Absolute Stereochemistry of Novel Isochromanone Derivatives from Leptosphaeria sp.KTC 727
Wilanfranco C.T AYONE ,1Saki K ANAMARU ,2Miho H ONMA ,2Kazuaki T ANAKA ,2Tatsuo N EHIRA ,3and Masaru H ASHIMOTO 1;2;y
1
The United Graduate School of Agricultural Sciences,Iwate University,3-18-8Ueda,Morioka 020-8550,Japan 2
Faculty of Agriculture and Life Science,Hirosaki University,3Bunkyo-cho,Hirosaki 036-8561,Japan 3
Graduate School of Integrated Arts and Sciences,Hiroshima University,1-7-1Kagamiyama,Higashi-Hiroshima 739-8521,Japan
Received August 19,2011;Accepted September 5,2011;Online Publication,December 7,2011[doi:10.1271/bbb.110621]
A novel isochromanone,(S )-8-hydroxy-6-methoxy-4,5-dimethyl-3-methylene-isochromen-1-one (1),known 2and previously reported metabolites from Leptosphae-ria sp.KTC 727(JCM 13076¼MAF
cvF 239586)were isolated from the same source by culturing for a relatively long period.The results of the prent study disclo their structures involving the absolute stereo-chemistry.The planar structures of the molecules were established by ESIMS and NMR spectral analys.The absolute configuration of 1was established by comparing its electronic circular dichroism (ECD)spectrum with that of structurally-related known com-pound 3.The relative stereochemistry of 2was revealed by a combination of nuclear Overhaur effect (NOE)experiments and thermodynamic discussions.Successful transformation of 1to 2led us to assign the config-uration of 2after comparing their ECD spectra.The compounds exhibited weak antifungal activities against Cochliobolus miyabeanus .Key words:
isochromanone;stereochemistry;Leptos-phaeria ;electronic circular dichroism;molecular modeling
During the cour of our investigation of novel natural products from ecologically unique fungi,1–4)we have recently reported (R )-4,8-dihydroxy-6-methoxy-4,5-dimethyl-3-methylene-isochromen-1-one (3)as well as veral other novel metabolites from Leptosphaeria sp.KTC 727(JCM 130765)¼MAFF 2395866))7)after 40d of incubation.We are developing this project with a hope that ecologically unique fungi will involve novel and unique metabolites.Further investigation led us to isolate additional novel C4deoxy analogue 1together with its dihydroxylated derivative 2when Leptos
phaeria sp.KTC 727was cultured for 55d.The results of the prent study disclo their structures involving the absolute configurations,details of this structural eluci-dation are being described here.
Results and Discussion
忠厚的近义词The ethyl acetate extract from a broth of Leptosphae-ria sp.KTC 727with a potato-sucro medium (1.6L)cultured for 55d was subjected to silica gel column chromatography to provide 1(1.5mg)and 2(13.9mg),together with previously isolated metabolites (R )-7-hydroxy-3-((S )-1-hydroxyethyl)-5-methoxy-3,4-dimethy-lisobenzofuran-1(3H )-one (47.7mg),(R )-3,4-dihydro-4,8-dihydroxy-6-methoxy-4,5-dimethyl-3-methyleneiso-chromen-1-one (2.8mg),and (R )-3-acetyl-7-hydroxy-5-methoxy-3,4-dimethylisobenzofuran-1(3H )-one (1.8mg).7)Only a trace amount of compound 1could be detected when the fungus was cultured for a shorter period of 40d under the same conditions,while the content of 2in that broth was below the detection level.Their 1H-NMR data are summarized in Table 1.
The ESIMS data for 1provided a protonated ion signal at m =z 235.0966to indicate its molecular formula as C 13H 14O 4(calcd.½M þH þ235.0965),being one oxygen less than that of 3which we had recently discovered from the same source.The 1H-NMR spec-trum in CDCl 3showed exomethyl
ene protons at C9as a pair of doublets at 4.56and 4.80ppm with 1.8Hz geminal spin coupling.An aromatic proton appeared at 6.38ppm as a singlet.Two methyl resonances C11H 3and C12H 3respectively appeared at 2.08and 3.86ppm,both as singlets.The foregoing signals rembled tho of 3in their chemical shifts and splitting patterns.However,C10H 3of 1was obrved as a doublet (J ¼7:2Hz)at 1.38ppm,while the corresponding signal for 3was a singlet at 1.74ppm.Additionally,a quartet signal for C4H in 1was newly detected at 3.82ppm,this signal being abnt in 3.The COSY spectrum revealed spin coupling between them.Further analys involving HMQC and HMBC disclod 1to be 8-hydroxy-6-methoxy-4,5-dimethyl-3-methylene-iso-chromen-1-one,a C4deoxy analogue of 3.The R f value of 1(ca.0.7)was in fact larger than that of 3(ca.0.4)by silica gel TLC (EtOAc:hexane ¼3:7).
y
To whom correspondence should be addresd.Tel/Fax:+81-172-39-3782;E-mail:hmasaru@cc.hirosaki-u.ac.jp
Abbreviations :m CPBA,m -chloroperoxybenzoic acid;THF,tetrahydrofuran;NOE,nuclear Overhaur effect;ECD,electronic circular dichroism;DFT,density functional
theory
Biosci.Biotechnol.Biochem.,75(12),2390–2393,2011
Since 1had a stereogenic center at C4,its chirality was next studied.We established this by comparing the ECD spectrum with that of structurally related 3,since 1possd no functional group available either for chiral assignment by the spectroscopic method or for chemical correlations.It has been well established that only the geometry of a UV-Vis absorbing chromophore will contribute to the ECD spectrum,8)so molecular model-ing calculations for 1and 3were performed prior to ECD spectral discussions about this compound.The computations enabled us to investigate the geometric relationship of their chromophores by conformational arches with mi-empirical AM1and subquent structural re-optimization with DFT B3LYP/6-31G Ãon Spartan 08(Wavefunction INC.,Wavefunction,Inc.18401Von Karman Avenue,Suite 370,Irvine,CA 92612,USA).The suggested that both 1and 3were constituted with more than 98%abundance by their most stable conformers at room temperature.The most stable conformers of the two molecul
es thus obtained were almost superimposable as shown in Fig.2.The C4alcohol should minimally absorb UV-Vis radiation,and this function would therefore hardly affect the ECD spectra.The configuration of 3had been established to be (R )in our previous study.7)If 1was in (S )-form,1and 3could be expected to show similar ECD spectra.9)It was found that 1and 3provided similar ECD spectra as shown in Fig.3,enabling us to establish the (S )-configuration for 1.
Theoretical ECD calculations for 1was also per-formed with time-dependent DFT at B3LYP/TZVP on Gaussian 09(Gaussian,Inc.,340Quinnipiac St Bldg 40,Wallingford,CT 06492,USA).The profile for the theoretical spectrum thus obtained showed good agree-ment with the experimental spectrum of 1,affirming the foregoing discussions.10,11)
The molecular formula of 2was determined to be C 13H 16O 6bad on the ESI mass spectrum (m =z 269.1023½M þH þcalcd.269.1025).Although the ESIMS data provided only a nominal protonated mo-
lecular ion,the 1H-NMR spectrum in CDCl 3disclod the sample to consist of a pair of components with approximately 7:1ratio.We have so far been unable to parate the two components by chromatography.A detailed analysis involving the HMQC and HMBC spectra led us to assign all reso
nances in CDCl 3as tho shown in Table 1.This information led us to assign the structure of the major component to be 3,8-dihydroxy-3-hydroxymethyl-6-methoxy-4,5-dimethylisochroman-1-one,this being depicted as 2.This molecule had been reported in 2011by Schu ¨ffler et al.from Allantopho-mopsis sp.12)Most 13
C-NMR resonances of the minor component appeared very cloly to tho of the major component or overlapped.For example,the resonances of C3for the major and minor components were respec-tively obrved at 103.1and 103.9ppm.Taking their chemical shifts and molecular formulae into account,the minor component was expected to be a stereomeric tautomer around the C3hemiacetal moiety.The keto form,another candidate for the minor component,should have given the C3resonance at around 200ppm in the 13
C-NMR spectrum,but this could not be detected.Although Schu ¨ffler et al.have mentioned considerable line broadening for the 1H-NMR signals in CD 3CN,their study reported neither the prence of another tautomer nor stereochemical discussions.
The relative stereochemistry of 2was studied by a combination of molecular modeling calculations and NOESY analys.Calculations were performed in a similar manner just described,revealing the
most stable conformers for both the (3R Ã,4S Ã)-and (3S Ã,4S Ã)-tautomers of 2as shown in Fig.4.The calculations also suggested that both tautomers were mostly con-stituted by their most stable conformers (>96%abun-dance for either isomer),which enabled us to discuss their properties.Methyl groups at C4(C10H 3)in both tautomers took on the quasi-axial conformation.Serious steric hindrance with the C11H 3group might have given ri to this.In both cas,the abnce of 1,3-diaxial repulsion for C10H 3may also have not destabilized this conformation (the C8a sp2carbon and oxygen atom located at the 1,3-position).The C3-hydroxy group in more stable (3R Ã,4S Ã)-tautomer was also in an axial-like conformation due to the so-called anomeric effect.Since the energy difference between the (3R Ã,4S Ã)-and (3S Ã,4S Ã)-tautomers was 3.39kJ/mol bad on the calculations,the Boltzmann distribution theory allowed us to expect the tautomeric ratio of ca.8:2at
银杏果的功效与作用及食用方法
room
Fig.2.Overlapped View of the Most Stable Conformers of 1and 3Obtained by B3LYP/6-31G Ã
.
Fig.3.Experimental ECD Spectra of 1and 3in CH 3CN,and the Theoretical Spectrum for (4S )-1.
人工养殖2H 331: R 1 = H, R 2 = 10CH 33: R 1 = OH, R 2 = 10CH 3
H 3312
3
2 (major tautomer):
R 1 = 9CH 2OH, R 2 = OH 2 (minor tautomer):
R 1 = OH, R 2 = 9CH 2OH
9
12
12
Fig.1.Structures of 1,2and 3.
日破苍穹
Novel Isochromanone from Leptosphaeria sp.2391
temperature.This accorded well with the experimental ratio (7:1)in the 1H-NMR spectrum.The NOESY spectrum provided correlation peaks at C4H/C9H and C10H 3/C9H for the major tautomer.The C9H 2signals in the major tautomer appeared at a higher frequency (3.64and 4.12ppm)than tho of the minor tautomer (3.38and 3.79ppm)in the 1H-NMR spectra.The obrvations can be explained by the shielding effect of the dihydropyrone ring.The NMR spectroscopic data all support the foregoing discussions.
The absolute configuration at the C4stereogenic center of 2was finally determined by chemical deriva-tization.Epoxidation of 1with m CPBA and subquent acidic hydrolysis successfully afforded 2after prepara-tive TLC.The chromatographic behavior on TLC and the UV spectrum of synthetic 2w
ere identical to tho of natural 2.The synthetic sample also showed an identical 1H-NMR spectrum with a similar tautomeric ratio to tho of natural compound.The data con-firmed their relative stereochemical relationship.Addi-tionally,the sample prepared in this way provided an identical ECD spectrum to that of natural 2in CH 3CN.Since we had already established the (S )-configuration for 1,the experiments led us to conclude that the configuration of C4in natural 2would also be (S )-form.Compounds 1and 2were the C4-deoxy forms of previously reported isochromenones 7)that had been isolated from the same fungus,but in a shorter period of cultivation.Since biological and/or chemical degra-dation of condary metabolites commonly involves oxidation,the compounds might have been derived
by some other pathways.We assume that prolonging the incubation period would change the circumstances around the fungus to initiate their production.Com-pounds 1and 2showed weak inhibition of fungal hyphal-growth with respective IC 50values of ca.100and 250m g/mL against Cochliobolus miyabeanus .The abnce of a hydroxyl group at C4in 1greatly diminished the antifungal activity when compared to its oxy-analogue (R )-3,4-dihydro-4,8-dihydroxy-6-methoxy-4,5-dimethyl-3-methyleneisochromen-1-one (IC 50of 0.5m g/mL)in the previous study,suggesting the impor-tance of C4OH to this activity.
Novel isochromanone derivative 1was isolated from the culture broth of Leptosphaeria sp.KTC 727as just described.The structure of 1involving its absolute configuration was established by spectroscopic and conformational analys.We also found 2from the same culture broth.Although the planar structure of 2has already been reported,its stereochemical informa-tion had remained unknown.The results of the prent study have disclod its absolute structure by analyzing the chemical and ECD spectral correlations.
Experimental
General experimental procedures.The specific rotation values were measured with a HORIBA SEP-700spectrometer,and UV spectra were obtained with a Hitachi U-2010spectrophotometer.The CD spectra were recorded with a JASCO J-725spectropolarimeter,and measurements of the IR spectra were performed with a HORIBA FT-720spectrometer in a KBr cell.The 1H-(500MHz)and 13C-(125MHz)NMR spectra were recorded with a JEOL
JNM-ECX500
Fig.4.Most Stable Conformers of the (3R Ã,4S Ã)-and (3S Ã,4S Ã)-Tautomers of 2.
Table 1.
1
H-and 13
C-NMR Spectral Data (500MHz for 1H and 125MHz for
13
C,in CDCl 3)for 1and 2
Position 1
(3R ,4S )-2(major tautomer)(3S ,4S )-2(minor tautomer) C H (J in Hz) C H (J in Hz) C H (J in Hz)1167.0—168.7—169.0—3157.1—
103.1—
103.9—
434.8 3.82,q (7.2)36.0 3.27,q (7.0)35.4 3.21,q (6.9)5114.0—115.4—115.5—6164.8—164.9—164.8—797.4 6.38,s 97.57 6.39,s 97.64 6.36,s 8163.0—
163.1—
163.3—
995.8 4.80,d (1.8)65.6 4.12,d (11.9)66.5 3.79,d (12.1)
4.56,d (1.8) 3.64,d (11.9) 3.38,dd (7.1,12.1)4a 142.0—141.7—141.5—8a 98.5—
99.2—
98.5—
informC10H 322.2 1.38,d (7.2)15.6 1.15,d (7.0)14.5 1.29,d (6.9)C11H 39.9 2.08,s 10.1 2.08,s 10.0 2.06,s C12OH 355.8 3.86,s 55.84 3.86,s 55.79 3.86,s C3OH ——
— 4.72,s ——
C8OH —11.02,s —11.18,s —11.22,s行楷字体大全
C9OH
—
—
—
2.50,s
—
1.26,dd (7.1,9.2)
2392
W.C.T AYONE et al.
spectrometer.The methyl proton signal for tetramethylsilane(0ppm) was ud as the standard when
CDCl3was ud as the solvent while CHD2CN(1.93ppm)was ud for CD3CN.For CDCl3,77.0ppm was ud as the standard in the13C-NMR spectra.Electrospray ionization (ESI)MS spectra were obtained by a Hitachi NanoFrontier LD spectrometer.
Fungal material.Leptosphaeria sp.KTC727(JCM 130765)¼MAFF2395866))was collected from woody debris in Akaiwa,Rebun Island in Hokkaido,Japan on August30,2001.The fungal isolate has been deposited at the Japan Collection of Micro-organisms(JCM)and the Ministry of Agriculture,Forestry and Fisheries,Japan(MAFF).
Fermentation and isolation.Leptosphaeria sp.KTC727was cultured in200mL of a potato-sucro medium,which had been prepared from a potato extract(40g of potato),4g of sucro,and H2O,in eight500-mL Erlenmeyerflasks at25 C for55d on a rotary shaker(100rpm).Afterfiltration by suction,thefiltrate was extracted with EtOAc(1.0LÂ2)and concentrated in vacuo to give an extract (380mg)which was fractionated by silica gel column chromatography. The fraction eluted with30%EtOAc/hexane was further parated by silica gel column chromatography(20%EtOAc/hexane)to give1 (1.5mg)and2(13.9mg).
(S)-8-Hydroxy-6-methoxy-4,5-dimethyl-3-methyleneisochromen-1-one (1).Compound1was obtained
as a colorless oil,½ D10þ143(c0.15, CHCl3).UV max(CH3CN)nm("):215(25,100),268(10,700),313 (6,300).CD ext(6:4Â10À5M,CH3CN)nm(Á"):266(þ12.6),235 (þ15.2),213(À10.0)nm.IR max(film)cmÀ1:2973(C–H),1685 (C=O),1654(C=C),1091(C–O).The1H-and13C-NMR spectro-scopic data in CDCl3are shown in Table1.ESIMS m=zðMþHÞþcalcd.for C13H15O4,235.0965;found,235.0966.
3,8-Dihydroxy-3-hydroxymethyl-6-methoxy-4,5-dimethyl-isochroman-1-one(2).Compound2was obtained as a colorless oil,½ D23þ61 (c0.68,CHCl3).UV max(CH3CN)nm("):209(31,600),257(8,700), 296(4,600).CD ext(4:1Â10À5M,CH3CN)nm(Á"):260(þ2.1), 215(À4.8)nm.IR max(film)cmÀ1:3392(O–H),2971(C–H),1658 (C=O),1617(C=C),1078(C–O).ESIMS m=zðMþHÞþcalcd.for C13H17O6,269.1025;found,269.1023.The1H-NMR spectrum in CD3CN was consistent with the data in the literature.12)1H-and 13C-NMR data in CDCl3are shown in Table1.The1H-NMR spectrum in CDCl3indicated that the sample consisted of a7:1tautomeric mixture(e Supplemental Information;Biosci.Biotechnol.Biochem. Web site).
Preparation of2from1.A solution of1(1.2mg,5.1m mol)in CH2Cl2/H2O(900m L/100m L)was stirred with mCPBA(3.5mg, 21m mol)for2d at0 C.An aqueous solution of Na2S2O3(13mg in 10mL)was add
ed to decompo the excess oxidant.The mixture was then poured into H2O(20mL)and extracted with diethyl ether (20mLÂ3).The combined ethereal solution was washed with brine (20mL),dried over MgSO4,and concentrated in vacuo.Silica gel column chromatography of the residue(5%EtOAc/hexane)gave the corresponding epoxide(ca.200m g).1H-NMR(CDCl3) :1.42(3H,d, J¼7:2Hz,C10H3),2.05(3H,s,C11H3),2.92(1H,q,J¼7:2Hz, C4H), 2.96(1H,d,J¼4:0Hz,C9H H), 3.30(1H,d,J¼4:0Hz, C9H H),3.87(3H,s,C12O H3),6.42(1H,s,C7H),10.98(1H,s, C8O H).The epoxide thus obtained(ca.200m g)in THF/H2O(0.5mL/ 0.5mL)was stirred with HCl(12mol/dm3,10m L)for4h at room temperature.After H2O(10mL)had been added,THF in the mixture was removed in vacuo.The resulting mixture was extracted with EtOAc(20mLÂ3),and the combined organic layer was concentrated in vacuo.Preparative TLC of the residue(EtOAc/hexane,1:1)gave2. The yield of the product was estimated to be80m g bad on the UV absorption of the natural product at258and297nm in CH3CN.The chromatographic behavior,1H-NMR,and ECD spectra in CH3CN were identical to tho of natural2.
Molecular modeling calculations.Conformational arches for1,3, and both tautomers of2were performed with AM1mi-empirical molecular orbital calculations to give a ries of stable conformers.All conformers thus obtained were further optimized by the DFT method with B3LYP/6-3
1GÃon Spartan08.A calculated ECD curve was obtained by subjecting the most stable conformation of(4S)-1to time-dependent energy calculations for48excited states with B3LYP/ TZVP on Gaussian09.
Biological assay.Solutions of Cochliobolus miyabeanus spores were prepared containing500,250,100,50,10and5m g/mL in two replicates for each metabolite with2%sucro in DMSO.Germination and the shapes of the spores were obrved under a microscope after 36h at25 C.The IC50values were determined by the concentration which showed approximately50%inhibition of germination. Acknowledgment
This work was supported by Naito Foundation. W.C.T.thanks Dr.G.G.Lopez,President of Davao Oriental State College of Science and Technology (DOSCST),for allowing him to study in Japan. References and Notes
1)Honma M,Kudo S,Takada N,Miura T,and Hashimoto M,
Bioorg.Med.Chem.Lett.,20,709–712(2010).
2)Tayone WC,Shindo S,Murakami T,Hashimoto M,Tanaka K,
and Takada N,Tetrahedron,65,7464–7467(2009).
3)Kudo S,Murakami T,Miyanishi J,Tanaka K,Takada N,and
Hashimoto M,Biosci.Biotechnol.Biochem.,73,203–204 (2009).
4)Murakami T,Sasaki A,Fukushi E,Kawabata J,Hashimoto M,
and Okuno T,Bioorg.Med.Chem.Lett.,15,2587–2590(2005).
5)Riken Bioresource Center Japan Collection of Microorganisms,
www.jp/JCM/genomelist.shtml.
6)MAFF Gene Bank,www.biotech-hou.jp/glossary/
glos110.html.
7)Tayone WC,Honma M,Kanamaru S,Noguchi S,Tanaka K,
Nehira T,and Hashimoto M,J.Nat.Prod.,74,425–429(2011).
对酒当歌人生几何譬如朝露去日苦多8)Harada N and Nakanishi K,‘‘Circular Dichroic Spectroscopy-
Exciton Coupling in Organic Stereochemistry,’’University Science Books,Mill Valley(1983).
9)The RS expression is reverd by replacing the hydrogen atom
with a hydroxy group.
10)Berova N,di Bari L,and Pescitelli G,Chem.Soc.Rev.,36,914–
931(2007).
11)Kanamaru S,Honma M,Murakami T,Tsushima T,Kudo S,
Tanaka K,Nihei K,Nehira T,and Hashimoto M,Chirality,in press.
12)Schu¨ffler A,Liermann JC,Opatz T,and Anke T,Chembiochem,
12,148–154(2011).
Novel Isochromanone from Leptosphaeria sp.2393
