Combinatorial chemistry

(organic chemistry) A method for reacting a small number of chemicals to produce simultaneously a very large number of compounds, called libraries, which are screened to identify useful products such as drug candidates.

A method in which very large numbers of chemical entities are synthesized by condensing a small number of reagents together in all combinations defined by a small set of reactions. The main objective of combinatorial chemistry is synthesis of arrays of chemical or biological compounds called libraries. These libraries are screened to identify useful components, such as drug candidates. Synthesis and screening are often treated as separate tasks because they require different conditions, instrumentation, and scientific expertise. Synthesis involves the development of new chemical reactions to produce the compounds, while screening aims to identify the biological effect of these compounds, such as strong binding to proteins and other biomolecular targets.

Combinatorial chemistry is sometimes referred to as matrix chemistry. If a chemical synthesis consists of three steps, each employing one class of reagent to accomplish the conversion, then employing one type of each reagent class will yield 1 × 1 × 1 = 1 product as the result of 1 + 1 + 1 = 3 total reactions. Combining 10 types of each reagent class will yield 10 × 10 × 10 = 1000 products as the result of as few as 10 + 10 + 10 = 30 total reactions; 100 types of each reagent will yield 1,000,000 products as the result of as few as 300 total reactions. While the concept is simple, considerable strategy is required to identify 1,000,000 products worth making and to carry out their synthesis in a manner that minimizes labor and maximizes the value of the resulting organized collection, called a chemical library.

The earliest work was motivated by a desire to discover novel ligands (that is, compounds that associate without the formation of covalent bonds) for biological macromolecules, such as proteins. Such ligands can be useful tools in understanding the structure and function of proteins; and if the ligand meets certain physiochemical constraints, it may be useful as a drug. For this reason, pharmaceutical applications provided early and strong motivation for the development of combinatorial chemistry. See also Ligand field theory.

In combinatorial chemistry, attention has been focused on the problem of how to identify the set of molecules that possess a desired combination of properties. In a drug-discovery effort, the library members that strongly bind to a particular biological receptor are of interest. In a search for new materials that behave as superconductors at relatively high temperatures, the special combination of elements yielding the best electrical properties is a goal. In each case, the library might consist of up to a million members, while the subset of target molecules might consist of several thousand contenders or just a single highly selective binder. This subset could then be studied in more detail by conventional means.

Several emerging strategies promise to address this problem. In the first case, a library is constructed in a spatial array such that the chemical composition of each location in the array is noted during the construction. The binding molecules, usually labeled with a fluorescent tag, are exposed to the entire assay. The locations that light up can then be immediately identified from their spatial location. This approach is being actively developed for libraries of proteins and nucleotides. A problem is that the chemistry required to attach various molecules to the solid surface, usually silicon, is quite tricky and difficult to generalize. The assaying strategy is intertwined with the available procedures for synthesizing the libraries themselves.

A conceptually straightforward approach is to first synthesize the library by using polystyrene beads as the solid support. The product molecules are then stripped from the support and pooled together into a master solution. This complex mixture consisting of a potentially large selection of ligand molecules could then be exposed to an excess of a target receptor. The next step is to devise a method for identifying the ligand-receptor pairs that point to molecularly specific binding. One approach is to examine a part of the mixture en masse by using affinity capillary electrophoresis. With this technique, the migration times of the ligand-receptor pair are significantly longer than the unreactive ligands, and can be interrogated by electrospray mass spectrometry.

The mass spectrometric method often provides a direct structural identification of the ligand, either by determination of its molecular weight or by collision-induced dissociation experiments. In the latter case, the molecular ion is selected by a primary mass spectrometer and is driven into a region of high-pressure inert gas for fragmentation. The fragment ions are then used to reconstruct the original molecular structure. This direct approach to screening and assaying has the advantage that the screening is carried out in solution rather than on a solid support, and it avoids steric problems associated with resin-bound molecules. At present the approach seems limited to libraries of about 1000 compounds because of interference from unbound ligands, and limited by sensitivity issues. New strategies using mass spectrometry may eliminate this limit.

A different tack involves assaying the polystyrene beads one by one after the resin-bound molecules are exposed to a receptor. With this approach, active beads may be identified by color or by fluorescence associated with the receptor, and are subsequently indexed in standard 96-well titer plates. Identification is then possible by using a variety of spectroscopic techniques; at present, the most popular methods are electrospray mass spectrometry and matrix-assisted laser desorption ionization mass spectrometry. See also Mass spectrometry.

Dynamic combinatorial chemistry integrates library synthesis and screening in one process, potentially accelerating the discovery of useful compounds. In the dynamic approach the libraries are not created as arrays of individual compounds, but are generated as mixtures of components, similar to natural pools of antibodies. One important requirement is that the mixture components exist in dynamic equilibrium with each other. According to basic laws of thermodynamics (Le Chatelier's principle), if one of the components (A_i) is removed from the equilibrated mixture, the system will respond by producing more of the removed component to maintain the equilibrium balance in the mixture. See also Chemical equilibrium.

The dynamic mixture, as any other combinatorial library, is so designed that some of the components have potentially high affinity to a biomolecular target. These high-affinity (effective) components can form strong complexes with the target. If the target is added to the equilibrated mixture, when the effective components form complexes with the target they are removed from the equilibrium. This forces the system to make more of these components at the expense of other ones that bind to the target with less strength. As a result of such an equilibrium shift, the combinatorial library reorganizes to increase the amount of strong binders and decrease the amount of the weaker ones. This reorganization leads to enrichment of the library with the effective components and simplifies their identification.

Combinatorial chemistry is a technology for creating a multitude of different compounds by reacting different combinations of interchangeable chemical "building blocks." The compounds are then screened for their ability to carry out a specified function, most commonly to act as drugs to treat a disease. Combinatorial chemistry allows the rapid synthesis and testing of many related compounds, greatly speeding the pace of drug discovery. Automated synthesis and screening systems are key to this approach.

The Combinatorial Approach

There are two general approaches for finding the correct answer to a question (besides asking someone who knows). One way is to learn everything relevant to the topic and then to use your knowledge to arrive logically at the answer. Scientists, and most other people, almost always use this method. A second approach is to keep guessing until you've guessed right! This seems like a foolhardy strategy, and usually is. What if it took a million guesses before you stumbled upon the right answer? But what if you could make a million or a billion guesses all at once? Through combinatorial chemistry, scientists can make and test millions, billions, or even quadrillions (10¹⁵) of guesses about which chemical compound might have a desirable function, such as the ability to bind to a specific molecule, or to serve as a drug.

Many chemicals are pieced together through combinations of smaller building blocks. For example, benzene is a chemical consisting of six carbon atoms connected in an aromatic ring structure, with a hydrogen atom bound to each carbon. Substituting one of the hydrogens with a hydroxyl (-OH) group forms the chemical phenol. Substituting a methyl (-CH³) group instead forms toluene, and substituting an amino (-NH²) group forms aniline. Because of their different "functional groups," or side groups, all of these compounds have very different physical and chemical properties. More variations can be synthesized by substituting additional side groups with more than one of the hydrogens. By substituting one of just these three groups (or by not adding any groups) for any of the six hydrogens in a benzene ring, there are 4⁶, or 4,096, possible combinations (the number of different compounds is much smaller, because benzene is symmetrical, and many of the combinations represent equivalent structures).

Side groups can also be placed onto other side groups. For example, a single chlorine atom can substitute for one of the hydrogens of the methyl group in toluene to form benzyl chloride. By using a moderately sized collection of side groups, placing them onto a "scaffold" molecule that is more complex than benzene (such as cholesterol, which has three six-carbon rings and a five-carbon ring), and by using additional levels of side groups, combinatorial chemists can synthesize vast numbers of distinct but related compounds.

Although the utility of combinatorial chemistry was not fully appreciated by scientists until the 1980s, nature uses this strategy over and over. Genes, after all, are composed of different combinations of only four different nucleotides, and just twenty different amino acids form the building blocks of all proteins. In the immune systems of mammals, B lymphocytes use an elaborate scheme for mutating and combining different segments of antibody genes to generate a diverse pool of antibody molecules that can recognize and bind a wide array of alien molecules that enter the body with a pathogen infection.

Drug Targets

Combinatorial chemistry is most often used to synthesize "small molecules," in contrast to macromolecules such as DNA, RNA, proteins, and polysaccharides, which are polymers containing long chains of monomer subunits. Because of their enormous size, macromolecules cannot easily enter cells, which is an important requirement for compounds intended for use as drugs.

In many cases the combinatorial chemist is looking for a compound that will bind tightly and specifically to a cellular molecule, such as the catalytic, or "active," site on the inside of an enzyme. Small molecules can fit into the holes and crevasses leading to the active site. By binding the enzyme, the synthetic compound may prevent it from binding to its natural substrate or from carrying out its catalytic reaction. Defective enzymes that resist normal cellular restraints on their activities are responsible for many diseases, including certain cancers. Chemical inhibitors of such rogue enzymes hold promise as powerful drugs. Alternatively, binding of a small molecule to an enzyme could enhance the enzyme's normal activity. Such molecules have potential as drugs for diseases caused by insufficient activity of a crucial enzyme.

For two molecules to bind to one another, they must have a proper fit, like a key in a lock. The fit depends on the shapes of the two molecules as well as on the chemical interactions between them. For example, two positively charged side groups will repel each other, but negative and positive groups can attract. Not surprisingly, a synthetic compound that binds a particular molecule often has chemical properties and a shape mimicking the natural ligand for the molecule. Such compounds are termed analogues.

When the drug target is known, its structure can be used as a template to create analogues with complementary shapes. Alternatively, if an analogue is already in hand that binds the target but has undesirable properties (such as weak binding, poor solubility, or serious side effects), this structure can be used as a starting point. Even without such clues, the speed and automation of the combinatorial approach makes it feasible to randomly synthesize and test millions of compounds.

High-Throughput Screening

A library of a billion or more different molecules is only useful if the molecules can be quickly and economically screened for the desired function. "High-throughput" techniques have been developed that automate most of the steps required to combine the molecules with their targets and evaluate the extent of any reaction.

Typically, the molecules are arrayed on a solid surface and the target is added. Unbound target is washed away. Fluorescent tags are often added to the target, to allow easy (and automated) visualization of the results. Robotic systems controlled by computers can react and evaluate billions of separate compounds in the time it would take a human to screen a dozen. One such approach is used in DNA microarrays, in which thousands of genes from a DNA library are attached to a solid base. These are reacted with messenger RNAs from a cell, and the results are visualized fluorescently.

Selex

In addition to its use in drug development, combinatorial chemistry can be applied to other areas of biomedical research, such as the design of molecules for diagnosing medical conditions. Compounds for these applications can be larger than pharmaceutical compounds, and do not have to be designed to enter the body. Using a novel combinatorial chemistry method called in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment), a short DNA or RNA molecule (termed an oligonucleotide) with a desired property, such as the ability to specifically recognize and bind a molecule associated with a particular disease, can be selected in a single experiment from a library containing approximately 10¹⁵ different compounds. First, a library of oligonucleotides is created in a machine called an oligonucleotide synthesizer. This apparatus can make oligonucleotides with either a defined or random sequence.

Oligonucleotides for SELEX are designed to have a central region containing random sequence and outer, flanking regions with defined sequences. These defined sequences will be used as primer-binding sites for the polymerase chain reaction (PCR). The oligonucleotide library is prepared as a mixture, usually containing about 10¹⁴ to 10¹⁵ different sequences. These specialized oligonucleotides, termed aptamers, are then exposed to target ligand molecules, which are typically attached to a solid support, such as a filter membrane. The unbound aptamers are then washed away, leaving only the rare aptamers that can bind the ligand adhering to the filter. These aptamers can then be recovered from the filter by washing it with a solution that disrupts the binding.

These binding candidate aptamers represent a minuscule fraction of the original library. Some may bind the target ligand tightly, but others may bind weakly. Since all the aptamers have defined primer-binding sites on the ends, this much-reduced population can now be amplified exponentially by PCR. After amplification, the aptamers can be subjected to another round of ligand binding, now using more stringent washing conditions, in which only the tightest-binding molecules will stay bound. These high-affinity binders can be recovered again subjected to still more cycles of PCR amplification, binding, washing, and recovery, until the population of aptamers consists exclusively of very tightly binding molecules.

For some applications, these molecules are useful directly. They can also be studied to design non-DNA molecules that have similar shapes but that will have more potential as drugs.

Bibliography

Alberts, Bruce, et al. Molecular Biology of the Cell, 4th ed. New York: Garland Science, 2002.

Borman, Stu. "Combinatorial Chemistry." Chemical and Engineering News 75, Feb. 24, 1997.

—Paul J. Muhlrad

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