods for reaction monitoring in a multistep solid-phase synthesis, including strategies for blocking unreacted substrates. Otherwise, the purification of the final products is too difficult. Advantages: - easy steps of synthesis as the addition of reagent solutions, washing, filtration, agitation and partitioning of compounds into multiple aliquots (in the case of split-pool synthesis) - acceleration of reactions with higher yields by using a large molar excess of reagents - good possibility of automation 4.2.2 Synthesis in Solution and Liquid-Phase Synthesis Only few examples exist in the literature with most being one- or two-step parallel synthesis of individual compounds or reagent-mixture synthesis of compound mixtures. It was shown that pools of dimeric compounds including esters, amides and carbamates have been successfully prepared without needing further purification. One extension of the combinatorial synthesis in solution is achieved by the use of soluble polymeric supports, which combines some of the advantages of chemistry in solution and on solid phase. The so-called liquid-phase combinatorial synthesis is based on the physical properties of poly(ethylene glycol) monomethyl ether. This polymer is soluble in a variety of aqueous and organic solvents reactions in homogeneous phases This polymer can crystallize in appropriate solvents possible isolation and purification of the compound at each step of the synthesis 6
5. Characterization of Combinatorial Libraries 5.1 Analytical Characterization - peptides and oligonucleotides: automatic microsequencing procedures (e.g. based on Edman degradation and DNA sequencing) - low-molecular mass organic compounds: - especially mass spectroscopic methods (femtomolar!) - novel IR and NMR methods 5.2 Hit Identification in Combinatorial Libraries by High-Throughput Screening Examples of bioassays that can be adapted to high-throughput screening of combinatorial libraries: - competitive receptor binding assays with radiolabeled ligands - functional assays - cell-based assays The trend in bioassays is clearly towards examining the test compounds in solution. The results are not influenced by undesired interactions with the solid support. Moreover, assaying compounds in solution is probably more relevant from a pharmacological standpoint. In addition, screening on solid support is not applicable to intracellular targets, when a whole-cell assay is used. 5.3 Strategies for Libraries of Compound Mixtures 5.3.1. On-Bead Screening - compounds are still attached to the resin beads - solid support and its linker must be soluble in water - for quantitative results (e.g. structure-activity relationsships) the beads must be uniform in size and substitution Execution: The solid-bound library is treated with a labeled (mostly fluorescent label) soluble biological target. The labeled receptor binds to those resin beads that are derivatized with compounds that have the highest affinity to the biological receptor. The labeled beads are then selected, followed by structural elucidation of the support-bound compound. One possibility of isolating the labeled beads is the use of a fluorescence-activated cell sorting instrument. Advantages: - useful for libraries of several thousand to a million compounds - isolation of a few bioactive compounds from many inactive ones. - remaining compounds can be reused for different biological assays Problems: Difficulties of screening on the solid support. It is well-known that compounds known to be biologically active in solution did not bind to the biological receptor when attached to a bead. 7
5.3.2. Deconvolution 5.3.2.1. Iterative Deconvolution The iterative deconvolution method involves preparation of a series of spatially separated sublibraries comprising compound mixtures (pools), in which the identity of the building block at least at one position is known, and in which at the remaining positions all combinations of building blocks are incorporated. This can be achieved, by omitting the final pooling step of the split-pool synthesis. Each pool is screened and the compound mixture with the highest biological activity indicates the importance of the building block at that defined position. On the basis of this result the split-pool synthesis is repeated with the selected building block at the initial defined position in order to prepare pools where the next defined position is introduced. Each pool is evaluated for biological activity in order to select the optimal building block at the additional defined position. The remaining positions are then identified sequentially through an iterative procedure of (re)synthesis and screening, as illustrated schematically in the figure below. 8
Problem: The compound mixture that shows the greatest biological activity does not necessarily contain the most potent compound, as the biological activity observed for a given compound mixture depends on both the activity of the compounds, which is generally a result of the sum of more than one biologically active compound, and the abundance of the active compounds in each pool. 5.3.2.2. Deconvolution by Positional Scanning In the positional scanning approach for deconvolution, separate sublibraries are prepared. Each sublibrary contains a single defined building block at one position and a mixture of building block at the others. The number of sublibraries is equal to the number of variable positions in the substitution pattern. Each positional sublibrary is then screened to directly determine the building block at each defined position that contributes most to biological activity. By combining the positive screening results from all series of sublibraries, the whole structure of a highly biologically active compound can be deduced directly. Advantage over iterative deconvolution: sublibrary syntheses are carried out at once. Disadvantage: In comparison with iterative deconvolution, there is an increased likelihood that the most potent compound will not be identified. 9
5.3.2.3. Deconvolution by Orthogonal Libraries The deconvolution by orthogonal libraries, also called indexed library approach, involves preparing two series of orthogonal sublibraries consisting of compound mixtures. In the first series, each of the building blocks An reacts separatey with a stoichiometric quantity of an equimolar mixture of the building blocks B 1 - Bx. In the second series of sublibraries, each building block Bm reacts separately with an equimolar mixture of the building blocks A 1 - Ax. This synthetic approach provides compound mixtures, in which the identity of either the building block A or the building block B is fixed. This synthetic procedure results in each compound being formed exactly twice, and every pair of mixture has exactly one compound in common. By combining the positive screening results from both series of sublibraries, the whole structure of an active component can be deduced directly. Moreover, this form of internal control allows the recognition of false positives in the biological evaluation process. 5.3.3. Chemical Encoding Strategies Here the strategy is to label the compound during the split-pool synthesis. Thus, instead of the structure of the biologically active compound the corresponding more accessible (chemical) code is determined. In general, resin linkers containing two protected functional groups are used to allow the concurrent synthesis of both the compound of interest and the encoding compounds (so-called tags) on the bead, which upon cleavage are sequenced or otherwise decoded to determine the structure of the compound of interest. The tags may be added to provide an encoding sequence whereby the structure of the tag encodes for the building block and the location in the sequence encodes for the library synthesis step. Oligonucleotides and peptides are often used as tags because of there sensitivity and possibility of sequencing many samples of codes in parallel. Alternatively, each tag can be added individually to the resin, encoding for both the building block structure and the step in the library synthesis. Problem: DNA and peptides have the tendency to break down under the very often very rough conditions of organic synthesis. 10
5.3.4. Multiple Cleavable Linkers The compound library is divided into mixtures of up to several hundred compounds. In the first step, about one-third of each respective compound bound on a bead is split off into solution for biological evaluation. Experiments indicate that an optimal mixture complexity is about 20 compounds per mixture. In a second step, the collection of beads corresponding to the greatest biological activity is then redistributed in smaller mixtures. A further aliquot of the respective compound is then released and assayed. Ideally, the beads are re-arrayed separately for direct identification of the compound responsible for biological activity. The structure of the bioactive compound can then be determined from the remaining resin-bound compound by common analytical methods. 11