Novel cerebrosides isolated from the fermentation mycelia of tuber indicum

Article abstract of DOI:10.1002/hlca.201200235

The four new cerebrosides 1-4 possessing a unique C₁₈ 9-methylsphinga-4,8-dienine-related moiety and a cyclic octapeptide, 5, possessing alternating proline and glycine moietes were isolated from the Tuber indicum fermentation mycelium. Their structures were established on the basis of a spectroscopic analysis including NMR and HR-ESI-MS, as well as an acidic methanolysis experiment. To the best of our knowledge, the cerebrosides identified in the present study are quite different from those isolated from Tuber indicum fruiting bodies. Additionally, it was the first time that a cyclic peptide was isolated from the Tuber genus. Copyright

Full text of DOI:10.1002/hlca.201200235

702
Helvetica Chimica Acta – Vol. 96 (2013)
Novel Cerebrosides Isolated from the Fermentation Mycelia of Tuber indicum
by Yuan-Yuan Lia)b), Ping Liua), Yang Tanga), Hong-Mei Lia), Ya-Ling Tangc), Xin-Hua Liangc), and
Ya-Jie Tang*a)d)
a) Key Laboratory of Fermentation Engineering (Minister of Education), Hubei University of
Technology, Wuhan 430068, P. R. China (phone/fax: þ 86-27-88015108; e-mail: yajietang@hotmail.com)
b) Research Group for Bioactive Products, Department of Biology & Chemistry, City University of
Hong Kong, Kowloon, Hong Kong SAR, P. R. China
c) State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University,
Chengdu 610041, P. R. China
d) Laboratory of Synthetic Biology, Shanghai Advanced Research Institute, Chinese Academy of
Sciences, Shanghai 201203, P. R. China
The four new cerebrosides 1 – 4 possessing a unique C18 9-methylsphinga-4,8-dienine-related moiety
and a cyclic octapeptide, 5, possessing alternating proline and glycine moietes were isolated from the
Tuber indicum fermentation mycelium. Their structures were established on the basis of a spectroscopic
analysis including NMR and HR-ESI-MS, as well as an acidic methanolysis experiment. To the best of
our knowledge, the cerebrosides identified in the present study are quite different from those isolated
from Tuber indicum fruiting bodies. Additionally, it was the first time that a cyclic peptide was isolated
from the Tuber genus.
Introduction. – Truffles, the hypogeous fruiting bodies belonging to the Tuber
species, are a precious and expensive delicacy in the famous French and Italian cuisines.
Because of the decrease in the natural production and worldwide increase of demand,
submerged fermentation is viewed as a potential way to produce Tuber mycelia, which
could be considered as an alternative resource for its fruiting bodies [1][2]. For a better
understanding of this alternative resource, our group has identified and assayed some
known compounds from the Tuber fermentation mycelia, such as volatile organic
compounds [3][4], hydrosoluble nucleosides [5], and fatty acids [6]. In this study, we
aim to phytochemically search for some new compounds with special chemical
structures from the Tuber fermentation mycelium. Through solvent partition and
repeated column chromatography, compounds 1 – 5 (Fig. 1) were isolated, and their
structures were identified. Compounds 1 – 41) were cerebrosides, while 5 was a
cyclopeptide. To the best of our knowledge, cerebrosides 1 – 4 are new compounds, and
a cyclic peptide, i.e., 5, was isolated from the Tuber genus for the first time.
Results and Discussion. – An AcOEt extract (100 g) of the cultured mycelia was
separated by column chromatography and semi-preparative HPLC to afford the new
compounds 1 – 4, which were supposed to have analogous chemical skeletons, besides
the known cyclopeptide 5.
1
)
Arbitrary atom numbering; for systematic names, see Exper. Part.
ꢀ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 96 (2013)
703
Fig. 1. Compounds 1 – 5 isolated from the fermentation mycelia of Tuber indicum
Compound 1 was a white amorphous powder, whose molecular formula was
determined as C43H79NO9 by HR-ESI-MS (m/z 776.6001 ([M þ Na]þ, C43H79NNa O9þ ).
The IR spectrum of 1 showed absorption bands of OH groups at 3396 cmꢀ1, a glycoside
moiety (CꢀO) at 1083 cmꢀ1, a secondary amide group at 1536 and 1640 cmꢀ1, long
aliphatic chains at 2921, 1468, and 721 cmꢀ1, and an amide carbonyl group at 1644 and
1
1678 cmꢀ1. The H- and 13C-NMR spectra (Table) including the DEPT data further
indicated that 1 contained one amide carbonyl group (d(C) 174.2), five olefinic CH
groups (d(C) 123.7, 127.8, 129.8, 133.3, and 133.6), one quaternary olefinic C-atom
(d(C) 135.6), a series of oxygenated C-atoms (d(C) 61.5, 68.8, 70.4, 71.7, 72.9, 73.8, 76.7,
76.7, and 103.5), two terminal Me groups (d(H) 0.88 (t, J ¼ 13.8 Hz, 6 H)), and one
allylic Me group (d(H) 1.58 (s). For a better understanding of the detailed planar
structure of 1, 2D-NMR experiments including HMBC and 1H,1H-COSY were
performed (Fig. 2). A series of HMBCs, including d(C) 103.5 (C(1’’))/d(H) 3.18
(HꢀC(2’’)), d(C) 76.7 (C(3’’))/d(H) 3.18 (HꢀC(2’’)), d(C) 73.8 (C(4’’))/d(H) 3.26
(HꢀC(5’’)), d(C) 76.7 (C(5’’))/d(H) 3.66 and 3.85 (HaꢀC(6’’) and HbꢀC(6’’)),
confirmed the hexose residue in the molecule. The cross-peak d(C) 103.5 (C(1’’))/
d(H) 4.11 and 3.69 (HaꢀC(1) and HbꢀC(1)) further suggested that the hexose residue
1
was attached to a long-chain base at C(1). The H,1H-COSY cross-peaks, including
d(H) 5.46 (HꢀC(4))/5.70 (HꢀC(5)), 5.70 (HꢀC(5))/2.02 (CH2(6)), 2.02 (CH2(6))/5.13
(HꢀC(8)), and 2.02 (CH2(7))/5.13 (HꢀC(8)), unambiguously assigned the position of
the C¼C bonds between C(4) and C(5) and C(8) and C(9) of the long-chain base.
Additionally, the position of a Me group at C(9) was also evidenced by the HMBC
spectrum, in which the allylic Me group at d(H) 1.58 (Me(18)) was correlated with the
olefinic C-atoms at d(C) 123.7 (C(8)) and 135.6 (C(9)) and the CH2 group at d(C) 39.6
(C(10)). The assignment of an OH group at C(3) was supported by the HMBCs d(H)
4.11 (HꢀC(3))/d(C) 133.6 (C(4)) and 129.8 (C(5)). Finally, because the OH group at
d(H) 4.42 (HꢀC(2’)) displayed an HMBC cross-peak with the C¼O group at d(C) 174.2

704
Helvetica Chimica Acta – Vol. 96 (2013)

Helvetica Chimica Acta – Vol. 96 (2013)
705
Fig. 2. Key features observed in the HMBC (H ! C) and 1H,1H-COSY (—) plot of compound 11)
(C(1’)) and 1H,1H-COSY cross-peaks with the olefinic CH groups at d(H) 5.49
(HꢀC(3’)) and 5.82 (HꢀC(4’)), a fatty 2-hydroxyall-3-enoic acid moiety was suggested
to exist in the molecule. Based on the above conclusions, compound 1 was considered as
a
cerebroside with
{(1S,2R,3E,7E)-1-[(b-d-glucopyranosyloxy)methyl]-2-hydroxy-8-methylnonadeca-3,7-
dien-1-yl}-2-hydroxyhexadec-3-enamide) [7] and cerebroside
(¼(2R,3E)-N-
a
constitution similar to asperamide
B
(¼(2R,3E)-N-
A
{(1S,2R,3E,7E)-1-[(b-d-glucopyranosyloxy)methyl]-2-hydroxy-8-methylheptadeca-
3,7-dien-1-yl}-2-hydroxyhexydec-3-enamide) [8], both of which were isolated from the
Pachybasium species. To establish the chain length of the fatty acid and sphingosine-
related moieties (sphingenine ¼ sphingosine ¼ (2S,3R)-2-aminooctadec-4-ene-1,3-diol)
and the configuration of the sugar moiety, 1 was subjected to acidic methanolysis to
provide three fractions containing a fatty acid methyl ester 1a, a sphingoid base 6, and a
methyl glycoside 7 (Scheme). The fraction containing 1a was analyzed by GC/MS
which revealed the characteristic fragment-ion peaks at m/z 326 (Mþ) and 267 ([M ꢀ
COOMe]þ), suggesting the presence of a C19 fatty acid moiety in 1. The fraction
Scheme. Methanolysis of Compounds 1 – 4

706
Helvetica Chimica Acta – Vol. 96 (2013)
containing 6, analyzed by LC/ESI-MS, showed the quasi-molecular ion of the main
peak at m/z 298 ([M þ H]þ), indicating that 18 C-atoms were contained in the
sphingoid-base moiety of 1. Compound 7 had an optical rotation [a]2D5 ¼ þ75.6 (c ¼ 0.2,
MeOH), which was quite near to that of methyl b-d-glucoside ([a]2D5 ¼ þ77.4 (c ¼ 0.2,
MeOH) [9], suggesting that the hexose contained in 1 was a b-d-glucose residue. The
coupling constant J ¼ 7.8 Hz ( Table) for the anomeric proton HꢀC(1’’) also supported
this conclusion [7][10]. The configuration of 1 was deduced by comparing the NMR
and optical rotation data with those of analogs in [7][9][11][12]. Except for the overlap
of the proton signals of HaꢀC(1) and HꢀC(3), HaꢀC(1) (d(H) 4.11 (overlap)) and
other H-atoms, including HbꢀC(1) (d(H) 3.69 (dd, J ¼ 10.3, 3.6 Hz)), HꢀC(2) ((d(H)
3.94 – 3.96 (m)), HꢀC(3) (d(H) 4.11 (overlap)), and HꢀC(2’) (d(H) 4.42 (d, J ¼
5.4 Hz)), presented chemical shifts and coupling constants very similar to those of
asperamide B [7]. Furthermore, the specific optical rotation of 1 ([a]2D5 ¼ ꢀ1.8) was
also very close to that of asperamide B ([a]2D5 ¼ ꢀ1.2) [7]. Therefore, 1 may contain the
same (2S,2’R,3R)1) moiety as that present in asperamide B [7]. The geometry of the
C¼C bonds of 1 was established by comparing its NMR data with literature: the values
of the vicinal coupling constants were J ¼ 15.0 Hz for the protons of CH(4)¼CH(5) and
CH(3’)¼CH(4’) compatible with (3’E,4E)1) configuration [12][13]. Additionally,
according to previous experiences, an Me group at an (E)-C¼C bond always appears
at d(C) 15.4, while that at to a (Z)-C¼C bond appears at d(C) 22.7 [9]. Thus, the
chemical shift of Me(18) at d(C) 14.9 (Table) indicated that the C(8)¼C(9) bond was
(E)-configured. Putting all these points together, the structure of 1 was established as
(2S,3R,4E,8E)-2-{[(2R,3E)-2-hydroxynonadec-3-enoyl]amino}-9-methylheptadeca-
4,8-diene-1,3-diol 1-(b-d-glucopyranoside)1).
The molecular formula of 2 was determined as C45H83NO9 by HR-ESI-MS (m/z
782.6108 ([M þ H]þ, C45H84NO9þ )). No significant difference was found in the IR and
NMR spectra of 1 and 2 (Table and Exper. Part). This indicates that 2 is an analogue of
1. Compounds 1 and 2 only differed from each other in the different number of C-atoms
in the aliphatic fatty acid chain. The acidic methanolysis of 2 (Scheme) revealed the
characteristic fragment 6 arsing from of the sphingoid-base moiety by LC/ESI-MS (m/z
298 ([M þ H]þ), while the characteristic fragment 2a arising from the fatty acid moiety
was determined by GC/MS (m/z 354 (Mþ) and 295 ([M ꢀ COOMe]þ)). These results
indicated that 2 contained the same sphingosine-related moiety as 1, while 21 C-atoms
were present in its fatty acid moiety. Therefore, compound 2 was established as
(2S,3R,4E,8E)-2-{(2R,3E)-2-hydroxyheneicos-3-enoyl]amino}-9-methylheptadeca-4,8-
diene-1,3-diol 1-(b-d-glucopyranoside)1).
The molecular formula of 3 was determined as C42H79NO9 by HR-ESI-MS (m/z
742.5345 ([M þ H]þ, C42H80NO9þ )). The H- and 13C-NMR spectra of 3 (Table) were
1
almost the same as that of 1 and 2, except for the absence of one C¼C bond. The acidic
methanolysis (Scheme) showed that compound 3 contained the same sphingosine-
related and sugar moiety as 1 and 2 and that the fatty acid moiety was derived from a
saturated aliphatic acid. GC/MS Analysis of 3a revealed characteristic ions at m/z 314
(Mþ) and 255 ([M ꢀ COOMe]þ), confirming proved the existence of a saturated fatty
acid moiety, i.e., 2-hydroxyoctadecanoyl, in 3. Therefore, compound 3 was established
as (2S,3R,4E,8E)-2-{(2R)-2-hydroxyoctadecanoyl]amino}-9-methylheptadeca-4,8-di-
ene-1,3-diol 1-(b-d-glucopyranoside)1).

Helvetica Chimica Acta – Vol. 96 (2013)
707
The molecular formula of 4 was determined as C45H85NO9 by HR-ESI-MS (m/z
1
784.6273 ([M þ H]þ, C45H86NO9þ )). As shown in the Table, the H- and 13C-NMR
spectra indicated that 4 was an analog of 1 – 3. In addition to that, compound 4
contained only two C¼C bonds. The acidic methanolysis (Scheme) showed that
compounds 3 and 4 were different from each other only in the chain length of the fatty
acid moiety. The identified characteristic ion of 4a in the GC/MS (m/z 356 (Mþ) and
297 ([M ꢀ COOMe]þ)) further indicated that the fatty acid moiety contained 21 C-
atoms. Therefore, the structure of 4 was established as (2S,3R,4E,8E)-2-{(2R)-2-
hydroxyheneicosanoyl]amino-9-methylheptadeca-4,8-diene-1,3-diol 1-(b-d-glucopyra-
noside)1).
Thus from the T. indicum fermentation mycelia, four novel cerebrosides were
isolated, and their structures were identified. Their common characteristic is that they
all contained a C18 9-methylsphinga-4,8-dienine-related and b-d-glucopyranosyl moiety
(sphinga-4,8-dienine ¼ (2S,3R,4E,8E)-2-aminooctadeca-4,8-diene-1,3-diol). They dif-
fered from each other in their fatty acid moiety with either a different number of C¼C
bonds or with a different chain length. Although some similar cerebrosides or
ceramides have been isolated from T. indicum fruiting bodies, their reported structures
are different from those of the cerebrosides investigated in this study by the number
and position of C¼C bonds and by the chain length of the sphingoid-base or fatty acid
moiety [14]. Besides cerebrosides, the cyclic octapeptide 5 composed of l-proline and
glycine was also isolated, and its structure was identified as cyclo(l-Pro-Gly)þ based on
the previously described spectral data [15]. To the best of our knowledge, it is the first
time that a cyclic peptide has been found in the Tuber genus. Furthermore, it is very
interesting that although Tuber indicum belongs to a higher fungus, compounds 1 – 5
were more similar to the constituents isolated from lower organisms, including the
unique marine protist Thraustochytrium globosum [16], the marine sponge Prosuber-
ites laughlini [17], the marine fungus Aspergillus niger EN-13 [7], an imperfect fungus
Pachybasium sp. [8], and the basidiomycetes Polyporus ellisii and Cortinarius
umidicola [12].
Financial support from the National Natural Science Foundation of China (NSFC, projects
No. 20976038 and 21176059), the Key Project of Chinese Ministry of Education (project No. 210132), the
Hubei Provincial Natural Science Foundation for Innovative Research Team (project No. 2008CDA002),
the Scientific Research Key Project of Hubei Provincial Department of Education (project
No. Z20101401), the Discipline Leader Project of Wuhan Municipality (project No. 200951830553),
the Key Technology R&D Program of Wuhan Municipality (project No. 201120822280-2), the Open
Project Programs for the Key Laboratory of Fermentation Engineering (Ministry of Education), the
National Key Laboratory of Biochemical Engineering (project No. 2010KF-06), and the State Key
Laboratory of Bioreactor Engineering is gratefully acknowledged. Y.-J.T. also thanks the Chutian Scholar
Program (Hubei Provincial Department of Education, China; 2006).
Experimental Part
General. All solvents of anal. grade were purchased from the BoDi Chemical Factory (Tianjin,
China). Colum chromatography (CC): silica gel (flash CC; SiO2; (200 – 300 mesh; Qingdao Marine
Chemical Factory (Qingdao, China)); Sephadex-LH-20 gel (Amersham Pharmacia Biotech China Ltd.
(Shanghai, China)). TLC: SiO2 G (Qingdao Marine Chemical Factory); pre-coated SiO2 G plates (Merck
Inc. (Darmstadt, Germany)). Prep. medium-pressure liquid chromatography (MPLC): Bꢀchi-B-608

708
Helvetica Chimica Acta – Vol. 96 (2013)
system (Flawil, Switzerland). Prep. HPLC: Waters-600 instrument coupled with a 2487 multiple-wave
detector; ODS column (10 ꢁ 250 mm, 5 mm; Agela Technologies Inc. (Beijing, China)); 100% MeOH as
mobile phase (4 ml/min). Optical rotations: Perkin-Elmer-314 polarimeter. IR (KBr) Spectra: Nicolet-
Nexus-670 FT-IR spectrometer; ˜n in cmꢀ1 1H-, 13C-, and 2D-NMR Spectra: Varian-Mercury NMR
.
instrument (600 and 400 MHz); d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Bruker-
Bio-TOF-Q-plus mass spectrometer (Bruker Daltonics Ltd., Coventry, UK). GC/ESI-MS: Shimadzu-
GC/MS-QP-2010-plus mass spectrometer (Tokyo, Japan) and Agilent-7890A GC system equipped with a
5975C quadrupole MS detector (Agilent Technologies, Inc., CA, USA). LC/ESI-MS: Agilent-1200 HPLC
system equipped with an ion-trap MS (Agilent Technologies, Inc., CA, USA).
Mycelia Material. The strain of Tuber indicum was purchased from the Mianyang Institute of Edible
Fungi (Sichuan, China). Fermentation mycelia were cultured in our laboratory according to the
procedure described previously [1][2]. The harvested mycelia were rinsed with deionized water to
remove the culture medium. The clean mycelia was stored at ꢀ 208 before it was used.
Extraction and Isolation. First, the mycelia (ca. 3 kg) was extracted by EtOH, and then the extract
was divided into three fractions by extracting with hexane, AcOEt, and 95% EtOH. The AcOEt fraction
(100 g) was subjected to CC (SiO2, CHCl3/MeOH 99 :1, 90 :10, 85 :15, 80 :20, and 70 :30): Fractions A – F
(TLC monitoring). Fr. D (ca. 5.0 g) was subjected to CC (SiO2, CHCl3/MeOH 85 :15): 5 (15.0 mg) and a
mixture of cerebrosides (ca. 35.3 mg). The cerebroside mixture was further separated by semi-prep.
HPLC: 1 (15.2 mg), 2 (10.2 mg), 3 (3.2 mg), and 4 (5.1 mg).
(2R,3E)-N-{(1S,2R,3E,7E)-1-[(b-d-Glucopyranosyloxy)methyl]-2-hydroxy-8-methylhexadeca-3,7-
dien-1-yl}-2-hydroxynonadec-3-enamide (1): Amorphous powder. [a]2D5 ¼ ꢀ1.8 (c ¼ 0.2, MeOH). IR
(KBr): 3396, 2921, 2985, 1640, 1536, 1467, 1082. 1H- and 13C-NMR: Table. HR-ESI-MS: 776.6001 ([M þ
Na]þ, C43H79NO9Naþ; calc. 776.5653).
(2R,3E)-N-{(1S,2R,3E,7E)-1-[(b-d-Glucopyranosyloxy)methyl]-2-hydroxy-8-methylhexadeca-3,7-
dien-1-yl}-2-hydroxyheneicos-3-enamide (2): Amorphous powder. [a]2D5 ¼ ꢀ2.0 (c ¼ 0.2, MeOH). IR
(KBr): almost identical to that of 1. 1H- and 13C-NMR: Table. HR-ESI-MS: 782.6108 ([M þ H]þ,
C45H84NOþ9 ; calc. 782.6146).
(2R)-N-{(1S,2R,3E,7E)-1-[(b-d-Glucopyranosyloxy)methyl]-2-hydroxy-8-methylhexadeca-3,7-di-
en-1-yl}-2-hydroxyoctadecanamide (3): Amorphous powder. [a]2D5 ¼ þ0.8 (c ¼ 0.2, MeOH). IR (KBr):
1
almost identical to that of 1. H- and 13C-NMR: Table. HR-ESI-MS: 742.5345 ([M þ H]þ, C42H80NOþ9 ;
calc. 742.5833).
(2R)-N-{(1S,2R,3E,7E)-1-[(b-d-Glucopyranosyloxy)methyl]-2-hydroxy-8-methylhexadeca-3,7-di-
en-1-yl}-2-hydroxyheneicosanamide (4): Amorphous powder. [a]2D5 ¼ þ1.3 (c ¼ 0.2, MeOH). IR (KBr):
1
almost identical to that of 1. H- and 13C-NMR: Table. HR-ESI-MS: 784.6273 ([M þ H]þ, C45H86NOþ9 ;
calc. 784.6303).
Cyclo(glycyl-l-prolylglycyl-l-prolylglycyl-l-prolylglycyl-l-prolyl) (Cyclo(l-Pro-Gly)4 ; 5): Colorless
amorphous powder. [a]2D5 ¼ ꢀ60.2 (c ¼ 0.2, CHCl3). IR (KBr): 3204, 3113, 1678, 1644, 1457, 1304, 1296,
1005, 794, 500. 1H-NMR: 1.86 (m); 2.01 (m); 2.32 (m); 3.50 (dd, J ¼ 9.3, 4.5); 3.57 (dd, J ¼ 9.6, 5.7); 3.83
(dd, J ¼ 8.4, 2.1); 4.02 (overlap). 13C-NMR: 22.6; 28.7; 45.6; 46.9; 58.8; 163.7; 170.1. HR-ESI-MS:
617.3036 ([M þ H]þ, C28H41N8Oþ8 ; calc. 617.3047).
REFERENCES
[1] Y. J. Tang, L. L. Zhu, D. S. Li, Z. Y. Mi, H. M. Li, Process Biochem. 2008, 43, 576.
[2] R. S. Liu, D. S. Li, H. M. Li, Y. J. Tang, Process Biochem. 2008, 43, 868.
[3] Y. Y. Li, G. Wang, H. M. Li, J. J. Zhong, Y. J. Tang, Food Chem. 2012, 135, 2628.
[4] Y. J. Tang, G. Wang, Y. Y. Li, J. J. Zhong, Anal. Chim. Acta 2009, 647, 40.
[5] P. Liu, Y. Y. Li, D. J. Wan, Y. J. Tang, Anal. Chim. Acta 2011, 687, 159.
[6] Y. Tang, Y.-Y. Li, H.-M. Li, D.-J. Wan, Y. J. Tang, J. Agric. Food Chem. 2011, 59, 4736.
[7] Y. Zhang, S. Wang, X. M. Li, C. M. Cui, C. Feng, B. G. Wang, Lipids 2007, 42, 759.
[8] R. D. Sitrin, G. Chan, J. Dingerdissen, C. DeBrosse, R. Mehta, G. Roberts, S. Rottschaefer, D.
Staiger, J. Valenta, K. M. Snader, R. J. Stedman, J. R. E. Hoover, J. Antibiot. 1988, 41, 469.

Helvetica Chimica Acta – Vol. 96 (2013)
[9] J. K. Liu, L. Hu, Z. J. Dong, Lipids 2003, 38, 669.
709
[10] X. Chen, Y. L. Wu, D. Chen, Tetrahedron Lett. 2002, 43, 3529.
[11] J. H. Chen, G. Y. Cui, J. Y. Liu, R. X. Tan, Phytochemistry 2003, 64, 903.
[12] J. M. Gao, Z. J. Dong, J. K. Liu, Lipids 2001, 36, 175.
[13] Z. J. Zhan, J. M. Yue, J. Nat. Prod. 2003, 66, 1013.
[14] J. M. Gao, W. M. Zhu, S. Q. Zhang, X. Zhang, A. L. Zhang, H. Chen, Y. Y. Sun, M. Tang, Eur. J.
Lipid Sci. Technol. 2004, 106, 815.
[15] V. Madison, M. Atreyi, C. M. Deber, E. R. Blout, J. Am. Chem. Soc. 1974, 96, 7566.
[16] K. M. Jenkins, P. R. Jensen, W. Fenical, Tetrahedron Lett. 1999, 40, 7637.
[17] J. Vicente, B. Vera, A. D. Rodrꢂguez, I. Rodrꢂguez-Escudero, R. G. Raptis, Tetrahedron Lett. 2009,
50, 4571.
Received May 8, 2012