COMMENTARY

Synthetic : lessons from the history of synthetic organic

Brian J Yeh & Wendell A Lim

The mid-nineteenth century saw the development of a radical new direction in chemistry: instead of simply analyzing existing , began to synthesize them—including molecules that did not exist in . The combination of this new synthetic approach with more traditional analytical approaches revolutionized chemistry, leading to a deep understanding of the fundamental principles of chemical structure and and to the emergence of the modern pharmaceutical and chemical industries. The history of synthetic chemistry offers a possible roadmap for the development and impact of synthetic biology, a nascent field in which the goal is to build novel biological systems.

In 1828, the German Friedrich Wöhler coffin of , and in the next few decades, fats3. These and other early syntheses demon- could hardly contain his excitement as he wrote chemists began to synthesize hundreds of other strated that chemists could indeed make ‘living’ to his former mentor, Jöns Jakob Berzelius, of organic molecules. In a particularly interesting molecules as well as new compounds that went a new finding1,2: “I must tell you that I can example in 1854, the French chemist Marcellin beyond those that naturally occurred, thus giv- prepare without requiring a kidney of an Berthelot synthesized the tristea- ing birth to synthetic . It was animal, either man or dog.” At the beginning rin from glycerol and stearic acid, a common unclear where this field would lead, and many of the nineteenth century, the synthesis of this naturally occurring fatty acid. Taking this a step feared these advances could lead to goals such small organic molecule was earth-shattering further, he realized that he could replace stea- as the creation of living beings. Today, however, news. At that time, chemists believed there ric acid with similar acids not found in natural nearly all aspects of our are touched by was a clear distinction between molecules , thus generating non-natural molecules synthetic molecules that mankind has learned from living beings (referred to as ‘organic’) that had properties similar to those of natural to make. and those from nonliving origin (‘inorganic’). It was known that organic substances could be easily converted to inorganic compounds through heating or other treatments; however, chemists could not perform the reverse trans- formation. Surely, a ‘vital force’ present only in living was required to convert the inorganic into organic. Wöhler’s discovery that ammonium cyanate could be converted to urea in the was a key nail in the

Brian J. Yeh is in the Chemistry and Graduate Program, University of California, San Francisco, 600–16th Street, San Francisco, California 94158-2517, USA. Wendell A. Lim is in the Department of Cellular and Molecular , University of California, San Francisco, 600–16th Street, San Francisco, California 94158-2517, USA, and is a member of the Propulsion Lab, USF/UCB National Institutes of Health Development Center, and the US National Science Foundation Synthetic Biology Research Center. Iwasa J. e-mail: [email protected] Synthetic approaches may transform biology just as they transformed chemistry.

NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 9 SEPTEMBER 2007 521 COMMENTARY

Advances in our ability to build and mod- Milestones of organic chemistry ify ‘organic’ molecules on increasingly larger scales have continued to push the frontier of Synthesis Theory our understanding of the physical principles Early 1800s underlying living systems. For example, chem- 1828 Empirical formulas ical synthesis of DNA (first Urea (Wöhler) performed by Gobind Khorana) led directly 1845 4 (Kolbe) to the elucidation of the . Bruce 1850s Methanol, , (Berthelot) Merrifield’s complete synthesis of RNase A 1852 1854 (Frankland) demonstrated that chemical structure (pri- Tristearin (Berthelot) mary sequence) is sufficient to confer tertiary 1856 1858 structure and the seemingly magical activity Purple (Perkin) Structural formulas (Kekulé) 5 1865 of . More recently, complete chemi- 1867 Ring structure for benzene (Kekulé) Indigo (Baeyer) cal synthesis of complementary 1874 Tetrahedral geometry for DNA was a vivid demonstration that genetic (Van’t Hoff) instructions are sufficient to specify an active biological system6. Over the last several years, this line of research has culminated in the emergence of 1908 a field known as ‘synthetic biology.’ Whether Sulfanilamide (IG Farbenindustrie) synthetic biology represents a truly new field is open to debate, but the boldness of the stated goals—to learn how to precisely and reliably Figure 1 and theories of structure emerged concurrently. Significant milestones in engineer and build self-organizing systems chemical synthesis (left of timeline, dates shown in green) and theories of chemical structure (right of that both recapitulate biological timeline, dates shown in red) are shown. and show new functions—is unquestionably novel. These goals hold promise for harnessing the efficiency and precision of living systems form logical computations9,11–13. Although analysis led to the discovery of isomers—the for diverse purposes: microbial factories that this comparison is useful, in some respects a shocking finding that compounds with very manufacture , materials or biofuels7; comparison with the development of synthetic different physical and chemical properties can cells that seek and destroy tumors8; cells that organic chemistry may be more appropriate10. have identical empirical formulas. Clearly, a can carry out rapid repair and regen- In this Commentary, we consider the historical more sophisticated understanding of chemical eration; cells that can direct the assembly of role of synthetic approaches in the development structure would be required. ; even living systems that can of modern organic chemistry in order to extract The nascent field of synthetic chemistry, compute. Synthetic biology, however, is more some lessons that might help guide the develop- with its many new reactions and molecules, than a broad set of visionary applications. ment of synthetic biology. provided a complementary body of infor- Much effort is going into developing a com- mation that contributed to the development mon toolkit of well-defined biological parts Synthesis and analysis transform chemistry of modern theories of chemical structure and devices as well as strategies to link them Before the time of Wöhler and Berthelot, and reactivity. It was not until after the mid- together into predictable systems9–12. These the understanding of even simple molecules nineteenth century—after synthesis of small foundational efforts are aimed at one day was as naïve as our current understanding of compounds was already becoming rou- making engineering cells as reliable and pre- complex biological systems. How the com- tine—that chemists began to develop models dictable as engineering an electronic device. position of organic compounds determined to explain bonding between atoms3. In 1852, Indeed, synthetic biology can be viewed as a their properties and reactivity was unknown, Edward Frankland proposed that each subdiscipline of bioengineering that is focused and the concept of molecules having defined had an ability to combine with a fixed num- on engineering self-organizing cellular devices structures was still undeveloped. Ultimately, ber of other . Kekulé used this ‘theory (as opposed to instruments that interrogate analytical and synthetic approaches synergized of valence’ to propose structures for many living systems or materials that interface with to produce an growth in our knowl- simple organic molecules in 1858, and in the organisms). edge of chemical principles, and fundamen- 1860s, Alexander Bulterov pointed out that At this threshold, where our view of biology tal theories of chemical structure developed these structural formulas could explain the as something to be observed is transitioning concurrently with the explosion of synthesis majority of isomers. This notion that atoms into a view of biology as something that can (Fig. 1). were held in fixed arrangements was a critical be engineered, there are many important ques- A critical early advance at the beginning advance. In 1865, Kekulé proposed the struc- tions. Why even attempt synthetic biology when of the nineteenth century was precise mea- tural formula for benzene. Although it had our understanding of biological systems is still surement and analysis of compounds as they already been synthesized by Berthelot in the incomplete? And should we choose to proceed, reacted3. For example, by collecting and pre- 1850s, benzene’s stability could not be ade- how should we go about it? Many reviews cisely weighing the carbon dioxide and water quately explained until Kekulé conceived the have compared synthetic biology to electrical that formed upon combustion of organic ring structure, thereby cementing the useful- engineering, noting that cellular networks, like molecules, it became possible to determine the ness of this paradigm. Later, these structures electronic circuits, are built in a hierarchical atomic compositions of these molecules, and were extended to three dimensions based on fashion from modular components that per- therefore their empirical formulas. This careful the notion that carbon bonds are arranged in

522 VOLUME 3 NUMBER 9 SEPTEMBER 2007 NATURE CHEMICAL BIOLOGY COMMENTARY a tetrahedral geometry, an idea proposed by can perform a behavior? How does that behav- Diverse and unexpected driving Jacobus Van’t Hoff in 1874. ior precisely vary as individual components, applications A critical lesson here is that a complete the linkages that connect them into larger If one important goal of synthetic biology is to understanding of chemical principles was not networks, and specific biochemical parameters create useful systems, then what applications a prerequisite for the emergence of synthetic are altered? For example, Suel et al. have stud- should we be targeting? Again, it is useful to chemistry. Rather, synthetic and analytical ied a simple genetic circuit in consider the early applications of synthetic approaches developed in parallel and syner- that underlies differentiation, both by quanti- organic chemistry. In today’s world, many tend gized to shape our modern understanding of tatively characterizing the existing circuit and to link synthetic chemistry with the produc- chemistry. The synthetic approach—empiri- by making new connections and analyzing the tion of drugs. Indeed, it was abundantly clear cally learning how to systematically manipulate changes that result14,15. to early chemists that synthetic products could and perturb molecules—directly contributed What can we expect to learn? Clearly there improve human health, but their initial efforts to developing theories of chemical structure will be more than a few simple rules explaining actually led to an industrial explosion in an and reactivity (Fig. 2a). At the simplest level, how living systems work. As in physical organic unexpected direction. August von Hofmann synthesizing a molecule was often the ulti- chemistry, however, it is likely that unifying pat- and his student William Perkin postulated that mate proof of the proposed structure. More terns will emerge, and an understanding of the it might be possible to synthesize the highly significantly, the ability to synthesize vari- logic underlying biological systems will develop. valuable antimalarial agent from ani- ants of known molecules allowed a rigorous It may even be possible to construct something line, a cheap of coal tar3. However, and systematic exploration of the principles analogous to the for biology that in attempting to synthesize quinine from ani- underlying chemical structure and reactivity, facilitates the systematic understanding of net- line in 1856, Perkin unexpectedly produced a thus beginning the field of physical organic work ‘elements’ and their properties16,17. brilliant purple compound—a . He soon chemistry. N Biology: synthesis redux a The convergence of analytical and synthetic NH H2N N N approaches seems to be replaying itself in mod- 2 H ern biology (Fig. 2b). Biology has historically been a field based almost entirely on observa- tion and analysis, and it is currently undergo- ing exponential growth in the accuracy and throughput of measurement. Modern experi- Analysis Complex ‘natural’ organic molecules mental techniques— sequencing, Simple chemical Applications DNA microarrays, molecular structure deter- precursors mination, and high-throughput microscopy Synthesis Complex synthetic coupled with in vivo —represent organic molecules major analytical advances, giving us extensive parts lists and descriptions of biological sys- tems and their behaviors, including the abun- • Synthetic methods dance, subcellular localization and interactions • Principles of chemical of entire proteomes. These developments are structures and reactivity akin to the advances in of the early nineteenth century. However, the his- b tory of organic chemistry suggests that synthe- sis will be a necessary complement to analysis in order for to truly understand the mechanisms of complex living systems. In many ways, synthetic biology can be Analysis ‘Natural’ viewed as in vivo reconstitution—an intel- biological systems Biological lectual descendant of simple . Applications Reconstitution methods (which essentially parts Synthesis Synthetic apply the synthetic philosophy to noncovalent biological systems systems) allow us to determine not only what is necessary, but also what is sufficient to build a system that carries out a particular function. The ability to build and systematically modify • Toolkit of parts biological systems will allow one to explore • Biological their design principles in much deeper ways. principles Thus, synthetic biological systems will allow Figure 2 Synthesis and analysis are complementary. (a) In organic chemistry, analysis and synthesis experimentation that is not possible with were both critical in determining fundamental principles of chemical structure and reactivity. Synthetic extant living systems, which are encumbered molecules have been used for a wide variety of applications. (b) Similarly, synthetic approaches will by eccentric evolutionary histories and con- complement analytical methods in elucidating biological principles, and synthetic cellular systems will straints. What are the minimal systems that prove highly useful.

NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 9 SEPTEMBER 2007 523 COMMENTARY opened a factory to synthesize this molecule, ogy from the traditional discipline of biology. another species, Lartigue et al. have shown that which he called “Aniline Purple,” and thus This philosophy may seem strange to many this will indeed be possible28. founded the synthetic dye industry. Along with academic biologists, but it is actually quite It is important to remember that early syn- other examples, such as the synthesis of indigo appropriate. A defining feature of thetic chemists did not always know what to by in 1867, these advances is the constant selection of organisms that expect in their reactions, only that something led to the explosive growth of the German achieve the best performance (fitness) in a interesting could happen18. Most impor- and Swiss dye industry, while simultaneously particular niche. Understanding how to build tantly, they were prepared to follow up these dismantling the import of indigo and other biological systems to achieve well-defined per- experiments to understand what did hap- natural from distant tropical locales. In formance specifications will force us to under- pen. Similarly, in these early days of synthetic fact, synthetic indigo remains an important stand biology at a far more quantitative level. biology, it will be very difficult to predict the commercial product and is used in today’s blue For example, efforts to engineer Escherichia behavior of novel biological systems. Therefore, jeans (Levi’s was founded in 1873). coli to synthesize isoprenoids have shown that and combinatorial methods Although dyes were the earliest economically it is not sufficient to simply express the right will be useful29; analyzing libraries of synthetic important synthetic compounds, the devel- collection of enzymes to create a new pathway. circuits and systems will maximize the prob- opment of the European dye industry would Rather, accumulation of metabolic intermedi- ability of obtaining the targeted biological ultimately lead to successes in . ates can greatly limit cell growth and overall behavior. Furthermore, systematically vary- Indeed, nearly all of the modern big phar- flux through the pathway21. Thus, precisely ing many parameters will produce structure- maceutical companies are in part descended balancing intermediate synthetic steps is criti- activity relationships at the biological network from German or Swiss dye manufacturers. cal for maximizing of the final product. level that will improve future . For example, the first effective antibacteri- Synthetic biology will also require sociological Developing educational initiatives will als were the sulfa drugs3,18. The first of these reorganization of how biologists work on prob- continue to be an important emphasis in syn- molecules, sulfanilamide, was synthesized lems. Achieving success will require close inte- thetic biology. Perkin was a teenager when he by IG Farbenindustrie in 1908 because of its gration of interdisciplinary teams of scientists initially synthesized Aniline Purple—during potential as a dye. In 1932, Gerhard Domagk focused on clear, practical goals. Many synthetic Easter vacation in a small laboratory in his discovered that sulfanilamide and related biology centers organized around target goals home3. Similarly, it is young scientists who compounds have bactericidal activity, and he have emerged over the last few years, such as are likely to truly view biology from this new was aided in his studies by chemists that could the US National Science Foundation Synthetic perspective, and who will shape the yet unfore- make a variety of related compounds. Biology Engineering Research Center, the US seen ‘killer applications’ of synthetic biology. The lesson here is very clear: synthetic biolo- National Institutes of Health Nanomedicine The innovative spirit of Perkin lives on today gists (and their funding agencies) must move Development Centers, and the US Department in the growing number of undergraduates forward with an open mind. The progress of of Joint Bioenergy Institute. and high school students participating in the synthetic biology cannot be myopically linked International Genetically Engineered Machine to only a few obvious targets; instead, we must The present and future of synthetic biology (iGEM) competition. Every summer, teams of be prepared for a variety of potential indus- Just as in the early days of synthetic chem- these students compete to design and build trial and therapeutic applications, including istry, it will be necessary to develop both an new synthetic biological systems. In 2007, 56 unexpected ones that we have not yet foreseen. intellectual framework and diverse tools for teams from North America, Europe and Asia Most of the successes in synthetic biology so synthetic biology; engineering a cell with have registered for the fourth year of the com- far have been in so-called toy systems; however, specific quantitative specifications will be petition (http://www.igem2007.com). we should not underestimate the importance challenging and will require development Like all , synthetic organic chem- of these achievements in laying a foundation on many fronts. First, we must understand istry also introduced its own set of problems. of parts and construction methods that will the different languages or currencies of biol- In addition to the plethora of beneficial drugs be useful for diverse applications of synthetic ogy. Early progress in synthetic biology was and that have significantly increased biology. For example, in synthetic chemistry, limited to transcriptional networks12,20, but our standard of living, harmful or ‘dual-use’ developing generic protecting-group strategies more recently, attention has turned to engi- compounds, including and chemi- or classes of reactions to make carbon-carbon neering networks based on phosphorylation22, cal weapons, have also been created. There is bonds allowed the synthesis of diverse organic GTPases23 and RNA interference24. Second, we no question that synthetic biological systems products with a wide array of applications. must maximize the efficiency of the cellular will also bring a mixed array of potential appli- Similarly, learning how to link a set of mol- “chasses” that will perform the desired func- cations. Synthetic biologists are not ignoring ecules into a generic type of regulatory circuit tions; therefore there are efforts to construct this possibility; discussions of and module, such as an ultrasensitive positive feed- cells that only have the minimal requirements security have been a major component of syn- back loop, could be useful for a diverse range for replication25. In addition, researchers are thetic biology conferences (http://synthetic of synthetic biology applications9,11,19,20. In engineering orthogonal components that only biology.org/SB2.0/Biosecurity_resolutions. the long term, synthetic biology is likely to interact with each other, and not existing cel- html). Initial attention has focused on prevent- have as broad and pervasive a role in our soci- lular molecules. These components, which ing commercial DNA synthesis companies from ety as the products of both the chemical and include novel -mRNA pairs26 and supplying pathogenic or otherwise dangerous electronic revolutions. -protein interactions27, should lead to sequences30. more reliable and predictable behavior when Goal-oriented biology used in cellular engineering. Ultimately, engi- Conclusions Focusing on specific goals and applications neering cells may require the ability to rewrite In the coming decades, we are likely to see a in the context of biological systems is a fea- entire . By successfully replacing revolution in biology akin to the revolution ture that clearly distinguishes synthetic biol- the genome of one bacterial cell with that of in chemistry that occurred in the latter half

524 VOLUME 3 NUMBER 9 SEPTEMBER 2007 NATURE CHEMICAL BIOLOGY COMMENTARY of the nineteenth century. The development and responsible way to foster productive 11. Endy, D. Nature 438, 449–453 (2005). 12. Voigt, C.A. Curr. Opin. Biotechnol. 17, 548–557 of increasingly sophisticated methods to alter growth of this exciting field. (2006). and build biological systems will provide 13. Arkin, A.P. & Fletcher, D.A. Genome Biol. 7, 114 essential synthetic tools that will synergize ACKNOWLEDGMENTS (2006). We thank A. Chau, M. Cohen, N. Helman, B. Rhau, 14. Suel, G.M., Garcia-Ojalvo, J., Liberman, L.M. & Elowitz, with analytical methods, which together will J. Taunton and A. Watters for their comments on M.B. Nature 440, 545–550 (2006). ultimately lead to a far deeper understand- this Commentary. This work was supported by the 15. Suel, G.M., Kulkarni, R.P., Dworkin, J., Garcia-Ojalvo, J. ing of the physical principles underlying US National Institutes of Health Roadmap, the US & Elowitz, M.B. Science 315, 1716–1719 (2007). 16. Alon, U. Nat. Rev. Genet. 8, 450–461 (2007). the behavior and design of cellular systems. National Science Foundation, the Packard Foundation and the Rogers Family Foundation. 17. Alon, U. Nature 446, 497 (2007). The applications of synthetic biology will be 18. Hoffmann, R. The Same and Not the Same (Columbia University Press, New York, 1995). highly varied, and progress and innovation COMPETING INTERESTS STATEMENT 19. Becskei, A., Seraphin, B. & Serrano, L. EMBO J. 20, is likely to come from unexpected areas. We The authors declare no competing financial interests. 2528–2535 (2001). might also expect that understanding the 20. Kaern, M., Blake, W.J. & Collins, J.J. Annu. Rev. Biomed. 1. Jaffe, B. Crucibles: The Story of Chemistry from Ancient engineering principles of biological systems Eng. 5, 179–206 (2003). to Nuclear Fission (Dover Publications, New 21. Pitera, D.J., Paddon, C.J., Newman, J.D. & Keasling, York, 1976). will have a transformative effect on other J.D. Metab. Eng. 9, 193–207 (2007). 2. Wöhler, F. Poggendorff’s Ann. Phys. 12, 253–256 22. Park, S.H., Zarrinpar, A. & Lim, W.A. Science 299, fields of science. For example, man-made (1828). 1061–1064 (2003). materials, even at the nanoscale, are currently 3. Asimov, I. A Short (Anchor Books, 23. Yeh, B.J., Rutigliano, R.J., Deb, A., Bar-Sagi, D. & Lim, Garden City, New York, USA, 1965). templated or built using directed assem- W.A. Nature 447, 596–600 (2007). 4. Khorana, H.G. Fed. Proc. 24, 1473–1487 (1965). bly—exactly the opposite of how biological 5. Gutte, B. & Merrifield, R.B. J. Biol. Chem. 246, 1922– 24. Rinaudo, K. et al. Nat. Biotechnol. 25, 795–801 molecules create structure and function. 1941 (1971). (2007). 6. Cello, J., Paul, A.V. & Wimmer, E. Science 297, 1016– 25. Forster, A.C. & Church, G.M. Mol. Syst. Biol. 2, 45 Biological molecules are self-assembling sys- 1018 (2002). (2006). tems that can adapt to change, show robust 7. Khosla, C. & Keasling, J.D. Nat. Rev. Discov. 2, 26. Rackham, O. & Chin, J.W. Biochem. Soc. Trans. 34, , and can self-repair. There may 1019–1025 (2003). 328–329 (2006). 8. Anderson, J.C., Clarke, E.J., Arkin, A.P. & Voigt, C.A. 27. Kortemme, T. & Baker, D. Curr. Opin. Chem. Biol. 8, come a day when man-made materials also J. Mol. Biol. 355, 619–627 (2006). 91–97 (2004). have these properties. Universities, industry, 9. Andrianantoandro, E., Basu, S., Karig, D.K. & Weiss, R. 28. Lartigue, C. et al. Science (in the press). Mol. Syst. Biol. 2, 2006.0028 (2006). 29. Haseltine, E.L. & Arnold, F.H. Annu. Rev. Biophys. governmental agencies and scientists will 10. Benner, S.A. & Sismour, A.M. Nat. Rev. Genet. 6, 533– Biomol. Struct. 36, 1–19 (2007). have to work together in an open-minded 543 (2005). 30. Bugl, H. et al. Nat. Biotechnol. 25, 627–629 (2007).

NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 9 SEPTEMBER 2007 525