Editing Combinatorial chemistry (section)

==Small molecules==
{{Review|section|date=July 2018}}
In the drug discovery process, the synthesis and biological evaluation of [[small molecule]]s of interest have typically been a long and laborious process. Combinatorial chemistry has emerged in recent decades as an approach to quickly and efficiently synthesize large numbers of potential
small molecule drug candidates. In a typical synthesis, only a single target molecule is produced at the end of a synthetic scheme, with each step in a synthesis producing only a single product. In a [[combinatorial synthesis]], when using only single starting material, it is possible to synthesize a large library of molecules using identical reaction conditions that can then be screened for their [[biological activity]]. This pool of products is then split into three equal portions containing each of the three products, and then each of the three individual pools is then reacted with another unit of reagent B, C, or D, producing 9 unique compounds from the previous 3. This process is then repeated until the desired number of building blocks is added, generating many compounds. When synthesizing a library of compounds by a multi-step synthesis, efficient reaction methods must be employed, and if traditional purification methods are used after each reaction step, yields and efficiency will suffer.

Solid-phase synthesis offers potential solutions to obviate the need for typical quenching and purification steps often used in synthetic chemistry. In general, a starting molecule is adhered to a solid support (typically an [[insoluble polymer]]), then additional reactions are performed, and the final product is purified and then cleaved from the solid support. Since the molecules of interest are attached to a solid support, it is possible to reduce the purification after each reaction to a single filtration/wash step, eliminating the need for tedious liquid-liquid extraction and solvent evaporation steps that most synthetic chemistry involves. Furthermore, by using heterogeneous reactants, excess reagents can be used to drive sluggish reactions to completion, which can further improve yields. Excess reagents can simply be washed away without the need for additional purification steps such as [[chromatography]].
[[File:Use of a solid-supported polyamine that is used to scavenge excess reagent.tif|thumb|Use of a solid-supported polyamine to scavenge excess reagent]]
Over the years, a variety of methods have been developed to refine the use of solid-phase organic synthesis in combinatorial chemistry, including efforts to increase the ease of synthesis and purification, as well as non-traditional methods to characterize intermediate products. Although
the majority of the examples described here will employ heterogeneous reaction media in every reaction step, Booth and Hodges provide an early example of using solid-supported reagents only during the purification step of traditional solution-phase syntheses.<ref>{{Cite journal |last1=Booth |first1=R. John |last2=Hodges |first2=John C. |year=1999–2001 |title=Solid-Supported Reagent Strategies for Rapid Purification of Combinatorial Synthesis Products |journal=Accounts of Chemical Research |language=en |volume=32 |issue=1 |pages=18–26 |doi=10.1021/ar970311n |issn=0001-4842}}</ref> In their view,
solution-phase chemistry offers the advantages of avoiding attachment and cleavage reactions necessary to anchor and remove molecules to resins as well as eliminating the need to recreate solid-phase analogues of established solution-phase reactions.

The single purification step at the end of a synthesis allows one or more impurities to be removed, assuming the chemical structure of the offending impurity is known. While the use of solid-supported reagents greatly simplifies the synthesis of compounds, many combinatorial syntheses require multiple steps, each of which still requires some form of purification. Armstrong, et al. describe a one-pot method for generating combinatorial libraries, called multiple-component condensations (MCCs).<ref>{{Cite journal |last1=Armstrong |first1=Robert W. |last2=Combs |first2=Andrew P. |last3=Tempest |first3=Paul A. |last4=Brown |first4=S. David |last5=Keating |first5=Thomas A. |s2cid=95815562 |year=1996–2001 |title=Multiple-Component Condensation Strategies for Combinatorial Library Synthesis |journal=Accounts of Chemical Research |language=en |volume=29 |issue=3 |pages=123–131 |doi=10.1021/ar9502083 |issn=0001-4842}}</ref> In this scheme, three or more reagents react such that each reagent is incorporated into the final product in a single step, eliminating the need for a multi-step synthesis that involves many purification steps. In MCCs, there is no deconvolution required to determine which compounds are biologically active because each synthesis in an array has only a single product, thus the identity of the compound should be unequivocally known.
[[File:Example of a solid-phase supported dye indicating ligand binding.tif|thumb|Example of a solid-phase supported dye to signal ligand binding]]
In another array synthesis, Still generated a large library of [[oligopeptides]] by split synthesis.<ref>{{Cite journal |last=Still |first=W. Clark |year=1996–2001 |title=Discovery of Sequence-Selective Peptide Binding by Synthetic Receptors Using Encoded Combinatorial Libraries |journal=Accounts of Chemical Research |language=en |volume=29 |issue=3 |pages=155–163 |doi=10.1021/ar950166i |issn=0001-4842}}</ref> The drawback to making many thousands of compounds is that it is difficult to determine the structure of the formed compounds. Their solution is to use molecular tags, where a tiny amount (1 pmol/bead) of a dye is attached to the beads, and the identity of a certain bead can be determined by analyzing which tags are present on the bead. Despite how easy attaching tags makes identification of receptors, it would be quite impossible to individually screen each compound for its receptor binding ability, so a dye was attached to each receptor, such that only those receptors that bind to their substrate produce a color change.

When many reactions need to be run in an array (such as the 96 reactions described in one of Armstrong's MCC arrays), some of the more tedious aspects of synthesis can be automated to improve efficiency. This work, the "[[DIVERSOMER method]]" was pioneered at [[Parke-Davis]] in the early 1990s to run up to 40 chemical reactions in parallel. These efforts led to the first commercially available equipment for combinatorial chemistry (Diversomer synthesizer which was sold by Chemglass) and the first use of liquid handling robotics within a chemistry labortory.<ref>{{Cite journal |last1=DeWitt |first1=S H |last2=Kiely |first2=J S |last3=Stankovic |first3=C J |last4=Schroeder |first4=M C |last5=Cody |first5=D M |last6=Pavia |first6=M R |date=1993 |title="Diversomers": an approach to nonpeptide, nonoligomeric chemical diversity. |journal=Proceedings of the National Academy of Sciences |language= |volume=90 |issue=15 |pages=6909–6913 |doi=10.1073/pnas.90.15.6909 |doi-access=free |issn=0027-8424 |pmc=47044 |pmid=8394002|bibcode=1993PNAS...90.6909D }}</ref><ref>{{Cite journal |last1=DeWitt |first1=Sheila Hobbs |last2=Czarnik |first2=Anthony W. |year=1996–2001 |title=Combinatorial Organic Synthesis Using Parke-Davis's DIVERSOMER Method |journal=Accounts of Chemical Research |language=en |volume=29 |issue=3 |pages=114–122 |doi=10.1021/ar950209v |pmid=39049427 |issn=0001-4842}}</ref> This method uses a device that automates the resin loading and wash cycles, as well as the reaction cycle monitoring and purification, and demonstrates the feasibility of their method and apparatus by using it to synthesize a variety of molecule classes, such as [[hydantoins]] and [[benzodiazepines]], running 8 or 40 individual reactions in parallel. This and several other pioneering efforts in combinatorial chemistry were featured as "classical" papers in the field in 1999.<ref>{{Cite journal |last=Lebl |first=Michal |date=1999-01-12 |title=Parallel Personal Comments on "Classical" Papers in Combinatorial Chemistry |journal=Journal of Combinatorial Chemistry |language=en |volume=1 |issue=1 |pages=3–24 |doi=10.1021/cc9800327 |pmid=10746012 |issn=1520-4766}}</ref>

Oftentimes, it is not possible to use expensive equipment, and Schwabacher, et al. describe a simple method of combining parallel synthesis of library members and evaluation of entire libraries of compounds.<ref>{{Cite journal |last1=Schwabacher |first1=Alan W. |last2=Shen |first2=Yixing |last3=Johnson |first3=Christopher W. |year=1999–2009 |title=Fourier Transform Combinatorial Chemistry |journal=Journal of the American Chemical Society |language=en |volume=121 |issue=37 |pages=8669–8670 |doi=10.1021/ja991452i |issn=0002-7863}}</ref> In their method, a thread that is partitioned into different regions is wrapped around a cylinder, where a different reagent is then coupled to each region which bears only a single species. The thread is then re-divided and wrapped around a cylinder of a different size, and this process is then repeated. The beauty of this method is that the identity of each product can be known simply by its location along the thread, and the corresponding biological activity is identified by [[Fourier transformation]] of fluorescence signals.
[[File:Using a traceless linker as described by Ellman.tif|thumb|Use of a traceless linker]]
In most of the syntheses described here, it is necessary to attach and remove the starting reagent to/from a solid support. This can lead to the generation of a hydroxyl group, which can potentially affect the biological activity of a target compound. Ellman uses solid phase supports in a multi-step synthesis scheme to obtain 192 individual 1,4-benzodiazepine derivatives, which are well-known therapeutic agents.<ref>{{Cite journal |last=Ellman |first=Jonathan A. |year=1996–2001 |title=Design, Synthesis, and Evaluation of Small-Molecule Libraries |journal=Accounts of Chemical Research |language=en |volume=29 |issue=3 |pages=132–143 |doi=10.1021/ar950190w |issn=0001-4842}}</ref> To eliminate the possibility of potential hydroxyl group interference, a novel method using silyl-aryl chemistry is used to link the molecules to the solid support which cleaves from the support and leaves no trace of the linker.
[[File:Products that can be synthesized from imines.tif|thumb|Compounds that can be synthesized from solid-phase bound imines]]
When anchoring a molecule to a solid support, intermediates cannot be isolated from one another without cleaving the molecule from the resin. Since many of the traditional characterization techniques used to track reaction progress and confirm product structure are solution-based,
different techniques must be used. Gel-phase <sup>13</sup>C NMR spectroscopy, MALDI mass spectrometry, and IR spectroscopy have been used to confirm structure and monitor the progress of solid-phase reactions.<ref name=accounts>{{Cite journal |last1=Gordon |first1=E. M. |last2=Gallop |first2=M. A. |last3=Patel |first3=D. V. |year=1996–2001 |title=Strategy and Tactics in Combinatorial Organic Synthesis. Applications to Drug Discovery |journal=Accounts of Chemical Research |language=en |volume=29 |issue=3 |pages=144–154 |doi=10.1021/ar950170u |issn=0001-4842}}</ref> Gordon et al., describe several case studies that utilize imines and peptidyl phosphonates to generate combinatorial libraries of small molecules.<ref name=accounts/> To generate the imine library, an amino acid tethered to a resin is reacted in the presence of an aldehyde. The authors demonstrate the use of fast <sup>13</sup>C gel phase NMR spectroscopy and magic angle spinning 1 H NMR spectroscopy to monitor the progress of reactions and showed that most imines could be formed in as little as 10 minutes at room temperature when trimethyl orthoformate was used as the solvent. The formed imines were then derivatized to generate 4-thiazolidinones, B-lactams, and pyrrolidines.

The use of solid-phase supports greatly simplifies the synthesis of large combinatorial libraries of compounds. This is done by anchoring a starting material to a solid support and then running subsequent reactions until a sufficiently large library is built, after which the products are cleaved from the support. The use of solid-phase purification has also been demonstrated for use in solution-phase synthesis schemes in conjunction with standard liquid-liquid extraction purification techniques.