Chemical Genetics Resulting from a
Passion for Synthetic Organic Chemistry

Stuart L. Schreiber, Ph.D.
Department of Chemistry & Chemical Biology
Howard Hughes Medical Institute, and
The Institute of Chemistry & Cell Biology
Harvard University, Cambridge, Massachusetts
Written in May, 1998

Stuart Schreiber's Personal Page | Schreiber Group Research Page | Schreiber Group Home Page

Seventeen years after completing my doctoral studies in synthetic organic chemistry at Harvard University with R. B. Woodward and Yoshi Kishi, I find myself back at Harvard, dreaming together with my students about a future where "chemical genetics" provides seamless connections between chemistry, biology, and medicine. It is certainly not a world I envisioned in 1981. My love of synthesis has grown in unanticipated ways, and I never imagined that it would cause me to explore the functions of proteins in cells.

When I initiated my independent studies in May 1981 in the Department of Chemistry at Yale University, I knew nothing of biology, having never taken a course in modern biology. However, the aspects of organic chemistry that drew me to natural product synthesis, especially conformational analysis and reaction mechanism, together with explosive advances in neighboring disciplines, led naturally to the exploration of cellular processes. It also led to the tackling of these problems in a way that was unfamiliar to biologists. I am grateful to Sir Derek Barton for encouraging me to use this forum, the article that traditionally accompanies the Tetrahedron Prize for Creativity in Organic Chemistry, to review the events that led to what is referred to as chemical genetics, where natural products and natural product-like compounds are used to understand and control the cellular and physiological functions of proteins.

The first phase of my research, where stereoselective synthesis was used to prepare natural products. Planning my first Yale experiments while still a graduate student at Harvard, I studied the remarkable structure of periplanone-B, recently defined stereochemically through the collaborative efforts of Koji Nakanishi, Clark Still, and Jon Clardy ¹. Inspired by the rearrangement of vinyl allene oxides to cyclopentenones, I considered cascade rearrangement reactions that would energetically funnel downwards to the skeleton of periplanone-B. The plan for a periplanone-B total synthesis was eventually realized by my first graduate student, Conrad Santini, on Christmas eve of 1982 (Figure 1) ^2,3. It was the crude "biological" assays with the synthetic pheromone that in the end would have its greatest impact on me. We searched successfully in the basement of the Chemistry building for females of Periplaneta americana (American cockroach), and then observed the remarkable influence of the pheromone on insect physiology. This experience reminded me of an earlier euphoric feeling, as an undergraduate at the University of Virginia, when I first became aware of molecules and chemical reactivity. Like envisioning a transition state in my mind's eye, I could imagine an intricate encounter of the pheromone with a (still) mysterious insect receptor, likely a protein. This idea was strengthened when Conrad and I witnessed an electrophysiological output on an oscilloscope following a puff of the synthetic pheromone onto a dissected cockroach antennae grasped with an alligator clip. Although our efforts (with Michael Lerner) to isolate the periplanone-B receptor proved unsuccessful, a seed had been planted.

My encounters with other natural products at Yale reinforced the research trail initiated by the periplanone-B project, as discussed below. While teaching a course in organic synthesis, a photocycloaddition reaction I had been discussing suddenly struck me as being related to the aldol reaction, which was attracting considerable interest in 1982. Immediately following the class, Amir Hoveyda, my second graduate student, and I brainstormed about the implications of the Paterno-Büchi reaction of furans and aldehydes to the aldol substructures contained within many natural products. Amir and Kunio Satake explored many facets of the "photo-aldol" reaction ⁴, eventually applying it to stereoselective syntheses of asteltoxin ^5,6 and avenaciolide ^7,3 (Figure 2). Other natural product-based projects were initiated at this time. Considerations of medium and large ring conformations led Tarek Sammakia to develop stereoselective polyepoxidation reactions ⁸ (Figure 3), Mike Klimas, Soroosh Shambayati, and Bill Crowe to develop stereoselective cyclooctanoid ring syntheses ^9,10 (a process applied by Tim Jamison to the synthesis of the diterpene epoxydictymene ¹¹ (Figure 4)), and Jack Taunton, John Wood, and John Porco to synthesize methylated variants of dynemicin^12,13 (Figure 5). Considerations of symmetry in natural product synthesis led Mark Goulet to determine the stereochemistry for the first time of members of the "skipped polyol", polyene macrolide antibiotics, mycoticins A and B^14,15, and Chris Poss and Scott Rychnovsky to develop a synthesis (+)-mycoticin A¹⁶ (Figure 6AB). These synthetic studies illustrated the "two-directional" chain synthesis strategy, where simultaneous double processing of chain termini and subsequent differentiation of the resulting homo-, enantio-, or diastereotopic groups at the chain termini are the defining features of the strategy ^17,18. The two-directional strategy has been used by a number of students and postdoctorals in studies resulting in syntheses of other natural products, including (+)-KDO¹⁹, (-)-hikizimycin ^20,21 (Figure 7) and (-)-FK506 ^22,23,24 (Figure 8).

Each of the syntheses above had a similar, almost mystical effect on me personally. Like the original experience with periplanone-B, building these beautiful and complex natural objects from simple building blocks invariably led to a fascination with the cellular receptors to which they bind. I reasoned that these would often be proteins, themselves natural products. I began to puzzle over the conformational properties of proteins, especially the conformations of their sidechains that I envisioned wrapping around the "small molecules" we had succeeded in synthesizing. This curiosity led me to a second phase of my synthetic chemistry research program. My move to Harvard in 1988 and my lab's research on FK506 catalyzed efforts in this area. The move forced me to think about the structure of the new labs I had been encouraged to design. Studying natural product interactions with proteins would surely require a lab equipped to handle proteins. Research on FK506 caused me to realize that an equally fascinating aspect of proteins concerns their functions inside of cells, and that natural products like FK506 (and now natural product-like compounds) could illuminate the mysteries of protein-mediated cellular processes in a powerful way²⁵. So my new labs were designed to facilitate the synthesis of complex molecules, the analysis of protein-small molecule interactions, and the functions of proteins in cells.

The second phase of my research, where synthesis was used to respond to fascinating challenges posed by biology. Our efforts to apply the two-directional strategy to the synthesis of FK506 were initiated while we were synthesizing compounds designed to probe the interaction of another immunosuppressive natural product, cyclosporin, with its then recently discovered protein receptor, cyclophilin. The cyclosporin studies had sensitized us to the importance of understanding how FK506 exerted its actions on cells of the immune system. Our early plans to synthesize the FK506-related reagents that eventually led to the co-discovery (with scientists at Merck) of the FK506 and rapamycin receptor^26,27 (named FKBP12) and to the realization of a novel mechanism of cellular action (through our studies of the designed macrolide 506BD^28,29, Figure 9) were foolishly delayed by well over a year as I was caught up in a "race" to synthesize the natural product. I learned from this mistake later when we were faced with the decision to either modify a protecting group strategy in order to finish a "race" to synthesize natural dynemicin (rather than the methylated variants that Jack Taunton had just made) or to initiate new synthetic efforts to prepare trapoxin and trapoxin-related reagents. Trapoxin is a natural product that had been shown to alter the morphology of mammalian cells. Jack was unequivocal in his assessment of the relative merits of these two projects, and he immediately launched synthetic efforts aimed to illuminate trapoxin's mysterious actions on cells. His synthesis of the reagent we call "K-trap" ³⁰(Figure 10) proved to be the key advance that led to his discovery of the receptor for trapoxin, which we named histone deacetylase-1³¹ (HDAC1), and then to an understanding of a novel mechanism for gene regulation involving this new family of HDAC proteins^{32,33,34,35,36,37}.

These two studies illustrate some of our early efforts to explore cell biology using the principles of organic chemistry, with special reliance on organic synthesis²⁵. It was an exciting period involving considerable change in the lab, and we were often faced with the need to learn new techniques, especially from molecular cell biology, in order to respond to research opportunities that our synthetic efforts had created. It was a period of time when we synthesized, among others, transmembrane ion channels having a gate that resulted from the rational incorporation of tartaric acid derivatives^38,39,40, designed ligands to MHC^41,42 and immunophilin proteins (seco506⁴³ and tricycloCsA⁴⁴, Figure 11), and biopolymer-inspired oligomers of vinylogous amino acids (what Samuel Gellman refers to as "foldamers"⁴⁵). But the most exciting moment came following a retrospective analysis of this research, when a general approach to the study of protein function was recognized and eventually formalized. This approach forms the basis of all of our current research and will likely be the focus of research in my laboratory for many years to come.

Introduction to chemical genetics Determining the cellular function of a protein generally requires a means to alter the function. The most common way of doing so is an indirect one involving the use of inactivating (e.g., deletion or "knock-out") or activating (e.g., oncogenic) mutations in the genes encoding proteins of interest. This is the genetic approach, and it has been widely used in biology. A complementary and direct approach involves the use of small molecules that alter the function of proteins to which they bind (Figure 12). Ligands exist that are capable of either inactivating (e.g., colchicine, which inactivates the function of tubulin) or activating (e.g., the steroid hormones, which activate the transcriptional properties of nuclear hormone receptors) protein function. This approach has been given a number of names, including the "pharmacological approach". So why do we give it a new name? I think the term chemical genetics is appropriate and important for three reasons. First, it "raises the bar" in terms of specificity. Biologists often believe that organic compounds lack the specificity inherent in the deletion of a gene. They are correct in some instances, but not all. We have initiated some experiments using chip-based hybridization technology where the influence of small molecules and gene knockouts can be compared directly (where the gene encodes the protein to which the small molecule binds). The preliminary results suggest that for some natural products, specificity can approach that of a gene knockout. The second reason is that the name chemical genetics raises the bar in terms of generality. The genetic approach to understanding protein function can be time-consuming, but it is general. The chemical genetic approach relies upon the existence of highly specific ligands, which at this point come primarily from Nature and exist for only a limited set of proteins. The third reason for using the name chemical genetics is that it points to a means to achieve specificity and generality -- by emulating the principles of genetics with chemistry. These three points are elaborated below.

Chemical genetic research During the past ten years, we synthesized numerous natural products and their variants in order to provide the means to explore the function of cellular proteins. FK506, cyclosporin, and rapamycin are used to study signal transduction^26,46,47,48 (Figure 13). Trapoxin^32,33 , trichostatin³³, and depudecin^37,49 are used to study gene regulation by HDAC-mediated chromatin remodeling^34,35,36,37 (Figure 14). Discodermolide^50,51,52,53 and lactacystin^54,55 are used to study the cell cycle and cell cycle checkpoints (Figure 15). However, these studies also illustrate the greater generality of genetics when compared to chemical genetics. Rapamycin was used to discover the protein FRAP, which mediates a cell cycle checkpoint⁴⁸. While searching for homologs of FRAP, Karlene Cimprich, Tae Bum Shin, and Curtis Keith discovered the protein ATR^56,57, which mediates a DNA damage checkpoint⁵⁸. Since a small molecule ligand to ATR is not known, in contrast to the situation with its family member FRAP, the approach we have used to explore ATR function thus far has relied on the use of mutant forms of ATR, produced by making mutations in the DNA encoding ATR⁵⁸.

The first illustration of a more general role for chemical genetics came from our studies of small molecule "dimerizers" (sometimes referred to as chemical inducers of dimerization, CIDs). During the past five years my lab has participated in an exciting and fruitful collaboration with Gerald Crabtree and his co-workers at Stanford to prepare small molecule dimerizers that activate the function of numerous proteins that regulate many important cellular processes⁵⁹. As I will discuss below, dimerizers allow the functions of proteins to be explored even when small molecule ligands are unknown. A limited number of such reagents have been synthesized that control the function of a much larger number of proteins (expressed as fusions of proteins of interest linked to a small molecule-responsive dimerization domain), and the list of proteins will undoubtedly grow without the need to make more dimerizers. These are unique features of the system our two labs has developed, and it is these features that provide the means to apply chemical genetics in situations where small molecules that bind to a protein of interest have not yet been identified.

At the outset of our studies, several experiments were beginning to suggest an important role for proximity and orientation effects in mediating information transfer in biology, in direct analogy to the role of these effects in determining the rates of reactions in organic chemistry^60,61 (Figure 16). In fact, the biological outcomes are due to an increased rate of chemical reactions between biological molecules, often an enzyme and its substrate. For example, a protein growth factor such as PDGF can transmit a signal across the cell membrane by causing the intracellular tail of its receptor to phosphorylate itself. This occurs because the tail has protein kinase activity, its substrate is another molecule of itself, and the growth factor dimerizes its receptor (Figure 16). The growth factor has rendered the phosphorylation reaction essentially intramolecular by inducing a proximal relationship between the enzyme and its substrate. Like the indicated transesterification, the growth factor (or bicyclic ring system) has increased the effective molarity of the reactants. In both cases, the result is an increase in the rate of the associated reaction. However, proximity alone does not ensure a high effective molarity and large reaction rate. This point is made by a famous reaction conceived and studied by Albert Eschenmoser⁶², where an improper orientation of reactants results in a slow rate of intramolecular reaction (Figure 16). This is reminiscent of the insulin receptor, which is a disulfide-bonded dimer in the absence of its activating ligand, insulin. Despite this enforced proximity, the receptor does not transphosphorylate itself in the absence of insulin. Presumably the kinase active sites are improperly oriented in the unliganded dimer, and insulin induces a reorientation that facilitates transphosphorylation. Effective molarity is controlled by proximity and orientation effects, and these are relevant to both chemistry and biology.

Our small molecule dimerizers have proved to be powerful reagents for controlling the cellular and physiological functions of proteins (usually by activation) and have illuminated the fundamental roles of proximity and orientation effects in biology in a variety of contexts^59,60,61. Signal transduction pathways have been activated and genes have been regulated in cells and transgenic animals by a variety of semisynthetic compounds. For example, our two labs have developed several methods to activate the Fas receptor-mediated signaling pathway that cells use to kill themselves (Figure 17)^63,64,65. The dimerizer procedure involves expressing in cells or transgenic mice target proteins fused to a second protein that serves as a small molecule-dependent dimerization site (e.g., Figure 18). We have synthesized both dumbbell-shaped (as in Figures 18, 19, 20, 21)^{65,66,67,68,63} and rapamycin-shaped (as in Figures 22, 23, 24)⁶⁹ dimerizers, and these molecules have been used to activate signaling pathways by either dimerizing receptors at the cell surface (e.g., the insulin⁷⁰, erythropoietin⁷¹, PDGF⁷⁰, TGFb^72,73, Fas^63,64,65, and T cell receptors^66,67), to recruit intracellular signaling proteins to the plasma membrane, allowing them to signal (ZAP⁷⁴, Raf^75,76, Sos⁷⁷, Src⁷⁸, Lck⁷⁸, Fas⁶³), to regulate gene transcription by recruiting activation domains to target genes^63,79, and to import⁶³ and export⁸⁰ target proteins to and from the nucleus. A partial listing of processes controlled by small molecule dimerizers is provided in Figure 25⁵⁹.

Originally to improve the specificity of our dimerizers, we developed a strategy for creating new protein-small molecule combinations from pre-existing ones, referred to as the "bump-hole" strategy⁸¹. This has proved to be an especially useful technique not only for preparing non-toxic dimerizers⁶⁹ (Figure 22), but also, as Peter Belshaw showed, for preparing highly specific inhibitors of phosphatases, even ones that target phosphatases in a cell-specific way⁸² (Figures 26, 27). Placing a "bump" alone on a protein target allows its small molecule ligand to inactivate the function of the unmodified, wild-type form and the bumped, recombinant form to complement the lost activity. In the first example, the "bump" was a methyl group added to serine 2035 of the FRAP protein (by site-directed mutagenesis) and this rapamycin-resistant mutant of FRAP was used to explore the precise function of cells whose endogenous FRAP had been inactivated by treatment with rapamycin⁸³. More recently, the bump-hole strategy was extended in Kevan Shokat’s laboratory to the synthesis of specific inhibitors of kinases⁸⁴.

The third and current phase of my research, where stereoselective synthesis of natural product-like compounds is the engine that drives biological research. The preceding examples provide illustrations of the equivalency of ligands and mutations in the study of cellular protein function. Thus far, with only few exceptions, the ligands have been either natural products or their synthetic variants (e.g., synthetic dimers of natural products). To extend the ligand-based approach, powerful methods of ligand discovery are required. The principles defined by geneticists to identify mutations that illuminate protein function are likely to be of value in the search for new ligands with similar properties. The geneticist generates large numbers of mutations, chooses from the myriad of methods to prepare the library of mutations, and selects the desired mutations through the use of an effective screen. Likewise, the chemical geneticist is now able to synthesize vast numbers of small molecules (using "split-and-pool" synthesis or, theoretically, massively parallel synthesis), choose from the myriad of methods to synthesize complex, natural product-like molecules, and select the desired ligands through the use of screens compatible with the split-and-pool method of small molecule generation (Figure 28). This personal view of chemical genetics is admittedly biased toward proteins, since they have been the object of my affection during the past fifteen years. Research in the area of small molecule recognition of nucleic acids could easily add another dimension to the field, a sentiment supported by the recent and remarkable progress made in this area, primarily in the laboratory of Peter Dervan⁸⁵.

Although a genetic-like approach to ligand discovery has only recently been tested in the laboratory, it has been used widely in nature to produce the natural product ligands described above. For example, bacterial geneticists have uncovered the global outline of polyketide synthesis, leading to polyketide natural products such as rapamycin and FK506 (Figure 29)⁸⁶. These molecules are synthesized by an iterative sequence involving a Claisen condensation, ketone reduction, dehydration, and enone reduction. The polyketide synthases containing the enzyme modules that perform these functions are encoded within single bacterial operons. These modules appear to have been shuffled throughout evolution by genetic recombination, which split-and-pool synthesis emulates. Other types of gene modifications, such as mutations in the ketoreductase modules, further enhance the structural complexity of the natural polyketide library. Finally, the process of natural selection leads to the existing members of this family of polyketide ligands. Their frequent use in present day cell biological studies stems from their selection, over a billion years, as protein ligands.

My lab has been developing a genetics-inspired research plan to synthesize natural product-like compounds and to assess their actions on a wide range of cellular processes during the past four years. Two years ago, it became evident that the plan was sound, yet it was depending more than ever on advances from several neighboring disciplines^87,88,89,90. These included disciplines not routinely encountered in a chemistry department. With the blessing of the President of Harvard University and the Deans of the Faculty of Arts and Sciences and the Medical School, my colleagues, Tim Mitchison, Marc Kirschner, Eric Jacobsen, Greg Verdine, Matthew Shair, Rebecca Ward, and I created the Harvard Institute of Chemistry and Cell Biology (ICCB)⁹¹. The ICCB is devoted to advancing the field of chemical genetics by creating a multidisciplinary environment involving synthetic organic chemistry, molecular cell biology, miniaturization and imaging sciences, and engineering. It offers a new training environment for students, postdoctorals, visiting scientists, and faculty interesting in developing chemical genetics and using it to explore protein function.

I believe that our major challenge will be to synthesize complex, natural product-like compounds, the types of compounds that Nature may have sampled but not selected on the billion year path that led to FK506. These would be polyketide-related compounds, but chemical synthesis does not limit us to the acetate, propionate, and butyrate building blocks used in Nature. It also does not limit us to polyketide-like natural products, and we in fact have projects aimed at making compounds related in structure to members of natural alkaloid and terpene families. I am excited by the group of bright and fearless synthetic chemists that have joined me in my efforts to undertake these challenges. We are gaining momentum, which I hope will be evident from the reports now being prepared for publication. For example, Derek Tan, Michael Foley, and Matthew Shair have synthesized over two million compounds having structural features both reminiscent of natural products and compatible with miniaturized cell-based assays⁹².

We also have the power to alter stereochemistry in a logical way. But the application of stereoselective methods to this new area of synthesis will demand careful planning, including a kind of planning not to my knowledge encountered in classical natural products synthesis. I will offer one example. We have recently initiated an effort to synthesize tens of millions of methymycin-related compounds. The natural product contains a twelve-membered macrolide. In designing a synthesis of the natural product, consideration might be given to a twelve-membered ring-closing step. Experience tells us that the efficiency of the ring closure will be dependent on the substitution pattern of the acyclic precursor, including the identity of protecting groups, and to the reaction conditions. Ad hoc solutions to the ring closure step, however, are not acceptable when the synthesis of tens of millions of methymycinoids is undertaken. However, a subset of these compounds can be designed that have as a common feature conformational preferences that ensure facile twelve-membered ring closure, as in the case of the designed twelve-membered macrolide shown in Figure 3⁸. These compounds can be viewed as expanded six-membered rings, where a two atom unsaturated linkage has been inserted into every other ring C-C bond.

It was the intellectual challenge of retrosynthetic analysis that first attracted me to the field of natural products synthesis. I am finding the field of natural product-like synthesis to be rich with these same challenges, and even new ones like that described above. Earlier, I stated that the major challenge in efforts to generalize chemical genetics will be in synthesis, but it is by no means the only one. Molecular cell biology will be needed to engineer cells to serve as reporters of many types of cellular processes, preferably by emitting light when a small molecule has inactivated or activated the function of its target protein⁶³ (Figure 30). Making many millions of compounds requires that chemists be able to run millions of experiments that assess the actions of their synthetic compounds on cells. Miniaturization is key to the success of this aspect of the research. An example is seen in the two techniques we have developed for creating tiny 50-200 nanoliter droplets of cell culture on a vast scale^93,94 (Figure 31, 32). These nanodroplets can be used to detect small molecules that bind proteins and that disrupt protein-protein interactions when generated with suitably engineered cells⁹⁵ (Figures 33, 34, 35, 36).

Conclusions I would like to conclude this essay with a view to the future of this third phase of research. There are many unanswered questions. Foremost in my mind is the question of whether we as synthetic chemists will ever be able to create in the laboratory compounds with the extraordinary specificity of natural products. Although I do not know the answer, I am optimistic for a variety of reasons. The DNA chip/hybridization array technology provides us with at least one method for determining the specificity of a synthetic compound when incubated with a living cell, with its enormous collection of proteins, and others are bound to emerge. This technique has shown that a natural product can have the absolute specificity previously only associated with a gene deletion mutation, so it proves the principle of small molecule specificity. New technology from a variety of disciplines has allowed every individual step of a classical genetic screen to be emulated with chemistry. Not one of the steps has yet been optimized, but there appears to be no theoretical impediments, no insurmountable activation barriers associated with any individual step. Will the full power of stereoselective methods in synthesis be brought to bare in split-pool or massively parallel syntheses of millions or billions of natural product-like compounds? Even more ambitiously, will we be able to recapture the many millions of presumed "transient" natural products that were evolutionarily de-selected along the paths that eventually led to the natural products synthesized on Earth today? It is true that even the first goal has not yet been demonstrated, but I cannot imagine that in a young synthetic chemist's lifetime, it will not be accomplished. And if it is realized, and effective means for assessing the properties of these compounds are perfected, the impact on the life sciences would be significant. Chemical genetics, like genetics, can be used to understand protein function. Although it is important to use small molecules that alter function with the specificity of a gene knockout, the discovery of such molecules will allow the instantaneous alteration of function, not possible in classical genetics, even in cells and animals not readily amenable to genetic analysis. Instantaneous alteration of function allows the kinetic timecourse of events to be determined, which can shed a bright light on complex cellular processes. Perhaps most importantly, in chemical genetics the tools that are used to alter function, small molecules, can be used to control the function of proteins. This promises to build more direct connections between biology and medicine, ones made possible by the awesome power of synthetic chemistry.

The original goal of the human genome project was "to sequence every gene." With that goal within sight, I suggest we consider a new goal for this project, one that can only be realized through the creative use of chemistry, "to identify a small molecule partner for every gene product".

Acknowledgement The research described in this article was performed by a remarkable group of co-workers, to whom I am indebted. Their dedication, creativity, and spirit have enriched my life in immeasurable ways. I hope their individual accomplishments will "come to life" for the readers by searching through a website prepared in part for this purpose: http://www-schreiber.chem.harvard.edu. I am also grateful to the National Institute of General Medical Sciences, which, since 1981, has served as a primary source of funding for the research described in this paper, and to the Howard Hughes Medical Institute, which has played a vital role in the growth of chemical genetics during the past four years.

References and Footnotes

¹ "Sex Pheromone of the American Cockroach: Absolute Configuration of Periplanone-B" Michael A. Adams, Koji Nakanishi, W. Clark Still, Edward V. Arnold, Jon Clardy, C.J. Persoons, J. Am. Chem. Soc. 1979, 101, 2495-2498.

² "Cyclobutene Bridgehead Olefin Route to the American Cockroach Sex Pheromone, Periplanone-B", Stuart L. Schreiber, Conrad Santini, J. Am. Chem. Soc., 1984, 106, 4038.

³ "[2+2] Photocycloadditions in the Synthesis of Chiral Molecules", Stuart L. Schreiber, Science, 1985, 227, 857.

⁴ "A Photochemical Route to the Formation of Threo-Aldols", Stuart L. Schreiber, Amir H. Hoveyda, Hsien-Jen Wu, J. Am. Chem. Soc., 1983, 105, 660.

⁵ "Application of the Furan-Carbonyl Photocycloaddition Reaction to the Synthesis of the Bis(tetrahydrofuran) Moiety of Asteltoxin", Stuart L. Schreiber, Kunio Satake, J. Am. Chem. Soc., 1983, 105, 6723.

⁶ "Total Synthesis of (±)-Asteltoxin", Stuart L. Schreiber, Kunio Satake, J. Am. Chem. Soc., 1984, 106, 4186.

⁷ "Synthetic Studies of the Furan-Carbonyl Photocycloaddition Reaction. Total Synthesis of (±)-Avenaciolide". Stuart L. Schreiber, Amir H. Hoveyda, J. Am. Chem. Soc., 1984, 106, 7200.

⁸ "Epoxidation of Unsaturated Macrolides: Stereocontrolled Routes to Ionophore Subunits", Stuart L. Schreiber, Tarek Sammakia, Bernard Hulin, J. Am. Chem. Soc., 1986, 108, 2106.

⁹ "A Lewis Acid Mediated Version of the Nicholas Reaction: Synthesis of Syn-Alkylated Products and Cobalt Complexed Cycloalkynes", Stuart L. Schreiber, Tarek Sammakia, William E. Crowe, J. Am. Chem. Soc., 1986, 108, 3128.

¹⁰ "Dynamic Behavior of Dicobalt Hexacarbonyl Propargyl Cations and Their Reactions with Chiral Nucleophiles", Stuart L. Schreiber, Michael T. Klimas, Tarek Sammakia, J. Am. Chem. Soc., 1987, 109, 5749.

¹¹ "Tandem Use of Cobalt-Mediated Reactions to Synthesize (+)-Epoxydictymene, a Diterpene Containing a trans-Fused 5-5 Ring System" Timothy F. Jamison, Soroosh Shambayati, William E. Crowe, Stuart L. Schreiber, J. Am. Chem. Soc. 1997, 119, 4353-4363.

¹² "Application of the Allylic Diazene Rearrangement: Synthesis of the Enediyne-Bridged Tricyclic Core of Dynemicin A" John L. Wood, John A. Porco, Jr., Jack W. Taunton, Angela J. Lee, Jon Clardy, Stuart L. Schreiber, J. Am. Chem. Soc. 1992, 114, 5900-5902.

¹³ "Total Syntheses of Di- and Tri-O-Methyl Dynemicin A Methyl Esters" Jack Taunton, John L. Wood, Stuart L. Schreiber, J. Am. Chem. Soc. 1993, 115, 10378-10379.

¹⁴ "Two-Directional Chain Synthesis: The Enantioselective Synthesis of Syn-Skipped Polyol Chains from Meso Precursors", Stuart L. Schreiber, Mark T. Goulet, Gayle Schulte, J. Am. Chem. Soc., 1987, 109 , 4718.

¹⁵ "Application of the Two-Directional Chain Synthesis Strategy to the First Stereochemical Assignment of Structure to Members of the Skipped-Polyol Polyene Macrolide Class: Mycoticin A and B", Stuart L. Schreiber, Mark T. Goulet, J. Am. Chem. Soc., 1987, 109, 8120.

¹⁶ "Two-Directional Chain Synthesis: An Application to the Synthesis of (+)-Mycoticin A" Christopher S. Poss, Scott D. Rychnovsky, Stuart L. Schreiber, J. Am. Chem. Soc. 1993, 115, 3360-3361.

¹⁷ "Two-Directional Chain Synthesis and Terminus Differentiation" Stuart L. Schreiber, Chemica Scripta, 1987, 27, 563.

¹⁸ "Two-Directional Chain Synthesis and Terminus Differentiation" Christopher S. Poss, Stuart L. Schreiber, Acc. Chem. Res. 1994, 27, 9-17.

¹⁹ "The Asymmetric Epoxidation of Divinyl Carbinols" David B. Smith, Zhaoyin Wang, Stuart L. Schreiber, Tetrahedron, 1990, 46, 4793.

²⁰ "Total Synthesis of the Anthelmintic Agent Hikizimycin" Norihiro Ikemoto, Stuart L. Schreiber, J. Am. Chem. Soc. 1990, 112, 9657.

²¹ "Total Synthesis of (-)-Hikizimycin Using the Strategy of Two-Directional Chain Synthesis" Norihiro Ikemoto, Stuart L. Schreiber, J. Am. Chem. Soc. 1992, 114, 2524-2536.

²² "Total Syntheses of FK506 and an FKBP Probe Reagent, (C8, C9-13C2)-FK506" Masashi Nakatsuka, John A. Ragan, Tarek Sammakia, David B. Smith, David E. Uehling, Stuart L. Schreiber J. Am. Chem. Soc., 1990, 112, 5583.

²³ "Total Synthesis of the FK506/FKBP Complex" Stuart L. Schreiber, John A. Ragan, Robert F. Standaert, In Strategies and Tactics in Organic Synthesis, Lindberg, T. Ed.; Academic Press:San Diego, 1991; Vol. 3, pp 417-461.

²⁴ "Chemistry and Biology of the Immunophilins and Their Immunosuppressive Ligands" Stuart L. Schreiber, Science 1991, 251, 283-287.

²⁵ "Using the Principles of Organic Chemistry to Explore Cell Biology" Stuart L. Schreiber C.& E. News 1992 (October 26), 22-32.

²⁶ "A Receptor for the Immunosuppressant FK506 is a cis-trans Peptidyl-Prolyl Isomerase" Matthew W. Harding, Andrzej Galat, David E. Uehling, Stuart L. Schreiber, Nature, 1989, 341, 758.

²⁷ "Molecular Cloning and Overexpression of the Human FK506-Binding Protein, FKBP" Robert F. Standaert, Andrzej Galat, Gregory L. Verdine, Stuart L. Schreiber, Nature, 1990, 346, 671.

²⁸ "Probing Immunosuppressant Action with a Nonnatural Immunophilin Ligand" Barbara E. Bierer, Patricia K. Somers, Thomas J. Wandless, Steven J. Burakoff, Stuart L. Schreiber, Science, 1990, 250, 556.

²⁹ "Synthesis and Analysis of 506BD, a High Affinity Ligand to the Immunophilin, FKBP" Patricia K. Somers, Thomas J. Wandless, Stuart L. Schreiber, J. Am. Chem. Soc. 1991, 113, 8045-8056.

³⁰ "Synthesis of Natural and Modified Trapoxins, Useful Reagents for Exploring the Histone Deacetylase Function" Jack Taunton, Jon L. Collins, Stuart L. Schreiber, J. Am. Chem. Soc. 1996, 118, 10412-10422.

³¹ "A Mammalian Histone Deacetylase Related to the Yeast Transcriptional Regulator Rpd3p" Jack Taunton, Christian A. Hassig, Stuart L. Schreiber, Science 1996, 272, 408-411.

³² "Histone Deacetylase Activity is Required for Full Transcriptional Repression by mSin3A" Christian A. Hassig, Tracey C. Fleischer, Andrew N. Billin, Stuart L. Schreiber, Donald E. Ayer, Cell 1997, 89, 341-347.

³³ "Nuclear Receptor Repression Mediated by a Complex Containing SMRT, Sin3 and Histone Deacetylase" L. Nagy, H.-Y. Kao, D. Chakravarti, R. Lin, Christian A. Hassig, Donald E. Ayer, S. L. Schreiber, Ronald M. Evans, Cell 1997, 89, 373-380.

³⁴ "Nuclear Histone Acetylases and Deacetylases and Transcriptional Regulation: HATs Off to HDACs" Christian A. Hassig, Stuart L. Schreiber, Curr. Opinion in Chem. Biol. 1997, 1, 300-308.

³⁵ "Fiber-derived Butyrate and the Prevention of Colon Cancer" Christian A. Hassig, Jeffrey K. Tong, Stuart L. Schreiber, Chem & Biol 1997, 4, 783-789.

³⁶ "A Role for Histone Deacetylase in HDAC1-Mediated Transcriptional Repression" Christian A. Hassig, Jeffrey K. Tong, Tracey C. Fleischer, Takashi Owa, Phyllis Grable, Donald E. Ayer, Stuart L. Schreiber Proc. Natl. Acad. Sci., U.S.A., 1998, 95, 3519-3524.

³⁷ "Depudecin Induces Morphological Reversion of Transformed Fibroblasts via the Inhibition of Histone Deacetylase" Ho Jeong Kwon, Takashi Owa, Christian A. Hassig, Junichi Shimada, Stuart L. Schreiber Proc. Natl. Acad. Sci., U.S.A. 1998, 95, 3356-3361.

³⁸ "Transmembrane Channels Based on Tartaric Acid-Gramicidin Hybrids" Stefan H. Heinemann, Frederic J. Sigworth, Charles J. Stankovic, Jose M. Delfino, Stuart L. Schreiber Science 1989, 244, 813.

³⁹ "Immobilizing the Gate of a Tartaric Acid-Gramacidin Hybrid Channel Molecule by Rational Design" Charles J. Stankovic, Stefan H. Heinemann, Stuart L. Schreiber, J. Am. Chem. Soc., 1990, 112, 3702.

⁴⁰ "Molecular Design of Transmembrane Ion Channels" Charles J. Stankovic, Stuart L. Schreiber, ChemTracts, Organic Chemistry, 1991, 4, 1-20.

⁴¹ "A Tricyclic Ring System Replaces the Variable Regions of Peptides Presented by Three Alleles of MHC Class I Molecules" Gregory A. Weiss, Edward J. Collins, David N. Garboczi, Don C. Wiley, Stuart L. Schreiber, Chem & Biol 1995, 2, 401-407.

⁴² "Covalent HLA-B27/Peptide Complex Induced by Specific Recognition of an Aziridine Mimic of Arginine" Gregory A. Weiss, Robert J. Valentekovich, Edward J. Collins, David N. Garboczi, William S. Lane, Stuart L. Schreiber, Don C. Wiley, Proc. Natl. Acad. Sci., U.S.A. 1996, 93, 10945-10948.

⁴³ "Structure-Based Design of an Acyclic Ligand That Bridges FKBP12 and Calcineurin" Merritt B. Andrus, Stuart L. Schreiber, J. Am. Chem. Soc. 1993, 115, 10420-10421.

⁴⁴ "Structure-Based Design of a Cyclophilin-Calcineurin Bridging Ligand" David G. Alberg, Stuart L. Schreiber, Science 1993, 262, 248-250.

⁴⁵ "Vinylogous Polypeptides: An Alternative Peptide Backbone" Masahiko Hagihara, Neville J. Anthony, Thomas J. Stout, Jon Clardy, Stuart L. Schreiber, J. Am. Chem. Soc. 1992, 114, 6568-6570.

⁴⁶ "The Mechanism of Action of Cyclosporin A and FK506" Stuart L. Schreiber, Gerald R. Crabtree, Immunology Today 1992, 13, 136-142.

⁴⁷ "Immunophilin-Sensitive Phosphatase Action in Cell Signaling Pathways" Stuart L. Schreiber, Cell 1992, 70, 365-368.

⁴⁸ "A Signaling Pathway to Translational Control" Eric J. Brown, Stuart L. Schreiber, Cell 1996, 86, 517-520.

⁴⁹ "Synthesis and Cellular Characterization of the Detransformation Agent, (-)-Depudecin" Junichi Shimada, Ho Jeong Kwon, Masaya Sawamura, Stuart L. Schreiber, Chem & Biol 1995, 2, 517-525.

⁵⁰ "Total Synthesis of the Immunosuppressive Agent (-)-Discodermolide" Jennie B. Nerenberg, Deborah T. Hung, Patricia K. Somers, Stuart L. Schreiber, J. Am. Chem. Soc. 1993, 115, 12621-12622.

⁵¹ "Distinct Binding and Cellular Properties of Synthetic (+)- and (-)-Discodermolide " Deborah T. Hung, Jennie B. Nerenberg, Stuart L. Schreiber, Chem. & Biol. 1994, 1, 67-71.

⁵² "(+)-Discodermolide Binds to Microtubules in Stoichiometric Ratio to Tubulin Dimers, Blocks Taxol Binding and Results in Mitotic Arrest" Deborah T. Hung, Jie Chen, Stuart L. Schreiber, Chem. & Biol. 1996, 3, 287-293.

⁵³ "Syntheses of Discodermolides Useful for Investigating Microtubule Binding and Stabilization" Deborah T. Hung, Jennie B. Nerenberg, Stuart L. Schreiber, J. Am. Chem. Soc. 1996, 118, 11054-11080.

⁵⁴ "Inhibition of Proteasome Activities and Subunit-Specific Amino-Terminal Threonine Modification by Lactacystin" Gabriel Fenteany, Robert F. Standaert, William S. Lane, Soongyu Choi, E. J. Corey, Stuart L. Schreiber, Science 1995, 268, 726-731.

⁵⁵ "Lactacystin, Proteasome Function, and Cell Fate" Gabriel Fenteany, Stuart L. Schreiber, J. Biol. Chem. 1998, 273, 8545-8548.

⁵⁶ "cDNA Cloning and Gene Mapping of a Candidate Human Cell Cycle Checkpoint Protein" Karlene A. Cimprich, Tae Bum Shin, Curtis T. Keith, Stuart L. Schreiber, Proc. Natl. Acad. Sci., U.S.A. 1996, 93, 2850-2855.

⁵⁷ "PIK-related Kinases: DNA Repair, Recombination, and Cell Cycle Checkpoints" Curtis T. Keith, Stuart L. Schreiber, Science 1995, 270, 50-51.

⁵⁸ "Overexpression of a Kinase-Inactive ATR Protein Causes Sensitivity to DNA-Damaging Agents and Defects in Cell Cycle Checkpoints" William A. Cliby, Christopher J. Roberts, Karlene A. Cimprich, Cheri M. Stringer, John R. Lamb, Stuart L. Schreiber, Stephen H. Friend, EMBO J 1998, 17, 159-169.

⁵⁹ "Three-Part Inventions: Intracellular Signaling and Induced Proximity" Gerald R. Crabtree, Stuart L. Schreiber, TIBS 1996, 21, 418-422.

⁶⁰ "Proximity vs Allostery: The Role of Regulated Protein Dimerization in Biology" David J. Austin, Gerald R. Crabtree, Stuart L. Schreiber, Chem. & Biol. 1994, 1, 131-136.

⁶¹ "Dimerization as a Regulatory Mechanism in Signal Transduction" Juli D. Klemm, Stuart L. Schreiber, Gerald R. Crabtree, Annual Review of Immunology, William e. Paul, Ed., Vol 16, 569-592 (1998).

⁶² "Endocyclische S_N-Reaktionen am gestättigten Kohlenstoff?" L. Tenud, S. Farooq, J. Seibl, A. Eschenmoser, Helvetica. Chim. Acta 1970, 53, 2059.

⁶³ "Controlling Protein Association and Subcellular Localization with a Synthetic Ligand that Induces Heterodimerization of Proteins" Peter J. Belshaw, Steffan Ho, Gerald R. Crabtree, Stuart L. Schreiber, Proc. Natl. Acad. Sci., U.S.A. 1996, 93, 4604-4607.

⁶⁴ "Functional Analysis of Fas Signaling in vivo Using Synthetic Dimerizers" David Spencer, Pete Belshaw, Lei Chen, Steffan Ho, Filippo Randazzo, Gerald R. Crabtree, Stuart L. Schreiber, Curr Biol, 1996, 6, 839-848.

⁶⁵ "Controlling Programmed Cell Death with a Cyclophilin-Cyclosporin-Based Chemical Inducer of Dimerization" Peter J. Belshaw, Gerald R. Crabtree, Stuart L. Schreiber, Chem & Biol 1996, 3, 731-738.

⁶⁶ "Controlling Signal Transduction with Synthetic Ligands" David Spencer, Thomas J. Wandless, Stuart L. Schreiber, Gerald R. Crabtree, Science 1993, 262, 1019-1024.

⁶⁷ "Mechanistic Studies of a Signaling Pathway Activated by the Organic Dimerizer FK1012" Martin N. Pruschy, David M. Spencer, Tarun M. Kapoor, Hiroshi Miyake, Gerald R. Crabtree, Stuart L. Schreiber, Chem. & Biol. 1994, 1, 163-172.

⁶⁸ "Single-Step Syntheses of Cell Permeable Protein Dimerizers that Activate Signal Transduction and Gene Expression" Steven T. Diver, Stuart L. Schreiber, J. Am. Chem. Soc. 1997, 119, 5106-5109.

⁶⁹ "Inducible Gene Expression and Protein Translocation Using Non-Toxic Ligands Identified by a Mammalian Three-Hybrid Screen" Stephen D. Liberles, Steven T. Diver, David J. Austin, Stuart L. Schreiber Proc. Natl. Acad. Sci., U.S.A. 1997, 94, 7825-7830.

⁷⁰ "Small Molecule Control of Insulin and PDGF Receptor Signaling and the Role of Membrane Attachment" Jian-xin Yang, Karen Symes, Mark Mercola, Stuart L. Schreiber, Curr Biol 1997, 8, 11-18.

⁷¹ "A Proliferation Switch for Genetically Modified Cells" C. Anthony Blau, Kenneth R. Peterson, Jonathan G. Drachman, David Spencer, Proc. Natl. Acad. Sci. 1997, 94, 3076-3081.

⁷² " Probing the Role Of Homomeric And Heteromeric Receptor Interactions in TGF-b Signaling Using Small Molecule Dimerizers" Brent R. Stockwell, Stuart L. Schreiber, Curr. Biol., 1998, 8, 761.

⁷³ "A Fusion Protein Between FKBP12 and the TGF-b Type I Receptor Intracellular Domain is Activated by FKBP Ligands" Brent R. Stockwell, Stuart L. Schreiber, Chem. & Biol., 1998, 5, 385.

⁷⁴ "Proximity and Orientation Underlie Signaling by the Non-Receptor Tyrosine Kinase ZAP70" Isabella A. Graef, Leslie J. Holsinger, Steve Diver, Stuart L. Schreiber, Gerald R. Crabtree, EMBO J. 1997, 16, 5618-5628.

⁷⁵ "Activation of the Raf-1 Kinase Cascade by Coumermycin-induced Dimerization" M. A. Farrar, J. Alberola-lla, R. M. Permutter, Nature 1996, 383, 178-180.

⁷⁶ "Oligomerization Activates c-Raf-1 Through a Ras-Dependent Mechanism" Z. Luo, G. Tzivion, P.J. Belshaw, D. Vavvas, M. Marshall, J. Avruch, Nature, 1996, 383, 181-185.

⁷⁷ "Signal Transduction in T Lymphocytes Using a Conditional Allele of Sos" Leslie J. Holsinger, David M. Spencer, David J. Austin, Stuart L. Schreiber, Gerald R. Crabtree, Proc. Natl. Acad. Sci., USA 1995, 92, 9810-9814.

⁷⁸ "A General Strategy for Producing Conditional Alleles of Src-Like Tyrosine Kinases" David M. Spencer, Isabella Graef, David J. Austin, Stuart L. Schreiber, Gerald R. Crabtree, Proc. Natl. Acad. Sci., USA 1995, 92, 9805-9809.

⁷⁹ "Dimeric Ligands Define a Role for Transcriptional Activation Domains in Reinitiation" Steffan N. Ho, Stephen R. Biggar, David M. Spencer, Stuart L. Schreiber, Gerald R. Crabtree, Nature 1996, 382, 822-826.

⁸⁰ "Rapid Targeting of Nuclear Proteins to the Cytoplasm" J. D. Klemm, C. R. Beals, Gerald R. Crabtree, Curr Biol 1997, 7, 638-44.

⁸¹ "Rational Design of Orthogonal Receptor-Ligand Combinations" Peter J. Belshaw, Joseph Schoepfer, Karen Liu, Kim Morrison, Stuart L. Schreiber Angew. Chem., Int. Ed. Eng. 1995, 34, 2129-2132.

⁸² "Cell-Specific Calcineurin Inhibition by a Modified Cyclosporin" Peter J. Belshaw, Stuart L. Schreiber, J. Am. Chem. Soc. 1997, 119, 1805-1806.

⁸³ "Control of p70 S6 Kinase by Kinase Activity of FRAP in vivo" Eric J. Brown, Peter Beal, Curtis T. Keith, Jie Chen, Tae Bum Shin, Stuart L. Schreiber Nature 1995, 377, 441-446.

⁸⁴ "Design of allele-specific inhibitors to probe protein kinase signaling" Anthony C. Bishop, Kavita Shah, Yi Liu, Laurie Witucki, Chi-yun Kung, Kevan M. Shokat Curr. Biol. 1998, 8, 257-266.

⁸⁵ "Regulation of gene expression by small molecules" Joel M. Gottesfeld, Laura Neely, John W. Trauger, Eldon E. Baird, Peter B. Dervan Nature 1997 387, 202-205.

⁸⁶ "Genetic Contributions to Understanding Polyketide Synthases" David A. Hopwood Chem. Rev. 1997 7, 2465 —2498.

⁸⁷ "Combinatorial Synthesis and Multidimensional NMR Spectroscopy: An Approach to Understanding Protein-Ligand Interactions" James K. Chen, Stuart L. Schreiber Angew Chemie, Int. Ed. Eng. 1995, 34, 953-969.

⁸⁸ "Protein Structure-Based Combinatorial Chemistry: Discovery of Non-Peptide Binding Elements to Src SH3 Domain" Andrew P. Combs, Tarun M. Kapoor, Sibo Feng, James K. Chen, Lygia F. Daude-Snow, Stuart L. Schreiber, J. Am. Chem. Soc. 1996, 118, 287-288.

⁸⁹ "Exploring the Specificity Pockets of Two Homologous SH3 Domains Using Structure-Based, Split-Pool Synthesis and Affinity-Based Selection" Tarun Kapoor, Amy Hamilton Andreotti, Stuart L. Schreiber, J. Am. Chem. Soc. 1998, 120, 23-29.

⁹⁰ "Exploring the Leucine-Proline-Binding Pocket of Src SH3 Domain Using Structure-Based, Split-Pool Synthesis and Affinity-Based Selection" James P. Morken, Tarun M. Kapoor, Sibo Feng, Fumiyuki Shirai, Stuart L. Schreiber, J. Am. Chem. Soc. 1998, 120, 30-36.

⁹¹ http://www.hms.harvard.edu/iccb

⁹² "Stereoselective Synthesis of Over Two Million Compounds Having Structural Features Both Reminiscent of Natural Products and Compatible with Miniaturized Cell-Based Assays" Derek S. Tan, Michael A. Foley, Matthew D. Shair, Stuart L. Schreiber, J. Am. Chem. Soc., in press.

⁹³"Small Molecule-Dependent Genetic Selection in Stochastic Nanodroplets as a Means to Detect Protein-Ligand Interactions on a Large Scale" Allen Borchardt, Stephen D. Liberles, Stephen R. Biggar, Gerald R. Crabtree, Stuart L. Schreiber Chem & Biol 1997, 4, 961-968.

⁹⁴ "Miniaturized Arrayed Assay Format for Detecting Small Molecule-Protein Interactions in Cells" Angie You, Rebecca J. Jackman, George M. Whitesides, Stuart L. Schreiber Chem & Biol 1997, 4, 969-975.

⁹⁵ "A Yeast Genetic System for Selecting Small Molecule Inhibitors of Protein-Protein Interactions in Nanodroplets" Jing Huang, Stuart L. Schreiber, Proc. Natl. Acad. Sci., U.S.A. 1997, 94, 13396-13401.