DESIGN, SYNTHESIS, AND EVALUATION OF NOVEL ANTIMICROBIALS FOR THE ERADICATION OF BIOFILMS by Danica Jade Walsh A dissertation submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Organic Chemistry MONTANA STATE UNIVERSITY Bozeman, Montana April 2020 © COPYRIGHT by Danica Jade Walsh 2020 All Rights Reserved ii DEDICATION This dissertation is dedicated to my father, Dr. Thomas J. Walsh. iii ACKNOWLEDGEMENTS First, I would like to thank Dr. Tom Livinghouse for his dedication to my success as a chemist and his unyielding commitment in lab. I would also like to thank my co- advisor, Dr. Phil Stewart for his constant support and advice. Committee members as well as MSU staff, past and present, who have kindly supported me and provided me with continuous inspiration and encouragement over the years; Professors Tom Livinghouse, Phil Stewart, Mary Cloninger, Sharon Neufeldt, Darla Goeres and other dedicated mentors, Betsey Pitts, Scott Busse, Steve Holmgren, and Doreen Brown. Ultimately, my success in graduate school could not have been achieved without the support of my thoughtful family & friends; Dr. Tom Walsh, Kathrine Walsh-Spurgin, Alison Walsh, Adrienne Arnold, Harrison Bly, Aoife Casey, Jonas David, Sharon Dorsey, Pieter Gerrits, Pieter’s mom, Alex Hintz, Casey Kennedy, Collin Miller, Sam Phalan, Billie Smith, Martha Welander, and Sam Ziegler. I would also like to thank past and present group members; Yenny Chase-Bayless, Greg Durling, Khoi Hong, Ky Mickelsen, Brian Pettygrove, Jeff Simkins and Sean Zabawa. Montana State University, National Institute of Health, and Juan Valdez for the Endowment of the Sciences have all contributed to this work. iv TABLE OF CONTENTS 1. GENERAL PROJECT BACKGROUND ............................................................. 1 1.1 Project Overview ............................................................................................ 1 1.2 General Background ....................................................................................... 3 1.2.1 Biofilm growth and development ..................................................... 3 1.2.2 Biofilms in industry ......................................................................... 5 1.2.3 Current anti-biofilm methods ........................................................... 6 1.2.4 Prodrug applications ........................................................................ 8 2. ANTIMICROBIAL ACTIVITY OF NATURALLY OCCURRING PHENOLS AND DERIVATIVES AGAINST BIOFILM AND PLANKTONIC BACTERIA .............................................................................. 11 Contribution of Authors and Co-Authors ............................................................ 11 Manuscript Information...................................................................................... 12 2.1 Abstract ........................................................................................................ 13 2.2 Introduction .................................................................................................. 13 2.3 Materials and Methods ................................................................................. 20 2.3.1 Experimental general information .................................................. 20 2.3.2 Efficacy of naturally occurring phenols and derivatives on inhibiting planktonic cells ....................................... 21 2.3.3 Efficacy of naturally occurring phenols and derivatives on killing planktonic cells ............................................. 22 2.3.4 Efficacy of naturally occurring phenols and derivatives on biofilms..................................................................................... 22 2.3.4.1 Biofilm eradication concentration assays ............................ 22 2.3.4.2 Center for disease control (CDC) biofilm reactor evaluation ............................................................... 23 2.3.5 Chemical synthesis procedures....................................................... 24 2.3.5.1 Preparation of 2-(2-propen-1-yl)-6- (1-methylethyl) 3-methylphenol (1b). Representative procedure ................................................... 24 2.3.5.2 Preparation of 2-(2-n-propyl)-6-(1-methylethyl)- 3-methylphenol (1c). Representative procedure .................. 25 2.4 Results and discussion .................................................................................. 26 2.5 Conclusion ................................................................................................... 39 2.6 Conflicts of Interest ...................................................................................... 40 2.7 Acknowledgements ...................................................................................... 40 3. SULFENATE ESTERS OF SIMPLE PHENOLS EXHIBIT ENHANCED ACTIVITY AGAINST BIOFILMS ............................................. 41 Contribution of Authors and Co-Authors ............................................................ 41 v TABLE OF CONTENTS CONTINUED Manuscript Information...................................................................................... 42 3.1 Abstract ........................................................................................................ 43 3. 1. Introduction ................................................................................................ 43 3.2. Materials and Methods ................................................................................ 46 3.2.1 Synthetic reagents and bacteria ...................................................... 46 3.2.2 Efficacy of phenols and derivatives on inhibiting planktonic cells .............................................................. 47 3.2.3 Efficacy of phenols and derivatives on biofilms ............................. 47 3.2.4 Measuring rate of hydrolysis of sulfenate derivatives ..................... 48 3.2.5 Synthesis of Preparation of ............................................................ 48 3.3 Results and discussion .................................................................................. 49 3.3.1 Disinfectant activities in the planktonic state...................................50 3.3.1.1 Parent phenols ....................................................................50 3.3.1.2 Trichloromethylsulfenate esters ....................................... 55 3.3.2 Disinfectant activity against biofilms .................................. 56 3.3.2.1 Parent phenols...................................... ..................58 3.3.2.2 Sulfenate esters .................................................... 60 3.3.2.3 Comparison of phenols and sulfenates ............................................................. 62 3.3.3 Analysis of sulfenate degradation ....................................... 63 3.4 Conclusion ................................................................................................... 65 3.5 Conflicts of Interest ...................................................................................... 66 3.6 Acknowledgements ...................................................................................... 66 4. ENHANCED ANTIMICROBIAL ACTIVITY OF PRODRUG PHENOLS AGAINST BIOFILMS AND PLANKTONIC BACTERIA ....................................................................................................... 67 Contribution of Authors and Co-Authors ............................................................ 67 Manuscript Information...................................................................................... 68 4.1 Abstract ........................................................................................................ 69 4.2 Introduction .................................................................................................. 69 4.3 Materials and Methods ................................................................................. 72 4.3.1 Synthetic reagents and bacteria ...................................................... 72 4.3.2 Efficacy of naturally occurring phenols and derivatives on inhibiting planktonic cells ......................................................... 73 4.3.3 Efficacy of naturally occurring phenols and derivatives on biofilms..................................................................................... 73 4.3.3.1 Biofilm eradication concentration assays: ........................ 73 4.3.3.2 Center for disease control (CDC) biofilm reactor evaluation: ...................................................................... 74 4.3.4 Enzyme assay ................................................................................ 75 4.3.5 General chemical synthesis procedure ............................................ 76 vi TABLE OF CONTENTS CONTINUED 4.3.5.1 Preparation of diethyl 2,2'-((5-allyl-2-hydroxy-3- methoxybenzyl)azanediyl)diacetate (1c) ............................. 76 4.3.5.2 Preparation of diethyl 2,2'-((5-allyl-2-hydroxy-3- methoxybenzyl)azanediyl)diacetic acid (1d) ....................... 76 4.3.5.3 Preparation of bis(acetoxymethyl) 2,2'-((5-allyl-2-hydroxy-3-methoxybenzyl) azanediyl)diacetate (1b) ...................................................... 76 4.4 Results and discussion .................................................................................. 77 4.5 Conclusion ................................................................................................... 90 4.6 Conflict of Interest ....................................................................................... 90 4.7 Acknowledgements