Another example of synergy between chemistry and biology in the generation of molecules with novel functions is the development and application of diversity-based synthetic strategies, an approach inspired by the sophisticated combinatorial and mutational mechanisms by which antibodies are evolved to recognize foreign antigens with high affinity and selectivity. The notion that this natural diversity can be used to create novel chemical function was first illustrated with the generation of catalytic antibodies. Rather than attempting to design a synthetic host that selectively binds a substrate of interest and then modify it with catalytic auxiliaries, it was realized that one could simply co-opt the immune system to generate a highly selective natural host in the form of an antibody combining site. To generate a selective catalyst rather than a selective receptor, stable transition-state analogues (rather than substrates) were used as antigens on the basis of the Pauling notion that enzymes evolve maximum binding affinity to the transition state of a reaction. The early experiments by Lerner and co-workers and in our own laboratory involved the generation of esterolytic antibodies using phosphonate/phosphate transition-state analogues. Other approaches have since been developed to generate catalytic antibodies, including covalent catalysis, proximity effects, and general acid-base catalysis (thereby allowing us to dissect the contribution of each of these factors to biological catalysis). Using these approaches, antibodies have been generated that catalyze a wide array of chemical reactions, from acyl transfer and redox reactions to pericyclic and photochemical reactions with specificities and, in some cases, rates rivaling those of enzymes.

The detailed characterization of the immunological evolution, three-dimensional structures, and mechanisms of catalytic antibodies has also helped to dissect and quantify the relationship between binding energy and catalysis in the evolution of catalytic function. Indeed the use of transition-state analogues to elicit catalytic antibodies provided “proof by synthesis” of the Pauling notion of enzymatic catalysis. In another example, a “ferrochelatase” antibody, which catalyzes the efficient insertion of metal ions into porphyrin (the last step in heme biosynthesis), was generated against an N-methyl porphyrin, which mimics the distorted porphyrin ring of the putative transition state for metalation. The crystal structure of the Michaelis complex indeed showed that the substrate is bound in a strained conformation, providing the first direct structural evidence for the theory of substrate strain proposed by Haldane over 70 years ago. The characterization of catalytic antibodies has also provided fundamental insight into the mechanisms by which the immune system itself evolves selective receptors. For example, the first detailed structural comparisons of germline and affinity-matured antibodies revealed the critical role of structural plasticity (in addition to genetic diversity) in determining the tremendous binding potential of the germline antibody repertoire. Germline antibodies appear to have a high degree of intrinsic combining site conformational flexibility (reminiscent of the chemical instruction theory of the immune response proposed by Haurowitz and Pauling) which allows them to bind multiple, distinct ligands in different conformational states. That conformational state which binds a specific antigen is then locked and further refined by somatic mutations which occur during affinity maturation (not protein folding as proposed by Pauling). Structural and biophysical analyses of the immunological evolution of catalytic antibodies also pointed to the critical role of mutations distal to the active site in controlling the binding and catalytic activity of proteins through complex networks of side chain and backbone interactions. Indeed these studies underscore a key aspect of diversity-based synthetic strategies—the fact that analyses of the relationship between molecular structure and properties in molecules obtained by combinatorial methods often lead to new chemical insights which further increase our ability to generate new molecular function from basic chemical principles.

The demonstration that the vast structural diversity of antibody molecules can be redirected with proper chemical instruction to generate selective catalysts illustrated the utility of molecular diversity (the antibody repertoire in this case) as a new, biologically inspired “synthetic strategy” to create novel chemical properties. Shortly thereafter, libraries of other biomolecules were designed and synthesized in order to identify molecules with new or enhanced functions. These included the use of phage display libraries to generate peptides, proteins, and antibody fragments with novel specificities, and libraries of random RNA sequences (including those containing unnatural bases with novel functional groups) to identify RNAs that selectively bind ligands with high affinity, that catalyze chemical reactions such as acyl or phosphoryl transfers, or whose structure and transcription is regulated by the binding of small synthetic molecules. Today, combinatorial strategies are impacting many areas of chemistry. This method is particularly valuable when theory has insufficient predictive power to guide molecular design with precision, and quickly provides large amounts of experimental data to guide additional experiments and/or theoretical predictions.

One particularly illustrative example is the application of diversity-based approaches to the generation of solid-state materials with novel properties. The properties of many functional materials, such as high-temperature superconductors, heterogeneous catalysts, ferroelectric materials, magnets, and even structural materials, arise from complex interactions involving the host structure, dopants, defects, and morphology, all of which are highly dependent on composition and processing. Unfortunately, our current level of theoretical understanding does not generally allow one to predict the structures and resulting properties of these materials. Given the large number of elements in the periodic table that can be used to make compositions consisting of up to six elements, the universe of possible new compounds with interesting physical and chemical properties remains largely unexplored; combinatorial synthetic methods represent a powerful way for experimentalists and theorists alike to more effectively mine this huge chemical space for interesting new materials properties. The first application of combinatorial methods to materials science involved the synthesis and screening of libraries of thin-film copper oxides to identify high-temperature superconductors. More recently, a variety of thin film, solution-based and physical methods (e.g., ball milling) have been used to make libraries of diverse solid state materials. In addition, a large number of scanning or parallel detection systems have been developed for rapidly screening materials libraries for optical, electronic, magnetic, adsorptive, or catalytic properties of interest. This combinatorial approach to materials discovery, is now practiced in many industries and has led to new olefin polymerization and oxidative catalysts, hydrogen storage materials, separations materials, dielectrics, phosphors, etc. and is now being applied to the optimization of complex integrated devices such as lithium ion batteries, solar cells, and computer chips. Indeed with the challenges we now face finding new environmentally friendly energy sources, combinatorial methods are likely to play a critical role in the development of enabling new materials. These include new hydrogen and methane storage materials, fuel cell catalysts, photovoltaic devices, CO₂ sequestrants, and high-energy-density batteries. This will likely be best achieved by a synergistic use of combinatorial approaches, more conventional solid-state chemistry, and theory.

Another particularly powerful application of combinatorial strategies, involves the synthesis of diverse libraries of nonoligomeric synthetic molecules. Just as large libraries of antibodies are genetically assembled from families of V, D, and J gene segments, it was realized that libraries of small organic molecules could be efficiently assembled from chemical building blocks. Although there are many examples of the rational design of biologically active small molecules, it remains a challenge to design a priori molecules that selectively activate or inhibit a desired enzyme or receptor, or modulate a specific cellular signaling pathway, regulatory circuit, or transcriptional program. As a consequence, the screening of synthetic chemical libraries offers a highly effective approach to identify biologically active molecules, especially molecules with novel cellular activities, which may not be predicted or even conceived of in hypothesis-driven experiments. However, with the increased availability and decreased cost of chemical libraries and the power of modern screening technologies, the question arises as to which opportunities should the academic chemistry community pursue with these new tools? One answer is to focus on those areas of biology which are still poorly understood and, as a consequence, there exists a real need for small molecules as in vitro and in vivo probes; another is to focus on major unmet medical needs that have been largely ignored by industrial research efforts due to perceived risk or financial considerations.

A timely example (of both) is regenerative medicine, in which new cells (e.g., neurons, muscle, chondrocytes, etc.) are generated to replace tissues lost to degenerative diseases or aging. To this end, we and others are carrying out cell-based screens to identify molecules that control cell fate. For example, we have carried out image-based screens with one class of adult stem cells, hematopoietic stem cells (HSCs), to identify molecules that control self-renewal and differentiation (HSCs are adult stem cells that give rise to all the blood lineages such as macrophages, B and T cells, platelets, red blood cells, etc.). Molecules have been identified that are able to significantly expand cord blood HSCs in an undifferentiated state by antagonizing the aryl hydrocarbon receptor. These compounds will likely produce a robust source of HSCs for the large number of cancer, blood, and autoimmune disease patients for which no matched donors exist. In another experiment we have used image-based screens to identify a molecule that induces the selective neurogenesis of neural progenitor cells in vitro and in vivo in the rat dentate gyrus. This compound acts by selectively binding the centrosomal protein TACC3, which has been previously implicated in regulating the balance between progenitor cell renewal and differentiation. This and other such molecules may ultimately lead to new treatments for neurodegenerative disease. We have also identified molecules that selectively induce mesenchymal stem cells (adult stem cells which normally give rise to osteoblasts, adipocytes, and chondrocytes) to undergo osteogenesis to form bone, or chondrogenesis to form cartilage. The chondrogenic molecule, kartogenin, functions by blocking the interaction of the cystolic protein filamin A with the transcriptional coactivator CBF-β. CBF-β then traffics to the nucleus and selectively upregulates the expression of the master transcriptional regulator, Runx1. These molecules have shown excellent efficiency upon intra-articular injection in rodent osteoarthritis models. More recently, a molecule has been identified from image-based screens of oligodendrocyte precursor cells that induce their selective differentiation to oligodendrocytes. These molecules show excellent efficacy and function in both in vitro and in vivo by inducing remyelination of axons, rather than by an immunosuppressive mechanism.

Another exciting opportunity for the academic community to exploit chemical libraries and screening technologies that is not generally competitive with pharmaceutical or biotechnology research interests is in the area of orphan and neglected diseases. For example, there exist both a large research opportunity and a major unmet medical need with respect to molecules that kill persistent Mycobacterium tuberculosis (the biology of persistors is largely unknown), or molecules that target nonessential host factors that are required for viral replication (HIV, HCV, Dengue, etc.), but which will not mutate rapidly. To this end, we recently identified a molecule from a cell-based biofilm screen using Myobacterium smegmatis that kills both replicating and nonreplicating Mtb as well as XDR drug resistant strains. These molecules downregulate key mycobacterial persistence genes, and function by inhibiting two activities—the cell wall biosynthetic enzyme DprE1 and the biosynthesis of an essential molybdenum cofactor. Analogues of this compound show excellent activity in chronic models of Mtb in rodents. In addition, there are a large number of orphan diseases (type I diabetes, muscular dystrophies, spinal muscular atrophy, childhood cancers, Rett syndrome, Fragile X, Huntington disease, etc.) for which no good treatments exist. The identification of molecules that modulate these disease processes may ultimately lead to new therapies as well as provide new insight into the biology of many of these diseases.

Chemistry continues to evolve from its historical focus on molecular structure, reactivity, and synthesis to take on the challenge of making small and large molecules and even systems of molecules with tailored properties and functions. This requires improved theoretical and analytical tools, as well as innovative new synthetic strategies. Given the remarkable array of functions found in biological molecules, Mother Nature offers help in this regard through an approach to synthesis that seamlessly interfaces biology and chemistry. Hopefully, the examples illustrated above from our work, and the many other elegant examples in the literature, convey the exciting and highly relevant opportunities that exist for chemical synthesis at the interface of the chemical and biological sciences.

Peter G. Schultz < > is professor at The Scripps Research Institute and Director at the California Institute for Biomedical Research, in La Jolla, California.