1. Which is the key????? Any biological process is controlled by chemical signals. By knowing of these signal we control any biological process in any organism.

2. Can we talk with microorganisms? Can we give them instructions?

3. Can we talk with plants? Can we give them instructions?

4. By Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology alrahman3@hotmail.com Tel: 002-01228188759

5.  Any biological process is controlled by chemical signals.  By knowing these chemical signals we can control, talk or give instructions to plants, microorganisms or insects.

6. Microbial products Microbial signals Quorum sensing Siderophores Antagonism Synergism Biosurfactants Enzymes and proteins

7. Slime molds plasmodium formation is controlled by c-AMP. This image from: http://2011.igem.org/Team:Grenoble/Projet/Modelling/Deterministic Quorum sensing control of P. aeruginosa Nikki Moran, http://blogs.rsc.org/mb/2011/01/25/lyngbyoic-acid-disrupts- quorum-sensing-in-p-aeruginosa/ c-AMP

8. Pests inhibitors Plant signals Root exudates Pests activators Antagonism Synergism Plant products Enzymes and proteins

9. Faba bean-Orobanche seeds germination stimulation A crucifer plant infected with S. sclerotiorum From the graphical abstract of M. Soledade C. Pedras and Mohammad Hossain, Org. Biomol. Chem., 2006, 4, 2581 LRR proteins Pectinases and cellulases inhibiting proteins belong to the leucine-rich repeat (LRR) family and are known to prevent fungal pathogen invasion. Phytoalexins Strigolatcgones

10. 1- The drug source selection 2- Extraction and purification 3- Biological activity determination 4- Chemical structure elucidation

11. So, The processes of new drug discovery requires co-work and expertise of a wide variety of scientific, technical and managerial groups

12. Plant (Plant tissues or root exudates) Microorganisms Bacteria, fungi, actinomycetes, algae 1- The drug source Proteineous compounds (From plant tissues, root exudates, microbial broth or cells) Non- proteineous compounds (From plant tissues, root exudates, microbial broth or cells) 2- Extraction 3- Testing of each obtained extract for a biological activity (Against bacteria, fungi, parasitic weeds, insects, nematodes, animal disease,……)

13. Proteineous compounds: - MW determination. - Subunits determination. - Amino acids sequencing. -Crystallization. -NMR ( 2D and 3D). non-proteineous compounds: - NMR analysis (one and two dimensions - IR analysis - Mass spectroscopy - X-ray crystallography 5- Chemical structure elucidation Purification of proteineous compounds: - Concentration (precipitation) - Fractionation (gel filtration, ion exchanger). - Electrophoresis (two- dimensional). Purification of non- proteineous compounds: - Liquid-liquid extraction. - Liquid- solid extraction. - Chromatographic methods (normal phase and reversed phase). - HPLC-MS analysis 4- Purification of the active compound from the obtained extract

14. 1) Plants: Several plants contain pharmaceutical compounds, pesticides, ….. Several plant extracts or root exudates showed a biological activity but the active compound is still not identified. You can found a grass grow on the train run-way contains a new antimicrobial or medicinal compound.

15. Root exudates collection: 1- Cultivate the plant in pots and irrigate with nutrient solution for a period vary with the plant type. 2- Incubate in a glasshouse or incubation chamber under the optimum growth conditions. 3- remove the plant carefully from the soil and wash the root by tap water. 4- Soak in a conical flask containing distilled water for one day only.

16. Isolation of microorganisms: Serial dilution method I the best. The used medium is: 1- PDA for fungi 2- Nutrient agar for bacteria. 3- Casein starch agar for actinomycetes. 2) Microorganisms:

17. 1- Against a pathogen Against hydrolytic enzymes of the pathogen Against the pathogen itself Nematodes Fungi Bacteria Insect Pectinases Cellulases Keratinase Hemicell- ulases Other Parasitic weeds

18. By the plant extract or microbial supernatant 1- Inhibition test in solid media 2- Inhibition test in liquid media Dual culture method Medium + an extract Control Inhibition tests against the pathogen itself

19. ...... 2- Tests against parasitic weeds (Germination inhibition or activation) Dual culture method By the extract Tissue culture method Control (Without the inhibitor or the activator) Magenta box Sterilized nutrient solution Parasitic weeds seeds Glass plate covered by filter paper

20.  For germination stimulation: The compound is added in the GFFR or in the tissue culture medium compared with two control treatments, why?  For germination inhibition: - A stimulator (such as strigol) is firstly added on two GFFR disks. The tested inhibitor is then added on one of them. - The same test can be performed by tissue culture.

21. The plant extract, microbial broth or a suspension of the isolated microorganism is mixed with the nutrient materials of the tested insect. The life and dead insects are calculated after a few minutes, hours to days (according to the insect type) and compared with control.

22. This is performed through three steps: 1- Production of the hydrolytic enzyme (from bacteria, fungi or a parasitic weed). 2- Determination of the enzyme activity in presence and absence of the tested substance.

23. The nature of the active drug can be: 1- Protein. 2- Non-proteineous. 3- Complex: Glycoside, glycolipid, lipoprotein, glycoprotein,…… 3- Extraction of the active drug

24. a) Extraction of the proteineous compounds: - Selection of the extraction buffer. - Some additives are required during extraction b) Extraction of the non-proteineous compounds: - Selection of the extraction solvent. - The extraction method: Liquid- liquid extraction Liquid solid extraction Solid phase extraction (SPE) Super Critical Fluid instrument (SCF)

25. Polarity indexSolvent Polarity indexSolvent Polarity indexSolvent 4.7 Methyl Ethyl Ketone 2.8Ethyl Ether0Pentane 4.81,4-Dioxane3.1Dichloromethane0Trichlorotrifluoroethane 5.1Acetone3.5 Ethylene Dichloride 0.1Cyclopentane 5.1Methanol3.9n-Butyl Alcohol0.1Heptane 5.3Pyridine3.9 Isopropyl Alcohol0.1Hexane 5.52-Methoxyethanol 4n-Butyl Acetate0.1Iso-Octane 5.8Acetonitrile4Isobutyl Alcohol0.1Petroleum Ether 6.1 Propylene Carbonate4 Methyl Isoamyl Ketone0.2Cyclohexane 6.4 Dimethylformamide 4n-Propyl Alcohol1n-Butyl Chloride 6.5 Dimethyl Acetamide4Tetrahydrofuran2.4Toluene 4.1Chloroform2.5Methyl t-Butyl Ether 7.2 Dimethyl Sulfoxide4.2 Methyl Isobutyl Ketone2.5o-Xylene 9Water4.4Ethyl Acetate2.7Chlorobenzene 2.7o-Dichlorobenzene

26.  1) Liquid- liquid extraction techniques: 1- Shake flasks. 2- Separatory funnel

27.  2) Solid-phase extraction (SPE) techniques:  The compounds in a mixture are separated according to their physical or chemical properties.  Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrix, including urine, blood, water, beverages, soil, animal tissue, plant extracts or microbial supernatants.

29. Types of solid phases in SPE:  Reversed phase (C8 or C18)  Normal phase (Silica gel or CN)  Ion exchanger (Cation or anion exchangers)  Affinity  Gel filtration.

30.  Supercritical Fluid Extraction (SCF or SFE) is the process of separating one component from a sample using supercritical fluids as the extracting solvent. Carbon dioxide (CO 2 is the most used supercritical fluid, sometimes modified by co-solvents such as ethanol or methanol

31.  Each of the obtained extracts must be tested for the biological activity.  The active extract is then analyzed for its purity determination: 1- Uv-Vis scanning is the first analysis to identify λ- max. Purity determination

32. 2- The HPLC is used by testing two columns: reversed phase and normal phase, and the detection wave length that was obtained from the scanning. 3- The HPLC-MS is performed utilizing the previous HPLC data. The HPLC tells us how many compounds in the active extract.

33.  The purification is the most important step. The purification is performed by:  TLC  Glass column  Preparative HPLC

34. The purity must be determined by HPLC/MS and the activity of final pure compound must be determined before chemical structure elucidation. HPLC chromatogram showed one pure compound

35. This is performed by spectroscopy techniques: 1- IR spectrophotometer: For identification of the functional groups. 2- Mass spectrophotometer: For determination of MW, base ion and mass fragmentation pattern. 3- One and two dimensions NMR: H 1, C 13, COSY, NOESY, HMABC, HMQC, APT, DEPT. 4- Crystallization and analysis by X-ray single crystal.

36. It identifies the function groups.

38. A.W.Atom 11H1H 1212 C 1414 N 1616 O Identifies the MW and fragmentation pattern M.W.Group 28C=O 45COOH 77 Benzene ring 29CHO

39. Mass spectrometry of organic compounds

40. One dimension: H 1 C 13 APT DEPT 45 DEPT 90 DEPT 135 Two dimensions: COSY NOESY TOCSY HMBC HMQC HSQC

42. Identifies number and types of hydrogen ions. Data interpretation: 1- Chemical shift values. 2- signals splitting. 3- Signals area. 4- j values

45. Identifies the number and type of carbon atoms. Types of C 13 NMR analysis: 1- 1D C 13 NMR 2- DEPT 3- APT C 13 data interpretation

46. C 13 NMR spectrum of acetophenone

47. The carbon attached with odd number of protons showed signals up. The carbon attached with even number of protons showed signals down.

48. DEPT 45 DEPT 90DEPT 135

49. Correlation between the proton with the adjacent proton (Three bonds correlation), (H-H correlation).

51. COSY spectrum of 2-chlorobutane

52.  it allows correlation of protons through space (H-H correlation in space).  it allows identification of the stereo-chemical structure.

53. Examples: H13 is coupled with H12, H8, H14. H15a is coupled with H16, H14, H15b Some C. atoms become neighbor to others in the three dimension structure, although it far in the carbon atom numbering, i.e. C23 is near to C12.

54. Correlates the protons to their directly attached carbon atoms (C-H correlation).

55. C14 correlates withH13, H15b, H20, H22 Detects heteronuclear correlations (C-H, N-H) over longer ranges of about 2–4 bonds.

57.  How many human pathogenic microorganisms that acquire resistance against the used anti-microbial agents every year?  How many plant pathogens that are controlled by biopesticide agents worldwide?  How many insects that infect plant or human worldwide?  How many signals are identified for control of each case?

58.  We can do that without high cost, but in presence serious support and kind partners in a multi-disciplinary work.

59. Thank you