The basic principles of combinatorial chemistry are often tied to, and sometimes confused with, the origins of combinatorial chemistry. The founding (but flawed)
principle was that, given a sufficiently large and diverse set of compounds to test, the discovery of an ideal drug for any given disease state would be statistically unavoidable. This principle was frequently popularized in metaphorical terms, such as the likelihood that any lock could be opened if a sufficiently large number of keys are tried, or that the pharmacological richness of natural products could be easily accessed and expanded through the laboratory equivalent of a rain forest. The anticipated surge in overall pharmaceutical R&D productivity has not materialized and this optimistic view of combinatorial chemistry has largely been abandoned. The contemporary view is more pragmatic and generally conforms to the principles outlined below.
1 The synthesis of many compounds simultaneously is more efficient than the synthesis of a single compound. In terms of simple time utilization, this principle was
intuitive to generations of chemists who set up two or more reactions in a hood each night. In the combinatorial context it has far greater significance as the number of compounds made from combinations of reagent sets increases geometrically with the number of reagents in these sets.
2 Any synthesis scheme can be executed in a manner that affords multiple products if individual reagents are replaced by complementary reagent sets used in different combinations. This perception is eponymous with combinatorial chemistry and lies at its heart. It can be readily illustrated with a Diels-Alder reaction (Fig. 2.3).
"diene" "dienophile" "Diels-Alder adduct"
Fig. 2.3. Combinatorial chemistry illustrated with a Diels-Alder reaction.
A chemist could easily execute this synthesis in a single workday. However within this same day, that chemist could weigh and prepare stock solutions of ten dienes and ten dienophiles. Then by making equimolar combinations of each diene with each of the ten dienophiles and running the corresponding Diels-Alder reactions, a total of 100 Diels-Alder adducts could be synthesized, thus achieving a 100-fold productivity gain. When a synthesis scheme provides for three reagent sets (incorporated into the products), the use often members in each set affords 1000 products. Similarly the use of four sets of ten reagents distributed over one (e.g. Ugi reaction) or several stages of a synthesis affords 10,000 possible combinations. The number of products increases geometrically (A x B x C) whereas the number of reagents increases arithmetically (A + B + C) and in this simple reality lies the true power of combinatorial chemistry.
3 Rigorous design of products and synthesis is critical and large numbers cannot compensate for poor design. Combinatorial chemistry, often viewed and applied solely as a tool to leverage serendipity, frequently fails to meet expectations. However when used to leverage the output of sound experimental designs based on synthetic and medicinal chemistry knowledge, the outcome is generally more satisfactory. There is probably a more fundamental principle behind this empirically derived view and it relates to the fact that the pharmacological effects of bioactive small molecules are derived from their ability to bind to and modulate the function of macromolecular targets, usually proteins. Proteins are built from a bounded set of amino acids. Their biological and biochemical functions have structural determinants derived from a bounded set of secondary structure motifs and these are usually associated with small molecule binding. Thus the number and types of small molecules that are likely to exhibit biological activity also represent bounded sets and combinatorial chemistry applied within this domain is far more likely to succeed than a totally random approach.
4 While synthetic organic chemistry evolved primarily in terms of serial processing, its productivity can be greatly enhanced by introducing parallel processing. Strong emphasis needs to be put on the importance of parallel processing as an explicit component of experimental design. The synthesis plan must comprehend the fact that hundreds or thousands of compounds are being synthesized simultaneously using hundreds or thousands of reagent combinations in each synthetic step. Each unit operation and each operating parameter - such as solvent used, temperature, and level of agitation - should encompass the conditions needed to bring each reagent combination to complete reaction while minimizing side-reactions and product decomposition. Sample tracking and in-process control data must also be managed at very high volume.
5 Laboratory automation, robotics, and mechanical devices enabling the simultaneous performance of multiple tasks are essential to combinatorial chemistry. The range of enabling tools goes from simple devices such as multichannel hand pipettes to high-performance, fully integrated systems such as the Irori NanoKan system. This issue will be revisited, but the basic principle is that the successful practice of combinatorial chemistry requires some level of commitment to and investment in laboratory automation.
6 Robust process chemistry is required to assure the desired outcome. In the pharmaceutical world, the importance of process chemistry lies at the two extremes. For single compound synthesis in a medicinal chemistry laboratory, a good process may be on the ''nice to have'' list, but as long as the synthesis scheme affords some of the desired compounds, intermediate and endstage purifications will overcome deficiencies in the process. For bulk pharmaceutical production, the process is everything: it must be extremely efficient, fully validated, and conform to regulated manufacturing practices. The importance of process chemistry in combinatorial synthesis is closer to the bulk production context.
For large compound libraries, the opportunity for product purification is very limited (although technologies for doing this are evolving nicely). Library members with poor-quality or outright failures are highly undesirable as they waste time and resources in biological testing laboratories. The best resolution of this dilemma is to develop the synthesis scheme to be used for production of a library into a robust, well-defined process. This requires careful optimization of reaction conditions, validating the individual members of the reagent sets, developing reliable analytical methods, and defining the process in something equivalent to a 'standard operating procedure'.
7 Electronic tracking and control systems are critical components of combinatorial chemistry. Every chemist has been exposed to the tedium of labeling samples by hand, filling out analytical request forms, and completing compound data sheets. Preserving the identity of samples and information about them by linkage to laboratory notebook pages is a cumbersome system that works when a few hundred samples per year are involved but it cannot function when individual experiments are producing thousands of individual compounds. The data management aspect of combinatorial chemistry requires access to electronic tracking and control systems for sorting and labeling samples (e.g. with machine-readable barcodes), for generating structure lists (SD files) that link sample codes to compound structures, and for collecting and processing analytical data and linking these data to sample codes and structure files. In addition to these sample and data management applications, electronic control systems embedded in the operating software of automated synthesis systems are necessary to ensure that each in-process material is in the right place at the right time for each step in order to ensure that the library plan is faithfully executed.
8 Analysis and quality control procedures are just as important in combinatorial chemistry as in other forms of synthetic chemistry. The shortcomings of early expressions of the combinatorial concept included the misperception that quality was not important. In fact many libraries were deliberately prepared as compound mixtures. It was taken for granted that biologically active samples could be separated, deconvoluted, or in some way dealt with after hits were detected. Two things resulted. The fact that synthesis without analysis is a prescription for poor science was reaffirmed and confidence in combinatorial chemistry as a productive discovery tool developed rather slowly. At its current state of development and acceptance, combinatorial chemistry is expected to provide samples of individual compounds with a purity level [by high-performance liquid chromatography (HPLC)] of at least 80%. The identity of compounds is routinely verified by mass spectrometry, using electronic comparison of probable molecular ion peaks with calculated molecular weights. Combined liquid chromatography/mass spectrometry (LC/MS) is preferable as it will confirm or deny that the major peak in an HPLC trace corresponds to the design intent for that particular library member. An aspect of quality control which needs further development is the application of in-process control procedures (IPCs) which are basic and routine in multistep synthesis of single compounds.
9 Since the purpose of combinatorial chemistry is to facilitate the discovery of useful compounds, combinatorial syntheses must be reproducible and scalable. This is not to say that the reaction sequence and mode of synthesis (solid or solution phase) must eventually serve to provide multikilogram supplies, but the compounds in a library have little value if interesting compounds cannot be conveniently resynthesized in small amounts for closer examination.
10 As a final basic principle it should be understood that combinatorial chemistry is a productivity-enhancing tool for chemists engaged in pharmaceutical research, agrochemicals, catalysis, and materials science - any field where the preparation and testing of new compositions of matter are the essential elements of discovery. However it does not displace or supplant established fields of synthetic chemistry such as medicinal chemistry - its best use is to leverage the productivity of existing fields.
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