J Basic Microbiol 42 (2002) 2, 133 – 136 Short Note (The Centre for Mycological Research of National Hanoi University, 334 Nguyen Trai Str., Dong Da – Hanoi, Vietnam; and Centre of Biotechnology, VNU, 144 Xuan Thuy, Hanoi, Vietnam; and 1HansKnöll-Institute for Natural Products Research, Beutenbergstr 11 a, D-07745 Jena, Germany) New homoserine lipids from Xerocomus langbianensis T T KIET, B SCHLEGEL1 and U GRÄFE1, * (Recieved 12 October 2001/Accepted 26 November 2001) New diacylglycerotrimethyl homoserine lipids (2 O,3 O-bislinolylglycero-4′-O-(N,N,N-trimethyl)homoserine) and (2 O,3 O-linolyl-palmitylglycero-4′-O-(N,N,N-trimethyl)-homoserine) were isolated from the fruiting body of a Vietnamese species of Xerocomus langbianensis The chemical structures were settled on the basis of mass spectrometry and NMR spectroscopy Edible mushrooms (basidiomycetes, ascomycetes) are known as a rich source of lipophilic material such as unsaturated fatty acids and terpenes (CAVAZZONI et al 1987, ANKE et al 1996, KIET et al 1999, PANG et al 1991) These cellular constituents are essential for the maintainance of cellular functions and may contribute to the flavour of the mushrooms and their nutritional value (BUCHBAUER et al 1993) In comparison with fatty acids and terpenoids much less information is available about polar fungal lipids As an example of nitrogen-containing lipids of basidiomycetes the cerebrosides A, B, C and D were reported to occur in Microsphaeropsis sp., Shizophyllum commune and Humicola sp (KEUSGEN et al 1996, SITRIN et al 1988, WICKLOW et al 1998) 2-O,3-O-diacylglycero-methyl-homoserines represent another rare class of nitrogencontaining lipids of marine algae (KATO et al 1996) and basidiomycetes (VASKOVSKY et al 1998) However, the structural diversity of homoserine-type lipids and their occurrence in other organisms was not investigated with the excerption of ulaline (O-(1-glyceryl)-N,N,Ntrimethylhomoserine) which was isolated as a non-acylated basic structure from both the marine plant Monostroma nitidum and the mushroom Lampteromyces japonicus (CHAPMAN and HALL 2001) A fruiting body of Xerocomus langbianensis was harvested near Da Lat (Southern of Central Vietnam; KIET et al 2001) and was lyophilized to yield 12 g dry material It was extracted for five consecutive times over a 24 hours period using 500 ml of CHCl3/MeOH (1 : 1; v/v) and the combined extracts were evaporated in vacuo The residue (2.4 g) was subjected to column chromatography on silica gel 60 (0.063 – 0.1 mm; column cm × 80 cm) The components of the mixture were eluted in order of their polarity by l CHCl3, 500 ml CHCl3/MeOH (9 : 1) and 500 ml CHCl3/MeOH (8 : 2), and 20 ml fractions were collected Samples of the eluate fractions were spotted on TLC sheets (Merck, silica gel 60), the chromatograms were developed by CHCl3/MeOH (9 : 1) and stained subsequently by 1% vanillin in conc H2SO4 Elution by CHCl3 and CHCl3/MeOH (9 : 1), respectively, furnished fractions containing terpenoids, sterols, fatty acids and cerebrosides and were eluted by CHCl3/MeOH (8 : 2; v/v) near the end of the chromatographic procedure They were detected by reddish staining with the above reagent and further purified by preparative TLC on silica gel 60 (aluminium sheets, Merck, CHCl3/MeOH, : 1, v/v) to yield 14.5 mg and 9.2 mg * Corresponding author Prof Dr U GRÄFE; e-mail: ugraefe@pmail.hki-jena.de WILEY-VCH Verlag Berlin GmbH, 13086 Berlin, 2002 0233-111X/02/0205-0133 $ 17.50+.50/0 134 T T KIET et al The chemical constitution of and as shown in Fig was elucidated by ESI-MS, HREI-MS and 1D/2D NMR spectroscopy The IR spectra of and showed the presence of carbonyl groups (1729 cm–1), double bonds (1622 cm–1) and hydroxyl (3386 cm–1) The negative ion ESI-MS of displayed m/z 794.1 [M + Cl– H]– and that of m/z 770.8 [M + Cl– H]– The chemical formulas [C46H82NO7]Cl for and [C44H82NO7]Cl for were afforded by HREI-MS (1: m/z 759.5989 [M–HCl]+, calcd 759.6013 for C46H81NO7; 2: m/z 735.6077 [M– HCl]+, calcd 735.6013 for C44H81NO7) The appearance of the chlorinecontaining cluster ion in the negative ion ESI-MS confirmed the occurrence of quaternary ammonium salts in both and Conclusive evidence for the chemical constitution of and was furnished by 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, NOESY, TOCSY, HSQC, HMBC) Thus in the 13C and DEPT NMR spectra of and signals of three carbonyl groups (166.7, 172.2 and 172.5 ppm), and six heteroatom-bonded singlet, doublet and triplet carbon signals were visible (50.5 (s), 62.5 (d), 67.9 (t), 68.2 (t), 69.7 (d), and 75.2 (d)) The signal intensity at 50.5 ppm was increased strongly due to the presence of three equivalent methyl O R2 R1 3' O 4' 1' 2' 5' H3C OH + N 7' CH CH3 6' R1 = A; R2 = A R1 = A, R2 = B or R1= B, R2 = A O A= 9" O 5" 3" 1" 2" 4" 10" 12" 13" 7" 6" 17" 15" 8" 11" 14" O B= O Fig Chemical constitution of homoserine lipids and from Xerocomus langbianensis 16" CH3 18" New homoserine lipids from Xerocomus langbianensis 135 groups Two strong olefinic carbon signals at 128.0 ppm (d) and 130.0 ppm (d) were attributable to the linolic acid residues A bulk of methylene carbon signals around 28 ppm was assignable to the aliphatic part of the side chains The 13C NMR spectrum of appeared as very similar except for the smaller intensity of the olefinic and carbon-bonded methylene signals The 1H NMR and COSY spectra unvailed the sequence of protons in the homoserine and fatty acid moieties of and The increased intensity of the proton signals at 3.08 ppm (H-5′, H-6′, H-7′, singlet), 5.28 ppm (multiplet H-9″, H-13″) and 5.31 ppm (H-10″, H-12″) in the 1H NMR spectrum of suggested the presence of identical moieties Instructive C,H-heteronuclear long-range couplings (HMBC) confirmed the chemical constitutions of and as shown in Fig Thus the observable 3JC, H couplings between C-1/H-4′ and C-4/H-1′, respectively, and the NOE’s between H-1/ H-4′ in the NOESY spectrum confirmed the ether linkage between homoserine and glycerol Supporting evidence for the chemical constitutions of and was furnished by positive ion ESI-CID-MS/MS spectra showing m/z 263 as a diagnostic fragment (C18H31O+) due to the carbon-heteroatom cleavage of the linolic acid ester However, due to the nearly identical chemical shift data of C-1″/H-2″ and C-1′′′/H-2′′′, respectively, assignment of the long-chain fatty acid residues A and B in to either O-2 or O-3 of the glycerol moiety was not possible Also, the absolute sterochemistry at C-2 and C-2′ in Fig was not determined and thus appear as new representatives of the homoserine-type glycerolipids They are distinguishable from the hitherto know structures by the presence of long-chain fatty acids such as linolic and palmitic acid Experimental IR spectra were recorded on a Mattson Sattelite FTIR instrument (Mattson, USA), HREIMS and HRESI-MS on a Finnigan MAT 95XL, ESI-MS and ESI-CID-MS/MS on a triple quadrupole mass spectrometer Quattro (VG Biotech, Altrincham, England) and NMR spectra on a Bruker Avance DRX 500 NMR spectrometer 1,2-Dilinolylglycero-N4′-(N,N,N-trimethyl)homoserine (1): Appearance: wax; Molecular weight: 795 Formula: C46H82NO7Cl HRESI-MS: m/z 794.5 ([M + Cl– H]–) HREI-MS: m/z 759.5989 ([M– HCl]+; calcd 759.6013 for C46H81NO7) Rf on TLC (silica gel aluminium sheets Merck, CHCl3/MeOH, :1): 0.2; [a]D22 (4.1 mg in ml MeOH; 0.5 cm): + 12.2° IR (film; λmax; cm–1): 802, 823, 963, 1085, 1117, 1172,1244, 1307, 1363, 1457, 1622, 1729, 2863, 2923, 3386 1H-NMR (500 Hz, in DMSO-d , TMS as internal standard; δ in ppm): 0.85 (H-18″, t, 7.1 Hz, H), 1.22 (H-4″, H-5″, H-6″, H-7″, H-15″, H-16″, m, 20 H, 1.25 (H-17″, m, H), 1.26 (H-3″, m, H), 1.50 (H-2A″, m, H), 1.80 (HA-3′, m, H), 2.00 (H-11″, m, H), 2.10 (HB-3′, m, H), 2.24 (H-2B″, m, H), 3.08 (H-5′, H-6′, H-7′, s, H), 3.38 (H-2′, dd, 15.2 Hz, 2.1 Hz, H), 3.48 (H-4′, dd, 16.1 Hz, 2.5 Hz, H), 3.50 (H-1, dd, 6.1 Hz, 12.0 Hz, H), 4.10 (HA-3, dd, 12.0 Hz, 7.1 Hz, H), 4.28 (HB-3, dd, 12.0 Hz, 3.1 Hz, H), 5.1 (H-2, m, H), 5.28 (C-9″ and C-13″, m, H), 5.30 (C-10″ and C-13″, m, H) 13 C-NMR (125 MHz, in DMSO-d6, TMS as internal standard, δ in ppm): 13.9 (C-18″, q), 21.9 (C-16″, C-15″, t), 24.8 (C-3″, t), 25.6 (C-8″, C-14″), 26.6 (C.-11″, t), 27.3 (C-3′, t), 28.2 (C-4″, C-5″, C-6″, C-7″, t), 28.8 (C-17″, t), 34.1 (C-2″, t), 50.5 (C-5′, q), 50.5 (C-6′, q), 50.5 (C-7′, q), 62.5 (C-3, t), 67.9 (C-4′, t), 68.2 (C-1, t), 69.7 (C-2, d), 75.2 (C-2′, d), 128,0 (C-10″, C-12″, d), 130.0 (C-9″, C-13″, d), 166.7 (C-1′, s), 172.2 (C-1″, s) 1(2)-linolyl-2(1)-palmityl-glycero-O-4'-(N,N,N-trimethyl)homoserine (2): Appearance: wax, Molecular weight: 771, formula [(C44H82NO7]+Cl ESI-MS: m/z 770.8 ([M + Cl– H]–) HREI-MS: m/z 735.6077 ([M-HCl]+, calcd 735.6013 for C44H81NO7) Rf on TLC (silica gel aluminium sheets Merck, CHCl3/MeOH, :1): 0.3 136 T T KIET et al H-NMR and 13C-NMR (conditions as described for 1): the chemical shift data of protons and carbons were identical with the data of However, the olefinic proton and carbon signals displayed reduced intensity due to the presence of only one linolic acid residue The bulk of aliphatic methylene proton signals at 1.25 ppm and of methylene carbons at 27.5 ppm was increated Acknowledgements Support of this work by DLR Bonn (project VIE 008-37) and FCI Frankfurt/Main is gratefully acknowledged References ANKE, H., MORALES, P and STERNER, O., 1996 Assays of the biological activities of two fatty acid derivatives formed in the edible mushrooms Cantharellus cibarius and C tubereformis is a response to injury Planta Med., 62, 181 – 183 BUCHBAUER, G., JIROVEK, L., WASICKY, M and NIKIFOROV, A., 1993 The aroma of edible mushrooms Headspace analysis with GC/FID and GC/FTIR/MS Z Lebensm Unters Forsch., 197, 429 – 433 CAVAZZONI, V., ADAMI, A., ARAGOZZINI, F and CRAVAI, R., 1987 Fatty acid composition of mycelial biomasses of some mushrooms Lebensmittel Wiss Technol., 20, 133 – 136 CHAPMAN and HALL Dictionary of Natural Products on CD-ROM 1982 – 2001, CHAPMAN and HALL Publishers KATO, M., SAKAI, M., ADACHI, K., IKEMOTO, H and SANO, H., 1996 Distribution of betaine lipids in marine algae Phytochemistry, 42, 1341 – 1345 KEUSGEN, M., CHAO-MEI, YU, CURTIS, J M., BREWER, D and AYER, S W., 1996 A cerebroside from the marine fungus Microsphaeropsis olivacea (BOUORD.) HÖHN Biochem System Ecology, 24, 465 – 468 KIET, T T., DÖRFELT, H., RITZAU, M., HEINZE, S and GRÄFE, U., 1999 14-Hydroxy-12-oxo10E,13E,15E-octadecatrienoic acid, a new fatty acid from a Vietnamese mushroom, Cantharellus friesii QUEL J Basic Microbiol., 39, 25 – 28 KIET, T T and DÖRFELT, H., 2001 Neue Pilzfunde von Vietnam und deren systematische und mykogeographische Bedeutung Intern Biol Congr Hanoi, 253 – 255 PANG, Z and STERNER, O., 1991 Cibaric acid, a new fatty acid derivative formed enzymatically in damaged fruit bodies of Cantharellus cibarius (CHANTERELLE) J Org Chem., 56, 1233 – 1235 SITRIN, R D CHANG, G., DINGERDISSEN, DEBROSSE, MEHTA, R., ROBERTS, G., ROTTSCHAEFER, S., STAIGER, D., VALENTA, J., SNADER, K M., STEDMAN, R J and HOOVER, J R E., 1988 Isolation and structure determination of Paclybasium cerebrosides which potentiate the antifungal activity of aculeacin J Antibiot., 41, 469 – 480 VASKOVSKY, V E., KHOTIMCHENKO, S V and BOOLUKH, E M., 1998 Distribution of diacylglycerotrimethylhomoserine and phosphatidylserine in mushrooms Phytochemistry, 47, 755 – 760 Mailing address: Prof Dr U GRÄFE, Hans-Knöll-Institut für Naturstoff-Forschung, Beutenbergstr 11 a, D-07745 Jena, Germany ... constitution of homoserine lipids and from Xerocomus langbianensis 16" CH3 18" New homoserine lipids from Xerocomus langbianensis 135 groups Two strong olefinic carbon signals at 128.0 ppm (d)... absolute sterochemistry at C-2 and C-2′ in Fig was not determined and thus appear as new representatives of the homoserine- type glycerolipids They are distinguishable from the hitherto know structures... long-chain fatty acids such as linolic and palmitic acid Experimental IR spectra were recorded on a Mattson Sattelite FTIR instrument (Mattson, USA), HREIMS and HRESI-MS on a Finnigan MAT 95XL,