Accep ed Manusc ip
Absolu e con igu a ion and an i- umo ac i i y o o ubiellin B
Ca alina F ancis Pé ez Hemphill, Geo gios Dale os, Alexand a Hamache ,
Ma hias Ull ich Kassack, Wenhan Lin, A ila Mándi, Tibo Ku án, Pe e
P oksch
PII: S0040-4039(15)00596-1
DOI: h p://dx.doi.o g/10.1016/j. e le .2015.03.126
Re e ence: TETL 46129
To appea in: Te ahed on Le e s
Recei ed Da e: 11 Feb ua y 2015
Re ised Da e: 24 Ma ch 2015
Accep ed Da e: 27 Ma ch 2015
Please ci e his a icle as: Hemphill, C.F.P., Dale os, G., Hamache , A., Kassack, M.U., Lin, W., Mándi, A.,
Ku án, T., P oksch, P., Absolu e con igu a ion and an i- umo ac i i y o o ubiellin B, Te ahed on Le e s (2015),
doi: h p://dx.doi.o g/10.1016/j. e le .2015.03.126
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G aphical abs ac
Absolu e con igu a ion and an i- umo ac i i y o o ubiellin B
Ca alina F ancis Pé ez Hemphilla, Geo gios Dale osa, Alexand a Hamache b, Ma hias Ull ich
Kassackb, Wenhan Linc, A ila Mándid, Tibo Ku ánd, and Pe e P okscha,*
aIns i u e o Pha maceu ical Biology and Bio echnology, Hein ich Heine Uni e si y
Duesseldo , Uni e si ae ss asse 1, Geb. 26.23, 40225 Duesseldo , Ge many
bIns i u e o Pha maceu ical and Medicinal Chemis y, Hein ich Heine Uni e si y
Duesseldo , Uni e si ae ss asse 1, Geb. 26.23, 40225 Duesseldo , Ge many
cS a e Key Labo a o y o Na u al and Biomime ic D ugs, Peking Uni e si y, 100191 Beijing,
PR China
dDepa men o O ganic Chemis y, Uni e si y o Deb ecen, POB 20, 4010 Deb ecen,
Hunga y
*Co esponding au ho . Tel.: +492118114170; ax: +492118111923; email: p oksch@uni-
duesseldo .de
Abs ac
The dime ic an h acene de i a i e o ubiellin B (1) was isola ed om he endophy ic ungus
Ac emonium sp. ha had been ob ained om lea es o he Mang o e plan Sonne a ia
caseola is. The absolu e con igu a ion o 1 was es ablished as (5'R,10'S,10a'R) o he i s
ime on he basis o i s elec onic ci cula dich oism (ECD) spec a aided wi h TDDFT-ECD
calcula ions. To ubiellin B (1) exhibi ed s ong an i- umo ac i i y when es ed in i o
agains se e al solid cance cell lines including cells ha a e esis an agains he widely used
cy os a ic d ug cispla in. The IC50 alues o 1 agains cispla in sensi i e and cispla in esis an
cells we e in he ange o 0.2 - 2.6 µM depending on cell line in es iga ed.
Keywo ds: To ubiellin B, Ac emonium sp., Absolu e con igu a ion, An i- umo ac i i y
In oduc ion
Endophy ic ungi a e known as a ich sou ce o s uc u ally di e se bioac i e compounds ha
a e o en unp eceden ed in o he o ganisms.1-7 Endophy es which inhabi plan s ha a e
subjec o se e e s ess condi ions a e especially in e es ing o biop ospec ing since he ungi
a e assumed o con ibu e o he su i al o hei hos s e.g. h ough accumula ion o de ense
me aboli es.8-10 We and o he s ha e epea edly shown ha Mang o e associa ed endophy es
a e aluable sou ces o new bioac i e me aboli es such as he an i- umo me aboli e
phomoxan hone A isola ed om he Mang o e endophy e Phomopsis longicolla which was
ound o s ongly inhibi umo cells.11 Mang o es a e p one o nume ous s ess ac o s such
as changing le els o salini y, pe iodical looding and a high incidence o he bi o es and o
pa hogenic mic obes. Appa en ly hese ac o s ha e shaped no only he seconda y
me aboli es o he hos s bu also ha o hei associa ed endophy es.12,13
In he cou se o ongoing s udies on na u al p oduc s om Mang o e de i ed
endophy es we in es iga ed he endophy ic ungus Ac emonium sp. ha was isola ed om
lea es o he Mang o e plan Sonne a ia caseola is om he island o Hainan (P. R. China).
Ou in e es in his ungus had been a oused by he s ong an i- umo ac i i y o i s c ude
E OAc ex ac when es ed in i o agains solid cance cell lines including cells ha a e
highly esis an agains he widely used cy os a ic d ug cispla in (se e e g ow h inhibi ion o
A2780sens cells obse ed a 10 µg/mL, and o A2780CisR cells a 100 µg/mL). Resis ance o
cance cells ha eme ges equen ly du ing chemo he apy is a se ious p oblem o he apy o
umo pa ien s and is esponsible o he ul ima e ailu e o his he apeu ic app oach in spi e
o educ ion o umo load du ing ea ly phases o ea men .14,15 The e o e, new he apeu ics
ha a e able o b eak esis ance agains cu en ly employed cy os a ic d ugs a e u gen ly
needed.
Du ing bioassay guided ac iona ion o he ex ac o Ac emonium sp. o ubiellin B
(1)16, an asymme ic an h acene de i a i e was isola ed as he ac i e ing edien ha is
esponsible o he an i- umo ac i i y o he ungal ex ac . To ubiellin B had i s been
epo ed om To ubiella sp. BCC 28517 and he ela i e con igu a ion o he compound had
been de e mined h ough NOE expe imen s.17 Howe e , he absolu e con igu a ion o he
compound was unknown so a . The de e mina ion o he absolu e con igu a ion o
o ubiellin B (1) especially in ligh o i s p ominen ac i i y agains cance cells is an
impo an issue du ing s uc u e de e mina ion. He e we epo o he i s ime he absolu e
con igu a ion o o ubiellin B (1) oge he wi h i s p onounced an i- umo ac i i y agains
cispla in sensi i e and cispla in esis an solid cance cell lines.
Resul s and discussion
The e hyl ace a e ex ac o he endophy ic ungus Ac emonium sp. g own on solid ice
medium was submi ed o a bioassay guided ch oma og aphic sepa a ion using di e en
s a iona y phases including silica gel, Sephadex LH-20 and semip epa a i e HPLC on a C18
e e sed phase column. To ubiellin B (1) was isola ed as he majo cons i uen o he ex ac
(Figu e 1). Compound 1 exhibi ed a UV/Vis spec um wi h h ee abso p ion maxima a 206,
283, and 397 nm ha is ypical o a conjuga ed sys em. In he HRESIMS he
pseudomolecula ion was de ec ed a m/z 541.1138 [M+H]+ and he molecula o mula was
de e mined as C30H20O10. Compa ison o he MS and NMR da a o 1 wi h hose p e iously
epo ed o o ubiellin B sugges ed ha hey a e iden ical.17 The s uc u e o 1 was inally
con i med as o ubiellin B by ca e ul analysis o COSY, HMBC and ROESY co ela ions.
In e p e a ion o he COSY and HMBC co ela ions shown in Figu e 2 e ealed wo

an h acene moie ies being used ia a cyclopen ene ing. The HMBC co ela ions om H-5’
o C-4, C-3, C-10a’ and o C-11 es ablished he connec i i y o bo h monome s. The
o ma ion o his a e linkage be ween C-4 and C-5’ and be ween C-3 and C-10a’ h ough C-
11 is a s uc u al ea u e ha is inhe en o o ubiellins. The linkage o he wo an h acene
uni s was u he co obo a ed h ough co ela ions om H2-11 o H-2 and om H-11 o H-4’
in he ROESY spec um o 1. The ela i e con igu a ion o C-5’ and C-10’ was deduced om
ROESY expe imen s (Figu e 4). H-10’ showed ROE co ela ions o H-5’ and H-11eq and
u he mo e H-11eq had a co ela ion o H-2, hus indica ing he (5’S*,10’R*,10a’S*) ela i e
con igu a ion o 1 as shown in Figu e 4, which is in ag eemen wi h ha p e iously
epo ed.17
To ubiellin B may be iewed as a 4,5’-linked he e odime ic bia yl o 7-hyd oxyemodin and
dihyd o-1,8,10- ihyd oxyan h acene-9(4H)-one, in which he o a ion along he bia yl axis is
blocked by he me hylene linke be ween C-3 and C-10a. In con as o axially chi al bia yls
o an h acene-9,10-dione monome s,18 o ubiellin B has only cen al chi ali y elemen s due o
he C-5’, C-10’ and C-10a’ chi ali y cen e s and he p ojec ion angle be ween he planes o
he wo monome s is go e ned by he cen al chi ali y. The solu ion ECD spec a o
o ubiellin B showed in ense nega i e Co on e ec (CE) a 435 nm and a posi i e one a 395
nm accompanied by shoulde s and qui e a numbe o o he o e lapping high-ene gy ECD
ansi ions (Figu e 3). Fo he con igu a ional assignmen , solu ion con o ma ional analysis
and TDDFT-ECD calcula ions we e pe o med. The ini ial MMFF con o ma ional analysis o
he a bi a ily selec ed (5’S,10’R,10a’S)-1 esul ed in 13 con o me s, which we e eop imized
a B3LYP/6-31G(d) le el in acuo. The eop imiza ion a o ded 2 con o me s (96.8% and
2.9% popula ions) abo e 1% Bol zmann-popula ion, which di e ed only in he o ien a ion o
hyd ogens o he 10’-OH and 3’-Me g oups (Figu e 4). The ωC-3,C-4,C-5’,C-6’ dihed al angle, a
pa ame e desc ibing he ela i e o ien a ion o he elec ic ansi ion momen s o he wo
subuni s, was ound +103.17°. TDDFT-ECD calcula ions o he wo low-ene gy con o me s
we e ca ied ou a 3 di e en le els o heo y and he Bol zmann-a e aged spec a we e
ound mi o -image o he expe imen al ECD cu e. The PBE0/TZVP me hod showed he
bes ag eemen , he esul o which is shown in Figu e 4. This allowed de e mining he
absolu e con igu a ion o o ubiellin B (1) as (+)-(5’R,10’S,10a’R).
The pu a i e biogene ic o ma ion o 1 and o he s uc u ally ela ed 6,7-dideshyd oxy
de i a i e o ubiellin A (2) has been sugges ed o occu h ough oxida i e coupling o
emodin (5), aloe-emodin (6) o o ch ysophanol (7) on he basis o he co-occu ence o hese
monome s in he espec i e in es iga ed ungal ex ac s.17 A e ho ough UV and LCMS
analysis o he c ude e hyl ace a e ex ac o Ac emonium sp. analyzed in his s udy ypical
UV abso p ion maxima a 223, 250, 273, 293 and 442 nm, as well as a p ominen
pseudomolecula ion peak a 269.5 [M-H]- we e de ec ed ha accoun o emodin, bu no
signals we e ound o aloe-emodin o o ch ysophanol. These indings sugges ha 1 may
biogene ically a ise om wo emodin (5) moie ies h ough subsequen educ ion and
hyd oxyla ion.
Compound 1 showed p onounced cy o oxic ac i i y agains se e al human solid
cance cell lines including Cal27 (head-neck cance ), Kyse510 (esophageal squamous cell
ca cinoma), HCC38 (b eas cance ), A2780 (o a ian cance ) and MDA-MB-231 (b eas
cance ) (Table 1). The cell lines included pai s o cells ha a e ei he sensi i e (sens) o
esis an (CisR) owa ds he well known cy os a ic d ug cispla in as indica ed by he
signi ican ly di e en IC50 alues o cispla in. To ubiellin B (1) p o ed o be s ongly ac i e
agains all cell lines in es iga ed wi h IC50 alues anging om 0.3 - 1.5 µM agains he
cispla in sensi i e cells and om 0.2 - 2.6 µM agains he cispla in esis an cells, dependen
on he cell line in es iga ed. Fo all cell lines in es iga ed he ac i i y o o ubiellin B (1) was
supe io o ha o cispla in which makes compound 1 an in e es ing candida e o u he
s udies.
Expe imen al
Gene al
The op ical o a ion was de e mined wi h a Pe kinElme 241MC Pola ime e . The 1H, 13C
and 2D NMR spec a we e eco ded in DMSO-d6 on a B uke A ance III 600 NMR
spec ome e . Mass spec a we e measu ed wi h a HP110 Agilen Finnigan LCQ Deca XP
The moques and high- esolu ion mass spec ome y (HRESIMS) spec a we e eco ded
using a UHR-TOF maxis 4G mass spec ome e . ECD spec a we e eco ded on a J-810
spec opola ime e . HPLC analysis was pe o med wi h a HPLC sys em Dionex p580 coupled
o a UVD340S pho odiode a ay de ec o ; ou ine de ec ion was a 235, 254, 280, and 340 nm.
The sepa a ion column was p e illed wi h Eu osphe e-10 C18 using a linea g adien o 0.1%
aqueous HCOOH and MeOH wi h a low a e o 1mL/min. Final pu i ica ion was pe o med
ia semi-p epa a i e HPLC using a linea g adien o 0.1% aqueous TFA and MeOH o e a
Eu osphe e-100 C18 RP column (5µm; 300x8mm) wi h a low a e o 5mL/min using a
Me ck Hi achi HPLC sys em (I-7400 UV de ec o ; L-7100 pump). Column ch oma og aphy
included Silica gel 60M (Mache ey.Nagel) and Sephadex LH20 (Sigma). P ecoa ed TLC
pla es (Silica gel 60 F254) we e used o moni o ac ions; de ec ion was unde UV a 254 and
366 nm. Sol en s we e dis illed be o e use, and spec al g ade sol en s we e used o
spec oscopic measu emen s.
Compu a ional sec ion
Mixed o sional/low mode con o ma ional sea ches we e ca ied ou by means o he
Mac omodel 9.9.22319 so wa e using Me ck Molecula Fo ce Field (MMFF) wi h implici
sol en model o chlo o o m applying a 42 kJ/mol ene gy window. Geome y
eop imiza ions o he esul an con o me s [B3LYP/6-31G(d) le el in acuo] and TDDFT
calcula ions we e pe o med wi h Gaussian 0920 using a ious unc ionals (B3LYP,
BH&HLYP, PBE0) and TZVP basis se . ECD spec a we e gene a ed as he sum o
Gaussians21 wi h 2400 cm–1 hal -heigh wid h (co esponding o ca. 29 nm a 350 nm), using
dipole- eloci y compu ed o a ional s eng hs. Bol zmann dis ibu ions we e es ima ed om
he ZPVE co ec ed B3LYP/6-31G(d) ene gies. The MOLEKEL22 so wa e package was used
o isualiza ion o he esul s.
Fungal ma e ial
Ac emonium sp. was isola ed om lea es o he mang o e plan Sonne a ia caseola is,
collec ed in Oc obe 2005 in Dong Zhai Gang Mang o e Ga den, Hainan, P. R. China, and
cul i a ed in la ge-scale ollowing he espec i e p o ocol desc ibed in he li e a u e.23 The
ungal s ain could be iden i ied a e DNA ex ac ion and ampli ica ion and a e sequencing
o o s ITS egion acco ding o he li e a u e.24 The sequence da a was submi ed o GenBank,
accession numbe FR822815.1.
Ex ac ion and isola ion
A e 4 weeks o cul i a ion he ungi we e ex ac ed wi h E OAc. The d ied c ude ex ac
(1g) was submi ed o VLC sepa a ion. The las ac ion (0.1% me hanolic TFA) con aining 1
was u he sepa a ed o e a Sephadex LH-20 column a o ding F1-F10 ac ions. F ac ions 6
and 7 we e combined and pu i ied using semip epa a i e HPLC wi h an elu ing g adien o
MeOH/0.1% aqueous TFA o yield 1 (20 mg).
Bioassay
Figu e legends
Figu e 1. S uc u e o o ubiellin B (1).
Figu e 2. Key COSY (bold lines) and HMBC (a ows) co ela ions o o ubiellin B (1).
Figu e 3. Expe imen al spec um o 1 in MeCN and PBE0/TZVP-calcula ed ECD spec um o
he wo low-ene gy con o me s (> 1%) o (5’S,10’R,10a’S)-1 in acuo. Ba s ep esen he
calcula ed o a ional s eng h alues o he lowes -ene gy con o me .
Figu e 4. Key long- ange ROE co ela ions shown on he o e lapped lowes -ene gy compu ed
con o me s o (5’R,10’S,10a’R)-1.

Figu e 1. S uc u e o o ubiellin B (1).
Figu e 2. Key COSY (bold lines) and HMBC (a ows) co ela ions o o ubiellin B (1).
Figu e 3. Expe imen al spec um o 1 in MeCN and PBE0/TZVP-calcula ed ECD spec um
o he wo low-ene gy con o me s (> 1%) o (5’S,10’R,10a’S)-1 in acuo. Ba s ep esen he
calcula ed o a ional s eng h alues o he lowes -ene gy con o me .
Figu e 4. Key long- ange ROE co ela ions shown on he o e lapped low-ene gy compu ed
con o me s o (5’R,10’S,10a’R)-1.
Highligh s
• A dime ic an h acene de i a i e was isola ed om he endophy e Ac emonium sp.
• The absolu e con igu a ion o o ubiellin B was es ablished by ECD calcula ions.
• To ubiellin B showed s ong an i- umo ac i i y agains se e al cance cell lines.