ZENDAH EL EUCH ___________________________________________ Isolation, Purification and Structure Elucidation of New Secondary Metabolites from Terrestrial, Marine, and Ruminal Microorganisms OH S N N N H H Dissertation Isolation, Purification and Structure Elucidation of New Secondary Metabolites from Terrestrial, Marine and Ruminal Microorganisms Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Imène ZENDAH EL EUCH aus Tunesien Göttingen 2012 D7 Referent: Prof. Dr. H. Laatsch Referent: Prof. Aly Raies Korreferent: Prof. Dr. A. Zeeck Tag der mündlichen Prüfung: 13. Juli 2012 Die vorliegende Arbeit wurde in der Zeit von Oktober 2005 bis März 2007 in der Faculté des Sciences de Tunis (Laboratoire des Microorganismes et des Bio- molécules Actives) unter der Leitung von Herrn Prof. RAIES Aly und von April 2007 bis Juli 2012 im Institut für Organische und Biomolekulare Chemie der Georg- August-Universität zu Göttingen unter der Leitung von Herrn Prof. Dr. H. Laatsch angefertigt. Herrn Prof. Dr. H. Laatsch danke ich für die Möglichkeit zur Durchführung dieser Arbeit sowie die ständige Bereitschaft, auftretende Probleme zu diskutieren. Für meine Eltern, meine Geschwister und meinen Ehemann Content I TABLE OF CONTENTS 1 INTRODUCTION ...................................................................................................................... 1 1.1 NATURE AS A SOURCE OF NATURAL PRODUCTS .................................................................... 1 1.2 NATURAL PRODUCTS AS PHARMACEUTICAL DRUGS FROM TERRESTRIAL BACTERIA ......... 10 1.3 MARINE NATURAL PRODUCTS AS PHARMACEUTICAL DRUGS ........................................... 16 1.4 RUMINAL BACTERIA AS NEW SOURCES OF POTENTIAL ANTIMICROBIALS ........................... 23 2 AIM OF THE PRESENT WORK ........................................................................................... 25 3 GENERAL TECHNIQUES ..................................................................................................... 27 3.1 COLLECTION OF STRAINS ................................................................................................... 27 3.2 WORK UP PROCEDURE FOR SELECTED BACTERIAL STRAINS ............................................... 28 3.3 PRE-SCREENING ................................................................................................................. 28 3.3.1 Biological screening ............................................................................................ 29 3.3.2 Chemical screening ............................................................................................. 29 3.3.3 Pharmacological and biological assays .............................................................. 30 3.4 CULTIVATION AND SCALE-UP ............................................................................................. 31 3.5 ISOLATION METHODS ......................................................................................................... 31 3.6 PARTIAL IDENTIFICATION AND DEREPLICATION ................................................................. 32 4 INVESTIGATION OF SELECTED BACTERIAL AND FUNGAL STRAINS: RUMINAL BACTERIA ......................................................................................................................................... 33 4.1 RUMINAL PSEUDOMONAS AERUGINOSA ZIO ....................................................................... 33 4.1.1 1H-Quinazoline-2,4-dione ................................................................................... 36 4.1.2 Barakacin ............................................................................................................. 38 4.1.3 Rhamnolipid A (Glycolipid A): ............................................................................ 49 4.1.4 Phenazine-1-carboxylic acid ............................................................................... 53 4.1.5 3-(Hydroxyacetyl)-indole ..................................................................................... 54 4.1.6 Indole-3-carbaldehyde ......................................................................................... 56 4.1.7 Cyclo(Phe,Pro) .................................................................................................... 56 4.1.8 Triethylamine ....................................................................................................... 58 4.2 RUMINAL BACTERIUM CITROBACTER FREUNDII ZIG ........................................................... 60 4.2.1 Oleic acid ............................................................................................................. 61 4.2.2 Isoxanthohumol.................................................................................................... 63 4.2.3 Cis-Cyclo(Pro,Leu) .............................................................................................. 65 4.2.4 Cyclo(Tyr,Pro) ..................................................................................................... 66 4.2.5 Phenol .................................................................................................................. 67 4.2.6 Adenine ................................................................................................................ 68 Content 4.3 RUMINAL BACTERIUM GEMELLA MORBILLORUM ZIK .......................................................... 70 4.3.1 Anthranilic acid ................................................................................................... 71 4.3.2 1-Hydroxy-4-methoxy-2-naphthoic acid .............................................................. 72 4.4 RUMINAL BACTERIUM ENTEROBACTER AMNIGENUS ZIH ..................................................... 74 4.4.1 Nß-Acetyltryptamine ............................................................................................. 75 4.4.2 Tyrosol ................................................................................................................. 76 4.4.3 Phenol .................................................................................................................. 78 4.4.4 Tryptophol ........................................................................................................... 78 4.4.5 Brevinic acid ........................................................................................................ 79 4.4.6 Indole-3-lactic acid .............................................................................................. 81 4.4.7 Butyl glycoside ..................................................................................................... 82 4.4.8 Other metabolites ................................................................................................ 84 4.5 RUMINAL SERRATIA RUBIDAE ZIE ........................................................................................ 85 4.5.1 Surfactin derivative .............................................................................................. 87 4.5.2 Prodigiosin .......................................................................................................... 90 4.5.3 Indole-3-acetic acid ............................................................................................. 91 4.5.4 1-Acetyl-β-carboline ............................................................................................ 92 4.5.5 4-Hydroxy-5-methyl-furan-3-one ........................................................................ 93 4.5.6 Actinomycin D ..................................................................................................... 94 4.6 RUMINAL KLEBSIELLA PNEUMONIAE ZIC .......................................................................... 96 4.6.1 4',5,7-Trihydroxyisoflavone (genisteine) ............................................................. 98 4.6.2 4-Hydroxybenzoic acid ...................................................................................... 100 4.7 RUMINAL BACTERIUM PSEUDOMONAS AERUGINOSA ZIL ................................................... 100 4.7.1 2-n-Heptyl-1-hydroxy-1H-quinolin-4-one ......................................................... 102 4.7.2 3-n-Heptyl-3-hydroxy-1,2,3,4-tetrahydroquinoline-2,4-dione ........................... 107 4.7.3 Polypropylenglycol ............................................................................................ 109 4.7.4 1-Phenazinol ...................................................................................................... 110 4.7.5 Phenazine-1-carboxamide ................................................................................. 112 4.7.6 Rhamnolipid A and rhamnolipid B .................................................................... 113 5 TERRESTRIAL BACTERIA ................................................................................................ 115 5.1 TERRESTRIAL BACILLUS SP. ZIR ....................................................................................... 115 5.1.1 Monensin B ........................................................................................................ 116 5.2 TERRESTRIAL PSEUDOMONAS SP. ZIPS ............................................................................. 120 5.2.1 Linoleic acid ...................................................................................................... 121 5.3 TERRESTRIAL STREPTOMYCES SP. 195 ............................................................................... 122 5.3.1 5-Hydroxymethyl-3-(1-hydroxy-6-methyl-heptyl)-dihydrofuran-2-one