Radboud University Nijmegen Department of Organic Chemistry Strigolactones

Radboud University Nijmegen Department of Organic Chemistry Strigolactones New plant hormones with a rich future and true challenges for organic chemists Binne Zwanenburg Radboud University Nijmegen The Netherlands Eur Organic Chem Congr Amsterdam March 3, 2017 1

Radboud University Nijmegen Department of Organic Chemistry Strigolactones are new plant hormones n 1. 2. 3. Strigolactones are new plant hormones with various bio-functions Germination stimulants for seeds of parasitic weeds Branching factors for AM fungi Inhibitors for bud outgrowth and branching Search for more biofunctions will continue Eur Organic Chem Congr Amsterdam March 3, 2017 2

Radboud University Nijmegen Parasitic weeds Department of Organic Chemistry Striga infested field of maize Our motivation to start our study on the molecular aspects of germination stimulants Eur Organic Chem Congr Amsterdam March 3, 2017 3

Radboud University Nijmegen Department of Organic Chemistry How parasites work Parasites take nutrient from hosts Host Parasite Nutrients Assimilates Water Relationship between a host and a root parasite Eur Organic Chem Congr Amsterdam March 3, 2017 4

Radboud University Nijmegen Department of Organic Chemistry Interaction between host and parasitic weed simplified scheme Host releases detrimental effect Attachment on host roots Chemical signal = germination stimulant interaction induction of germination Seeds of the parasite Occurrence of Striga spp: tropical and subtropical areas in Africa and Azia (India). Food crops: maize, sorghum, millet and rice; yield losses 50 -90%; 100 million African people suffer. Occurrence of Orobanche spp: mediterranean area, Eastern Europe and Middle East. Food crops: legumes (tomato, fava beans), sunflower and tabacco; damage severe. Eur Organic Chem Congr Amsterdam March 3, 2017 5

Radboud University Nijmegen Department of Organic Chemistry Striga seed germination Germination test of stimulants Eur Organic Chem Congr Amsterdam March 3, 2017 Seed germination 6

Radboud University Nijmegen Department of Organic Chemistry Striga seed attached to root of host plant Striga seed attaches to root of host plant and draws nutrition from host Eur Organic Chem Congr Amsterdam March 3, 2017 7

Radboud University Nijmegen Department of Organic Chemistry Naturally occurring germination stimulants Strigol w isolated from cotton (Cook et al. , 1966) w absolute configuration (Brooks et al. , 1985) w isolated from maize (Siame et al. , 1993) w activity 10 -7 -10 -12 M Sorgolactone w isolated from sorghum (Hauck et al. , 1992) w absolute configuration: confirmed by total synthesis (Zwanenburg et al. , 1997) w activity 10 -9 -10 -12 M Eur Organic Chem Congr Amsterdam March 3, 2017 Alectrol w isolated from cowpea (Müller et al. , 1992) w exact structure unknown w for a long time w activity 10 -12 M w. Correct structure: 2011 Orobanchol w isolated from red clover (Yokota et al. , 1999) w absolute configuration: confirmed by total synthesis (Mori et al. , 1999) w activity: high w. LATER: Not correct 8

Radboud University Nijmegen Department of Organic Chemistry SLs as germination stimulants Current state of affairs *Several natural SLs have been isolated and their structures have been elucidated. Examples: strigol, sorgolactone, orobanchol *Plants produce tiny amounts: 15 pg/day. hampers isolation and identification *Recently several structures were corrected: orobanchol, alectrol, solanacol Needs *Need for sophisticated chromatographic and spectroscopic techniques *Need for the total synthesis of new SLs to confirm structures and to prepare all possible stereoisomers to establish influence of stereochemistry on the bioactivity n • Eur Organic Chem Congr Amsterdam March 3, 2017 9

Radboud University Nijmegen Department of Organic Chemistry SLs as germination stimulants Two SL families Helps to understand the biosynthesis of SLs Eur Organic Chem Congr Amsterdam March 3, 2017 10

Radboud University Nijmegen Department of Organic Chemistry Roles of organic chemist in parasitic weed research n n n n n What can an organic chemist do? Elucidate structure of naturally occurring stimulants Total synthesis of natural stimulants Structure activity relationship Identify bioactiphore Design and prepare bioactive analogues Mode of action How can we control weed pests? ? See what happens when we start Eur Organic Chem Congr Amsterdam March 3, 2017 11

Radboud University Nijmegen Department of Organic Chemistry Identification of the Bioactiphore (+)-strigol GR 5 GR 24 Not active GR 7 Not active ED 50 10 -7 S. hermonthica Conclusion: bioactiphore resides in the CD-part Eur Organic Chem Congr Amsterdam March 3, 2017 12

Radboud University Nijmegen Department of Organic Chemistry Tentative Molecular Mechanism (Mode of Action) • Characteristics of the proposed mechanism: Addition/Elimination of nucleophilic site of the receptor (-) l Nucleophilic site e. g. : HS , H 2 N l D-ring serves as a good leaving group E. M. Mangnus, B. Zwanenburg; J. Agric. Food Chem. , 1992, 40, 1066 -1070. Eur Organic Chem Congr Amsterdam March 3, 2017 13

Radboud University Nijmegen Department of Organic Chemistry Relevant Structural Features of SLs Stereochemistry is (very )important A-ring modifications reduce activity not very much a, b-unsaturated system and D-ring are essential Bioactiphore resides here! This model is the basis for designing SL analogues Eur Organic Chem Congr Amsterdam March 3, 2017 14

Radboud University Nijmegen Department of Organic Chemistry New Strigolactones from simple Ketones Strategy: *Incorporate essential structural feature, using simple scaffolds. *Obey the molecular mechanism. *Use simple starting materials. *D-ring must be present Two step synthesis, one pot. Eur Organic Chem Congr Amsterdam March 3, 2017 15

Radboud University Nijmegen Department of Organic Chemistry Bioassays of new Strigolactone Analogues from Ketones 100 % germination 10 0 80 60 40 20 20 0 0 indanone tetralone GR 24 S. hermontica Eur Organic Chem Congr Amsterdam March 3, 2017 indanone 1 ppm 0. 01 ppm tetralone GR 24 O. crenata 16

Radboud University Nijmegen Department of Organic Chemistry SLs analogues based on the model Many more options, allows structural fine tuning Eur Organic Chem Congr Amsterdam March 3, 2017 17

Radboud University Nijmegen Department of Organic Chemistry Parasitic weed control • Hand weeding: removal of mature parasites may reduce seedbank, damage to host has already occurred, laborious method. • Crop rotation: fallow period; trap crops e. g. cotton (induce germination without being affected reduction of seedbank); catch crops also attacked, only to reduce seedbank. Requires strict agricultural measures. • Resistent crops: breeding is slow process, some examples known, loss of resistence. • Biological control: little success so far. § Chemical control: some herbicides are effective, e. g. 2, 4 -D, dicamba, glyphosate Can be used under strict control only. Also damage to crop. § Suicidal germination: treat field with stimulant in absence of host, parasitic seed will germinate, cannot survive without host. Problem: find suitable germinating agent. Advantage: highly specific There is a great need for methods to control parasitic weeds! Eur Organic Chem Congr Amsterdam March 3, 2017 18

Radboud University Nijmegen Department of Organic Chemistry Nijmegen-1 The essential features of the bioactiphore are connected to a simple scaffold, i. e. phthalimide Nijmegen-1 ED 50: 10 -7 M Striga hermonthica 10 -6 M Orobanche crenata Easy synthesis; potential candidate for use in suicidal germination Suical germination: germination in absence of host: seed will die! Nefkens, G. H. L. , Thuring, J. W. J. F. , Beenakkers, M. F. M. , Zwanenburg, B. J. Agric. Food Chem. , 1997, 45, 2273 -2277. Eur Organic Chem Congr Amsterdam March 3, 2017 19

Radboud University Nijmegen Department of Organic Chemistry Field test with SL analogue Nijmegen-1 example of successful suicidal germination tobacco field treated with Nijmegen-1 untreated tobacco field 20

Radboud University Nijmegen Department of Organic Chemistry Serendipitous finding of SL mimics Disobedient action: violation of the rules of the design model Surprise: Active!! Germination: model S. hermonthica: 0. 1 mg/L: 18. 5 %; 0. 01 mg/L: 11% O. cernua: I mg/L : 70%; 0. 1 mg/L 31% BZ & Mwakaboko, Bioorg Med Chem 7394 (2012) Germination: S. Hermonthica R = H 0. 1 mg/L: 9%; 0. 01 mg/L: 7% R =OH 7%; 21% O. cernua R = H 1 mg/L: 87%; 0. 1 mg/L: 78% R = OH 52%; 82% Eur Organic Chem Congr Amsterdam March 3, 2017 21

Radboud University Nijmegen Department of Organic Chemistry SL mimics New development: SL mimics, highly active germination stimulants. However, with Methyl group at C-4: no activity!! Molecular mechanism Interaction with protein receptor probably different from SL analogues Many more options: new avenue!! Eur Organic Chem Congr Amsterdam March 3, 2017 22

Radboud University Nijmegen Department of Organic Chemistry A new approach for weed control A novel concept for the control of parasitic weeds by decomposing germination stimulants prior to action Chinnaswamy Kannan and Binne Zwanenburg* Crop Prot. 61, 11 -15 (2014) Use of simple chemicals to decompose the stimulant as soon as they are produced by the host root Results are very promising Experiments are ongoing in India Eur Organic Chem Congr Amsterdam March 3, 2017 23

Radboud University Nijmegen Department of Organic Chemistry Biosynthesis of strigolactones, carlactone is key Tentative Synthesis of CL: very difficult, world record: <1% yield Many ? ? ? Hot topic Eur Organic Chem Congr Amsterdam March 3, 2017 24

Radboud University Nijmegen Department of Organic Chemistry SYNTHESIS Synthesis of Natural SLs Synthesis of Analogues Eur Organic Chem Congr Amsterdam March 3, 2017 25

Radboud University Nijmegen Department of Organic Chemistry Synthesis of all eight Stereoisomers of Strigol (1) Step 1: Synthesis of ABC part Eur Organic Chem Congr Amsterdam March 3, 2017 26

Radboud University Nijmegen Department of Organic Chemistry Synthesis of all eight Stereoisomers of Strigol (2) Step 2: Introduction of the D-ring Eur Organic Chem Congr Amsterdam March 3, 2017 27

Radboud University Nijmegen Department of Organic Chemistry Synthesis of all eight Stereoisomers of Strigol (3) Step 3: Introduction of the OH in the A-ring Step 4: Separation on a chiral column Eur Organic Chem Congr Amsterdam March 3, 2017 28

Radboud University Nijmegen Department of Organic Chemistry Summary of Synthesis Reizelman, Synthesis 2000, 1944 All 8 stereoisomers were bio-assayed Eur Organic Chem Congr Amsterdam March 3, 2017 29

Radboud University Nijmegen Department of Organic Chemistry Natural stimulant with an aromatic A-ring Note the assignment of stereochemistry Motivation to prepare aromatic orobanchol (+)-strigol Eur Organic Chem Congr Amsterdam March 3, 2017 Important; stereochemistry is related to (+)-strigol as reference 30

Radboud University Nijmegen Department of Organic Chemistry Synthesis of Aromatic Orobanchol, introduction of OH in B-ring Ratio: 82: 3 The oxidation of the B-ring is tricky Inversion of configuration gives 9 b Reagents and conditions: (i) Fe. Cl 3 (2 mol %), aq t. Bu. OOH (70%, 3 equiv) pyridine, 82 ºC, 24 h, (ii) Na. BH 4, Ce. Cl 3. 7 H 2 O, Et. OH, (iii) PPh 3, Ph. CO 2 H, Et. O 2 CN=NCO 2 Et, toluene, (iv) K 2 CO 3, Me. OH. Eur Organic Chem Congr Amsterdam March 3, 2017 31

Radboud University Nijmegen Department of Organic Chemistry Synthesis of Aromatic Orobanchol, introduction of D-ring, OH down, NO protection needed Reagents and conditions: HCO 2 Me, t-Bu. OK, THF, then butenolide 10, DME (ii) Ac 2 O, pyridine, CH 2 Cl 2. Malik, Kohlen, Jamil, Rutjes, BZ, Org. Biomol. Chem. 9, 2286 -2293 (2011) Eur Organic Chem Congr Amsterdam March 3, 2017 32

Radboud University Nijmegen Department of Organic Chemistry Other biofunctions of SLs: n Branching factor of AM fungi n Control of Plant Growth Eur Organic Chem Congr Amsterdam March 3, 2017 33

Radboud University Nijmegen Department of Organic Chemistry SLs as branching factors for AM fungi Understanding of the communication between plants, arbuscular mycorrhizal (AM) fungi and parasitic plants, a symbiotic pair This interaction with AM fungi needs much attention, lack of understanding on the molecular level, AM fungi improve P and N content in the soil Eur Organic Chem Congr Amsterdam March 3, 2017 34

Radboud University Nijmegen Department of Organic Chemistry SLs as branching factor of AM fungi n SL model structure for the branching factor of AM fungi Eur Organic Chem Congr Amsterdam March 3, 2017 35

Radboud University Nijmegen Department of Organic Chemistry SLs as inhibitors for bud outgrowth and shoot branching • Control of plant architecture is of great practical importance in agriculture • Horticultural studies are a challenge for STREAM • Little is know of the this inhibition process on a molecular level SL model structure for shoot inhibition, one SL analogue and one SL mimic, many possibilities for molecular fine tuning Eur Organic Chem Congr Amsterdam March 3, 2017 36

Radboud University Nijmegen Concluding remarks n n n n Department of Organic Chemistry SLs are new plant hormones with a rich future They are active at a very low concentration, that is a highly relevant property They are present in root exudates; insight in the biosynthetic origin is very relevant Various biofunctions have been discovered, each has it own mode of action on a molecular level New biofunctions of SLs will be discovered Some compounds with claimed activity have not been tested correctly, reinvestigation might be of interest. Total synthesis of natural SLs is a challenge, emphasis on stereochemistry is a must Identification of the receptor protein is a challenge Detailing of the mode of action for the various biofunctions is important Practical applications are a true challenge Production of stimulants using root culture is an interesting option. Suicidal germination has promise in the control of the parasitic weeds. Search for inhibition of germination needs attention. Would open new avenues for parasitic weed control. Sophisticated equipment and instrumentation opens many new windows Many problems and challenges in this rapidly evolving field, multidisciplinary research is the future direction for moving the field forward. Eur Organic Chem Congr Amsterdam March 3, 2017 37

Radboud University Nijmegen Department of Organic Chemistry Radboud University Nijmegen, Faculty of Science The Huygens Building 2004 (first wings); Finished 2006 Official opening May 2007 by Queen Beatrix Eur Organic Chem Congr Amsterdam March 3, 2017 38

Radboud University Nijmegen Department of Organic Chemistry Beautiful flowers but a real pest Our ultimate goal: Get rid of the flowers! Molecules with dedicated functions are our hope Thank you for attention Eur Organic Chem Congr Amsterdam March 3, 2017 39