Secondary metabolites from the endophytic fungus Pestalotiopsis virgatula isolated from the mangrove plant Sonneratia caseolaris

Accep ed Manusc ip Seconda y Me aboli es om he Endophy ic Fungus Pes alo iopsis i ga ula Isola ed om he Mang o e Plan Sonne a ia caseola is Da id Rönsbe g, Abdessamad Debbab, A ila Mándi, Vic o W ay, Hao u Dai, Tibo Ku án, Pe e P oksch, Amal H. Aly PII: S0040-4039(13)00605-9 DOI: h p://dx.doi.o g/10.1016/j. e le .2013.04.031 Re e ence: TETL 42798 To appea in: Te ahed on Le e s Recei ed Da e: 27 Feb ua y 2013 Re ised Da e: 4 Ap il 2013 Accep ed Da e: 8 Ap il 2013 Please ci e his a icle as: Rönsbe g, D., Debbab, A., Mándi, A., W ay, V., Dai, H., Ku án, T., P oksch, P., Aly, A.H., Seconda y Me aboli es om he Endophy ic Fungus Pes alo iopsis i ga ula Isola ed om he Mang o e Plan Sonne a ia caseola is, Te ahed on Le e s (2013), doi: h p://dx.doi.o g/10.1016/j. e le .2013.04.031 This is a PDF ile o an unedi ed manusc ip ha has been accep ed o publica ion. As a se ice o ou cus ome s we a e p o iding his ea ly e sion o he manusc ip . The manusc ip will unde go copyedi ing, ypese ing, and e iew o he esul ing p oo be o e i is published in i s inal o m. Please no e ha du ing he p oduc ion p ocess e o s may be disco e ed which could a ec he con en , and all legal disclaime s ha apply o he jou nal pe ain. 1 G aphical Abs ac 2 Seconda y Me aboli es om he Endophy ic Fungus Pes alo iopsis i ga ula Isola ed om he Mang o e Plan Sonne a ia caseola is Da id Rönsbe ga, Abdessamad Debbaba, A ila Mándib, Vic o W ayc, Hao u Daid, Tibo Ku ánb, Pe e P okscha, Amal H. Alya,∗ aIns i u ü Pha mazeu ische Biologie und Bio echnologie, Hein ich-Heine-Uni e si ä , Uni e si ä ss asse 1, 40225 Düsseldo , Ge many bDepa men o O ganic Chemis y, Uni e si y o Deb ecen, POB 20, 4010 Deb ecen, Hunga y cHelmhol z Cen e o In ec ion Resea ch, Inho ens aße 7, 38124 B aunschweig, Ge many dIns i u e o T opical Bioscience and Bio echnology, Chinese Academy o T opical Ag icul u al Sciences, Haikou 571101, Hainan, P. R. China ∗ Co esponding au ho . Tel.: +49-211-81-14173; ax: +49-211-81-11923; e-mail: amal[email p o ec ed] 3 Abs ac Ch oma og aphic analysis o an e hyl ace a e ex ac o Pes alo iopsis i ga ula ice cul u es yielded ou new -py one de i a i es, pes alo iopy ones I-L, and he new (6S,1'S,2'S)-hyd oxypes alo in, in addi ion o h ee known compounds. The plana s uc u es o he new compounds we e elucida ed on he basis o ex ensi e NMR spec oscopic and mass spec ome ic analyses. The absolu e con igu a ions o he new compounds we e de e mined on he basis o biosyn he ic conside a ions, coupling cons an s and o pes alo iopy ones L by TDDFT ECD calcula ions o solu ion con o me s. Keywo ds Sonne a ia caseola is, endophy ic ungi, Pes alo iopsis i ga ula, -py ones 1. In oduc ion The sea ch o new d ugs om na u al sou ces is nowadays becoming mo e indispensable han e e , due o a comp ehensi e knowledge on he mode o ac ion in e ms o speci ici y and e iciency, possible side e ec s and he g owing isk o de eloping esis ance. Acco dingly, na u al p oduc s and d ugs de i ed om na u al sou ces inc easingly ep esen a signi ican p opo ion among newly de eloped d ugs.1-3 Bioac i e seconda y me aboli es om endophy ic ungi, isola ed om highe plan s, a e a majo ocus o na u al p oduc esea ch. O pa icula in e es a e plan sou ces o endophy ic ungi, which a e able o su i e in ex eme condi ions such as d ough , salini y, hea o cold, and wi h a a ie y o compe ing o ganisms, which li e in hei na u al en i onmen s. Examples o such na u al habi a s whe e all o ganisms ha e o adap mo phologically and physiologically o wi hs and he exis ing e olu iona y p essu e a e mang o e o es s. Chemical a ia ions a e equi ed, in e alia, o hese 4 o ganisms o ob ain e olu iona y bene i s. These ac s make endophy es om such en i onmen s e y p omising a ge s in he sea ch o new bioac i e seconda y me aboli es.4-8 Membe s o he genus Pes alo iopsis ha e a ac ed conside able a en ion in ecen yea s. Many impo an seconda y me aboli es ha a e po en ial leads o he ea men o human diseases and con ol o plan diseases ha e been iden i ied om his genus.9,10 A p ominen example is he disco e y o pacli axel as an endophy ic seconda y me aboli e om he ungal s ain P. mic ospo a.9 The genus is cha ac e ized by i s ex ensi e dis ibu ion and he wide gene ic and biological a iabili y o i s membe s, which make i a as ese oi o bioac i e na u al p oduc s s ill o be explo ed.11 In his s udy ou new α-py one de i a i es, pes alo iopy ones I-L (1-4), a new hyd oxypes alo in dias e eome (5), as well as h ee known compounds, hyd oxypes alo in, pes alo in and pes alopy one, we e isola ed om he mang o e-de i ed endophy ic ungus P. i ga ula by ch oma og aphic me hods. The s uc u e elucida ion o he new compounds (1-5) (Figu e 1) is desc ibed in de ail. 5 Figu e 1. Chemical s uc u es o compounds 1-5. 2. Resul s and Discussion The e hyl ace a e ex ac was ac iona ed by column ch oma og aphy ini ially using silica gel and hen Sephadex LH-20 o Diaion HP20 as s a iona y phases. Each subs ance was pu i ied by subsequen semi-p epa a i e HPLC. Compound 1 was ound o ha e he molecula o mula C13H16O7 based on he p ominen signal a m/z 285.0968 [M+H]+ in he HRESIMS. The 1H NMR spec um (Table 1) indica ed he p esence o one ole inic me hyl g oup a δ(H) 2.42 ppm (1’-CH3), one me hoxy g oup a δ(H) 3.94 ppm (4-OCH3), wo oxygena ed me hylene g oups a δ(H) 3.63 (CH2-3’’) and 4.21/4.30 ppm (CH2-1’’), an oxygena ed alipha ic p o on a δ(H) 3.92 ppm (H-2’’), wo me a-coupled a oma ic p o ons a δ(H) 5.78 (H-3) and 6.6 ppm (H-5), and one ole inic p o on single a δ(H) 6.74 ppm (H-2’). In o al, 6 hi een signals we e de ec ed in he 13C NMR spec um (Table 1) including signals o wo es e ca bonyl g oups, six ole inic and h ee oxygena ed alipha ic ca bons, one me hoxy and one me hyl g oup. The unsa u a ed δ-lac one pa ound in 1 was con i med by he HMBC co ela ions o H-3 o C-4 and C-5, and o H-5 o C-3, C-4 and C-6 (Table S1). The posi ion o he me hoxy unc ion was p o en by i s co ela ion o C-4. Th ough wo-dimensional NMR analyses, i was u he mo e shown ha he α-py one co e s uc u e o 1 was a ached o a side chain a C-6, which consis s o isoc o onic acid es e i ied wi h glyce ol. The p esence o he glyce ol moie y was indica ed by he COSY co ela ions es ablishing he down ield shi ed spin sys em CH2(1’’)CH(2’’)CH2(3’’). HMBC co ela ions o CH2-1’’ o C-2’’, o H-2’’ o C-1’’ and C-3’’, and o CH2-3’’ o C-2’’ o e ed u he e idence (Table S1). The isoc o onic acid moie y was con i med by he HMBC co ela ions o H-2’ o C-1’, 1’-CH3 and C-6, and o 1’-CH3 o C-1’, C-2’ and C-6 (Table S1). The HMBC co ela ion o H-5 o he sp2 ca bon C-1’ o he side chain con i med he linkage o C-6 o he β-posi ion o he ca boxylic acid. Mo eo e , es e i ica ion wi h he glyce yl moie y was shown by he co ela ion o H-1’’ o he ca boxyla e es e a om C-3’. The (E) geome y o he isoc o onic acid pa was de e mined by he obse ed co ela ion om H-5 o H-2’ in he ROESY spec um. Thus, he s uc u e o 1 was he e o e unambiguously de e mined as 2,3-dihyd oxyp opyl (2E)-3-(4-me hoxy-2-oxo-2H-py an-6-yl)bu -2-enoa e o which we sugges he name pes alo iopy one I. Compa ison o he [α]D alue o 1 (–13.1, MeOH) wi h epo ed alues o s uc u ally ela ed compounds as (2S)-1-O-p-couma oylglyce ol (+12.8, MeOH),12 (2R)-glyce ol monoace a e (–9.2, py idine) and (2R)-glyce ol monobenzoa e (–13.3, py idine)13 sugges ed ha he C-2’’ chi ali y cen e o 1 may ha e (R) absolu e con igu a ion. 7 Table 1. 1H and 13C NMR da a o 1-3 (MeOH-d4, δ in ppm, J in Hz) N . δH aδC bδH cδC dδH aδC b 2 165.6 165.7 165.6 3 5.78, d (1.9) 91.3 5.73, d (2.0) 91.4 5.78, d (2.0) 91.3 4172.9 173.1 173.0 5 6.6, d (1.9) 104.0 6.55, d (2.0) 104.2 6.6, d (2.1) 104.0 6160.2 160.3 160.2 1' 144.1 144.1 144.1 2' 6.74, bs 121.0 6.70, d (1.1) 121.3 6.76, d (1.2) 121.1 3' 167.3 167.6 167.4 4' --- 5' --- 4.30, dd (4.3, 11.5) 66.7 4.40, dd (2.8, 11.6) 67.5 4.46, dd (2.4, 11.4) 67.1 4.21, dd (6.3, 11.5) 4.22, dd (6.5, 11.6) 4.28, dd (5.5, 11.5) 2'' 3.92, m 71.1 3.79, m 71.3 3.82, m 71.0 3'' 3.63, dd (1.7, 5.4) 64.1 3.60, m 73.6 3.82, m 71.0 -3.59, dd (6.0, 14.4) 64.7 4.37, dd (2.8, 11.6) 67.1 3.76, dd (3.4, 14.4) 4.20, dd (5.5, 11.5) 1''' - - 172.9 2''' - ---2.12, s 20.8 1'-CH32.42, d (0.8) 13.7 2.37, d (1.2) 13.8 2.43, d (1.2) 13.7 1''-OCH3--- 4-OCH33.94, s 57.2 3.89, s 57.4 3.94, s 57.2 312 1'' 4'' - a600 MHz b150 MHz c500 MHz d125 MHz Compound 2 had he molecula o mula C14H18O8 as indica ed by he HRESIMS signal a m/z 315.1072 [M+H]+. The 1H and 13C NMR da a o 2 (Table 1) esembled hose o 1, excep o he exis ence o one addi ional hyd oxy me hine g oup esona ing a δ(H) 3.60 ppm (δ(C) 73.6 ppm, CH-3’’), which was in acco dance wi h he inc ease in molecula weigh o 2 by 30 amu compa ed o 1. Fu he con i ma ion was achie ed by he obse ed COSY co ela ions o CH2-1’’ o H-2’’, o H-2’’ o H-3’’, and o H-3’’ o CH2-4’’ (Table S1). Acco dingly, he side chain o 2 is composed o isoc o onic acid es e i ied wi h a bu ane-1,2,3,4- e ol uni . Thus, 2 was iden i ied as he new na u al p oduc 2,3,4- ihyd oxybu yl (2E)-3-(4-me hoxy-2-oxo-2H-py an-6-yl)bu -2-enoa e and gi en he name pes alo iopy one J. 8 The HRESIMS o 3 exhibi ed a p ominen peak a m/z 357.1180 [M+H]+ which is in acco dance wi h he molecula o mula C16H20O9. The NMR spec a o 3 showed he same signals de ec ed o 2 as well as signals co esponding o an addi ional ace yl g oup (Table 1). The me hyl g oup o he la e was de ec ed a δ(H) 2.12 ppm (δ(C) 20.8 ppm, CH3-2’’’) and he es e ca bonyl ca bon a δ(C) 172.9 ppm (C-1’’’). This was u he con i med by he down ield chemical shi o CH2-4’’ (δ(H) 4.20/4.37 and δ(C) 67.1 ppm) and he HMBC co ela ion obse ed o CH2-4’’ o he ca boxyla e es e ca bon C-1’’’. Compound 3 was hus iden i ied as he new na u al p oduc pes alo iopy one K (4-(ace yloxy)-2,3-dihyd oxybu yl (2E)-3-(4-me hoxy-2-oxo-2H-py an-6-yl)bu - 2-enoa e). In o de o de e mine he ela i e con igu a ion in combina ion wi h he NMR da a, con o ma ional analysis o he dias e eome ic (R,R)- and (R,S)-2 we e ca ied ou . Thei ini ial MMFF sea ch p o ided 700 and 806 con o me s, espec i ely, which we e op imized a B3LYP/6-31G(d) wi h PCM sol en model o chlo o o m o p oduce 11 and 18 con o me s, espec i ely (Figu es S1 and S2). Since he e ol moie y showed la ge con o ma ional lexibili y o bo h dias e eome s wi h e sa ile dihed al angles and hyd ogen bondings, he co ela ion o he compu ed con o me s wi h he 3JH,H da a did no allow he de e mina ion o he ela i e con igu a ion o 2. Howe e , biosyn he ic conside a ions indica e ha he bu ane-1,2,3,4- e ol uni in 2 and 3 may a ise om ei he one o he wo na u al e ols, also known as suga alcohols, h ei ol [(2R,3R)-bu ane-1,2,3,4- e ol] and e y h i ol [(2R,3S)-bu ane-1,2,3,4- e aol]. E y h i ol is op ically inac i e due o he p esence o a plane o symme y in i s s uc u e (meso o m), whe eas h ei ol is op ically ac i e.14,15 The [α]D alues o 2 and 3, measu ed in MeOH, we e –22.5 and –20.1, espec i ely, sugges ing h ei ol (–14.0 in E OH)16 as he e ol uni o 2 and 3 p o ided ha he acyla ion o he e ol uni did no in ol e an asymme ic desymme iza ion. Al hough 2 showed a