Activity and Structure Elucidation of Ceramides

Send Orders of Reprints at [email protected] Current Bioactive Compounds 2012, 8, 370-409

Activity and Structure Elucidation of Ceramides

E. S. Elkhayata*, G. A. Mohameda and S. R. M. Ibrahimb

aFaculty of Pharmacy, Department of Pharmacognosy, Al–Azhar University, Assiut, 71524 Egypt; bFaculty of Phar-macy, Department of Pharmacognosy, Assiut University, Assiut,71526 Egypt

Abstract: Ceramide, a derivative of sphingolipid breakdown products, acts as second messenger for multiple extracellular stimuli including growth factors, chemical agents, and environmental stresses. They have been shown to be crtically in-volved in various biological processes, including differentiation, senescence, cell-cycle arrest, proliferation, and apoptosis.Ceramide molecules form distinct domains in the cell membrane, which may serve to re-organize cellular receptors and signaling molecules. Because of their promising biological activities and applications, here we focus on: biosynthesis, ex-istence, importance, and structure elucidation, as well as representative examples concerning their structure and activity.

Keywords: Ceramide, sphingolipid, biological activity, identification.

INTRODUCTION

Ceramide is a family of lipids that consist of sphingosine linked to fatty acid via amide linkage [1]. The vast majority of fatty acids are -hydroxylated. The polyunsaturated fatty acids exist in certain testicular cells [2,3]. Ceramide plays important role in organizing membrane structure as it has the tendency to self-aggregate and segregate into membrane microdomains [4]. For years, it was assumed that ceramide was purely structural elements. This is now known to be not completely true. Ceramide attracted substantial concern be-cause of its contribution in vital biological processes such as cell cycle arrest, apoptotic cell death, cellular proliferation and inflammatory responses [5,6]. Such effects have been attributed to the second messenger signaling capabilities of this lipid. With a small hydroxy head group and two long saturated hydrophobic chains, in addition to intermolecular hydrogen bonding, ceramide packed tightly in bilayers and promotes membrane rigidity [7]. Ceramide represent precursor of major sphingolipids, such as sphingomyelin (SM), ceramides 1-phosphate (C1P), and glucosylceramides (GlcCer) [8,9]. Sphingolipids them-selves are the precursors to generate series of glycosphin-golipids and gangliosides [10,11] (Fig. 1).

BIOSYNTHESIS OF CERAMIDES

New approaches in cell biology and the development of in vivo models (e.g. yeast [12], Dorsophila [13], and geneti-cally modified mice [14]), afforded the identification of dif-ferent biosynthetic pathways for ceramide [15,16]. Several efforts have described the basic engagement of major phos-pholipids in biosynthesis and degradation of ceramide [4]. Ceramide could be generated by one of three main pathways; de novo synthesis, through SM hydrolysis, or through sal-vage pathway.

*Address correspondence to this author at the Faculty of Pharmacy, De-partment of Pharmacognosy, Al–Azhar University, Assiut, 71524 Egypt; E-mail: [email protected]

HO

NH3

OH

Sphingosine

O

NH3

OH

PHO

O

O

Sphingosine-1-phosphate

O

HN

OH

PHO

O

O

O Ceramide-1-phosphate

HO

HN

OH

O

Amide linked fatty acyl chain

O

HN

OH

PO

O

O

O Sphingomyelin

N

Cerebroside

O

NH

O

OH

OHO

HOOH

OH

Fig. (1). Bioactive Sphingolipids

a. Sphingomyelin Hydrolysis (Sphingomyelinase)

In this pathway, ceramide was generated from hydrolysis of SM induced by the effect of sphingomyelinase (SMase) [17,18], which cleave SM to ceramide and phosphocholine. SMases are stimulated in response to TNF- [19,20], fatty acids ligand [21], or oxidative stress [22]. The SM hydroly-sis has emerged as a major pathway of stress-induced cera-mide generation. This pathway has been suggested to regu-late SM and ceramide levels [23], as well as the activation of NF B [24-26].

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Activity and Structure Elucidation of Ceramides Current Bioactive Compounds 2012, Vol. 8 No. 4 371

b. De novo Pathway

The de novo pathway is localized to the endoplasmic reticulum (ER) and continues in the Golgi apparatus where the enzymes glucosylceramides synthase and SM synthase exist [27-29]. The de novo pathway is an anabolic route that begins with condensation of serine and palmitoyl-CoA to form 3-ketosphinganine (3KSn) via serine palmitoyltrans-ferase (SPT). Then ceramide is produced by the action of 3KSn reductase, dihydroceramide synthases (CerS), and di-hydroceramide desaturases (Des1 and -2) [30]. Sphingosine could be liberated from ceramide by ceramidases (CDases), which is phosphorylated to sphingosine-1-phosphate (S1P) [28,29]. Once ceramide was generated, it could be glycosy-lated to glucosylceramide by glucosylceramide synthase [31,32].

c. Salvage Pathway

Ceramide can also be produced by the catabolism of other complex sphingolipids, even indirectly. A series of events referred to as sphingolipid recycling or the salvage pathway, higher-order sphingolipid (SM and glycosphin-golipid) are degraded within acidic cellular compartments by resident enzymes (acidic SMase and acid -glucosidase 1, respectively) to form ceramide [33-35], which is itself de-graded into sphingosine and free fatty acids that are able to enter the cytosol [36]. Once in the cytosol, sphingosine is converted back to ceramide via ceramidase. Salvage pathway may account for over half of the sphingolipid pool within the cell [37]. It should be noted that the treatment of cells with exogenous ceramide may result in generation of endogenous

long chain ceramide via the salvage pathway. In fact, exoge-nous C6-ceramides were subject to deacylation by cerami-dase, releasing free sphingosine which in turn undergoes reacylation in a ceramides synthase-dependent manner [30].

ACTIVITY OF CERAMIDE

Sphingolipids, especially ceramides and sphingomyelin, play important roles in maintaining membrane function and integrity. Generation of ceramide within the cell membrane dramatically alters membrane properties. Sphingolipids in-teract with each other and with cholesterol molecules via hydrophilic linkage. In addition, hydrophobic van der Waal interactions bind ceramide moieties to the sterol ring system. The tight interaction between sphingolipids and cholesterol promotes the transition of membrane lipids into a liquid or-dered status and the formation of minute distinct domains within cell membrane, referred to as rafts. Hydrolysis of SM generates ceramide within cell membrane, which alters these rafts and lead to the formation of large ceramide-enriched membrane domains. Ceramide molecules are self-associated into ceramide-enriched membrane microdomains, which tend to fuse spontaneously into larger domains [38,39]. These ceramide-enriched platforms serve to organize recep-tor and signaling molecules in the cell. Thus, provide the spatial and temporal organization of the cell’s signaling ma-chinery [40]. Ceramide has been implicated in a variety of biological activities. Roles of ceramide and its downstream metabolites have been suggested in many pathological events including cancer, neurodegeneration, diabetes, microbial pathogenesis, obesity, and inflammation [41-43].

OHN

OH

POO

O

O Sphingomyelin (SM)

N

Sphingomyelinase+ Phospatidylcholine

Sphingomyelin synthase

HOHN

OH

O Ceramide (Cer)

CeramidaseHO

NH2

OH

SphingosineCeramide synthase

Sphingosine kinase

ONH3

OH

PHOO

O

Sphingosine-1-phosphate

O

O

NH3

HO

O

CoSA

SerinePalmitoyl-CoA

Serine palmitoyl-transferase

HONH2

O

3-Ketosphinganine

3-Ketosphinganinereductase

HONH2

OH

Dihydrosphingosine

Ceramide synthase

HOHN

OH

O Dihydroceramide

Dihydroceramidedesaturase

De novo synthesis

Ceramide-1-phosphate phosphotase

Ceramide kinase

OHN

OH

PHOO

O

O Ceramide-1-phosphate(Cer-1-P)

Sphingomyelin metabolism

Glucosyl ceramide synthase

Glucosylceramides

1-Acylceramide synthase1-O-Acylceramide

Fig. (2). Ceramide synthesis and metabolism. Ceramide is generated from sphingomyelin or via the de novo synthesis. Ceramide is further converted into other sphingolipids such as ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate.

372 Current Bioactive Compounds 2012, Vol. 8, No. 4 Elkhayat et al.

CERAMIDE CHANNELS AND THEIR ROLE IN RE-LEASING PROTEINS FROM MITOCHONDRIA

Protein translocation through membranes can occur in a variety of ways: protein translocation via carrier-type mechanism, endo/exocytosis, membrane damage, and chan-nel formation. Ceramide channels are speculated to be formed by a ring of columns each consisting of six cera-mides interconnected by the hydrogen bonds of the amide linkage [44]. Mitochondria contain the enzymes responsible for ceramide synthesis and hydrolysis, namely, ceramide synthase and ceramidase. Both mitochondrial outer and inner membranes are capable of generating ceramide [45]. Moreo-ver, several studies demonstrated that CD95-, TNF -radiation, and UV-induced apoptosis occur via an increase in mitochondrial ceramide levels [46-48]. Ceramide causes change of mitochondrial transmembrane potential (MTP) by targeting MTP-controlling proteins, such as Bcl-2 family members, resulting in the release of cytochrome C and apop-tosis-inducing factor (AIF) from mitochondria to cytoplasm, followed by caspase-3 activation [45,46]. Ceramide has also been reported to have numerous effects on mitochondria, including enhanced generation of reactive oxygen species (ROS), alteration of calcium homeostasis at the mitochon-drial and endoplasmic reticulum, ATP depletion, collapse of the inner mitochondrial membrane potential, and inhibition and/or activation of various components involved in the mi-tochondrial electron transport chain [45,46]. Furthermore, C2- and C16-ceramides increased the permeability of the mitochondrial outer membrane through the formation of large protein permeable channels that would allow the re-lease of inter membrane space small proteins, including cy-tochrome C [48,49]. It is noteworthy that ceramide channel formation requires the presence of the 4-5 trans double bond as dihydroceramide does not form channels and does not induce cytochrome C release [49].

1- Role of Ceramides in Cancer Therapy

Ceramides regulates a myriad of cellular pathways in-cluding apoptosis, cell senescence, the cell cycle, and differ-entiation [50]. Apoptosis can be induced by various factors including chemotherapeutic agents, CD95, tumor necrosis factor-1, growth factor withdrawal, hypoxia, or DNA dam-age. Many of these apoptosis mediators are regulators of ceramides generation, suggesting a role for ceramides in apoptosis [27]. Increasing the endogenous levels of cera-mide, together with inhibitors of ceramide metabolizing en-zymes or over-expression of ceramide-generating enzymes which leads to apoptosis and/or growth arrest [51,52]. Some anticancers as vincristine, paclitaxel, and etoposide, or after radiation treatment work by elevating cellular cera-mide level, which result from stimulation of ceramides de novo synthesis, increase in SMase activity, or by a disruption of ceramide catabolism leading to apoptosis [52-58]. Herin we report representative of anticancer ceramides: Strepsiamides A-C (1-3) which have been isolated from the Strepsichordaia lendenfeldi sponge showed cytotoxic activity against mouse lymphoma (L5178Y) and human cer-vix carcinoma (Hela) cell lines [59]. The new ceramides, glumoamide (4) and glumoside (5) isolated from the stem

bark of Ficus glumosa exhibited anticancer activity against prostate cancer PC-3 cells [60]. Three gangliosides (6-8) were isolated from the starfish Astropecten latespinosus. Compounds 6 and 7 showed neuritogenic and growth inhibi-tory activities towards the mouse neuroblastoma cell line [61]. While compound 8 showed in vitro antitumor activity against murine lymphoma L1210 cell line [62]. The anhy-drophytosphingosine derivative, Jaspine B (9) was isolated from Jaspis sp. sponge. It reduced the viability of murine B16 and human SK-Mel28 melanoma cells. Jaspine B trig-gered cell death by apoptosis. It was able to kill melanoma cells by inhibiting the activity of SM synthase (SMS) and consequently on ceramide formation. It may represent a new class of cytotoxic compounds with potential applications in anticancer melanoma therapy [63]. The ceramides analogue, B13 (10) showed potent anticancer activity through cerami-dase inhibition and induces apoptosis. A series of thiourea B13 analogues were evaluated for their in vitro cytotoxic activities against human renal cancer Caki-2 and leukemic cancer HL-60 in the MTT assay [64]. A new glycosyl cera-mides, 1-O- -D-glucopyranosyl-(2S,3S,4R,9Z)-2-[(2R)-2-hydroxydocosanoylamino]-9-octadecene 1,3,4-triol (11) was isolated from the roots of Livistona chinensis. It showed sig-nificant antiproliferative effects against the human tumor cell lines (K562, HL-60, HepG2, and CNE-1) with the IC50 of 10-65 M [65]. Two cytotoxic ceramides were isolated from the pollen of Brassica napus L; 1-O-( -D-glucopyranosyl)-(2S,3S,4R,8E)-2-[(2R)-2-hydroxytetracosenoylamino]-8-octadecene-1,3,4-triol (12) and (2S,3S,4R,8E)-2-[(2R)-2-hydroxytetracosenoylamino]-8-octadecene-1,3,4-triol (13). They showed cytotoxic activity against human tongue squamous carcinoma cell line (Tca8113) [66]. The suillu-mide (14) isolated from the basidiomycete Suillus luteus L.. It showed cytotoxic activity against human melanoma cell line (SK-MEL-1) with IC50 value of 10 M [67]. Bathymo-diolamides A and B (15 and 16) were isolated from the in-vertebrate mussel bathymodiolus thermophilus. They inhib-ited the growth of HeLa (IC50 0.4 M and 0.5 M, respec-tively) and MCF7 (IC50 0.1 M and IC50 0.2 M, respec-tively) cell lines [68]. C16-serinol and (2S,3R)-(4E,6E)-2-octanoylamidooctadecadiene-1,3-diol (4,6-diene-ceramides), were novel structural analogs of ceramides. They have in-duced apoptosis in various human cancer cells [69,70].

2- Role of Ceramides in Pathogenesis

Ceramides has been shown to facilitate the entry of a variety of pathogens. Thus, it was demonstrated that differ-ent bacterial, parasitic, and viral infections activate the acid SMase. They induce the translocation of the enzyme onto the cell surface and trigger the release of ceramides and forma-tion of ceramides-enriched membrane platforms that facili-tate invasion [71,72]. Receptors for certain pathogens were found to be enriched within ceramides domains which facili-tate microbial entry [42,73]. Ceramides-enriched membrane domains were also shown to mediate Pseudomonas aerugi-nosa internalization, is a critical step to clear this pathogen by bronchial epithelial cells. Internalization of P. aeruginosa leads to apoptosis of the infected cells, limiting systemic inflammatory responses, and interleukin-1-induced septic death of infected mice [74]. Furthermore, viruses as HIV and


NH

HO

OH

OHO

R2R1

10

R3

R3 =R1 = H R2 = CH3

R1 = OH

R1 = HR2 = CH3 R3 =

R3 =R2 = H

1

23

CH2-CH-(CH3)2

CH2-CH-(CH3)2

(CH2)3-CH-(CH3)2

17

HO

NH

O

OH

OH

OH

4

15

8

O

NH

O

OH

OH

OHO

HOHO

OH

OH

4

20

5

O

NH

O

OH

OH

OHO

HOOH

OH

x

19

6x+y = 15

yOOH

NHO

COOHHO

OHOH

H3C

O

O

OH

OHOH

O

O

NH

O

OH

OH

OHO

HOOH

OH

x

19

7x+y = 15

yO

O

OH

OH

O

HN

OHHOOC

HO

H3CO

HO O

O

OH

OHN

HO

COOHHO

OCH3

OH

O

OO

O

OH

OHHO

HO


O

NH

O

OH

OH

OHO

HOOH

OH

10

17

8

OOHN

O

COOHHO

OHOH

H3C

O

O

OHOH

O

O

HO

OH

HOO

O

OH

OH

HO

O

H2N OH

C(CH2)12CH3

9

O2N

OH

NH

OH

O 109

O

NH

O

OH

OH

OHO

HOOH

OH

7

17

11

HO

RO

NH

O

OH

OH

OH

12 R = Glu13 R = H

7

11 6 O

NH

OH

8

O

OH

13

14


8

OH OH

NH O

O

O

O

O

15

12

OH OH

NH O

O

O

O

O

16

8

10

rhinoviruses were speculated to utilize ceramides-enriched microdomains to invade epithelial cells and consequently manipulate the host machinery to replicate, survive, and fi-nally infect neighboring cells [75]. Complementary studies have reported that treatment with fenretinide, which in-creases de novo ceramides biosynthesis or with exogenous SMase, increased ceramides and inhibits HIV infection [76,77]. Recent evidence indicated that certain pathogens continue to modulate enzymes of the sphingolipid metabo-lism after the internalization step. Whereas, other pathogens like Cryptococcus neoformans utilized their own sphin-golipid metabolism as virulence factors [73]. The essential functions of sphingolipids coupled with the divergence of the biosynthetic pathway between mammals and eukaryotic pathogens have resulted in the investigation of the biosyn-thetic enzymes as possible drug targets for antifungal and antiprotozoals therapy. Where, inhibitors sphingolipid bio-synthesis have been described [78,79]. Unfortunately, the inhibitors targeted the fungal enzymes were non selective and inhibited the mammalian orthologues [79]. This has cur-tailed their clinical application as antifungal agents. For ex-ample, the mammalian toxicity associated with fumonisin B1, which inhibited the fungal phytoceramides synthase [80]. The absence of inositol phosphorylceramides (IPC) synthase and inositol-based sphingolipids in mammalian cells highlights the therapeutic potential of inhibitors target-ing fungal IPC synthases. Such inhibitors could provide se-lective antifungal drugs with minimal host toxicity. Addi-tionally, the identification and isolation of functional orthologues of the fungal enzyme in the parasites Leishma-nia spp., Trypanosoma brucei, and T. cruzi, indicated that

IPC synthase is a potential target for antiprotozoal com-pounds [82]. Trypanosoma cruzi (Chaga`s disease), synthe-size surface glycosylphosphatidylinositol (GPI) anchored glycoconjugates, meaning that the biosynthesis of GPI an-chors is attractive target for new therapies against Chaga`s disease [82]. Antiviral sphingosine derivatives (17 and 18) were iso-lated from the green algae Ulva fasciata [82,83]. Several glycosphingolipids, halicylindrosides A1-A4 (19-22), and B1-B6 (23-28) having -N-acetylglucosamine sugar moiety, were isolated from Halichondria cylindrata sponge. These compounds showed moderate antifungal activity against Mortierella remanniana and cytotoxicity against P388 mur-ine leukemia cell line [84]. (2S,3S,4R,10E)-2-[(2'R)-2-hydroxytetracosanoylamino]-1,3,4-octadecanetriol-10-ene (29), 1-O- -D-Glucopyranosyl-(2S,3S,4R,10E)-2-[(2'R)-2-hydroxytetracosanoylamino]-1,3,4-octadecanetriol-10-ene (30), and 1-O- -D-glucopyranosyl-(2S,3R,4E,8E)-2-N-2'-hydroxypalmitoyl-4,8-sphingadienine (31), were isolated from the stems of cucumber (Cucumis sativus L.). They ex-hibited strong inhibitory activity on Pseudomonas lachry-mans with IC50 values being 15.3 g/mL, 17.4 g/mL and 37.3 g/mL, respectively [85]. Ceramides analogs (32 and 33) were potent inhibitors of Plasmodium falciparum culture growth. Interestingly, the nature of the linkage between the fatty acid part and the sphingoid core considerably influ-ences the antiplasmodial activity and the selectivity of ana-logs when compared to their cytotoxicity on mammalian cells. The ceramides analogs might inhibit P. falciparum growth through modulation of the endogenous ceramides level [86].

RO

NH

O

OH

OH

OH

17

RO

NH

O

OH18

2

11

10

12

6


NH

O

R

OH

OH

m

n

19 m = 16; n = 10; R = H20 m = 16; n = 11; R = H21 m = 17; n = 11; R = H22 m = 18; n = 11; R = H23 m = 16; n = 8; R = OH

24 m = 17; n = 8; R = OH25 m = 16; n = 9; R = OH26 m = 16; n = 10; R = OH27 m = 17; n = 9; R = OH 28 m = 16; n = 11; R = OH

OO

HONHAc

OH

HO

HO

NH

O

OH

OH 29

19

5

O

NH

O

OH

OH 30

OHO

HOOH

OH 19

5

O

NH

O

OH

OH 31

OHO

HOOH

OH 12

6

CH

OHHC CH2

NH

N O

CO(H2C)14H3C

32

CH

OH

HC CH2OH

NH

CO(H2C)13H3C

33

No2

3- Role of Ceramides in Inflammation

The acute inflammatory response triggered by infection or tissue injury involves the coordinated delivery of blood components to the site of injury or infection [87]. Inflamma-tory mediators include chemokines, cytokines, products of proteolytic cascades, phospholipases, or lipids such as ei-cosanoids and sphingolipids. Concerning phospholipases, a major mediator of inflammatory responses is phospholipase A2 (PLA2). Ceramides have been described as key media-tors of inflammatory responses [88-91]. The role of cera-mides in the development of allergic asthmatic responses and airway inflammation was established recently [92]. It was proposed that at least some of the pro-inflammatory effects of ceramides may be mediated by its conversion to C1P, which was demonstrated to be potent and specific stimulant for arachidonic acid (AA) release and prostanoid synthesis in

A549 lung adenocarcinoma cells [93]. Moreover, C1P could be generated intracellularly through stimulation of ceramides kinase (CerK) by interleukin 1 . The mechanism whereby C1P stimulates AA release occurs through direct activation of cPLA2 [94]. Subsequently, C1P is a positive allosteric activator of cPLA2. It enhances the interaction of the cPLA2 with phosphatidylcholine. Finally, C1P may function to re-cruit cPLA2 to intracellular membranes and allosterically increases the catalytic ability of the membrane-associated enzyme [95]. Recent studies have demonstrated that activa-tion of group IV cPLA2 by C1P is chain length-specific. In particular, C1P bearing acyl chains equal or higher than 6 carbons were able to efficiently activate cPLA2 in vitro,whereas shorter acyl chains (particularly C2-C1P) were un-able to activate the enzyme [96]. C1P was shown to act in coordination with S1P to ensure maximal production of pros-taglandins. Specifically, S1P induces cyclooxygenase-2


(COX-2) activity, which then uses cPLA2-derived AA as substrate to synthesize prostaglandins [97]. It should be pointed out that C1P is involved in other inflammation proc-esses including stimulation of phagocytosis in neutrophils [98,99], and activation of degranulation in mast cells [100]. The prosurvival and pro-inflammatory actions of C1P, the enzyme responsible for C1P formation. CerK might be an important target for development of novel therapeutic com-pounds for treatment of inflammatory illnesses [94]. Phosphoceramides analog-1 (PCERA-1) (34) has been described as a potent in vivo suppressor of the pro-inflammatory cytokine TNF- , and thus as a putative drug for the treatment of inflammatory diseases [101]. A new sphingosine derivative (35) isolated from the soft corals of Sinularia crassa and Lobophytum sp. It showed activity against carrageenin-induced rat hind paw edema model for acute inflammation and cotton pellet granuloma model for chronic inflammation at 10 mg/kg and was comparable to indomethacin (2 mg/kg) [102]. (2R,3S)-2-Formamido-1,3-dihydroxyoctadecane (36) was isolated from the red alga Gracilari verrucosal. It inhibited the production of pro-inflammatory mediators (NO, IL-6, and TNF-R) in lipopolysaccharide (LPS)-activated murine macrophage cells (RAW 264.7) [103].

4- Role of Ceramides in Cardiovascular Diseases

Ceramides have been shown to regulate cardiovascular functions, including cardiac contractility, vasomotor re-sponses, and endothelial function [104-106]. In general, they mediate the detrimental actions of many cardiovascular pathogenic factors [107-109]. Although in only a very few conditions ceramides were found to exert a protective effect [110]. There is evidence that treatment with cell membrane-permeable short-chain ceramides, SMase, produce contrac-tions in canine cerebral arteries, bovine coronary resistance arteries, and capacitance vessels [111-113]. In contrast, some studies reported thoracic aorta ceramides-induced relaxation associated with decreased intracellular calcium mobilization [114,115]. In small coronary arteries, ceramides signaling contributes to the impairment of endothelium-dependent vasodilatation by reducing NO bioavailability in coronary arteries [104,116]. Ceramides decrease endothelial NO lev-els, thereby inhibit NO-mediated endothelium-dependent

relaxation in response to various endothelium-dependent vasodilator agonists. In this respect, ceramides may be a pathogenic factor resulting in endothelial dysfunction in the coronary circulation. The reduction of NO bioavailability is associated with ceramides-induced oxidative stress [117-119]. Recent studies have suggested that the generation of reactive oxygen species (ROS) in particular O2

–• by endothe-lial cells is both physiologically and pathophysiologically relevant to the function of these cells [112]. ROS levels are tightly regulated by antioxidants such as superoxide dismu-tase (SOD), catalase, thioredoxin, glutathione, antioxidant vitamins, and other small molecules [116-119]. The mis-match between the production and the elimination of ROS will result in the increased bioavailability of ROS and a state of oxidative stress. One of the pathogenic outcomes of oxi-dative stress is oxidative damage resulting in endothelial dysfunction. Ceramides-mediated oxidative stress is primar-ily due to the activation of endothelial NADPH oxidase [120]. Ceramides-enriched membrane domains serve as re-dox signaling platforms. They facilitate the aggregation and activation of NADPH oxidase [120,121]. It is plausible that the formation of this redox signaling platform provides im-portant contributions to the endothelial dysfunction involved in the pathogenesis of various vascular diseases, such as atherosclerosis, hypertension, and ischemia/reperfusion in-jury. The formation of this ceramides-enriched redox signal-ing platform on the membrane of endothelial cells may be implicated in the development of these diseases.

5- Role of ceramides in Atherosclerosis

Atherosclerosis is characterized by the deposition of atheromatous plaques containing cholesterol and other lipids on the innermost layer of arterial walls. Aggregation of lipoproteins is a fundamental step in the formation of atherosclerotic lesions. Modulation of sphingolipid levels prevents plaque formation in apoE–deficient mice

The most abundant lipids within lipoproteins include cholesterol, cholesterol esters, triglycerides, and SM. Noting that plasma SM levels correlate with coronary artery disease independently of cholesterol levels [122,123], and that atherosclerotic lesions contained much higher concentrations of ceramides when compared with plasma low–density lipoproteins (LDLs) [124,125]. Study showed that in

HN

OOP

O

HO

OHO

34

HO

NH

O

OH

8

13

35

HO

NHCHO

OH

836


apolipoprotein E (apoE)–deficient mice, which are a commonly used rodent model of atherosclerosis, SPT activity and plasma SM levels increased markedly during high-fat feeding. Treating these animals with the SPT inhibitor myriocin dramatically lowered SPT activity and reduced plasma SM levels by 64%, bringing it to the level of standard chow-fed animals. Interestingly, it also caused a reduction in circulating cholesterol, very low-density lipoproteins, and LDLs. Ultimately the treatment strategy led to a 93% reduction in atherosclerotic lesion coverage within the aorta, as well as substantial decrease in plaques in the brachiocephalic artery and aortic valve area [126].

6- Ceramides and Obesity

The de novo synthesis of ceramides occurs on the outer leaflet of the endoplasmic reticulum. Ceramides are then modified into complex sphingolipids in the golgi apparateus. However, evidences suggest that sphingolipid metabolism can also occur in the mitochondria [45]. A. Sphingolipids in the Diet

Sphingolipids are particularly prevalent in meat, eggs, and dairy products and may have significant roles within the digestive tract [125]. Rich diet of sphingolipids can increase serum SM concentrations and can be proatherogenic [128,129]. However, it is unlikely that increased intake of sphingolipids promotes their accumulation during obesity. The vast majority of sphingolipids are degraded in the gut by glucoceramidases, SMases, and ceramidases. About 25% of consumed sphingolipids resist degradation, only to be se-creted in the feaces, predominantly in the form of ceramides [130]. B. Regulation of Sphingolipid Synthesis and Metabolism During Obesity

Number of factors associated with obesity selectively alter rates of ceramides synthesis. Long-chain saturated fats are required for formation of the sphingoid backbone and are sufficient to drive the formation of ceramides [131,132]. Thus, increased saturated acyl chains within circulating lipo-protein particles likely contribute to the induction of cera-mides in peripheral tissues. In addition, obesity is associated with a state of chronic low-level inflammation [133,134], which likely contributes to the induction of ceramides accu-mulation.

7- Ceramides and Cosmetic

Comprising more than 40% of lipid components in the stratum corneum ceramides serve as a barrier to suppress loss of moisture and protects against hazardous external sub-stances as allergens and viruses [135,136]. But they are gradually decreased with aging [136]. From the pharmaceu-tical and cosmetic point of view, there is a good reason to believe that ceramides are among the most important com-pounds for skin protection. Therefore, they have become important cosmetic ingredients [137]. Lots of efforts in skin research pointed out that ceramides are key players in main-taining the epidermal integrity and health. These findings lead to the concept that ceramides are high performance ac-tive ingredients for skin care products. Since their topical application can replenish low levels of stratum corneum lip-

ids [138]. Recent studies showed that phytosphingosine (PS) has anti-microbial properties against Propionibacterium ac-nes and Staphylococcus aureus as well as anti-inflammatory function. PS is able to inhibit the growth of this micro-organism with MIC of 0.020%. Furthermore, this study demonstrates that PS effectively reduces the release of IL-1 after UVB irradia-tion, thereby preventing skin inflammation [139].

METHODS FOR SEPARATION OF CERAMIDES

- Acetylation

Acetylation of ceramides afforded efficient mean to iso-late and purify individual ceramides. As it is difficulty to deale with genuine ceramides due to similarities in behavior and polarity, especially within the -hydroxyceramides. The separation occurs because acetylation appears to nullify the effect of the -hydroxy on polarity. Acetylation of the cera-mides was also useful for the NMR analysis as it has elimi-nated irrelevant exchangeable hydroxyl protons, that other-wise might complicate the spectra, in addition to improve the solubility in pure deuterated CHCl3 [140]. To acetylate a ceramide, the sample was dissolved in a 1:1 mixture of pyridine/acetic anhydride and allowed to react for 2 h at room temperature. The reagents were then evapo-rated under nitrogen. The obtained acetylated ceramides were subjected to further chromatographic techniques [141].

- The Oxidative-reduction-hydrolysis Method

This method for ceramides preparation is relatively labo-rious, still important since all N-fatty acyl linkages of the original glycosphingolipid were preserved. Moreover, this method can be indirectly extended to other neutral and acidic glycosphingolipids [142]. This method has two restrictions: (i) different steps are not quantitative; (ii) the phytosphingos-ine ceramides are destroyed in the periodate oxidation step. But it can be improved by the incidence of enzymatic proc-esses. Endoglycoceramidase enzyme cleaves the linkage between the oligosaccharide and ceramides of neutral and acidic glycosphingolipids [143]. Cerebrosides (40-50 mg) were solved in 1 mL of CHCl3and 3.5 mL EtOH and oxidized with 2 mL solution of NaIO4 (0.5 M) for 22 hr at room temperature and in dark. Excess periodate was removed by adding ethylene glycol (6 mL in H2O). Upper phase, containing salts and formic acid was discarded. To the lower phase, a solution consisting of 6 mL MeOH, 3.5 mg NaBH4, and 2 mL 0.1 N NaOH was added and left for 22 hr at room temperature. Partial acidic hy-drolysis was made by adding 0.5 mL 6 M HCl and preserv-ing overnight at room temperature. By mixing the latter solu-tion with 10 mL CHCl3 and 15 mL H2O, two layers ap-peared, the lower one containing the ceramides. The cera-mides solution was dried, filtered, evaporated to dryness and the residue was chromatographed on a column of silica gel. Finally, a peracetylated ceramides was prepared and NMR spectra were registered [143].

STRUCTURE ELUCIDATION OF CERAMIDES

There are four types of sphingoid bases associated with ceramide structure; sphinganine (Sa), sphingosine (S), phy-


tosphingosine (PS), and 6-hydroxysphingosine (HS) (Fig. 3)[144,145]. In order to identify ceramide and sphingolipid structure, various spectroscopic analysis techniques are em-ployed; e.g. mass-spectroscopy, 1D and 2D-NMR, and IR analyses have been used as a key tool for the structural elu-cidation. The most widespread and studied sphingosine is 4-sphingenine trans 1,3-dihydroxy 2-amino 4-octadecene [(2S,3R,4E)-1,3-dihydroxy 2-amino 4-octadecene]. Addition of a molecule of water to 4-sphingenine produces the so called phytosphingosine, which was found also in animal tissues [144]. The sphingosine chain length is also variable, 14-20 carbon atoms. The degree of unsaturation oscillated from complete saturation (phytosphingosine) to two or even three double bonds, usually situated on C-4, C-8, and C-10.

I- Mass Analysis

The new approaches in mass spectroscopy afforded ma-jor analytical method to define ceramide and other sphigolip-ids:a) Electron impact (EI): Derivatiztion of samples prior

analysis (acetylated or methylated derivatives), repre-sents a major drawback of this ionization method. How-ever, this approach in addition to the uncetainity about location of isolated double bonds in the fatty acid acyl moiety [145,146]. Although the EI is quite sensitive in comparison to conventional chemical methods, the mo-lecular ions are often of low abundance and amount of 8

g or more of samples are still needed to observe struc-turally diagnostic fragment ions [146].

b) Fast atom bombardment mass spectrometry (FABMS) and FABMS/MS have been established to be useful for structural elucidation of ceramides, that are relatively large, and thermally labile [147-149]. But FAB has a distinct disadvantage concerning the sensitivity to com-plex multicomponent analysis.

c) Electrospray ionization (ESIMS) was advantagous over the FABMS/MS, regarding the lower background sig-nals due to absence of matrix ions, the longer lasting and stable primary ion currents, the ease of sampling, and compatibility with liquid chromatography. More re-

cently, small quantities of biological or synthesized ce-ramides have been examined using ESIMS and ESIMS/MS [150-153]. Positive-ion mode ESI/MS em-ploying quadrupole instruments has been used exten-sively for fungal cerebroside analysis [154].

d) Reversed-phase LC/ESIMS with in-source collision in-duced dissociation (CID) has been shown to be valuable for qualitative and semi-quantitative analysis of different ceramides molecular species. Molecular ions and adduct species ([M+H]+, [M+Na]+, [M-H]–, [M+HCOO-]-, and [M+AcO-]-) of ceramides, obtained by using both posi-tive and negative ion modes. They were used to charac-terize the molecular weights of ceramides. The CID fragmentation patterns of molecular anion mode make it possible to obtain information on the nature of the sphingoid base and fatty acyl chains. The product ion spectra in negative ion mode of ceramides species pro-vide more structural information than those obtained in positive ion mode. It was proved to be useful method for quality control of products contained in commercial cosmetic ceramides. It is possible to apply this method to the routine analysis of ceramides in different samples, such as natural products and cosmetics [155].

e) LCAPCI-MS is a coupling between atmospheric pressure chemical ionization and quadruple mass spectrometer for the determination of ceramides. The thermal decom-position of the ceramides at temperatures characteristic of APCI sources is possible via cleavage of the N-acyl moiety [145].

f) Gas chromatography/mass spectrometry (GC/MS), used after hydrolysing the ceramide and/or cerebroside sam-ple to their components of sugar, fatty acids, and Long chain bases (LCB) [156]. Fatty acids as well as sphin-goid bases can be separately analyzed by GC/MS. How-ever, the data do not reveal information about the com-bination of the two moieties. The GC/MS analysis of in-tact ceramides requires prior trimethylsilylation [156,157]. The required derivatization is considered to be a disadvantage of GC/MS, especially in comparison with the recently developed LC/MS methods. The pres-ence of the trimethylsilylated ceramides in a surplus of

OH

NH2

OHSphingosine (S)

H2N

OH

OH

OH6-Hydroxy sphingosine (HS)

H2N

OH

OH

Phytosphingosine (PS)OH

nn

n

H2N

OH

OH

Sphinganine (Sa)

n

Fig. (3). Sphingoid bases.


derivatization reagents may cause contamination of the GC/MS system and thereby affect quantification stabil-ity [158]. To overcome the requirements for volatiliza-tion, the ceramides and/or cerebrosides were metha-nolyzed and their components analyzed as fatty acid methyl esters (FAMEs) [159]. The monosaccharide components of cerebroside were converted to alditol acetate derivatives after hydrolysis [160]. LCBs were re-leased (LCB or sphingoid base) from cerebrosides by methanolysis then analyzed by GC/MS as the TMS de-rivatives after treatment with bis-(trimethylsilyl)-trifluoroacetamide/pyridine [161,162].

g) Matrix assisted laser desorption ionization (MALDI) is a desorption ionization (DI) method with high sensitivity, allowing the analysis of biomolecules (biopolymers such as DNA, proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. It is similar in character to ESI in that both techniques are relatively soft ways of obtaining large ions in the gas phase. Though MALDI produces many fewer multiply charged ions depending on their chemical characteristics. However, it is also common to find ions produced by the combination of the analyte with different cations or ani-ons, such as Na+, K+, and Cl-, which are referred to as ad-ducts. These ions are commonly present in the solvent and could aid the ionization of several polar molecules such as neutral cerebrosides [163-165].

The fragments obtained due to loss of the N-linked fatty acid moiety and one or two molecules of water, are equiva-lent to the number of hydroxyl groups in the structure. The following figure illustrates the possible fragmentation pattern of ceramides and/or cerebroside (Fig. 4).

Methods for Detection of FA and LCB

I- Methanolysis of Ceramides

Ceramides (10-15 mg) were refluxed with 3 mL 5% HCl-MeOH at 60 C for 6 h. 10 mL of cold water was poured into

the reaction mixture, which was extracted with n-hexane for three times (5 mL 3). The combined organic layer is then dried over anhydrous Na2SO4 and concentrated under re-duced pressure. The residue (FAME) is analyzed by GC/MS [166].

II- IR of Sphingolipids

Ceramides and/or cerebrosides showed IR absorption bands for hydroxyl group (OH) at 3400-3500 cm-1, secon-dary amide (-NH) at 3100-3300 cm-1, in addition to strong absorption bands for amidic carbonyl group of fatty acid at 1640-1680 and 1530-1540 cm-1 [167].

III- Nuclear Magnetic Resonance (NMR) Spectroscopy

The NMR spectroscopy is capable of furnishing informa-tion concerning the primary structure of cerebrosides, such as monosaccharide composition, anomeric configuration, and size of the sugar rings. The assignments of all ceramides signals could also be obtained without interference of the sugar moiety. The proton corresponding to a -glucopyranosyl was recognized as sharp doublet at H 4.4-5.2 with a coupling constant J1,2 = 7.9 Hz due to anomeric nuclei. The remaining sugar protons appear at H 3.2-5.5 [168]. The chemical shifts of H 4.30-5.30 and 4.5-5.50 were assigned to H-3 and H-2 of hydroxy fatty acid, respectively. A triplet signal at H 2.2-2.5 due to the methylene protons connected to amide carbonyl in non-hydroxylated fatty acids [169]. A large methylene cluster at H 1.20-1.35 and C 29.1-29.9 in 13C NMR is due to long aliphatic chains [170]. In addition to, signals at H 5.3-5.76 and C 128.7-134.4 were assigned to olefinic double bond. The geometry of the double bond could be deduced from coupling constant (J). Where J of 14.0-16.0 Hz indicated trans-double bond (E) [171], while J of 7-11 Hz refers to cis configuration (Z). The characteristic amide NH signal at H6.0-9.5 was confirmed by the downfield signals at C 174.0 (C-1 of fatty acid) and 54.5 (C-2 of sphingosine). The hydro-

O

NH

O

OH

OH

O

HOHO

OH

OH

-H2O

-H2O

O

NH

O

OH

OHa

-H2O

-H2OH

-H2O

b

Fig. (4). Possible mass fragmentation patterns of ceramide (a) and cerebroside (b).


carbon chain of sphingosine is alternatively linear, iso, an-teiso or 9-methyl. Anteiso hypostasis is associated with a supplementary chiral center while 9-methyl group is found on an unsaturated carbon [172,173]. The terminal isopropyl moiety of either fatty acid and/or sphingosine was deduced by the presence of two doublet methyl signals at H 0.86 (J = 6.0-7.0 Hz). The triplet signal at H 0.86-0.88 assigned to fatty acid and/or sphingosine terminal methyl. The stereochemistry at C-2 and C-3 was determined to be D-erythro (2S,3R), since 1H NMR spectral data [H-1A at H3.95 (dd, J = 3.7, 11.0 Hz), H-1B at H 3.70 (dd, J = 3.2, 11.0 Hz), H-2 at H 3.92 (m) and H-3 at H 4.30 (t, J = 5.0 Hz)], together with 13C NMR signals of C-2 ( C 54.5-54.6) and C-3 ( C 72.3) [174]. 13C NMR spectroscopy has been shown to be very help-ful tool for the elucidation of glycolipid structure, e.g. with respect to ring size and anomeric attachment. One aspect should be mentioned concerning linear, iso, anteiso forms: while 1H NMR spectra cannot distinguish between the three types, this can be done by measuring value of terminal carbons in 13C NMR spectra: linear, 14.2; iso, 22.8, both methyl groups; anteiso, 11.4 (terminal Me) and 19.3 (anteiso CH3) [172]. Even a cyclopropanated hydrocarbon chain has been found in sphingosine. Fatty acid constituents of cera-mide showed wide variations: chain length (14-26 or even 34-36 C), hydroxylation (2-hydroxy or -hydroxy), unsatu-ration (saturated or mono-, di-, tri-unsaturated), ramification (linear, iso-, anteiso) [172,175]. Tables 1 and 2 showed the chemical shift differences between L-threo and D-erythro-sphingosine, ceramide, and glucosylceramide. Table 1. 13C NMR of D-erythro-sphingosine and L-threo-

sphingosine in CDC13/CD3OD

Chemical shift (ppm)

Carbon atom D-erythro-sphingosine L-threo-sphingosine

C-1 63.73 63.73

C-2 51.02 57.45

C-3 74.75 73.18

C-4 134.97 134.41

C-5 129.93 130.38

C-6 32.86 32.86

C-7-C-15 29.35-30.32 29.35-30.3

C-16 32.37 32.37

C-17 23.07 23.07

C-18 14.22 14.22

From the Tables

1- The carbonyl carbon resonance in threo compound was at C 175.63 (c.f. 175.09 ppm in the erythro derivative).

2- The signals of the two olefinic carbons, particularly C-4 of the threo derivative was at C 133.4 (c.f. 134.72 ppm of the erythro derivative), and C-5 at C 129.50 (c.f.

129.75ppm). Thus the signal positions of the carbonyl carbon and the olefinic carbons of the sphingoid moiety were diagnostic for the erythro or threo configuration in glucosylceramides.

3- The -linked glucosyl moiety resonates at C 103.80 ppm with a very small downfield shift of 0.09 ppm with regard to erythro derivative ( C 103.71).

4- The C-3 D-glucosyl-D-erythro-ceramide resonates at C72.58 (c.f. C 70.70 in threo derivative).

5- C-1 of Threo sphingosine at C 69.55, which showed a downfield shift of 0.45 ppm in comparison to C-1of erythro derivative.

6- No significant shift differences between the signals of the threo and erythro isomers have been observed below 50 ppm where the alkyl-chain carbons resonate [174].

D-erythro- and L-threo-ceramide

1- The carbonyl of L-threo-ceramide was at at C 175.38 (c.f. 175.11 ppm in D-erythro–ceramide).

2- The C-4 of threo derivative resonated at C 133.35, in comparison to 134.27 in D-erythro-ceramide.

3- C-3 of threo-ceramide resonated at C 71.24 (c.f. 73.48 in erythro-ceramide). Thus, both C-4 and C-3 afforded strong guide for the stereoisomery of the sphingoid moi-ety.

4- The resonence at C 62.57 is assigned C-1 of threo-derivative (c.f. 61.87 in erythro- derivative).

On comparing D-glucosyl-L-threo-ceramide and L-threo-ceramide with their corresponding D-erythro deriva-tives, it is noticeable that the most evident differences are upfield shifts in the spectra of the L-threo isomers. An im-portant feature of ceramide and glucosylceramides of both stereoisomeric forms is the shift difference of the signals of sphingosine C-4 and C-5. These signals, although shifted a little when ceramides is glucosylated, are apart by nearly 5.0 ppm in the erythro isomers, but only about 4.0 ppm in the threo isomers. Indeed, all natural glycosphingolipids pub-lished to date and including gangliosides, the signals for C-4 and C-5 are found to be apart by 5.0 ppm, with no indication of appropriate signals being apart by 4.0 ppm. Thus, we con-clude that the natural glycosphingolipids analyzed so far contain the erythro form (2S,3R) of sphingosine base [174].

2D NMR

2D NMR spectroscopy has been used to characterize ceramide/cerebrosides as genuine (underivatized) or perace-tylated samples. Series of 2-D homonuclear and heteronu-clear NMR experiments (1H-1H TOCSY, NOESY, HSQC, HMQC, and HMBC) are used to identify and elucidate struc-ture. Disolving samples in DMSO-d6/2% D2O afforded supe-rior solubility characteristics as compared with CDCl3/CD3OD, and chemical shift reproducibility for cere-broside without derivatization. 1H-1H COSY and TOCSY experiments were used for assignments of spin system con-nectivities of LCB and FA and 1H resonance chemical shifts, followed by identification of monosaccharide configura-tional using relative stereochemical assignments extracted


from the values of three-bond scalar coupling constants be-tween vicinal sugar protons. Assignment of all protons to their directly bonded carbons using 1H and 1H-13C HSQC spectra for 13C chemical shift assignments via one-bond 1H-13C connectivities. The determination of glycosidic linkages via strong through space dipolar cross relaxations could be extracted from 1H-1H NOESY and/or 1H-detected 1H-13CHMBC experiments. Also, the positions of the hydroxyl groups, double bonds, sugar moiety, and connectivity of FA with LCB were elucidated and confirmed through HMBC correlations. The relative stereochemistry of the conjugated

diene and sugar moieties was deduced by NOESY measure-ment [169,176].

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENTS

Declared none.

Table 2. 13C NMR of Ceramides and Glucosylceramides in CDC13/CD3OD

Chemical shift (ppm) Carbon atom

Glucosyl-erythro-ceramides Glucosyl-threo-ceramides Erythro-ceramides Threo-ceramides

1 69.10 69.55 61.87 62.57

2 53.84 53.74 55.16 55.16

3 72.58 70.70 73.58 71.24

4 134.72 133.48 134.27 133.35

5 129.75 129.50 129.32 129.37

C-6 32.71 32.76 32.56 32.51

C7-C15 30.37-29.38 30.37-29.38 30.0-29.40 30.10-29.40

16 32.26 32.31 32.13 32.16

17 22.96 22.97 22.85 22.85

18 14.22 14.22 14.20 14.20

1´ 175.09 175.63 175.11 175.38

2´ 36.90 36.89 36.84 36.79

3´ 26.35 26.35 26.05 26.10

4´ 30.37-29.38 30.37-29.38 30.10-29.40 30.10-29.40

5´ 30.37-29.38 30.37-29.38 30.10-29.40 30.10-29.40

6´ 30.37-29.38 30.37-29.38 30.10-29.40 30.10-29.40

7-15´ 30.37-29.38 30.37-29.38 30.10-29.40 30.10-29.40

16´ 32.26 32.31 32.13 32.16

17´ 22.96 22.97 22.85 22.85

18´ 14.22 14.22 14.20 14.20

1´´ 103.71 103.80

2´´ 74.15 74.12

3´´ 77.01 77.01

4´´ 70.69 70.70

5´´ 76.80 76.81

6´´ 61.99 62.15


Appendix 1: Structure elucidation examples As noted above, ceramides are physiologically active compounds. They possess great scientific and practical interest. The study of ceramides structures is important for resolving issues connected with chemotaxonomy and the search for new physio-logically active compounds. The isolation, chemical transformations, synthesis, and biological conversions in addition to in-formation about the physiological activity of natural ceramides compounds have been reviewed in monographs and articles. Therefore, we considered it advisable to include brief spectral data of some isolated ceramides and cerebrosides from natural sources.

HO

HN

OH

OH

O

OH

10-23

H 7.38 (d, 8.7)

H 3.66 (dd, 4.8, 11.4), 3.75 (brd, 11.2)C 61.3

H 4.03 (m)C 51.8

H 3.44 (brs)C 75.8

H 3.47 (brs)C 72.4

H 1.33 (m), 1.51 (m)C 33.1

H 1.10-1.30 (brs)C 25.7

7-13

H 1.10-1.30 (brs)C 29.3-29.6

H 1.10-1.30 (brs)C 32.5

H 1.10-1.30 (brs)C 22.6

H 0.82 (t, 6.3)C 14.0

C 175.5

H 3.98 (dd, 3.6, 8.4)C 72.0

H 1.50 (m), 1.73 (m)C 34.4

H 1.35 (m)C 25.2

H 1.35 (m)C 25.2

H 1.61 (m), 1.94 (m)C 32.7

H 5.34 (brs)C 129.7

H 5.34 (brs)C 130.8

H 1.33 (m), 1.87 (m)C 32.5

H 1.10-1.30 (brs)C 29.3-29.6

H 1.10-1.30 (brs)C 32.5

H 1.10-1.30 (brs)C 22.6

H 0.82 (t, 6.3)C 14.0

Glumoamide (1)

Glumoamide ((2R,7E)–2–hydroxy–N–[(2S,3S,4R)–1,3,4–trihydroxy–hexadecan–2–yl]hexacos–7–enamide) (1):White powder, mp 127.0–127.3 °C. [ ]D +10.5 (c 0.03, HF). IR (KBr) max: 3330, 3203, 2918, 2850, 1620, 1544, 1263 cm–1. HRE-SIMS m/z: 682.6319 [M+H]+, 668.6182 [M–CH2+H]+, 654.6033 [M–C2H4+ H]+, 40.5844 [M–C3H6+H]+. NMR data (CDCl3+CD3OD, 300 and 75 MHz) [1].

H 7.53 (d, 8.8)

H 3.73 (m), 4.01 (dd, 5.5, 11.4)C 68.6 H 3.52 (t, 10.4)

C 74.1

H 3.46 (brd, 10.3)C 72.0

H 1.10-1.30C 32.5

C 175.6

H 3.96 (dd, 3.6, 8.0)C 72.0

O

HN

OH

OH

O

OH

5-22

OHO

HOOH

OH

H 4.14 (brd, 3.6)C 50.0 H 1.10-1.30

C 25.9

H 1.10 -1.30 (brs)C 29.2-29.6

H 1.98 (m)C 27.0

H 5.27 (brs)C 129.2

H 5.27 (brs)C 130.1

H 1.98 (m)C 27.1

H 1.10 -1.30 (brs)C 29.2-29.6

H 1.10 -1.30 (brs)C 31.8

H 1.10 -1.30 (brs)C 22.5

H 0.80 (t, 6.4)C 13.9

H 1.10 -1.30 (brs)C 34.2

H 1.10 -1.30 (brs)C 25.2

H 1.10 -1.30 (brs)C 32.0

H 1.10 -1.30 (brs)C 22.5

H 0.80 (t, 6.4)C 13.9

H 4.22 (d, 7.6)C102.9

H 3.21 (brd, 7.2)C 73.2H 3.36 (t, 8.0)

C 76.1

H 3.36 (t, 8.0)C 69.5

H 3.22 (brs)C 76.0

H 3.69 (m), 3.78 (brd, 11.2)C 61.1

Glumoside (2)

Glumoside (2R)–N–((2S,3S,4R,9Z)–1–O–[( –D–glucopyranosyl]–3,4–dihydroxy heptadec–9–en–2–yl)–2–hydroxypentacosanamide) (2). White powder, mp 199.6–199.8 °C. [ ]D +17 (c 0.035, THF). IR (KBr) max: 3402, 1620, 1544 cm–1. HRESIMS m/z: 844.6897 [M+H]+. NMR data (CDCl3+CD3OD, 300 and 75 MHz) [1].


NH

HO

OH

O

10

4-20

H 3.95 (dd, 3.7, 11.0), 3.70 (dd, 3.2, 11.2)

C 62.5

C 174.0

H 5.52 (dd, 6.3, 15.4)C 128.7 Iotrochotamide I (3)

H 3.92 (m)C 54.5

H 4.30 (t, 5.0)C 74.6

H 5.76 (dd, 6.6, 15.4)C 134.4

H 2.07 (q, 6.9)C 32.2

H 1.20-1.35 (m)C 29.1-29.9

H 1.15 (q, 6.6)C 39.0

H 1.52 (septet, 6.9)C 27.9

H 0.86 (d, 6.6)C 22.6

H 0.86 (d, 6.6)C 22.6

H 2.23 (t, 8.2)C 36.8

H 1.64 (m)C 25.7

H 1.23 (m)C 22.7

H 1.20-1.35 (m)C 29.1-29.9

H 0.88 (t, 6.5)C 14.0

H 6.25 (d, 7.3)

Iotrochotamide I ((2S,3R,4E)–N–docosanoyl–2–amino–19–methyl–icosa–4–ene–1,3–diol) (3). Colorless amorphous powder. [ ]D –8.3 (c 0.1, CHCl3). EIMS m/z (rel. int.): 663 [M]

+ (30.7), 365 (17.9), 281 (3.5), 97 (37.9), 57 (84.9), 43 (100). IR (KBr) max: 3550, 3360, 1650, 2930, 2850, 1395 cm–1. [2].

NH

HO

OH

O

11

17

H 4.46 (m)C 62.4

C 173.4

H 4.28 (m)C 73.1

Strepsiamide A (4)H 5.06 (m)

C 53.8H 4.37 (m)

C 76.8

H 2.22 (m), 1.95 (m)C 33.8

H 1.68 (m)C 26.4

H 1.20-1.42 (m)C 29.0-30.0

H 1.41 (m)C 22.6

H 0.85 (d, 7.5)C 22.7

H 2.46 (t, 7.2)C 369

H 1.78 (m)C 26.5

H 1.23 (m)C 31.7

H 1.20-1.42 (m)C 29.0-30.0

H 0.84 (t, 6.6)C 14.4

OH

H 0.85 (d, 7.5)C 22.7

H 8.46 (d, 8.5)

H 1.27 (m)C 22.8

Strepsiamide A ((2S,3S,4R)–D–erythro–N–tricosanoyl–2–amino–18–methyl–isocosan–1,3,4–triol) (4): White amor-phous powder. [ ]D +14° (c 0.1, CHCl3). IR (KBr) max: 3400, 2940, 1640, 1535 cm–1. NMR data (C5D5N, 600 and 150 MHz). APCIMS m/z: 682 [M+H]+, 664, 654, 646, 640, 368, 296 [3].


NH

HO

OH

O

11-15

5-21

H 4.48 (dd, 6.7, 10.7),4.43 (dd, 4.5, 10.7)

C 62.0

C 175.2

H 4.31 (m)C 72.9

Trolliamide (5)

H 5.11 (m)C 52.9

H 4.61 (m)C 76.8

H 2.00 C 33.8

H 1.74 (m)C 26.7

H 5.53 (m)C 130.8

H 4.35 (m)C 72.4

H 1.98 (m)C 35.7

H1.76 (m)C 25.8

H 1.24-1.30 (m)C 29.5-30.3

H 0.87 (t, 6.0)C 14.3

OH

H 0.87 (t, 6.0)C 14.3H 8.58 (d, 8.6)

H 2.16 (m)C 33.3

HO

H 1.24-1.30 (m)C 32.1

H 1.24-1.30 (m)C 22.9

H 5.53 (m)C 130.7

H 1.99 (m)C 33.0

H 1.24-1.30 (m)C 29.5-30.3

H 1.24-1.30 (m)C 32.1

H 1.24-1.30 (m)C 22.9

Trolliamide (5): White powder, mp 116–118 °C. (–) HRESIMS m/z: 680.6279 [M+H]– (calcd for C42H82NO5, 680.6271); EIMS m/z: 681 [M]+, 663 [M–H2O]+, 456, 439, 408, 384, 356, 280, 262, 239, 185, 155, 139, 129, 125, 98, 97, 83, 60, 57. UV (CDCl3) max: 243 nm. IR (KBr) max: 3334 (NH), 3215 (OH), 2919, 2850 (CH), 1622 (C=O), 1545, 1467 (–CH=CH–), 1357, 1271, 1067, 1021, 964, 723 cm–1. [4].

NH

HO

OH

O

10

H 4.51 (dd, 4.1, 10.5),4.42 (dd, 4.7, 10.5)

C 62.1

C 175.3

H 4.28 (m)C 73.0

H 5.11 (m)C 53.0

H 4.36 (dd, 4.0, 6.5)C 76.8

H 1.91 (m)C 34.2

H 1.71 (m)C 25.9

H 1.45 (m)C 32.4

H 4.62 (dd, 3.8, 7.7)C 72.5

H 2.03 (m)C 35.8

H 2.26 (m)C 34.6

OHH 8.57 (d, 8.8)

HO

H 0.86 (t, 6.7)C 14.3H 1.21

C 29.6_

H 1.96 (m)C 27.6

H 1.75 (m)C 23.0

H 1.25 (m)C 23.0

H 2.11 (m)C 26.8

H 5.49 (brs)C 130.3

H 5.49 (brs)C 130.3

H 2.11 (m)C 26.8

11

H 1.45 (m)C 32.4

H 0.86 (t, 6.7)C 14.3

Hygrophamide (6)

Hygrophamide (6): mp 121 123 C. [ ]D +7.65 (c 0.3, pyridine). UV (MeOH). max (log ): 205 nm (3.43), 194 nm (3.09). IR (KBr) max: 3435 (OH), 2921, 2849, 1651, 1540, 1468, 1261, 1077 cm–1. HRFABMS m/z: 680.6168 [M–1]– (calcd. for C46H82NO5 680.6189). EIMS m/z (rel. int.): 682 (35) [M+1]+, 664 (55) [M+1]+ [5].


C 174.7

H 4.44 (d, 7.1)C 73.3

H 1.10 (m)C 40.1

H 1.25-1.35 (m)C 20.8

H 0.88 (t, 6.7)C 13.8

H 2.05 (m)

H 5.71 (dt, 5.9, 15.3)C 133.3

H 2.12 (m)H 2.05 (m)

H 1.25-1.35 (m)C 23.2

8H 0.88 (t, 6.7)

C 14.2

HN

OH

O

OH

HO

H 7.48 (d, 8.5)

H 6.00 (m)C 131.6

H 6.00 (m)C 131.6

H 5.84 (dtd, 1.6, 6.8, 15.5)C 134.3

H 5.52 (m)C 128.5

H 1.38 (m)

H 1.38 (m)

H1.25-1.35C 29.2-32.5

H 0.85 (t, 6.8)C 19.6

H 5.55 (m)C 131.1

H 5.55 (m)C 132.8

H 5.49 (m)C 130.8

H 4.08 (t, 4.4)C 72.7

H 3.83 (m)C 55.5

H 2.08 (m)

H 3.80 (dd, 3.5, 11.1),3.68 (dd, 3.1, 11.1)

C 61.1

H 1.38 (m)Ceramide 1 (7)

Ceramides 1 (7): Amorphous solid. IR (KBr) max: 3360, 2852, 1650, 1538, 1463, 1050 cm–1. NMR (CD3OD, 500 and 125 MHz) [6].

O

NH

O

OH

OH

OHO

HOOH

OH

Lutaoside (8)

H 2.25 (m)C 33.5

C 175.0

H 5.20 (m)C 53.0 H 4.50 (m)

C 75.4

H 4.58 (dd, 4.9, 10.7),4.40 (dd, 4.3, 10.7)

C 68.8

H 4.49 (d, 7.8)C 102.9

H 8.50 (d, 8.2)

H 0.90 (t, 6.4)C 13.0

H 5.50 (dt, 5.3, 15.4)C 128.7

H 5.00 (dt, 4.7, 15.4)C 128.1

H 2.25 (m)C 32.8

H 1.28 (brs)C 24.0-26.5

H 2.11 (m)C 33.0

H 1.99 (m)C 32.1

H 5.54 (dd, 4.8, 16.2)C 129.8

H 5.40 (dd, 4.6, 16.2)C 1291

H 1.28 (brs)C 24.0-26.5

H 4.20 (t, 7.3)C 72.5 H 1.80 (m)

C 31.0

4-14

H 1.70 (m)C 21.9

H 0.90 (t, 6.4)C 13.0

H 4.48 (m)C 73.5

H 4.00 (m)C 71.9

H 4.11 (m)C 70.9

H 4.55 (m)C 70.0

H 3.98 (dd, 5.5, 11.5),3.70 (dd, 2.4, 11.5)

C 61.8

O

NH

O

OH

OH

OHO

HOOH

OH

m/z 197m/z 277

m/z 459

m/z 254

m/z 71m/z 97m/z 207

m/z 237

m/z 477

m/z 163 EIMS fragmentation pattern of 8.

Lutaoside (1–O– –D–Glucopyranosyl–(2S,3R,5E,12E)–2N–[(2´R)–hydroxyhexad– ecanoyl]–octadecasphinga–5,12–dienine) (8): Crystalline compound with. mp 203–205 °C. [ ]D +8.12 (c 0.04, MeOH). IR (KBr) max: 3445 (OH), 3209 (NH), 2924, 2853, 1647, 1586, 1427, 1388, 1217, 1167, 1078, 1047, 1040, 881 cm–1. NMR data (C5D5N, 400 and 100 MHz). FABMS m/z: 736 [M+Na]+, 714 [M+H]+, 696 [M+H–H2O]+, 678 [M+H–2H2O]+, 552 [M+H–C6H11O5 (sugar moiety)]+; HRFABMS: 736.5339 [M+Na]+ [7].


HO

NH

O

OH

OH

18

5

9

H 8.57 (d, 8.8)

H 5.52 (m)C130.8

H 5.50 (m)C130.7

H 5.10 (d, 4.3)C 52.9

H 4.61 (m)C 72.5

H 4.51 (brs),4.42 (m)

C 62.0

H 4.33 (m)C 76.9 H 4.27 (m)

C 73.0

OHC 33.0

C 26.8

H 1.25-1.30C 29.6-30.3

C 32.2

C 33.0

C 175.3

H 1.25-1.30C 29.6-30.3

C 22.9H 0.85 (t, 6.7)C 14.3

C 35.7

C 25.8

H 1.25-1.30C 29.6-30.3

C 22.9H 0.85 (t, 6.7)C 14.3

(2S,3S,4R,10E)–2–[(2'R)–2–hydroxytetracosanoylamino]–1,3,4–octadecanetriol–10–ene (9): White amorphous powder (MeOH); mp 138–139 °C. (–) FABMS m/z: 680 [M–H]–. NMR data (C5D5N, 500 and 125 MHz) [8].

O

NH

O

OH

OH

OHO

HOOH

OH

C 72.5

C 175.7

H 5.27 (d, 4.3)C 51.9

C 75.9

C 70.4

H 4.93 (d, 6.9)C 105.5

H 8.54 (d, 8.8)

H 0.85 (t, 6.7)C 14.3

H 5.50 (m)C 130.9

H 5.49 (m)C 130.7

C 32.9

13-16

C 33.0

C 33.3

H 1.24-1.31C 29.6-30.0

C 72.5C 35.6

H 0.85 (t, 6.7)C 14.3

C 75.2

C 78.5

C 71.6C 78.6

C 62.7

OH

10

C 32.8

C 32.2

C 25.9

5-22

H 1.24-1.31C 29.6-30.0

C 23.0

C 23.0

1–O– –D–glucopyranosyl–(2S,3S,4R,10E)–2–[(2'R)–2–hydroxytetracosanoyl–amino]–1,3,4–octadecane–triol–10–ene (10): White amorphous powder (MeOH); mp 203–205 °C. (–) FABMS m/z: 842 [M–H]–. NMR data (C5D5N, 500 and 125 MHz) [8].

O

NH

O

OH

C 172.2

H 3.90 (m)C 55.2H 4.3 (d, 8.5)

C 102.0

H 7.43 (d, 9.3)H 0.85 (t, 6.9)

C 14.3

9-20

5.45 (ddd, 6.4, 8.2, 15.1)

C 128.1

1.60

C 31.7

H 3.50 (m)C 71.0

21

H 2.40 (m)C 33.0

H 1.90 (m)C 34.3

H 3.43 (m)C 74.1

H 3.32 (m), 3.25 (m)C 63.5

H 3.63 (m)C 69.5

H 4.77 (d, 4.2)C 99.0

H 340C 68.4

H 4.35 (m), 4.20 (m)

C 63.5

H 3.55C 75.0

H 2.30C 35.8

1.20

C 29.0-33.0

H 3.70 (m)C 83.2

OH

5.71 (ddd, 6.6, 8.5, 15.1)

C 131.5

H 0.85 (t, 6.9)C 14.3

1.20

C 29.0-33.0

O

HOOH

OHO

OH

HOOH

O

OH

H 3.20 (m)C 70.5

H 3.60 (m)C 69.2

H 3.28 (m)C 80.7

H 3.40 (m)C 68.2

H 3.30 (m), 3.40 (m)C 62.2

Petrocerebroside 1 (11)

Petrocerebroside 1 (11): Amorphous powder. ESIMS m/z: 1032.1 [M+H]+, 1031.2 [M]+. NMR data (DMSO–d6) [9].


O

NH

O

OH

C 170.0

H 3.40 (m)C 54.5H 4.3 (d, 8.5)

C 102.0

H 7.72 (d, 8.2)

H 0.85 (t, 6.9)C 14.3

10

5.45 (ddd, 6.4, 8.2, 15.1)

C 128.1

1.60

C 31.7

H 3.50 (m)C 71.0

20

H 2.40 (m)C 33.0

H 1.90 (m)C 34.3

H 3.43 (m)C 74.1

H 3.32 (m), 3.25 (m)C 63.5

H 3.63 (m)C 69.5

H 4.77 (d, 4.2)C 99.0

H 340C 68.4

H 4.35 (m), 4.20 (m)

C 63.5

H 3.55C 75.0

H 2.30C 35.8

1.20

C 29.0-33.0

H 3.70 (m)C 83.2

OH

5.71 (ddd, 6.6, 8.5, 15.1)

C 131.5

H 0.85 (t, 6.9)C 14.3

1.20

C 29.0-33.0

O

HOOH

OHO

OH

HOOH

O

OH

H 3.20 (m)C 70.5

H 3.60 (m)C 69.2

H 3.28 (m)C 80.7

H 3.40 (m)C 68.2

H 3.30 (m), 3.40 (m)C 62.2


Petrocerebroside 2 (12): Amorphous powder. ESIMS m/z: 1018.1 [M+H]+, 1017.2 [M]+. NMR data (DMSO–d6) [9].

O

HN

O

OH

OHO

HOOH

OH

C 175.4

H 3.93 (4.5, 8.0)C 54.6

H 0.90 (t, 6.0)C 14.6

H 6.02 (d, 15.5)C 136.3

1.71 (s)

C 12.9

3

2.08 (td, 6.5, 7.5)

C 33.7

H 1.29 (m)C 30.4-31.1

H 4.87 (d, 4.5)C 103.6

H 3.96 (dd, 4.5, 7.5)C 79.1

2.03 (td, 6.5, 7.5)

C 33.6

H 5.50 (dd, 6.0, 15.5) C 129.1

H 1.29 (m)C 23.9

H 5.47 (8.0, 15.5)C 131.6

H 3.63 (m), 3.56 (m)C 64.3

H 4.10 (t, 7.5)C 76.3

H 3.63 (m)C 74.4

H 3.69 (m)C 83.6

H 3.88 (dd, 4.5, 1.5), 3.83 (4.5, 10.5)

C 68.8

H 4.14 (t, 8.0)C 72.9

H 4.45 (d, 6.0)C 74.3

2.08 (td, 7.0, 7.5)

C 34.1

OH

H 5.72 (6.5, 15.5)C 134.3

2.20 (td, 7.0, 7.5)

C 29.0H 5.35 (t, 7.0)

C 130.6

C 135.3

H 5.55 (d, 7.0, 15.5)C 128.7

1.38 (m)

C 30.4

H 5.83 (dd, 6.5, 15.5) C 135.0

10

H 1.29 (m)C 30.4-31.1

1.38 (m)

C 30.4

H 1.29 (m)C 23.9

H 0.90 (t, 6.0)C 14.6

Sarcoehrenoside A (13)

Sarcoehrenoside A (13): White amorphous powder. [ ]D +77.0 (c 0.2, MeOH). IR (KBr) max: 3387, 2951, 2859, 1638, 1547, 1456, 1387, 1241, 1131, 1035, 737 cm–1. UV max (MeOH): (log ) 224 (3.97) nm. NMR data (CD3OD, 500 and 125 MHz). HRESIMS m/z: 774.5490 [M+Na]+ (calcd for C43H77NO9Na, 744.5496) [10].

HO

HN

O

O

C 175.7

H 5.11 (m)C 53.5

H 0.85 (t, 6.5)C 14.7

H 8.55 (d, 8.8)

8

H 5.48 (dt, 6.0, 14.5)C 131.2

H 1.70 (m)C 34.7

H 4.28 (m)C 73.5

H 4.51 (dd, 4.5, 10.5)4.41 (dd, 4.5, 10.5)

C 62.5

H 4.34 (dd, 4.5, 6.5)C 77.4

H 4.62 (4.0, 8.0)C 73.0

OH

H 1.86 (m)C 33.4

1.73 (m)

C 32.6

11

H 1.30 (s)C 30.1

H 0.85 (t, 6.5)C 14.7

H 1.58 (m)C 26.3

OH

H 5.56 (dt, 6.0, 14.5)C 131.3

C 23.4

H 1.95-2.10 (m)C 33.8

H 1.95-2.10 (m)C 33.8

H 1.30 (s)C 30.1

Tithoniamide B (14)


Fragmentation pattern of tithoniamide B

HO

HN

O

OH

OHOH

11

8

m/z 67m/z 83

m/z 97

m/z 225m/z 283

m/z 255

m/z 356m/z 298

m/z 309m/z 339m/z 280

Tithoniamide B (14): Colorless powder. mp 135°C. [ ]D +10.23 (c 0.92, CHCl3+MeOH). IR (KBr) max: 3610, 2940, 2860, 1640, 1297 cm–1. NMR data (CDCl3, 500 and 125 MHz). HREIMS m/z: 681.6252 (calcd. 681.6271 for C42H83NO5). CIMS m/z:682.4 [M+1]+ [11].

Tanacetumide A (15)

HO

HN

OHH 5.10 (m)C 53.0

H 8.56 (d, 8.7)

H 0.86 (t, 6.1)C 14.2

C 25.8

7-18

H 5.36 (dt, 6.1, 15.1)C 129.1

H 4.33 (dd, 5.5, 10.7)H 4.42 (dd, 3.8, 10.0)

C 62.1

H 4.52 (m)C 72.5

C 175.2

O

OH

6-18

H 4.60 (m)C 73.0

H 5.57 (dt, 6.0, 15.3)C 129.5

H 5.44 (dt, 6.2, 15.5)C 130.8 H 2.05 (m)

C 32.9

H 1.25 (m)

C 22.9

H 5.52 (dt, 5.8, 15.1)C 130.2

H 2.19 (m)C 32.1

C 25.8

C 22.9

H 0.86 (t, 6.1)C 14.2

HO

HN

OH

7-18

O

OH

6-18

m/z 235

m/z 211

m/z 340

m/z 325

m/z 365

m/z 281

Fragmentation pattern of 15.

Tanacetumide A ((2S,2R,3R,3E,4E)–N–[2´–hydroxy–3´–henicosenoyl]–2–amino–4–henicosene–1,3–diol) (15): Color-less oil. [ ]D –6.00 (c 0.01, CHCl3 + MeOH). IR max (C5D5N): 3605, 2940, 2850, 1630, 1455 cm–1. NMR (C5D5N). HREIMS m/z: 663.6157 (calcd. 663.6144 for C42H81NO4), 382.3311 (calcd. 382.3321 for C23H44NO3), 365.3282 (calcd. 365.3293 for C23H43NO2), 340.3229 (calcd. 340.3215 for C21H42NO2), 323.2936 (calcd. 323.2949 for C21H39NO2); EIMS m/z (rel. int.): 663 [M]+ (1), 382 [M–CH3(CH2)15CH=CHCH(OH)]+, 365 [M–CH3(CH2)15CH=CHCH(OH)2]+, 340 (56), 325 (54), 323[CH3(CH2)16CH=CHCH(OH)CO]+, 340[CH3(CH2)16CH=CHCH(OH)CONH2 +H]+, 281 (32), 211 (26) [12].

HO

HN

OHH 4.00 (m)C 51.3

H 7.42 (d, 8.6)

H 0.80 (t, 6.7)C 13.5C 25.5

5.35 (dt, 6.5, 15.5)

C 130.4

7-18H 3.65 (dd, 4.8, 11.7)H 3.72 (dd, 4.1, 11.7)

C 60.8

H 4.50 (m)C 71.9

C 171.0

O

H 5.27 (dt, 6.0, 15.2)C 129.6

C 24.8

1.23 (brs)

H 1.95 (m)C 32.2

C 22.2

Tanacetumide B (16)

Tanacetumide B ((2S,3R,4E)–N–[acetoxy]–2–amino–4–henicosene–1,3–diol) (16): Colorless oil. [ ]D –6.67 (c 0.03, CHCl3+MeOH). IR max (C5D5N): 3340, 2930, 2850, 1620, 1540 cm–1. NMR (CDCl3). HREIMS m/z: 383.3383 (calcd. for C23H45NO3, 383.3399); EIMS m/z (rel. int.): 383 [M]+ (1), 298 [CH3(CH2)15CH=CHCH(OH)2]+, 281 [CH3(CH2)15CH= CHCH(OH)]+, 225 (34), 39 (22), 97 (34) [12].


HN

OH

O

OH

H 6.20 (d, 7.3)C 173.8

H 2.26 (t, 7.5)C 36.88

H 1.63 (m)C 25.78

4-20

H 1.23 (m)C 22.70

H 0.89 (t, 6.8)C 14.1

H 1.23C 29.72

H 3.68 (dd, 3.0, 11.0),3.94 (dd, 3.8, 11.0)

C 62.58

H 3.89 (m)C 54.57

H 4.30 (dd, 3.4, 6.6)C 74.76

H 1.23 (m)C 29.96

H 5.51 (dd, 6.6, 15.4)C 128.87

H 5.77 (dd, 7.2, 15.4)C 134.34

H 2.04 (m)C 32.29

H 1.83 (m)C 31.94

8-13

H 1.23C 29.72

H 0.85 (d, 6.6)C 22.67

H 0.85 (d, 6.6)C 22.67

C22 ceramide (17)

C22 ceramides (N–docosanoyl–D–erythro–(2S,3R)–16–methyl–heptadecasphing 4(E)–enine) (17): Colorless powder. [ ]D –6.0° (c 0.01, MeOH). IR (KBr) max: 3418, 2950, 2922, 2854, 1634, 1386, 1060 cm–1. UV (MeOH) max: 206 nm. FABMS m/z: 621 [M–H]–; HRFABMS m/z: 621.6134 [M–H]– (calcd for C40H79NO3, 621.6138). NMR data (CDCl3) [13].

18

HO

NH

O

OH

4-12

11-18H 3.70 (dd, 3.6, 10.8),3.95 (dd, 3.6, 11.4)

C 62.4 H 3.91(m)C 54.4

H 4.32 (brs)C 74.6

H 5.54 (dt, 15.0, 6.0)C 128.9

H 5.78 (dt, 15.0, 6.6)C 130.9

H 2.15 (q, 6.6)C 32.4

H 2.08 (q, 6.6)C 32.2

H 5.36 (dt, 6.6, 15.0)

C 134.3

H 1.97 (q, 7.8)C 32.6

H 1.30 (m)C 29.0

H 0.88 (t, 7.2)C 14.2

H 6.28 (d, 7.2)

C 174.1

H 2.23 (t, 7.2)C 36.8

H 1.64 (m)C 32.3

H 1.30 (m)C 29.0

H 0.88 (t, 7.2)C 14.2

H 5.43 (dt, 6.6, 15.0)

C 129.1

H 1.30 (m)C 25.8

H 1.30 (m)C 22.7

(2S,3S,4E,8E)–2N–[tetradecanoyl]–4(E),8(E)–icosadiene–1,3–diol (18): Colorless amorphous powder, mp 123–125 °C. [ ]D +83 (c 0.1, CHCl3). IR (KBr) max: 3340–3200 (OH and amide NH), 1640 (amide carbonyl), 1620 (olefinic double bond) cm–1. NMR data (CDCl3). HRFABMS m/z: 558.4876 [M+Na]+ (Calcd 558.4862); EIMS m/z: 535 [M, C34H65NO3]+ [14].

m/z 280

Newbouldiamide (19)

HO

NH

O

OH

4-12

8-21H 4.50 (dd, 6.0, 10.5),4.35 (dd, 6.0, 10.5)

C 62.9H 5.11 (m)

C 553.8H 4.42 (m)

C 77.6

H 4.61 (m)C 73.3

H 1.99 (m)C 33.0

H 1.72 (m)C 33.0

C 27.5

C 23.8H 0.86 (t, 6.8)

C 15.1

H 8.57 (d, 9.0)

C 176.1

H 2.23 (t, 7.2)C 36.6

H 1.76 (m)C 35.0

HO

NH

O

OH

11

OH

OH

14

m/z 226

m/z 267

m/z 284

m/z 384

m/z 357m/z 327

m/z 297

OH

OH

H 4.27 (m)C 73.9

H 1.29 (brs)C 29.9

H 1.29 (brs)C 29.9

C 27.5

C 23.8H 0.86 (t, 6.8)

C 15.1


Newbouldiamide (1,3,4,5–tetrahydroxy–2–octadecanoyl–amino–tetracosane) (19): Colorless powder. mp 129 °C. [ ]D+12.0° (c 0.001). IR (KBr) max: 3610, 2930, 2860, 1640, 1297 cm–1. NMR data (C5D5N). CIMS (CH4) m/z: 684.4 [M+1]+;EIMS m/z (rel. int.): 683.4 [M]+ (10) [15].


H 4.33 (dd, 4.8, 6.2)C 74.9

C 173.9

H 2.23 (t, 7.7)C 36.8

H 3.92 (m)C 54.4

H 6.23 (d, 7.7)

4-13

H 3.70 (3.3, 11.0)H 3.96 (dd, 3.7, 11.4)

C 62.5

7HO

HN

O

OH

H 5.56 (dd, 6.2, 15.8)C 129.0

H 5.80 (dt, 5.9, 15.8)C 133.8

H 5.09 (t, 6.6)C 123.0

H 1.25-1.36 (brs)C 28.0-29.7

H 1.64 (m)C 25.8

H 0.88 (d, 7.3)C 14.1

20

H 1.58 (brs)C 16.0

C 22.7

C 136.3

H 2.08 (m)C 27.5

H 2.11 (m)C 32.5

C 31.9C 22.7

H 0.88 (d, 7.3)C 14.1

(2S,3R,4E,8E)–N–hexadecanoyl–2–amino–9–methyl–4,8–octadecadiene–1,3–diol (20): Amorphous powder. [ ]D –11.6° (c 0.09, CHCl3). IR (CHCl3) max: 3605, 3434, 2928, 2855, 1657, 1510, 1466 cm–1. NMR data (CDCl3). HREIMS m/z: 549.5113 (M+, Calcd for C35H67NO3: 549.5121). EIMS m/z (rel. int. %): 549 [M]+ (2), 298 (15), 281 (43). (–) FABMS m/z: 548 [M–H]–

[16].

H 4.30 (brs)C 74.9

C 173.8

H 2.24 (t, 7.7)C 36.8

H 3.91 (m)C 54.3

H 6.25 (d, 7.3)

4-7

H 3.70 (m)H 3.96 (ddd, 3.3, 3.3, 11.4)

C 62.5

7HO

HN

O

OH

H 5.56 (dd, 6.6, 15.4)C 129.0

H 5.80 (dt, 6.6, 15.4)C 133.8

H 5.09 (t, 6.6)C 123.0

H 1.26-1.32 (brs)C 28.0-29.6

H 2.05 (m)C 27.2

H 1.65 (m)C 25.7

H 0.88 (d, 7.0)C 14.1

21

H 1.58 (d, 0.7)C 16.0

C 22.7

C 136.3

H 2.09 (m)C 27.5

H 2.11 (m)C 32.5

C 22.6

H 1.26-1.32 (brs)C 28.0-29.6

H 5.39 (m)C 130.1

H 5.33 (m)C 128.1

H 2.77 (t, 7.0)C 25.6

H 5.33 (m)C 127.9

H 5.39 (m)C 130.2

H 2.05 (m)C 27.5

C 28.0-29.6

C 31.5 H 0.88 (d, 7.0)C 14.1

(2S,3R,4E,8E,9´Z,12´Z)–N–9´,12´–octadecadienoyl–2–amino–9–methyl–4,8 octadecadiene–1,3–diol (21): Amorphous powder. [ ]D –13.3° (c 0.08, CHCl3). IR (CHCl3) max: 3603, 3435, 2929, 2856, 1656, 1510, 1466 cm–1. HREIMS m/z: 573.5092 (M+, Calcd for C37H67NO3: 573.5121). EIMS m/z (rel. int. %): 573 [M]+ (4), 444 (M+–C8H15–H2O) (3), 322 (26), 305 (100). (–) FABMS m/z: 572 [M–H]– [16].

NH

HO

OH

O

5

10

H 3.93 (dd, 3.3, 10.5),3.69 (brd, 10.5)

C 62.4

C 175.2

H 5.54 (dt, 6.4, 15.5)C129.3

H 3.91 (m)C 54.8

H 4.31 (m)C 74.3

H 5.78 (dt, 6.4, 15.5)C 133.4

H 2.10 (m)C 32.3

H 5.36 (dt, 5.8, 15.0)C 131.3

H 2.23 (t, 7.6)C36.8

H 1.64 (m)C 25.7

H1.76 (m)C 25.8

C 29.3-29.6

H 0.88 (t, 6.8)C 13.9

H 0.88 (t, 6.6)C 13.9H 6.26 (d, 7.0)

H 2.10 (m)C 32.5

C 31.9

C 22.6

H 5.42 (dt, 5.8, 15.0)C 129.0

H 1.95 (m)C 32.1

H 1.25 (brs)C 29.3-29.6

C 31.9

C 22.6

22

D (-)-2S,3R-2-Aminooctadeca-4E,8E-diene-1,3-diol-N-palmitate (22). Amorphous powder, [ ]D - 6.8° (c 0.21, MeOH). [ ]D -4.0° (c 0.32, CHCl3). IR max: 3500-3100, 2960, 2930, 2860, 1650, 1630, 1550, 1470, 1380, 1060 and 970 cm-1. EIMS m/z(rel. int.): 535 (< 1%), 534 (< 1), 517 (2), 504 (1), 486 (1), 368 (1.5), 366 (2), 352 (3), 351 (3), 337 (1), 320 (2), 299 (8), 298 (45), 281 (100), 280 (10), 262 (6), 250 (10), 239 (5) and 238 (4) [17].


O

NH

O

OH

OH

OHO

HOOH

OH C 175.7

H 4.05 (m)C 54.6

H 4.92 (d, 7.7)C 105.7

H 8.38 (d, 8.8) H 0.86 (t, 6.9)C 14.3

H 5.49 (m)C 132.1

2.14

C 32.1

11-16

2.14

C 32.2

H 1.25-1.35C 29.6-30.0 1.71

C 35.7

H 0.85 (t, 6.9)C 14.3

H 4.05C 75.2

1.73

C 25.9

5-14

C 23.0

Soya-cerebroside I (23)

H 5.77 (m)C 132.1

H 5.49 (m)C 130.0

H 5.49 (m)C 131.1

H 4.51 (m), 4.38 (m)C 62.7

H 4.25C 78.5

H 4.21C 71.5

H 3.92C 78.6

H 4.25 (m), 4.05C 70.2

H 4.05C 71.5

H 3.92C 72.3

1.99

C 32.9

C 23.0

Soya-cerebroside I (23): White amorphous powder (MeOH). mp 194-195 °C. (-) FABMS m/z: 712 [M-H]- [8].

O

NH

O

OH

C 172.2

H 3.90 (m)C 55.2H 4.3 (d, 8.5)

C 102.0

H 7.43 (d, 9.3)

H 0.85 (t, 6.9)C 14.3

9-20

5.45 (ddd, 6.4, 8.2, 15.1)

C 128.1

1.60

C 31.7

H 3.50 (m)C 71.0

21

H 2.40 (m)C 33.0

H 1.90 (m)C 34.3

H 3.43 (m)C 74.1

H 3.32 (m), 3.25 (m)C 63.5

H 3.63 (m)C 69.5

H 4.77 (d, 4.2)C 99.0

H 340C 68.4

H 4.35 (m), 4.20 (m)

C 63.5

H 3.55C 75.0

H 2.30C 35.8

1.20

C 29.0-33.0

H 3.70 (m)C 83.2

OH

5.71 (ddd, 6.6, 8.5, 15.1)

C 131.5

H 0.85 (t, 6.9)C 14.3

1.20

C 29.0-33.0

O

HOOH

OHO

OH

HOOH

O

OH

H 3.20 (m)C 70.5

H 3.60 (m)C 69.2

H 3.28 (m)C 80.7

H 3.40 (m)C 68.2

H 3.30 (m), 3.40 (m)C 62.2


Petrocerebroside 1 (24): An amorphous powder. ESIMS m/z: 1032.1 [M+H]+, 1031.2 [M]+ [9].

O

NH

O

OH

C 170.0

H 3.40 (m)C 54.5H 4.3 (d, 8.5)

C 102.0

H 7.72 (d, 8.2)

H 0.85 (t, 6.9)C 14.3

10

5.45 (ddd, 6.4, 8.2, 15.1)

C 128.1

1.60

C 31.7

H 3.50 (m)C 71.0

20

H 2.40 (m)C 33.0

H 1.90 (m)C 34.3

H 3.43 (m)C 74.1

H 3.32 (m), 3.25 (m)C 63.5

H 3.63 (m)C 69.5

H 4.77 (d, 4.2)C 99.0

H 340C 68.4

H 4.35 (m), 4.20 (m)

C 63.5

H 3.55C 75.0

H 2.30C 35.8

1.20

C 29.0-33.0

H 3.70 (m)C 83.2

OH

5.71 (ddd, 6.6, 8.5, 15.1)

C 131.5

H 0.85 (t, 6.9)C 14.3

1.20

C 29.0-33.0

O

HOOH

OHO

OH

HOOH

O

OH

H 3.20 (m)C 70.5

H 3.60 (m)C 69.2

H 3.28 (m)C 80.7

H 3.40 (m)C 68.2

H 3.30 (m), 3.40 (m)C 62.2


Petrocerebroside 2 (25): Amorphous powder. ESIMS m/z: 1018.1 [M+H]+, 1017.2 [M]+ [9].


O

HN

O

OH

OHO

HOOH

OH

C 175.4

H 3.93 (4.5, 8.0)C 54.6

H 0.90 (t, 6.0)C 14.6

H 6.02 (d, 15.5)C 136.3

1.71 (s)

C 12.9

3

2.08 (td, 6.5, 7.5)

C 33.7

H 1.29 (m)C 30.4-31.1

H 4.87 (d, 4.5)C 103.6

H 3.96 (dd, 4.5, 7.5)C 79.1

2.03 (td, 6.5, 7.5)

C 33.6

H 5.50 (dd, 6.0, 15.5) C 129.1

H 1.29 (m)C 23.9

H 5.47 (8.0, 15.5)C 131.6

H 3.63 (m), 3.56 (m)C 64.3

H 4.10 (t, 7.5)C 76.3

H 3.63 (m)C 74.4

H 3.69 (m)C 83.6

H 3.88 (dd, 4.5, 1.5), 3.83 (4.5, 10.5)

C 68.8

H 4.14 (t, 8.0)C 72.9

H 4.45 (d, 6.0)C 74.3

2.08 (td, 7.0, 7.5)

C 34.1

OH

H 5.72 (6.5, 15.5)C 134.3

2.20 (td, 7.0, 7.5)

C 29.0H 5.35 (t, 7.0)

C 130.6

C 135.3

H 5.55 (d, 7.0, 15.5)C 128.7

1.38 (m)

C 30.4

H 5.83 (dd, 6.5, 15.5) C 135.0

10

H 1.29 (m)C 30.4-31.1

1.38 (m)

C 30.4

H 1.29 (m)C 23.9

H 0.90 (t, 6.0)C 14.6

Sarcoehrenoside A (26)

Sarcoehrenoside A (26): White amorphous powder. [ ]D +77.0 (c 0.2, MeOH). IR (KBr) max: 3387, 2951, 2859, 1638, 1547, 1456, 1387, 1241, 1131, 1035, 737 cm-1. UV max (MeOH): (log ) 224 (3.97) nm. NMR data (CD3OD, 500 and 125 MHz). HRESIMS m/z: 774.5490 [M+Na]+ (calcd for C43H77NO9Na, 744.5496) [10].

H 1.29 (m)C 33.2

O

HN

O

OH

OHO

HOOH

OH C 177.4

H 3.99 (3.5, 7.5)C 54.7

H 0.90 (t, 6.0)C 14.6

H 1.97 (t, 7.5)C 40.9

1.59 (s)

C 16.3

4

2.06 (m)

C 34.0

H 1.29 (m)C 30.5-30.9

H 4.27 (d, 8.0)C 104.9

H 3.19 (dd,8.0, 9.5)C 75.1

1.29 (m)

C 30.5-30.9

H 1.70 (m), 1.55 (m) C 36.0

H 1.29 (m)C 23.9

H 5.48 (7.5, 15.5)C 131.3

H 3.87 (dd, 1.5, 12.0), 3.67 (5.0, 12.0)

C 62.8

H 3.34 (m)C 78.1

H 3.30 (m)C 71.7

H 3.28 (m)C 78.1

H 4.12 (dd, 3.5, 10.5), 3.71 (3.5, 10.5)

C 69.9

H 4.13 (t, 7.5)C 73.0

H 3.99 (dd, 4.0, 8.0)C 73.2

OH

H 5.74 (6.0, 15.5)C 134.8

2.07 (m)

C 28.8H 5.14 (t, 6.5)

C 125.0

C 136.9

H 1.39 (m)C 29.3

H 1.41 (m)C 26.3

15

H 1.29 (m)C 23.9

H 0.90 (t, 6.0)C 14.6

Sarcoehrenoside B (27)

Sarcoehrenoside B (27): White amorphous powder. [ ]D +51.3 (c 0.3, MeOH). IR (KBr) max: 3396, 2951, 2857, 1638, 1546, 1457, 1385, 1242, 1178, 1131, 1036, 736 cm-1. NMR data (CD3OD, 500 and 125 MHz). HRESIMS m/z: 820.6274 [M+Na]+ (calcd for C46H87NO9Na, 820.6278) [10].


Fragmentation pattern of tithoniamide B

HO

HN

O

OH

OHOH

11

8

m/z 67m/z 83

m/z 97

m/z 225m/z 283

m/z 255

m/z 356m/z 298

m/z 309m/z 339m/z 280

HO

HN

O

O

C 175.7

H 5.11 (m)C 53.5

H 0.85 (t, 6.5)C 14.7

H 8.55 (d, 8.8)

8

H 5.48 (dt, 6.0, 14.5)C 131.2

H 1.70 (m)C 34.7

H 4.28 (m)C 73.5

H 4.51 (dd, 4.5, 10.5)4.41 (dd, 4.5, 10.5)

C 62.5

H 4.34 (dd, 4.5, 6.5)C 77.4

H 4.62 (4.0, 8.0)C 73.0

OH

H 1.86 (m)C 33.4

1.73 (m)

C 32.6

11

H 1.30 (s)C 30.1

H 0.85 (t, 6.5)C 14.7

H 1.58 (m)C 26.3

OH

H 5.56 (dt, 6.0, 14.5)C 131.3

C 23.4

H 1.95-2.10 (m)C 33.8

H 1.95-2.10 (m)C 33.8

H 1.30 (s)C 30.1

Tithoniamide B (28)

Tithoniamide B (28): Colorless powder. mp 135 °C. [ ]D +10.23 (c 0.92, CHCl3+MeOH). IR (KBr) max: 3610, 2940, 2860, 1640, 1297 cm-1. NMR data (CDCl3, 500 and 125 MHz). HREIMS m/z: 681.6252 (calcd. 681.6271 for C42H83NO5). CIMS m/z:682.4 [M+1]+ [11].

H 4.50 (dd, 5.0, 10.8), 4.60 (dd, 4.5, 10.8)

C 62.2H 5.20 (m)

C 53.1

H 8.65 (d, 9.9)

H 4.45 (dd, 4.7, 6.5)C 76.9

C 175.4

H 4.75 (dd, 3.8, 7.7)C 72.6

H 2.18 (m),2.28 (m)

C 35.8H 0.90 (t, 7.2)

C 14.4

HN

O

OH

O

H H7-15

5-21OH

H 4.35 (td, 2.3, 7.7)C 73.2

H 2.35 (m), 2.08 (m)

C 34.3

H 1.51 (m), 1.80 (m)C 26.8

H 1.33-1.37 (m)C 29.7-30.6

H 1.25-1.45 (m)C 32.3

H 1.25-1.45 (m)C 23.1

H 1.49 (m), 1.90 (m)

C 26.5

H 1.25-1.45 (m)C 29.7-30.6

H 1.25-1.45 (m)C 32.3

H 1.25-1.45 (m)C 32.1

H 0.90 (t, 7.2)C 14.4

Lactariamide A (29) Lactariamide A (N-2 -hydroxytetracosanoyl-2-amino-3,4-epoxyoctadecan-1-ol) (29): White amorphous powder. [ ]D+11.4° (c 0.51, CHCl3-MeOH, 1:1). UV max (MeOH): 210, 600 nm. IR (KBr) max: 3334, 3216, 2918, 1625, 1544, 1467, 723 cm-1. EIMS m/z (rel. int.): 665 [M]+ (12), 384 (36), 357 (10) 57 (100). NMR (C5D5N). (+) FABMS m/z (rel. int.): 670 [M+Na-H2O]+ (100), 384 (7), 286 (5). HREIMS m/z: 665.6329 (calcd for C42H83NO4, 665.6322) [18].


HO

HN

O

OH

C 175.1

H 4.22 (m)C 54.6

H 0.90 (t, 7.3)C 14.1

H 1.95 (t, 6.8)C 39.7

1.60 (s)

C 16.0

5 2.05 (m)

C 32.5

H 1.22-1.32 (m)C 31.9

H 1.55 (m), 1.75 (m) C 34.9

H 5.10 (m)C 134.1

H 3.72 (m), 3.78 (m)

C 62.2

H 4.08 (dd, 4.4, 7.2)C 74.2

H 3.90 (dd, 4.0, 10.8)C 72.5

OH

H 5.78 (m)C 128.9

2.05 (m)

C 28.7

H 5.50 (dd, 6.4, 15.6)C 125.0

C 136.3

H 1.22-1.32 (m)C 29.4-29.7

H 1.39 (m)C 25.1

11

H 1.22-1.32 (m)C 22.7

H 7.21 (d, 7.8)

H 1.22-1.32 (m)C 29.4-29.7

H 1.22-1.32 (m)C 31.9

H 1.22-1.32 (m)C 22.7

H 0.90 (t, 7.3)C 14.1

Lactariamide B (30)

Lactariamide B [(4E,8E)-N-2 -hydroxyoctadecanoyl-2-amino-9-methyl-4,8-octadecadine-1,3-diol] (30): White amor-phous powder. [ ]D +8.38° (c 0.39, MeOH). UV max (MeOH): 210, 600 nm. IR (KBr) max: 3350, 3274, 2919, 1652, 1538, 1467, 721 cm-1. EIMS m/z (rel. int.): 594 [M]+ (3), 396 (23), 342 (45), 325 (81), 60 (100) [18].

HO

NH

O

OH

4-14

6-16

H 5.97 (d, 6.0)

H 4.38 (m)C 51.9

H 2.22 (t, 7.5)C 36.7

H 4.18 (dd, 7.0, 9.0),3.51 (dd, 7.0, 9.0)

C 70.4

H 3.97 (dd, 4.0, 6.0)C 75.2

H 3.70 (dd, 6.0, 11.5)C 85.2

OSO3H

C 22.7

H 1.20-1.34C 29.3-29.7

C 174.0

H 0.88 (t, 7.0)C 14.1

H 1.56 (m)C 33.4

H 1.63 (m)C 25.8

H 1.20-1.34C 29.3-29.7

C 22.7H 0.88 (t, 7.0)C 14.1

Ircisulfamide (31)

Ircisulfamide (N-[(1S,2S,3R)-2-hydroxy-1-(hydroxymethyl)-3-(sulfooxy)hepta- decyl]hexadecanamide) (31): Colorless amorphous powder. [ ]D +16.7° (c 0.24, CHCl3). IR (KBr) max: 3383, 3312, 2919, 2850, 1645, 1545, 1470, 1382, 1234, 1082, 718 cm-1. FABMS m/z: 636 [M+H]+; HRFAMS/MS: 636.4865 ([M+H]+, C34H70NO7S+; calc.636.4873) [19].


O

NH

O

OH

OH

OHO

HOOH

OH C 177.1

H 3.99 (m)C 54.5

H 4.26 (d, 9.0)C 104.6

H 7.73 (d, 8.0)

H 0.90 (t, 7.0)C 14.5

C 136.6

2.04 (m)

C 35.4

11-16

2.06 (m)

C 33.8

H 1.25-1.35C 29.6-30.0

1.55 (m),1.69 (m)

C 35.8

H 3.19 (dd, 8.0, 9.5)C 74.9

1.38 (m)

C 26.2

5-18

H 5.73 (dt, 6.0, 15.5)C 134.6

H 5.14 (t, 6.5)C 124.8

H 5.47 (dd, 7.5, 15.5)C 131.1

H 3.87 (dd, 2.0, 10.5), 3.67 (dd, 3.5, 10.5)

C 62.6

H 3.36 (dd, 8.0, 9.5)C 77.8

H 3.26 (m)C 71.5

H 3.31 (m)C 77.9

H 4.10 (dd, 5.0, 10.0),3.69 (dd, 3.5, 10.0)

C 69.7 H 4.14 (dd, 5.0, 7.5)C 72.8

H 3.97 (m)C 73.0

1.98 (t, 7.5)

C 40.8

H 1.59 (s)C 16.2

H 1.20-1.35C 30.4-30.8

H 1.20-1.35C 23.7

H 1.20-1.35C 23.7

H 0.90 (t, 7.0)C 14.4

Ircicerebroside (32)

Ircicerebroside (2R)-N-{(1S,2S,3E,7E)-1-[ -D-glucopyranosyloxy)methyl]-2-hydroxy-8-methylheptadeca-3,7-dienyl}-2-hydroxyeicosanamide) (32): Colorless amorphous powder. [ ]D +9.1° (c 0.02, MeOH). IR (KBr) max: 3378, 2921, 2851, 1643, 1536, 1456, 1080, 1036 cm-1. FABMS m/z: 784 ([M+H]+), 605 ([M+H-C6H12O6]+); HRFAM/MS: 784.6280 ([M+H]+,C45H86NO9

+; calc.784.6303) [19].

HO

HN

OH

7-18

O

OH

6-18

Tanacetumide A (33)

m/z 235

m/z 211

m/z 340

m/z 325

m/z 365

m/z 281

HO

HN

OHH 5.10 (m)C 53.0

H 8.56 (d, 8.7)

H 0.86 (t, 6.1)C 14.2

C 25.8

7-18

H 5.36 (dt, 6.1, 15.1)C 129.1

H 4.33 (dd, 5.5, 10.7)H 4.42 (dd, 3.8, 10.0)

C 62.1

H 4.52 (m)C 72.5

C 175.2

O

OH

6-18

H 4.60 (m)C 73.0

H 5.57 (dt, 6.0, 15.3)C 129.5

H 5.44 (dt, 6.2, 15.5)C 130.8 H 2.05 (m)

C 32.9

H 1.25 (m)

C 22.9

H 5.52 (dt, 5.8, 15.1)C 130.2

H 2.19 (m)C 32.1

C 25.8

C 22.9

H 0.86 (t, 6.1)C 14.2


Tanacetumide A (2S,2R,3R,3E,4E)-N-[2´-hydroxy-3´-henicosenoyl]-2-amino-4-henicosene-1,3-diol) (33): Colorless oil. [ ]D -6.00 (c 0.01, CHCl3+ MeOH). IR max (C5D5N): 3605, 2940, 2850, 1630, 1455 cm-1. NMR (C5D5N). HREIMS m/z:663.6157 (calcd. 663.6144 for C42H81NO4), 382.3311 (calcd. 382.3321 for C23H44NO3), 365.3282 (calcd. 365.3293 for C23H43NO2); EIMS (rel. int.): 663 [M]+ (1), 382 [M-CH3(CH2)15CH=CHCH(OH)]+, 365 [M-CH3(CH2)15CH=CHCH(OH)2]+,340 (56), 325 (54), 323[CH3(CH2)16CH=CHCH(OH)CO]+, 340 [CH3(CH2)16CH=CHCH(OH)CO NH2+H]+ , 281 (32), 211 (26) [20].


H 4.42 (m)C 51.2

H 3.94 (m)3.85 (m)

C 60.3

HO

NH

O

OH

OH

OH

H 4.36 (m)C 74.4

H 4.10 (m)C 70.9

H 1.68 (m)C 31.8

H1.46 (m)C 24.9

7-10

C 24.9-30.8

H 1.25 (brs)C 30.8

C 21.5

H 0.88 (t, 6.0)C 13.3

H 7.30 (d, 6.0)

C 173.1

H 5.23 (m)C 71.0

H 2.12 (m)C 34.0

H 1.68 (m)C 24.1

H 1.55 (m)C 28.2

6-20

C 24.9-30.8

H 1.25 (brs)C 30.8

C 21.5H 0.88 (t, 6.0)

C 13.3

34

(2S,3S,4R)-2-[(R)-2´-hydroxytricosanoylamino]-1,3,4-tridecanetriol (34): [ ]D +8.2° (c 0.5, MeOH). IR max: 3250-3400, 2924, 2868, 1680, 1350, 1060 cm-1. NMR (DMSO-d6). (+) ESIMS m/z (rel. int.): 1221.8 [2M+Na]+ (100), 622.9 [M+Na]+ (4); (-) ESIMS/MS m/z (rel. int.): 598.6 [M-H]- (30), 354.5 (15), 342.6 (10), 299.6 (95) [21].

HO

HN

OHH 5.12C 53.7

H 8.63

1.94, 2.16

C 33.9

10

H 4.28C 73.0

H 4.50, H 4.48C 62.2

H 4.42C 76.7

C 173.4

OH

O

19

H 2.49C 36.9

H 1.87C 26.4 H 1.20-1.32

C 29.6-32.1H 0.87C 14.3

H 1.48C 22.7 H 0.88

C 22.7

H 0.88C 22.7

Ceramide 1 (35)

H 1.25-1.32C 29.6-32.1

Ceramides 1 (35): White amorphous solid, mp 193-1958 °C. [ ]D +10.0 (c 0.1, MeOH). IR (KBr) max: 3400, 1040, 2940, 1640, 1540, and 1435 cm-1. FABMS m/z: 654 [M+H]+ [22].

H 5.12C 52.9

H 8.58

1.91, 2.18

C 34.1

10

H 4.28C 72.9

H 4.50, H 4.39C 61.9

H 4.35C 76.6

C 175.3

H 1.48C 28.2

H 0.86C 22.7

H 0.86C 22.7

HO

NH

O

OH

OH

OH

H 4.62C 72.4

H 1.97C 35.6

H 1.25-1.32C 29.6-32.1

20H 0.86C 14.3

Ceramide 2 (36)

H 1.25-1.32C 29.6-32.1

Ceramides 2 (36): White amorphous solid, mp 105-107 °C, [ ]D +28.0 (c 0.1, MeOH). IR (KBr) max: 3400, 1040, 1640, 2950, 1640, 1540, and 1450 cm-1, NMR data (C5D5N). ESIMS m/z: 684 [M+H]+, 707 [M+Na]+ [22].


H 4.32 (brt, 5.0)C 74.9

C 174.1

H 2.23 (t, 7.5)C 37.0

4-10

7-15HO

HN

O

OH

m/z 255

m/z 296 m/z 237

m/z 280

m/z 263

H 3.91 (dt, 3.5, 7.1)C 54.6

H 6.22 (d, 7.1)

4-10

H 3.71 (3.4, 11.0)H 3.95 (dd, 3.4, 11.0

C 62.7

7-15HO

HN

O

OH

H 5.53 (dd, 6.4, 15.4)C 129.0

H 5.79 (dd, 6.6, 15.4)C 134.5

H 2.06 (p, 6.6)C 32.4

H 1.26 (brs)C 29.3-30.1

H 1.26 (brs)C 32.1

H 0.86 (m)C 22.9

H 0.89 (t, 7.0)C 14.3

H 1.63 (m)C 25.9

H 1.26 (brs)C 29.3-30.1

H 1.26 (brs)C 30.1

H 1.26 (brs)C 27.6 H 1.14 (m)

C 39.2

H 1.51 (m)C 28.1

H 0.86 (d, 7.0)C 22.8

H 0.86 (d, 7.0)C 22.8

37


(2S,3R,4E)-2-(14´-methyl-pentadecanoylamino)-4-octadecene-l,3-diol (37): White amorphous powder, mp 80-828°C. [ ]D -5.9 (c 0.11, CHCl3). IR (KBr) max: 3400, 1040, 1640, 2950, 1640, 1540, 1450 cm-1. NMR data (CDCl3). (+) ESIMS m/z:560 [M+Na]+, 1098 [2M+Na]+; (-) ESIMS m/z: 536 [M-H]-, 506 [M-2CH3-H]-, 1073 [2M-H]-. (+) HRESIMS m/z: 560.5018 [M+Na]+, (calcd for C34H67NO3Na, 560.5021) [23].

HO

HN

OHH 5.11 (dt, 4.5, 13.5)

C 53.0

H 8.58 (d, 8.7)

H 5.46 (dd, 5.7, 15.3)C 130.7

C 33.8

6

H 4.28 (m)C 72.9

H 4.51 (dd, 4.5, 11.1)H 4.42 (dd, 5.1, 11.1)

C 62.0

H 4.33 (dd, 5.1, 6.3)C 76.8

C 175.3

OH

O

OH

n

H 4.62 (m)C 72.5

C 35.7

C 26.7

H 1.24-1.30C 22.9-35.7

C 22.9 H 0.85 (t, 6.6)C 14.3

H 5.56 (dd, 5.7, 15.3)C 130.8

C 33.3C 25.8

C 33.0

C 22.9

H 0.85 (t, 6.6)C 14.3

Urtica ceramides (38)

n = 20 (major), n = 18, 19, 21, 22 (minor)

Urtica ceramides (38): Amorphous powder (MeOH). NMR (C5D5N). HRFABMS m/z: 704.6624. Calcd for C42H83O5N+Na: 704.6169, m/z: 690.6045. Calcd for C41H81O5N+Na: 690.6013, m/z: 676.5859. Calcd for C40H79O5N+Na: 676.5856, m/z: 338 [C18H35O3N+Na]+ [24].


H 5.18 (m)C 53.0

H 4.41 (dd, 4.8, 11.1),4.49 (dd, 4.8, 11.1)

C 62.0

HO

NH

O

OH

OH

OH

H 4.33 (m)C 76.9

H 4.29 (m)C 73.0

H 4.27 (m)C 72.9

3H 0.84 (t, 7.2)

C 14.3

H 8.57 (d, 9.0)

C 175.2

H 4.61 (m)C 72.5

H 2.20 (m),1.99 (m)

C 35.7H 1.22-1.29 (m)C 22.9-33.0

5-23

OH

H 2.19 (m),2.02 (m)

C 34.0

H 5.51 (m)C 130.2

H 5.48 (m)C 130.8

H 2.00 (m)C 32.1

H 2.00 (m)C 32.1

H 5.48 (m)C 130.8

H 5.51 (m)C 130.2

H 2.22 (m)C 33.3

H 1.22-1.29 (m)C 22.9-33.0

H 0.84 (t, 7.2)C 14.3

Mucusamide (39)

Mucusamide (2S,3S,4R,5R,7E,11E)-2-{[(2R)-2-Hydroxytetracosanoyl]-amino }-heptadeca-7,11-diene-1,3,4,5-tetrol) (39): Colourless powder, mp 125-126 °C. [ ]D +29° (c 0.11, pyridine). IR (KBr) max: 3200-3500 (OH and NH), 1660 (NH-C=O) cm-1. NMR data (C5D5N). HRFABMS m/z: 682.5931 (Calcd for C41H80NO6: 682.5986 [M+H]+ [25].

O

NH

O

OH

OH

OHO

HOOH

OHC 175.7

H 5.27 (m)C 51.7H 4.92 (d, 7.8)

C 105.4

H 8.55 (d, 9.0)

2.27 (m),1.90 (m)

C 33.9

7-10

1.90 (m)

C 26.6

2.17 (m),1.98 (m)

C 35.5

H 3.98 (dd, 7.5, 7.8)C 75.1

1.90 (m)

C 26.7

5-23

H 5.45 (m)C 130.2

H 4.18 (m)C 72.5

H 4.30 (dd, 4.8, 11.4), 4.45 (dd, 4.8, 10.8)

C 62.6

H 3.85 (m)C 78.5

H 4.49 (dd, 6.6, 9.9),4.69 (dd, 6.6, 9.9)

C 70.3H 4.44 (m)

C 75.9

H 4.55 (m)C 72.4

Mucusoside (40)

OH

15-17

1.29 (m)

C 29.6-33.9

2.04 (m)

C 27.5 H 5.48 (m)C 130.4

H 2.20 (m)C 27.9

1.22-1.29 (m)

C 22.9-33.0

H 0.84 (t, 7.2)C 14.3

1.22-1.29 (m)

C 22.9-33.0

H 0.84 (t, 7.2)C 14.2

H 4.15 (m)C 78.4

H 4.18 (m)C 71.5

Mucusoside (1-O-( -D-Glucopyranosyl)-(2S,3S,4R,12Z)-2-{[(2R)-2-hydroxy tetracosanoyl]amino}octadec-12-ene-1,3,4-triol) (40): Colourless powder, mp 209-210 °C. [ ]D +34° (c 0.08, pyridine). IR (KBr) max: 3437 (OH), 1670 (NH-C=O), 1643 (C=C) cm-1. NMR data (C5D5N). HRFABMS m/z: 844.6849 (Calcd for C48H94NO10: 844.6878 [M+H]+ [25].


Flavuside A (41)

O

NH

O

OH

OH

OHO

HOOH

OH 5-16

13-16

H 3.52 (dd, 3.8, 10.5),3.92 (dd, 5.6, 10.5)

C 68.7

H 3.81 (m)C 52.8

H 3.98 (ddd, 6.0, 6.5, 6.5)C 70.5

H 5.39 (dd, 15.3, 6.5)C 131.0

H 5.59 (dt, 15.3, 6.0)C 131.1

H 1.91 (m)C 32.1

H 1.93 (m)C 27.2

H 1.96 (m)C 32.1

H 5.08 (t, 6.2)C 123.5

C 134.9

H 1.90 (t, 7.3)C 39.0

H 1.33 (m)C 27.4

H 1.23 (m)C 28.7-31.3

H 1.23 (m)C 22.1

H 0.85 (dd, 5.5, 7.5)C 13.9

H 7.38 (d, 9.4)

C 173.7H 1.53 (s)

C 15.7

H 3.81 (m)C 71.0

H 1.43 (m)C 34.5

H 1.22 (m)C 24.5

H 1.22 (m)C 28.7-31.3

H 1.22 (m)C 22.1

H 0.85 (dd, 5.5, 7.5)C 13.9

H 4.11 (d, 7.8)C 103.5

H 2.95 (ddd, 4.0, 8.0, 8.5)C 73.4

H 3.13 (m)C 76.5

H 3.02 (m)C 70.0

H 3.06 (m)C 76.9

H 3.44 (m), 3.66 (dd, 5.9, 11.3)

C 61.1

Flavuside A (1-O- -D-glucopyranosyl-(2S,3S,4E,9E)-2-[(2´R)-2´-hydroxy octa- decanoylamino]-10-methyl-octadeca-4,9-dien-1,3-diol) (41): A colorless amorphous solid. [ ]D +5 (c 0.55, MeOH). IR (KBr) max: 3333, 2921, 2851, 1733, 1641, 1541, 1468,1278, 1248, 1079, 1029, 966, 721 cm-1. UV max (MeOH): (log ) 199 (4.1) nm. LRFABMS m/z (rel. int.): 778 [M+Na]+ (100), 760 [M+Na-H2O]+ (0.4), 496 [M+Na-fatty acid]+ (5), 396 (5), 348 (2), 244 (0.3), 203 (0.1), and 185 (0.3); HRFABMS m/z: 778.5823 [M+Na]+ (Calcd for C43H81NO9Na, 778.5809) [26].

Flavuside B (42)

O

NH

O

OH

OH

OHO

HOOH

OH 5-16

13-16

H 3.50 (dd, 3.8, 10.5),3.93 (dd, 5.4, 10.2)

C 68.6

H 3.79 (m)C 52.9

H 3.98 (ddd, 5.5, 6.7, 6.7)C 70.5

H 5.36 (dd, 15.3, 6.7)C 130.9

H 5.56 (dt, 15.3, 6.0)C 130.9

H 1.91 (m)C 32.1

H 1.93 (m)C 27.3

H 1.95 (m)C 31.3

H 5.09 (t, 6.2)C 123.5

C 134.9

H 1.90 (m)C 39.3

H 1.32 (m)C 27.4

H 1.23 (m)C 28.7-31.3

H 1.23 (m)C 22.1

H 0.84 (dd, 6.5, 7.0)C 13.9

H 7.38 (d, 9.4)

C 172.0H 1.53 (s)

C 15.7

H 4.29 (m)C 71.9

H 5.43 (dd, 5.5, 15.5)C 129.0 H 1.95-123 (m)

C 28.7-31.6

H 1.23 (m)C 22.1

H 0.85 (dd, 6.5, 7.0)C 13.9

H 4.11 (d, 7.8)C 103.5

H 2.95 brdd, 8.0, 8.5)C 73.4

H 3.13 (m)C 76.5

H 3.03 (m)C 70.0

H 3.06 (m)C 76.9

H 3.44 (m), 3.66 (dd, 5.5, 11.5)

C 61.1

H 5.43 (dd, 6.7, 15.5)C 130.9

Flavuside B (1-O- -D-glucopyranosyl-(2S,3S,4E,9E)-2-[(2´R,3´E)-2´-hydroxy octadec-3´-enoylamino]-10-methyl-octadeca-4,9-dien-1,3-diol) (42): A colorless amorphous solid. [ ]D -8.3 (c 0.4, MeOH). IR (KBr) max: 3327, 2923, 2853, 1739, 1651, 1538, 1467, 1378, 1241, 1078, 1032, 966, 721 cm-1. UV max (MeOH) (log ) 199 (4.1) nm. LRFABMS m/z (rel. int.): 776 [M+Na]+ (100), 758 [M+Na-H2O]+ (0.7), 522 (1.0), 496 [M+Na-fatty acid]+ (3), 346 (2), 348 (2), 244 (0.5), 203 (1), 185 (0.3); HRFABMS m/z: 776.5644 [M+Na]+ (Calcd for C43H79NO9Na, 776.5653) [26].


43

HO

NH

O

OH

4-12

11-18H 3.70 (dd, 3.6, 10.8),3.95 (dd, 3.6, 11.4)

C 62.4 H 3.91(m)C 54.4

H 4.32 (brs)C 74.6

H 5.54 (dt, 15.0, 6.0)C 128.9

H 5.78 (dt, 15.0, 6.6)C 130.9

H 2.15 (q, 6.6)C 32.4

H 2.08 (q, 6.6)C 32.2

H 5.36 (dt, 6.6, 15.0)

C 134.3

H 1.97 (q, 7.8)C 32.6

H 1.30 (m)C 29.0

H 0.88 (t, 7.2)C 14.2

H 6.28 (d, 7.2)

C 174.1

H 2.23 (t, 7.2)C 36.8

H 1.64 (m)C 32.3

H 1.30 (m)C 29.0

H 0.88 (t, 7.2)C 14.2

H 5.43 (dt, 6.6, 15.0)

C 129.1

H 1.30 (m)C 25.8

H 1.30 (m)C 22.7

(2S,3S,4E,8E)-2N-[tetradecanoyl]-4(E),8(E)-icosadiene-1,3-diol (43): Colorless amorphous powder, mp 123-125 °C (30 mg, 0.002%). [ ]D +83 (c 0.1, CHCl3). IR (KBr) max: 3340-3200 (OH and amide NH), 1640 (amide carbonyl), 1620 (olefinic dou-ble bond) cm-1. NMR data (CDCl3). HRFABMS m/z: 558.4876 [M+Na]+ (Calcd 558.4862); EIMS m/z: 535 [M, C34H65NO3]+ [14].

LMCer-1 (44)

HO

NH

O

OH

OH

3-12

6-13H 3.74 (m), 3.87 (m)C 62.2 H 3.91 (m)

C 54.6

H 4.27 (m)C 74.3

H 7.21 (d, 7.9)

C 175.1

H 4.11 (dd, 3.8, 7.8)C 71.9

H 0.88 (m)C 14.2

H 5.49 (dd, 6.1, 15.3)C 128.6

H 5.77 (dd, 7.9, 15.3)C 134.4

H 0.88 (m)C 11.4

H 0.88 (m)C 19.4

LMCer-1 ((2S,3R,4E,2R)-2-(2-hydroxyhexadecanoylamino)-16-methyl-4-octa-decene-1,3-diol) (44): Amorphous pow-der. IR (KBr) max: 3413 (hydroxyl), 1646, 1540 (amide) cm-1. (+) FABMS m/z: 590, 616, 630, 644, 646, 656, 660, 670, 674, 688 [M+Na]+ [27].

LMCer-2 (45)

HO

NH

O

OH

OH

3-12

6-13H 4.40 (dd, 5.1, 10.7)4.49 (dd, 4.5, 10.7)

C 62.2 H 5.09 (m)C 52.9

H 4.34 (dd, 4.6, 6.6)C 76.9

H 8.56 (d, 9.2)

C 175.4

H 4.60 (dd, 4.0, 7.6)C 72.5

H 0.88 (m)C 14.2

H 4.26 (dd, 6.2, 6.6)C 73.2

H 0.88 (m)C 11.4

H 0.88 (m)C 19.4

OH

LMCer-2 (2S,3S,4R,2R)-2-(2-hydroxyhexadecanoylamino)-16-methyl-octadec ane-1,3,4-triol (45): Amorphous pow-der, mp 125-128 °C. [ ]D +10.1° (c 0.13, pyridine). (+) FABMS: m/z 608 [M+Na]+ [27].


LMCer-3 (46)

HO

NH

O

OH

OH

3-18

6-11

C 62.0

C 53.0

C 76.8

C 175.3

C 72.4

C 14.2

C 73.0 C 14.2

OH

LMCer-3 (2S,3S,4R,2R)-2-(2-hydroxydocosanoylamino)-hexadecane-1,3,4-triol (46): Amorphous powder, mp 127-137 °C. [ ]D +12.8° (c 0.33, pyridine). (+) FABMS: m/z 650 [M+Na]+ [27].

JCer-1 (47)

HO

NH

O

OH

3-12

11-17

C 62.5

C 54.5

C 74.7

C 173.9

C 36.8

C 129.0

C 133.6 C 131.4

C 129.2

C 14.0

C 19.7

C 14.4

C 32.3

C 32.3

JCer-1 (47): Amorphous powder, mp 83-85 °C. [ ]D +7.8° (c 0.43, n-PrOH). (+) FABMS m/z: 764 [M+Na]+. NMR data (CDCl3) [28,29].

Newbouldiamide (48)

HO

NH

O

OH

4-12

8-21H 4.50 (dd, 6.0, 10.5),4.35 (dd, 6.0, 10.5)

C 62.9H 5.11 (m)

C 553.8H 4.42 (m)

C 77.6

H 4.61 (m)C 73.3

H 1.99 (m)C 33.0

H 1.72 (m)C 33.0

C 27.5

C 23.8H 0.86 (t, 6.8)

C 15.1

H 8.57 (d, 9.0)

C 176.1

H 2.23 (t, 7.2)C 36.6

H 1.76 (m)C 35.0

OH

OH

H 4.27 (m)C 73.9

H 1.29 (brs)C 29.9

H 1.29 (brs)C 29.9

C 27.5

C 23.8H 0.86 (t, 6.8)

C 15.1

(48). Contd….


m/z 280

HO

NH

O

OH

11

OH

OH

14

m/z 226

m/z 267

m/z 284

m/z 384

m/z 357m/z 327

m/z 297

Fragmentation pattern of 48. Newbouldiamide (1,3,4,5-tetrahydroxy-2-octadecanoyl-amino-tetracosane) (48): Colorless powder, mp 129 °C. [ ]D

+12.0° (c 0.001). IR (KBr) max: 3610, 2930, 2860, 1640, 1297 cm-1. NMR data (C5D5N). CIMS (CH4) m/z: 684.4 [M+1]+;EIMS m/z (rel. int.): 683.4 [M]+ (10) [15].

H 4.33 (dd, 4.8, 6.2)C 74.9

C 173.9

H 2.23 (t, 7.7)C 36.8

H 3.92 (m)C 54.4

H 6.23 (d, 7.7)

4-13

H 3.70 (3.3, 11.0)H 3.96 (dd, 3.7, 11.4)

C 62.5

7HO

HN

O

OH

H 5.56 (dd, 6.2, 15.8)C 129.0

H 5.80 (dt, 5.9, 15.8)C 133.8

H 5.09 (t, 6.6)C 123.0

H 1.25-1.36 (brs)C 28.0-29.7

H 1.64 (m)C 25.8

H 0.88 (d, 7.3)C 14.1

49

H 1.58 (brs)C 16.0

C 22.7

C 136.3

H 2.08 (m)C 27.5

H 2.11 (m)C 32.5

C 31.9C 22.7

H 0.88 (d, 7.3)C 14.1

(2S,3R,4E,8E)-N-hexadecanoyl-2-amino-9-methyl-4,8-octadecadiene-1,3-diol (49): Amorphous powder, [ ]D -11.6° (c0.09, CHCl3). IR (CHCl3) max: 3605, 3434, 2928, 2855, 1657, 1510, 1466 cm-1. NMR data (CDCl3). HREIMS m/z: 549.5113 (M+, Calcd for C35H67NO3: 549.5121). EIMS m/z: (rel. int. %): 549 [M]+ (2), 298 (15), 281 (43). (-) FABMS m/z: 548 [M-H]-

[30].

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Appendix 2: LIST OF ABBREVIATIONS AND DEFINITIONS

AA = Arachidonic Acid APCI = Atmospheric Pressure Chemical Ionization apoE = Apolipoprotein E C1P = Ceramide-1-Phosphate CDases = Ceramidases Cer = Ceramide CerK = Ceramide Kinase CID = Collision Induced Dissociation COX 2 = Cyclooxygenase-2 DES = Dihydroceramide Desaturase dhCer = Dihydroceramide


DI = Desorption Ionization EI = Electron Impact ER = Endoplasmic Reticulum ESIMS = Electrospray Ionization FABMS = Fast Atom Bombardment Mass Spectrometry FAMEs = Fatty Acid Methyl Esters GlcCer = Glucosylceramide GPI = Glycosylphosphatidylinositol HIV = Human Immunodeficiency Virus HS = 6-Hydroxysphingosine IPC = Inositol Phosphorylceramide LCB = Long-Chain Bases LDLs = Low-Density Lipoproteins LPS = Lipopolysaccharide MALDI = Matrix Assisted Laser Desorption Ionization NMR = Nuclear Magnetic Resonance PCERA-1 = Phosphoceramide Analog-1 PLA2 = Phospholipase A2 PS = Phytosphingosine PS = Phytosphingosine ROS = Reactive Oxygen Species S = Sphingosine Sa = Sphinganine S1P = Sphingosine -1-Phosphate SM = Sphingomylin SMS = SM Synthase SOD = Superoxide Dismutase SPT = Serine Palmitoyl Transferase TNF = Tumor Necrosis Factor

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Received: November 26, 2012 Revised: January 12, 2013 Accepted: January 29, 2013

Activity and Structure Elucidation of Ceramides

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