Erylusamides: Novel Atypical Glycolipids from Erylus cf. deficiens

marine drugs Article Erylusamides: Novel Atypical Glycolipids from Erylus cf.deficiens Helena Gaspar 1,2,*, Adele Cutignano 3, Laura Grauso 3, Nuno Neng 1, Vasco Cachatra 1, Angelo Fontana 3, Joana Xavier 4, Marta Cerejo 5, Helena Vieira 6and Susana Santos 1,* 1Centro de Química e Bioquímica (CQB), Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Lisboa 1749-016, Portugal; [email protected] (N.N.); [email protected] (V.C.) 2MARE-Centro de Ciências do Mar e do Ambiente, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Lisboa 1749-016, Portugal 3CNR-Istituto di Chimica Biomolecolare, Bio-Organic Chemistry Unit, via Campi Flegrei 34, Pozzuoli (NA) 80078, Italy; [email protected].it (A.C.); [email protected].it (L.G.); [email protected].it (A.F.) 4Department of Biology and Centre for Geobiology, University of Bergen, P.O. Box 7803, Bergen N-5020, Norway; [email protected] 5Research & Innovation Accelerator, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, Caparica 2829-516, Portugal; [email protected] 6BioISI, Instituto de Biociências e Ciências Integrativas, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Lisboa 1749-016, Portugal; [email protected] *Correspondence: [email protected] (H.G.); [email protected] (S.S.); Tel.: +351-217500563 (H.G.); +351-217500948 (S.S.) Academic Editor: Danielle Skropeta Received: 11 July 2016; Accepted: 15 September 2016; Published: 11 October 2016 Abstract: Among marine organisms, sponges are the richest sources of pharmacologically-active compounds. Stemming from a previous lead discovery program that gathered a comprehensive library of organic extracts of marine sponges from the off-shore region of Portugal, crude extracts of Erylus cf.deficiens collected in the Gorringe Bank (Atlantic Ocean) were tested in the innovative high throughput screening (HTS) assay for inhibitors of indoleamine 2,3-dioxygenase (IDO) and showed activity. Bioassay guided fractionation of the dichloromethane extract led to the isolation of four new glycolipids, named erylusamide A – D . The structures of the isolated compounds were established by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and chemical derivatization. The metabolites shared a pentasaccharide moiety constituted by unusual highly acetylated D-glucose moieties as well as D-xylose and D-galactose. The aglycones were unprecedented long chain dihydroxyketo amides. Erylusamides A , B and D differ in the length of the hydrocarbon chain, while erylusamide C is a structural isomer of erylusamide B. Keywords: Erylus; indoleamine 2,3 dioxygenase; glycolipids; marine natural products; sponges; anti-cancer; erylusamides 1. Introduction The secondary metabolites found in marine invertebrates represent a rich source of novel chemical diversity for lead compounds, with sponges being the most prolific source of new molecules. Between these structurally unique metabolites, glycolipids play an important role. Glycolipids belong to the broad class of glycoconjugates and are characterized by having one or more monosaccharide residues linked by a glycosidic bond to a hydrophobic moiety, such as an acylglycerol, a sphingoid, Mar. Drugs 2016,14, 179; doi:10.3390/md14100179 www.mdpi.com/journal/marinedrugs Mar. Drugs 2016,14, 179 2 of 14 or a prenyl phosphate [ 1 ]. Glycolipids, including glycosphingolipids and gangliosides, are widely found in marine invertebrates, especially in echinoderms (sea stars, sea cucumbers) and sponges, and show a large variety of biological activities such as antitumor, immunomodulatory and nitric oxide release-inhibiting activities [2]. Sponges of the genus Erylus Gray, 1867 (Tetractinellida, Geodiidae) were reported to produce uncommon phospholipid methyl branched fatty and unusual glycolipids, some of which have interesting pharmacological activities, such as anticancer and interleukin-6 (IL-6) receptor antagonists (Table 1). The same type of glycolipids found in Pachymatisma johnstonias, a species that belongs to the same family of Erylus, showed inhibitory activity of bacterial type III secretion [3]. Table 1. Glycolipid and lipid content of Erylus and Pachymatisma species. Sponge/Origin Compounds Activity Erylus formosus La Parguera, Puerto Rico [4] Fatty acid: Tetradecanoic 13-Methyltetradecanoic 12-Methyltetradecanoic 3-Methylpentadecanoic Hexadecenoic Methylpentadecanoic Hexadecanoic 3-Methylhexadecanoic 15-Methylhexadecanoic 14-Methylhexadecanoic 5,9-Octadecadienoic Octadecenoic Octadecanoic Methyloctadecanoic 5,9-Icosadienoic 19-Methyl-5,9-icosadienoic 18-Methyl-5,9-icosadienoic Heneicosanoic Tricosanoic Tetracosanoic Pentacosanoic 24-Methyl-5,9-pentacosadienoic 5,9-Hexacosadienoic 25-Methyl-5,9-hexacosadienoic 24-Methyl-5,9-hexacosadienoic 5,9-Octacosadienoic 5,9-Nonacosadienoic NR Erylus goffrilleri Mona Island (Puerto Rico) [5] Fatty acid: Tridecanoic 12-Methyltridecanoic Tetradecanoic 3-Methyltetradecanoic 13-Methyltetradecanoic 12-Methyltetradecanoic 9-Pentadecenoic Pentadecanoic 3-Methylpentadecanoic 14-Methylpentadecanoic 13-Methylpentadecanoic (Z)-9-Hexadecenoic (Z)-11-Hexadecenoic Hexadecanoic (Z)-15-Methyl-9-hexadecenoic 10-Methylhexadecanoic 15-Methylhexadecanoic 14-Methylhexadecanoic (5Z,9Z)-2-Methoxy-5,9-hexadecadienoic (Z)-9-Heptadecenoic (Z)-11-Heptadecenoic Heptadecanoic (5Z,9Z)-2-Methoxy-15-methyl5,9-hexadeca-dienoic Methylheptadecanoic (5Z,9Z)-5,9-Octadecadienoic (9Z)-2-Methoxy-15-methyl-9-hexadecenoic (Z)-9-Octadecenoic (Z)-11-Octadecenoic 2-Methoxy-14-methylhexadecanoic Octadecanoic Methyl-6-octadecenoic (5Z,9Z)-17-Methyl-5,9-octadecadienoic 11-Methyloctadecanoic (5Z,9Z)-2-Methoxy-5,9-octadecadienoic (5Z,9Z)-2-Methoxy-5,9-nonadecadienoic 11-Eicosenoic Eicosanoic (5Z,9Z)-19-Methyl-5,9-eicosadienoic (5Z,9Z)-18-Methyl-5,9-eicosadienoic Methyleicosanoic (5Z,9Z)-5,9-Heneicosadienoic 19-Methyleicosanoic 18-Methyleicosanoic (5Z,9Z)-2-Methoxy-5,9-eicosadienoic 11-Nonadecenoic Nonadecanoic 5,8,11,14-Eicosatetraenoic Docosanoic 16-Methyldocosanoic 21-Methyldocosanoic 20-Methyldocosanoic Tricosanoic Methyltricosanoic Tetracosanoic Methyltetracosanoic (5Z,9Z)-24-Methyl-5,9-pentacosadienoic (5Z,9Z)-23-Methyl-5,9-pentacosadienoic (5Z,9Z)-5,9-Hexacosadienoic (5Z,9Z)-25-Methyl-5,9-hexacosadienoic (5Z,9Z)-24-Methyl-5,9-hexacosadienoic (5Z,9Z)-5,9-Heptacosadienoic (5Z,9Z)-26-Methyl-5,9-heptacosadienoic (5Z,9Z)-25-Methyl-5,9-heptacosadienoic (5Z,9Z)-5,9-Octacosadienoic (5Z,9Z)-5,9-Nonacosadienoic Methylnonadecanoic 17-Methyloctadecanoic 16-Methyloctadecanoic NR Mar. Drugs 2016,14, 179 3 of 14 Table 1. Cont. Sponge/Origin Compounds Activity Erylus placenta Hachijojima Island (Japan) [6,7] Erylusamine A: R1= CH2CH2CH3, R2= H Erylusamine B: R1= CH2CH(CH3)2, R2= H Erylusamine C: R1= CH2CH(CH3)2R2= Ac Erylusamine D: R1= CH2CH2CH2CH2CH3R2= Ac Mar.Drugs2016,14,1773of14 (Z)‐9‐Octadecenoic (Z)‐11‐Octadecenoic 2‐Methoxy‐14‐methylhexadecanoic Octadecanoic Methyl‐6‐octadecenoic (5Z,9Z)‐17‐Methyl‐5,9‐octadecadienoic 11‐Methyloctadecanoic (5Z,9Z)‐2‐Methoxy‐5,9‐octadecadienoic  (5Z,9Z)‐25‐Methyl‐5,9‐heptacosadienoic (5Z,9Z)‐5,9‐Octacosadienoic (5Z,9Z)‐5,9‐Nonacosadienoic Methylnonadecanoic 17‐Methyloctadecanoic 16‐Methyloctadecanoic NR Erylusplacenta Hachijojima Island(Japan) [6,7] ErylusamineA: R1=CH2CH2CH3,R2=H ErylusamineB: R1=CH2CH(CH3)2,R2=H ErylusamineC: R1=CH2CH(CH3)2R2=Ac ErylusamineD: R1=CH2CH2CH2CH2CH3R2=Ac Interleukin‐6(IL‐6)receptorantagonists Eryluscf. LendenfeidiGulf ofEilat(Redsea) [8] ErylusamineTA: R1=Ac;R2=(CH2)5N(CH3)2;R3=H,n=8,m=2 Erylusine: R1=Ac;R2=(CH2)3NCH3(CH2)4N(CH3)2;R3=H,n=8,m=2 Erylusidine R1=H;R2=(CH2)4NHC=NH(NH2);R3=COCH2CH(CH3)2, n=8,m=3 NR Erylustrisphaerus Dominica[9] Trisphaerolide LowinvitrocytotoxicityagainstMCF7humanbreastcancercells Pachymatisma johnstoniaIsleof Mann(UK)[3] PachymosideA CrudeextractshowedinhibitoryactivityofbacterialtypeIIIsecretion NR:notreported Indoleamine2,3‐dioxygenase(IDO1),formerlyknownasIDObeforethediscoveryofasecond isoform,isthefirstandrate‐limitingenzymeintheoxidativedegradationoftheessentialaminoacid tryptophanthroughthekynureninepathwayandplaysaroleinthecontrolofinfectionandin evasionofT‐cell‐mediatedimmunerejection[10].ItisbelievedthatIDO1inhibitstheproliferation anddifferentiationofTcells,whicharesensitivetothedegradationoftryptophanandaccumulation ofitscatabolites.IDO1isoverexpressedinavarietyoftumorcelltypesandactsagainsttheT‐cell attack,thusfacilitatingthegrowthandsurvivalofmalignantcells[11].Forthesereasons,IDO1has emergedasakeytargetincancerimmunotherapy.Severalinhibitorshavebeensynthesizedand provedtobeefficient,aloneorincombinationwithothertherapeutics.However,by2014,the pipelineofIDOinhibitorscomprisedonlyfourdrugcandidates:indoximod,epacadostat,NLG919 andanIDOderivedpeptide[12].Indoximod(D‐1‐methyl‐tryptophan)isbeingtestedincombination withotherdrugsinseveralphaseIandIIclinicaltrials.Epacadostat(INCB024360),an hydroxyamidinethattargetsandbindstoIDO1isnowinseveralphaseIandIIclinicaltrials[13]. NLG919isanimidazoleisoindolederivativeundergoingphaseIclinicaltrialsinthetreatmentof recurrentadvancedsolidtumorsaloneorincombinationwithotherdrugs.AfterthehumanIDO1 structurewasdeterminedbyX‐raycrystallographyin2006,severalsyntheticinhibitorswere O R 3 O C5H11 O O O HO OH O O HO OH O O N H R2 n m OR1 OH HO OH OH OH  Interleukin-6 (IL-6) receptor antagonists Erylus cf. Lendenfeidi Gulf of Eilat (Red sea) [8] Erylusamine TA: R1= Ac; R2= (CH2)5N(CH3)2; R3= H, n= 8, m= 2 Erylusine: R1= Ac; R2= (CH2)3NCH3(CH2)4N(CH3)2; R3= H, n= 8, m= 2 Erylusidine R1= H; R2= (CH2)4NHC = NH(NH2); R3= COCH2CH(CH3)2, n= 8, m= 3 Mar.Drugs2016,14,1773of14 (Z)‐9‐Octadecenoic (Z)‐11‐Octadecenoic 2‐Methoxy‐14‐methylhexadecanoic Octadecanoic Methyl‐6‐octadecenoic (5Z,9Z)‐17‐Methyl‐5,9‐octadecadienoic 11‐Methyloctadecanoic (5Z,9Z)‐2‐Methoxy‐5,9‐octadecadienoic  (5Z,9Z)‐25‐Methyl‐5,9‐heptacosadienoic (5Z,9Z)‐5,9‐Octacosadienoic (5Z,9Z)‐5,9‐Nonacosadienoic Methylnonadecanoic 17‐Methyloctadecanoic 16‐Methyloctadecanoic NR Erylusplacenta Hachijojima Island(Japan) [6,7] ErylusamineA: R1=CH2CH2CH3,R2=H ErylusamineB: R1=CH2CH(CH3)2,R2=H ErylusamineC: R1=CH2CH(CH3)2R2=Ac ErylusamineD: R1=CH2CH2CH2CH2CH3R2=Ac Interleukin‐6(IL‐6)receptorantagonists Eryluscf. LendenfeidiGulf ofEilat(Redsea) [8] ErylusamineTA: R1=Ac;R2=(CH2)5N(CH3)2;R3=H,n=8,m=2 Erylusine: R1=Ac;R2=(CH2)3NCH3(CH2)4N(CH3)2;R3=H,n=8,m=2 Erylusidine R1=H;R2=(CH2)4NHC=NH(NH2);R3=COCH2CH(CH3)2, n=8,m=3 NR Erylustrisphaerus Dominica[9] Trisphaerolide LowinvitrocytotoxicityagainstMCF7humanbreastcancercells Pachymatisma johnstoniaIsleof Mann(UK)[3] PachymosideA CrudeextractshowedinhibitoryactivityofbacterialtypeIIIsecretion NR:notreported Indoleamine2,3‐dioxygenase(IDO1),formerlyknownasIDObeforethediscoveryofasecond isoform,isthefirstandrate‐limitingenzymeintheoxidativedegradationoftheessentialaminoacid tryptophanthroughthekynureninepathwayandplaysaroleinthecontrolofinfectionandin evasionofT‐cell‐mediatedimmunerejection[10].ItisbelievedthatIDO1inhibitstheproliferation anddifferentiationofTcells,whicharesensitivetothedegradationoftryptophanandaccumulation ofitscatabolites.IDO1isoverexpressedinavarietyoftumorcelltypesandactsagainsttheT‐cell attack,thusfacilitatingthegrowthandsurvivalofmalignantcells[11].Forthesereasons,IDO1has emergedasakeytargetincancerimmunotherapy.Severalinhibitorshavebeensynthesizedand provedtobeefficient,aloneorincombinationwithothertherapeutics.However,by2014,the pipelineofIDOinhibitorscomprisedonlyfourdrugcandidates:indoximod,epacadostat,NLG919 andanIDOderivedpeptide[12].Indoximod(D‐1‐methyl‐tryptophan)isbeingtestedincombination withotherdrugsinseveralphaseIandIIclinicaltrials.Epacadostat(INCB024360),an hydroxyamidinethattargetsandbindstoIDO1isnowinseveralphaseIandIIclinicaltrials[13]. NLG919isanimidazoleisoindolederivativeundergoingphaseIclinicaltrialsinthetreatmentof recurrentadvancedsolidtumorsaloneorincombinationwithotherdrugs.AfterthehumanIDO1 structurewasdeterminedbyX‐raycrystallographyin2006,severalsyntheticinhibitorswere O R 3 O C5H11 O O O HO OH O O HO OH O O N H R2 n m OR1 OH HO OH OH OH  NR Erylus trisphaerus Dominica [9] Trisphaerolide Mar.Drugs2016,14,1773of14 (Z)‐9‐Octadecenoic (Z)‐11‐Octadecenoic 2‐Methoxy‐14‐methylhexadecanoic Octadecanoic Methyl‐6‐octadecenoic (5Z,9Z)‐17‐Methyl‐5,9‐octadecadienoic 11‐Methyloctadecanoic (5Z,9Z)‐2‐Methoxy‐5,9‐octadecadienoic  (5Z,9Z)‐25‐Methyl‐5,9‐heptacosadienoic (5Z,9Z)‐5,9‐Octacosadienoic (5Z,9Z)‐5,9‐Nonacosadienoic Methylnonadecanoic 17‐Methyloctadecanoic 16‐Methyloctadecanoic NR Erylusplacenta Hachijojima Island(Japan) [6,7] ErylusamineA: R1=CH2CH2CH3,R2=H ErylusamineB: R1=CH2CH(CH3)2,R2=H ErylusamineC: R1=CH2CH(CH3)2R2=Ac ErylusamineD: R1=CH2CH2CH2CH2CH3R2=Ac Interleukin‐6(IL‐6)receptorantagonists Eryluscf. LendenfeidiGulf ofEilat(Redsea) [8] ErylusamineTA: R1=Ac;R2=(CH2)5N(CH3)2;R3=H,n=8,m=2 Erylusine: R1=Ac;R2=(CH2)3NCH3(CH2)4N(CH3)2;R3=H,n=8,m=2 Erylusidine R1=H;R2=(CH2)4NHC=NH(NH2);R3=COCH2CH(CH3)2, n=8,m=3 NR Erylustrisphaerus Dominica[9] Trisphaerolide LowinvitrocytotoxicityagainstMCF7humanbreastcancercells Pachymatisma johnstoniaIsleof Mann(UK)[3] PachymosideA CrudeextractshowedinhibitoryactivityofbacterialtypeIIIsecretion NR:notreported Indoleamine2,3‐dioxygenase(IDO1),formerlyknownasIDObeforethediscoveryofasecond isoform,isthefirstandrate‐limitingenzymeintheoxidativedegradationoftheessentialaminoacid tryptophanthroughthekynureninepathwayandplaysaroleinthecontrolofinfectionandin evasionofT‐cell‐mediatedimmunerejection[10].ItisbelievedthatIDO1inhibitstheproliferation anddifferentiationofTcells,whicharesensitivetothedegradationoftryptophanandaccumulation ofitscatabolites.IDO1isoverexpressedinavarietyoftumorcelltypesandactsagainsttheT‐cell attack,thusfacilitatingthegrowthandsurvivalofmalignantcells[11].Forthesereasons,IDO1has emergedasakeytargetincancerimmunotherapy.Severalinhibitorshavebeensynthesizedand provedtobeefficient,aloneorincombinationwithothertherapeutics.However,by2014,the pipelineofIDOinhibitorscomprisedonlyfourdrugcandidates:indoximod,epacadostat,NLG919 andanIDOderivedpeptide[12].Indoximod(D‐1‐methyl‐tryptophan)isbeingtestedincombination withotherdrugsinseveralphaseIandIIclinicaltrials.Epacadostat(INCB024360),an hydroxyamidinethattargetsandbindstoIDO1isnowinseveralphaseIandIIclinicaltrials[13]. NLG919isanimidazoleisoindolederivativeundergoingphaseIclinicaltrialsinthetreatmentof recurrentadvancedsolidtumorsaloneorincombinationwithotherdrugs.AfterthehumanIDO1 structurewasdeterminedbyX‐raycrystallographyin2006,severalsyntheticinhibitorswere O R 3 O C5H11 O O O HO OH O O HO OH O O N H R2 n m OR1 OH HO OH OH OH  Low in vitro cytotoxicity against MCF7 human breast cancer cells Pachymatisma johnstonia Isle of Mann (UK) [3] Pachymoside A Mar.Drugs2016,14,1773of14 (Z)‐9‐Octadecenoic (Z)‐11‐Octadecenoic 2‐Methoxy‐14‐methylhexadecanoic Octadecanoic Methyl‐6‐octadecenoic (5Z,9Z)‐17‐Methyl‐5,9‐octadecadienoic 11‐Methyloctadecanoic (5Z,9Z)‐2‐Methoxy‐5,9‐octadecadienoic  (5Z,9Z)‐25‐Methyl‐5,9‐heptacosadienoic (5Z,9Z)‐5,9‐Octacosadienoic (5Z,9Z)‐5,9‐Nonacosadienoic Methylnonadecanoic 17‐Methyloctadecanoic 16‐Methyloctadecanoic NR Erylusplacenta Hachijojima Island(Japan) [6,7] ErylusamineA: R1=CH2CH2CH3,R2=H ErylusamineB: R1=CH2CH(CH3)2,R2=H ErylusamineC: R1=CH2CH(CH3)2R2=Ac ErylusamineD: R1=CH2CH2CH2CH2CH3R2=Ac Interleukin‐6(IL‐6)receptorantagonists Eryluscf. LendenfeidiGulf ofEilat(Redsea) [8] ErylusamineTA: R1=Ac;R2=(CH2)5N(CH3)2;R3=H,n=8,m=2 Erylusine: R1=Ac;R2=(CH2)3NCH3(CH2)4N(CH3)2;R3=H,n=8,m=2 Erylusidine R1=H;R2=(CH2)4NHC=NH(NH2);R3=COCH2CH(CH3)2, n=8,m=3 NR Erylustrisphaerus Dominica[9] Trisphaerolide LowinvitrocytotoxicityagainstMCF7humanbreastcancercells Pachymatisma johnstoniaIsleof Mann(UK)[3] PachymosideA CrudeextractshowedinhibitoryactivityofbacterialtypeIIIsecretion NR:notreported Indoleamine2,3‐dioxygenase(IDO1),formerlyknownasIDObeforethediscoveryofasecond isoform,isthefirstandrate‐limitingenzymeintheoxidativedegradationoftheessentialaminoacid tryptophanthroughthekynureninepathwayandplaysaroleinthecontrolofinfectionandin evasionofT‐cell‐mediatedimmunerejection[10].ItisbelievedthatIDO1inhibitstheproliferation anddifferentiationofTcells,whicharesensitivetothedegradationoftryptophanandaccumulation ofitscatabolites.IDO1isoverexpressedinavarietyoftumorcelltypesandactsagainsttheT‐cell attack,thusfacilitatingthegrowthandsurvivalofmalignantcells[11].Forthesereasons,IDO1has emergedasakeytargetincancerimmunotherapy.Severalinhibitorshavebeensynthesizedand provedtobeefficient,aloneorincombinationwithothertherapeutics.However,by2014,the pipelineofIDOinhibitorscomprisedonlyfourdrugcandidates:indoximod,epacadostat,NLG919 andanIDOderivedpeptide[12].Indoximod(D‐1‐methyl‐tryptophan)isbeingtestedincombination withotherdrugsinseveralphaseIandIIclinicaltrials.Epacadostat(INCB024360),an hydroxyamidinethattargetsandbindstoIDO1isnowinseveralphaseIandIIclinicaltrials[13]. NLG919isanimidazoleisoindolederivativeundergoingphaseIclinicaltrialsinthetreatmentof recurrentadvancedsolidtumorsaloneorincombinationwithotherdrugs.AfterthehumanIDO1 structurewasdeterminedbyX‐raycrystallographyin2006,severalsyntheticinhibitorswere O R 3 O C5H11 O O O HO OH O O HO OH O O N H R2 n m OR1 OH HO OH OH OH  Crude extract showed inhibitory activity of bacterial type III secretion NR: not reported. Indoleamine 2,3-dioxygenase (IDO1), formerly known as IDO before the discovery of a second isoform, is the first and rate-limiting enzyme in the oxidative degradation of the essential amino acid tryptophan through the kynurenine pathway and plays a role in the control of infection and in evasion of T-cell-mediated immune rejection [ 10 ]. It is believed that IDO1 inhibits the proliferation and differentiation of T cells, which are sensitive to the degradation of tryptophan and accumulation of its catabolites. IDO1 is overexpressed in a variety of tumor cell types and acts against the T-cell attack, thus facilitating the growth and survival of malignant cells [ 11 ]. For these reasons, IDO1 has emerged as a key target in cancer immunotherapy. Several inhibitors have been synthesized and proved to be efficient, alone or in combination with other therapeutics. However, by 2014, the pipeline of IDO inhibitors comprised only four drug candidates: indoximod, epacadostat, NLG919 and an IDO derived peptide [ 12 ]. Indoximod (D-1-methyl-tryptophan) is being tested in combination with other drugs in several phase I and II clinical trials. Epacadostat (INCB024360), an hydroxyamidine that targets and binds to IDO1 is now in several phase I and II clinical trials [ 13 ]. NLG919 is an imidazoleisoindole derivative undergoing phase I clinical trials in the treatment of recurrent advanced solid tumors alone or in combination with other drugs. After the human IDO1 structure was determined by X-ray crystallography in 2006, several synthetic inhibitors were developed based on the structure of the active-site [ 14 ]; however, to the best of our knowledge, no comprehensive screening of compounds (or extracts) from marine origin was ever undertaken. With that background in view, in a previous project, we have undertaken a comprehensive screening of crude extracts of sponges from the Portuguese coast using the Blockade application of GPS D 2 High Throughput Screening (HTS) system that uses the human version of indoleamine 2,3-dioxygenase 1 (IDO1) as therapeutic target [ 15 ]. This paper describes the isolation and structure Mar. Drugs 2016,14, 179 4 of 14 determination of four new glycolipids, named erylusamides A – D , compounds 1 – 4 (Figure 1), found in the IDO’s inhibitor organic extract of Erylus cf.deficiens Topsent, 1927. Mar.Drugs2016,14,1774of14 developedbasedonthestructureoftheactive‐site[14];however,tothebestofourknowledge,no comprehensivescreeningofcompounds(orextracts)frommarineoriginwaseverundertaken. Withthatbackgroundinview,inapreviousproject,wehaveundertakenacomprehensive screeningofcrudeextractsofspongesfromthePortuguesecoastusingtheBlockadeapplicationof GPSD2HighThroughputScreening(HTS)systemthatusesthehumanversionofindoleamine 2,3‐dioxygenase1(IDO1)astherapeutictarget[15].Thispaperdescribestheisolationandstructure determinationoffournewglycolipids,namederylusamidesA–D,compounds1–4(Figure1),found intheIDO’sinhibitororganicextractofEryluscf.deficiensTopsent,1927.  Figure1.StructuresoferylusamidesA–D. 2.ResultsandDiscussion Withinthescopeofapreviousdrugdiscoverycampaign,acomprehensivelibraryof185 organicextractsofspongespecimenscollectedinseveraloff‐shorePortugueselocations(Berlengas, AzoresandGorringebank)wasconstructed.Theextractswerescreenedasmodulatorsofproteins involvedincancerandneurodegenerativediseasesusingtheGlobalPlatformScreeningforDrug Discovery(GPSD2)technologydevelopedbythePortuguesebiotechcompanyBIOALVO(Lisbon, Portugal),whichusesmodifiedSaccharomycescerevisiaestrainsdesignedtoexpressspecifictargets involvedindiseaseswithatremendoussocialandeconomicburden.BIOALVO’sBLOCKADE application,whichtargetscompoundsabletoinhibittheenzymeindoleamine2,3dioxygenase (IDO‐1),wasselectedtofirsttesttheextracts.Extractswereconsideredpositiveiftheyinhibitedthe growthofBLOCKADEyeast>60%[15].IntheBLOCKADEscreening,thedichloromethaneextract ofthemarinespongeEryluscf.deficienscollectedintheGorringeBank(AtlanticOcean)tested positiveataconcentrationof0.125mg/mL.Theactivityofthisextractwasconfirmedusinganadditional assaywithAfricangreenmonkeykidneyfibroblastCOS7cellstransfectedwithIDO,revealinganIDO inhibitoryactivityof80%.TheorganicextractwasfurtherseparatedbyflashchromatographyonC18 reversephasesilicagel(RP‐18)intoelevenfractions,oneofwhich(fraction2)conservedtheactivity oftheoriginalextract,inhibitingkynurenineproductionby80%atthesameconcentration. 1Hnuclearmagneticresonance(NMR)spectrumoffraction2(150mg)revealedcomplex signalsbelongingtosugarcomponentsbetweenδ6.4and3.5ppm,togetherwithaliphatic resonances,duetoalipidmoietyintheupfieldregionofthespectrum,thussuggestingthe occurrenceofaseriesofglycoconjugates.Hence,asafirststepinthestructureelucidationofthe bioactivecomponents,amethanolysisreactionwasperformedonanaliquotofthemixtureto liberatetheaglyconefromthemonosaccharidepool.Methylglycosideswereconvertedintothe correspondingtrimethylsilyl(TMS)derivatives[16]andanalysedbyGC‐MSincomparisonwith authenticstandards.AccordingtoretentiontimeandcharacteristicMSfragmentationpatterns, monosaccharideunitswereidentifiedasD‐xylose,D‐glucoseandD‐galactose.Ontheotherhand, aglyconesshowedIRbandsat3349,1740,1701and1636cm−1,suggestingthepresenceofhydroxyl, ester,ketoneandamidefunctionalities,whichwereconfirmedbyNMRdata.Separationof individualcomponentswasachievedbyRP‐HPLConaphenyl‐hexylcolumn(Phenomenex) Figure 1. Structures of erylusamides A–D. 2. Results and Discussion Within the scope of a previous drug discovery campaign, a comprehensive library of 185 organic extracts of sponge specimens collected in several off-shore Portuguese locations (Berlengas, Azores and Gorringe bank) was constructed. The extracts were screened as modulators of proteins involved in cancer and neurodegenerative diseases using the Global Platform Screening for Drug Discovery (GPS D2) technology developed by the Portuguese biotech company BIOALVO (Lisbon, Portugal), which uses modified Saccharomyces cerevisiae strains designed to express specific targets involved in diseases with a tremendous social and economic burden. BIOALVO’s BLOCKADE application, which targets compounds able to inhibit the enzyme indoleamine 2,3 dioxygenase (IDO-1), was selected to first test the extracts. Extracts were considered positive if they inhibited the growth of BLOCKADE yeast >60% [ 15 ]. In the BLOCKADE screening, the dichloromethane extract of the marine sponge Erylus cf.deficiens collected in the Gorringe Bank (Atlantic Ocean) tested positive at a concentration of 0.125 mg/mL. The activity of this extract was confirmed using an additional assay with African green monkey kidney fibroblast COS7 cells transfected with IDO, revealing an IDO inhibitory activity of 80%. The organic extract was further separated by flash chromatography on C18 reverse phase silica gel (RP-18) into eleven fractions, one of which (fraction 2) conserved the activity of the original extract, inhibiting kynurenine production by 80% at the same concentration. 1 H nuclear magnetic resonance (NMR) spectrum of fraction 2 (150 mg) revealed complex signals belonging to sugar components between δ 6.4 and 3.5 ppm, together with aliphatic resonances, due to a lipid moiety in the upfield region of the spectrum, thus suggesting the occurrence of a series of glycoconjugates. Hence, as a first step in the structure elucidation of the bioactive components, a methanolysis reaction was performed on an aliquot of the mixture to liberate the aglycone from the monosaccharide pool. Methyl glycosides were converted into the corresponding trimethylsilyl (TMS) derivatives [ 16 ] and analysed by GC-MS in comparison with authentic standards. According to retention time and characteristic MS fragmentation patterns, monosaccharide units were identified as D-xylose, D-glucose and D-galactose. On the other hand, aglycones showed IR bands at 3349, 1740, 1701 and 1636 cm −1 , suggesting the presence of hydroxyl, ester, ketone and amide functionalities, which were confirmed by NMR data. Separation of individual components was achieved by RP-HPLC on a phenyl-hexyl column (Phenomenex) affording 1 – 4 (Figure 1), as pure compounds, here named erylusamides A – D . High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analysis in negative ionization polarity revealed that compounds 1 – 4 constituted a series of homologous compounds displaying molecular mass ions at m/z1782.8345, 1796.8515, 1796.8434 and 180.8644. Mar. Drugs 2016,14, 179 5 of 14 Erylusamide A ( 1 ) gave a molecular ion [M − H] − at m/z1782.8345, which accounts for the molecular formula C 83 H 133 NO 40 requiring 18 degrees of formal unsaturation. 1D and 2D-NMR data (Tables 2and 3) revealed diagnostic signals of an oligosaccharide moiety composed of five sugar residues, and of a polyketide aglycone displaying three carbonyl signals at δ 210.5, 174.9 and 173.2 ppm in the 13 C NMR spectrum. Several different spin systems were identified in the aglycone moiety through COSY and HSQC-TOCSY connectivities, and joined by HMBC correlations (Figure 2). In particular, one terminal end of the aglycone polyketide chain was assigned to a N-methylalanine substructure. In fact, a deshielded signal at δ 5.75 (H-2 0 , q) was coupled in the COSY spectra with a methyl doublet at δ 1.54 (H 3 -4 0 ), as well as in the HMBC spectra, and showed correlations with a carboxyl function at δ 174.9 ppm (C-1 0 ) and a methyl carbon on a nitrogen atom at δ 31.5 ppm (C-3 0 ). In turn, the corresponding proton of this later signal was coupled to the carbonyl group at δ 173.2 ppm. The N-methylalanine moiety displayed two sets of signals (ratio 3:1) in 1 H NMR spectrum of 1 , consistent with a syn/anti rotamer equilibrium typically observed with tertiary amides [ 17 ], the major conformer being the syn one as deducted from the NOESY correlation H-2 and H-3 0 . Indeed, this phenomenon was also observed for structurally related pachymoside A, a glycolipid isolated from the marine sponge Pachymatisma johnstonia [3]. Table 2. NMR data for the aglycone amoieties of erylusamides A–D(1–4) in pyridine-d5. 1 2 3 4 N◦δ13Cδ1H, m (J, Hz) N◦δ13Cδ1H, m (J, Hz) N◦δ13Cδ1H, m (J, Hz) N◦δ13Cδ1H, m (J, Hz) 1173.2 - 1173.2 - 1173.2 - 1173.2 - 173.1 - 173.1 - 173.1 - 173.1 - 233.8 2.43, m 233.8 2.43, m 233.8 2.43, m 233.8 2.43, m 325.5 1.81, m 325.5 1.81, m 325.5 1.81, m 325.5 1.82, m 429.7 1.38, m 429.8 1.38, m 429.8 1.39, m 429.7 1.38, m 5–12 29.6–29.9 1.18–1.33 Overlap. 5–12 29.6–29.9 1.18–1.33 Overlap. 5–12 29.7–29.9 1.18–1.32 Overlap. 5–12 29.6–29.9 1.19–1.33 Overlap. 13 29.6 1.28, m 13 29.6 1.28, m 13 29.6 1.28, m 13 29.6 1.28, m 14 24.2 1.64, m 14 24.2 1.64, m 14 24.2 1.64, m 14 24.2 1.65, m 15 42.8 2.42, m 15 42.7 2.42, m 15 42.8 2.42, m 15 42.8 2.42, m 16 210.5 - 16 210.5 - 16 210.5 - 16 210.5 - 17 42.8 2.42, m 17 42.7 2.42, m 17 42.8 2.42, m 17 42.8 2.42, m 18 24.2 1.64, m 18 24.2 1.64, m 18 24.2 1.64, m 18 24.2 1.65, m 19–23 29.6–29.9 1.19–132 Overlap 19–24 29.6–29.9 1.18–1.33 Overlap 19–24 29.7–29.9 1.18–1.32 Overlap 19–25 29.7–29.9 1.19–1.33 Overlap 24 29.7 1.83, m 25 29.7 1.82, m 25 29.7 1.81, m 26 29.7 1.83, m 25 74.9 5.53, m 26 74.9 5.53, m 26 74.9 5.54, m 27 74.9 5.54, m AcO 169.7 * AcO 169.7 * AcO 169.7* AcO 169.7 * 26 80.7 4.03, m 27 80.8 4.03, m 27 80.7 4.03, m 28 80.8 4.05, m 27 30.9 1.79, m 28 30.9 1.79, m 28 39.1 1.16, m 29 30.9 1.80, m 28 32.0 1.23, m 29 32.0 1.23, m 29 28.2 1.51, m 30 32.0 1.25, m 29 22.8 1.28, m 30 22.8 1.28, m 30 22.8 0.85, d (6.0) 31 22.9 1.27, m 30 14.7 0.84, t (7.0) 31 14.2 0.86, t (6.5) 31 22.8 0.85, d (6.0) 32 14.2 0.86, t (6.6) 32 22.8 0.85, d (6.0) 1’ 174.9 - 1’ 174.9 - 1’ 174.9 - 1’ 175.0 - 174.5 - 174.5 - 174.5 - 174.6 - 2’ 52.7 5.75, q (7.2) 2’ 52.6 5.74, q (7.3) 2’ 52.7 5.74, q (7.3) 2’ 52.7 5.75, q (7.3) 55.8 4.97, q (7.3) 4.97, q (7.4) 55.7 4.97, q (7.3) 55.8 4.97, q (7.2) 3’ 31.5 3.06, s 3’ 31.5 3.06, s 3’ 31.5 3.06, s 3’ 31.5 3.07, s 28.9 3.14, s 28.9 3.13, s 28.9 3.14,s 28.9 3.15,s 4’ 15.0 1.54, d (7.3) 4’ 15.0 1.55, d (7.3) 4’ 15.0 1.55, d (7.3) 4’ 15.0 1.54, d (7.4) 16.0 1.60, d (7.2) 16.0 1.69, d (7.1) 16.0 1.60, d (7.3) 16.0 1.61, d (7.2) a duplicated values correspond to the major syn and minor anti rotamers respectively; * Overlapped with C-4 Glc3. Mar. Drugs 2016,14, 179 6 of 14 Table 3. NMR data for the carbohydrate moieties of erylusamides A–D(1–4) in pyridine-d5. Position 1 2 3 4 δ13Cδ1H, m (J, Hz) δ13Cδ1H, m (J, Hz) δ13Cδ1H, m (J, Hz) δ13Cδ1H, m (J, Hz) Gal 1104.4 5.16, d (7.5) 104.4 5.16, d (7.7) 104.4 5.15, d (7.6) 104.4 5.16, d (7.7) 270.7 4.54, m 70.7 4.53, m 70.7 4.53, m 70.72 4.53, m 384.4 4.24, m 84.3 4.25, m 84.3 4.25, m 84.32 4.24, m 469.0 4.70, brs 68.9 4.71, brs 68.7 4.71, brs 68.91 4.72, brs 577.3 4.21, m 77.4 4.20, m 77.3 4.21, m 77.27 4.20, m 661.6 4.38, dd 61.6 4.38, dd 61.6 4.38, dd 61.62 4.38, dd (5.1; 10.6) (5.2; 10.6) (4.9; 10.9) (5.2; 10.6) 4.44, dd 4.43, dd 4.44, dd 4.44, dd (6.8; 10.6) (6.9; 10.6) (6.7; 10.9) (6.9; 10.6) Xyl 1103.2 4.90,d (7.3) 103.2 4.89, d (7.3) 103.2 4.90, d (7.6) 103.2 4.90, d (7.4) 278.3 4.22, m 78.2 4.22, m 78.3 4.21, m 78.3 4.21, m 388.2 4.25, m 88.1 4.24, m 88.0 4.24, m 88.0 4.26, m 469.0 4.00, m 68.9 3.99, m 69.0 3.99, m 69.0 4.00, m 565.9 3.51, t (10.7) 65.9 3.50, t (9.8) 65.9 3.51, t (10.3) 65.9 3.51, t (10.7) 4.14, m 4.14, m 4.14, m 4.13, m Glc1 1104.6 4.86, d (7.9) 104.6 4.87, d (7.8) 104.6 4.88, d (8.0) 104.6 4.87, d (8.0) 274.9 3.97, m 74.6 3.96, m 74.6 3.97, m 74.6 3.98, m 376.6 4.18, m 76.6 4.18, m 76.6 4.18, m 76.6 4.18, m 481.9 3.97, m 81.8 3.97, m 81.9 3.97, m 81.9 3.97, m 573.0 4.09, m 72.9 4.08, m 73.0 4.10, m 73.0 4.11, m 664.3 4.92, m 64.3 4.93, m 64.1 4.93, m 64.3 4.93, m 5.16, m 5.14, m 5.16, m 5.15, m Ac (C-6) 171.0 - - 171.0 171.0 - Glc2 1102.8 5.55, d (8.0) 102.8 5.55, d (8.2) 102.8 5.55, d (8.5) 102.8 5.55, d (8.1) 272.2 5.48, dd 72.2 5.47,dd 72.2 5.48, t 72.1 5.48, dd (8.2; 9.5) (8.4; 9.3) (9.3) (8.4;9.3) 373.5 5.78, t (9.6) 73.5 5.78, t (9.6) 73.5 5.78, t (9.9) 73.4 5.78, t (9.5) 469.4 5.42, t (9.8) 69.4 5.42, t (9.7) 69.4 5.42, t (9.7) 69.4 5.42, t (9.7) 572.1 4.22, m 72.1 4.21, m 72.1 4.22, m 72.1 4.22, m 6 62.7 4.32, dd 62.7 4.32, dd 62.6 4.31, dd 62.7 4.31, dd (2.3; 12.1) (2.1; 11.8) (~2; 11.8) (2.3;12.1) 4.51, dd 4.50,dd, 4.52, dd 4.51, dd (5.4; 12.1) (5.3; 12.0) (4.8; 11.8) (5.4;12.1) Ac (C-2) 170.2 - 170.2 - 170.2 - 170.2 - Ac (C-3) 170.1 - 170.1 - 172.2 - 170.2 - Ac (C4) 169.8 - 169.9 - 169.9 - 169.9 - Ac (C-6) 170.4 - 170.5 - 170.5 - 170.5 - Glc3 199.6 6.37, d (7.9) 99.6 6.35, d (8.0) 99.6 6.36, d (7.6) 99.6 6.36, d (7.9) 272.3 5.62, m 72.2 5.61, t (10) 72.2 5.62, m 72.2 5.62, m 373.7 5.90, t (9.4) 73.7 5.89, t (9.5) 73.7 5.9, t (9.3) 73.7 5.90, t (9.5) 469.7 5.58, m 69.7 5.56, m 69.7 5.56, m 69.7 5.56, m 572.3 3.93, m 72.2 3.92, m 72.2 3.92, m 662.5 4.19, m 62.5 4.56, m 62.4 4.19, m 62.4 4.18, m 4.57, m 4.18, m 4.56, m 4.57, m Ac(C-2) 169.6 - 169.6 - 169.7 - 169.7 - Ac(C-3) 170.5 - 170.5 - 170.5 - 170.5 - Ac(C-4) 169.7 - 169.7 - 169.8 - 169.7 - Ac(C-6) 170.2 - 170.4 - 170.4 - 170.4 - Mar. Drugs 2016,14, 179 7 of 14 Mar.Drugs2016,14,1777of14  Figure2.KeyHMBC(redashes)andHSQC‐TOCSY(bluelines)correlationsestablishingthe structureoftheaglyconemoiety. Thepresenceoftwovicinaloxymethinegroupsconstitutinganisolatedstereoclusterwasthe moststrikingfeatureoftheaglyconemoiety.IntheHSQCspectrum,thecrosspeaksatδ80.7/δ4.03 andδ74.9/δ5.53suggestedthepresenceoftwonon‐equivalentsecondaryO‐substitutedalcohols. AnHMBCcross‐peakwasobservedbetweentheprotonatδ4.03andthecarbonatδ74.8ppm. However,noCOSYcorrelationwasobservedbetweenthetwooxymethinesignalssuggestingthat thedihedralanglebetweenthetwoprotonsshouldbearound90°[18].Thesedatawereconsistent withavicinaldiol,withonehydroxylgroupacylatedandtheotheronelinkedtoasugarmoiety [19,20].Furthermore,aconnectioncouldbeassignedbetweenthisdiolmoietyandterminaln‐butyl, asdepictedfromtheH2BcrosspeakbetweenC‐26(δ80.7)andtheprotonatδ1.79ppm(H‐27),as wellasHSQC‐TOCSYlongrangecorrelations30.9→ 32.0→ 22.8→ 14.7→ 0.84.Theremaining deshieldedsignalatδ210.5correspondedtoanaliphaticsymmetricalketone,asdeducedfromthe HMBCcorrelationwithtwoseparatedCH2signalsatδ2.42(4H)and1.64(4H)ppm.Compound1 wasmethanolysedtoliberatetheaglyconemethylester(compound5,Figure3),whichwasfurther convertedinthecorrespondingacetonide,andtheirMSandNMRspectra(seeSections3.4and3.7) analysedandcomparedwiththoseofcompound1.Compound5showedamolecularadduction [M+Na]+atm/z620.5(Figure3)compatiblewiththemethylesterofthedeacetylatedfreeaglycone. ComparisonofthisresultwiththeonesobtainedfromMSanalysisofcompound1confirmedthe presenceofanacetylgroupontheaglyconemoiety:theMS/MSdataonmolecularion[M−H]−at m/z1782.8ofcompound1showedafragmentionatm/z624.5,duetothelossoftheoligosaccharide portion,compatiblewithanmonoacetylatedaglyconemoiety.Additionally,adetailedanalysisof tandemmassspectrometry(ESI+‐MS/MS)data(Figure3)obtainedontheaglyconemethylester5,at m/z620.5,suggestedthelocationofthecarbonylfunctionatC‐16inthealiphaticchain.Infact, productionspectracontaineddiagnosticionsatm/z265.3[C15H30O2+Na]+and390.3formally arisingfromα‐cleavageofthecarbonylgroup.Furthermore,afragmentionatm/z138.1confirmed thepresenceoftheN‐methylalaninemoiety. Finally,analysisofNMRspectraoftheacetonide6(seeSection3.7)confirmedtheoccurrence andrelativestereochemistryofthe1,2diolsystem:thetwooxymethineprotonsatδ3.72and3.74 werecoupledbyH2BCtothedownfieldshiftedcarbonsatδ81.4and1.5,respectively,aswellasby HMBCwiththeoxygenatedcarbonsignalatδ107.8,bearing,inturn,thetwoacetonidemethyl groupsatδ1.50).Accordingtothecarbonchemicalshiftsofthesemethylgroupsof6,overlappingat 27.0ppm,therelativestereochemistryofthe1,2‐diolwasproposedasthreo.[21–23].  Figure3.ESI‐MS/MSanalysisoftheaglyconemethylester5atm/z620.5[M+Na]+. Theaglyconepartasdescribedaboveaccountedforfouroutofthe18formalunsaturations predictedbythemolecularformulaof1.Thus,theremaining14doublebondequivalentswere 560.6 390.3 ‐H 2 O 138.1 N H 3 CO O (CH 2 ) 4 (CH 2 ) 4 (CH 2 ) 5 OO 265.3 OH OH 503.5 Figure 2. Key HMBC (red ashes) and HSQC-TOCSY (blue lines) correlations establishing the structure of the aglycone moiety. The presence of two vicinal oxymethine groups constituting an isolated stereocluster was the most striking feature of the aglycone moiety. In the HSQC spectrum, the crosspeaks at δ 80.7/ δ 4.03 and δ 74.9/ δ 5.53 suggested the presence of two non-equivalent secondary O-substituted alcohols. An HMBC cross-peak was observed between the proton at δ 4.03 and the carbon at δ 74.8 ppm. However, no COSY correlation was observed between the two oxymethine signals suggesting that the dihedral angle between the two protons should be around 90 ◦ [ 18 ]. These data were consistent with a vicinal diol, with one hydroxyl group acylated and the other one linked to a sugar moiety [ 19 , 20 ]. Furthermore, a connection could be assigned between this diol moiety and terminal n-butyl, as depicted from the H2B crosspeak between C-26 ( δ 80.7) and the proton at δ 1.79 ppm (H-27), as well as HSQC-TOCSY long range correlations 30.9 → 32.0 → 22.8 → 14.7 → 0.84. The remaining deshielded signal at δ 210.5 corresponded to an aliphatic symmetrical ketone, as deduced from the HMBC correlation with two separated CH 2 signals at δ 2.42 (4H) and 1.64 (4H) ppm. Compound 1 was methanolysed to liberate the aglycone methyl ester (compound 5 , Figure 3), which was further converted in the corresponding acetonide, and their MS and NMR spectra (see Sections 3.4 and 3.7) analysed and compared with those of compound 1 . Compound 5 showed a molecular adduct ion [M + Na] + at m/z620.5 (Figure 3) compatible with the methyl ester of the deacetylated free aglycone. Comparison of this result with the ones obtained from MS analysis of compound 1 confirmed the presence of an acetyl group on the aglycone moiety: the MS/MS data on molecular ion [M − H] − at m/z1782.8 of compound 1 showed a fragment ion at m/z624.5, due to the loss of the oligosaccharide portion, compatible with an monoacetylated aglycone moiety. Additionally, a detailed analysis of tandem mass spectrometry (ESI + -MS/MS) data (Figure 3) obtained on the aglycone methyl ester 5 , at m/z620.5, suggested the location of the carbonyl function at C-16 in the aliphatic chain. In fact, product ion spectra contained diagnostic ions at m/z265.3 [C 15 H 30 O 2 + Na] + and 390.3 formally arising from α -cleavage of the carbonyl group. Furthermore, a fragment ion at m/z138.1 confirmed the presence of the N-methylalanine moiety. Finally, analysis of NMR spectra of the acetonide 6 (see Section 3.7) confirmed the occurrence and relative stereochemistry of the 1,2 diol system: the two oxymethine protons at δ 3.72 and 3.74 were coupled by H2BC to the downfield shifted carbons at δ 81.4 and 1.5, respectively, as well as by HMBC with the oxygenated carbon signal at δ 107.8, bearing, in turn, the two acetonide methyl groups at δ 1.50). According to the carbon chemical shifts of these methyl groups of 6 , overlapping at 27.0 ppm, the relative stereochemistry of the 1,2-diol was proposed as threo. [21–23]. Mar.Drugs2016,14,1777of14  Figure2.KeyHMBC(redashes)andHSQC‐TOCSY(bluelines)correlationsestablishingthe structureoftheaglyconemoiety. Thepresenceoftwovicinaloxymethinegroupsconstitutinganisolatedstereoclusterwasthe moststrikingfeatureoftheaglyconemoiety.IntheHSQCspectrum,thecrosspeaksatδ80.7/δ4.03 andδ74.9/δ5.53suggestedthepresenceoftwonon‐equivalentsecondaryO‐substitutedalcohols. AnHMBCcross‐peakwasobservedbetweentheprotonatδ4.03andthecarbonatδ74.8ppm. However,noCOSYcorrelationwasobservedbetweenthetwooxymethinesignalssuggestingthat thedihedralanglebetweenthetwoprotonsshouldbearound90°[18].Thesedatawereconsistent withavicinaldiol,withonehydroxylgroupacylatedandtheotheronelinkedtoasugarmoiety [19,20].Furthermore,aconnectioncouldbeassignedbetweenthisdiolmoietyandterminaln‐butyl, asdepictedfromtheH2BcrosspeakbetweenC‐26(δ80.7)andtheprotonatδ1.79ppm(H‐27),as wellasHSQC‐TOCSYlongrangecorrelations30.9→ 32.0→ 22.8→ 14.7→ 0.84.Theremaining deshieldedsignalatδ210.5correspondedtoanaliphaticsymmetricalketone,asdeducedfromthe HMBCcorrelationwithtwoseparatedCH2signalsatδ2.42(4H)and1.64(4H)ppm.Compound1 wasmethanolysedtoliberatetheaglyconemethylester(compound5,Figure3),whichwasfurther convertedinthecorrespondingacetonide,andtheirMSandNMRspectra(seeSections3.4and3.7) analysedandcomparedwiththoseofcompound1.Compound5showedamolecularadduction [M+Na]+atm/z620.5(Figure3)compatiblewiththemethylesterofthedeacetylatedfreeaglycone. ComparisonofthisresultwiththeonesobtainedfromMSanalysisofcompound1confirmedthe presenceofanacetylgroupontheaglyconemoiety:theMS/MSdataonmolecularion[M−H]−at m/z1782.8ofcompound1showedafragmentionatm/z624.5,duetothelossoftheoligosaccharide portion,compatiblewithanmonoacetylatedaglyconemoiety.Additionally,adetailedanalysisof tandemmassspectrometry(ESI+‐MS/MS)data(Figure3)obtainedontheaglyconemethylester5,at m/z620.5,suggestedthelocationofthecarbonylfunctionatC‐16inthealiphaticchain.Infact, productionspectracontaineddiagnosticionsatm/z265.3[C15H30O2+Na]+and390.3formally arisingfromα‐cleavageofthecarbonylgroup.Furthermore,afragmentionatm/z138.1confirmed thepresenceoftheN‐methylalaninemoiety. Finally,analysisofNMRspectraoftheacetonide6(seeSection3.7)confirmedtheoccurrence andrelativestereochemistryofthe1,2diolsystem:thetwooxymethineprotonsatδ3.72and3.74 werecoupledbyH2BCtothedownfieldshiftedcarbonsatδ81.4and1.5,respectively,aswellasby HMBCwiththeoxygenatedcarbonsignalatδ107.8,bearing,inturn,thetwoacetonidemethyl groupsatδ1.50).Accordingtothecarbonchemicalshiftsofthesemethylgroupsof6,overlappingat 27.0ppm,therelativestereochemistryofthe1,2‐diolwasproposedasthreo.[21–23].  Figure3.ESI‐MS/MSanalysisoftheaglyconemethylester5atm/z620.5[M+Na]+. Theaglyconepartasdescribedaboveaccountedforfouroutofthe18formalunsaturations predictedbythemolecularformulaof1.Thus,theremaining14doublebondequivalentswere 560.6 390.3 ‐H 2 O 138.1 N H 3 CO O (CH 2 ) 4 (CH 2 ) 4 (CH 2 ) 5 OO 265.3 OH OH 503.5 Figure 3. ESI-MS/MS analysis of the aglycone methyl ester 5at m/z620.5 [M + Na]+. Mar. Drugs 2016,14, 179 8 of 14 The aglycone part as described above accounted for four out of the 18 formal unsaturations predicted by the molecular formula of 1 . Thus, the remaining 14 double bond equivalents were attributable to the glucosidic portion. The analysis of the 1 H, 13 C and HSQC spectra revealed five anomeric carbons, accounting for five sugar rings. The remaining formal unsaturations were assigned to nine acetate residues, which fulfilled the observed [M −H]−ion peak at m/z 1782.8345. Hydrolysis of compound 1 showed that D-xylose, D-galactose and D-glucose were the only monomers present with a ration 1:1:3. The sequence of these sugar residues was determined by extensive NMR study, especially based on 2D techniques (COSY-45, HSQC, HSQC–TOCSY, H2BC, HMBC and NOESY) (Table 2). The five anomeric carbons and their attached protons were unequivocally identified at δ13 C/ 1 H: 104.6/4.86 (d, J= 7.9 Hz); 104.4/5.16 (d, J= 7.5 Hz); 103.2/4.90 (d, J= 7.3 Hz); 102.8/5.55 (d, J= 8.0 Hz ) and 99.6/6.37 (d, J= 7.9 Hz) (Figure 4). The anomeric configurations were assigned as β from the magnitude of the 3 J 1,2 , values, all within the 7–9 Hz interval, typical of diaxial proton coupling [ 24 ]. Moreover, the 13 C NMR shifts of the anomeric carbons, approximatively 100 ppm, also indicate that the corresponding sugars are connected through β-glycosidic bonds [25,26]. Mar.Drugs2016,14,1778of14 attributabletotheglucosidicportion.Theanalysisofthe 1 H, 13 CandHSQCspectrarevealedfive anomericcarbons,accountingforfivesugarrings.Theremainingformalunsaturationswere assignedtonineacetateresidues,whichfulfilledtheobserved[M−H] − ionpeakatm/z1782.8345. Hydrolysisofcompound1showedthat D ‐xylose, D ‐galactoseand D ‐glucoseweretheonly monomerspresentwitharation1:1:3.Thesequenceofthesesugarresidueswasdeterminedby extensiveNMRstudy,especiallybasedon2Dtechniques(COSY‐45,HSQC,HSQC–TOCSY,H2BC, HMBCandNOESY)(Table2). Thefiveanomericcarbonsandtheirattachedprotonswereunequivocallyidentifiedatδ 13 C/ 1 H: 104.6/4.86(d,J=7.9Hz);104.4/5.16(d,J=7.5Hz);103.2/4.90(d,J=7.3Hz);102.8/5.55(d,J=8.0Hz) and99.6/6.37(d,J=7.9Hz)(Figure4).Theanomericconfigurationswereassignedasβfromthe magnitudeofthe 3 J 1,2 ,values,allwithinthe7–9Hzinterval,typicalofdiaxialprotoncoupling[24]. Moreover,the 13 CNMRshiftsoftheanomericcarbons,approximatively100ppm,alsoindicatethat thecorrespondingsugarsareconnectedthroughβ‐glycosidicbonds[25,26].  Figure4.ExpansionofHSQCspectrumoferylusamideA(1)showingtheanomericcarbon,the oxymethinesandoxymethylenescorrelations. Sixoftheoxymethines(δH5.90,5.78,5.62,5.58,5.48and5.42)andthreeoftheoxymethylenes (δH4.51/4.32,4.57/4.19,5.16/4.92)hadprotonresonatingat1–2ppmdownfieldwithrespecttofree hydroxylgroups[19],whichindicatedthesitesofacetylation(Figure4).Thepositionofacetyl groupswasascertainedbyHMBCcorrelationsbetweentheacetylcarbonylsandthecorresponding oxymethineprotons(Figure5). Thelong‐rangeHMBCcorrelationbetweenC‐26(δ80.7ppm)andtheβ‐anomericprotonatδ 4.86disclosedthelinkagebetweentheaglyconeportionandthefirstunitofthepentasaccharide chain,whichwasassignedtoamonoacetylatedglucoseresidue(Glc1).Infact,startingfromthe anomericprotonTOCSYexperimentsallowedtodelineatetheentirespinsystemwhilerelative configurationwasachievedbyanalysisofNOESYdataandJcouplings.Furthermore,H‐6methylene resulteddeshieldedthussuggestingthefirstacetylationsite.TheMS/MSfragmentatm/z828.5 [aglyconeGlc1−H] − )fromtheion[M−H] − atm/z1782.8iscompatiblewithamonoacetylated glucose. Figure 4. Expansion of HSQC spectrum of erylusamide A ( 1 ) showing the anomeric carbon, the oxymethines and oxymethylenes correlations. Six of the oxymethines ( δ H 5.90, 5.78, 5.62, 5.58, 5.48 and 5.42) and three of the oxymethylenes ( δ H 4.51/4.32, 4.57/4.19, 5.16/4.92) had proton resonating at 1–2 ppm downfield with respect to free hydroxyl groups [ 19 ], which indicated the sites of acetylation (Figure 4). The position of acetyl groups was ascertained by HMBC correlations between the acetyl carbonyls and the corresponding oxymethine protons (Figure 5). The long-range HMBC correlation between C-26 ( δ 80.7 ppm) and the β -anomeric proton at δ 4.86 disclosed the linkage between the aglycone portion and the first unit of the pentasaccharide chain, which was assigned to a monoacetylated glucose residue (Glc1). In fact, starting from the anomeric proton TOCSY experiments allowed to delineate the entire spin system while relative configuration was achieved by analysis of NOESY data and Jcouplings. Furthermore, H-6 methylene resulted deshielded thus suggesting the first acetylation site. The MS/MS fragment at m/z828.5 [aglyconeGlc1 −H]− ) from the ion [M −H]−at m/z1782.8 is compatible with a monoacetylated glucose. Mar. Drugs 2016,14, 179 9 of 14 Mar.Drugs2016,14,1779of14  Figure5.ExpansionofHMBCspectrumshowingcorrelationstoacetatecarbonylsinsugarmoiety. TheHMBCcrosspeakbetweenC‐4Glc1andtheanomericprotonat4.90identifiedthe glycosidicbondbetweenthisglucoseandthexyloseresidue,confirmedbythecorrelationbetween C‐1XylandH‐4Glc1.Xyloseshowedanotherglycosidicbondwithanotherglucoseresidue,which wasdepictedfromcrosspeaksC‐2Xyl/H‐1Glc3andC‐1Glc3/H‐2Xyl.Athirdβ‐glycosidicbond betweenxyloseandagalactoseresiduewasapparentfromthelongrangecorrelationC‐3Xyl/H‐1 GalandtheNOESYcorrelationH‐3Xyl/H‐1Gal.Finally,thegalactoseresiduewasconnectedto anotherglucoseunitthroughthecrosspeakbetweenC‐3GalandH‐1Glc2(Figure6). HR‐ESI‐MSoferylusamidesB(2)andC(3)showed[M−H] − ionsatm/z1796.8515and 1796.8434respectively,consistentwiththeempiricalformulaC 84 H 135 NO 40 ,suggestinganisomeric relationship,whichwasreflectedinadifferentbehaviourofthetwometabolitesinHPLCanalysis. AcarefulcomparisonofNMRspectraindicatedstructureswithaglyconescloselyrelatedto erylusamideA,whichdifferedforanextramethylene,alsoconfirmedbythepeakatm/z638.5 [aglycone−H] − (C 37 H 68 O 7 N)inbothESI − ‐MS/MSanalyses.  Figure6.KeyHMBC(redashes),NOESY(dashedashes)andHSQC‐TOCSY(bluelines)correlations establishingthestructureofthepentasaccharidemoiety. 1 Hand 13 CNMRspectraofcompound2werealmostsuperimposablewiththoseof1, suggestingthattheadditionalmethyleneshouldbepositionedwithinthelonghydrocarbonchain. Furthermore,NMRdatashowedthattheonlydifferencebetweentheisomericcompounds2and3 wasatonechainendoftheaglyconemoiety,whereanisobutylgroupin3replacedtheterminal n‐butylresidueof2.Infact,the 1 HNMRspectrumof3showedthepresenceofadoubletatδ0.85 ppm(6H,J=6.0Hz)andamultipletsignalatδ1.51assigned,respectively,tothemethyland methineprotonsoftheisobutylmoiety.Thesignalatδ1.16wasattributedtotheremaining Figure 5. Expansion of HMBC spectrum showing correlations to acetate carbonyls in sugar moiety. The HMBC cross peak between C-4 Glc1 and the anomeric proton at 4.90 identified the glycosidic bond between this glucose and the xylose residue, confirmed by the correlation between C-1 Xyl and H-4 Glc1. Xylose showed another glycosidic bond with another glucose residue, which was depicted from cross peaks C-2 Xyl/H-1 Glc3 and C-1 Glc3/H-2 Xyl. A third β -glycosidic bond between xylose and a galactose residue was apparent from the long range correlation C-3 Xyl/ H-1 Gal and the NOESY correlation H-3 Xyl/H-1Gal. Finally, the galactose residue was connected to another glucose unit through the cross peak between C-3 Gal and H-1 Glc2 (Figure 6). HR-ESI-MS of erylusamides B ( 2 ) and C ( 3 ) showed [M − H] − ions at m/z1796.8515 and 1796.8434 respectively, consistent with the empirical formula C 84 H 135 NO 40 , suggesting an isomeric relationship, which was reflected in a different behaviour of the two metabolites in HPLC analysis. A careful comparison of NMR spectra indicated structures with aglycones closely related to erylusamide A, which differed for an extra methylene, also confirmed by the peak at m/z638.5 [aglycone − H] − (C37H68O7N) in both ESI−-MS/MS analyses. Mar.Drugs2016,14,1779of14  Figure5.ExpansionofHMBCspectrumshowingcorrelationstoacetatecarbonylsinsugarmoiety. TheHMBCcrosspeakbetweenC‐4Glc1andtheanomericprotonat4.90identifiedthe glycosidicbondbetweenthisglucoseandthexyloseresidue,confirmedbythecorrelationbetween C‐1XylandH‐4Glc1.Xyloseshowedanotherglycosidicbondwithanotherglucoseresidue,which wasdepictedfromcrosspeaksC‐2Xyl/H‐1Glc3andC‐1Glc3/H‐2Xyl.Athirdβ‐glycosidicbond betweenxyloseandagalactoseresiduewasapparentfromthelongrangecorrelationC‐3Xyl/H‐1 GalandtheNOESYcorrelationH‐3Xyl/H‐1Gal.Finally,thegalactoseresiduewasconnectedto anotherglucoseunitthroughthecrosspeakbetweenC‐3GalandH‐1Glc2(Figure6). HR‐ESI‐MSoferylusamidesB(2)andC(3)showed[M−H] − ionsatm/z1796.8515and 1796.8434respectively,consistentwiththeempiricalformulaC 84 H 135 NO 40 ,suggestinganisomeric relationship,whichwasreflectedinadifferentbehaviourofthetwometabolitesinHPLCanalysis. AcarefulcomparisonofNMRspectraindicatedstructureswithaglyconescloselyrelatedto erylusamideA,whichdifferedforanextramethylene,alsoconfirmedbythepeakatm/z638.5 [aglycone−H] − (C 37 H 68 O 7 N)inbothESI − ‐MS/MSanalyses.  Figure6.KeyHMBC(redashes),NOESY(dashedashes)andHSQC‐TOCSY(bluelines)correlations establishingthestructureofthepentasaccharidemoiety. 1 Hand 13 CNMRspectraofcompound2werealmostsuperimposablewiththoseof1, suggestingthattheadditionalmethyleneshouldbepositionedwithinthelonghydrocarbonchain. Furthermore,NMRdatashowedthattheonlydifferencebetweentheisomericcompounds2and3 wasatonechainendoftheaglyconemoiety,whereanisobutylgroupin3replacedtheterminal n‐butylresidueof2.Infact,the 1 HNMRspectrumof3showedthepresenceofadoubletatδ0.85 ppm(6H,J=6.0Hz)andamultipletsignalatδ1.51assigned,respectively,tothemethyland methineprotonsoftheisobutylmoiety.Thesignalatδ1.16wasattributedtotheremaining Figure 6. Key HMBC (red ashes), NOESY (dashed ashes) and HSQC-TOCSY (blue lines) correlations establishing the structure of the pentasaccharide moiety. 1 H and 13 C NMR spectra of compound 2 were almost superimposable with those of 1 , suggesting that the additional methylene should be positioned within the long hydrocarbon chain. Furthermore, NMR data showed that the only difference between the isomeric compounds 2 and 3 was at one chain end of the aglycone moiety, where an isobutyl group in 3 replaced the terminal n-butyl residue of 2 .