Proliferin, a new sesterterpene from Fusarium proliferatum

Tetrahedron Vol. 49.

No. 41. Pp. 10883-10896.1993 Printedin Great Britain

004OdO20/93 $6.00&O 0 1993 Perpmon Press L

Proliferin,

Giacomino

a New

Sesterterpene

from

Randazzo,a*, Vincenzo Foglianoa, Albert0 Ritienia, Luisa Manninab Enrico Rossin, Angela ScaralloC and Anna Laura Segred.

aistituto

di Industrie

Agrarie dell’Universitd

II”, Parco Gussone, 80055 Roma, cCO.E.PO.;

Portia,

degli Studi di NapoIi “Federico

Napoli, Italy; k.N.R.

Area della Ricerca di

dlstituto di Strutturistica Chimica C .N .R ., Roma.

(Received in UK 30 July 1993; accepted 17 September 1993)

A new

ABSTRACT: characterized activity.

The

data from

from

m

structure 2D-NMR

bicyclic

sesterterpene,

orollferalum. elucidation strategies.

of

the

Prohferin

named

proliferin,

The pure compound molecula was

shown

was

has

been

exhibit

toxic

accomplished

to have

a novel

using ring

skeleton and molecular formula C27H4005

INTRODUCTION Our laboratory has a continuing interest in the chemistry of mycotoxin produced by fungi. In this paper we report the structure determination of a new sesterterpene isolated from Fusarium oroliferatum (Matsushima) Nirenberg, a member of the Liseola Woll. section, that occurs worldwide as a pathogen on shows toxic activity on -ia Salru L. host plantsl92. The new compound This fungal strain is known to produce other toxin3*4*5 whose chemical bioassay. nature is quite different from terpenes. The usual spectral methodologies (UV. IR, MS, and 1D 1H and 1% NMR) do not allow one to obtain an univocal structure, so the molecular structure is achieved by the new generation of 2D-NMR spectroscopy; in particular the inv4lplrnd sequence has proved to be a very helpful tool to gather the microstructure.

10883

G. Rwxzo

et al.

RESULTS AND DISCUSSION . F. is a fungus that makes maize ear rot. It was recently isolated and identified in northern Italy. Methanolic extract of P. ~tollfetatum. grown on autoclavated corn kernels, was highly toxic on brine shrimp bioassay, causing 100% of mortality of larvae within 48 h. fn the water phase of colture, fumonisin was found4, while two different toxic compounds were isolated in methanolic extract. The former was identified as beauvericins, while the latter, rather hydrophobic, was purified as described in experimental section obtaining the pure compound.

A

Figure 1. Bottom A: 50.3 MHz, (H) Waltz broad band decoupled, t3C NMR spectrum of proliferin. Top B: the DEFT 135’ is shown; in the small insert, the CH resonance at 76.51. The CDC13 is zeroed by the DEPT sequence.

Proliferin. a new sesterterpene

A preliminary characterization was achieved using traditional spectroscopic methods (MS, UV, JR) togheter with preliminary NMR spectra run on a Bruker AC200 spectrometer. t3C { IH) spectrum Waltz broad band decoupled (Fig. 1A)e and 135’ DEPT edited (Fig.lB)T showed the presence of 6 CH3 groups, 7 CH2 groups, 6 CH groups and 8 non-bearing protons C atoms. The 1H spectrum, see Fig. 2, showed the presence of one OH group at 5.56 ppm and a sharp signal at 2.02 ppm (3H) that well account for the presence of an acetyl group. This group was also confirmed by IR (vruax 1728 cm-t), t3C spectrum (peak at 171.91 ppm) and MS that gave M+-60 signal. Moreover t3C gave a peak at 207.86 ppm due to a keto group, Vmax 1708 cm-*. Data obtained up to now clearly have proved the presence of 27 C, 39 H and 4 0. The reasonable ipothesis of another OH group arose to account for the MS data that gave M+ 444 m/z. The presence of two OH groups was confirmed by MS of acetylated derivative (m/z 528; M+ + 2CHjCO). Thus, the molecular formula was C27H4005; it suggested the occurence of 8 unsaturation: in fact two C=O groups were present, and 1H and *3C spectra suggested the presence of four double bounds (one, detectable only in the t3C spectra, must be fully substituted); thus considering the observed molecular formula the molecula must have two rings. The assignment of proton signals between 1.65 ppm and 2.40 ppm was quite of six CH2 with non-equivalent proton hard as the presence in this region maked the tH spectrum too overlapped. That is why, a Bruker AMX600 spectrometer was used for the following experiments.

Figure

2. 600.13 MHz, tH NMR spectrum of proliferin.

10885

10886

G.

RANDAZZO et al.

To get the complete microstructure it was necessary to perform 2D-NMR experiments. tH-tH COSY8, not shown, allows one to identify five different spin systems, which were confirmed by TOCSYo spectrum, Fig. 3. Five independent spin systems, Fig. 4, were outlined as follows: (in parenthesis the final assignment is shown) 1,70 (1’); 2.38 (1”); $24 (2’); 1,64 (20). 1) ABC X3 : 2) ABCD XY3: 2,30(4’); 2,01(4”); 2,30(5’); 2,13(5”); 5,12(6’); 1,64(21). 3)ABCDX: 2,11(8’); 1.78 (8”); 1,77 (9’); 1,68 (9”); 4,05(10’). 2,67(14’); 2,40(13’);1,92(13”); 5,38(U); I,56 (22) 4) ABC XY3: 5) ABC X3: 2.78 (19’); 4.28 (24’); 4,25 (24”); I,31 (25)

I

I

(0

Q

5

PPm

mm-

5

DPB

Figure

3.

4

3

2

600.13 MHz, IH-IH TOCSY map of proliferin.

1

Proliferin, a new sesterterpene

10887

I9

iBHi /l”\c$H ,C

1

H

m

CH3

lc13 “i,, /pcdll 14

4

Figure

4.

Sketch

of

5

/

$2

spin

\

/z 19”\

system

obtained

from

tH-IH

COSY

H

and

tH-1H

TOCSY. The

t3C

resonances,

the not shown

corresponding

tH-r3C

correlated

to the five

map, obtained

heteronuclear

shift

correlation

with

pulse10~1t712;

thus

all protons

bearing

in Table

worth

number assignment

and

of

and

the

assign

and

well

in

assigned

be found

in

i.e. a 2D

Fl,

using

and

were

C-15.

quaternary

C atoms

quaternary that,

peak

notwithstanding

a bird reported

of the proton

groups,

the peak

assignment of

the

detection

carbon

2D cross spectrum,

is

easier;

CH2

triplet,

in

fact

the is

to

the

able note

to that

J filter

connections

does

cross

was

It is also worth

a low-pass

bond

which, of a projection.

relative

systems

heteronuclear was

to the

to clarify For each peak

(J

can be

not correspond

but it corresponds

equal

the

group

experiment

neighbour;

spectrum,

5 instead

which

peaks

full

about spin

via

6 Hzt5.

and

a

keto

sequence

o third

was only

of decoupling

undecoupled of Fig.

user

second

the

different

inv4lplrnd

5. The

of

anything

bond,

the

with two o three

shift of some

decoupled

know

double an

Fig.

the absence

The chemical

not

suggestions

due to the high

necessity

in reverse

proton

150 Hz), CH groups

literature

the

to connect

in the experiment

of the component phase” assignment, is reported in Fl central

see

to any

used

we did

resonances,

correlation

C atoms

atoms,

substituted

In order

coherence,

constant

assumed

observed.

full

matches

for our molecula,

C

In fact,

the

quaternary

i.e. a 2D lH-33C

the J coupling one bond

i.e.

strictly

13; however

quaternary

arose.

groups:

doublequantum

connect noting

were

can

sequence,

*H-tH decoupling C atoms

approach

and

C spectrum

three

perfomedlb, zero

this

structures

unsaturation

of

to

that

unknown

of

position

any

noting

unraveling

(C-16)

systems,

I.

It is for

spin

with hxcobicp

matches

correspondent

to “in the CH2 the fully

10888

G. ILumm

et al.

Table I 1H-and 13C NMR data

cnr I vpc I 1

cH2

114@pm)

13C(ppm)

I

I

I 39.14

JO

I I-w,70 H”-238

2

CH

3

C

4

CH2

121,38

$24

1382 4w3

H’=2#30 w-2,01

5

CH2

H’q30 H”=2,13

6

CH

7

C

13293

8

CH2

34,93

124,31

5,12 x-r-2,11 H”S1.78

9

CH2

29‘72

H-l,77 H”=1#68

10

CH

11

C

12

a-i

13

a-l2

76,Sl

9’-lo’: 9”_1(y*’

co5

136,54 128889 28n

53 l-r-2&l I-r-1 I92

w-12’= W-P-6 13-w=:

14

CH

49,56

15

C

49,Ol

2,667

14’-u-2 w-w-11

16

C

207,86

17

C

147,27

18

C

146,71

19

CH

33.71

20

CH3

E&55

W

21

CH3

15.32

1.64

2078

22

CH3

1438

1,56

23

CH3

16,19

W9

24

u-u

66‘43

I-I+28

24’-19’=7,

H”=q?5

24”-w-6, 24’-24”=10,

25

CH3

l/52

26

C

170.91

27

CH3

20,91

I 1

OH (17) OH (lO,l

1s 202 5.56 1,611

10889

Proliferin, a new sesterterpene

-

-

* 4

=

4 -

-

-

-

= -

-

. . . .

r) .

.

,..~.,‘~.~I.‘~~“~~~,~“‘~“~~,.‘~...”.I~”~~~’~’I”~’ 4 5 DP.

0

2

1

Figure 5. 2D tH-1% correlation via heteronuclear zero and double quantum coherence optimized on long range couplings reverse detection, no decoupling during acquisition. Instead than Fl projection a *3C undecoupled is reported, and in top of this a fully decoupled spectrum is also reported. The proton frequency is 600.13 MHz.

10890

G. RANDAZZO et al.

decoupled spectrum, which is reported in top of the fully coupled carbon spectrum, both obtained at 150.92 MHz. In this way, it was possible to connect all previously mentioned spin systems (Fig. 6). Anyway the main goal of this experiment was to assign the position of non-bearing protons C atoms. As shown in Fig. 6 the H-14’ plays an essential role, being the only proton on the five membered ring. This proton gave a very helpful contact with C-15, C-17 and C18.

Figure 6. Sketch of structure of proliferin shown the main between the five spin systems obtained from the 2D map of Fig. 5.

connection

It is important to observe that each CH3 group gave contact with the two second neighbours allowing to link the spin systems and to position the C=O group near the quaternary carbon bearing the CH3-23. The structure of the five membered ring was also in full agreement with UV (h,,, nm 261 &= 6000)16 and IR data (=C(OH)-C=O) 1663 cm-t 17. From all this data a chemical structure can be proposed, see Fig. 8. A NOESY experimentts, Fig. 7, fully confirms previous findings. Three double bonds are present in a fifteen membered ring. Noesy 2D map clearly shows that in the double bonds C(2)=C(3) and C(l l)=C(12) the CH3 and the proton are in a E

Proliferin. a new sesterterpene

configuration (cross peak 5.24-1.64 ppm and 5.38-1.56 ppm respectively), while in the double bond C(6)=C(7) (cross peak 5.12-1.64 ppm) the CH3 and the proton are in a Z configuration. Full interpration of NOESY data is under progress, in order to gain the relative The results of all 2-D configuration of chiral C atoms, distances and angles. experiments are summarized in Table II. We proposed for this compound the trivial name of proliferin. As it was units this molecula is a sesterterpene. possible to identify five isoprenic However proliferin is an unusual sesterterpene, since its skeleton ring is definitively new. Further studies are in progress to clarify the biosynthetic pathway and the absolute configuration of the four chiral centers.

Figure

7.

600.13 MHz NOESY map, contact time 300 ms.

10891

10892

G.

RANDAZZO et al.

Table II tH and 1% 2D correlation

Cnr. H I

2

mv4LPLRND

l-r

1”;:

l-r

1’;:

H

1’;1’; (2C

H

4yy

H H”

6

2 2(

H

2o;t

I’; 5’;5 5%4b4%( I I ,

9

10

14’;IT; 23;4”;t

2’;t 21

5’;4; 4”;6; 2

5’; s’(21

4’;4: 2’

H’

8”; 9’; 9”;2

1c

H”

8’; 9’; 9

1C

H

9”; 8”; 8’; l(

H”

10’;9’; r;a

H

21

2’;5’;20

2:

5’;5”;I’; 4”;8”;9’;21 23; 10’;2’; 12’;4”; 8’; ! 5’;5”;8”;.;y;g

6’; l(

lo’; 9’; 9

23; 22 lo’; 8

23; t I

9’; 9

8’; 8”

9’; 9”; 2i

12’; 22; 6’; t

13’;13’:9’; 9”%“;2;

11 12

H

13

H H”

14

1’; 1’;4’; 20; 13

20;t

7

8

14

20; 1’;1”;I’; 4”

H” 5

NOESY

2’; 14

3 4

I

H’

13’; w;2:

14

13’;12’;14 13’; 13

lo’; 11’;13’; 2s

10’;14’;23;2’;22; t

14

13”; 12’;I#; 2:

19’;25;23;2 23; 2.

z 12’; 2:

1’; 13’;13”;23; 19 12y; 22;21;19’;1’;23% 23;19’;14’;13’;13”;1”;1

15 16

23; 1’

17

5,56(OH) 24’;14

18

5,56(OH) 24’;14

24”; 15”;2!

21”. , 19-r , H 3H

i

I’; 4

2

21

3H

8’; 5

5’; I’; I”

&Y;9

22

3H

13’; 12

1l’;lY

23

3H

24 25 26

13’;II’; 21

24’;21:14’;2t

24’. , 24”. I 2’-

19 20

10 1”. I 1

H

19’; 24

2!

H”

19’; 24

2!

3H

IS

2#; 2l’

19’; 2! 24’; 24”;19 24’; PI”; 2;

4’; ! 14 13”;13’;14’; 8”;lo’; Ii 1”.~2bl3.L , ,I’.I 8

,

l

2’; t 2 2! 13’; 24’; 24

10893

Proliferin, a new sesterterpene

Figure

8.

Sketch of structure of proliferin. The configuration is assigned with a noesy experiment, see Fig 7. EXPERIMENTAL

of double bond

SECTION

Proton and Carbon NMR spectra were run in CDC13 (5 mg/ml) on a Bruker AM200 and on a Bruker AMX600 spectrometers operating at 200.13 and 50.33 and 600.13 and 150.92 MHz respectively. Literature pulse sequences were used for the ID and 2D experiments: lH-tH COSY: 512x512 data matrix size; time domain 512 in Fl and 1024 in F2; relaxation delay (rd)= 2s; number of scans (ns)=8; dummy scans (ds)= 4; lH-1H TOCSY: data matrix size 512x512; time domain 512 in Fl and 1024 in F2; rd= 3s; ns= 8; ds= 8; mixing time 87 ms. NOESY: data matrix size 512x512; time domain 512 in Fl and 1024 in F2; rd= 2s; ns= 40; ds= 32, contact time 0.3 s. Inv4lplrnd: 512x512 matrix size, time domain in Fl JK, in F2 512; ds= 4, ns= 96, rd=ls, low pass filter 0.0033s. delay for evolution 0.1s; hxcobicp same as inv4lplrnd with delay corresponding to a J=l50 Hz. Low resolution electronic impact mass spectrometry data were obtained by TRIO 2000 Fisons at 70 eV, 400 jtA and source temperature 200°C. All solvents were spectral grade for spectroscopy. The Mps was uncorrected and was perfomed using a Gallenkamp Melting Point Apparatus; the IR spectra was recorded on a Perkin-Elmer 399 instrument in CHC13 solution; UV spectra was

G.RANLXZOet al.

10894

measured optical and

on

a spectrophometer

rotatory

was

preparative

20

cm

TLC

thickness

F254,

northern

di

and

the

Tossine

e

identification Fusarium

was

Isolation material

NaCl

was lipidic

on v/v)

by

inactive

fraction.

25 mm id.) TLC,

The

omogenus

bioassayed

for

residue

80.7

of

their

CHCl3-MeOH(955 the

yielded

plates,

Fractions

MeOH

was

collection

of

Bari.

The

combined,

three

uv

times

detected

gave

g,

were

and

iodine and

a crude

run

at 254

was

(350

vacuum

TLC

vacuum.

times

fractions light

activity

to

residue

under

mycotoxic

the

water

three

The

The

by

under

and the

to SiOz column

dried

was

filtered

extracted mg).

by preparative

dried

with L g of fresh

Then

for

solvent.

visualized

band

in

1,2 1 of methanolwas

ml)

(426

the same

and

rot

out

nm,

with

scraped

This

procedure

of pure

compound

(MeOH

c=O.255).

product. n-hexane

was obtained

NMR spetra

are described

1708 (C=O ketone),

to a solution

acetyl-proliferin (500 pl)

acetate

“C; [a]~

and discussion.

1663 (=C(OH)-C=O).

Ac20

in ethyl

with mp 142-147

in the results

The with

the

pressure.

phase

residue

purified

air dried

ear

(ITEM),

with

(250

showed

fluorescent

Acetyl-proliferin:

corn

at

suspension

reduced

and

were

The

solid

(5 mg)

with

v/v).

Adding

1494

v/v) was applied

eluent,

further

an amorfous

proliferin

(95-5

was

48 mg of pure

Proliferin:

oily

that

by

infected

extracted

aqueous

an

fractions

extracted

after

who deposited the strain in as accession number M-7308.

n-hexane

The

same

activity.

mg

(Whatman

visualized

vegetali

The

under

and eluted

in the

and

with

obtaining

100 cm high,

number

for 48 hours.

distillation

extracted

ml),

F254, 20 x

Rp18

vapours.

parassiti

Nelson Park, USA

The

Analytical

plates

phase

from

P.E.

solution.

Proliferin was product by inoculation autoclaved yellow corn kernels. 250

in 1 ml of CHCl3-iPrOH by

with

in a Waring-blender

monitored

ester),

by

gel, Merck

were

isolated

MeOH polarimeter.

reversed

by iodine

da

in

141

the spots

deposited

was

(250

and

exposition

University

(55:45

ml)

CHzCl2

vapours.

mm);

Micotossine

removed

(500

dissolved

mm)

was

strain

Center,

grinded

1% aqueous methanol separate

and/or

Procedures: ITEM 1494

was

residue

0,25

930

on SiOz (Silica

05

confirmed

Research

proliferatum

from

and

Uvikon

a Perkin-Elmer

F Droliferatum

Italy

Istituto

on

perfomed

mm

to uv light

Identification:

with

were

0,25

20 x 20 cm thickness

by exposure

Kontron

measured

h,,, was

and Pyridine

-35”

IR v max cm-*:

1728 (C=O

nm 261 (E= 6000) obtained

by

(500 ~1) at room

reaction temperature

of

10895

Proliferin, a new sesterterpene

for

12 hours.

pressure (MeOH

After

with

addition

the

formation

c=O,135).

(=C(OH)-C=O).

IR h,,,

of cold

of azeotropic

v max cm-t:

also

“Chimica

1728

the mixture moisture

(C=O

Fine

This II”; NMR

research

service

was dried

under

benzene/pyridine:

ester),

nm 228 (&= 6500) and h,,,

ACKNOWLEDGMENTS: program

MeOH,

1708

(C=O

reduced

[a]~

-44O

ketone),

1663

nm 207 (E= 7200).

was

supported

by

C.N.R.

of the Area of the research

special

of C.N.R.

acknowledged.

REFERENCES

AND NOTES

1)

Nirenberg, H.; “Mitt. Biol. Dahlem, 1976,169, 1.

Bundesanst”

2)

Gerlach, W. and Nirenberg, Forstwirtsch; Berlin-Dahlem,

H.; “Mitt. Biol. Bundesanst” 1982, 209, 1.

3)

Marasas, W.F.O.; Thiel, P.G; Rabie, C.J.; Nelson, P.E. and Toussoun, Mycologla; 1986, 78, 242.

4)

Ross, P.F.; Nelson, P.E.; Richard, J.L.; Osweiler, J.F.; Rice, L.G.; Plattner R.D. and Wilson T.M.; Appl. Environ. Microbial.; 1990,56, 3225.

5)

Moretti, A.; Logrieco, A.; Bottalico, A.; Ritieni, A.; Randazzo, G.; Abs. of 6th International Congress of Plant Pathology, Montreal, 1993, 442.

6)

Neuhas

7)

Morris, G.A. “Topics vol. 4.

8)

Jeener, J.; Yugoslavia

9)

Bax, A. and

10)

Bax, A; J. Magn. Res. 1983,53,

11)

Rutar,

V.; J

12)

Wilde,

J.A. and Bolton,

13)

Crews,

P.; Jimenez,

,D.; Keeler,

Ampere 1971.

Land-Forstwirtsch;

Land-

R.; J. Magtz. Res. 1985,61,

J.; Freeman,

in 1% NMR Spectroscopy”

International

Summer

School,

Davis, D.G.; J. Magn Res. 1985.65,

Wiley,

T.A.;

553.

New York 1984;

Basko

Polje,

355.

517.

Magtt. Res. 1984,58, P.H.;

Berlin-

306.

J. Mapn. Res. 1984,59,

C.; O’Neil-Johnson,

343.

M.; Tetrahedron,

1991.47,

585.

is

10896

G. RAND-

er al.

14)

Bax, A and Summers, M.F.; J. Am. Chem. Sot., 1986,108,

2093.

1%

Stothers, J.B. in “Carbon-13 NMR Spectroscopy”; Acad. Press, New 1972, 348.

16)

Silverstein, R.M.; Bassler C.G; and Morril C.T. in” Spectrometric identification of organic compounds”; Wiley & Sons, New York, 1981, Fourth Edition

17)

Nakanishi, K. in “Infrared Absorption Spectroscopy”; Francisco, 1962, 17.

18)

Ernst R.R.; Bodenhausen and Wokaun A. in “Principles of Nuclear Magnetic Resonance in One and Two Dimensions”; Oxford Sci., New York, 1987

Holden-Day,

York,

San