# Structural Biochemistry/Bioinformatics/Combinatorial Chemistry

Combinatorial Chemistry represents a mixture of chemistry and biology. It involves the use of computers and technology to create a massive array of different, but structurally related/similar, molecules in order to investigate a specific molecule and its derivatives. In common use, a "virtual library" is first created via computer in order to create a compilation of the different molecules that are to be investigated. From this library will specific molecules be chosen to be synthesized in the lab and further analyzed for desired characteristics. This technique is extremely useful in that it can mass produce the variety of diverse collection of molecules with limited condition variability needed to conduct the mass scale assays required to screen for specific characteristics. You can imitate the process of evolution by creating large sets of molecules and select for a specific function.
Combinatorial chemistry is a method used to synthesize different substances rapidly and at the same time. Compared to those time-consuming methods of traditional chemistry where compounds are synthesized individually one at a time, combinatorial chemistry is a very useful and faster method.
Combinatorial Chemistry is used to synthesize large number of chemical compounds by combining sets of building blocks. It synthesizes different substances rapidly and at the same time.
Combinatorial chemistry creates a large sets of molecules and select for a specific function. It requires three steps in process: - the generation of a diverse population - selection of members based on some criterion of fitness - reproduction to enrich the population in these more-fit members
Combinatorial Chemistry has had the largest impact and is mostly used in the pharmaceutical industries such as drug discovery and research, as well as in agrochemical industries. In an area of research in which the optimization of a certain activity or characteristic is the goal, combinatorial chemistry can be used to track down molecules and reactions that will accomplish this task.
Pharmaceutical companies had the need of improving drugs and being able to offer them to the public faster. Usually only 1 of 10 000 synthesized drugs are considered for marketing and the process may take up to 12 years to be settled, meaning millions of dollars spent. Since only few compounds could be synthesized by a scientist per year, a new technique to synthesize drugs faster was needed, and combinatorial chemistry was born.$^1$
Combinatorial chemistry is considered the synthesis of large numbers of molecules reacting together all possible combinations of certain reagents at the same time.$^2$ These combinatorial reactions are done on microtiter plates, which have several short-test-tube-like wells. By making note of exactly which variation of the reagents was placed in each well, one can determine the separate simultaneous reactions that took place to give the products obtained in each cell.$^1$
The execution of combinatorial reactions in these microtiter plates give way to chemical libraries that are screened and analyzed to determine the properties of each of the compounds in it. Computational chemistry then comes into play in order to maintain an organized database of the different characteristics revolving around each of the compounds in the library, such as the structures of the reagents and their quantities, reaction features, the location of the cell in the well, results of tests done, among other information.$^1$

## Combinatorial Chemistry Technology

At first, combinatorial chemistry was conceived as a technology for synthesizing and characterizing collections of compounds and screening them for useful properties. Its main focus was on the synthesis of peptide and oligonucleotide libraries. Then after a while, the focus of field changed to synthesis of small drugs as organic compounds, rather than large chemical compounds. Over past years, the combinatorial chemistry has emerged as an exciting new paradigm for the drug discovery for many pharmaceutical companies.
Meanwhile, researchers continue to find ways to improve the capabilities of combinatorial chemistry, with developments such as:
1. A growing trend toward the synthesis of complex natural-product-like libraries
2. An increased focus on "phase trafficking" techniques aimed at integrating synthesis with purification
3. strategies for purification and analysis, like use of supercritical fluid chromatography
4. the application of combinatorial chemistry to new targets, such as nuclear receptors
Even though combinatorial chemistry is more efficient in both cost and time wise, it, similar to traditional drug design, still relies on organic synthesis methods. Yet, the large libraries of compounds used for combinatorial chemistry do not produce active compounds, resulting in the necessary straightforward method to locate the active components. Such method is called combinatorial organic synthesis (COS), systematic and repetitive method that uses chemical building blocks to form numerous chemical compounds. There are three different COS approaches: arrayed, spatially addressable synthesis, encoded mixture synthesis, and deconvolution.
The first approach, arrayed, spatially addressable synthesis, groups chemical building blocks according to their individual positions; thus, allowing active compounds to be identified by their locations. The second strategy, encoded mixture synthesis is used to identify each compound using inert chemical tags such as nucleotides or peptides. The third technique, deconvolution, combinatorially synthesizes numerous compound mixtures in order to pursue the most active combination.

## Soft Organic Biomolecules

Drug research and design has made significant advancements within the past few decades, and still remains an industry in progress. With many different sub-categories in the world of drug design, one particular branch has received a growing amount of attention: creating disease-specific drug delivery vehicles. Drug delivery devices would be predominantly used to treat diseases such as cancer, because current treatments (chemotherapy) also cause harm to essential and non-pathogenic bodily tissues.
Soft, amphiphilic polymers may hold much utility in creating efficient drug delivery vehicles. The desired characteristics of the polymers are as follows: biodegradable, biocompatible, and high molecular weight. Being so, the widely abundant Poly Lactic Acid polymer serves a viable foundation for modeling the polymers after. In utilizing several known synthetic methodologies, researchers have proposed a way in creating potentially novel amphiphilic biomolecules [5].
Reaction of an aldehyde with a specifically designed isocyanide yields the necessary α-hydroxy acid monomer needed to create a Poly(α-hydroxy acid) polymer [6]. Since any aldehyde can be used, a lot of functionality can be incorporated into the polymer backbone. In particular, a norbornene moiety can be used. Norbornene can participate in a widely used Ring Opening Metathesis Polymerization (ROMP). Using two modes of polymerization in tandem opens up a vast amount of functionalization and drug incorporation, as well as fine-tuned adjustments on amphiphilicity. The picture at the right shows a synthetic scheme in obtaining the bifunctionalized α-hydroxy acid monomer.
Amphiphilic polymers will create micelle aggregates with hydrophilic components exposed in the body’s aqueous environment. Poly Ethylene Glycol can be used for the amphiphilic zone, which will allow the drug-encapsulated aggregates to dissolve and be compatible with a patient’s biochemistry. With disease-specific antibodies incorporated into the polymer’s functionality, the micelle aggregates can seek out the cancer, and inflict a lethal dose of medicine.

## RNA Synthesis

A useful application of RNA analysis can allow scientists to synthesis specific RNA sequences for intended functions. The process itself is rather simple. First, a very large amount of completely random RNA sequences are synthesized with really no thought to actual use. Then the collection of RNA are eluted down a column that specifically selects for RNA that fits a desired function such as ATP-binding where the column will just be filled with ATP bonded molecules. Naturally, only the RNA that bind to the function will stay in the column and those can be isolated for further testing by just eluting the rest of the column. Through this random selection process, scientists can pretty much create RNA to conduct any function. The following synthesis can be summarized in a few steps:
1. Initially, a randomized RNA pool containing of any variety molecules is present in a given population inserted into an ATP affinity column.
2. A selection process occurs in which only specific molecules are isolated with desired binding or reactivity properties.
3. The remaining RNA population that have remained the isolation process are then intensely amplified through Polymerase Chain Reaction (PCR).
4. Errors that occur within the course of replication are seen as additional variations introduced into this generation of RNA. Errors are induced through reverse transcription of RNA to DNA and back to RNA to increase the likelihood of error and mutation. The new population would consequently be introduced back into the column for further ATP analysis. Eventually structures that emerge out of the tube are assumed to be plausible structures of what the RNA could have existed in the past.

## Amplified Ancient DNA

The use of PCR to amplify DNA has been used to sequence the DNA of ancient organisms, such as the Neanderthal. Results showed that the DNA of homo sapiens have between 22 and 36 substitutions, as opposed to 55 from a chimpanzee. These results showed that the Neanderthal and homo sapiens shared a common ancestor.

## Types of Libraries

The first combinatorial libraries where based on peptides (consisting of few of their monomeric units, the 20 animo acids). Other oligomeric libraries (from carbohydrates, nucleic acids, etc.) were also made out of adding component A to B, resulting in AB, then adding C to obtain ABC, adding D...$^1$
This was the main technique for creating combinatorial libraries until the synthesis of benzodiazepine (fusion of benzene ring with diazepine ring) libraries was performed by DeWitt$^3$. Instead of using the same technique as the oligomeric libraries, these libraries were based on the attachment of several R substituent chains to a molecule in different positions. The number of products obtained in the library is calculated d by multiplying the number of substituent chains used on the main molecule, in this case benzodiazepine. The R groups are referred to as functionalities, while the points where they attach in the central molecule are called points of diversity.$^1$

## Library Synthesis

There are two ways of synthesizing libraries through combinatorial quemistry: 1. Using solid-phase synthesis 2. Through solution-phase synthesis
Solid-phase synthesis This technique allows an easy isolation and manipulation of the product, as well as having simple control systems (automation). Excess reagents can be used to complete reactions. Still it has certain disadvantages, such as the specificity of the solid support that don’t allow certain chemical reactions to be performed.
Solution-phase synthesis On the contrary, solution-phase synthesis allows a better manipulation of the sample (once isolated) in any amounts without being concerned about the solid support. Its disadvantage is difficult sample isolation, reason why scientists are working on implementing the use of reactants that give pure products (or at least products that can be easily purified).$^1$

## Diversity-oriented Libraries

Even though combinatorial chemistry has been an essential part of early drug discovery for more than two decades, so far only one combinatorial chemistry-synthesized chemical has been approved for clinical use by FDA (sorafenib, a multikinase inhibitor used for renal cancer) (Newman & Cragg 2007). The analysis of poor success rate of the approach has been suggested to connect with the limited chemical space covered by products of combinatorial chemistry. When comparing the properties of compounds in combinatorial chemistry libraries to those of approved drugs and natural products, it was noted that combinatorial chemistry libraries suffer particularly from the lack of chirality, as well as structure rigidity, which are both widely regarded as drug-like properties. Even though natural product drug discovery has not been the most fashionable trend in pharmaceutical industry in recent times, a large proportion of new chemical entities still is nature-derived compounds, so it has been suggested that effectiveness of combinatorial chemistry could be improved by enhancing the chemical diversity of screening libraries. As chirality and rigidity are the two most important features distinguishing approved drugs and natural products from compounds in combinatorial chemistry libraries, these are the two issues emphasized in so-called diversity oriented libraries, for example compound collections that aim at coverage of the chemical space, instead of just huge number of compounds.

## Library management

As we all know, integrating all types of chemistry, biology, and mixed information is exceptionary difficult and important combinatorial chemistry techniques. Thus, without managing all the information, library can not be function as one expected. For instance, if one scientist are trying to synthesize product "A," library must be able to show all the information necessary to synthesize the "A" such as, key reagent, cost, or if the reaction procedures are reliable etc... it is because the question that one will ask is different depends on the researchers.

## Molecular Evolution

Evolution requires three processes:
1. The generation of a diverse population
2. The selection of members based on some criterion of fitness
3. Reproduction to enrich the population in these more-fit members.
Molecular evolution primarily occurs due to mutations, which are changes in the genetic material of a cell. They can occur during copying errors or due to exposure to chemicals and radiation. Natural selection removes the less favorable mutations. Key topics under molecular evolution are the study of evolution of enzyme function and the study of nucleic acids. Nucleic acids have shown the divergence of species because they are denoted as the "molecular clock."

## References

* W.A. Warr, Encyclopedia of Computational Chemistry. Combinatorial Chemistry, John Wiley & Sons, Ltd.
* S. R. Wilson and A. W. Czarnik (eds.), Combinatorial Chemistry. Synthesis and Applications, Wiley, New York, 1997.
* S. H. DeWitt, J. S. Kiely, C. J. Stankovic, M. C. Schroeder, D. M. R. Cody, and M. R. Pavia, Proc. Natl. Acad. Sci. USA, 1993, 90, 6909–6913
* http://www.netsci.org/Science/Combichem/feature02.html
* Gianneschi, C. N., Rubinshtein, M., James, R.C., Kobayashi, Y., Yang, J., Young, J., Yanyan, J.M. Org. Lett., 2010, 12 (15), pp 3560–3563
* Kobayashi, Y., Buller, J.M., Gilley, B.C., Org. Lett., 2007, 9 (18), pp 3631–3634