new potent fluorescent analogues of strigolactones ...new potent fluorescent analogues of...
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New Potent Fluorescent Analogues of
Strigolactones: Synthesis and Biological Activity
in Parasitic Weed Germination and Hyphal
Branching in AM Fungi
Cristina Prandi
Dipartimento di Chimica Generale e Chimica Organica
Università degli Studi di Torino
IPPS 2011, 7-12 June Martina Franca, Italy
Root Development
Koltai H. and Kapulnik Y. (2011),
Biosynthesis STRIGOLACTONES
Root exudates
Plant hormones
NMe
O
O
OO
Inhibition of
Shoot branching
Gomez-Roldan 2008
Umehara 2008
N
O
O
CH3
O
(H3C)2N
O
NMe
O
O
OO
NMe
O
O
OO
S
Hyphal branching
Akiyama 2005
Germination stimulants
Cook, 1966
Myc Factor/s LCO
J.Denaire, G. Becard, Nature 2011
Signalling events in the rhizosphere
Akiyama et al., Nature June 2005
Plant sesquiterpenes induce
hyphal branching in AM fungi
Gomez-Roldan et al., Nature 2008
Strigolactone inhibition of shoot branching
Besserer et al, PLOS 2006
Plant Physiol., 2008
O O
O OO
5-deoxy Strigol
IPPS 2011, 7-12 June Martina Franca, Italy
Introduction
Natural Strigolactones (SLs)
OH
O O
O OO
(+) Strigol
OO
O O
OSorgolactone
OO
O O
O
Orobanchol
OH
OO
O O
O
OH
CH3
CH3
Solanacol
OO
O O
O2'-Epi-orobanchol
OH
OO
O O
O
Orobanchyl acetate
OH
OO
O O
O
OH
O
7-oxo Orobanchylacetate
OO
O O
O
A BC
D
1
2
3a
3
44a5
6
87
8a 8b
9 10
1'
2'
3'
4'
5'
6'
7'
IPPS 2011, 7-12 June Martina Franca, Italy
Introduction
enol ether bridge
stereochemistry at C2’
IPPS 2011, 7-12 June Martina Franca, Italy
Introduction
Synthetic analogues
Zwanenburg, B. Pest Manag Sci 2009, and references therein
OH
OO
O O
O
(+) Strigol
OO
O O
O
GR 24
ED50 10-12 S. hermontica
10-10 O. crenata
ED50 10-9 S. hermontica
10-7 O. crenata
OO
O O
OGR 7
ED50 10-7 S. hermontica
10-7 O. crenata
O O
O O
OGR 5
ED50 10-7 S. hermontica
10-6 O. crenata
O O
OMe
inactive
EtOO
O
inactive
N
O
O
OOMe
OO
ONijmegen-1
O O
O OO
A BC
D
IPPS 2011, 7-12 June Martina Franca, Italy
Introduction
Structure-Activity: an overview
Bioactiphore
Molecular mechanism for the interaction with the receptor protein
Michael addition
Zwanenburg, B. Pest Manag Sci 2009, and references therein
O
O
OO
O
Nu
O
O
OO
ONu
O
O
Nu+
OOC
DC C
O
O
OO
O
Nu
O
O
OO
ONu
O
O
Nu+
OOC
DC C
IPPS 2011, 7-12 June Martina Franca, Italy
Introduction
Structure-Activity: non-SL germination stimulants
S. hermontica 10-5 M, 79%
O. minor 10-5 M, 80%
HO
H
OHOH
OH
OCH3Fusicoccin deacetyl aglycone
S. hermontica 10-5 M, 79%
O. minor 10-5 M, 79%
HO
H
OH
OCH3Cotylenin
HO
OO
O
11ββββ,13-Dihydroparthenolide
activity 10-2 10-21 M
OH
OH
CH2
OH
H3CO
Dihydrosorgoleone
O
O
OH
C15H25H3CO
Sorgoleone
O
CO2CH3
Methyl jasmonate
activity 10-3
-10-4
M
IPPS 2011, 7-12 June Martina Franca, Italy
Introduction
Structure-Activity: non-SL germination stimulants
Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD Science 305: 977 (2004)
Mechanism
O OO
Nu
O OO
Nu
HO OO
Nu
O OO
Nu
H
addition elimination tautomer
O OO
O OO
O O
O O O
GR24
KAR1
KAR2
IPPS 2011, 7-12 June Martina Franca, Italy
Introduction
Structure-Activity: seed germination on Orobanche minor
OH in 4 and 9 OH in 4 and 9 OH in 4 and 9 OH in 4 and 9 enhancesenhancesenhancesenhances
the the the the activityactivityactivityactivity
StereochemistryStereochemistryStereochemistryStereochemistry at Cat Cat Cat C----2222’’’’: : : :
R R R R isisisis the the the the naturalnaturalnaturalnatural configurationconfigurationconfigurationconfiguration
OO
O O
O
sorgolactone (C)
OO
O O
O
5-deoxystrigol (C)
LipophilicLipophilicLipophilicLipophilic structuresstructuresstructuresstructures are are are are lesslesslessless activeactiveactiveactive
Yoneyama et al. J. Pestic. Sci., 2010 35 (3), 344-347.
OH
OO
O O
O
(+) Strigol (B)
OO
O O
O
Orobanchol (A)
OH5
4 OO
O O
O
sorgomol (A)
HO
9
OO
O O
OOH
4
Solanacol (B)O O
O OO
2'-epiorobanchol (A)
OH
4
IPPS 2011, 7-12 June Martina Franca, Italy
Introduction
Structure-Activity: an overview
K. Akiyama, Plant Cell Physiol. 51(7): 1104–1117 (2010)
O O
O OO
OO
NO O
O
O O
O
OO
O O
O
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
X
OO O
O
R
X
OO O
O
R
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Structure-Reactivity: our approach
X = O, S, N, C
n = 0, 1, 2
Heterocyclic B ring conjugated with the bioactive part of the molecule
A C
D
B Strigol
α,β-unsaturated ketone
Heterocyclic fragment
O O
O OOOH
A BC
D
Prandi C., Kapulnik Y. et al. Org. Biomol. Chem. 2009 7, 3413-3420; Zwanenburg B. Mwakaboko S. Plant Cell Physiol. 2011, 699-715
X OTf
R B
OEt
OO
+ XOEt
R
R = H, 70 %R = Me, 78 %
PdCl2(PPh3)2
K2CO3 2M, 3 eq
THF, rt, 3hX
R
O
R = H, 85 %R = Me, 88 %
o-BDSI
X = O, N, S
Structure-Reactivity: synthesis
Discussion
IPPS 2011, 7-12 June Martina Franca, Italy
X
R
O
R = HR = Me
t-BuOK, HCOOEt
THF, 0 °C to rt, 2hX
R
O
O-K+
OO NBS, AIBN 1%
CCl4, reflux, on
OO
Br
X
O
OO
R
O
DCE
0 °C, 3h
SO2
NH
SO2
o-BDSI
Prandi C., Kapulnik Y. et al. Org. Biomol. Chem. 2009 7, 3413-3420
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Structure-Reactivity: synthesis
NMe
O
O
OO
PLN655
O
OO
O
PLC655
O
O
O
O O
PLO65
S
O
O
O O
PLS65
N
O
OMeOOC
O O
PLN65
N
MeO
O
OO
PLN655a
Prandi C., Kapulnik Y. et al. Org. Biomol. Chem. 2009 7, 3413-3420
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Strong bioactive SLs analogues for agricoltural applications (Patent)
• 3 synthetic steps from common and cheap reagents
• access to functionalized framework
• scale-up process
• active on seeds and fungi
Synthetic designIn field applications
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Strong bioactive SLs analogues for agricoltural applications (Patent)
Germination on Seeds of Orobanche aegyptiaca
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Strong bioactive SLs analogues for agricoltural applications (Patent)
O O
OR O O
OR
(+)-MB (-)-MB
b
b
b
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Strong bioactive SLs analogues for agricoltural applications (Patent)
O O
OR O O
OR
(+)-MB (-)-MB
a
b b
a,c
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Chasing the receptor
The computational-based structure of Dwarf14 provides evidence for its role as potential strigolactone receptor in plants
Biochemistry
Chemistry Genetics
Bioinformatic and
computational
NMe
O
R O
OO
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Synthesis of fluorescent SLs analogues
NMe
O
O
OO
NMe
O
O
OO
NMe
O
R O
OO
λλλλexexexex = = = = 320 nmλλλλem = em = em = em = 425 nm
Built-in Labell
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Chasing the receptor
HN NH
S
O
H H
(CH2)4
O O
NH
O
O
O
O
O
NH
O O NH
Biotin-labelled GR24
O
NH
O
O
O
O
O
HN
HN
SOOC
O
(H3C)2N
N(CH3)2
Tetramethyl-rhodamine-labelled GR 24
Zwanenburg et al. Org. Biomol. Chem. 950-959 (2003)
NMe
O
R O
OO
XNMe
O-
R O
OO
XN O
Me
Br6
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
N O
Me
NBS (1.1 eq.)
CH3CN, rtN O
Me
Br
98%
ArB(OH)2
Pd(0)N O
Me
Ar
N OTf
Me
Ar
OEt
NMe
Ar
O NMe
Ar
O
O
OO
BOO
78%
58% overall yield
Suzuki Buchwald-Hartwig
In print, EJOC, Special Issue “Women in Chemistry” in occasion of the centenary of the Nobel Prize to Marie Curie
New synthetic strategy See Poster
Silvia Tabasso
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
N
O
O
CH3
O
(H3C)2N
O
ST23b
ST23c
N
O
O
CH3
O
O
H3CO
N
O
O
CH3
O
OS
ST23d
N
O
O
CH3
O
OS
N OO
H3C
O
O
S
Sample λex/nm λem/nm Φf[a] τ1/ns τ2/nsST 23a 335 480 0.10 7.26 (100%) -
ST 23b 300 560 0.03 4.37 (56%) 11.9 (44%)
ST 23c 325 506 0.06 4.69 (47%) 5.78 (53%)
ST 23d 335 480 0.12 5.99 (57%) 7.96 (43%)
EGO 15 365 437 0.02 0.80 (100%) -
EGO 9c 320 484 0.14 4.64 (100%) -
In press, EJOC, Special Issue “Women in Chemistry” in occasion of the centenary of the Nobel Prize to Marie Curie
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Bar graph representation of percentages of germinated seeds of Orobanche aegyptiacaafter exposure to diastereomeric mixtures of new generation of fluorescent molecules
NCH3
O
S
22c
In print, EJOC, Special Issue “Women in Chemistry” in occasion of the centenary of the Nobel Prize to Marie Curie
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
Bar graph representation of percentages of hyphal branching of Gigasporamargarita after exposure to diastereomeric mixtures of new fluorescent molecules
NCH3
O
S
22c
In print, EJOC, Special Issue “Women in Chemistry” in occasion of the centenary of the Nobel Prize to Marie Curie
Seeds SLs Fungi
α-MB
β-MB
Seeds SLs Fungi
MB-15
MB 5
OO
O O
OGR 24
N
O
O
CH3
O
O
H3CO
23d
NCH3
O
S
22c
N
O
O
CH3
O
O
23c
S
N
O
O
CH3
O
O
23b
DMAP
N
O
O
CH3
O
O
23d
PMP
strongstrongstrongstrong
GR24GR24GR24GR24
inactiveinactiveinactiveinactive
IPPS 2011, 7-12 June Martina Franca, Italy
Discussion
In print, EJOC, Special Issue “Women in Chemistry” in occasion of the centenary of the Nobel Prize to Marie Curie
N
O
O
CH3
O
O
H3CO
23d N
O
O
CH3
O
O
23d
PMP
N
O
O
CH3
O
O
23b
DMAP
NCH3
O
S
22c
• New synthetic sequence leading to a new class of potent SLs analogues
• Different structural requirements for bioactivity in seeds and fungi
• Molecules lacking the D-ring is active on seeds and inactive on fungi
• Large scale synthesis of SLs active analogues
• Fluorescent analogues to be used as biological probes
IPPS 2011, 7-12 June Martina Franca, Italy
Summary
NCH3
O
S
Paolo Larini
Ernesto G. Occhiato, University of Florence
Paolo Venturello
Silvia Tabasso
Acknowledgements
Paola Bonfante
Mara Novero
IPPS 2011, 7-12 June Martina Franca, Italy
Gabriele Pupo
Alberto Oppedisano
IPPS 2011, 7-12 June Martina Franca, Italy
Aula Magna “Primo Levi”
Chemistry Department, Turin
Department of Plant Biology
Turin
IPPS 2011, 7-12 June Martina Franca, Italy