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
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G aphical Abs ac
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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]
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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
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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.

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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,
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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.
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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.
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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