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

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Page 1: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 2: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 3: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 4: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 5: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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’

Page 6: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 7: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 8: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 9: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 10: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 11: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 12: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

IPPS 2011, 7-12 June Martina Franca, Italy

Discussion

Page 13: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 14: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 15: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 16: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 17: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

IPPS 2011, 7-12 June Martina Franca, Italy

Discussion

Strong bioactive SLs analogues for agricoltural applications (Patent)

Germination on Seeds of Orobanche aegyptiaca

Page 18: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 19: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 20: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 21: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 22: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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)

Page 23: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 24: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 25: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 26: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 27: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 28: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

• 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

Page 29: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

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

Page 30: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

IPPS 2011, 7-12 June Martina Franca, Italy

Aula Magna “Primo Levi”

Chemistry Department, Turin

Department of Plant Biology

Turin

Page 31: New Potent Fluorescent Analogues of Strigolactones ...New Potent Fluorescent Analogues of Strigolactones: Synthesis and Biological Activity in Parasitic Weed Germination and Hyphal

IPPS 2011, 7-12 June Martina Franca, Italy