PHOTOPOLYMERIZABLE "ROUNDUP" SYNTHESIS, HERBICIDAL ACTIVITY AND COATING FORMULATION Victoria Piunova A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2006 Committee: Douglas C. Neckers, Advisor Tomas H. Kinstle Neocles B. Leontis iii ABSTRACT Douglas Neckers, Advisor Marine biofouling is a worldwide problem for all seagoing vessels. It causes a roughness of a ship's hull, a decrease in its speed and maneuverability and thereby increasing fuel consumption and emission of waste products into the atmosphere. As long as 2000 years ago, people attempted to prevent biofouling by covering ship’s hulls with copper and lead sheets. Since then a large variety of methods have been tried but none proved ideal. The current research project tests incorporation a glyphosate-based biocide into a model marine coating to prevent the formation of biofilms- one of the first steps in marine fouling and thereby block biofouling process. This work describes a synthetic route for a novel compound - acrylated glyphosate - and characterization by chemical, analytical and physical methods. Polymerization, photopolymerization and copolymerization experiments proved the novel compound efficiently polymerizes and copolymerizes during reasonably short time intervals (120sec-10 min). Biological assays based on the Kirby-Bauer test and monitoring of growth inhibition showed that the acrylated glyphosate derivative, as well as its polymer, possess strong herbicidal activity against model and common biofouling organisms. Incorporation of acrylated glyphosate into model acrylic resin yielded a highly cross- linked coating which proved to be toxic toward the microorganisms. Release experiments showed no leaching of copolymerized acrylated glyphosate from the coating over 21 days. This indicates that the compound, incorporated into the backbone structure of the iv coating, retains herbicidal activity against common fouling organisms. Therefore, acrylated glyphosate is a promising component for antifouling coatings for seagoing vessels. v To my beloved parents vi ACKNOWLEDGMENTS I would like first express my gratitude to my advisor Dr. Douglas Neckers for his guidance, encouragement and understanding. I would like to thank Dr. Tomas Kinstle and Dr. Neocles Leontis for being on my thesis committee. My special thanks to Dr. Andrei Federov for valuable advises and discussions. Also, I would like to acknowledge Dr. Aneta Bogdanova, Dr. Dan Berger, Dr. Bullerjahn, Maria Baranova and my lab mates for they support and help. vii TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION AND BACKGROUND INFORMATION ....................... 1 CHAPTER II. MATERIALS AND METHODS.................................................................. 7 Materials ............................................................................................................ 7 General procedure...................................................................................................... 7 Polymerization experiments and polymer characterization....................................... 7 Thermal polymerization ............................................................................................ 7 Photopolymerization.................................................................................................. 8 Photo-copolimerization experiment........................................................................... 8 Molecular mass determination for poly(acrylated glyphosate) ................................. 8 Formulation and characterization of model acylic matrixes loaded with acrylated glyphosate or glyphosate ........................................................................................... 9 Release experiment ........................................................................... 9 Coating formulation........................................................................... 10 Coating characterization ........................................................................................... 10 Solid state NMR ............................................................................................ 10 MALDI-TOF Mass Spectrometry ................................................................. 10 SEM ............................................................................................... 11 MALDI-TOF Mass Spectrometry ................................................................. 11 Synthesis of N-Methacryloyl-N-(phosphonomethyl)glycine (MA) .......................... 11 Synthesis of N-Acryloyl-N-(phosphonomethyl)glycine (AA) .................................. 12 Computational methods ............................................................................................ 13 viii Biological activity tests ............................................................................................. 14 Media Preparation and Cell Growth Measurements ..................................... 14 Agar experiments .......................................................................................... 17 E. Coli Agar experiments .............................................................................. 17 Kirby-Bauer test ............................................................................................ 18 Biological Activity of Coating Pellets .......................................................... 18 CHAPTER III. SYNTHESIS AND CHARACTERIZATION OF ACRYLIC AND METHACRYLIC DERIVATIVES OF GLYPHOSATE...................................................... 20 CHAPTER IV. POLYMERIZATION OF ACRYLATED AND METHACRYLATED DERIVATIVES OF GLYPHOSATE.................................................................................... 31 Molecular mass determination by SEC ..................................................................... 39 CHAPTER V. COMPUTATIONAL STUDIES OF ACRYLATED AND METHACRYLATED DERIVATIVES OF GLYPHOSATE .............................................. 41 CHAPTER VI. BIOLOGICAL ACTIVITY OF ACRYLATED GLYPHOSATE AND POLY(ACRYLATED GLYPHOSATE) .............................................................................. 44 CHAPTER VII. COATING FORMULATION, CHARACTERIZATION AND ELUCIDATION OF HERBICIDAL ACTIVITY ................................................................ 53 CONCLUSIONS ......................................................................................................... 61 REFERENCES ...................................................................................................................... 63 ix LIST OF FIGURES Figure Page 1.1 Sequential steps in biofouling ............................................................................... 2 3.1 1H NMR spectrum of N-methacryloyl-N-(phosphonomethyl)glycine ..................... 24 3.2 13C NMR spectra of N-methacryloyl-N-(phosphonomethyl)glycine ....................... 25 3.3 Two different isomers of N-methacryloyl-N-(phosphonomethyl)glycine ................ 26 3.4 1H NMR spectra of N-acryloyl-N-(phosphonomethyl)glycine ................................. 27 3.5 13C NMR spectra of N-acryloyl-N-(phosphonomethyl)glycine ................................ 27 3.6 Temperature-dependent 1H NMR spectra of N-methacryloyl-N- phosphonomethyl)glycine.......................................................................................... 29 3.7 Temperature-dependent 1H NMR spectra N-acryloyl-N- (phosphonomethyl)glycine ....................................................................................... 30 4.1 IR spectra of acrylated glyphosate and poly(acrylated glyphosate) .......................... 32 4.2 Photopolymerization of AA in the presence of Darocure 1173................................. 33 4.3 Photopolymerization of AA in the presence of Irgacure 651 .................................... 33 4.4 Polymerization profiles for N-acryloyl-N-(phosphonomethyl)glycine with different initiators: VA 057, VA 085, VA 086 .......................................................... 35 4.5 Absorption spectra of camphorquinone in 1-hexanol ............................................... 36 4.6 Double bond conversion versus time in polymerization experiment induced by visible light with CQ/EDAB as an initiating system ................................................. 37 4.7 Copolymerization profile for acrylated glyphosate and 2-hydroxyethylene acrylate mass determination by SEC ......................................................................... 38 4.8 SEC profile of poly(acrylated glyphosate) ................................................................ 39 x 4.9 Calibration curve for molecular mass determination of poly(acrylated glyposate) .. 40 5.1 Results of C(P)-N-C=C Amide bond rotational analysis for both AA and MA ....... 41 5.2 Results of C=C-C=O acryloyl bond rotational analysis for both AA and MA ....... 43 5.3 B3LYP/6-31++G(d,p) optimized structures of the lowest energy isomers of AA and MA ............................................................................................................ 43 6.1 DH 5α growth curve ................................................................................................. 45 6.2 Number of colonies on M9 agar plates loaded with glyphosate , acrylated glyphosate, and pure M9 agar ................................................................................... 46 6.3 Growth response of Synechococcus 7002 to the treatment with glyphosate and acrylated glyphosate ................................................................................................