Isolation and Characterization of Two New Fusaric Acid Analogs from Fusarllun Lnonilijonne NRRL 13,163

Isolation and Characterization of Two New Fusaric Acid Analogs from FusarlLun lnonilijonne NRRL 13,163

H. R. BURMEISTER." M. D. GROYE.t R. E. PETERSON. D. WEIS LEDER. AND R. D. PLATTNER Northern Regional Research Center, Agricultural Research Service. U.S. Department (~rAgricultllre, Peoria. Illinois 61604

Received 28 January 19851Accepted 14 May 1985

Fusarium monilijorme NRRL 13,163 produced two new fusaric acid analogs, a 10,11-dihydroxyfusaric acid and a diacid of fusaric acid in which the C-ll methyl was oxidized to a carboxyl. Several hundred milligrams of the 10,11-dihydroxyfusaric acid were routinely recovered from a kilogram of corn grit medium. It crystallized as white, irregularly shaped rectangles that melted at 153 to 154°C. The diacid analog of fusaric acid crystallized as white rods that melted at 210 to 211°C. Unlike the consistent recovery experienced with the 1O,11-dihydroxyfusaric acid, the diacid analog proved difficult to purify after the initial discovery and was detectable in subsequent fermentations only by mass spectrometry.

Fusarium monilijorme Sheldon, a field fungus common on 3 days at ambient temperature followed by incubation for 18 many crops. including corn, small grains. millet. sorghum. days at 15°C. After incubation. 250 ml of methanol was and citrus fruits (1, 9), produces a number of interesting, added to each flask before the culture medium was macer biologically active secondary metabolites. The more studied ated in a Waring blender jar. The extract was recovered by products of this species are the plant growth hormone filtration. and the aqueous methanol was removed on a gibberellin and its analogs (8), along with certain rotary evaporator. The residual gummy material was dis mycotoxins, moniliformin (3). fusarin C (20), fusariocin m. solved in water. and any water-insoluble solids were dis and fusaric acid (17). Fusaric acid is not only moderately carded. After reconcentration, the gummy material from 10 toxic to animals (intraperitoneal 50% lethal dose. of 100 flasks was combined and dissolved in 500 ml of methanol. to mglkg ofbody weight in mice) (18), but also has antibiotic (4. which 300 ml of acetone was then added. The precipitate 15), pharmacological (6, 11, 12), insecticidal (2), and formed on the addition of the acetone was discarded and the phytotoxic properties (4. 15). solution was then dried. The acetone-methanol solubles During a survey for antibiotics and mycotoxins produced were redissolved in 50 to 75 ml of water and were applied to by fusaria. several crystalline products appeared in fractions a column (4.5 by 20 cm) of Silica Gel 60 silianized with of the culture extract of F. monilijorme NRRL 13,163 dimethylsilane (RP-2. 70-230 mesh: EM Science). The col separated by high-pressure liquid chromatography (HPLC). umn was eluted with water until the eluate no longer Two of the products had infrared (lR) spectra similar to the quenched the fluorescence in thin-layer chromatographic spectrum of fusaric acid. Mass spectrometric and nuclear (TLC) plates containing indicator. After removal of water magnetic resonance (NMR) spectra of the two products from the column eluate. about 20 to 30 g of solids was established their structures as 10.11-dihydroxyfusaric acid obtained from 0.5 kg of culture medium. [5-(3' ,4'-dihydroxybutyl)-2-pyridinecarboxylic acid: DHFA] HPLC. Preparative HPLC separations of the fusaric acid and a fusaric acid analog differing from fusaric acid by the analogs were carried out on a Prep LC/System 500 (Waters oxidation of the C-ll methyl to a carboxyl [5-(3'-carboxy Associates, Milford, Mass.) fitted with a Prep PAK-500/C I8 propyl)-2-pyridinecarboxylic acid: DAOFA]. cartridge. Samples were eluted with water-acetonitrile (90:10) at 100 mllmin, and the eluant was monitored with a MATERIALS AND METHODS refractive index detector at a sensitivity of 20. Approxi Microorganism. F. monilijorme NRRL 13.163 is main mately 50 g (the residue from 1 kg of WCG medium) of solids tained by the Agricultural Research Service Culture from the silanized silica gel column was divided into two Collection (NRRL). Peoria. III. For this study. the strain was equal portions. Each fraction was dissolved in 100 ml water cultured on Bacto YM Agar (Difco Laboratories) slants and for preparative HPLC fractionation. After elution, DHFA transferred weekly. and DAOFA were found primarily in the fractions collected Production and concentration of fusaric acid analogs. A between 1,500 and 1,900 ml and 2,350 and 2,850 ml. respec stock culture ofF. monilijorme NRRL 13,163 was incubated tively. Fractions were evaporated to dryness and dissolved at ambient temperature until sporulation (ca. 3 days) and in warm methanol. On evaporation, crystals contaminated then transferred to a refrigerator. Subcultures were made with yellow, water-soluble impurities were obtained. weekly. The conidia-bearing surface of a 7-day-old slant was Thin-layer chromatography. Crystalline fusaric acid. scraped into 10 ml of water, and about 0.5 ml of the conidial DHFA. and the diacid analog of fusaric acid. DAOFA. used suspension was added to each 50 g ofwhite corn grits (WCG) as TLC standards were either purchased. as was fusaric acid in 300-ml Erlenmeyer flasks. Before autoclaving 15 ml of (Aldrich Chemical Co.), or were purified by HPLC and water was added to the WCG. and 10 ml of water was added recrystallization until they gave a single spot on TLC plates after autoclaving. The inoculated medium was incubated for coated with 0.25-mm-thick Silica Gel 60 F-254 with fluores cent indicator (E. M. Merck). " Corresponding author. TLC plates were developed either in isopropyl alcohol t Deceased: 24 April 1984. ethyl acetate-water-acetic acid (4.0:3.8:2.0:0.2) (solution A)

311 312 BURMEISTER ET AL. Appl. ENVIRON. MICROBlOl. or in isopropyl alcohol-butanol-water-ammonium hydroxide TABLE 2. DC NMR data for fusaric acid and analogs" (6.0:2.0:1.5:0.5) (solution B). The compounds were identi Chemical shifts (ppml of the following compound": fied either by quenching of the indicator when observed Carbon assignment under shortwave UV light (254 nm) or by spraying with 50% 2 sulfuric acid followed by charring at 130°e. 2 146.6s 146.6s 147.8s Melting point determinations. Melting points (uncorrected) 3 12S.Sd 129.0d 12S.Sd were determined with a Fisher-Johns melting block appara 4 143.4d 143.1d 143.1d tus. 5 146.4s 146.3s 146.15 Crystallization. Fractions obtained by HPLC containing 6 149.6d 150.1d 149.7d DHFA or DAOFA crystallized from methanol solutions on 7 166.Ss 166.35 166.6s evaporation. The crystals were washed with acetone and S 30.9t 35.7t 34.4t recrystallized by dissolving crude crystals in hot methanol 9 35.5t 27.5t 34.3t 10 73.4d 33.St 24.1t until saturation was nearly reached. To the nearly saturated 11 67.9t lS1.0s 15.7q solution. an equal volume of acetone was added. and the solution was placed in a freezer. " Chemical shifts (0) are expressed in parts per million from internal sodium DHFA methyl ester. DHFA (10 mg) dissolved in 1 ml of 3-trimethylsilylpropionate-2.2.3.3.d". Spectra were recorded in 0,0 on a Bruker WM-300 spectrometer. MUltiplicities were determined by DEPT methanolic boron trifluoride (14%) was heated at 6Q°C for 1 (distortionless enhancement by polarization transfer) experiments. In some h. The reaction mixture was concentrated at reduced pres instances assignments were verified by specific proton decoupling. sure. taken up in dichloromethane-methanol (95:5), neutral b See Fig. 1 for structures. Abbreviations: s. singlet: d. doublet: t. triplet: q. quartet. ized with NaHC03 , and dried (NalS04). The organic solu tion was concentrated and chromatographed on a column of silica gel (5 g). Elution with dichloromethane-methanol killed mice within 24 h after intraperitoneal injection. Al (95:5) gave DHFA methyl ester: IR (KRS-5 plate) 1,725 though attempts at the separation of a product that could I cm- , MS 1Il1::. 225.1009 (M"), CllH IS N04 requires 225.1001, explain the toxicity of this strain remain unsuccessful, Infrared spectra. IR spectra were recorded with a Perkin NRRL 13,163 grown on a WCG medium yielded a number of Elmer 1320 spectrophotometer from KBr pellets. crystalline products when fractionated by HPLC. These UV spectra. UV spectra were recorded in ethanol with a included DHFA. DAOFA, 10-hydroxyfusaric acid, choline Unicam SP800 spectrophotometer. sulfate, and several unidentified products that were neither Mass spectrometry. Low resolution mass spectra (MS) antibiotic nor toxic. Because fusaric acid itself is a weak were obtained by chemical ionization with isobutane as the toxin (intraperitoneal 50% lethal dose, 100 mglkg of body reagent gas in a Finnigan 45351TSQ. High resolution electron weight in mice [18]) and has other interesting biological impact MS were determined at 70 eV with a Nuclide 12-90 properties. the fusaric acid analogs were studied and char DF instrument. Samples were introduced with a direct acterized. insertion probe. Crystalline DHFA was recovered from WCG medium at Nuclear magnetic resonance. Proton (IH) and carbon-13 the rate of 2 to 300 mg per kg. Separation of DAOFA, (BC) NMR spectra were recorded at 300 and 75 MHz, however. was another matter. After the initial recovery of 88 respectively, in deuterium oxide on a Bruker WM-300 mg from 1 kg of WCG medium we were unable to separate instrument with sodium 3-trimethylsilylpropionate any additional crystallizable quantities of product by con 2,2,3.3,d4 as the internal standard. ventional chromatography or by HPLe. even though small amounts of DAOFA were still produced by the fungus as RESULTS AND DISCUSSION indicated by TLC and confirmed by mass spectrometric analyses. Since production and purification procedures were F. lIlonilZ!(JI'Ine NRRL 13.163 was isolated from corn and not altered after discovery of the two new fusaric acid deposited in the Agricultural Research Service Culture Col analogs, it was assumed that the fungus no longer produced lection in 1971. This strain was reported to be toxic, and sufficient amounts of DAOFA required for purification or it methanol extracts. obtained by culturing the fungus on corn. was producing substances that interfered with purification. Upon placing a warm, nearly saturated methanolic solu tion of DHFA, crystallized and rechromatographed by TABLE 1. 'H NMR chemical shifts and coupling constants for HPLe. in a freezer at 18°e. white. irregularly shaped. fusaric acid and its analogs" rectangular crystals formed that melted at 153 to 154°e. The

b crystals were soluble in water. methanol. and ethanol. but Chemical shifts (ppm) of the following compound : Proton they were essentially insoluble in nonpolar solvents. Crys assignment tals of DAOFA formed when a nearly saturated warm 3 8.31d (8.01 S.32d (8.1) 8.26d (8.2) methanolic solution was cooled. The crystals were white and 4 8.54dd (8.0. 1.51 S.54dd (S.1. 1.51 S.47dd (S.2. l.S) rod-shaped, and they melted at 210 to 211°C. Like DHFA, 6 8.65bs S.63bs S.57bs DAOFA was soluble in water and alcohols. but it was not 8 3.0301 2.96t (7.51 2.S7t 0.61 readily soluble in nonpolar solvents. On TLC plates, DHFA 9 1.8801 2.0201 1.6601 migrated to an Rrof 0.22 in developing solution A and an RJ 10 3.7301 2.45t 0.31 1.3201 of 0.24 in developing solution B. In solution A, DAOFA 11 3.5701 (7.31 0.89t migrated faster than DHFA, reaching an Rrof O.4L but in " Chemical shifts (01 are expressed in parts per million from internal sodium solution B it migrated slower than DHFA, r'eaching an Rrof 3-trimethylsilylpropionate-2.2.3.3.d". and coupling constants (J: in parenthe 0.10. . ses) are expressed in Hertz. Extensive decoupling was lIsed to verify' Like the UV spectrum of fusaric acid (10), the UV assignments. Spectra were recorded in 0,0 on a Bruker W"I-300 spectrom (E eter. spectrum of DHFA showed maxima (in ethanol) at 228 nm b See Fig. 1 for structures. Abbreviations: d. doublet: dd. double doublet: = 9.420) and 268 nm (E = 5,124). and DAOFA had maxima bs. broad singlet: m. multiplet: t. triplet. at 223 nm (E = 7.756) and 268 nm (E = 5,089). A comparison VOL. 50. 1985 NEW FUSARIC ACID ANALOGS FROM F. MONILIFORl'vlE 313

methylene carbon bearing oxygen at 0 67.9, a methine 4 carbon bearing oxygen at 0 73.4, five aromatic carbons at 0 128.8 to 0146.9, and a carbonyl carbon at 0 166.8. The NMR 3 data reduced the structural possibilities for the compound to either 10.11-dihydroxyfusaric acid or 9,1l-dihydroxyfusaric 7 2 acid. However, the 9,11-dihydroxyfusaric acid possibility HOOC was eliminated from consideration because the 0 3.58 signal was due to the AB portion of an ABC system and not of an ABC2 system. That the primary alcohol methylene is the 0 1 3.57 signal and not the more complex pattern at 03.03 was established by selective heteronuclear decoupling experi ments. Irradiation at 0 3.57 resulted in collapse of the triplet at 0 67.9 in the l3C spectrum to a singlet. Likewise, irradia tion of the methylene proton at 0 3.03 resulted in collapse of the high-field methylene carbon triplet at 0 30.8 to a singlet. These data establish the structure of DHFA (Fig. 1, struc ture 1). The IR of DAOFA showed, in addition to the absorptions HOOC exhibited by DHFA, a band at 1,710 cm-1 (carbonyl). That it is a fusaric acid derivative was indicated by UV absorption at 268 nm and the even mass MH+ ion at II1/Z 210 by chemical 2 ionization-MS. The IH NMR spectrum exhibited signals at 0 2.02, 0 2.45, and 0 2.96 as a multiplet and two triplets, respectively, for a -CH2-CH2-CH2 - grouping, plus three aromatic protons at 0 8.32, 0 8.54, and 08.63. The l3C NMR spectrum consisted of signals for three methylene carbons at 027.5,033.8, and 0 35.7, five aromatic carbons at 0 129 to 150 and two carbonyl carbons at 0 166.3 and 0 181,0. These data establish the compound as DAOFA (Fig. 1, structure 2). HOOC Fusaric acid (Fig. 1, structure 3), a compound with diverse biological activities, affects animals, insects, plants, and microbial cells. It was first isolated by Yabuta et al. in 1934 3 (21) and was subsequently implicated in wilt diseases of plants (4. 15). In plants, the presence of fusaric acid impairs FIG. 1. Structures of 10.11-dihydroxyfusaric acid (trivial name). water permeability and exaggerates transpiration, resulting 5-(3'.4'.-dihydroxybutyl)-2-pyridinecarboxylic acid (International in loss ofturgor and ionic imbalance (15). Whether these new Union of Pure and Applied Chemistry. nomenclature) (structure 1): fusaric acid analogs contribute to phytotoxicity is not diacid analog of fusaric acid (trivial name). 5-(3'-carboxypropyl)-2 known. but results of preliminary tests in our laboratory pyridinecarboxylic acid (International Union of Pure and Applied indicate that they have no effect on the germination or Chemistry nomenclature) (structure 2): and fusaric acid (trivial development of grass, cucumber, or velvet leaf seeds at 50 name). 5-(butyl)-2-pyridinecarboxylic acid (International Union of f-Lg/ml in water. Pure and Applied Chemistry nomenclature) (structure 3). Hidaka et al. (6) have found fusaric acid to inhibit dopamine-f3-hydroxylase, one of the enzymes involved in of the NMR data (Tables 1 and 2) of fusaric acid with the the biosynthesis of norepinephrine, and they showed that it fusaric acid analogs support the described chemical charac had hypotensive effects in mice, rabbits, and dogs. These terizations of the analogs. and other biological activities have created an interest in the The IR spectrum of DHFA exhibited absorptions at 3.310 pharmacological properties of fusaric acid and synthetic cm-1 (hydroxyl). 1.640 cm- 1 (carboxylate ion), and 1.580 derivatives made from it (11, 12, 19). Before this report, and 1,525 cm-1 (aromatic ring). The abnormal band appear DAOFA was identified from MS analyses by Miyazaki et al. ing at 1,640 cm-1 instead of 1,705 cm-1 likely is due to the (13) as a catabolic product in the urine of rats dosed with formation ofa zwitter ion with KBr in the presence of a small 5-(4'-chloro-n-butyl) picolinic acid. They were able to sepa amount of moisture, as explained previously by Taylor (16). rate the urine products on an amberlite column and estab The MH+ ion at II1/Z 212 in chemical ionization MS indicated lished the structure of DAOFA by the presence of a molec a compound with an odd number of nitrogen atoms and is ular ion of the dimethyl ester at m/e 237 and by comparison consistent with a derivative of fusaric acid. This assignment of a synthetic product with the one recovered from urine. was supported by the UV spectrum which exhibited a The facts that DAOFA occurred in urine as a catabolite of characteristic absorption at 268 nm (2, 5, 14). High chlorinated fusaric acid and that it also appeared in culture resolution MS of the methyl ester showed a molecular ion at media that supported the growth of F. lI1onilifomze suggest 1I1/z 225.1009 with an empirical formula of CllH 1S N04 (con that fusaric acid undergoes biological oxidation with sistent with a dihydroxyfusaric acid derivative). The IH DAOFA as the likely end product. Pitel and Vining (14) were NMR spectrum showed two aliphatic methylene protons at able to demonstrate that dehydrofusaric acid is interconvert chemical shifts (0) 1,88: two benzylic protons at 0 3.03: a ible to either fusaric acid or 10-hydroxyfusaric acid, suggest methylene group attached to oxygen at 0 3.57: a methine ing a biological oxidation of fusaric acid through proton adjacent to oxygen at 0 3.73: and three aromatic dehydrofusaric acid, 10-hydroxyfusaric acid, DHFA, and protons at 0 8.31, 08.54, and 0 8.65. The l3C NMR spectrum DAOFA. exhibited two methylene carbons at 0 30.9 and 0 35.5, a Because fusaric acid and numerous derivatives of fusaric 314 BURMEISTER ET AL. AppL. ENVIRON. MICROBIOL. acid are being extensively studied for their efficacy as 10. Kuo, M. S., and R. P. Scheffer. 1964. Evaluation of fusaric acid blockers of dopamine 13-hydroxylase, these newly character as a factor in development of fusarium wilt. Phytopathology ized fusaric acid analogs, DHFA and DAOFA, could be 64:1041-1044. useful in evaluating the catabolism of fusaric acid in treated 11. Miyano, K., T. Koshigoe, T. Sakasai, and H. Hamano. 1978. Synthesis of 5-(substituted alkyl) picolinic acids. the dopamine animals. l3-hydroxylase inhibitors I. Chern. Pharm. Bull. 26:1465-1472. 12. Miyano, K., T. Sakasai, and H. Hamano. 1978. Synthesis of ACKNOWLEDGMENTS 5-(substituted alkyl) picolinic acids. The dopamine 13 We thank Scott Taylor for technical assistance and William hydroxylase inhibitor II. Chern. Pharm. Bull. 26:2328-2333. Rohwedder for high resolution mass spectrometry. and H.R.B. 13. Miyazaki, H., H. Takayama, Y. Minatogawa, and K. Miyano. especially thanks Ronald Vesonder for advice regarding chemical 1976. A novel metabolic pathway in the metabolism of 5 interpretations that arose after the death of Dr. G;ove. - (4'chloro-n-butyi) picolinic acid. Biochem. Mass Spectrosc. 3:140-145. 14. Pitel, D. W., and L. C. Vining. 1970. Accumulation of LITERATURE CITED dehydrofusaric acid and its conversion to fusaric and 10 1. Christensen, C. M., and H. H. Kaufmann. 1969. Grain storage: hydroxyfusaric acid in cultures of Giberella jujikuroi. Can. J. the role offungi in quality loss, p. 159. University of Minnes~ta Biochem. 48:623-630. Press. Minneapolis. Minn. 15. Sadasivan, T. S. 1961. Physiology of wilt diseases. Annu. Rev. 2. Claydon, N., J. F. Grove, and M. Pople. 1977. Insecticidal Plant Physiol. 12:449-468. secondary metabolic products from the entomogenous fungus. 16. Taylor, L. D. 1962. The infrared spectrum of nicotinic acid. J. Fusarium solani. J. Invertebrate Pathol. 30:216-223. Org. Chern. 27:4064-4065. 3. Cole, R. J., J. W. Kirksey, H. G. Cutter, B. L. Doupnik, and 17. Tirunarayan, M. 0., and 1\1. Sirsi. 1961. The elaboration of J. C. Peckham. 1973. Toxin from Fusariummonilijorme: effects anti-fungal substances by Fusarium. Ind. J. Med. Res. 49: on plants and animals. Science 179:1324-1326. 819-827. 4. Gaumann, E. 1957. Fusaric acid as a wilt toxin. Phytopathology 18. Ueno, Y., and I. Ueno. 1978. Toxicology and biochemistry of 47:342-357. mycotoxins. p. 107-188. In K. Uraguchi and M. Yamazaki (ed.). 5. Grove, J. F., P. W. Jeffs, and T. P. C. Mulholland. 1958. Toxicology. biochemistry and pathology of mycotoxins. John Gibberellic acid. Part V. The relation between gibberellin A, Wiley & Sons, Inc .. New York. and gibberellic acid. J. Chern. Soc. 1958:1236-1240. 19. Umezawa, H., T. Takeuchi, K. Miyano, T. Koshigo, and H. 6. Hidaka, H., T. Nagatsu, K. Takeya, T. Takeuchi, H. Suda, K. Hamano. 1973. Fusaric acid derivatives: the effect on dopamine Kojiri, M. Matsuzaki, and H. Umezawa. 1969. Fusaric acid. a l3-hydroxylase. J. Antibiot. 26:189. hypotensive agent produced by fungi. J. Antibiot. 22:228-230. 20. Wiebe, L. A., and L. F. Bjeldames. 1981. Fusarin C. a mutagen 7. Ito, T. 1979. Fusariocin C. a new cytotoxic substance produced produced by Fusarium monilijorme. grown on corn. Agric. by Fusarium monilijorme. Agric. BioI. Chern. 43:1237-1242. BioI. Chern. 43:1237-1242. 8. Jefferys, E. G. 1970. The gibberellin fermentation. Adv. App!. 21. Yabuta, T., K. Kambe. and T. Havaski. 1934. Biochemistrv of Microbiol. 13:283-316. the bakanae-fungus. r: Fusarinic 'acid, a new product or" the 9. Joffee, A. Z. 1984. Contamination of Arizona corn. Science bakanae-fungus. J. Agric. Chern. Soc. Japan 10:1059-1068. 224:340. (Chern. Abstr. 29:1132. 1935.)