➢More than 18.000 New metabolites. ➢0,1% lead structures. ➢More than 400
patents. Marine Natural Products. Therapeutical. Potential. (1) FUSETAMI N (ed),
...
Natural Products Chemistry of Marine
[email protected] Organisms
CHO H
OH
H
OH
HO H
OHOH OH O H H
H OH CH2OH
GLUCOSIO
H HO
OH H H
Emil Fisher Premio Nobel 1902
NH
O
O HO
O
O OH
O
OH
H
OH
NH
OH
NH2
discodermolide
N
N O
OMe
MeO
HO N
NH
O
HN O
N
cyclomarine
HN
HN
H
HN
NH
O
N
H
O
O
OH O
isogranulatimide
H
OCOCH3
O
O OCH3
eleutherobin
H
O
O N H3C
N
O
O O
O
H
O
NH OH
O O
O
HO
O
H
O
HN
Lamellarin I O
O
O
O
O OMe
granulatimide O
OMe MeO
N
N
O
O MeO
O
O
H
O
O
OH OH
OH
O
H
H
O H
Halichondrin B
H
Drugs from the Sea
Marine Natural Products From 1967:
(1)
¾More than 18.000 New metabolites ¾0,1% lead structures ¾More than 400 patents
Therapeutical Potential (1) FUSETAMI N (ed), DRUGS FROM THE SEA, Basel, Karger, 2000, 1-15
®
Zalypsis
OMe O O MeO
N
O
N O
OH OAc
Jorunna funebris
jorumycin
•Zalypsis® (PM00104/50) is PharmaMar's fifth new marine derived compound that has begun Phase I clinical trials for the treatment of solid tumours. •Zalypsis® is a novel chemical entity related to the marine natural compounds Jorumycin and the family of Renieramycins, obtained from molluscs and sponges, respectively. Zalypsis binds to DNA and is cytotoxic; however, it does not activate the “DNA damage checkpoint” response. Thus, Zalypsis has cytotoxic effects dependent on DNA binding that are not associated with DNA damage. •In pre-clinical trials, Zalypsis demonstrated strong in vitro and in vivo antitumoural activity in a wide variety of solid and haematological tumour cell lines and human transplantable breast, gastric, prostate and renal xenografted tumours. Zalypsis also demonstrated a manageable and reversible preclinical toxicology profile.
Transcription
DNA
Translation
RNA
Protein
Transcription
DNA
Translation
RNA
Protein
Metabolites
The metabolite pattern (metabolome) reflects the cell’s status. It is the totality of metabolic processes including anabolism and catabolism, as well as all the related cellular processes such as absorption, distribution, and detoxification of natural and xenobiotic materials, energy utilization, signal transduction, and regulation. It results from the expression of the genome and proteome in response to the cellular environment. While the Genome is representative of what might be, and the Proteome is what is expressed, it is the Metabolome that represents the current status of the cell or tissue.
Predicting Life Processes: Reverse Engineering Living Systems (storage)
DNA
Transcription Gene Expression Translation
Proteins Environment
Proteomics
Biochemical Circuitry Metabolomics
Phenotypes (Traits)
CNR - Institute of Biomolecular Chemistry
Guido Cimino
Margherita Gavagnin Emiliano Manzo Maria Letizia Ciavatta Marianna Carbone (PhD Student) Guido Villani Ernesto Mollo
Adele Cutignano Giuliana d’Ippolito (PhD student) Antonio Maiello Angelo Fontana Consiglio Nazionale delle Ricerche
Memorie storiche sugli opistobranchi 1890
Il prof. Herdman pose alcuni esemplari vivi di Ancula cristata sulla lingua del suo assistente dr. Moore
1960
T.E. Thompson J. Mar. Biol. U.K. 39, 115 a) b)
mollusc with shell mollusc without shell
c) opisthobranch
1976
J. Ros Oecologia aquatica 3, 153 "bad taste of the nudibranchs" "ignorance of the chemical mechanisms"
spugne cnidari
briozoi
alghe
tunicati
- bio-accumulation - bio-transformation - bio-synthesis
Multifunctional metabolites in Dendrodoris limbata AcO
O
CHO CHO
OAc
olepupuane AcO
O
polygodial R C O O
O
7
7-deacetoxy-olepupuane anche da una spugna del genere Dysidea Garson et al. J. Nat. Prod.. 55, 364, 1992
Experientia 47, 56, 1991
Ecological activity: aposematic colours of marine opisthobranchs
Ecologica activity: aposematic colours in marine opisthobranchs Thalassoma Thalassomapavo pavo 10 10esemplari esemplari
25 modelli
a) non aposematici b) aposematici con tossine c) aposematici senza tossine
O
Hypselodoris
+ furodysinin
% 100
a
b
c
a
b
c
attacca rifiuta 1° giorno
12° giorno
T. pavo remembers the external features of ptentially dangerous preys
Dietary origin of bioactive molecules Alicante
SPAIN Mar Menor
gg.
Hypselodoris webbi (nudibranco)
O O
longifolin O
Dysidea fragilis (spugna)
(-)-furodysinin longifolin
0
0 (pura longifolin)
7
3,8
35
13,0
(-)-furodysinin
MDF
Experientia 50, 510, 1994
Bio-transformation of dietary metabolites
MDF 0.4 mm
C. mollior AcO
CHO C HO
AcO
HO
17
H H
H. orsini MDF
H. orsini O
AcO
HO
H
H
H
H H
6
O
H
H
H
H
O Experientia 49, 482, 1993
Biosynthesis in Tethys fimbria
cerata
Mucus, mantle, cerata O
O O 15
HO
17
1
OH
O
15
HO
defense defense
1
COOH
17
muscle muscle contraction contraction PGE-lattone
PGE
HO 11
O
O 15
C H3C O
17
1
O
O 9 11
PGF-lattone
HO
O 15 17
mantle PGF-lattone
ovotestis, eggs
1
O
Biosynthesis
the way in which a natural product is produced by in a living organism O HO
acetate
G U A C C G U G A G
DNA
OH O O
O
OH
O
O
O
O
O
O O
HO O
O
O
O
O
O
HO
HO
O OH O
O OH
O
OH
O
O
HO
NaO
HO
S
O O
HO
OH
OH O
O
OH
O
OH HO
O O
O
ONa OH
OH O OH
O HO
S
HO
O
O HO
O HO
O
O
O HO
HO HO HO
HO
Biosynthetic Capacity and Chemical Markers order
Cephalaspidea
chemical class
chemicals
ω-aryl-methylchetones
navenone-B
O
Sacoglossa
polypropionates
O
O
OCH3
O
elysione O
acetogenins
O HO
Nudibranchia
O
PGE2- 1,15-lactone
terpenes
OHC O
luteone
Biosynthetic Capacity and Evolutionary Scenario polypropionates
C. cristallina P. dendritica C. mediterranea E. funerea E. timida
polypropionates
T. hopei E. viridis diterpenoids
B. mimetica
E. translucens
O. olivacea sesquiterpenoids L. serradifalci A. fragilis Cimino & Ghiselin, Chemoecology, 8, 51, 1998
Isolation and structure elucidation •Faulkner DJ Stallard MO Fayos J Clardy J 1973 A novel monoterpene from the sea hare; Aplysia californica. J. Am. Chem. Soc. 95 3413 •Kato Y Scheuer PJ 1974 Aplysiatoxin and debromoaplysiatoxin; constituents of the marine mollusk Stylocheilus longicauda. J. Am. Chem. Soc. 96 2245
Chemical ecology Scheuer PJ 1977 Chemical communication of marine invertebrates. Bioscience 27 664 •Kinnel RB Dieter RK Meinwald J Van Engen D Clardy J Eisner T Stallard MO Fenical W 1979 Brasilenyne and cis-dihydrorhodophytin; antifeedant mediumring haloethers from a sea hare (Aplysia brasiliana) Proc. Natl. Acad. Sci. U. S. A. 76 3576
Biosynthetic studies •Ireland CM Scheuer PJ 1979 Photosynthetic marine mollusks: in vivo 14C incorporation into metabolites of the sacoglossan Placobranchus ocellatus Science (Washington DC) 205 922 •Sleeper HL Fenical W 1977 Navenones A-C: trail-breaking alarm pheromones from the marine opisthobranch Navanax inermis J. Am. Chem. Soc. 99 2367
Biosynthesis of Secondary Metabolites in Opisthobranchs Phylum
Mollusca
Class
Gastropoda Opisthobranchia
Subclass
Order
Gymnosomata Acochlidea Thecosomata Rhodopemorpha Cephalaspidea Sacoglossa Navanax inermis Haminoea cymbalum Bulla striata
Anaspidea
Placobranchus ocellatus Elysia viridis Cyerce cristallina Ercolania funerea Oxynoe viridis
Notaspidea Nudibranchia
Biotransformation of Caulerpenyne? mucus Oxynoe olivacea
Caulerpa prolifera AcO
10 10 ppm ppm
AcO
AcO
13
13
11 ppm ppm H
caulerpenyne
1
OAc
AcO
0.1 0.1 ppm ppm CHO
H
2
CHO CHO
1
oxytoxin-1
1
13
oxytoxin-2
toxicity toxicity to to G. G. affinis affinis Experientia 46, 767, 1990
Could the Direct Observations be Misleading? • selective bio-accumulation of minor dietary metabolites Pietra Pietra et et al., al., Helv. Helv. Chim. Chim. Acta Acta 78, 78, 1759, 1759, 1995 1995
• enzymatic structural modifications in the injuried tissues of the prey Pohnert Pohnert and and Jung, Jung, Org. Org. Lett. Lett. 26, 26, 509, 509, 2003 2003
Addition of Caulerpenyne to Oxynoe homogenate caulerpenyne
OAc 1 13
AcO H OAc
preoxytoxin-2 POXY-2
15’’
Oxynoe homogenate
OAc
CHO OXY-2
CHO CHO
15’ POXY-2 OXY-2
oxytoxin-2
Org. Org. Biomol. Biomol. Chem. Chem. 2004, 2004, 2, 2, 001 001 -- 005 005
Wound-activated Hydrolysis in Caulerpa prolifera OAc 1
CYN
13
AcO H OAc
15’’
after grinding of the alga
CYN
OXY-1
OXY-2
POXY-2
15’ OXY-2
CYN POXY-2
Selective Lipases are Active in Oxynoe olivacea OAc
CHO
1 13
LIP -2
AcO
AcO H OAc
H OAc
oxytoxin-1
caulerpenyne LIP -1
LIP -1
OAc 1
LIP -2
CHO
CHO CHO
13
preoxytoxin-2
oxytoxin-2 Org. Biomol. Chem., 2004, 2, 001-005
Metabolite Compartmentalization in Cephalaspideans Les glandes palléales de défense chez le Scaphander lignarius Perrier and Fisher Séanc. Ac. Sci. Paris 146, 1163, 1908
OH
N
3-alkyl-pyridines
Haminoea fusari
O
O O OH
polypropionates
Bulla striata
O
ω-aryl-methyl-chetones Scaphander lignarius
6 CoA-SH O 6x
O
SCoA
CO2H
o
N
2
11
1
CoA N O
N
11
2
1
CO2
Assembly of haminol on Haminoea’s modular enzyme KR ER KS
KS DH O
PKS
KR
KR
10
O
PKS
O
PKS
KR ER O
TE
PKS
O
PKS
O
AcO
2
2
O
CO2H O
O 10
N
2 x KS DH
KS DH
PKS
O
DH KS
10
N
release N
O N
10 10
10
10
N N
N
N
haminol-2
2
H3C CH2
H C HO O
C C H2N C
O SH
ACP
C S
C
PCP A
H CH3
H R SH
SH
KS
ACP AT
KR
H3C CH2
H C HO O
C C H2N C
O SH
ACP
C A
C
H CH3
H R
SH
S
SH
PCP
KS
ACP AT
KR
H3C CH2
H C HO O
C C
H CH3 O
H2N C O SH
ACP
C A
C
O
H
C C
R
O
C
SH
S
S
PCP
KS
ACP AT
KR
H CH3 H
H3C CH2
H C HO O
C C H2N C
O
C
H CH3
H R
O O
C C
O
SH
ACP
C A
C
SH
S
S
PCP
KS
ACP AT
KR
CH3 H
Biosynthesis in Bulla striata
9µCi [1-14C]-propionic acid 3 specimens – 3 days 3 chromatographic steps
O
O
O
O O
aglajne-1 520 cpm/mg
O OH
aglajne-3 394 cpm/mg Tetrahedron Lett 45, 6847, 2004
Bulla O
O
O
release decarboxylation
O O O
PKS
O
O
PKS
O
PKS
O O
PKS
O
2 cycles KR ER
KR KS
KS DH
PKS
KR
ER
DH
O
KS
KS DH
KS
KR
DH
2x
KS
2x
KS
3 cycles KS
O
KR ER
KR
KR KS
DH
KS DH
PKS
O
KR
PKS
KS
KR
KS
DH
O
PKS
O
DH
PKS
O
PKS
O
O HO
O
O
release
HO
Smaragdinella
decarboxylation
HO
HO
HO
Current Org. Chem., 2005, in press
Metabolites from: Mediterranean Pleurobranchoidea
Intertidal Molluscs O
O
O
O
Pleurobranchus membranaceous
O
Tetrah. Lett.
O
O
O
Siphonaria maura Manker and Faulkner J. Org. Chem. 54, 5374, 1989
34, 6791, 1993
membrenone-C
vallartanone-B
CHO
OH
13
Pleurobranchaea meckelii
Trimusculus reticulatus Rice J. Exp. Mar. Biol. 93, 83, 1985
OH
Tetrahedron 36, 8673, 1995
8
OH O 8β-hydroxy- labd-13E-en-15-al
Pleurobranchus testudinarius
H
O
O
O Lottia limatula (limpet) Albizati et al. J. Org. Chem. 50, 3428, 1985
H O
OH
OH
HO testudinariol-A
HO
O
limatulone 6
Biosynthetic studies of Nudibranchs Nudibranchia
Order:
Doridina
Suborder:
Superfamily:
Family:
Arminina
Dendronotina
Anadoridoidea
Eudoridoidea
Dorididae Chromodorididae Dendrodorididae
Sclerodoris tanya Diaulula sandigensis Cadlina Doris verrucosa luteomarginata Archidoris montereyensis Archidoris odhneri Austrodoris keguelenensis
Aeolidina
Dendrodoris limbata Dendrodoris grandiflora Dendrodoris arborescens Doriopsilla areolata
Onchidorididae
Triopha catalinae Acanthodoris nanaimoensis
Tethydidae
Tethys fimbria Melibe leonina
Doriopsilla areolata biosynthetizes typical sponge sesquiterpenoids with opposite A/B ring junction AcylO O
A
OH
H
drimane esters
th n CO2- osy i b o v o n mevalonate OH
B
de
s esi
O
A
B H
(-)-pallescensin-A Tetrahedron 57: 8913, 2001 J Org Chem 68, 2405, 2003
lateral gene transfer (Faulkner) retrosynthetic mode (Ghiselin)
Purification of the enzyme is currently planned in order to address the molecular aspects of the process in the nudibranch (and in the sponge)
O
Experientia 49, 268, 1993 Tetrahedron 21, 797, 1980; Tetrahedron 25, 11, 1984;
O O O
OH O
OH
OAc
H
O
OH
O
OH
Archidoris tubercolata
Tetrahedron Lett. 11, 6093, 1990; Tetrahedron 53, 797, 1491;
O
Doris verrucosa Archidoris odhneri A. montereyensis Doris freterae Doris fontaini Austrodoris kerguelenensis
OAc 15 O
OAc O O Experientia 49, 268, 1993
O 3'
OH
O O 3'
S
1'
OH
1'
OAc OH
Tetrahedron 47, 9743, 1991; Tetrahedron 59, 5579, 2003
before
after OAc
H
O
OH
O
Hydra vulgaris
Terpenoid acyglycerols are feeding-deterrent and ichthyotoxic. Like the classical diacyglycerols, these compounds are activators of protein kinase C and, in vivo, show morphogenic effects Experientia, 52, 874, 1996
Glycerol Origin in Verrucosins
OAc
[6 -1313C] -glucose [6C]-glucose
O O
C-2’
C-3’
C-1’
OH
C-1’ C-3’
Natural intensity of glycerol 13C signals
Eur J Org Chem 16, 3104, 2003
Experiment with Antarctic Austrodoris kerguelenensis
H HO H H
CHO OH H OH OH OH
CHO H OH OP
OH O OP
[6-13C]-glucose 45 mg/specimen
O O
3'
1'
OAc
OH labelling position to determine
HO
OR H OP Unpublished results
Experiment with Antarctic Austrodoris kerguelenensis Despite the de novo biosynthesis, specimens of the same population show different metabolites
O O 3'
OAc
OAc
1'
OAc OH
15 O
O 3'
S
15 O
1'
O 3'
OH
S
1'
OH
Such a diversity is analogous to that described in a restricted group of terrestrial plants (e.g., Polyathia longifolia), thus suggesting a similar enzymatic arsenal Tetrahedron, 59, 5579, 2003
Opisthobranchs possess the ability to produce al large number of unique secondary metabolites, some of which (e.g. 3-alkylpyridine and polypropionates) are unlike those found in any terrestrial species. ; Our knowledge of the biosynthetic processes leading to these products is still in its infancy – only a few genera have been investigated; The tremendous progress of genetic and genomic techniques is expected to bring a crucial contribution, although these studies alone will not provide all the answers since the expression of a gene may have no definitive relationship to the ultimate production of the metabolites. This emphasizes the need for traditional biosynthetic work as prerequisite for the full exploration of the biochemical aspects of the secondary metabolism in these organisms; Characterization of enzymes involved in synthesis of ecological mediators is likely the next step for the comprehension of secondary metabolic pathways and ecological interactions (Biochemical Ecology) in marine organisms, either betnhic and planktonic;
