Adding Aliphatic CH Bond Oxidations to Synthesis M. Christina White Science 335, 807 (2012); DOI: 10.1126/science.

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PERSPECTIVES
them from increasing to high frequencies. Furthermore, some have already been associated with severe human diseases, supporting the less-is-less hypothesis. Other LoF variants, which can reach higher population frequencies, fall into poorly evolutionarily conserved genes or belong to multigene families displaying high paralogous sequence identity. This suggests that the functions of the corresponding genes are highly redundant, explaining their greater tolerance for LoF variants and supporting a less-is-nothing scenario. Also, although no substantial enrichment in positive selection signals was observed among LoF variants at the genomewide level, 20 of them fell into regions displaying signatures of positive selection, as predicted by the less-is-more hypothesis, suggesting that they may have conferred a selective advantage in human evolution. The key question is whether LoF variants and other variants located both in coding and noncoding regions of the genome have a true impact on human phenotypes. The study of the relationship between genetic variation and intermediate molecular phenotypes, such as expression quantitative trait loci, offers new functional insights into the etiology of complex human traits and diseases (8). Genetic variants located in regulatory regions across the genome control gene expression (9, 10), but their effects are not straightforward (8). Indeed, their impact on gene expression can differ between cell types and tissues (8, 11, 12). Even in the same cell type, genetic variants can modulate gene expression differently depending on the presence or absence of external stimuli, such as drugs or infectious agents (1315). MacArthur et al. provide a glimpse of this by studying how a fraction of LoF variants affect gene expression, and show that a minority are associated with reduced expression of the corresponding gene. The intricate relationships observed between genetic variation and gene expression emphasize the complex ways in which genotypic variation may underlie phenotypic consequences at the physiological or pathological levels. Whole-genome sequencing data from diverse human populations exposed to different environments and presenting different life-styles will provide a realistic picture of the extent of human genetic variation, including low-frequency and population-specific variants. Such information, combined with epigenomics, transcriptomics, and proteomics data from the same individuals and from different cell types, will help to determine the contribution of genetic variants (and the epistatic interactions among them) and environmental variables to variation in organismal traits (see the gure).
1. The 1000 Genomes Project Consortium, Nature 467, 1061 (2010). 2. C. Stewart et al., PLoS Genet. 7, e1002236 (2011). 3. P. H. Sudmant et al., Science 330, 641 (2010). 4. D. G. MacArthur et al., Science 335, 823 (2012). 5. M. V. Olson, Am. J. Hum. Genet. 64, 18 (1999). 6. P. C. Ng et al., PLoS Genet. 4, e1000160 (2008). 7. K. Pelak et al., PLoS Genet. 6, e1001111 (2010). 8. S. B. Montgomery, E. T. Dermitzakis, Nat. Rev. Genet. 12, 277 (2011). 9. S. B. Montgomery et al., Nature 464, 773 (2010). 10. J. K. Pickrell et al., Nature 464, 768 (2010). 11. A. S. Dimas et al., Science 325, 1246 (2009). 12. J. E. Powell et al., Genome Res.; 10.1101/gr.126540.111 (2011). 13. L. B. Barreiro et al., Proc. Natl. Acad. Sci. U.S.A. 109, 1204 (2012). 14. E. Choy et al., PLoS Genet. 4, e1000287 (2008). 15. J. C. Maranville et al., PLoS Genet. 7, e1002162 (2011). 10.1126/science.1219299

References

CHEMISTRY

M. Christina White Oxidations of aliphatic CH bonds, known since the 1800s, have only recently been considered for use in organic synthesis.

liphatic CH bonds are among the least reactive in organic chemistry, yet enzymes have evolved that not only oxidize them, but can discriminate between individual tertiary (3) and secondary (2) CH bonds in complex molecules. Chemists considered reactivity differences between these inert bonds too minor for a small-molecule catalyst to discriminate (1); bio-inspired catalysts with intricate binding pockets were thought to be the only viable solution (2). This view pervaded because the reported examples of selective aliphatic CH oxidations [halogenations (3), alkylations (4), aminations (5), hydroxylations (6), and dehydrogenations (7)] were not sufciently high in yield or predictable in site selectivity to be useful in synthesis, except in some special cases such as steroids (see below). In the past several years, however, aliphatic CH oxidations and simple rules for predicting their selectivities have been emerging prominently in synthetic planning. Powerful small-molecule catalysts have been invented that furnish high enough yields (>50%) to be preparatively useful and can predictably discriminate between aliphatic CH bonds even within complex molecules that have many possible sites of oxidation. In contrast to these newest methods, the majority of earlier intermolecular transformations were run with large excesses of subDepartment of Chemistry, Roger Adams Laboratory, University of Illinois, Urbana, IL 61801, USA. E-mail: white@scs. uiuc.edu

strate (generally solvent quantities), generated <1% oxidized products, and showed unexplained and/or inconsistent site selectivity trends (see the gure, panels A and B). For example, CH hydroxylations with iron porphyrin catalysts like 1 showed poor yields and minor, unexplained site selectivities for oxidizing the most distal sites from the electron-withdrawing group on substrates (6). Stoichiometric organic oxidants such as methyl(trifluoromethyl)dioxirane (TFDO) (8) oxidized 3 CH bonds in select substrates, such as steroids, in good yields (see the gure, panel F). However, the operational difculty of generating stoichiometric amounts of perfluorinated reagents and, in the case of TFDO, rapid decomposition under ambient conditions (temperatures above 20C and visible light) to reactive free radicals (to form CH3 and CF3)limited their reproducibility and use (9). In certain cases, higher reactivity and control of site selectivity were obtained for intramolecular reactions (see the figure, panel C). Substrate tethers that generated carbene or nitrene species upon reacting with oxidant and rhodium (Rh) catalysts led to powerful reactions in synthesis for CH alkylations (10, 11) and aminations (12). Substrate-directed metalations with palladium furnished reactions for acetoxylation and iodinations (13, 14). For carbene and nitrene insertion reactions, the rank order of reactivity between different CH bond types (for example, 2 versus 3 ali-

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Adding Aliphatic CH Bond Oxidations to Synthesis

PERSPECTIVES
A
33 equiv. Fe(TPP)Cl 1 (16 mol%) PhI(O) (1 equiv.) CO2Et <1% yield based on substrate OAc Isomer distributions (GC ratios)

OH

(17) (21) (24)

(<2) (15) (22)

Rh (OAc)4 n = 3 (1 2 mol%)

CO2Et 2 + CO2Et 3 2 > 3 = 11:1 OH

C
O

CO2Me N2

50 equiv.

N2 n=2 EtO2C 1 equiv.

MeO2C Rh2(OAc)4

CO2Me 2 25%

2 (6 mol%) 3

<1% GC yield based on substrate

+ C 3H 7 3 59% 3 > 2

D
PivO

Catalyst 2 or 3 AcOH, H2O2 PivO

E Electronics
HO Me H Me EWG 3 n m 2 O HH n

Sterics O BG H
8 1

Stereoelectronics t-Bu 3 O 59% O O

4 1 equiv. N N

NCCH3 C F O O Fe 6 5 S NCCH3 N N O N F 3C Fe(PDP) 2 3

25

O EWG MeO

26 Examples avg. 50% yields OH O OH OH OH

2 52% BG BG = H, 17% (3 major) Me, 41% H OH 3 H HO O 2 H HO 7 O O

F
C A AcO H H H D OH O O H 3C CF3 TFDO (3 equiv.) 66% yield

G CH

H
O H O O O

alkylation O HO HO HN + H 2N NH

CH amination 77% yield

O H 54% yield

O 62% yield at C7

Aliphatic CH bonds as a new functional group in synthesis. The emergence of reactions with preparative utility and rules for predictable selectivity in aliphatic CH bond oxidations is illustrated. Unless otherwise noted, one equivalent of substrate is used, and the CH functionalization site is shown with a yellow circle. (A and B) Intermolecular aliphatic CH bond reactivities and site selectivities for (A) hydroxylations [Ac = acetyl, Fe(TPP)Cl 1 = iron tetraphenylporphyrin chloride; selectivities using FePPIX-DME = ferriprotoporphyrin(IX) dimethylester] and (B) alkylations (Et = ethyl); yields are based on gas chromatography (GC). (C) Intramolecular alkylation with improved reactivity and different site selectivity (Me = methyl). (D) Newer examples of intermolecular aliphatic CH hydroxylations that achieve high yields, site selectivity, and stereoretention (Piv = pivaloyl). (E) Examples of how selectivity rules predict reaction sites in hydroxylations with Fe(PDP) (EWG = electron-withdrawing group; t-Bu = tert-butyl). (F) A 20th-century example of CH oxidation in a steroid substrate. (G) New catalysts enable selective intramolecular CH aminations [using Rh2(HNCOCF3)4.] and alkylations for synthesis of complex alkaloids such as tetrodotoxin (bonds formed shown in red). For example, in the alkylation, complex mixtures resulted with Rh2(OAc)4 catalyst, whereas only desired product was seen with Rh2(HNCOCPh3)4 (Ph = phenyl). (H) Intermolecular hydroxylations of complex terpenes made possible with Fe(PDP); the 3 case is artemisinin and the 2 case is dihydropleuromutilone (C-7 oxidation: 42% hydroxyl, 20% ketone).

phatic) was delineated for intramolecular reactions; however, factors governing selectivities among one bond type (for example, 2 aliphatic) were unclear. Moreover, these bond type selectivities were often inconsistent. For example, selectivities for intermolecular Rh-carbene insertion into the less sterically hindered 2 versus 3 CH bond conflict with those reported for an analogous intramolecular reaction (see the gure, panels B and C) (4, 11). The lack of systematic studies on selectivity effects suggested that examples of site selectivity were anomalous, rather than a general reactivity trend. Moreover, it was thought that attempts to boost yields in the remarkable cases of intermolecular aliphatic CH oxi-

dations would diminish their site selectivity. Preparatively useful intermolecular reactions are still lacking for many types of aliphatic CH oxidations. However, in 2007, it was shown that a nonheme, nitrogen-coordinated iron catalyst, Fe(PDP) 2, enabled preparative hydroxylations of aliphatic 3 CH bonds (15). One equivalent of substrate could be reacted with Fe(PDP), acetic acid (AcOH), and hydrogen peroxide (H2O2) under ambient conditions to furnish 50% isolated yields of mono-oxidized products (see the gure, panel D). Fe(PDP) was later shown to catalyze preparative oxidations of 2 aliphatic CH bonds, arguably the most difcult CH bonds to oxidize selectively because of their bond strength and ubiquity (16, 17). In 2009,

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(SbF6)2

5 51 to 61% yield

50% OH

a stable organocatalyst 3 was also reported that used AcOH and H2O2 for the preparative hydroxylation of 3 aliphatic CH bonds (18). Rules that reliably predict site selectivities of oxidation are crucial for such reactions to be adopted into late-stage synthetic planning, where CH oxidations are most strategically powerful (19). With preparative utility achieved under Fe(PDP) catalysis, systematic studies could now be performed on simple substrates designed to test individually the effects of electron-withdrawing groups, sterically bulky groups, and stereoelectronic activating groups on the site selectivity of oxidation among CH bonds of the same relative bond strengths [2 or 3 ( see the gure, panel E)]. Using the highly electrophilic, bulky oxidant generated with Fe(PDP) and H2O2, electron-withdrawing functionality on the substrate could effectively prevent oxidation even at remote sites (up to four carbons away), and steric bulk on the substrate could shield proximal sites from oxidation. Additionally, Fe(PDP) could be directed to oxidize sites that were activated through stereoelectronic effects, such as hyperconjugative activation and relief of 1,3-diaxial interactions and torsional strain. Perhaps the most important nding with Fe(PDP) was that the same selectivity rules that govern the differential reactivity of traditional functional groups (i.e., electronics, sterics, and stereoelectronics) also govern the reactivity of relatively inert CH bonds (1517). The generality of these predictable selectivity rules for other electrophilic, bulky oxidants has now begun to emerge (18, 20). Many of the earlier examples of selectivity can now be easily, retrospectively, rationalized with these rules (21). It was uncertain whether the reactivity and selectivity observed with simple substrates would translate over a range of complexmolecule settings. In addition to many more possible sites of CH oxidation for intermolecular reactions, the presence of dense functionality and topological complexity on a substrate can markedly retard reaction rates and erode selectivities. The majority of 20th-century examples of intra- and intermolecular aliphatic CH oxidations in complex-molecule settings were limited to steroids (see the gure, panel F). For example, with weaker organic oxidants such as TFDO, where competing 2 oxidation is not an issue, intermolecular reactions showed a striking preference for oxidation at the C-25 3 CH bond of the C- and D-ring side chain across a range of A and B rings (8). However, the privileged nature of the steroid core (highly rigid and sparsely functionalized) and conicting

PERSPECTIVES
rationales given for selectivities (sterics and electronics) made it difcult to see that such selectivity might be general. Newer methods have provided extraordinary examples of predictably selective aliphatic CH oxidations in a wide range of complex-molecule settings. In the synthesis of the alkaloid tetrodotoxin, selective intramolecular 3 aliphatic CH alkylation and amination reactions, made possible by newer Rh-acetamide catalysts, were used to install a section of the carbon skeleton and a key amine functionality (see the figure, panel G) (10, 12). The advent of Fe(PDP) catalysis enabled intermolecular aliphatic CH oxidations to occur at both 3 and 2 sites in a wide range of complex terpenes (see the gure, panel H). Despite dense functionality, one equivalent of substrate could be used to afford preparatively useful amounts of monooxidized products (1517).
EVOLUTION

In essence, CH bonds have emerged as a new functional group for organic synthesis: They can now be specically targeted for reactivity in a synthetic sequence. Many challenges remain, however, including the development of chemoselective catalysts that can oxidize aliphatic CH bonds in the presence of more electron-rich functional groups (such as olens, aromatic rings, and nitrogen atoms); preparative catalysts for intermolecular aminations, alkylations, and halogenations; and catalysts that can override biases induced by the substrate itself.
1. R. G. Bergman, Nature 446, 391 (2007). 2. K. Godula, D. Sames, Science 312, 67 (2006). 3. G. A. Russell, H. C. Brown, J. Am. Chem. Soc. 77, 4578 (1955). 4. A. Demonceau, A. F. Noels, A. J. Hubert, P. Teyssie, Bull. Soc. Chim. Belg. 93, 945 (1984). 5. R. Breslow, S. H. Gellman, J. Chem. Soc. Chem. Commun. 1982, 1400 (1982). 6. J. T. Groves et al., J. Am. Chem. Soc. 101, 1032 (1979).

References

10.1126/science.1207661

Contemplating the First Plantae

Frederick W. Spiegel

What characterized the rst photosynthetic eukaryotes?

he cells of photosynthetic eukaryotes such as plants and algae contain organelles, called plastids or chloroplasts, responsible for photosynthesis. Plastids contain some DNA, a vestige of their origin from free-living cyanobacteria. On page 843 of this issue, Price et al. (1) use comparative genomics with the obscure algal protist, Cyanophora paradoxa, to propose a resolution to the question of how plastids have spread through the eukaryotes. By understanding the implications of their results, we may be able to picture the overall characteristics of the very rst alga. In recent decades, it has become clear that photosynthetic eukaryotes rose through endosymbiosis (2, 3). In this process, a eukaryotic cell becomes inhabited by a photosynthetic cell. A complex set of events ensues, including transfer of genes from the colonizers genome to the hosts nuclear genome. This is followed by the evolution of mechanisms that allow proteins encoded by these transferred genes back into the colonizer so that it can still photosynthesize. The result is a chimeric organism in which the colonizer has become the plastid. If the colonizer was a cyanobacteDepartment of Biological Sciences, SCEN 601, 1 University of Arkansas, Fayetteville, AR 72701, USA. E-mail: fspiegel@uark.edu

rium, the chimera is said to be the product of primary endosymbiosis. If the colonizer was a photosynthetic eukaryote, the chimera is the product of secondary or higher-order endosymbiosis (2, 3). Because of their complex genetic histories, the relationships among chimeric organisms can be difcult to interpret (3). It can also be challenging to infer relationships between photosynthetic eukaryotes and their closest relatives that never had plastids. Phylogenetic data suggest that all plastids in todays photosynthetic eukaryotes can be traced back to just two different cyanobacterial colonizers (3). One of these colonizers led to the primary plastids in the photosynthetic amoeba Paulinella (3), but this is not the focus of Price et al., who investigate primary endosymbiotic events thought to have occurred more than a billion years ago. Todays Glaucophyta (~13 species, including C. paradoxa), red algae (~6000 species), and green algae and land plants (over 300,000 species) all have primary plastids descended from one cyanobacterial ancestor (1, 2). All other eukaryotic algae have secondary or higher-order plastids derived from either red or green algae (13). The data have been equivocal as to how many times that cyanobacterium colonized eukaryotic hosts. According to the Plantae hypothesis, it entered only one protist; the resulting chi-

mera became the sole ancestor to the eukaryotic supergroup Plantae (or Archaeplastida), which contains the glaucophytes, red algae, and land plants and green algae. Alternative non-Plantae hypotheses suggest that the cyanobacterium that was ancestral to the plastids of glaucophytes, red algae, and land plants and green algae must have colonized different protists more than once (4-7). Acceptance of the Plantae hypothesis requires that Plantae be monophyletic and that it cannot include any secondarily photosynthetic algae or any primitively plastid-free eukaryotes. In other words, a well-supported phylogeny must show that only the glaucophytes, red algae, and land plants and green algae evolved from one chimeric ancestor. To test this hypothesis, Price et al. compare genomes from the most complete set of photosynthetic eukaryotes to date. They provide a number of independent lines of evidence, including data about genes that are independent of the plastids. All their data strongly support the Plantae hypothesis. The glaucophytes are the most speciespoor Plantae. Many phylogenies show them branching most basally in Plantae, that is, diverging before the separation of the reds and the greens (2). In such cases, we often have a careless tendency to think of the species-poor branch as primitive in all

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