Synthetic studies of roquefortine C: SPECIAL FEATURE Synthesis of isoroquefortine C and a heterocycle

David J. Richard, Bruno Schiavi, and Madeleine M. Joullie´ *

Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323

Edited by Kyriacos C. Nicolaou, The Scripps Research Institute, La Jolla, CA, and approved April 14, 2004 (received for review April 9, 2004)

The syntheses of isoroquefortine C and a related heterocycle were achieved by implementation of both intra- and intermolecular vinyl amidation reactions. These accomplishments represent a signifi- cant advance in the use of these strategies in the generation of complex molecules.

etabolites produced by fungi represent one of the largest Mclasses of natural products. Such compounds vary widely in their structural composition and have found application in pharmaceuticals such as antibiotics, immunosuppressants, anti- fungal agents, and growth promoters (1). Other congeners Fig. 1. The roquefortine class of natural products. within this class display biological properties harmful to humans and other animals. These natural products have been classified

as and are produced as secondary metabolites by an containing hemins but no potency toward Gram-negative or- CHEMISTRY array of soilborne and airborne fungi (2). Mycotoxins have been ganisms (15). Additional studies led to the finding that roque- isolated as contaminants of a variety of grain products and have fortine C inhibited bacterial RNA synthesis but only modestly been a topic of great interest for scientists concerned with affected DNA and protein production (16). veterinary health and agricultural safety. The lack of consistent toxicological data, along with the In most cases, eradication of the fungal infection responsible ubiquitous nature of P. roqueforti in human and animal food for production is the most reasonable method for products, establishes roquefortine C as a natural product worthy elimination of these compounds from commercial foodstuff. of synthetic investigation. The compound is also interesting from However, an interesting exception to this generalization is found a synthetic chemistry standpoint, because it possesses distinctive in the roquefortine family of natural products (Fig. 1). Roque- structural characteristics. Roquefortine C contains the unusual fortine C (1) was isolated independently by researchers in Japan E-dehydrohistidine moiety, a system that typically undergoes (3, 4) and France (5, 6) from the roqueforti Thom facile isomerization under acidic, basic, or photochemical con- strain. This finding was significant due to the fact that this ditions (17–21). This functional group is found in only two is essential for the production of Roquefort and a number of natural products, the other being oxaline (22). The total syn- other blue-veined cheeses. In subsequent communications, ro- theses of these compounds have yet to be accomplished. quefortines have been found as metabolites of additional P. Isoroquefortine C (3), or the 3,12 double-bond isomer of roqueforti strains as well as other Penicillium species isolated roquefortine C (Fig. 2), was obtained by photochemical irradi- from a variety of contaminated food products such as feed grain ation of the natural product (23). The biological activity of (7, 8), wine (9), and beer (10). The presumed biosynthetic isoroquefortine C has yet to be investigated. The goals of the precursor of roquefortine C (1), the dihydro compound roque- current synthetic efforts have been to develop a strategy appli- fortine D (2), has also been isolated from cultures of the P. cable to both roquefortine C and isoroquefortine C. A successful roqueforti fungal species (11). route to either of these compounds would allow for investigation Roquefortine C has received attention because of its neuro- of their interconversion and stability. toxic properties. Wagener and coworkers (12) described para- We previously reported the synthesis of roquefortine D (24) lytic activities in cockerels, and Frayssinet and Frayssinet found as well as the generation of isoroquefortine C using Wittig– ͞ the LD50 in mice to be 15–20 mg kg after i.p. injection (5). Horner olefination to establish the enamide stereochemistry Symptoms included prostration, seizures, and death. Support for (25). This latter method was not applicable to the synthesis of these findings was provided by Ohmomo (13), who used a similar roquefortine C. We now disclose recent efforts toward genera- ͞ mouse assay and reported an LD50 of 20 mg kg. However, tion of the dehydrohistidine system using copper-catalyzed vinyl Arnold et al. (14) were unable to reproduce this activity and amidation chemistry. This method has allowed for the synthesis found the lethal dose to be an order of magnitude larger than this of both isoroquefortine C and a polycyclic heterocycle (26). value. A nonalkaloid natural product also produced by P. Studies on the stability of isoroquefortine C and efforts to roqueforti, PR toxin, was found to possess greater toxicity. accomplish isomerization to the natural product are described A subsequent publication by Ha¨ggblom (8) further compli- also. cated the biological activity profile of roquefortine C. In this Due to the questionable stability of the dehydrohistidine finding, roquefortine C, but not PR toxin or other mycotoxins, moiety in the roquefortine system, palladium- or copper- was isolated from a grain sample that produced paralytic symp- toms in cows. These effects disappeared as soon as the cows were no longer fed moldy grain. Another report investigated the This paper was submitted directly (Track II) to the PNAS office. effects of this metabolite on bacterial growth and showed it to *To whom correspondence should be addressed. E-mail: [email protected]. possess inhibitory properties toward Gram-positive bacteria © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401407101 PNAS ͉ August 17, 2004 ͉ vol. 101 ͉ no. 33 ͉ 11971–11976 Downloaded by guest on September 25, 2021 6.6 Hz, 1H), 4.18 (dd, J ϭ 9.6, 6.8 Hz, 1H), 5.09 (d, J ϭ 17.3 Hz, 1H), 5.16 (d, J ϭ 10.7 Hz, 1H), 5.39 (s, 1H), 5.47 (broad s, 2H), 6.01 (dd, J ϭ 17.3, 10.7 Hz, 1H), 6.83 (broad s, 1H), 7.00–7.05 (m, 2H), 7.12–7.40 (m, 17H), 7.52 (s, 1H), 8.21 (s, 1H); 13C NMR ␦ (125 MHz, CDCl3) 23.0, 23.1, 39.1, 41.0, 59.1, 62.3, 76.2, 86.8, 114.7, 114.9, 123.5, 125.5, 125.6, 128.2, 128.3, 129.2, 129.7, 133.4, 134.3, 138.4, 141.9, 143.3, 151.8, 169.5, 172.9; IR (neat) 3,331 (w, broad), 3,170 (w, broad), 2,966 (w), 1,690 (s), 1,661 (m), 1,595 (w), 1,472 (m), 1,449 (m), 1,384 (m) cmϪ1; high-resolution MS ͞ ϩ ϩ (electrospray) m z calculated for C41H37N5O2Na (M Na) : Fig. 2. Photochemical isomerization of roquefortine C to isoroquefortine C. ͓␣͔20 ϭϩ 654.284495; found: 654.283253; D 21.5 (c 0.55, CHCl3).

Hexahydropyrroloindoleimidazolidinone (22). A solution of trityl- catalyzed vinyl amidation offered great promise (Fig. 3). The protected imidazole 21 (0.020 g, 0.032 mmol) and hydroxyben- transformation proceeds with stereospecificity and under rela- zotriazole (0.0129 g, 0.095 mmol) in trifluoroethanol (0.5 ml) was tively mild conditions (27, 28). A large number of catalyst stirred at room temperature for 48 h. The solution was diluted O systems have been reported for the analogous nitrogen carbon with water and extracted with ethyl acetate (10 ml). The organic bond-forming reactions involving amines or amides with aryl layer was dried over sodium sulfate and concentrated under halides or triflates. In contrast, the use of vinyl systems with reduced pressure. The crude product was purified by column amines (29, 30) or amides (31, 32) has only recently received chromatography (80% acetone͞hexanes) to yield 22 as a white ϭ ͞ attention. Therefore, investigation offered the potential for foam (0.0099 g, 80%). Rf 0.22 (10% methanol methylene 1 ␦ significant extension of this methodology. chloride); H NMR (500 MHz, CDCl3) 1.01 (s, 3H), 1.13 (s, 3H), 2.64 (s, 1H), 2.69 (dd, J ϭ 14.0, 9.6 Hz, 1H), 3.25 (dd, J ϭ Materials and Methods 14.0, 6.4 Hz, 1H), 4.32 (dd, J ϭ 9.6, 6.4 Hz, 1H), 5.11 (dd, J ϭ Experimental data for compounds 5–9, 11–14, 16, 17, 19, 20, and 17.6, 1.1 Hz, 1H), 5.19 (dd, J ϭ 11.0, 1.1 Hz, 1H), 5.47 (s., 1H), 23–26 are available in Supporting Materials and Methods, which 5.52 (broad s, 1H), 6.02 (dd, J ϭ 17.6, 11.0 Hz, 1H), 6.79 (broad is published as supporting information on the PNAS web site. s, 1H), 6.94 (d, J ϭ 7.7 Hz, 1H), 6.99 (s, 1H), 7.09 (dt, J ϭ 7.7, 1.0 Hz, 1H), 7.26 (m, 1H), 7.29 (m, 1H), 7.34 and 7.43 (m, 1H, Trityl-hexahydropyrroloindoleimidazolidinone (21). Vinyl bromide conformers), 7.69 and 7.94 (m, 1H, conformers); 13C NMR (125 ␦ 20 (0.175 g, 0.238 mmol), cuprous iodide (0.0045 g, 0.024 mmol), MHz, CDCl3) 23.0, 23.1, 39.2, 41.0, 58.9, 62.2, 87.6, 114.8, 115.0, and finely powdered potassium carbonate (0.066 g, 0.476 mmol) 116.8, 124.0, 125.8, 125.9, 129.4, 134.1, 134.4, 136.6, 137.2, 143.1, were added to a thick-walled pressure tube, and the vessel was 151.7, 170.4, 171.9; IR (neat) 3,203 (m, broad), 2,956 (m), 2,923 fitted with a rubber septum. The tube was evacuated and (m), 1,693 (s), 1,682 (s), 1,642 (s), 1,593 (m), 1,392 (s) cmϪ1; back-filled with argon three times. Dioxane (2 ml) and N, NЈ- high-resolution MS (electrospray) m͞z calculated for ϩ ϩ ͓␣͔20 ϭ dimethylethylenediamine (0.0052 ml, 0.048 mmol) were added, C22H24N5O2 (M H) : 390.193000; found: 390.193970; D Ϫ and the reaction mixture was flushed with argon and sealed by 57.8 (c 0.56, CHCl3). replacement of the septum with a Teflon screw cap. The reaction was heated to 100°C for 14 h and then cooled to room temper- (2S,3aR,8aS)-10b-(1,1-Dimethylallyl)-3(Z)-(1H-imidazol-4-ylmethylene)- -ature, diluted with ethyl acetate (10 ml), and filtered through a 2,3,6,10b,11,11a-hexahydro-5aH-pyrazino[1؅,2؅:1,5]pyrrolo[2,3-b]indole plug of silica. The silica plug was flushed with additional ethyl 1,4-dione (3) (Isoroquefortine C). Tosylated imidazole 26 (0.040 g, acetate (10 ml), and the filtrate then was concentrated under 0.074 mmol) was dissolved in tetrahydrofuran (2.5 ml) and 1 M reduced pressure. The crude product was purified by column aqueous sodium hydroxide (0.4 ml) was added. The reaction was chromatography (1–2% methanol͞methylene chloride) to yield stirred at room temperature for 12 h, concentrated under ϭ ͞ 21 as a white foam (0.111 g, 74%). Rf 0.48 (5% methanol reduced pressure, and diluted with water (5 ml). The solution 1 ␦ methylene chloride); H NMR (500 MHz, CDCl3) 0.90 (s, 3H), was acidified to pH 3 by the addition of 0.2 M aqueous hydrogen 1.10 (s, 3H), 2.59 (dd, J ϭ 15.0, 9.6 Hz, 1H), 31.4 (dd, J ϭ 14.0, chloride and then extracted with ethyl acetate (3 ϫ 10 ml). The combined organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (2–3% methanol͞methylene chloride) to yield isoroquefortine C (3)as a white foam (0.0157 g, 55%). All physical data were in agree- ϭ ment with that reported in the literature (23, 33). Rf 0.55 (10% ͞ 1 ␦ methanol methylene chloride); H NMR (500 MHz, CDCl3) 1.04 (s, 3H), 1.15 (s, 3H), 2.48 (dd, J ϭ 12.2, 11.5 Hz, 1H), 2.60 (dd, J ϭ 12.2, 5.7 Hz, 1H), 4.11 (dd, J ϭ 11.5, 5.7 Hz, 1H), 4.98 (broad s, 1H), 5.09 (d, J ϭ 17.4 Hz, 1H), 5.13 (d, J ϭ 10.8 Hz, 1H), 5.67 (s, 1H), 6.00 (dd, J ϭ 17.4, 10.8 Hz, 1H), 6.59 (d, J ϭ 7.5 Hz, 1H), 6.71 (s, 1H), 6.76 (t, J ϭ 7.5 Hz, 1H), 7.09 (s, 1H), 7.10 (t, J ϭ 7.5 Hz, 1H), 7.17 (m, 1H), 7.68 (s, 1H), 10.44 (broad 13 ␦ s, 1H), 11.72 (broad s, 1H); C NMR (125 MHz, CDCl3) 22.5, 23.0, 37.3, 40.9, 59.1, 61.6, 78.0, 105.4, 109.0, 114.5, 117.6, 118.8, 125.2, 126.3, 128.9, 135.4, 137.2, 143.5, 150.4, 158.4, 165.6; IR (neat) 3,210 (m, broad), 2,962 (m), 1,682 (s), 1,665 (s), 1,622 (s), Ϫ1 ͓␣͔20 ϭϪ 1,435 (s), 1,384 (m) cm ; D 329.0 (c 0.8, CHCl3). Results Fig. 3. Retrosynthetic analysis of roquefortine C involving intramolecular Implementation of the intramolecular vinyl amidation reaction vinyl amidation. required the synthesis of two fragments: a hexahydropyrroloin-

11972 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401407101 Richard et al. Downloaded by guest on September 25, 2021 SPECIAL FEATURE

Scheme 1. Synthesis of the hexahydropyrroloindole moiety by a selenation͞prenylation sequence. Reagents and conditions: a, fluorenylmethoxycarbonyl-Cl, Na2CO3, dioxane, H2O, 99%; b,(tert-butyloxycarbonyl)2O, dimethylallyl monophosphate, CH3CN, 83%; c, N-(phenylseleno)phthalimide, pyridinium p- toluenesulfonate, CH2Cl2, 72%; d, prenyl tributylstannane, methyl triflate, 2,6-di-tert-butyl-4-methylpyridine, CH2Cl2, 60%; e,NH3, MeOH, 89%.

dole moiety and a dehydrohistidine derivative (Fig. 3). The chromatography. Treatment of the crude product with 1,8- synthesis of the former compound can be found in Scheme 1. diazabicyclo[5.4.0]undec-7-ene, however, successfully promoted Sequential protection of the primary and secondary amines of hydrogen bromide elimination and provided a separable mixture L-tryptophan methyl ester (4) provided the fully protected amino of E and Z vinyl bromides in a 6:1 ratio of desired (14)to acid derivative (6). A two-step procedure developed by Dan- undesired (15) compound. The structures of these compounds ishefsky and coworkers (34, 35) was then used for conversion of were confirmed by x-ray crystallography. Saponification of the the resulting compound to the prenylated, tricyclic system. The methyl ester produced the unsaturated carboxylic acid (16).

first step of this sequence involves formation of a 3-selenenylated With the two desired components in hand, efforts then CHEMISTRY pyrroloindole (7a and 7b) by treatment with N-phenylselenoph- focused on amide bond formation and generation of the vinyl thalimide (36). This inseparable set of diastereomers then was amidation precursor (Scheme 3). Initial attempts led to produc- treated with methyl triflate and prenyl tributylstannane to tion of the desired amide (17) in only low yields, and extensive provide the alkylated compound (8). As desired, ammonolysis of investigation of alternative procedures resulted in no improve- the methyl ester resulted in simultaneous removal of the flu- ment. Despite this setback, sufficient material was available for orenylmethoxycarbonyl protecting group, producing the requi- investigation of the key diketopiperazine ring closure. A large site amino amide (9). number of palladium catalysts, phosphine ligands, bases, and Our attention then turned to formation of the vinyl halide solvents were screened for this transformation. Two methods dehydrohistidine fragment. Although a number of methods were seemed to produce the desired compound (18) successfully with investigated, elaboration of commercially available urocanic acid crude yields of 13% and 45%, as indicated by high-resolution MS (10) proved most efficient (Scheme 2). Methylation and trityl and NMR data. However, attempts to purify this product by a protection of the imidazole nitrogen provided the ester 12 in variety of methods led to decomposition. Removal of the good yields (37). A previously reported bromination͞elimination acid-labile protecting groups also proved unsuccessful. Modifi- sequence then was used to introduce the halogen substituent cation of the pyrroloindole fragment by use of the sterically less (38). Bromination of this enone in carbon tetrachloride resulted demanding methoxycarbonyl protecting group did not result in in formation of the enantiomeric dibromides (13a and 13b), any improvements in the amide bond-forming reaction. Because although the compounds were unstable to silica gel column of the difficulties obtaining the required starting material and

Scheme 2. Synthesis of the bromo-dehydrohistidine fragment. Reagents and conditions: a,H2SO4,Na2SO4, MeOH, 99%; b, trityl chloride, triethylamine, dimethylformamide, 91%; c,Br2, CCl4; d, diazabicycloundecane, CH2Cl2, 74%, two steps; e, LiOH, tetrahydrofuran, H2O, 94%.

Richard et al. PNAS ͉ August 17, 2004 ͉ vol. 101 ͉ no. 33 ͉ 11973 Downloaded by guest on September 25, 2021 Scheme 3. Diketopiperazine ring closure by palladium-catalyzed intramolecular vinyl amidation. Reagents and conditions: a, N,N-bis(2-oxo-3-oxazolidinyl)- phosphinic chloride, triethylamine, CH2Cl2, trace; b, Pd(OAc)2 (10 mol %), (ϩ͞Ϫ)-2,2Ј-bis(diphenylphosphino)-1,1Ј-binaphthyl (15 mol %), K2CO3, toluene, 13% or Pd2(dba)3 (12.5 mol %), P(o-tolyl)3 (37.5 mol %), K2CO3, toluene, 45% crude yields.

the apparent instability of the intermediate produced by this than the amide nitrogen. This finding was of interest in that it route, an alternative method was examined. represents only the second report of reaction of a substrate The problems associated with generation of the amidation containing both amide and amine functional groups under vinyl precursor were believed to be due to an unfavorable steric or aryl amidation conditions. The sole other example to date interaction with the indoline protecting group. Therefore, we involves preferential intermolecular reaction of the primary examined amide bond formation with a fully unprotected com- amide of 4-aminobenzamide (32). In the intramolecular reaction pound (Scheme 4). Removal of the tert-butoxycarbonyl moiety of 20, the preference for a six-membered over a seven-membered provided the diamine intermediate (19). Amide bond formation metallocycle intermediate may be used to rationalize the results. proceeded smoothly to yield the primary amide 20. Reaction of Removal of the triphenylmethyl protecting group by traditional this compound under the previously optimized palladium con- acid-catalyzed methods proved problematic; however, this trans- ditions led only to the recovery of starting material and decom- formation was accomplished in excellent yield by using triflu- position products. Use of a copper catalyst with the protocol oroethanol and hydroxybenzotriazole (39). This heterocycle (22) developed by Buchwald and coworkers (32), however, led to possesses an unknown tetracyclic ring system. formation of a cyclized product (21) in good yields. Examination With the reactivity of the unprotected amidation precursor of this product by 15N NMR (26) was extremely helpful in (20) (Scheme 4) clearly established, efforts were made to protect determining its structure to be the imidazolidinone shown, the aniline nitrogen after N,N-bis(2-oxo-3-oxazolidinyl)- resulting from preferential attack of the indoline nitrogen rather phosphinic chloride-mediated coupling. Unfortunately, none of

Scheme 4. Formation of an imidazolidinone by intramolecular copper-catalyzed vinyl amidation. Reagents and conditions: a, iodotrimethylsilane, CH3CN, 70%; b, N,N-bis(2-oxo-3-oxazolidinyl)-phosphinic chloride, triethylamine, CH2Cl2, 74%; c, CuI (10 mol %), N,NЈ-dimethylethylenediamine (20 mol %), K2CO3, dioxane, 74%; d, hydroxybenzotriazole, trifluoroethanol, 80%.

11974 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401407101 Richard et al. Downloaded by guest on September 25, 2021 SPECIAL FEATURE

Scheme 5. Formation of isoroquefortine C by intermolecular copper-catalyzed vinyl amidation. Reagents and conditions: a, CuI (20 mol %), N,NЈ- dimethylethylenediamine (40 mol %), K2CO3, toluene, 41%; b, iodotrimethylsilane, 2,6-lutidine, CH2Cl2,CH3CN, 68%; c, NaOH, tetrahydrofuran, H2O, 55%.

a wide variety of protecting groups and reaction conditions With an efficient method developed for the synthesis of proved successful. In all cases, recovery of starting material isoroquefortine C, examination of its potential isomerization (triethylsilyl, triisopropylsilyl, methyl carbamate), protection of to the natural product proceeded. A wide variety of conditions CHEMISTRY the amide nitrogen only [(2-(trimethylsilyl)ethoxymethyl, meth- (thermal, Lewis and protic acid, basic, photochemical) failed oxymethyl, tosyl], or decomposition (trifluoroacetyl, formyl) to produce the desired compound and without exception were observed. Use of strong bases (NaH) or increased temper- resulted in the recovery of starting material or the generation atures also invariably led to decomposition and recovery of of decomposition products. Although unsuccessful in produc- compounds lacking the reverse prenyl moiety. These results led ing roquefortine C, the results demonstrate the stability of the us to explore the intermolecular variant of the copper-catalyzed isoroquefortine C conformation toward a wide variety of amidation. reagents. The hexahydropyrroloindole (23) and tosyl-protected dehy- The syntheses of isoroquefortine C (3) and a related het- drohistidine compound (24) were prepared by methods analo- erocycle (22) were achieved by implementation of both intra- gous to those described above (Scheme 5). Closure of the and intermolecular vinyl amidation reactions. These accom- diketopiperazine ring proceeded under copper catalysis to pro- plishments represent a significant advance in the use of these vide enamide 25, although reaction required increased catalyst strategies in the generation of complex molecules. The stability loading and afforded a lower yield than the intramolecular of the dehydrohistidine moiety of isoroquefortine C to a variant. Removal of both indoline and imidazole protecting variety of conditions has been demonstrated. This result groups provided isoroquefortine C (3). Treatment of a sample of supports the hypothesis that the biosynthesis of the natural roquefortine C under these deprotection conditions failed to product roquefortine C (1) is achieved by dehydrogenation of yield any isoroquefortine C. We concluded that the vinyl ami- roquefortine D (2) and does not involve dehydrohistidine dation procedure led to isomerization of the enamide double isomerization (40). bond after coupling. As additional evidence, treatment of pri- mary amide 23 with the isomeric brominated dehydrohistidine This work was supported by National Science Foundation Grant CHE 15 (Scheme 2) also led to the formation of compound 25. 01-30958.

1. Jang, Z. & An, Z. (2000) in Bioactive Natural Products (Part C), ed. Atta-Ur- 15. Kopp, B. & Rehm, H. J. (1979) Eur. J. Appl. Microbiol. Biotechnol. 6, 397–401. Rahman (Elsevier, New York), Vol. 22, pp. 245–272. 16. Kopp, B. & Rehm, H. J. (1981) Eur. J. Appl. Microbiol. Biotechnol. 13, 232–235. 2. Bennett, J. W. & Klich, M. (2003) Clin. Microbiol. Rev. 16, 497–516. 17. Wild, J. (1982) Ph.D. thesis (University of Stuttgart, Stuttgart). 3. Ohmomo, S., Sato, T., Utagawa, T. & Abe, M. (1975) Agric. Biol. Chem. 39, 18. Poisel, H. & Schmidt, U. (1975) Chem. Ber. 108, 2547–2553. 1333–1334. 19. Schmidt, U., Griesser, H., Leitenberger, V., Lieberknecht, A., Mangold, R., 4. Ohmomo, S., Utagawa, S. & Abe, M. (1977) Agric. Biol. Chem. 41, 2097–2098. Meyer, R. & Riedl, H. (1992) Synthesis, 487–490. 5. Scott, P. M., Merrien, M.-A. & Polonsky, J. (1976) Experientia 32, 140–142. 20. Kim, D., Li, Y., Horenstein, B. A. & Nakanishi, K. (1990) Tetrahedron Lett. 49, 6. Scott, P. M. & Kennedy, B. P. C. (1976) J. Agric. Food Chem. 24, 868–868. 7119–7122. 7. Ohmomo, S. & Kitamoto, H. K. (1994) J. Sci. Food Agric. 64, 211–215. 21. Horenstein, B. A. & Nakanishi, K. (1989) J. Am. Chem. Soc. 111, 6242–6246. 8. Ha¨ggblom, P. (1990) Appl. Environ. Microbiol. 56, 2924–2926. 22. Nagel, D. W., Pachler, K. G. R., Steyn, P. S., Vleggaar, R. & Wessels, P. L. 9. Mo¨ller, T., Åkerstrand, K. & Massoud, T. (1997) Nat. Toxins 5, 86–89. (1976) Tetrahedron 32, 2625–2631. 10. Cole, R. J., Dorner, J. W., Cox, R. H. & Raymond, L. W. (1983) J. Agric. Food 23. Scott, P. M., Polonsky, J. & Merrien, M.-A. (1979) J. Agric. Food Chem. 27, Chem. 31, 657–659. 201–202. 11. Ohmomo, S., Oguma, K., Ohashi, T. & Abe, M. (1978) Agric. Biol. Chem. 42, 24. Chen, W.-C. & Joullie´, M. M. (1998) Tetrahedron Lett. 39, 8401–8404. 2387–2389. 25. Schiavi, B. M., Richard, D. J. & Joullie´, M. M. (2002) J. Org. Chem. 67, 620–624. 12. Wagener, R. E., Davis, N. D. & Diener, U. L. (1980) Appl. Environ. Microbiol. 26. Hadden, C. E., Richard, D. J., Joullie´, M. M. & Martin, G. E. (2003) J. 39, 882–887. Heterocycl. Chem. 40, 359–362. 13. Ohmomo, S. (1982) J. Antibact. Antifung. Agents 10, 253–264. 27. Wolfe, J. P., Wagaw, S., Marcouw, J.-F. & Buchwald, S. L. (1998) Acc. Chem. 14. Arnold, D. L., Scott, P. M., McGuire, P. F., Harwig, J. & Nera, E. A. (1978) Res. 31, 805–818. Food Cosmet. Toxicol. 16, 369–371. 28. Hartwig, J. F. (1998) Angew. Chem. Int. Ed. Engl. 37, 2046–2067.

Richard et al. PNAS ͉ August 17, 2004 ͉ vol. 101 ͉ no. 33 ͉ 11975 Downloaded by guest on September 25, 2021 29. Lebedev, A. Y., Izmer, V. V., Kazyul’kin, D. N., Beletskaya, I. P. & Voskoboy- 35. Depew, K. M., Marsden, S. P., Zatorska, D., Zatorski, A., Bornmann, W. G. nikov, A. Z. (2002) Org. Lett. 4, 623–626. & Danishefsky, S. J. (1999) J. Am. Chem. Soc. 121, 11953–11963. 30. Willis, M. C. & Brace, G. N. (2002) Tetrahedron Lett. 43, 9085–9088. 36. Nicolaou, K. C., Claremon, D. A., Barnette, W. E. & Seitz, S. P. (1979) J. Am. 31. Shen, R. & Porco, J. A., Jr. (2000) Org. Lett. 2, 1333–1336. Chem. Soc. 101, 3704–3706. 32. Jiang, L., Job, G. E., Klapars, A. & Buchwald, S. L. (2003) Org. Lett. 5, 3667– 37. Pirrung, M. C. & Pei, T. (2000) J. Org. Chem. 65, 2229–2230. 3669. 38. Cloninger, M. J. & Frey, P. A. (1998) Bioorg. Chem. 26, 323–333. 33. Vleggaar, R. & Wessels, P. L. (1980) J. Chem. Soc. Chem. Commun., 160–162. 39. Bodanszky, M., Bednarek, M. A. & Bodanszky, A. (1982) Int. J. Pept. Protein 34. Marsden, S. P., Depew, K. M. & Danishefsky, S. J. (1994) J. Am. Chem. Soc. Res. 20, 387–395. 116, 11143–11144. 40. Ohmomo, S., Ohashi, T. & Abe, M. (1979) Agric. Biol. Chem. 10, 2035–2038.

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