32 www.hxyswgc.com 2016Vol.33No.05 Chemistry & Bioengineering doi:10.3969/j.issn.1672-5425.2016.05.008 dl-34 123 1 13 1 1 (1. 264003; 2. 264001;3. 100049) : dl-34 7 1 2 1 (+)-wickerola 2 harziandione 3~7 1 2 1 : ; ; ;(+)-wickerola;harziandione :Q58 :A :1672-5425(2016)05-0032-05 DiterpenesandSteroidsfrom MarineAlga-Endophytic FungusTrichoderma Asperelum dl-34 LIANGXiao-rui 123 MIAOFeng-ping 1 SONGYin-ping 13 GUOZhan-yong 1 JINai-yun 1 (1.YantaiInstituteof CoastalZone ResearchChinese Academyof SciencesYantai264003China; 2.Naval AeronauticalEngineeringInstituteYantai264001China; 3.Universityof Chinese Academyof SciencesBeijing100049China) Abstract:Thebioactivemetabolitesfromamarinebrownalga-endophyticTrichodermaasperelum strain (dl-34) wereseparatedandpurifiedbychromatographytlcand HPLC.Sevenkindsofcompoundswereobtainedandtheirstruc- tureswereidentifiedbynmrmsandir.theabsoluteconformersofcompound1andcompound2wereconfirmedby quantumchemicalcalculations.resultsshowedthatcompound1was(+)-wickerolaanewditerpeneantipodeandcom- pound2washarziandioneaknownditerpeneandcompounds3~7wereergosterolderivatives.bioactivityexperimentalre- sultsshowedthatbothcompounds1and2exhibitedlethalactivityagainstartemiasalinaandcompound1alsoshowedan- tibacterialactivityagainstescherichiacoliandstaphylococusaureus. Keywords:Trichodermaasperelum;diterpene;steroid;(+)-wickerolA;harziandione (Trichodermaspp.) [2-5] [1] dl-34 [1] 1 1.1 200~300 GF-254 : (41106136) :2016-01-29 : (1979-) : E-mail:xrliang@yic.ac.cn; : E-mail:nyji@yic.ac.cn
: /2016 5 33 ;SephadexLH-20 ( - 1 1) ( - GE Healthcare SephadexLH-20 ( - 1 1) HPLC ChirascanCD SephadexLH-20 ( - 1 1) HPLC ;P-1020 FTIR4100 ( - 9 1) 5(4.0mg) 6(2.7mg) JASCO ;Avance Ⅲ 500 19 HPLC( - 2 3) 7(5.3 (TMS ) Bruker ;AutospecPremier P776 Waters ;Agilent1260 (EclipseSB-C 18 9.4 mm 250mm5μm) Agilent 1.2 1.2.1 2010 9 rvinsketch (2014) [7] Gaussian09 dl-34 Trichodermaasperel- [8] lum GenBank KR023953 (TD-DFT) (PDA) ECD SpecDic (sigma=0.3) [9] (-)-wickerola [α] D 100 1L B3LYP/6-311+ +G(2d2p) 300mL : 500 ml L -1 10g L -1 NaNO 32g L -1 500 ml L -1 25 30d 1.2.2 2 100 40 2.1 1~7 13 CNMR ( 1) 14.1g 2 1) 3(3.8mg) 14 ( - 9 1) 4(2.0mg) 16 mg) 1.2.3 IR 1 HNMR 13 CNMR EIMS HREIMS B3LYP/6-31G(d) B3LYP/6-31G(d) 2 1.2.5 1 2 1 2 1: ;[α] 21 D1.2(c0.2CHCl 3 );IR 1.2.4 2 (-)-wickerola 2 (-)-wickerola Ma- - (1 1 ) (KBr)νcm -1 :33672931287714581381 44.1g 1099; 1 HNMR(500 MHzacetone-d 6 )δ:1.49(1h (TLC) (HPLC) mh-2)1.62(1hmh-3a)2.10(1hmh- 3b)1.48(1HmH-4a)2.01(1HmH-4b)2. 58.2g 15(1HmH-5)1.23(1HdddJ=14.0 Hz 14.0 - Hz 3.7 HzH -7a)1.67(1HmH -7b)1.44 TLC 22 (1HmH-8a)1.53(1HmH-8b)1.90(1H - (10 1) 8 dddj=13.4 Hz 10.0 Hz 6.3 HzH-10)1.58 SephadexLH-20 8-1 8-2 (1HmH-11a)1.73(1HmH-11b)1.02(1H 8-1 - (10 mh-12a)1.37(1hddj=11.1 Hz 8.4 HzH- 1) 1(5.4mg) 8-2 12b)1.32(1HdJ=13.4 HzH-14)1.50(1H - (5 1) mh-15a)1.72(1hmh-15b)0.97(3hsh- 2(15.0mg) - (2 1) 16)1.07(3HsH-17)1.04(3HdJ=7.0 HzH 13 SephadexLH-20-18)1.07(3HsH-19)1.12(3HsH-
34 : /2016 5 1 Tab.1 1~7 13 CNMR (125 MHzδ) 13 CNMRDataforcompounds1~7 (125 MHzδ) 1 a 2 b 3 b 4 b 5 c 6 b 7 b 1 38.6C 49.6C 34.7CH2 32.6CH2 32.9CH2 32.8CH2 25.5CH2 2 41.2CH 59.3CH 30.1CH2 30.6CH2 31.7CH2 30.9CH2 30.0CH2 3 25.5CH2 214.3C 66.5CH 66.4CH 66.4CH 67.8CH 67.2CH 4 28.7CH2 42.6CH2 37.0CH2 36.1CH2 40.7CH2 39.6CH2 36.9CH2 5 26.6CH 29.9CH 82.2C 82.7C 74.9C 76.4C 79.6C 6 38.6C 51.6C 135.4CH 135.5CH 72.6CH 82.5CH 198.2C 7 26.1CH2 29.8CH2 130.8CH 130.8CH 119.9CH 115.0CH 119.8CH 8 40.9CH2 29.5CH2 79.4C 78.4C 140.1C 143.7C 164.8C 9 71.9C 146.4C 51.1CH 142.6C 42.7CH 43.9CH 74.7C 10 44.2CH 149.4C 37.0C 38.0C 37.1C 37.3C 41.8C 11 21.7CH2 197.8C 23.4CH2 119.7CH 23.1CH2 22.9CH2 28.7CH2 12 43.7CH2 60.0CH2 39.4CH2 41.2CH2 39.4CH2 39.4CH2 34.9CH2 13 39.0C 39.9C 44.6C 43.6C 43.5C 43.9C 45.3C 14 51.6CH 53.0CH 51.7CH 48.2CH 54.6CH 54.9CH 51.8CH 15 42.9CH2 26.7CH2 20.6CH2 20.9CH2 21.8CH2 22.2CH2 22.4CH2 16 25.1CH3 25.0CH3 28.6CH2 28.6CH2 28.2CH2 27.9CH2 27.9CH2 17 24.2CH3 23.2CH3 56.2CH 55.9CH 55.8CH 56.0CH 56.0CH 18 22.4CH3 20.9CH3 12.9CH3 13.0CH3 12.5CH3 12.3CH3 12.3CH3 19 19.4CH3 20.7CH3 18.2CH3 25.6CH3 18.2CH3 18.4CH3 20.4CH3 20 19.8CH3 22.5CH3 39.7CH 39.9CH 40.4CH 40.4CH 40.3CH 21 20.9CH3 20.7CH3 21.4CH3 21.1CH3 21.1CH3 22 135.2CH 135.1CH 135.9CH 135.4CH 135.1CH 23 132.3CH 132.4CH 131.8CH 132.1CH 132.5CH 24 42.8CH 42.8CH 42.5CH 42.8CH 42.8CH 25 33.1CH 33.1CH 32.9CH 33.1CH 33.1CH 26 19.6CH3 19.6CH3 19.9CH3 19.6CH3 19.6CH3 27 20.0CH3 19.9CH3 20.2CH3 20.0CH3 20.0CH3 28 17.6CH3 17.6CH3 17.8CH3 17.6CH3 17.6CH3 29 58.3CH3 : a b CDCl3 c DMSO-d6 20); 13 CNMR 1;EIMSm/z(%):290(24) 275(57)272(100)257(41)247(38)232(60)217 (37)189(56)176(51)162(53)148(66)136(43) 126(56)110(55)98(53)85(51)73(46)59(50); HREIMSm/z:290.2615[M] + ( C 20 H 34 O 290.2610) 2.2 1~7 1 ([α] 21 D ) 1.2 C 20H 34O290.2610) C 20H 34O 4 1 HNMR 4 δ:0.97(3h sh-16) 1.07(3HsH-17) 1.07(3HsH-19) 1.12(3HsH-20)1 δ:1.04(3h dj=7.0hzh-18) 13 CNMR( 1) DEPT 5 7 4 4 [10] wicker- ola HMBC H-16 C-1 C- 3367cm -1 2 C-6 C-17 H-17 C-1 C-2 C-6 C (HREIMS)(m/z290.2615[M] + -16 H-18 C-4 C-5 C-6 H-19
: /2016 5 35 NOE H-19 H-5 H-10 10 H-5 C-19 H-14 H-16 H -20 H-14 C-1 C-20 C-12 C-13 C-14 C-15 H-20 C-8 C harzianone 292nm Coton -9 C-10 1 wick- C-3 erola H-10 H-14 B3LYP/6-31G(d) (J=13.4 Hz) 2 ECD 2 1 wickerola 1 [α] 21 [10] D wick- erola (-2.8) 1 (-)-wickerola (+)-wick- erola (-)-wickerola (+)-wicker- ola B3LYP/6-311++G(2d2p) 2S5R 6R9S10S13S 14R [α] 21 D ([α] 21 D -3.7) (-)-wickerola (+)- wickerola 2R5S6S9R10R13R 14S (-)-wickerola 1 2 2S5R6R13S [13-14] 14S 13 CNMR 28 NMR 1 2 (-)-wickerola 3~7 NMR Fig.1 Energy-minimizedconformersofcompound2 [15-16] 3 and(-)-wickerola 4 5α8α- -69(11)22E- 2 1 HNMR 4 δ:0.98(3hsh-17)1.00(3hsh-16) π * ) 292nm(n π * ) 2 Coton 251nm(π π * ) Coton 337nm 251nm Coton 2 harzianone 2 2 ECD Fig.2 ExperimentalandcalculatedECDspectraofcompound2 2 2 1 3~7 1 HNMR δ0.5~1.1 2 4 δ3.7~4.1 1 δ5.1~5.3 dd 13 CNMR ( 1) 6-3β- [15-17] 5 (22E24R)- - 722- -6β- -3β 5α- [18] 6 [16] 1.52(3HsH-19) 2.13(3HsH-20);1 7 5α9α- - δ1.12(3hdj=7.2 HzH-17) 13 CNMR 722- -6- [18-19] 1~7 ( 1) DEPT 20 5 5 3 3 7 ; 2.3 1 2 2 δ197.8(c-11) 1 2 2 1 5 1 α β 1 8.0 [11-12] harziandione mm( 30μg) NMR 2 harzianone (ECD) 337nm(n 2 1 2 LC 50 12.0μg ml -1 38.2μg ml -1
36 : /2016 5 3 1~7 Fig.3 Structuresofcompounds1~7 3 dl-34 2 5 : [1] REINOJLGUERRERO R FHERN NDEZ-GAL N Ret al.secondary metabolitesfrom speciesofthebiocontrolagent Trichoderma[J].PhytochemistryReviews20087(1):89-123. [2] CARO ESTARKSC MJENSEN PRetal.Trichodermamides AandBcytotoxicmodifieddipeptidesfromthemarine-derived fungustrichodermavirens[j].journalof NaturalProducts 200366(3):423-426. [3] SUN YTIAN LHUANGJetal.TrichodermatidesA-Dnovel polyketidesfromthemarine-derivedfungustrichodermareesei [J].OrganicLeters200810(3):393-396. [4] SONGFDAIHTONG Yetal.TrichodermaketonesA-Dand 7-O-methylkoningininDfromthemarinefungusTrichoderma koningi[j].journalofnaturalproducts201073(5):806-810. 1 1 2 (-)- wickerola 1 2 1 dl-34 wickerane harziane [5] CHEN LZHONGPPANJRetal.AsperelinesGand Htwo newpeptaibolsfromthemarine-derivedfungustrichodermaas- perelum[j].heterocycles201387(3):645-655. MIAOFPLIANG X RYIN XLetal.Absoluteconfigurations ofuniqueharzianediterpenesfrom Trichodermaspecies[J].Or- ganicleters201214(15):3815-3817. dictionandcalculation[z/ol].2014.chemaxonmarvin6.2.2 [2014-04-19].htp://www.chemaxon.com. [8] FRISCH MJTRUCKSG WSCHLEGEL H Betal.Gaussian 09RevisionC.012010[Z/OL].[2012-01-0].htp://www. gaussian.com. [9] BRUHN TSCHAUML FFEL AHEMBERGER Yetal.SpecDis: quantifyingthecomparisonofcalculatedandexperimentalelectronic circulardichroismspectra[j].chirality201325(4):243-249. [7] Marvinsketchwithcalculatorpluginsforstructurepropertypre- [10] YAMAMOTO TIZUMINUIHetal.WickerolsAandB:novelanti -influenzavirusditerpenesproducedbytrichodermaatroviridefki- 3849[J].Tetrahedron201268(45):9267-9271. [11] GHISALBERTIELHOCKLESSDCRROWLANDCetal.Harzi- andioneanewclassofditerpenefrom Trichodermaharzianum [J]. JournalofNaturalProducts199255(11):1690-1694. [12] ADELIN ESERVY CMARTIN M Tetal.Bicyclicandtetra- cyclicditerpenesfromatrichodermasymbiontoftaxusbaccata [J].Phytochemistry201497:55-61. [13] MANNINA LSEGRE A LRITIENIAetal.A newfungal growthinhibitorfrom Trhichodermaviride[J].Tetrahedron 199753(9):3135-3144. [14] KOBAYASHINMURANAKA AMACKJ.CircularDichroismand MagneticCircularDichroism SpectroscopyforOrganicChemists[M]. London:RoyalSocietyofChemistry2012:42-47. [15] GRECA M DMANGONILMALINARO Aetal.5β 8β- Epidioxyergosta-622-dien-3β-olfromTyphalatifolia[J]. GazzetaChimicaItaliana1990120(6):391-392. [16] KWON H CZEES DCHO S Yetal.Cytotoxicergosterols frompaecilomycessp.j300[j].archivesofpharmacalresearch 200225(6):851-855. [17] CATENIGDOLJAKBZACCHIGNA Metal.Newbiological- lyactiveepidioxysterolsfromstereum hirsutum[j].bioorganic& MedicinalChemistryLeters200717(22):6330-6334. [18] KAWAGISHIHKATSUMIRSAZAWA Tetal.Cytotoxic steroidsfromthe mushroom Agaricusblazei[J].Phytochemis- try198827(9):2777-2779. [19] XIONG H YFEID QZHOUJSetal.Steroidsandothercon- stituentsfromthemushroom Armilarialueovirens[J].Chemis- tryofnaturalcompounds200945(5):759-761.