4-Oxo fatty acids Hygrophorus Natural Product Communications Vol.1(12)2006 1081 protons and carbons counted from the NMR spectra from a three spin system at 6 4.95,4.99,and 5.83. H NMR spectrum of 3 did not almost identical to that of acid 2 (see above)wer d.th double ond.On th the h inte ng for 2H compo ted fatty a id- n,as in H NMR n82.60-282. nd y attributed three different methylene ps adiacent to ei saturated carbonyl or carboxylic groups This t120.160 d to the as in the assignment was confirmed by the resonances at chain.From the mass spectral data,the lengt of the 6 177.4 and 209.7 in the C NMR spectrum chain in both compounds 4 and 5 could be attributed to the carbons of a carboxylic acid and a determined as Ci6 thus permitting assignment of the saturated ketone,respectively.In addition,a broac structure of 6-hydroxy-4-oxo-hexadec-15-enoic acid multiplet at4.10(1H).which was correlated to a to 4.and of 6-hydroxy-4-oxo-hexadecanoic acid to 5 carbon at 67.7 in the HSQC spectrum,was firmly disubsti he pre The absolute configuration arbinols 3-5 has yet to onjuga was s1-5h 218 rate end isolated from a natural sou acid 5 was obtained the UV ith EC NMR previously as a racemate by synthesis at6129.3 1301 elated to A preliminary qualitative test indicated that acids 1 the HSOC spectrum.The AB coupling constant of and 2 are moderately fungicidal against the 10 5 Hz of these ved genic fungus Cladosporium cucumerinum 2-configuration of the double bond.A homonuclear COSY experiment,and two and three bonds HMBC correlations(Figure 3)allowed establishment of the The structures 1-5 are closely related to othe 4-relations oxidized C fatty acids and their derivatives L.3-re the cart ionship of recently isolated from a few Hygrophorus species [3-5],for which hypothetical biogenetic relationships have been proposed [3,5].A rare feature of all these NMR data alone left the position of the internal structures i he ox to a ketone o the C-4 o double hond undetermined Therefore comnound 3 th atty ac a le ddit compound was exposed to ozone and,after work-up,the crude shte-spec reaction mixture was directly subjected to GC to Comparison control.Indeed.6-hyd with an authentic sample heptanal to be formed by ozonolysis of acids like 3-5 are.to our knowledge,unprecedented in nature.They can be considered advanced biogenetic precursors of hygrophorones F and G 11-enoic acid. [4a].Examining the literature data,it was conc each Hygr by Its atty acid de may marke More e to h OH O fungicidal and bactericidal pr erties 13-51 thes metabolites likely function as "chemical deterrents" protecting Aygrophorus fruiting bodies against the attack of parasites and predators. related to 3 as Experimental tha nd sdid sigals of an internal double bond.Instead,in the H e determined on a ID ded R po NMR spectrum of compound 4,the pattem of signals Pa ragon 1000 PCs spectromete as neat films on Nac
4-Oxo fatty acids Hygrophorus discoxanthus Natural Product Communications Vol. 1 (12) 2006 1081 protons and carbons counted from the NMR spectra. Remarkably, the 1 H NMR spectrum of 3 did not contain the characteristic signals of the cross conjugated dienone system of 1 and 2; instead, three overlapping multiplets, each integrating for 2H, were found between δ 2.60-2.82, and were attributed to three different methylene groups adjacent to either saturated carbonyl or carboxylic groups. This assignment was confirmed by the resonances at δ 177.4 and 209.7 in the 13C NMR spectrum, attributed to the carbons of a carboxylic acid and a saturated ketone, respectively. In addition, a broad multiplet at δ 4.10 (1H), which was correlated to a carbon at δ 67.7 in the HSQC spectrum, was firmly assigned to a secondary alcohol. The presence of an internal, non-conjugated, disubstituted olefin was demonstrated by an end absorption band at λmax = 218 nm in the UV spectrum, along with the 13C NMR signals of two methines at δ 129.3 and 130.1, which were correlated to an NMR signal at δ 5.25-5.45 in the HSQC spectrum. The AB coupling constant of 10.5 Hz of these two protons proved the Z-configuration of the double bond. A homonuclear COSY experiment, and two and three bonds HMBC correlations (Figure 3) allowed establishment of the 1,4-relationship of the carboxylic group with the ketone, and the 1,3-relationship of the hydroxyl and carbonyl groups. NMR data alone left the position of the internal double bond undetermined. Therefore, compound 3 was exposed to ozone and, after work-up, the crude reaction mixture was directly subjected to GC analysis. Comparison with an authentic sample revealed heptanal to be formed by ozonolysis of olefin 3. From all results, the structure of compound 3 was established as (Z)-6-hydroxy-4-oxo-octadec- 11-enoic acid. OH O OH O Figure 3: Selected HMBC correlations of compound 3. The NMR data of compounds 4 and 5 were closely related to 3 as regards to the 6-hydroxy-4-oxocarboxylic acid [C(1)–C(6)] unit. In contrast, other than compound 3, the acids 4 and 5 did not show the signals of an internal double bond. Instead, in the 1 H NMR spectrum of compound 4, the pattern of signals from a three spin system at δ 4.95, 4.99, and 5.83, almost identical to that of acid 2 (see above) were due to a terminal double bond. On the other hand, compound 5 contains a fully saturated fatty acid-like chain, as indicated, in the 1 H NMR spectrum, by the characteristic distorted triplet (J = 6.8 Hz) at δ 0.88, assigned to the terminal methyl group, and by a broad peak at δ 1.20-1.60, assigned to the methylenes in the chain. From the mass spectral data, the length of the chain in both compounds 4 and 5 could be determined as C16, thus permitting assignment of the structure of 6-hydroxy-4-oxo-hexadec-15-enoic acid to 4, and of 6-hydroxy-4-oxo-hexadecanoic acid to 5. The absolute configuration of carbinols 3-5 has yet to be determined. Compounds 1-5 have never been isolated from a natural source; acid 5 was obtained previously as a racemate by synthesis [11]. A preliminary qualitative test indicated that acids 1 and 2 are moderately fungicidal against the phytopathogenic fungus Cladosporium cucumerinum Ell. et Arth.. The structures 1-5 are closely related to other oxidized C16-C22 fatty acids and their derivatives recently isolated from a few Hygrophorus species [3-5], for which hypothetical biogenetic relationships have been proposed [3,5]. A rare feature of all these structures is the oxidation to a ketone of the C-4 of the parent fatty acid; a few compounds show an additional site-specific oxidation at C-6, which the optically active alcohols 3-5 indicate to occur under enzyme control. Indeed, 6-hydroxy-4-oxo-carboxylic acids like 3-5 are, to our knowledge, unprecedented in nature. They can be considered advanced biogenetic precursors of hygrophorones F12 and G12 [4a]. Examining the literature data, it was concluded that each Hygrophorus species is characterized by its own pattern of oxidized C16-C22 fatty acid derivatives, which may thus be considered a significant chemotaxonomic marker. Moreover, due to the fungicidal and bactericidal properties [3-5], these metabolites likely function as “chemical deterrents”, protecting Hygrophorus fruiting bodies against the attack of parasites and predators. Experimental General experimental procedures: Optical rotations were determined on a Perkin-Elmer 241 polarimeter; IR spectra were recorded on an FT-IR Perkin Elmer Paragon 1000 PC spectrometer as neat films on NaCl
1082 Natural Product Commnications vol 1 (12)2006 Gilardonieral. dises.UV spectra were obtained in spectromete V.550 on a LiChroprep RP-18 on a Bruker CXP 300 spectrometer operating at 300 increasing MeCN re 100mL MHz (H)and 75 MHz (C).respectively.Hand C final mixture of MeCN-HO.10:1.v/y.The column chemical shifts (ppm)are relative to residual was then washed with MeCN(100 mL),followed by CHCls signals [n 7.26 c (central line of t)77.1. Me2CO(100 mL).Thirty-four fractions(A1-A34).of spectra (COSY,HSQC 5 The ord by using 2 5125 3203 d ultiple orded acid 34 4.6103g are oupli tants (D 胸 c acid (107 m reported in Hz.ESIMS experiments were carried out 49106 using a Finnigan LCQ Advantage MS 14 were obtained by evaporation of fractions A23,A25 spectrometer, equipped with the Xcalibur 1.4 and A27,respectively.Fraction A15 (76 mg)was software. High-res were Bruke Apex FI-ICR mas nt ol 120 spectro it油eithe sheet mixtur with RP-18 MeCN-10 B1-B13 were yisualized under UV light (254 and 366 nm) of 40 mL each were collected Acid 2 (10 mg 12 and by spraying with a 0.5%solution of vanillin in 103%)was isolated by evaporation of fraction B7 HSO-EtOH (4:1).followed by heating.Preparative Fractions A19 and A20 were pooled together and the column raphy was out or residue (110 mg)was further separated on 04m, Merck) Reagent RP column (20 g)elutec with a grad use,were ng ed for nti a final mi of MeCN-HO 10: erformed with a Perkin fractions (C1-C14).each of 35 mL.were co ted Fraction C5 (61 me)afforded comnound I (10 mg.1.4 10%)on successive separation on a material: Fresh fruiting bodies of LiChroprep RP-18 column(15 g)eluted with MeOH. Hygrophoru (Batsch. Fr.)Fr. wer HO.4:1.v. e00 6 an lo the province of Pavia (2E,5E,9Z)-4-Oxo-octadeca-2,5,9-trienoic acid (1) of th ors (M.C.) Whitish sticky solid specimen has heen den ited at the RE0.45(RP18,MeCN-H2O.7:1) Dipartimento di Chimica Organica.University of IR(6lm:3600-2800.3090,3050,2920,2852,1693. Pavia,Italy. 1666.1613.1278.1216,1000,975,950cm UV/Vis Lmax(CHCl3)nm (log c):234 (4.34) NMR:0.88(3H .8 Hz,Me),1.10-1.45 27 172 2H,d 52 (41 and H-o s00 zH-8 ad /1T J=10.3.65HH.9.5.42(0H.dd.=10.3.65Hz EtoAc solution was concentrated to dryness in vac at <30C to produce an oily residue (2.1 g),which H-10),6.39(1H,d,J-15.9HzH5),6.75(1H,d J=15.7HzH-3).7.06(1HdtJ=15.9.6.7Hz was partitioned between MeCN(0.5 L)and n-hexane (0.5 L).Evaporation of the two layers gave crude H-6,7.48(1Hd,J=15.7z,H-2. CNMR:13.9(CHC-18),22.5(CH,C-17),25.5 extrac 8272 H07:1 V/V) which 2549 was (CH,C-5)
1082 Natural Product Communications Vol. 1 (12) 2006 Gilardoni et al. discs. UV spectra were obtained in spectrometer grade CHCl3 from a Jasco V-550 spectrophotometer. 1 H and 13C NMR spectra were determined in CDCl3 on a Bruker CXP 300 spectrometer operating at 300 MHz (1 H) and 75 MHz (13C), respectively. 1 H and 13C chemical shifts (δ, ppm) are relative to residual CHCl3 signals [δH 7.26; δC (central line of t) 77.1, respectively]. 2D NMR spectra (COSY, HSQC, HMBC) were recorded by using standard pulse sequences. The abbreviation s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad are used throughout; coupling constants (J) are reported in Hz. ESIMS experiments were carried out using a Finnigan LCQ Advantage MS 1.4 spectrometer, equipped with the Xcalibur 1.4 software. High-resolution ESI mass spectra were determined on a Bruker Apex II FT-ICR mass spectrometer. TLC was performed on sheets precoated with either silica gel F254 (Polygram) or with RP-18 F254 (Merck, Germany). Compounds were visualized under UV light (254 and 366 nm) and by spraying with a 0.5% solution of vanillin in H2SO4-EtOH (4:1), followed by heating. Preparative column chromatography was carried out on LiChroprep RP-18 (25-40 μm, Merck). Reagent grade solvents, redistilled just before use, were employed for extraction; HPLC grade solvents were used for chromatographic separations. GC analysis was performed with a Perkin Elmer Autosystem gaschromatograph. Fungal material: Fresh fruiting bodies of Hygrophorus discoxanthus (Batsch.: Fr.) Fr. were collected on 16 October 2005 in a mixed conifer and beech wood near Brallo, in the province of Pavia, Italy, at an altitude of 1050 m. The mushroom was identified by one of the authors (M.C.) and a frozen voucher specimen has been deposited at the Dipartimento di Chimica Organica, University of Pavia, Italy. Extraction and isolation: Fruiting bodies (750 g) were frozen at –20 °C, minced, and extracted at –20 °C with EtOAc (3 x 1.5 L), followed by MeOH–H2O (4:1, 1 L), and H2O (1 L) at 0°C. The light yellow EtOAc solution was concentrated to dryness in vacuo at <30 °C to produce an oily residue (2.1 g), which was partitioned between MeCN (0.5 L) and n-hexane (0.5 L). Evaporation of the two layers gave crude residues of 1.02 g and 1.08 g, respectively. Acids 1-5 were contained in the MeCN extract (TLC: Rf = 0.55-0.70; RP-18 F254, solvent system: MeCNH2O, 7:1 v/v), which was subjected to column chromatography on a LiChroprep RP-18 column (100 g). Elution was performed with a gradient of MeCN-H2O, starting from a mixture 1:1, v/v, and increasing MeCN regularly every 100 mL, until a final mixture of MeCN-H2O, 10:1, v/v. The column was then washed with MeCN (100 mL), followed by Me2CO (100 mL). Thirty-four fractions (A1-A34), of 35 mL each, were collected. Fraction A9 gave acid 4 (12 mg, 1.6 10-3 % of fresh fruiting bodies), fraction A13 gave acid 5 (25 mg, 3.2 10-3 %), and fraction A14 afforded acid 3 (34 mg, 4.6 10-3 %). Linoleic acid (36 mg, 4.8 10-3 %), oleic acid (107 mg, 14 10-3 %), and methyl linoleate (37 mg, 4.9 10-3 %) were obtained by evaporation of fractions A23, A25, and A27, respectively. Fraction A15 (76 mg) was further separated on a LiChroprep RP-18 column (20 g) eluted with a gradient of MeCN-H2O, starting from a mixture 1:1, v/v, and increasing MeCN regularly every 50 mL, until a final mixture of MeCN-H2O, 10:1, v/v. Thirteen fractions (B1-B13), of 40 mL each, were collected. Acid 2 (10 mg, 1.4 10-3 %) was isolated by evaporation of fraction B7. Fractions A19 and A20 were pooled together and the residue (110 mg) was further separated on a LiChroprep RP-18 column (20 g) eluted with a gradient of MeCN-H2O, starting from a mixture 1:1, v/v, and increasing MeCN regularly every 50 mL, until a final mixture of MeCN-H2O, 10:1, v/v; 14 fractions (C1-C14), each of 35 mL, were collected. Fraction C5 (61 mg) afforded compound 1 (10 mg, 1.4 10-3 %) on successive separation on a LiChroprep RP-18 column (15 g) eluted with MeOHH2O, 4:1, v/v. (2E, 5E, 9Z)-4-Oxo-octadeca-2,5,9-trienoic acid (1) Whitish sticky solid. Rf: 0.45 (RP18, MeCN-H2O, 7:1). IR (film): 3600-2800, 3090, 3050, 2920, 2852, 1693, 1666, 1613, 1278, 1216, 1000, 975, 950 cm-1. UV/Vis λmax (CHCl3) nm (log ε): 234 (4.34). 1 H NMR: 0.88 (3H, t, J = 6.8 Hz, Me), 1.10-1.45 (12H, brs H2-12–H2-17), 2.05 (2H, q, J = 6.8 Hz, H2-11), 2.27 (2H, distorted q, J = 7.0 Hz, H2-8), 2.38 (2H, distorted q, J = 7.0 Hz, H2-7), 5.30 (1H, dd, J = 10.3, 6.5 Hz, H-9), 5.42 (1H, dd, J = 10.3, 6.5 Hz, H-10), 6.39 (1H, d, J = 15.9 Hz, H-5), 6.75 (1H, d, J = 15.7 Hz, H-3), 7.06 (1H, dt, J = 15.9, 6.7 Hz, H-6), 7.48 (1H, d, J = 15.7 Hz, H-2). 13C NMR: 13.9 (CH3, C-18), 22.5 (CH2, C-17), 25.5 (CH2, C-8), 27.2 (CH2, C-11), 29.2, 29.3, 29.4, 29.5 (4CH2, C-12, C-13, C-14, C-15), 31.6 (CH2, C-16), 32.8 (CH2, C-7), 127.1 (CH, C-9), 129.4 (CH, C-5)
4-Oxo fatty acids Hygrophorus Natural product commnunications vol 1 02)2006 1083 129.8(CHC-3),131.6(CH,C-10),139.7(CHC-2), R(flm:3600-3200,3010,2917,2850,1702,1412, (CH C-SLTO2CC) 4. 025655204 H. (2E,5E)-4-Oxo-hexadeca-2,5,15-trienoic acid(2) J=10.3.1.8.1.5Hz,H-16E,4.99(1HddJ =170 Whitish sticky solid. 1.81.5HH-162,5.83iH,dd,J=17.0.10.3 Rf:0.5(RP18,MeCN-H2O,7:1). 67HzH-15) IR(ilm:3600-3200,3050,2923,2851,1690,1664, NMR:27.4(CH,C-2),253,288,29.0,292 ,1000,915cm 2 C.10.C-11.C-1 235410 (CH2 。c-6 0T-68HzH.14231H a I= 72 Hz.H-7) 4.95(1Hdtd.j=10.3.1.8.1.5HzH-16E.502 (C,C4 (1H,dtd,J=17.0,1.8.1.5HzH-16☑，5.83(1H,ddt Negative ion ESI-FT-ICR-MS:m/z [M-H]calcd forC,H04283.1909.found283.1911 J=17.0.10.3.6.7Hz.H-15).6.39(1HdJ=15.9 H,H-5),6.75(1H d,J 57HH-3),7.06(1H 6-Hydroxy-4-oxo-hexadecanoic acid (5) a 15.9,6.7HzH-6),7.48(1HdJ 15.7Hz 照R2吸87829965 CH RE0.65 (RP18,Me 33.7(CH2. 14 114.0(CH2, C.16 101 3600-3200.2920.2855.1710.1415 (CH,C-5.129.7(CH,C-3).139.0(CH,C-15),139.7 (CH,C-2),151.7(CH,C-6),169.5(C,C-1),188.2 'HNMR0.88(3H,t,J-6.8Hz,Me),1.20-1.60 (C,C-4) (18H,brsH2-7-H2-15),2.60-2.80(6Hm,H2-2 Negative ion ESI-FT-ICR-MS m/z [M-H']caled forC16H30:263.1647,found:263.1649 14.0 (Z)-6-Hydroxy-4-oxo-octadec-11-enoic acid(3) 13C.14.C-15. 49 1 (CH Whitish sticky solid 20g5.4 :-340(c=10 mg/mL,CHCl3). Rf.0.6(RP18,MeCN-H2O.7:1). Negative ion ESI-FT-ICR-MS:m/z [M-H]caled IR(film:3600-3200,3010,2928.2856.1713,1406 forC,Hg04285.2066.found285.2064 2601201,100cm MR: 088(31 8 1.65 d solution of O3 in H-10.H-131.2.60-2.82（6H dissolved in CH-C H-3.H-5).4.10(1Hbrm.H-6).5.25-5.452H addin Me.s and the mixture was left at oc ove H-11,H-12. 3CNMR:14.0(CH3,C-18),22.5(CH2,C-17),25.0, sample was directly analyzed by GC under the following conditions:column HP-5(25 mx0.25 mm C-13,C-14,C-15),27.4(CH2,C-2),31. 0.33 thickness),injection temperatur .nC-703 ector(FID)temperature 280C,carrier gas rate ,27m /min,consta How mode 09.7(cC.4 splitle on ESI-FT-ICR-MS:m/z [M-H]calcd forC,H0.3112222.found3112225 rate of C/min.then rai sed to 2808C at rate of 109C/min then isothermal at 2809C for 5 min 6-Hydroxy-4-oxo-hexadec-15-enoic acid(4) Enrichment of the peak eluted at 9.69 min with an authentic sample of heptanal,confirmed its identity. Whitish sticky solid MeCN-O
4-Oxo fatty acids Hygrophorus discoxanthus Natural Product Communications Vol. 1 (12) 2006 1083 129.8 (CH, C-3), 131.6 (CH, C-10), 139.7 (CH, C-2), 150.8 (CH, C-6), 170.2 (C, C-1), 188.1 (C, C-4). Negative ion ESI-FT-ICR-MS: m/z [M – H- ] calcd for C18H27O3 291.1960, found 291.1962. (2E, 5E)-4-Oxo-hexadeca-2,5,15-trienoic acid (2) Whitish sticky solid. Rf : 0.5 (RP18, MeCN-H2O, 7:1). IR (film): 3600-3200, 3050, 2923, 2851, 1690, 1664, 1625, 1279, 1215, 1000, 915 cm-1. UV/Vis λmax (CHCl3) nm (log ε): 235 (4.19). 1 H NMR: 1.30-1.65 (12H, brs H2-8–H2-13), 2.06 (2H, q, J = 6.8 Hz, H2-14), 2.31 (2H, q, J = 7.2 Hz, H2-7), 4.95 (1H, dtd, J = 10.3, 1.8, 1.5 Hz, H-16E), 5.02 (1H, dtd, J = 17.0, 1.8, 1.5 Hz, H-16Z), 5.83 (1H, ddt, J = 17.0, 10.3, 6.7 Hz, H-15), 6.39 (1H, d, J = 15.9 Hz, H-5), 6.75 (1H, d, J = 15.7 Hz, H-3), 7.06 (1H, dt, J = 15.9, 6.7 Hz, H-6), 7.48 (1H, d, J = 15.7 Hz, H-2). 13C NMR: 27.8, 28.7, 28.9, 29.2, 29.3, 29.4 (6 x CH2, C-8, C-9, C-10, C-11, C-12, C-13), 32.8 (CH2, C-7), 33.7 (CH2, C-14), 114.0 (CH2, C-16), 129.2 (CH, C-5), 129.7 (CH, C-3), 139.0 (CH, C-15), 139.7 (CH, C-2), 151.7 (CH, C-6), 169.5 (C, C-1), 188.2 (C, C-4). Negative ion ESI-FT-ICR-MS: m/z [M – H- ] calcd for C16H23O3: 263.1647; found: 263.1649. (Z)-6-Hydroxy-4-oxo-octadec-11-enoic acid (3) Whitish sticky solid. [α]D 25: -340º (c = 10 mg/mL, CHCl3). Rf: 0.6 (RP18, MeCN-H2O, 7:1). IR (film): 3600-3200, 3010, 2928, 2856, 1713, 1406, 1260, 1201, 1100 cm-1. 1 H NMR: 0.88 (3H, t, J = 6.8 Hz, Me), 1.20-1.65 (14H, brs, H2-14–H2-17, H2-7– H2-9), 2.05 (4H, q, J = 7.0 Hz, H2-10, H2-13), 2.60-2.82 (6H, m, H2-2, H2-3, H2-5), 4.10 (1H, brm, H-6), 5.25-5.45 (2H, m, H-11, H-12). 13C NMR: 14.0 (CH3, C-18), 22.5 (CH2, C-17), 25.0, 27.0, 27.1, 28.9, 29.5, 29.6 (6 x CH2, C-8, C-9, C-10, C-13, C-14, C-15), 27.4 (CH2, C-2), 31.7 (CH2, C-16), 36.3 (CH2, C-7), 37.5 (CH2, C-3), 49.1 (CH2, C-5), 67.7 (CH, C-6), 129.3, 130.1 (2 x CH, C-11, C-12), 177.4 (C, C-1), 209.7 (C, C-4). Negative ion ESI-FT-ICR-MS: m/z [M – H- ] calcd for C18H31O4 311.2222, found 311.2225. 6-Hydroxy-4-oxo-hexadec-15-enoic acid (4) Whitish sticky solid. [α]D 25: -109º (c = 11 mg/mL, CHCl3). Rf: 0.7 (RP18, MeCN-H2O, 7:1). IR (film): 3600-3200, 3010, 2917, 2850, 1702, 1412, 1250, 1080, 1000, 913 cm-1. 1 H NMR: 1.25-1.65 (14H, brs, H2-7–H2-13), 2.04 (2H, q, J = 6.7 Hz, H2-14), 2.60-2.80 (6H, m, H2-2, H2-3, H2-5), 4.10 (1H, brm, H-6), 4.95 (1H, dtd, J = 10.3, 1.8, 1.5 Hz, H-16E), 4.99 (1H, dtd, J = 17.0, 1.8, 1.5 Hz, H-16Z), 5.83 (1H, ddt, J = 17.0, 10.3, 6.7 Hz, H-15). 13C NMR: 27.4 (CH2, C-2), 25.3, 28.8, 29.0, 29.2, 29.3, 29.4 (6 x CH2 C-8, C-9, C-10, C-11, C-12, C-13), 33.7 (CH2, C-14), 36.3 (CH2, C-7), 37.6 (CH2, C-3), 49.1 (CH2, C-5), 67.8 (CH, C-6), 114.0 (CH2, C-16), 139.1 (CH, C-15), 177.1 (C, C-1), 209.7 (C, C-4). Negative ion ESI-FT-ICR-MS: m/z [M – H- ] calcd for C16H27O4 283.1909, found 283.1911. 6-Hydroxy-4-oxo-hexadecanoic acid (5) Whitish sticky solid. [α]D 25: -95º (c = 10 mg/mL, CHCl3). Rf: 0.65 (RP18, MeCN-H2O, 7:1). IR (film): 3600-3200, 2920, 2855, 1710, 1415, 1255 cm-1. 1 H NMR: 0.88 (3H, t, J = 6.8 Hz, Me), 1.20-1.60 (18H, brs, H2-7–H2-15), 2.60-2.80 (6H, m, H2-2, H2-3, H2-5), 4.10 (1H, brm, H-6). 13C NMR: 14.0 (CH3, C-16), 27.6 (CH2, C-2), 22.8-29.6 (8 x CH2, C-8, C-9, C-10, C-11, C-12, C-13, C-14, C-15), 36.3 (CH2, C-7), 37.6 (CH2, C-3), 49.1 (CH2, C-5), 67.7 (CH, C-6), 177.2 (C, C-1), 209.5 (C, C-4). Negative ion ESI-FT-ICR-MS: m/z [M – H- ] calcd for C16H29O4 285.2066, found 285.2064. Ozonolysis of acid 3: A saturated solution of O3 in CH2Cl2-MeOH, 4:1 v/v, was added to compound 3 (3 mg) dissolved in CH2Cl2, (0.5 mL) at –78°C. The reaction was quenched after 3 h by adding excess Me2S and the mixture was left at –20°C overnight. A sample was directly analyzed by GC under the following conditions: column HP-5 (25 m×0.25 mm, 0.33 μm film thickness), injection temperature 250°C, detector (FID) temperature 280°C, carrier gas nitrogen, flow rate 1.27 mL/min, constant flow mode, split splitless injection, ratio 1:35, column temperature program: 40°C for 5 min, then raised to 100°C at a rate of 2°C/min, then raised to 280°C at a rate of 10°C/min, then isothermal at 280°C for 5 min. Enrichment of the peak eluted at 9.69 min with an authentic sample of heptanal, confirmed its identity. Fungicidal activity: A simple test, adapted from the literature [4a, 12], was carried out to reveal the
1084 Natural Product Communications Vol.1(12)2006 Gilardonietal d1-5.Five with 15 inhi in were sprayed with a conidal suspension of Acknowledgments-The authors thank Prof Cladosporium cucumerinum Ell.et Arth spores in a Mariella Mella and Prof.Giorgio Mellerio for NMR glucose mineral medium (Czapek broth).The plates and MS spectra measurements, respectively ere days,when they by h University o co References 21 BoeMC92nbereMcomogdEuropreL.LshygropiorswesnoptoraceaeLotes.hDoaimieomsA6calbgsqaesmeamote (a)Lubken T Schmidt J Porzel a arold N.Wessiohann L (2004)Hys ones fron cetes).Phytoch aCi7hgph (b)Lubke aph7elaeherC tandem mass spectrometry.Journal of Mass Spectrometry.41.361-371. [51 Gilardoni G.Clericuzio M,VidariG.Chrysotriones A and B from Hygrophorsdnunpublished results. 6 Food Chemistry.45.831 ation and structure of a new ceramide from the basidiomycete Hygrophorus eburneus. agreeable odor of 101-108,(d)Ros81R, Quir Baraldi PG.Barco A.Bene pSaytCeio&SmeiR,ecdkeaaeo35-dsubstmod2souoinstymotdcmm 12
1084 Natural Product Communications Vol. 1 (12) 2006 Gilardoni et al. possible fungicidal activity of compounds 1-5. Five solutions of compounds 1-5 in MeOH, each containing approximately 20 μg of substance, were spotted on F254 Merck silica gel plastic sheets, which were sprayed with a conidal suspension of Cladosporium cucumerinum Ell. et Arth spores in a glucose mineral medium (Czapek broth). The plates were then incubated at 25°C in the dark in a wet chamber (> 95% humidity) for 5 days, when they were overgrown with a dark gray colored mycelium. White spots (inhibition zones), signaling fungicidal activity, were found, in particular in correspondence with compounds 1 and 2; they were about eight times smaller than the inhibition area of the reference compound pseudomycin A (20 μg). Acknowledgments - The authors thank Prof. Mariella Mella and Prof. Giorgio Mellerio for NMR and MS spectra measurements, respectively. Fungicidal tests were carried out by Dr Solveig Tosi. Financial support by the Italian MIUR (Grants COFIN and FIRB) and the University of Pavia (Grant FAR) is acknowledged. References [1] This paper is Part 51 of the series “Fungal Metabolites”. Part 50: Clericuzio M, Tabasso S, Bianco MA, Pratesi G, Beretta G, Tinelli S, Zunino F, Vidari G. (2006) Cucurbitane triterpenes from fruiting bodies and cultivated mycelia of Leucopaxillus gentianeus. Journal of Natural Products, submitted. [2] Bon M (1990) Flore Mycologique d’Europe. 1. Les Hygrophores Hygrophoraceae Lotsy. In Documents Mycologiques mémoire hors série 1. CRDP, Amiens, 1-99. [3] Teichert A, Lübken T, Schmidt J, Porzel A, Arnold N, Wessjohann L. (2004) Unusual bioactive 4-oxo-2-alkenoic fatty acids from Hygrophorus eburneus. Zeitschrift fur Naturforschung, 60B, 25-32. [4] (a) Lübken T, Schmidt J, Porzel A, Arnold N, Wessjohann L. (2004) Hygrophorones A-G: fungicidal cyclopentenones from Hygrophorus species (Basidiomycetes). Phytochemistry, 65, 1061-1071; (b) Lübken T, Arnold N, Wessjohann L, Bőttcher C, Schmidt J. (2006) Analysis of fungal cyclopentenone derivatives from Hygrophorus spp. by liquid chromatography/electrospraytandem mass spectrometry. Journal of Mass Spectrometry, 41, 361-371. [5] Gilardoni G, Clericuzio M, Vidari G. Chrysotriones A and B from Hygrophorus chrysodon, unpublished results. [6] Breheret S, Talou T, Rapior S, Bessiere JM. (1997) Monoterpenes in the aromas of fresh wild mushrooms (Basidiomycetes). Journal of Agricultural and Food Chemistry, 45, 831-836. [7] Qu Y, Zhang H, Liu J. (2004) Isolation and structure of a new ceramide from the basidiomycete Hygrophorus eburneus. Zeitschrift fur Naturforschuns, 59B, 241-244. [8] Wood WF, Smith J, Wayman K, Largent DL. (2003) Indole and 3-chloroindole: the source of the disagreeable odor of Hygrophorus paupertinus. Mycologia, 95, 807-808. [9] Gill M, Steglich W. (1987) Pigments of fungi (macromycetes). In Progress in the Chemistry of Organic Natural Products. Vol 51, Herz W, Grisebach H, Kirby GW, Tamm Ch. (Eds). Springer Verlag, Wien, New York. 1-317. [10] (a) Williamson RT, Carney JR, Gerwick WH. (2000) Application of the BIRD sandwich for the rapid and accurate determination of 1 H-1 H NMR coupling constants in higher order spin systems. Journal of Natural Products, 63, 876-878; (b) Stamatov SD, Stawinski J. (2000) A simple and efficient method for direct acylation of acetals with long alkyl-chain carboxylic acid anhydrides. Tetrahedron, 56, 9697-9703; (c) Vieville C, Mouloungui Z, Gaset A. (1995) Synthesis and analysis of the C1-C18 alkyl oleates. Chemistry and Physics of Lipids, 75, 101-108; (d) Rossi R, Carpita A, Quirici MG, Verancini CA. (1982) Insect pheromone components. Use of carbon-13 NMR spectroscopy for assigning the configuration of carbon-carbon double bonds of monoenic or dienic pheromone components and for quantitative determination of Z/E mixtures. Tetrahedron, 38, 639-644. [11] Baraldi PG, Barco A, Benetti S, Manfredini S, Simoni D. (1987) Ring cleavage of 3,5-disubstituted 2-isoxazolines by molybdenum hexacarbonyl and water to β-hydroxy ketones. Synthesis, 3, 276-278. [12] Gottstein D, Gross D, Lehmann H. (1982) Mikrobiotest mit Cladosporium cucumerinum Ell. et Arth. zum Nachweis fungitoxischer Verbidungen auf Dűnnschichtplatten. Archiv fűr Phytopatologie und Pflanzenschutz, 20, 111-116
NPC Natural Product Communications 2006 Vol.1 No.12 Kenyaloside,a Novel O,0,0-Triglycosylated Naphthalene 1085-1088 Derivative from the Exudate of Kenyan Aloe Species oDaniela Moeri Cripp Poiro Calo Moreid Di Istituto di Chimica del Riconoscimento Molecolare,C.N.R.via Mario Bianco 9.20131 Milano,Italy giovanna.speran-a@unimi.it Received:;Accepted: Dedicated to the memory of Professor Ivano Morelli. valoside (1). isolated from the dried exudate of kem Aloe 1-(B-D-glucopyranosyloxy)-8-(a-L-rhamnopyranosyloxy)-3-(B-D-xylopyranosyloxymethyl)naphthalene. Keywords:aloes,Aloe ferox,naphthalene..O-triglycoside,kenyaloside. elucidation of a ne that exudate of Kenyan Mloe species is reported here.This chromatographed successively on silica gel and exudate,flowing from the cut leaves of Aloe ferox Sephadex LH-20 columns.Kenyaloside (1)was Miller and of its hybrids with A. obtained in ca.0.1%yield (based on the starting i-s(n was derived m/2 o rug has +Na']m/z. The presence of three )belong to the families of che mical shifts and coupin onstants in the H and 6-phenvl-2-p ones,5-methyl-7-hvdrox C NMR spectra of 1 (Table 1).in addition,the and 1,8-dihydroxyanthrones (see Ref.3 for a NOESY spectrum revealed two significant complete list of such compounds). associations between the anomeric proton at 85.77 and the upfield aromatic proton,and between another The structure of the new product,named kenyaloside anomeric proton (at 6 431)and both the aromatic (（hods g股8749吧e four aromatic C-H groups,together with the values of residues [4.51. nucleus Part 19 in the series"Studies on Aloe".For Part 18,see Ref.1
Kenyaloside, a Novel O,O,O-Triglycosylated Naphthalene Derivative from the Exudate of Kenyan Aloe Species¶* Giovanna Speranzaa,*, Daniela Montib , Sergio Crippaa , Paola Cairolia , Carlo F. Morellia and Paolo Manittoa a Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, via Venezian 21, 20133 Milano, Italy b Istituto di Chimica del Riconoscimento Molecolare, C.N.R., via Mario Bianco 9, 20131 Milano, Italy giovanna.speranza@unimi.it Received: July 11th, 2006; Accepted: September 2nd, 2006 Dedicated to the memory of Professor Ivano Morelli. A new naphthalene O,O,O-triglycoside, kenyaloside (1), was isolated from the dried exudate of Kenyan Aloe species, a bittering and laxative agent. Its structure was established by combined spectral and chemical methods as 1-(β-D-glucopyranosyloxy)-8-(α-L-rhamnopyranosyloxy)-3-(β-D-xylopyranosyloxymethyl)naphthalene. Keywords: aloes, Aloe ferox, naphthalene O,O,O-triglycoside, kenyaloside. As part of a systematic chemical investigation into Aloe exudates (bitter aloes) [1], the structural elucidation of a new water-soluble constituent of the exudate of Kenyan Aloe species is reported here. This exudate, flowing from the cut leaves of Aloe ferox Miller and of its hybrids with A. spicata and A. africana growing in Kenya [2, 3], when dried, is used as a bittering agent and as a purgative, similarly to Cape aloes [4, 5]. The drug has been reported to contain a number of polyketide metabolites (such as O- and/or C-glucosides) belonging to the families of 6-phenyl-2-pyrones, 5-methyl-7-hydroxychromones, and 1,8-dihydroxyanthrones (see Ref. 3 for a complete list of such compounds). The structure of the new product, named kenyaloside (1), was determined by spectral and chemical methods. To our knowledge, it represents the first example of a naphthalene glycoside both occurring in Aloe species and bearing three different O-glycosyl residues [4, 5]. ¶ Part 19 in the series “Studies on Aloe”. For Part 18, see Ref. 1 The aqueous extract of the dried exudate of Kenyan Aloe species, after partitioning with ethyl acetate, was lyophilized to afford a residue that was chromatographed successively on silica gel and Sephadex LH-20 columns. Kenyaloside (1) was obtained in ca. 0.1% yield (based on the starting drug). Its molecular formula, C30H40O17, was derived from ESI-HRMS (found: m/z 695.21326, calcd for [M+Na+ ] m/z: 695.21577). The presence of three O-glycosyl residues was suggested by inspection of chemical shifts and coupling constants in the 1 H and 13C NMR spectra of 1 (Table 1); in addition, the NOESY spectrum revealed two significant associations between the anomeric proton at δ 5.77 and the upfield aromatic proton, and between another anomeric proton (at δ 4.31) and both the aromatic proton at δ 7.48 and an Ar-CH2 group (AB system: δ 4.73, 4.94, J = 12.4 Hz). 1 H and 13C signals due to four aromatic C-H groups, together with the values of 1 H-1 H coupling constants and mutual NOEs, were indicative of a 1,2,3,8-tetrasubstituted naphthalene nucleus. NPC Natural Product Communications 2006 Vol. 1 No. 12 1085 - 1088