ABSTRACT Title of Dissertation: SYNTHESIS AND MOLECULAR RECOGNITION PROPERTIES OF ACYCLIC CUCURBIT[N]URIL AND ITS DERIVATIVES Sandra A. Zebaze Ndendjio, Doctor of Philosophy, 2020 Dissertation directed by: Professor Lyle Isaacs, Department of Chemistry & Biochemistry The study of molecular containers has accelerated dramatically since the 1960's. The introduction of cucurbit[n]urils has contributed tremendously and continues to contribute to the expansion of the field. Chapter 1 introduces cucurbit[n]urils and their molecular recognition properties toward amino acids, peptides, and Insulin. Followed by an overview of the history of CB[n] and their analogs made with modified glycoluril backbone, solubilizing groups, alkyl arm linker, and type of aromatic arms. Chapter 2 describes the application of the acyclic CB[n], Calabadion 1 and 2 for the molecular recognition of amino acids, peptides, and Insulin. The results show that 1 and 2 have preferential binding affinity toward aromatic (e.g. H-Phe-NH2) and di-cationic (e.g. H-Lys-NH2) amino acid amides. Electrostatic interactions between the tetraanionic 1 and 2 with the amino acid guests (in their N-acetylated, zwitterionic, or CO-NH2 forms) was demonstrated to dramatically influence the strength of the recognition process. The binding affinity of 1 and 2 toward insulin was compared to that of CB[7], respectively (Ka= 1.32 ? 105 M-1 for 1, 3.47 ? 105 M-1 for 2, and 5.59 ? 105 M-1 for CB[7]) which showed comparable levels of affinity between these three hosts. Chapter 3 introduces a new acyclic CB[n] featuring a central glycoluril trimer with sulfonated triptycene aromatic sidewalls. It was observed that the binding affinity increases as the alkyl chain length of the guest increases. An x-ray crystal structure reveals an overall out-of-plane distortion of the aromatic sidewalls and intermolecular packing driven by interactions between the external faces of the triptycene sidewalls. Finally, the trimer host was shown to bind strongly to fentanyl which suggests potential usage as in vivo reversal agent. In chapter 4, an analog of 1 was synthesized in which the (CH2)3 linking group was removed. This structural change brings the anionic sulfate substituents closer to the electrostatically negative ureidyl C=O portals. We find that this new host displays preferential binding affinity toward quaternary ammonium ioins and a higher binding affinity toward dications compared to 1. Most impressive are the nanomolar binding affinities toward rocuronium and vecuronium which suggests potential application for in vivo reversal agent. SYNTHESIS AND MOLECULAR RECOGNITION PROPERTIES OF ACYCLIC CUCURBIT[N]URIL AND ITS DERIVATIVES by Sandra A. Zebaze Ndendjio Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2020 Advisory Committee: Professor Lyle Isaacs, Chair Professor Jeffrey Davis Professor Philip DeShong Professor Myles Poulin Professor Akua Asa-Awuku, Dean's representative ? Copyright by Sandra A. Zebaze Ndendjio 2020 Dedication To my parents, particularly, my mother Marie T. Lekane, but also my three siblings, Sonia Ndeloa, Wilfried Zebaze, and Rolling Zebaze, and my aunt Madeleine Dongmo. ii Acknowledgements I would like to thank my Ph.D. advisor Dr. Lyle Isaacs for his support, guidance and mentoring in the past five years. I'm forever thankful for the amazing opportunity that was offered to me by Dr. Isaacs, the chance to work in the forefront of scientific research. I experience many challenges, setbacks, and mental fatigues, but throughout it all Dr. Isaacs never gave up on me even when I was ready to abandon the project. Thanks to him, I learned to be resilient and rigorous. His strong work ethics became an example for me, as I learned to raise my own standard and develop a higher self-confidence. These priceless skills have made me into a better chemist. I would like to acknowledge Dr. Philip DeShong who not only encouraged me during my undergraduate studies but continued to inspire and push me to reach my best during my graduate studies. Additionally, Dr. Herman Sintim who first introduced me to the world of research as an undergraduate student at the University of Maryland in College Park. My successful journey to my Ph.D. was made possible because of the financial support from the Ronald E. McNair program and the Louis- Stokes Alliance for Minority program (LSAMP), the National Science Foundation (CHE-1404911, CHE-1807486), the National Institutes of Health (GM132345, GM124270). I would also like to thank former and current Isaacs group members, namely, Brittany Vinciguerra, Shweta Ganapati, David Sigwalt, Soumen Samanta, Xiaoyong Lu, Wenjin Liu, Weijian Xue, Nicholas Yvanez, Steven Murkli, Kimberly Brady, David King, Delaney Patterson, Ming Cheng, and Chunlin Deng. My labmates have iii helped me tremendously when dealing with technical difficulties particularly with instrumentations and literature reviews for my research project. A special thank you to my undergraduate student Rohan Shah who has assisted me in setting up multiple experiments and running Isothermal Titration Calorimetric studies. I would like to extend my sincere gratitude to Dr. Peter Zavalij and Dr. Fu Chen for assistance with X-ray crystallography and Nuclear Magnetic Resonance (NMR). I would also like to thank Dolores Jackson for emotional support and tips on time management, Dr. Theodore Kwaku-Dayie and all the members of the National Organization of Black Chemists and Chemical Engineers (NOBCChE) organization at the University of Maryland. Finally, I would like to thank my family members in the United States, Cameroon, and Gabon. My mother and my aunt who have supported me from the beginning and brought me food when I was overwhelmed with work. My siblings for providing me with entertainment when I was stressing out with my research project. Jude Ndeloa who fixed my computer and my two nephews Ethan and Liam Ndeloa. iv Table of Contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Table of Contents .......................................................................................................... v List of Tables ............................................................................................................... iv List of Figures ............................................................................................................ viii List of Schemes??????????????????????????... xii List of Abbreviations ................................................................................................. xiii Chapter 1: Introduction to Molecular Containers ......................................................... 1 1.1 Introduction to Molecular Recognition ............................................................... 1 1.2 Introductioin to Macrocyclic Hosts .................................................................... 2 1.3 The Cucurbit[n]uril Family of Molecular Containers ....................................... 11 1.3.1 Molecular recognition of Cucurbit[n]uril (CB[n]) towards amino acids ?14 1.3.2 Molecular recognition of Cucurbit[n]urils toward peptides ...................... 17 1.3.3 Molecular recognition of Cucurbit[n]urils toward proteins ....................... 18 1.4 Acyclic Cucurbit[n]urils .................................................................................. 20 1.5 Acyclic Cucurbit[n]urils derivatives ................................................................. 23 1.5.1 Modification of the solubilizing groups .................................................... 24 1.5.2 Modifications of aromatic arms ................................................................. 26 1.5.3 Modifications of glycoluril backbone ........................................................ 29 1.5.4 Modifications of alkyl linker on aromatic arms ...???????...?...31 1.6 Conclusion ........................................................................................................ 32 Chapter 2: Molecular Recognition Properties of Acyclic Cucurbit[n]urils Toward Amino Acids, Peptides, and a Protein ........................................................................ 33 2.1 Introduction ....................................................................................................... 33 2.2 Results and discussion ...................................................................................... 37 2.2.1 Goals of the study ...................................................................................... 37 2.3 1H NMR investigations of calabadion?guest binding ....................................... 38 2.4 Isothermal titration calorimetry (ITC) determination of host?guest energetics ............................................................................................................. 43 2.4.1 Discussion of trends in the thermodynamic parameters ............................ 44 2.4.2 Influence of N-acetylation and C-amidation .............................................. 49 2.5 Influence of neighbouring residues ................................................................... 50 2.6 Molecular recognition of insulin by 1, 2, and CB[7] ........................................ 52 2.7 Conclusion ........................................................................................................ 53 Chapter 3: Triptycene Walled Glycoluril Trimer: Synthesis and Recognition Properties .................................................................................................................... 54 3.1 Introduction ....................................................................................................... 54 3.2 Results and discussion ...................................................................................... 57 3.2.1 Design, synthesis, and characterization of host 1 ...................................... 57 3.2.2 Self-association properties of host 1 .......................................................... 58 3.2.3 X-ray crystal structure of 1 ........................................................................ 59 3.2.4 Qualitative 1H NMR host?guest recognition study ................................... 61 3.3 Measurement and discussion of the thermodynamic parameters of complex formation ................................................................................................................ 63 v 3.3.1 Magnitude of binding constants and enthalpies ......................................... 65 3.3.2 Influence of diammonium ion length ......................................................... 66 3.3.3 Drugs of abuse ........................................................................................... 67 3.3.4 Influence of guest charge ........................................................................... 67 3.3.5 Influence of the cationic headgroup ........................................................... 68 3.3.6 Influence of guest hydrophobic residue ..................................................... 68 3.3.7 Comparisons between hosts ?????????????????....69 3.4 Conclusion ........................................................................................................ 70 Chapter 4: Acyclic Cucurbit[n]uril-Type Receptors: Optimization of Electrostatic Interactions for Dicationic Guests .............................................................................. 71 4.1 Introduction ....................................................................................................... 71 4.2 Synthesis and characterization .......................................................................... 72 4.3 1H NMR investigation....................................................................................... 75 4.4 Isothermal Titration Calorimetry investigation ................................................ 77 4.4.1 binding to drug of abuse ............................................................................ 81 4.4.2 Serving as reversal agents for neuromuscular blocking molecules ........... 82 4.5 Conclusion ........................................................................................................ 82 Chapter 5: Conclusion................................................................................................ 84 5.1 Summary .......................................................................................................... 84 5.2 Future Work ...................................................................................................... 86 Appendix I .................................................................................................................. 89 Appendix II.??...??????????????????????.?...... 221 Appendix III??????????????????????????..? 277 Bibliography ............................................................................................................. 333 vi List of Tables Chapter 1 Table I-1. Thermodynamic parameters for the complexes CB[6] with amino acids Chapter 2 Table II-1. Thermodynamic parameters obtained by ITC for the interaction of 1 and 2 with the amino acid amides, N-acetyl amino amides, and amino acids. Table II-2. Thermodynamic parameters obtained by ITC for the interaction of 1 and 2 with tripeptides. Table II-3. Thermodynamic parameters obtained by ITC for the interaction of CB [7], 1, and 2 with Insulin. Chapter 3 Table III-1. Binding constants (K -1a, M ) and binding enthalpies (?H, kcal mol-1) measured for 1?guest. Binding constants (Ka, M-1) measured for DimerTrip?guest, and M1?guest complexes (298 K, 20 mM NaH2PO4 buffered water, pH 7.4) Chapter 4 Table IV-1. Binding constants measured by ITC for host?guest complexes of 1. Comparative data for M1 are drawn from the literature. Conditions: 20 mM sodium phosphate buffered H2O, pH 7.4, 25?C. vii List of Figures Chapter 1 Figure I-1. Illustration of host? guest complexation. a) complexation between a C- shaped container and a guest. b) complexation between a cyclic shaped container and a guest. P4 Figure I-2. Chemical structure of Spherand and Podand. P5 Figure I-3. Chemical structures of selected molecular container molecules including ?-Cyclodextrins, CB[n], 4-sulfo Calix [4]arene, and Klaerners phosphorylated molecular tweezers and Nolte's molecular clips. P7 Figure I-4. Chemical structure of Sugammadex. P9 Figure I-5. Chemical structure of Blue box and general structure Pillarenes. P11 Figure I-6. a) Recognition properties of CB[n]. b) Typical alkyl ammonium guests for CB[n]. P14 Figure I-7. a) Illustration of CB[8]?MV complex. b) Chemical structures of tryptophan (Trp) and Trp derivatives. c) CB[8] promoted dimerization of Trp and Methyl Viologen. P16 Figure I-8. a) Structures of CB[7] and b) Illustration of non-covalent interactions (ion-dipole interactions and hydrophobic effect) governing the complexation of CB[7] and N-terminal phenylalanine. P18 Figure I-9. Illustration of N- terminal residue of phenylalanine of insulin binding to CB[7]. P20 Figure I-10. Synthesis of C-shaped glycoluril oligomers 2-6. P20 viii Figure I-11. Chemical structures of Calabadion 1 and 2 also known as M1 and M2. P21 Figure I-12. Chemical structures of acyclic CB[n] derivatives with different solubilizing groups; 1 (SO3Na), 2 (NH3Cl), and 3 (OH). X-ray crystal structure of 2 and 3 in self-complexation form and open form. P24 Figure I-13. Chemical structure of Zhang 1 5 and triptycene walled acyclic cucurbit[n]uril 6. P29 Figure I-14. Chemical structures Trimer acyclic CB[n] host 1 and 2. P30 Figure I-15. General structure of acyclic CB[n] with 2 or 4 carbon chain alkyl linkers. P32 Chapter 2 Figure II-1. Chemical structures of CB[n] and Calabadions 1 and 2. P33 Figure II-2. Chemical structure of amino acids, amino amides, and N-acetyl amino amides used as guests in this study and illustration of the geometries and driving forces involved in their complexation with macrocyclic CB[n]. P35 Figure II-3. 1H NMR spectra recorded (400 MHz, RT, 20 mM NaH2 PO4 buffered D2O , pD 7.40) for: a) H-Phe-NH2 (5 mM), (b) 1 (1 mM) and H-Phe-NH2 (2 mM), c) 1 (1 mM) and H-Phe-NH? (1.5 mM), (d) 1 (1 mM) and H-Phe-NH2 (1 mM), e) 1 (1 mM) and H-Phe-NH2 (0.5 mM), (f) 1 (2 mM). P40 Figure II-4. (Color online) Schematic illustrations of the geometry of (a) 1?H-Phe- NH2 and (b) 1?H-Lys-NH2 complexes. P41 ix Figure II-5. Job plot constructed for the interaction of 1 with H-Lys-NH2 ([1] + [H- Lys-NH2] = 1 mM) monitoring the chemical shift of Hf on 1 by 1H NMR spectroscopy (600 MHz, RT, 20 mM NaH2PO4 buffered D2O, pD 7.40). The solid line serves as a guide for the eye. P43 Figure II-6. (a) Thermogram obtained during the titration of 1 (100 ?M) in the cell with H-Phe-NH2 (1 mM) in the syringe (298.0 K, 20 mM sodium phosphate buffered H2O, pH 7.4) and (b) fitting of the data to a 1:1 binding model with K? = 2.62 x 106 M-1 and ?H = ?17.1 kcal mol-1. P44 Figure II-7. (Colour online) Schematic illustration of the complexes of 1 with H-Phe- CO2?, H-Phe-NH2, and Ac-Phe-NH2 along with their driving forces and relative stabilities. P50 Chapter 3 Figure III-1. Structure of CB[n] (n = 5, 6, 7, 8, 10, 14), acyclic CB[n]-type receptor M1, and DimerTrip. P55 Figure III-2. Plot of chemical shift of Hb versus [1] used to determine the self- association constant Ks = 480 ? 81 M-1 for 1. P59 Figure III-3. Cross-eyed stereoviews of the crystal structure of 1. (a and b) Two independent molecules of 1. (c) Packing of the two independent molecules of 1 into a dimeric unit. Color code: C, grey; H, white; N, blue; O, red; S, yellow; F, green. P60 Figure III-4. Structures of guests 4?26 used in this study. P61 Figure III-5. 1H NMR spectra recorded (600 MHz, RT, 20 mM sodium phosphate x buffered D2O , pH 7.0) for: (a) guest 8 (2 mM), (b) a mixture of 1 (250 mM) and 8 (500 mM), (c) a mixture of 1 (250 mM) and 8 (250 mM), and (d) host 1 (250 mM). P63 Figure III-6. (a) ITC thermogram recorded during the titration of host 1 (100 mM) in the cell with guest 5 (1.0 mM) in the syringe, (b) fitting of the data to a 1: 1 binding model with K = 1.33?106 M-1a . P64 Chapter 4 Figure IV-1. Cross eyed stereoviews of the x-ray crystal structures of a) 1?6d, and b) 1?6a. Color code: C, gray; H, white; N, blue; O, red; H-bonds red-yellow striped. P74 Figure IV-2. Structures of guests 5 ? 23 used in this study. P76 Figure IV-3. 1H NMR spectra (D2O, 600 MHz) recorded for: a) 1 (1 mM), b) a mixture of 1 (1 mM) and 6d (1 mM), c) a mixture of 1 (1 mM) and 6d (2 mM), and d) 6d (1 mM). Resonances for bound guests as marked with an asterisk (*). P77 Figure IV-4. a) Thermogram recorded during the titration of a mixture of 1 (100 ?M) and 13 (2 mM) in the cell with a solution of 6d (1.0 mM) in the syringe, and b) fitting of the data to a competition binding model to extract Ka = 6.79 ? 109 M-1 and ?H = - 12.1 kcal mol-1. P79 Chapter 5 Figure V-1. Series of sulfate hosts composed of trimer sulfate host 10, dimer sulfate host 11, monomer sulfate host 12. P88 xi List of Schemes Chapter 1 Scheme I-1. Synthesis of CB[n]. Scheme I-2. Synthesis of Acyclic CB[n] (M 1). Chapter 3 Scheme III-1. Synthesis of host 1 Chapter 4 Scheme IV-1. Synthesis of host 1. Conditions: a) TFA, 25 ?C, N2, 16 h; b) pyridine sulfur trioxide, pyridine, 90 ?C, N2, 18 h. Chapter 5 Scheme V-I. Proposed synthesis of a new acyclic CB[n] 8 which would lead to 9. xii List of Abbreviations 1H NMR proton nuclear magnetic resonance 13C NMR carbon-13 nuclear magnetic resonance ? angstrom Ach acetylcholine br broad BSA bovine serum albumin CB[n] cucurbit[n]uril CD cyclodextrin d doublet DPT 2,7-dimethyldiazaphenanthrenium D2O deuterium oxide DMSO dimethyl sulfoxide DOSY diffusion-ordered spectroscopy ESI-MS electrospray ionization-mass spectrometry EtOH ethanol g gram H hydrogen h hour HCl hydrochloric acid H2SO4 sulfuric acid HP-?-CD hydroxypropyl-?-cyclodextrin xiii Hz hertz IR infrared ITC isothermal titration calorimetry J coupling constant K kelvin Ka Binding affinity kg kilogram M molar m multiplet M.p. melting point m/z mass to charge ratio M+ molecular ion Me methyl MeOH methanol MHz megahertz mg milligram min minute mL milliliter mM millimolar MW molecular weight M1 Motor 1 M2 Motor 2 xiv M2C2 Motor 2 with a two-carbon long alkyl chain linker M2C4 Motor 2 with a four-carbon long alkyl chain linker MBBI tetramethylbenzobis(imidazolium MV Methyl viologen N binding stoichiometry NaOH sodium hydroxide NaH2PO4 sodium monophosphate NMR nuclear magnetic resonance o ortho OH Hydroxyl group p para Ph phenyl ppm parts per million PXDA para-xylylene diammonium ion RT room temperature s singlet t triplet TFA trifluoroacetic acid USFDA Food and Drug Administration UV/Vis Ultraviolet/ visible ?? change in chemical shift ?H change in enthalpy xv ?G Change in free energy ?S change in entropy ?M micromolar ? pi xvi Chapter 1: Introduction to Molecular Containers 1.1 Introduction to Molecular Recognition Supramolecular chemistry also known as "Chemistry beyond molecule" was first conceptualized by Prof. Dr. Jean-Marie Lehn. Prof. Lehn describes supramolecular chemistry as the study of intermolecular forces such as non-covalent interactions (Hydrogen bonding, ion-dipole interactions, and ?-? interactions) between molecules that can be used to bring molecular entities together to form a supramolecular complex that often exhibit properties that are distinct from their molecular components.to form a new complex.1 Underneath the overarching theme of supramolecular chemistry, lies multiple conceptual and application areas including molecular self-assembly, molecular recognition, molecular interlocking, catalysis, drug solubilization and delivery2. Within the realm of biology, it is clear that many biological functions cellular transport, cell-cell communications and enzymatic functions are driven by high affinity and high selectivity molecular recognition events that are carefully orchestrated in space and time. The main focus of this dissertation is the synthesis and molecular recognition properties of a new class of supramolecular hosts known as cucurbit[n]urils (CB[n]) that also exhibit high binding affinity and high selectivity in aqueous solution toward hydrophobic ammonium ions. As the target guests we have focused, in part, on those guests that are components of or display interesting biological functions. Beyond the molecular recognition properties of CB[n]-type hosts lies interest in using supramolecular systems to control catalytic processes of designed systems.3 1 Molecular recognition is the specific association of two or more guests driven by non- covalent interactions including hydrophobic effect, hydrogen bonding, and shape and size complementarity, polarity etc.4 Enzymes use two mechanisms for molecular recognitions the "lock and key" mechanism and the "induced fit" mechanism5. The binding of the substrate to the active site of the enzyme is critical for the efficiency of the enzymatic activity. In the lock and key mechanism, the substrate fits precisely in the enzyme active site through shape and size complementarity. In the induced fit model, the conformation of the substrate and the enzyme changes to simultaneously to complement each other in order to induce the binding. Of major interest to supramolecular chemist is the synthesis of molecular artificial receptors able to mimic the functioning of enzymes in biological conditions.6 This introduction section discusses molecular hosts and their applications with a focus on cucurbit[n]urils, acyclic cucurbit[n]uril and its derivatives. 1.2 Introduction to Macrocyclic Hosts Macrocyclic receptors are host molecules that are able to bind to and sequester smaller molecule typically within a central cavity. Such macrocyclic receptors are often referred to as molecular container compounds because they are able to hold onto their guests in much the same way that real-world containers hold onto other objects. The encapsulation of guest molecules within these molecular containers results in new entities known as host?guest complexes that can display properties that are different from those of the components. For example, host?guest complexes may enhance the solubility of poorly soluble guests, can prolong the lifetime of guests that are prone to decomposition reactions, can suppress the vapor pressure of guests that 2 are malodorous, and can even influence the in vitro and in vivo activity of biological active guests.7 Of course, the successful creation of such functional systems requires the availability of high affinity host systems to effectively sequester the guests. Below, I present examples of other popular host systems that are used in these and related applications, along with some of the guiding principles in the creation or discovery of high affinity host systems. The 1987 Nobel Prize in Chemistry was awarded in part to Professor Donald J. Cram for his elucidation of the principle of preorganization,8-10 which is one route to enables the formation of stable host-guest complexes. The principle of preorganization states that the closer the geometry and solvation of the uncomplexed host and uncomplexed guest resemble their host?guest complex the more stable their host?guest complex will be.10 Accordingly, a key question in the field of supramolecular chemistry and more specifically molecular container chemistry is how to generate a preorganized host? Host?guest complexes generally feature a smaller guest molecule that is more or less surrounded by the larger host molecule (Figure I-1). 3 a) + Guest Acylic CB[n] Container Host guest complex b) + Guest cyclic CB[n] container Host guest complex Figure I-1. Illustration of host? guest complexation. a) complexation between a C- shaped container and a guest. b) complexation between a cyclic shaped container and a guest For example, Figure I-1a shows the geometry of the hypothetic complex formed between a roughly spherical guest and an acyclic C-shaped host. In order to assume the C-shaped conformation required for binding the hypothetical acyclic hosts typically must restrict many rotational degrees of freedom (e.g. (CH2)n linkers) which is energetically costly and thereby reduces the observed free energy of complexation. Accordingly, one way to reduce the energetic penalty incurred upon host?guest complexation is simply to pay the energetic price of conformational restriction during the synthesis by the formation of a macrocyclic host that has fewer conformational degrees of freedom. 4 Me Me Me Me Me O O O O H Me H Me O O Me O Me Me Me Me Me 6 Podand Me Spherand Figure I-2. Chemical structure of Spherand and Podand. A seminal example of the power of preorganization comes from the work of Cram who studied the binding of spherand and its acyclic analogue toward Li+ and observed a >17 kcal mol-1 decrease in affinity due to the cleavage of a single carbon- carbon bond (Figure I-2). Popular examples of macrocyclic host systems also include CB[n], cyclodextrins, 4-sulfo-calix[4]arene (Figure I-3). However, acyclic systems are also capable of exhibiting variable degrees of preorganization. For example, in 1978 Whitlock, the term "Molecular Tweezers" to refer to a class of acyclic molecular receptors with two flat arms, generally aromatic, separated by a more or less rigid spacer, that converge on one another and define a pocket for guest inclusion.11 The group of Professor Steven Zimmerman has made large contributions toward the development of the field of molecular tweezers. Of more direct relevance to the work described in this dissertation is the work of Prof. Roeland Nolte and Prof. Frank-Gerrit Klaerner on the glycoluril derived molecular clips and benzonorbornene derived molecular tweezers, respectively (Figure I-3). Klaerner?s phosphorylated 5 molecular tweezers are water soluble, exhibit high binding affinities, and selectivity toward arginine and lysine peptide residues up to 104 M-1.12-14 It was further demonstrated that they are able to function as inhibitors of amyloid plaque formation due to their specific interaction to the lysine residue of the amyloid ? protein.13, 15 Prof. Roeland Nolte?s group developed molecular clips composed of one central diphenyl glycoluril ring and two aromatic o-xylylene rings.16 By virtue of the conformational preferences of the seven-membered rings connecting the central bicyclic glycoluril unit to the aromatic sidewalls, these molecular clips feature a binding cleft. Nolte?s molecular clips are pre-organized despite the lack of a macrocycle, relying instead on the conformational preferences of the fused polycyclic ring system. 6 Figure I-3. Chemical structures of selected molecular container molecules including ?-Cyclodextrins, CB[n], 4-sulfo Calix [4] arene, and Klaerners phosphorylated molecular tweezers and Nolte's molecular clips. A brief description of the structures and binding properties of calixarenes, cyclodextrins, and various cyclophanes are given below to serve as an introduction to the broader field of molecular containers. Calixarenes are composed of n aromatic rings (n = 4 ? 8 mainly) ? typically phenols ? linked in the 2,6-positions (e.g. meta linkages) by methylene bridges.17, 18 The synthetic chemistry of calixarenes is particularly well developed which allows the preparation of numerous variants for 7 molecular recognition of cations, anions, and molecular species in organic solvents and in water.19, 20 Calixarenes are commercially available and have shown applications as ionophores for membrane transport, extractants, stationary phases, electrode ionophores and in optical, electrochemical, biological and chemical sensors.21 Calixarenes can exist in four different conformational forms, namely the cone, partial cone, 1,2-alternate, and 1,3-alternate conformers. Of highest relevance to molecular recognition of molecular guests is the cone conformation which features a bowl-shaped hydrophobic cavity. Of highest relevance to the research described in this dissertation is the molecular recognition properties of sulfo calix[4]arene (Figure I-3) that displays high solubility in water. Sulfo calix[4]arene is able to bind to hydrophobic molecules in water and particularly hydrophobic cations that benefit from ion-ion interactions and the hydrophobic effect upon complexation. The binding affinity of sulfo calix[4]arene toward such hydrophobic cations in water is typically in the range of 104 ? 105 M-1 which relatively low selectivity.22 Despite this low affinity and selectivity, sulfo calix[4]arene has a variety of biological applications including as an in vivo reversal agents for the toxicity of methyl viologen.23, 24 Cyclodextrins are another type of widely used and commercially available molecular hosts. Cyclodextrins are macrocyclic oligomers composed of n glucose rings (?- n = 6, ?- n = 7, ?- n= 8) connected by ?-1,4-glycosidic bonds. Cyclodextrins are cheap, commercially available, nicely soluble in water, and non- toxic. Cyclodextrins exhibit molecular recognition properties toward hydrophobic cations, anions, and neutral molecules with K values up to ? 104 M-1a . Cyclodextrins have been used for a variety of academic purposes including supramolecular 8 catalysis,25, 26 chemical sensors,27 stationary phases for chiral separations,28 and supramolecular polymers and materials.29 Cyclodextrins also have a variety of more practical everyday uses. For example hydroxypropyl-?-CD (HP-?-CD) is the active ingredient in the household product FebrezeTM where the cyclodextrin serves to release a fragrance molecule and take up odorant molecules when sprayed in the air.30 Cyclodextrins also have a variety of applications in pharmaceutical science. For example, HP-b-CD and sulfobutyl ether b-CD (SBE-CD, CaptisolTM ) are used to increase the solubility and thereby formulate for human delivery > $5 billion per year of various active pharmaceutical ingredients.31 Precise molecular engineering of ?- cyclodextrin allowed the creation of Sugammadex (figure I-4) which exhibits 107 M-1 affinity toward the clinically important neuromuscular blocking agents rocuronium and vecuronium.32 Sugammadex is a billion dollar per year drug that is marketed by Merck under the trade name BridionTM and is used as a reversal agent for these neuromuscular blockers post-surgically. SR RS O O OOH HO OH OHO O O OH SR HO RS O OH OH O Sugammadex O OH OH O OH SR OH O RS OOH OHO O OH OHO R = (CH2)2CO2Na O RS SR Figure I-4. Chemical structure of Sugammadex 9 The other most popular class of molecular container compounds are the cyclophanes which are macrocycles composed of aromatic building blocks and come in a variety of sizes and shapes. A complete description of all the different types of cyclophanes are beyond the scope of this introduction; the reader is referred to several reviews of aspects of this area.33-38 An increasingly popular class of cyclophanes are the pillar[n]arenes (Figure I-5) which are composed of n phenylene or arylene units which are linked by methylene bridges in the para positions. The synthetic chemistry of pillararenes has rapidly developed in the past decade and pillararenes soluble in both organic solvents and water are available. Pillararenes have been used for a wide variety of applications including the purification of gases, membrane transport, supramolecular polymers, chemical sensors, and an increasing number of biomedical applications.39, 40 Another class of cyclophanes that have drawn intense interest and ultimately garnered the 2016 Nobel Prize in Chemistry for the development of molecular machines for Professor Fraser Stoddart is the cationic cyclophane known as the blue box (Figure I-5).41 By virtue of its cationic viologen walls, the cavity of the blue box is highly electron deficient and constitutes a binding site for electron ring aromatic rings. Furthermore, the viologen units are electrochemically active and can form the radical cations upon electrochemical reduction. This change in the electronic properties of the aromatic walls changes its molecular recognition properties and allows the construction of a variety of molecular devices like molecular switches, shuttles, elevators, and the like.42-44 Most recently the Stoddart 10 group has greatly expanded the potential of this system by expanding the aromatic sidewalls and going into the third dimension to create cage systems.41, 43 n OH N N H2 C 4Cl HO n N N Pillar[n]arenes n=0 or n=1 n= 5 - 15 Figure I-5. Chemical structure of Blue box and general structure Pillar[n]arenes 1.3 The Cucurbit[n]uril Family of Molecular Containers The molecular containers described above (e.g. calixarenes, cyclodextrins, cyclophanes) were constructed from aromatic or sugar building blocks. An alternative building block known as glycoluril (1, Scheme I-1) has also become popular in supramolecular chemistry. Because glycoluril is a bicyclic ring system and because it is concave its incorporation into molecular hosts facilitates the curvature needed for macrocyclization to cucurbiturils, for the preparation of C- shaped compounds that form dimeric assemblies by H-bond driven self-assembly,45 and for the creation of molecular clips.46 In 1905 Prof. Robert Behrend reported the condensation reaction of glycoluril with formaldehyde (2 equivalents) in concentrated HCl followed by recrystallization from conc. H2SO4 yielded a crystalline substance whose structure was not elucidated, but which possessed the ability to form 11 complexes with inorganic salts and dye stuffs (Scheme I-1).47 The structural elucidation of the product of Behrend?s reaction would need to wait until 1981 when Mock and co-workers structurally characterized it and dubbed it cucurbituril due to its resemblance to the segmented geometry of pumpkins which are members of the cucurbitaceae family of plants. Subsequently, cucurbituril has been referred to as cucurbit[6]uril (CB[6]) because it is composed of six glycoluril units. When the groups of Kim and co-workers and Day and co-workers conducted the cucurbituril forming reaction under milder conditions there were able to form and isolate the key members of the cucurbit[n]uril (n = 5, 6, 7, 8) family of molecular containers.47-52 O O O O OO O N N HN NH N N N N NN N N NNO H H + HCl HN NH H H NN N N <100 oC N N N N NN NN O O O O OO O 1eq 2eq Cucurbit[n]uril (CB[n]) n-5 n = 5, 6, 7, 8, 10 1 Scheme I-1. Synthesis of CB[n]. The structure of CB[n] molecular containers feature glycoluril monomers linked by pairs of methylene bridges. CB[n] molecular containers are structurally rigid, highly symmetric (Dnh), and contain a hydrophobic cavity surrounded by two electrostatically negative ureidyl carbonyl portals. The height of each CB[n] homologue is generally 9.1? however, the volume of the cavity varies with the number of glycoluril repeat units. CB[5], the smallest homologue, has a volume of 68?3 making it optimal for inclusion of small gases such as N2. CB[6] has a volume 12 of 142?3 and CB[7] has a large volume of 242?3 and CB[8] has an even larger volume of 367?3. The even numbered homologues (CB[6] and CB[8]) have low solubility in water (< 100 ?M) whereas the odd numbered homologues (CB[5] and CB[7]) display higher (> 10 mM) solubility in water. The synthesis, molecular recognition properties, and applications of macrocyclic CB[n] hosts have been the subject of more than 2000 publications to date and the reader is referred to various authoritative reviews on the subject.47, 49, 53-55 The most unique feature of the molecular recognition properties of CB[n] molecular containers is that they exhibit remarkably high binding affinities (Ka routinely 106 M-1, often 109 M-1, and even up to 1017 M-1 in special cases)55, 56 hydrophobic (di)cations in water (Figure I-6). This remarkably tight binding is attributed to a combination of the ion-dipole interactions at each portal and the hydrophobic effect which is enhanced due to the presence of high energy waters in the cavity of the uncomplexed CB[n] hosts.55, 57-60 Equally importantly, because CB[n] are such selective hosts they often display large changes in binding affinity due to changes in pH as well as electrochemical, photochemical, or chemical stimuli.50 These unique properties of CB[n] have been harnessed to create CB[n] derived functional systems including drug delivery, supramolecular catalysts, chemical sensing, and drug solubilization.7, 61-64 Somewhat outside the scope of this introduction is the recognition behavior of CB[8] which is large enough to encapsulate two aromatic rings simultaneously which has been used by the Kim Scherman, and Urbach groups extensively to create molecular machines, for the 13 recognition of amino acids, peptides and proteins, and for supramolecular materials chemistry.4, 63, 65-69 a) Ion-dipole interactions O O O NH OO3 O N N Polar portal N N N N N N N N N N N N N N N N Hydrophobic cavityN N N N N N O O O O OH3N O Hydrogen bonding b) Br NH2 N I N N I-4 I-5 HCl I-7 2II-3 H N NH H N N2 2 2 I-2 2HCl NH I-6 2HCl 2 Figure I-6. a) Recognition properties of CB[n]. b) Typical alkyl ammonium guests for CB[n] 1.3.1 Molecular recognition of cucurbit[n]uril (CB[n]) towards amino acids Buschman et al. were the first to perform binding studies of CB[6] with amino acids and amino alcohols using isothermal titration calorimetry (ITC) (Table I-1).70, 71 The study was performed in 50% (v/v) aqueous formic acid at 25?C with select amino acids including Glycine (Gly), Alanine (Ala), Valine (Val), and phenylalanine (Phe). 14 There was little change in binding strength despite the difference in size and hydrophobicity of the amino acids. It is believed that the amino acids formed exclusion complexes at the ureidyl carbonyl portal of CB[6] instead of going inside its hydrophobic cavity. However, this study opened the door for further investigations of CB[n]s with amino acids. Table I-1. Thermodynamic parameters for the complexes CB[6] with amino acids.71 Amino acids K (M-1) ?H (kcal?mol-1a ) T?S (kcal?mol-1) Gly 4.7 ? 103 -3.1 1.9 Ala 1.0 ? 103 -1.7 2.4 Val 1.4 ? 103 -1.0 3.2 Phe 1.4 ? 103 -1.6 2.7 Kim et al. demonstrated that CB[8] could simultaneously bind two guests, namely methyl viologen (MV) and 2,6-dihydroxynaphthalene (HN).72 Subsequently, they also showed that the CB[8]?MV complex was able to bind with the amino acids tryptophan (Trp) and tyrosine (Tyr) by the insertion of the aromatic sidechain into the remaining cavity to form the CB[8]?MV?amino acid ternary complexes.73 A complete study of the binding of CB[8]?MV toward all 20 amino acids was subsequently performed by the Urbach group, which revealed the selectivity of CB[8]?MV toward Trp, Phe, and Tyr. No binding was observed for the remaining amino acids.74-76 Urbach and co-workers also studied the influence of overall guest charge on their ability to successfully form CB[8]?MV?amino acids complexes (Figure I-7).77 Postively charged guests 1 and 2 bind more strongly than zwitterionic free amino acid Trp, which in turn binds more strongly than negatively charged guests 3 and 4 by at least one order of magnitude.77 The enhancement of binding is due to the fact that the 15 ureidyl C=O portal of CB[n] is electrostatically negative and therefore a cation binding site; conversely the ureidyl C=O portals of CB[n] reject guests that place additional negative charge at the portal.48, 78, 79 Other studies demonstrated that DPT or MBBI could be used instead of MV to form a complex with CB[8] which display similar binding to Trp but with enhanced optical outputs.61, 80 These studies of CB[8] heteroternary complex formation has been most useful in the context of sensing applications and electrochemical molecular machines. In a separate line of inquiry, Prof. Kim and Inoue found that CB[8] could bind two equivalents of Trp or Phe on their own with binding constants of 6.9 ? 107 and 1.1 ? 108 M-1 respectively which has subsequently been used to promote peptide and protein dimerization as described below.74 An elongated CB[6] derivative was able to bind the aromatic amino acids including His and the (?)-bis-nor-seco-cucurbit[6]uril host studied in the Isaacs lab was found to bind Phe with a XX:1 diastereoselectivity.81, 82 The binding of CB[7] toward amino acids was performed by the Kim and Urbach group,59, 83 which revealed analogous binding preferences with higher binding affinities. a) b) HN H N NH3 NH3 O N H ON 1 2 NH3 H O N H O NN Trp NHAc CB[8]?MV O O OO 3 4 16 c) + + + + + CB[8] Methyl Viologen Trp CB[8] MV Trp Figure I-7. a) Illustration of CB[8]?MV complex. b) Chemical structures of tryptophan (Trp) and Trp derivatives.77 c) CB[8] promoted dimerization of Trp and Methyl Viologen.4 1.3.2 Molecular recognition of cucurbit[n]urils toward peptides Since peptides consist of multiple amino acid residues, molecular recognition of peptides adds another layer of challenges and complexity. Peptide contain an oligoamide backbone, which can have different structures based on the amino residue sequence and have multiple sidechains and charges. Binding studies were performed with CB[6] and various dipeptides and tripeptides.70, 71, 84 The binding constants ranged from 3.7 ? 102 to 1.5 ? 103 M-1 concluding the peptides formed exclusion complexes with CB[6].84, 85 As previously mentioned, CB[8]?MV forms a stronger binding complex with positively charged Trp derivatives. Based on this information it was hypothesized that CB[8]?MV would selectively bind to N-terminal Trp in peptides due to a combination of N-terminal NH +3 ???O=C ion-dipole interactions and the hydrophobic effect. A binding study of CB[8]?MV toward a series of tripeptides containing Trp residues at the N-terminus, internal position, and C-terminus were used to prove this hypothesis.77 The results showed the application of CB[8] as a 17 synthetic receptor which is capable of sequence specific peptide recognition.77 CB[8] has been used in multiple applications such as self-assembled multivalent receptors for peptides,86 peptide dimerization,87 and selective capture and release of N-terminal Trp peptide.69 a) b) ion-dipole N ON N N interactionsN O +H peptide N N 3 N N O O NN O O N N O N N CB[7] O O N N N O O N N N O O N O N Hydrophobic effectN O N N N N N CB[7]?Phe-peptide Figure I-8. a) Structures of CB[7] and b) Illustration of non-covalent interactions (ion-dipole interactions and hydrophobic effect) governing the complexation of CB[7] and N-terminal phenylalanine.88 In 2006, Kim et al. reported that CB[7] was able to selectively recognize N- terminal Phe in the context of dipeptides (Figure I-8).88 This study focused on the ability of CB[7] to discriminate between different zwitterionic peptides and cationic peptides compared to their sequence diastereomers.67, 88 Kim et al., also demonstrated that CB[7] had selective binding towards other N-terminal aromatic peptides.88 1.3.3 Molecular recognition of cucurbit[n]urils toward proteins The information obtained from the previously mentioned studies demonstrates the potential use of CB[n]s for the molecular recognition of proteins. Multiple 18 research groups have taken steps toward this goal including the Wang research group, that use CB[8] to mediate the binding of a photodynamic therapy sensitizer to bovine serum albumin (BSA)89 and Nau and coworkers, that use CB[7] to enhance the fluorescence and binding of a dye (Brilliant Green) to BSA.90 Most importantly, however, the Urbach research group has shown that CB[7] exhibits a high, selective binding affinity (Ka = 1.5 x 10? M-1) toward the N-terminal phenylalanine residue of Insulin (Figure I-6).63 Interestingly, the x-ray crystallographic results show that the N- terminus needs to unravel slightly to accommodate the CB[7] unit. By virtue of this ability to unwind, Urbach asserts that the N-terminus of proteins constitutes a privileged recognition domain. Subsequently, a collaboration between Urbach and Isaacs should that immobilized CB[7] could be used to perform affinity purification of proteins (insulin and human growth hormone) from within complex mixtures by a catch and release process.63 This preceding sections have shown that CB[n] possess a variety of interesting molecular recognition properties and can be used in a variety of application areas. However, CB[n] do possess some limitations including their low water solubility and the difficulty of modifying them. Chapter 2 of this dissertation details the molecular recognition properties of two prototypical acyclic CB[n]-type receptors toward amino acids, peptides, and the protein insulin. 19 Figure I-9. Illustration of N- terminal residue of phenylalanine of insulin binding to CB[7].63 1. 4 Acyclic cucurbit[n]urils O O O O n= 0 HN NH CH O HN N N N N NH 2 3 n= 12 H H H H H H H H 4 n= 2 HN NH HCl HN N N N N NH 5 n= 3 6 n= 4 O O O n O 1 Figure I-10. Synthesis of C-shaped glycoluril oligomers 2-6. The research theme of the Isaacs group is centered around developing an understanding of the mechanism of CB[n] formation and using that knowledge to create new CB[n]-type receptors with enhanced functions. As part of these mechanistic studies, the Isaacs group isolated methylene bridged glycoluril dimer ? hexamer (2 ? 6) and studied their molecular recognition properties toward hydrophobic dications (Figure I-10).91, 92 They found that the acyclic hexamer still 20 bound quite nicely to the hydrophobic dications with a loss of only about 100-fold in binding affinity relative to macrocyclic CB[6]. Based on this result, the Isaacs group realized that acyclic CB[n]-type receptors would retain the essential recognition elements of macrocyclic CB[n], but unfortunately, hexamer is not very soluble and can undergo decomposition reactions. Accordingly, the Isaacs groups saw the opportunity to create acyclic CB[n]-type receptors to address issues of solubility and enable more straightforward functionalization. O O O O OR OR N N N N N N N N H HH H N N N N N N N N OR OR O O O O R = (CH Motor 1 2)3SO3Na O O O O OR OR N N N N N N N N H HH H N N N N N N N N OR OR O O O O R = (CH Motor 2 2)3SO3Na Figure I-11. Chemical structures of Calabadion 1 and 2 also known as M1 and M2 Figure I-11 shows that structure of the two prototypical acyclic CB[n]-type receptors (M1 and M2) studied extensively by the Isaacs group over the past decade for use in biomedical applications.7 The structure of M1 and M2 feature a central glycoluril tetramer which gives the molecular an overall C-shape and the ability to bind to hydrophobic cations, two terminal aromatic walls which allow it to engage in ??? interactions with guests that contain aromatic rings in their structure, and four 21 sodium sulfonate groups which dramatically enhance their solubility in water and electrostatic interactions with positively charged guests.93, 94 Compared to macrocyclic CB[n], M1 (356 mM) and M2 (18 mM) are much more soluble in pure water. Scheme I-2 shows the convergent building block based synthetic route used to prepare the Motor1 host on scales up to 50 grams. Methylene bridged glycoluril dimer is obtained by the condensation of glycoluril with formaldehyde (1 equiv.) in HCl. Subsequently, 2 is allows to undergo controlled condensation with glycoluril bis(cyclic ether) in MeSO3H to give glycoluril tetramer 7 as a key glycoluril oligomer building block. Subsequently, 7 reacts with aromatic wall 8 by a double electrophilic aromatic substitution process in acidic conditions (Scheme I-2) to yield Motor1.7 A related route can be used to synthetize M2 simply by changing the aromatic wall building block in the final step. M1 and M2 can be easily synthesized on large scale making them a good candidate for industrial applications. O O O O O O O HN N N NH N N N N N N N N N N H HH H + O O MeSO3H O H HH H O HN N N NH N N 50oC N N N N N N N N O O O O O O O 2 7 OR O O O OOR OR 7 + TFA/Ac N N N N N N N N 2O H HH H 70oC N N N N N N N N OR 8 R = (CH OR OR O O O O 2)3SO3Na Motor 1 Scheme I-2. Synthesis of Acyclic CB[n] (M 1). 22 Due to their high binding affinity, high water solubility, and ease of synthesis, M1 and M2 have been used for a multiple applications including as solubilizing agents for insoluble drugs, hydrocarbons and carbon nanotubes, reversal agents for neuromuscular blocking agents, reversal agents for rat intoxication with Methamphetamine, and components of sensor arrays.95-98 Not only do acyclic CB[n]s show multiple applications, when compared to other already commercialized host molecules they show higher efficiency. Take for example Sugammadex, a type of cyclodextrin currently used clinically as a post-surgical reversal agent for rocuronium and vecuronium. M2 binds to Rocuronium and Vecuronium a 100-fold stronger than Sugammadex and maintains a high level of discrimination against acetylcholine.97 In vivo studies with rats were conducted with M2 confirming their efficiency and effectiveness.98 1.5 Acyclic cucurbit[n]uril derivatives As described above, the synthesis of M1 and M2 proceeds by a building block approach using glycoluril oligomer and aromatic wall building blocks. The Isaacs group is in the process of performing a medicinal chemistry type structure-binding affinity optimization of the properties of acyclic CB[n]-type receptors. Modifications to M1 and M2 have been made with the goal of improving binding affinity, water solubility, binding selectivity, glycoluril backbone, enhancing aromatic walls ?-? interactions, and even introduction new properties such as fluorescence.95, 99 The following section will focus on four type of modifications made to acyclic CB[n]. First analogs with different solubilizing groups attached to the aromatic wall. Second, 23 analogs with different length alkyl linker to sulfonate groups. Third, modifications were made using different type of aromatic arms. Fourth, analogs with different length glycoluril backbone, notably a trimer glycoluril backbone. 1.5.1. Modification of the solubilizing groups. One of the key structural features of the structure of M1 and M2 are the (CH -2)3SO3 arms which function in part as solubilizing groups. In order to assess the scope of functional groups that would confer high solubility in water of acyclic CB[n]-type receptors while maintaining high binding affinity, the Isaacs group designed and studied new acyclic CB[n] featuring anionic (SO3?), neutral (OH), and cationic (NH 1004?) (Figure I-12) solubilizing groups. O O O O OR OR N N N N N N N N H HH H N N N N N N N N OR OR O O O O 9 R = (CH R = (CH R = (CH2)2SO3Na, 10 2)2NH3Cl, 11 2)2OH 10 24 11a 11b Figure I-12. Chemical structures of acyclic CB[n] derivatives with different solubilizing groups; 9 (SO3Na), 10 (NH3Cl), and 11 (OH). X-ray crystal structure of 10 and 11 in self-complexation form and open form. It was determined that 9 (346 mM) and 10 (250 mM) displayed high solubility in water but that 11 (<2.00 mM) with its OH groups displayed insufficient water solubility to be considered for use as a solubilizing excipient for insoluble drugs.100, 101 According to x-ray crystallography all three analogs (9 ? 11) assume an overall C- shape featuring CH-? and ?-? interactions between their aromatic walls. Similar geometric features were seen previously for M1 and M2. In contrast to the behavior of Motor1, however, the ammonium solubilizing groups of 10 were found to undergo self-complexation with its own C=O portals driven by intramolecular ion-dipole interactions (-NH +3 ???O=C). This self-folding of the arms of 10 results in a smaller cavity and reduces the binding affinity of 10 toward its guests. In the context of the 25 potential use of 10 as a solubilizing agent for insoluble drugs, this lower binding affinity dramatically reduces its utility.100 Compound 11 with its OH solubilizing groups also displayed self-complexation between the hydroxyl group and the carbonyl portal (OH???O=C) which also reduced guest binding affinity, but not so significantly as that of 10. Interestingly, the x-ray crystal structure of 11 shows two different conformations of 10 in the crystal that differ in the degree of curvature of the glycoluril oligomer backbone which provides direct evidence of the ability of acyclic CB[n] to adapt their shape to accommodate different sized guests.101 The binding properties of 9 ? 11 toward hydrophobic ammonium ions and three insoluble drugs (tamoxifen, 17?-ethynylestradiol, and indomethacin) were determined by 1H NMR, UV/vis spectroscopy, and phase solubility diagrams. It was found that tetra- anionic host 9 was the superior host for cationic guests compared to the neutral host 11 or the cationic host 10 which can be understood based on electrostatic interactions and the presence of self-folded conformations for 10 and 11 that feature intramolecular H-bonds or ion-dipole interactions. The Isaacs group concluded that the SO -3 groups are not merely solubilizing groups but also enhance the affinity of acyclic CB[n]-type receptors toward cationic guests electrostatically. Chapter 4 describes a modification to the solubilizing arms that further enhances binding affinity in particular toward dicationic guests. 1.5.2 Modifications of aromatic arms The aromatic walls of acyclic CB[n]-type hosts represent one of the key synthetic building blocks used in their synthesis. Over the years, the Isaacs group has 26 studied a number of different aromatic sidewalls including the benzene and naphthalene walls of M1 and M2, an isomer of M2 known as Zhang1 (12), and methylated and cyclohexane fused versions.102 These ability of these hosts to function as solubilizing excipients for insoluble drug molecules was tested by constructing phase solubility diagrams. From this work, the Isaacs group concluded that M1 with its high solubility and very good binding affinity is the most general purpose host whereas M2 displays highest affinity toward its guests. By virtue of their aromatic sidewalls, acyclic CB[n] have excellent potential to construct fluorescence sensors arrays.103-107 A collaboration between the Isaacs Lab and Prof. Anzenbacher showed that a mixture of 12 and a naphthyl derivative of CB[6] function as a supramolecular sensing array for cancer associated nitrosamines, over-the-counter drugs, and opiates and their metabolites.106 In this study, the naphthalene substituted cucurbit[6]uril has high affinity and selectivity toward small guests whereas acyclic cucurbit[n]uril derivative 12 with its flexible methylene bridged glycoluril backbone functions a cross-reactive component of the sensor array. 105 Because of the stimuli responsive properties of (acyclic) CB[n] hosts, this sensing array is sensitive to the presence of metal ions such as Eu3+, because of its interaction with the carbonyl portals. When a guest reduced or removes this interaction between the metal ion and the carbonyl portal, a change in fluorescence is observed which further enhances the sensitivity of the assay.105, 106 Significantly, such sensors are able to quantify the presence of drugs within the urine of with a single fluorescence reading.106 More recently, inspired by the work of Swager and others,108 the Isaacs research group synthesized the triptycene walled acyclic CB[n] host 13.109 Triptycene 27 have been used as building blocks for polymeric material and fluorescent sensor applications.109-112 Host 13 features a central glycoluril tetramer unit and O(CH2)3SO3Na solubilizing groups which are identical to those of M1 and M2 (Figure I-13). Host 13 has good water solubility (3.00 mM) due to the sulfonate groups. However, the triptycene walls each possess two benzene rings that help define the cavity which in theory makes 13 a host that has eight subunits (e.g. 4 glycolurils and 4 benzene rings) and may create a more voluminous cavity akin to CB[8]. Unfortunately, the x-ray crystal structure of 13 showed the presence of ??? interactions between the blades of the opposing triptycene walls in a self- complexed geometry which reduced overall cavity volume. Quite interestingly, however, the researchers found that 13 could bind to large guests such as the Fujita square and Stoddarts blue box. However, in these cases the intertwined structures of the complexes blurred the lines between what molecules constituted the host and which constituted the guest. Host 13 also displays changes in fluorescence upon 13?guest complexation presumably due to changes in orientation between triptycene walls and undergoes complete quenching in the presence of electron deficient pyridinium guests.108 The triptycene walled acyclic CB[n] is just one example of the interesting features and properties that can be observed when modifications are made to the original acyclic CB[n]. In Chapter 3 of this thesis I present my work on the synthesis and molecular recognition properties of a triptycene walled glycoluril trimer that we hoped would circumvent the issue of cavity self-inclusion and ??? stacking between the opposing triptycene walls. 28 OR O O O O RO N N N N N N N N H HH H N N N N N N N N OR O O O O RO 12 R= (CH2)3SO3Na O O O O OR OR N N N N N N N N H HH H N N N N N N N N OR OR O O O O 13 R= (CH2)3SO3Na 13 Figure I-13. Chemical structure of Zhang 1 12 and triptycene walled acyclic cucurbit[n]uril 13. 1.5.3 Modifications of glycoluril backbone Whereas supramolecular hosts containing glycoluril monomer46 or methylene bridged glycoluril dimer113 have been known for many years, systems based on glycoluril trimer are uncommon. Accordingly, a collaboration between the groups of Profs. Vladimir Sindelar and Lyle Isaacs sought to study the influence of glycoluril 29 oligomer length on the hosts ability to act as solubilizing excipients for insoluble drugs.114 Dr. Laura Gilberg created glycoluril trimer hosts (Trimer Host 1 and Trimer Host 2, Figure I-14) analogous to Motor1 and Motor2 using our building block approach. For this purpose, she first condensed glycoluril with dimethyl glycoluril bis(cyclic ether) to give methylene bridged glycoluril trimer building block (2, Scheme III-1). Subsequently, a double electrophilic aromatic substitution reaction between (2, Scheme III-1) and the appropriate aromatic wall was performed which delivered Trimer Host 1 (48%) and Trimer Host 2 (59%) in good yields.114 Trimer host 1 and Trimer Host 2 possess good water solubility (102 and 336 mM) but possess smaller cavities than the M1 and M2. The Isaacs group studied the ability of Trimer Host 1 and Trimer Host 2 as solubilizing excipients for the insoluble drugs camptothecin, ziprasidone, PBS-1086, and b-estradiol by means of phase solubility diagrams. The researchers found that the glycoluril trimers host performed better than analogous hosts based on glycoluril monomer or dimer but less well than M1 or M2.114 Trimer Host 1 and Trimer Host 2 display lower binding affinity that M1 and M2 toward a common guest presumably due to the smaller cavity volume and the presence of fewer high energy waters78 in the cavity of the trimer derived hosts. N N N N N N O N N N O N NN N N O N O O N O N O O N N ON O N O N N O N OR RO OR RO RO RO OROR R= CH2CH2CH2SO3Na R= CH2CH2CH2SO3Na Trimer host 1 Trimer host 2 30 Figure I-14. Chemical structures Trimer acyclic CB[n] host 1 and 2 1.5.4 Modifications of alkyl linker on aromatic arms A final structural variable of acyclic CB[n] that has been considered by the Isaacs lab is the length of the O(CH2)nSO3Na linking group. Two acyclic CB[n] were made, M2C2 and M2C4 with (CH2)n (n=2 and n=4 respectively) and studied in comparison to M2 (n = 3) itself (Figure I-14).115 Just like M2 the hydrophobic cavity and carbonyl portal of M2C2 and M2C4 allow for the encapsulation of hydrophobic cations as good guests. Compared to M2 (water solubility 14 mM), M2C2 and M2C4 have higher water solubility (68 mM or 196 mM, respectively). It is however, worth noting, that M2C4 become a viscous solution as concentration increases. After investigation, it was determined that M2C4 forms a gel in the presence of insoluble drugs, however, M2C2 was determined increase water solubility of insoluble drugs. The slopes of the phase solubility diagrams show that the binding strength of the three hosts toward a panel of 15 insoluble drugs generally follows the order: Motor2 > M2C2 > M2C4. Therefore, M2C2 with its higher aqueous solubility has untapped potential as a potential solubilizing excipient. In Chapter 4 of this dissertation I present the synthesis and molecular recognition properties of an analogue of M1 where the O(CH2)nSO3Na alkylene linker has been completed removed and becomes OSO3Na groups instead. 31 O O O O OR OR N N N N N N N N H HH H N N N N N N N N OR OR O O O O R=(CH M2C2 R= (CH2)2SO3Na and M2C4 2)4SO3Na Figure I-15. General structure of acyclic CB[n] with 2 or 4 carbon chain alkyl linkers 1.6 conclusion This introductory chapter has presented some of the background of the field of molecular recognition using molecular containers compounds (e.g. cyclodextrins, calixarenes, pillararenes, cyclophanes). These macrocyclic molecular containers are preorganized for binding and therefore display high binding affinity at selectivity. In this thesis, I focus on the molecular recognition properties of molecular containers that are acyclic ? acyclic CB[n]-type receptors ? which have limited degrees of freedom and are therefore preorganized and display very good levels of binding affinity and selectivity. The influence of different structural variables (e.g. aromatic wall, glycoluril oligomer length, solubilizing group, linker length) on the recognition properties of acyclic CB[n]-type receptors conducted prior to my work was reviewed. In the three research chapters of this dissertation I study the: 1) molecular recognition properties of Motor 1 and Motor2 toward amino acids, peptides, and insulin in comparison to CB[n], 2) the molecular recognition properties of a triptycene walled glycoluril trimer, and 3) an analog of Motor 1 whose (CH2)3 linking groups have been removed to create sulfate functional groups. 32 Chapter 2: Molecular Recognition Properties of Acyclic Cucurbit[n]urils Toward Amino Acids, Peptides, and a Protein The work presented in this chapter was take from Zebaze Ndendjio, S.; and Isaacs, L. Molecular recognition properties of acyclic cucurbiturils toward amino acids, peptides, and a protein. Supramol. Chem. 2019. This work was entirely done by me. 2. 1 Introduction. The synthetic and supramolecular chemistry of the CB[n] family has played a leading role in the field since the preparation of cucurbit[n]uril homologues example, the Ka for CB[7]?guest complexes routinely exceed 106 M- 1, often exceed 109 M-1, and in special cases (e.g. cationic adamantanes and diamantanes) can even exceed 1012 M-1 in water due to the combined effects of ion?dipole interactions and the hydrophobic (CB[n], Figure II-1) at the turn of the millennium.52, 78 m k,ln n m k,l N N N N N N N O N N NO N i,j N O N O N i,j N O N N N N N O O O o N N N O O o N O O N NN N N N N NN N N N O O pO O p O O O O O O O O N N CB[n] N N N N N N N Nq,r,s N Nq,r,s g,h OR RO g,h OR RO N N O O NO O N t N O N N N RO f u N O N OR RO OR N N n-6 Calabadion 1 R = (CH2)3SO3Na Calabadion 2 R = (CH2)3SO3Na Figure II-1. Chemical structures of CB[n] and Calabadions 1 and 2. CB[n] compounds are powerful receptors for hydrophobic (di)cations in aqueous solution due to their two-symmetry equivalent ureidyl carbonyl-lined portals 33 which guard entry to a hydrophobic cavity58. For example, the Ka for CB[7]?guest complexes routinely exceed 106 M-1, often exceed 109 M-1, and in special cases (e.g. cationic adamantanes and diamantanes) can even exceed 1012 M-1 in water due to the combined effects of ion?dipole interactions and the hydrophobic effect. 57, 58 In addition to high-affinity binding, CB[n]?guest complexes also display high stimuli responsiveness (e.g. pH, electrochemistry, photochemistry, competing guest) which allows for the use of CB[n] in a variety of applications.50, 53 For example, CB[7]?guest binary complexes and CB[8]?guest1?guest2 ternary complexes are used to construct molecular switches and machines116, as supramolecular catalysts54, 117, to solubilize, protect, sequester, and even create targeted pharmaceuticals118, as components of chemical sensors119, 120, and to promote the assembly of supramolecular polymers, materials, and frameworks121. CB[n] have also found wide applicability in peptide and protein chemistry. For example, in pioneering work, the Urbach group showed that CB[8] promotes the cooperative dimerisation of the Phe-Gly-Gly peptide in water via formation of the CB[8]?(Phe-Gly-Gly)2 complex with K =1.5 x 1011 M-2 4a . This motif has been capitalized upon by Brunsveld and Liu to promote the dimerisation of proteins tagged with N-terminal FGG units and thereby control biological functions.122 For example, Brunsveld engineered a split luciferase123 that could be reconstituted by addition of CB[8], and similarly CB[8] was found to promote the dimerisation of monomeric caspase-9 into the active dimer.124 Scherman has used the FGG motif to promote hydrogel formation.68 34 O H O O H3N N H NNH NH 32 2 O R O R R Amino amide N-Acetyl amino amide Amino acid Repulsive Ion-dipole H-bonding Ion-dipole electrostatic interaction interaction interaction interaction O O O H N NHR O H N NHR3 H N O3 R R R Hydrophobic effect Hydrophobic effect Hydrophobic effect Figure II-2. Chemical structure of amino acids, amino amides, and N-acetyl amino amides used as guests in this study and illustration of the geometries and driving forces involved in their complexation with macrocyclic CB[n]. In a lovely series of papers, Urbach and collaborators demonstrated that the N-terminus of peptides and proteins is a privileged site for host complexation due to simultaneous ammonium ion and side chain binding (Figure II-2). In particular, they found that CB[7] displays a selectivity toward N-terminal Phe over other N-terminal residues presumably because they display suboptimal fit for the CB[7] cavity (e.g. other aromatic or hydrophobic amino acids may not fully release cavity waters of solvation) or because they cannot sterically accommodate N-terminus and cationic side chain binding (e.g. Lys, His, Arg). The CB[7]?N-terminal Phe motif was used to recognize Insulin in solution and on resin, to determine protease substrate selectivity, to impose sequence-specific inhibition on a non-specific protease, and for supramolecular enhancement of protein analysis.125 Recently, Langer, Anderson, and 35 Isaacs used a monofunctionalised CB[7] derivative to non-covalently PEGylate the N-terminal Phe residue of Insulin and thereby prolong its in vivo function.126 In recent years, the Isaacs group has synthesized and investigated the molecular recognition properties of acyclic CB[n]-type receptors that feature a central glycoluril oligomer, two terminal aromatic walls, and four sulphonate solubilising groups.95 Two prototypical acyclic CB[n] (Calabadion 1 and Calabadion 2) are shown in Figure 1 although numerous variants are known.102 The Calabadions retain the essential molecular recognition features of CB[n], but because they are acyclic, they are able to flex their methylene bridged glycoluril oligomer backbone to accommodate more voluminous guests. Additionally, the acyclic structure of the Calabadions may allow guest substituents to protrude through the side of the host?guest complex rather than through the portals as required for macrocyclic CB[n]. The Calabadions have several important biomedical applications including the solubilization of insoluble anticancer drugs for in vivo application127, 128, as agents to reverse neuromuscular block in vivo62, and most recently to modulate the hyper locomotor activity of rats treated with methamphetamine.98 Recently the Ma group has been using acyclic CB[n] for acid-sensitive controlled release and bioimaging applications.129 By virtue of their aromatic walls, Calabadions undergo changes in their UV/Vis and fluorescence properties upon guest binding and have therefore been use as sensors for nitrosamines, over-the counter drugs, amino acids, and opioids.105, 107 Given the ability of CB[7] and CB[8] to interact with peptide N-termini and internal residues, protein N-termini, and to promote dimerization and the fact that Calabadions retain the essential recognition properties of macrocyclic CB[n] we 36 hypothesized that 1 and 2 would perform well in peptide and protein recognition perhaps with selectivity that is complementary to that observed for CB[n]. 2.2 Results and discussion This results and discussion section is organized as follows. First, we lay out the goals and the hypotheses to be tested in this study. Next, we describe 1H NMR investigations of the binding properties of 1 and 2 toward 19 amino acid amides to confirm the 1:1 binding stoichiometry and shed light on the geometrical features of the complexes. Next, we present the energetics (?G, ?H, ?S) of binding of 1 and 2 toward the various amino amides by isothermal titration calorimetry. Subsequently, we detail the influence of electrostatics on the energetics of host?guest binding as probed through mutation of amino acid to amino acid amide to N-acetyl amino acid amide. Next, we present the binding of 1 and 2 toward selected tripeptides to delineate the influence of neighboring residues. Finally, we present the results of binding of 1 and 2 toward insulin compared to CB[7]. 2.2.1 Goals of the study Macrocyclic CB[7] selectively recognizes peptides that feature an N-terminal hydrophobic aromatic residue like Phe or Trp due to a combination of ion?dipole interactions and the hydrophobic effect and discriminates against residues containing hydrophobic aliphatic side chains, polar neutral side chains, acidic (anionic) side chains, and even basic (cationic) side chains.4, 67, 130 The discrimination of CB[7] against the residues with cationic side chains (e.g. Lys and Arg) is due to the fact that the side chain cannot thread through the cavity to form ion?dipole interactions at both 37 portals without creating steric interactions between the wall of CB[7] and the adjacent CONHR group. We hypothesized that acyclic CB[n] 1 and 2 ? with their acyclic structure ? would display higher affinity toward Lys and Arg and because the steric constraints of these amino acids might be better accommodated.131 Accordingly, a primary goal of the work is the measurement of the binding affinity of 1 and 2 toward the amino acid amides which mimics the context of the amino acid in a longer peptide. Within this realm, we also sought to compare the peptide recognition properties of 1 and 2 which differ in the nature of their aromatic walls, cavity size, and presumably their selectivity toward aromatic amino acids in particular. Given the ability of CB[8] to bind two residues simultaneously4, 68, we were aware of the possibility of similar behavior with 2 (or 1) and were careful to verify the binding stoichiometry. Given that 1 and 2 are tetra-anionic in pH 7.4 water whereas CB[7] is neutral at this pH we sought, as a secondary goal, to understand the role of electrostatics on the molecular recognition properties of 1 and 2 toward amino amides, N-acetyl amino amides, and amino acids themselves. Finally, we wanted to examine the recognition properties of 1 or 2 toward N-terminal amino acid in the context of a protein (insulin). 2.3 1H NMR investigations of calabadion?guest binding First, we investigated the binding interactions between 1 and 2 toward amino acid amides by 1H NMR spectroscopy. We performed these experiments in biologically relevant 20mM sodium phosphate buffered D2O at pD7.4 to ensure that the N-terminus of each amino acid amide is present as its NH +3 form which provides a primary binding site for hosts 1 and 2. As documented in the Supporting 38 Information for 19 amino acid amides (excluding cysteine), we measured 1H NMR spectra at different host:guest ratios (generally 1:2, 1:1.5, 1:1, and 1:0.5). We monitored the change in guest chemical shifts upon complexation to provide crude information on host?guest stoichiometry and to determine whether the kinetics of guest exchange is in the fast, intermediate, or slow exchange regime on the 1H NMR timescale. Rather than discuss the precise changes for each host?guest complex, we discuss here some general trends that are observed in the chemical shifts of both host and guest upon complexation. For example, Figure II-3 shows the 1H NMR spectra recorded for mixtures of 1 and H-Phe-NH2. A comparison of Figure II-3(a,d) shows that the protons on the aromatic ring of H-Phe-NH2 (Hc ? He) and the adjacent benzylic CH2 group (Hb) undergo a substantial upfield change in chemical shift upon complexation (Hc ? He; 1?1.5 ppm; Hb ? 0.8 ppm) which strongly suggests that the Arring and benzylic CH2 of H-Phe-NH2 is bound within the cavity of 1 within the 1?H-Phe-NH2 complex. Conversely, the ?-proton Ha of H-Phe-NH2 undergoes a slight downfield shift (? 0.2 ppm) upon complex formation which indicates that H? is located nearby the deshielding region defined by the ureidyl C = O groups of the host. When an excess of H-Phe-NH2 is present (e.g. 1:2, Figure II-3(b)) the resonances for Hc ? He shift back toward the position observed for uncomplexed H-Phe-NH2 and broaden which indicates that this complex displays intermediate exchange kinetics on the 1H NMR timescale and likely has 1:1 stoichiometry. In accord with this data and based on the known binding preferences of CB[n] and acyclic CB[n], we formulate the geometry of the 1?H-Phe-NH2 complex as illustrated in Figure II-4(a). 39 We believe that the amide (C = O)NH2 group of H-Phe-NH2 is hydrogen bonded to the ureidyl C=O group of host 1 in the complex.87 Figure II-3. 1H NMR spectra recorded (400 MHz, RT, 20 mM NaH2PO4 buffered D2O , pD 7.40) for: a) H-Phe-NH2 (5 mM), (b) 1 (1 mM) and H-Phe-NH2 (2 mM), c) 1 (1 mM) and H-Phe-NH? (1.5 mM), (d) 1 (1 mM) and H-Phe-NH2 (1 mM), e) 1 (1 mM) and H-Phe-NH2 (0.5 mM), (f) 1 (2 mM). Related changes are observed during the complexation of the hydrophobic amino acid amides (Appendix 1) indicating that side chain cavity inclusion and NH +3 portal binding is the dominant geometry. Changes are also observed in the 1H NMR 40 resonance for host 1 in the 1?H-Phe-NH2 complex. For example, the Hf protons in C2v-symmetric 1 are symmetry equivalent and display a sharp singlet (Figure II-3(f)) but become broadened and split upon formation of the C1- symmetric (e.g. no symmetry) 1?H-Phe-NH2 complex. This observation can be explained by the fact that the chiral host?guest complex renders all four Hf atoms diastereotopic. In the case of fast kinetics of exchange, typified beautifully by the 1?H-Leu-NH2 complex, two doublets are observed for Hf (Appendix 1, Figure S83). Related observations are observed with host 2. None of the complexes between 1 or 2 and the amino acid amides displayed slow kinetics of exchange on the 1H NMR timescale. Interestingly, for complexes between 1 (2) and the hydrophobic non-aromatic amino acid amides, we typically observe a small downfield shift for host aromatic sidewall resonance Hf, Ht, Hu (1: up to 0.3 ppm; 2: up to 0.5 ppm). Ion-dipole H-bonding Alleviated steric clash interaction O interaction - SO3 H N N H - - O 3 H SO3 OH3N SO3 - SO Ion-dipole3 interaction CONH2 Hydrophobic effect - - SO3 SO3 - O H N a) SO 33 1 -?H-Phe-NH b)2 1?H-Lys-NH2 SO3 Figure II-4. (Color online) Schematic illustrations of the geometry of (a) 1?H-Phe- NH2 and (b) 1?H-Lys-NH2 complexes. As described previously in related systems, we believe this is due to a conformational change that hosts 1 and 2 undergo upon complexation that removes edge-to-face C-H???? interactions that occur in the uncomplexed host that result in 41 upfield shifting.95 In contrast, for the aromatic amino acid amides (e.g. H-Trp-NH2, H-Tyr-NH2, H-Phe-NH2) only very small changes in chemical shift are observed for Hf which we believe reflects a balance between the expected downfield shift due to conformational change and upfield shift due to the shielding effect of the aromatic ring of the guest. Interestingly, we found no evidence by 1H NMR of binding of 1 or 2 (at mM concentrations) toward Asp and Glu which contain CO2H groups in their side chains that are expected to be present in their anionic CO2? form at pD 7.4. This result is in accord with previous observations from CB[n] molecular recognition that the electrostatically negative ureidyl C = O portals do not tolerate the presence of guest negative charge at the portals.59, 132 Furthermore, the presence of the SO3? solubilizing groups on 1 and 2 would be expected to electrostatically destabilize complexes with Asp and Glu. Finally, for H-Gly-NH2, H-Ala-NH2, and H-Ser-NH2 which have no, small, or hydrophilic side chains we observe only small changes (e.g. ?? and broadening) in the 1H NMR which probably indicates very weak binding or non-inclusion binding (e.g. portal binding) or a combination thereof. In the 1H NMR experiments, we often observed substantial upfield shifts for guest resonances at 1:1 host:guest stoichiometry which moved back toward the position for complexed guest at 1:2 host:guest ratios which suggested the formation of 1:1 complexes. We constructed Job plots133 to further established the 1:1 host:guest stoichiometry in select cases where the resonances were sharp enough to be easily monitored. For example, Figure II-5 shows a Job plot constructed for mixtures of 1 and H-Lys-NH2 at a constant total concentration of 1 mM in phosphate buffered D2O (pD 7.40). The chemical shift of Hf of the host was monitored. As can readily be seen, the Job plot 42 displays a maximum at a mole fraction of 0.5 which firmly establishes the 1:1 absolute stoichiometry of the 1?H-Lys- NH2 complex. A related Job plot was constructed to confirm the 1:1 stoichiometry of the 1?H-Arg-NH2 complex. Figure II-5. Job plot constructed for the interaction of 1 with H-Lys-NH2 ([1] + [H- Lys-NH2] = 1 mM) monitoring the chemical shift of Hf on 1 by 1H NMR spectroscopy (600 MHz, RT, 20 mM NaH2PO4 buffered D2O, pD 7.40). The solid line serves as a guide for the eye. 2.4 Isothermal titration calorimetry (ITC) determination of host?guest energetics The substantial broadening and intermediate exchange kinetics observed in the 1H NMR spectra of the complexes of 1 and 2 with amino acid amide guests complicates the use of NMR to determine binding constants. Accordingly, we turned to ITC to determine the thermodynamic parameters for binding. Figure II-6(a) shows a representative ITC Thermogram recorded for the titration of 1 (100 ?M) in the ITC cell with a solution of H-Phe-NH2 (1 mM) in the ITC syringe and the fitting of the data to a 1:1 binding model using the PEAQ ITC analysis software (Figure II-6(b)). In this manner, we were able to extract K? = 2.62 x 106 M-1 and ?H = ?17.1 kcal mol- 43 1 for the 1?H-Phe-NH2 complex. Analogous ITC titrations were performed for the complexation between hosts 1 and 2 and the remainder of the amino acid amides, amino acids, and N-acetyl amino acid amides (Appendix 1), and the results are presented in Table 1. Figure II-6. (a) Thermogram obtained during the titration of 1 (100 ?M) in the cell with H-Phe-NH2 (1 mM) in the syringe (298.0 K, 20 mM sodium phosphate buffered H2O, pH 7.4) and (b) fitting of the data to a 1:1 binding model with K? = 2.62 x 106 M-1 and ?H = ?17.1 kcal mol-1. 2.4.1 Discussion of trends in the thermodynamic parameters The dynamic range of binding constants presented in Table 1 spans the range from102 M-1 to above 106 M-1. Given the constraints of this relatively narrow range, we have been able to discern some trends in the thermodynamic parameters and 44 present them here grouped according to the chemical nature of the amino acid amide side chain (e.g. anionic, cationic, aromatic, hydrophobic aliphatic, polar). Table II-1. Thermodynamic parameters obtained by ITC for the interaction of 1 and 2 with the amino acid amides, N-acetyl amino amides, and amino acids. Host 1 Host 2 Guest K -1 a / M ?G? ?H? -T?S? K / M-1 a ?G? ?H? -T?S? kcal mol-1 kcal mol-1 kcal mol-1 kcal mol-1 kcal mol-1 kcal mol-1 Aromatic Sidechain H- 2.62 ? -8.76 -17.1 ? 8.36 3.24 ? -8.88 -17.3 ? 8.45 Phe- 106 0.04 106 0.07 NH2 H- 7.87 -6.68 -12.5 ? 5.83 9.61 ? -6.80 -7.23 ? 0.432 Phe- ? 104 0.153 104 0.074 CO2? Ac- 4.17 -4.94 -8.95 ? 4.01 2.99 ? -6.11 -10.1 ? 4.04 Phe- ? 103 0.448 104 0.239 NH2 H- 2.56 -7.38 -14.8 ? 7.43 3.98 ? -9.00 -17.7 ? 8.68 Trp- ? 105 0.064 106 0.07 NH2 H- 1.06 -5.50 -7.41 ? 1.91 1.14 ? -6.90 -6.93 ? 0.026 Trp- ? 104 0.132 105 0.026 CO2? AcTrp 8.40 -3.99 -10.4 ? 6.43 3.56 ? -6.21 -5.38 ? -0.828 -NH2 ? 102 0.854 104 0.30 H- 1.01 -8.19 -15.1 ? 6.89 9.52 ? -8.16 -15.6 ? 7.40 Tyr- ? 106 0.03 105 0.03 NH2 H- 1.01 -5.46 -8.60 ? 3.14 2.03 ? -5.88 -8.76 ? 2.88 Tyr- ? 104 0.226 104 0.111 CO2? AcTyr 1.26 -4.23 -9.36 ? 5.12 3.06 ? -4.76 -8.21 ? 3.45 -NH 32 ? 10 0.371 103 0.079 H-His- 2.35 -5.97 -9.72 ? 3.75 2.61 ? -6.03 -7.18 ? 1.15 NH2 ? 104 0.128 104 0.104 Cationic Sidechains H- 7.43 -8.01 -10.9 ? 2.90 1.41 ? -7.03 -9.02 ? 1.99 Lys- ? 105 0.02 105 0.12 NH2 H- 3.33 -3.44 -11.6 ? 8.18 497 -3.68 -2.19 ? -1.49 Lys- ? 102 5.02 0.036 CO2? 45 Ac- 9.80 -5.45 -7.13 ? 1.68 8.06 ? -5.33 -2.84 ? -2.49 Lys- ? 103 0.220 103 0.078 NH2 H- 7.09 -7.98 -11.6 ? 3.57 8.26 ? -6.71 -8.84 ? 2.13 Arg- ? 105 0.02 104 0.058 NH2 H- 1.51 -4.34 -3.93 ? -0.416 2.39 ? -4.61 -9.82 ? 5.21 Arg- ? 103 0.099 103 0.386 CO2? AcArg 8.13 -5.34 -6.84 ? 1.50 7.87 ? -5.32 -5.44 ? 0.119 -NH2 ? 103 0.178 103 0.248 Non-aromatic polar sidechains H- 1.47 -5.69 -5.71 ? 0.02 7.25 ? -5.27 -2.82 ? -2.44 Gln- ? 104 0.213 103 0.69 NH2 H-Ser- 1.25 -5.59 -0.867 ? -4.73 1.90 ? -4.47 0.944 -5.42 NH2 ? 104 0.007 103 ?0.185 H- 8.33 -5.35 -0.546 ? -4.80 n.b n.b n.b n.b Thr- ? 103 0.112 NH2 H- 1.44 -4.31 1.42 ? -5.73 1.35 ? -4.27 -1.62 ? -2.66 Asn- ? 103 1.22 103 0.012 NH2 H- 1.32 -6.99 -13.1 ? 6.08 8.85 ? -6.75 -11.7 ? 4.97 Met- ? 105 0.04 104 0.04 NH2 Hydrophobic aliphatic sidechains H-Ile- 9.43 -6.79 -10.8 ? 4.02 1.68 ? -7.13 -9.18 ? 2.05 NH2 ? 104 0.04 105 0.085 H- 1.40 -5.66 -10.1 ? 4.40 2.15 ? -5.91 -4.66 ? -1.26 Val- ? 104 0.085 104 0.142 NH2 H- 2.85 -7.44 -9.80 ? 2.35 2.29 ? -7.31 -8.20 ? 0.891 Leu- ? 105 0.027 105 0.43 NH2 H-Pro- 5.75 -5.13 -5.43 ? 0.299 1.70 ? -5.77 -5.26 ? -0.510 NH 3 2 ? 10 0.149 104 0.146 H- 408 -3.56 -5.32 ? 1.76 515 -3.70 -2.77 ? -0.930 Ala- 0.545 0.130 NH2 H- 80.6 -2.60 -3.88 ? 1.28 115 -2.81 -1.36 ? -1.46 Gly- 0.742 0.125 NH2 Firstly, in agreement with our 1H NMR measurements, no heat is evolved during the titration of 1 or 2 with the H-Asp-NH2 and H-Glu-NH2 and we therefore conclude that no binding occurs with these amino acid amides that contain anionic side chains. This result is in accord with the well-known preference of the CB[n] 46 cavity for hydrophobic and neutral binding epitopes over hydrophilic and anionic groups.58, 132 Second, we find that the aromatic amino acid amides (H-Phe-NH2, H- Trp-NH2, H-Tyr-NH2 form the tightest complexes with 1 and 2 with Ka values that exceed 106 M-1 and with large enthalpic driving forces in the range of ?15 ? ?18 kcal mol-1. This trend is not so surprising in light of the hydrophobicity of the aromatic rings of the amino acid amides, the ability of aromatic walls of 1 and 2 to engage in ?-? interactions with the guest, and of course the precedent from the work of Kim, Inoue, Nau and Urbach that showed that peptides with aromatic side chains at the N- terminus are bound selectively by CB[7].4, 67, 88, 130 Macrocyclic CB[7] has been reported to bind Phe-Gly-Gly (K = 2.8 x 106a M-1), Phe-Gly (Ka = 3 x 107 M-1), Tyr- Gly (K = 3.6 x 106 M-1a ), and Trp-Gly (K 5a = 5.6 x 10 M-1) with affinities that are comparable to those observed for 1 and 2.4, 88 For H-Phe-NH2 and H-Trp-NH2, the larger host 2 binds stronger than 1 as expected based on its larger cavity size.62, 102 H- His-NH2 with its hydrophilic and charged aromatic side chain is bound significantly weaker to both 1 and 2 (Ka ? 104 M-1) and with significantly lower enthalpic driving force. Third, H-Lys-NH2 and H-Arg-NH2 which are dicationic at neutral pH and contain 4?5 C-atoms between cationic N-atoms bind toward 1 with affinities (Ka ? 7 ? 105 M-1) that are only slightly lower that the aromatic amino acid amides. They do not reach the very high affinity typically observed for diammonium ion complexes with cucurbiturils presumably because of the presence of the CONH2 group. Host 1 displays higher affinity than 2 toward H-Lys-NH2 (5.3-fold) and H-Arg-NH2 (8.6- fold) because the cavity of 1 is smaller than 2 and prefers narrower (e.g. alkylene) guests as observed previously.95 Fourth, among the amino acid amides with polar side 47 chains, we find that H-Met-NH2 binds most strongly to 1 (Ka = 1.32 x 105 M-1) and 2 (Ka = 8.85 x 104 M-1) with substantial enthalpic driving forces. This is not surprising given that methionine has both the largest side chain surface area and the least favorable free energy of transfer from cyclohexane to water which enhances the hydrophobic driving force for complexation.4 The remaining polar amino acid amides (H-Gln-NH2, H-Asn-NH2, H-Thr-NH2, H-Ser-NH2) bind more weakly with Ka in the 102 ?104 M-1 range which reflects the need to desolvate the side chains polar OH and CONH2 functional groups as they enter the host cavity. As expected, a comparison of the binding constant of 1 toward H-Gln-NH2 versus H-Asn-NH2 and H-Thr-NH2 versus H-Ser-NH2 reveals that the compound with the additional CH2-group in the side chain is the stronger binder.58 Finally, the amino acid amides with hydrophobic aliphatic side chains (H-Ile-NH2, H-Val-NH2, H-Leu-NH2, H-Pro-NH2) bind to 1 and 2 with K 3a values in the 6 ? 10 to 3 ? 105 M-1 range and ?H values in the?5 to?7.5 kcalmol-1 range with; host 2 is generally the tighter binder. The order of binding affinity toward 1 and 2 follows the order H-Pro-NH240 mM). Before proceeding to investigate the host?guest properties of 1 we investigated its self-association properties to ensure that self-association does not impinge upon the planned binding constant measurements.34 Accordingly, we measured the 1H NMR spectrum for aqueous solutions of 1 upon dilution from 40 mM to 1 mM. We did not observe any significant changes in chemical shifts (?? < 0.02 ppm) over this concentration range and therefore conclude that host 1 is monomeric in aqueous solution (Appendix 3). Figure IV-1. Cross eyed stereoviews of the x-ray crystal structures of a) 1?6d, and b) 1?6a. Color code: C, gray; H, white; N, blue; O, red; H-bonds red-yellow striped. We were fortunate to obtain the x-ray crystal structures of the 1?6d and 1?6a complexes (Figure IV-1). Figure IV-1a shows the 1?6d complex adopts a geometry that optimizes Me N+3 ???O=C electrostatic interactions at both portals and displays only small out-of-plane skewing of the terminal aromatic rings. The geometry of 1?6d is reminiscent of those of CB[n]?guest complexes where the Me N+3 ???O=C 74 distances cluster in the 3.810 ? 4.690 ? range to spread the positive charge to the carbonyl portals.60 Quite interestingly, a second molecule of di-cationic guest 6d fits nicely into a cleft created by the aromatic sidewalls and the outward pointing OSO3? groups to balance the overall 4- charges of host 1. These OSO ?3 groups also engage in electrostatic interactions with 6d with the Me +3N ???O-S shortest distances in the 3.808 ? 4.722 ? range. The crystal structure of 1?6a (Figure IV-1) also shows one intracavity and one extra cavity molecule of 6a but displays significant out-of-plane twisting of the aromatic termini. Interestingly, one of the four OSO3? groups turns inward toward the ammonium ion guest which establishes that this group can directly participate in the guest recognition process. 4.3 1H NMR spectrum investigation 75 NH2R3 NNHR3 NMe NR3 3 NH NRa + 3 3 NH3(CH +2)n b (CH2)5 - c NHM2 Mee+ 11a?2Cl NH R d NMe 2 CH3 -+ 2 3 3 6Q - 11d?2Cl12a? H n = 4 Cl- 3 N - 5a 12d?I 13a?2Cl - N O NR6a d n = 6 3 7d n = 7 N O N 8d n = 8 Ph 9d n = 9 OAc - O 23?Cl N HN - 16 (PC PPh) 10d n = 10 O 22?2Cl 14 (meth) OAc 15 (fentanyl) Ph morphine N + Ph N O (17) H N N H H H N AcO 20 vecuroniumH O HO O H OH N OAc N naloxone O (18) O = R HO H N + O N (MeO)2Ph H H 21 OR O HO (cisatracurium) (CH2)5 OH O H 19 (rocuronium) Figure IV-2. Structures of guests 5 ? 23 used in this study. Next, we performed qualitative host?guest binding studies of 1 with guests 5- 13 (Figure IV-2, Appendix 3) as monitored by 1H NMR spectroscopy. Figure IV-3 shows the 1H NMR spectra recorded for 1, 6d, and 1:1 and 1:2 mixtures of 1:6d. As expected the methylene resonances for guest 6d (Hm, Hn, Ho) within the 1?6d complex (Figure IV-3b) experience a sizable upfield shift upon complexation due to the anisotropic shielding effects of the aromatic walls and the glycoluril concavity.45, 58 At a 1:2 1:6d ratio, resonances are observed for both free 6d and 1?6d which indicates slow exchange on the 1H NMR chemical shift time scale which is usually observed only for tight host?guest complexes. Similar 1H NMR measurements were made for 76 the remainder of the guests (Appendix 3). We find that the narrow guests (e.g. 11a, d and 6a-d) display slow exchange kinetics whereas the bulkier guests 12a,d display intermediate to fast ex-change on the chemical shift timescale. We attribute this to their lower binding constants (vide infra) as a result of the expansion of the cavity of 1 required to accommodate the larger adamantane framework Figure IV-3. 1H NMR spectra (D2O, 600 MHz) recorded for: a) 1 (1 mM), b) a mixture of 1 (1 mM) and 6d (1 mM), c) a mixture of 1 (1 mM) and 6d (2 mM), and d) 6d (1 mM). Resonances for bound guests as marked with an asterisk (*). 77 4.4 Isothermal Titration Calorimetry Given the high binding constants typically observed for host?guest complexes of CB[n] and acyclic CB[n]-type receptors,95, 154 we elected to use isothermal titration calorimetry (ITC) to measure the Ka values between host 1 and guests 5 ? 23. For the weaker binding complexes (Ka ? 107 M-1), we performed the direct titration of host 1 in the ITC cell with a solution of guest in the syringe and fitted the data to a 1:1 binding model implemented by the PEAQ ITC software to obtain Ka and ?H values (kcal mol-1). Table IV-1 reports the thermodynamic data for 1?5, 1?6Q, 1?12a, 1?13a, 1?14 ? 1?18, and 1?21 ? 1?23 that were obtained by direct ITC titrations (Appendix 3). Complexes with K values that exceed 107a M-1 cannot be measured accurately by direct titrations, so we turned to ITC competitive titrations.155 In competitive titrations, a solution of host and an excess of a weak guest of known ?H and Ka is titrated with a solution of a tighter binding guest. Fitting of the heat released during the displacement process is analyzed by a competitive binding model in the PEAQ ITC data analysis software which delivers ?H and Ka for the tighter binding complex. Figure IV-4a shows the thermogram recorded when a mixture of 1 and 13 was titrated with 6d and Figure IV-4b shows the fitting of the integrated heats to a competitive binding model to determine Ka = 6.79 ? 109 M-1 and ?H = -12.1 kcal mol-1. Table IV-1 reports Ka and ?H values for the remaining 1?guest complexes obtained in an analogous manner (Appendix 3). 78 Figure IV-4. a) Thermogram recorded during the titration of a mixture of 1 (100 ?M) and 13 (2 mM) in the cell with a solution of 6d (1.0 mM) in the syringe, and b) fitting of the data to a competition binding model to extract Ka = 6.79 ? 109 M-1 and ?H = - 12.1 kcal mol-1. The binding constant data reported in Table IV-1 allows us to draw some conclusions about the molecular recognition preferences of host 1 in comparison to M1. As expected, we find that the 1?guest complexes are uniformly driven by favorable enthalpic (?H) contributions. In the CB[n] series of hosts these favorable enthalpy values are attributed to the presence of high energy host intracavity water molecules that are released upon guest binding.57, 79 Host 1 displays high affinity toward hexane diammonium ion guests 6a ? 6d with Kd values in the single digit nM to sub-nM range. Host 1 prefers the quaternary ammonium ion guest 6d by ?10-fold 79 over the primary ? tertiary ammonium ions 6a ? 6c. In selected contexts, related preferences have been seen for CB[7]60 where they are attributed to the more efficient spreading of positive charge to the entire ureidyl C=O portal. Host 1 binds quaternary monoammonium ion guest 6Q 890-fold weaker than the corresponding quaternary diammonium 6d; this ?103 M-1 difference in affinity is also noted for CB[n]-type receptors.20 Importantly, we find that 1 binds to guests 6a ? 6c 5.6 ? 11.9-fold stronger than M1, but 75-fold stronger than M1 toward bis(quaternary) guest 6d. Similar preferences are observed for di-cationic guests 11a and 11d but not for mono-cationic guests 12a and 12d which suggests that the defined separation between OSO ?3 groups in 1 makes it especially complementary to diammonium ion guests. Host 1 also binds with high affinity (single digit nM to sub nM Kd values) toward the longer alkane di-ammonium ions 7d ? 10d although 6d is the tightest binder in this series which reflects the ability of acyclic CB[n] to flex their cavity to accommodate larger guests and optimize binding affinity. Related preferences have been seen for M1 and related receptors toward primary alkane diammonium ion guests previously.94 Table IV-1. Binding constants measured by ITC for host?guest complexes of 1. Comparative data for M1 are drawn from the literature. Conditions: 20 mM sodium phosphate buffered H2O, pH 7.4, 25?C. K [M-1 a ]; ?H (kcal mol -1) 1 M1e) 5 1.68 ? 106; -6.76 ? 0.020a) ? 6a 3.70 ? 108; -8.60 ? 0.021b) 5.05 ? 107; -6.23 ? 0.014 6b 5.26 ? 108; -9.82 ? 0.038c) 9.43 ? 107; -7.15 ? 0.025 6c 5.74 ? 108; -10.5 ? 0.028c) 4.81 ? 107; -7.66 ? 0.073 6d 6.71 ? 109; -12.1 ? 0.042c) 8.93 ? 107; -9.35 ? 0.021 6Q 7.57 ? 106; -9.68 ? 0.063a) 1.24 ? 106; -5.67 ? 0.033 80 7d 6.06 ? 109; -12.2 ? 0.041c) ? 8d 1.75 ? 109; -10.5 ? 0.032c) ? 9d 7.57 ? 108; -10.2 ? 0.030c) ? 10d 5.43 ? 108; -10.3 ? 0.088c) ? 11a 9.71 ? 108; -9.69 ? 0.014b) 1.67 ? 108; -8.09 ? 0.018 11d 1.05 ? 109; -12.0 ? 0.030b) 1.78 ? 108; -11.4 ? 0.022 12a 9.90 ? 105; -4.45 ? 0.021a) 9.62 ? 105; -6.55 ? 0.029 12d 6.66 ? 106; -7.36 ? 0.030d) 1.70 ? 107; -9.09 ? 0.027 13a 3.41 ? 106; -2.92 ? 0.019a) 1.95 ? 106; -5.70 ? 0.027 14 3.02 ? 106; -9.28 ? 0.058a) 7.5 ? 106 15 3.64 ? 106; -12.2 ? 0.076a) 1.1 ? 107 16 1.89 ? 105; -6.18 ? 0.069a) 4.7 ? 104 17 7.69 ? 105; -8.03 ? 0.07a) 5.3 ? 105 18 4.85 ? 106; -5.90 ? 0.205a) ? 19 6.29 ? 108; -12.9 ? 0.056b) 8.4 ? 106 20 1.00 ? 109; -9.62 ? 0.036c) 5.8 ? 106 21 5.32 ? 105; -15.4 ? 0.174a) 9.7 ? 105 22 2.41 ? 104; -5.26 ? 0.372a) ? 23 2.31 ? 105; -8.54 ? 0.063a) 2.4 ? 104 ? not reported in the literature. a) Direct titration, b) competitive ITC with 5 as competitor, c) competitive ITC with 13a as competitor, d) competitive ITC with 6d as competitor, e) Taken from the literature. 6,12 4.4.1 binding to drug of abuse Previously, we have shown that M1 and a naphthalene walled analogue known as M2 function as in vivo sequestration agents for drugs of abuse (e.g. methamphetamine (14)).156 Accordingly, we decided to measure the binding affinities of some compounds (14 ? 18) relevant to counteracting the effects of drugs of abuse. We find that host 1 binds less tightly than M1 toward 14 and 15. In contrast, host 1 binds somewhat tighter to PCP (16) and morphine (17) than M1 does, but the single digit ?M dissociation constants are unlikely to render 1 an efficient in vivo sequestration agent for 16 and 17. Accordingly, 1 is not an improved lead compound for the sequestration of drugs of abuse (14 ? 17). This is perhaps not surprising given that 1 has a distinct preference for bis(quaternary) di-ammonium ions whereas 14 ? 17 are secondary and tertiary ammonium ions. 81 4.4.2 Serving as reversal agents for neuromuscular blocking molecules In a separate line of inquiry, we have previously shown that M1 and M2 act as in vivo reversal agents for neuromuscular block induced by rocuronium (19), vecuronium (20), and cisatracurium (21).95, 129 Accordingly, we measured the binding constants of 1 toward a panel of compounds relevant to its potential use as an in vivo reversal agent. Table 1 shows that 1 possesses higher binding affinity toward 19 (75-fold) and 20 (172-fold) than M1 does. Importantly, 1 binds >2700-fold tighter to 19 or 20 than to acetylcholine (23). Acetylcholine is also present in the neuromuscular junction and must not be sequestered. The affinity of 1?19 (6.29 ? 108 M-1) and 1?20 (1.00 ? 109 M-1) are comparable to those of M2?19 (3.4 ? 109 M-1) and M2?20 (1.6 ? 109 M-1) which function very well in vivo.129 Host 1, however, possesses superior aqueous solubility (>40 mM) compared to M2 (18 mM) which might prove advantageous for formulation purposes. 4.5 Conclusion In summary, we have presented the synthesis of a new acyclic CB[n]-type receptor (1) with OSO3? groups directly connected to the aromatic walls. Host 1 has excellent aqueous solubility (40 mM), does not undergo self-association, and binds more tightly to quaternary diammonium ions than analogue M1 that features propylene linking chains. The X-ray crystal structures of 1?6a and 1?6d show the usual cavity encapsulation of the diammonium guest but also show an external diammonium ion that balances the overall charge of the tetra-anionic host 1. In conclusion, we find that the negatively charged OSO3? groups do not merely function as solubilizing group, but rather than their close proximity to the ureidyl C=O portals 82 of 1 results in enhanced binding affinity toward quaternary diammonium ions including important neuromuscular blocking agents (19 and 20). This suggests that 1 should be considered alongside M1 and M2 as in vivo reversal agents for neuromuscular blockers. 83 Chapter 5: Conclusion Section 5.1 Summary Supramolecular concepts have been applied for molecular recognition, catalysis, drug delivery and reversal, solubilization, spectroscopic sensor probes, carbon nanotubes and more. The development of molecular containers has played a major role in this expansion. Molecular containers such as ?-cyclodextrins are already used commercially for pharmaceutical purposes and industrial uses such as in FebreezeTM. The discovery of glycoluril based molecular containers such as cucurbit[n]urils and acyclic cucurbit[n]urls opened the door for more innovations. In particular, acyclic cucurbit[n]urils which display higher water solubility due to the sulfonate groups on its aromatic walls, it has a flexible backbone to encapsulate a wider variety of guests. The Isaacs research group did a tremendous work in studying and reporting the applications of acylic cucurbit[n]uril, M1 and M2, in solubilizing drugs, drug reversal toward neuromuscular blocking agents such as Rocuronium and Vecuronium, and recently, reversal of drugs such as Methamphetamine and Fentanyl. Chapter 2 described the yet another property of acyclic Cucubit[n]uril which is recognition properties toward amino acids, peptides and Insulin. It was found that acyclic CB[n] has high binding affinity toward amino acid amides due to ion-dipole interactions and hydrophobic effect. M1 and M2 show preferential binding affinity toward di-cationic and aromatic amino acid amides particularly phenylalanine amide. A deeper analysis shows the energetic cost or benefit in the formation of the complexation of acyclic CB[n] with amino acids or amino acid amides or amino acetyl amides can be linked to ion-dipole and ion-ion interactions between host and 84 guest. Like cucurbit[n]urils, the acyclic CB[n] can also bind to peptides however they are unable to encapsulate two amino acid residues at once. Finally, M1 and M2 show recognition ability toward Insulin in physiological conditions. In chapter 3, a new acyclic CB[n] host ? sulfonated trimer triptycene ? was synthesized, characterized by spectroscopic methods, and its molecular recognition properties examined. An X-ray crystal structure of the host revealed its helical structure and an intriguing packing dynamic. This host displayed interesting recognition properties toward alkyl ammonium guests. It was found that the binding affinity increases as the length of the alkyl ammonium guest increases. The determined Ka values served as a blinded experimental dataset for the SAMPL series of challenges for computational chemists looking to improve their methods to calculate binding free energies in water. Finally, the trimer triptycene host shows strong preferential binding affinity toward Fentanyl. Chapter 4 introduced yet another acyclic CB[n] host, sulfate substituted acyclic CB[n] host. In this new host the alkyl linker connecting the aromatic ring to the sulfonate group is eliminated in order to optimize the binding affinity and selectivity of acyclic CB[n] toward guests. This new host displayed preferential binding affinity toward quaternary ammonium guests. Additionally, the new host demonstrated stronger binding affinity toward Rocuronium and Vecuronium than M1. This indicates its potential application as an in vivo reversal agent for neuromuscular blocking agents. 85 Section 5.2 Future Work Acyclic cucurbit[n]uril hosts although they were just recently developed have already demonstrated outstanding applications in the field of pharmaceuticals. Although we have shown that M1 and M2 bind to a protein (Insulin) there are other proteins that could be targeted by acyclic CB[n]. For example, Klaerner and co- workers have previously shown that benzonorbornene derived molecular tweezers can bind to amyloid protein and prevent aggregation. I suggest that it might be fruitful to perform a similar study with acyclic CB[n]s to evaluate their ability to binding to amyloid protein and their impact on aggregation in physiological conditions. Amyloid proteins are pertinent in the development of Alzheimer's disease which according to the National Institute of Health (NIH) affects over 5.5 million Americans over the age of 65,157 so this has potentially high impact on human health. 86 O O O O OH N N N N N N N N + TFA O H HH H O 8 N N N N N N N N 16h, rt, N2OH O O O O O O O O OH OH N N N N N N N N H HH H N N N N N N N N OH OH O O O O 8 90oC, Pyridine dry pyridine sulfur trioxide N2, 18h O O O O OR OR N N N N N N N N H HH H N N N N N N N N OR OR O O O O 9 R= SO3Na Scheme V-I. Proposed synthesis of a new acyclic CB[n] 8 which would lead to 9. Another project to focus on for future work, is the synthesis of a new acyclic host which combined strong binding and selectivity toward quaternary guests and fluorescent sensing properties. To achieve this goal, host 8 can be obtained by reacting dihydroxy triptycene with glycoluril tetramer by double electrophilic aromatic substitution. Once 8 is obtained it can be transformed into 9 by reaction with pyridine sulfur trioxide under dry conditions. 87 Finally, a series of new sulfate hosts can be produced in order to complete the study of the impact of glycoluril backbone on the molecular recognition properties toward dicationic guests. Now that it is established that the sulfate substituted host with a glycoluril tetramer backbone has a strong binding affinity and selectivity toward quaternary ammonium guests. The new sulfate host 11 and 12 would have smaller cavities and would be selective toward smaller quaternary guests. Host 10 with its trimer glycoluril backbone would most likely feature a helical structure which would allow it to bind to longer quaternary ammonium guests. Accordingly, by creation of a library of acyclic CB[n] we should be able to optimize their binding affinity toward a wide variety of chemically and biologically relevant guests. OR O O O O OR OR O OR N N N N N N N N N N H H N N N N N N N N N N OR O O O OR OR O O OR 10 R= SO3Na 11 R= SO3Na O OR OR N N N N OR OR 12 R= OSO 3Na Figure V-1. Series of sulfate hosts composed of trimer sulfate host 10, dimer sulfate host 11, monomer sulfate host 12 88 Appendix I Molecular Recognition Properties of Acyclic Cucurbiturils Toward Amino Acids, Peptides, and a Protein Supporting Information By Sandra A. Zebaze Ndendjio, and Lyle Isaacs Department of Chemistry and Biochemistry, University of Maryland, College Park, College Park, MD 20742, USA Table of Contents Pages General experimental details ?????????????????????.90 ITC for Motor 2 and amino acid amides ?????????????.........91-109 ITC for Motor 1 and amino acid amides ??????????????...110-127 ITC for Motor 2 and acetyl amino acid amides???????????......128-132 ITC for Motor 1 and acetyl amino acid amides???????????..?133-137 ITC for Motor 2 and amino acids ??????????????..??... 138-142 ITC for Motor 1 and amino acids ??????????????..??. 143-147 ITC for Motor 2 and tripeptides ????????????????..?. 148-154 ITC for Motor 1 and tripeptides ???????????????..??..155-161 ITC for Motor 2 and Insulin, Motor 1 and Insulin, CB [7] and Insulin ?..?..162-164 1H NMR spectra for Motor 1 with amino acid amides ??.................?.?. 165-182 1H NMR spectra for Motor 2 with amino acid amides ??????...?? 183-200 1H NMR spectra for Motor 1 with acetyl amino amides ?????..??.. 201-205 1H NMR spectra for Motor 2 with acetyl amino amides ?????..??.. 206-210 1H NMR spectra for Motor 1 with amino acids ?????...?????... 211-215 1H NMR spectra for Motor 2 with amino acids ?????...?????... 216-220 ????????????????????????????????????????????????????????????????????? 89 General experimental details. The amino acid amides, amino acids, and acetyl amino amides were commercially available and obtained from Bachem, Chem- Impex, Alfa Aeser, and Acros Organic. The acyclic CB[n] were synthesized, purified and characterized as described in a previous publication from the Isaacs group62. The tripeptides were commercially available and obtained from peptide2go and native human insulin recombinant from Saccharomyces cerevisiae (SAFC Biosciences). Calorimetric Titration using microcal PEAQ-ITC from Malvern were performed to obtain stability constants and reaction thermodynamics information for the formation of the complex between acyclic CB[n] and amino acid amides, amino acids and acetyl amino amides. Binding studies were also performed using 400 MHz 1H NMR at room temperature in 20 mM sodium phosphate (NaH2PO4) buffered D2O at pD 7.40 with 64 scans. Details for Isothermal Titration Calorimetry (ITC) measurements. All measurements were performed in High Performance Liquid Chromatography (HPLC) NaH2PO4 buffered water at pH 7.40. The Isothermal Titration Calorimetry (ITC) instrument were set at 19 injections with a volume of 2.00 ?l per injections, 298.0 K temperature, with a reference power of 5.00 ?cal/sec, stir speed of 750 rpm and the feedback was set on high. The measurements for Insulin and the three hosts were done in 20mM sodium phosphate buffered water (NaH2PO4) at pH 7.40, 4mM EDTA at room temperature. 90 Figure II-S1. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Threonine amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= No binding 91 Figure II-S2. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Serine amide (7.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1897.5 M-1 92 Figure II-S3. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (5.00 mM) in the cell was titrated with Glycine amide (50.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 115.0 M-1 93 Figure II-S4. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Arginine amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 8.26 ? 104 M-1 94 Figure II-S5. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Glutamine amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 7.25 ? 103 M-1 95 Figure II-S6. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Leucine amide (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 2.29 ? 105 M-1 96 Figure II-S7. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (5.00 mM) in the cell was titrated with Asparagine amide (50.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1.35 ? 103 M-1 97 Figure II-S8. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Tyrosine amide (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 9.52 ? 105 M-1 98 Figure II-S9. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (1.00 mM) in the cell was titrated with Valine amide (10.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 2.15 ? 104 M-1 99 Figure II-S10. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (1.00 mM) in the cell was titrated with Proline amide (10.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1.70 ? 104 M-1 100 Figure II-S11. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (1.00 mM) in the cell was titrated with Histidine amide (10.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 2.61 ? 104 M-1 101 Figure II-S12. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Lysine amide (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1.41 ? 105 M-1 102 Figure II-S13. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Methionine amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 8.85 ? 104 M-1 103 Figure II-S14. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Phenylalanine amide (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 3.24 ? 106 M-1 104 Figure II-S15. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Tryptophan amide (1.20 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 3.98 ? 106 M-1 105 Figure II-S16. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Aspartic acid amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= No binding 106 Figure II-S17. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Isoleucine amide (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1.68 ? 105 M-1 107 Figure II-S18. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (5.00 mM) in the cell was titrated with Alanine amide (1.00 ? 102 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 515 M-1 108 Figure II-S19. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Glutamic amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= No binding 109 Figure II-S20. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (1.00 mM) in the cell was titrated with Proline amide (10.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 5.75 ? 103 M-1 110 Figure II-S21. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (2.00 mM) in the cell was titrated with Serine amide (18.5 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1.25 ? 104 M-1 111 Figure II-S22. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (1.00 mM) in the cell was titrated with Valine amide (10.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1.40 ? 104 M-1 112 Figure II-S23. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (100 ?M) in the cell was titrated with Glutamic amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= No binding 113 Figure II-S24. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (5.00 mM) in the cell was titrated with Glycine amide (50.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 80.6 M-1 114 Figure II-S25. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (100 ?M) in the cell was titrated with Threonine amide (4 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 8333.3 M-1 115 Figure II-S26. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (1.00 mM) in the cell was titrated with Glutamine amide (25.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1.47 ? 104 M-1 116 Figure II-S27. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (5.00 mM) in the cell was titrated with Alanine amide (1.00 ? 102 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 4.08 ? 102 M-1 117 Figure II-S28. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (100 ?M) in the cell was titrated with Arginine amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 7.09 ? 105 M-1 118 Figure II-S29. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (200 ?M) in the cell was titrated with Asparagine amide (5 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1.44 ? 103 M-1 119 Figure II-S30. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (100 ?M) in the cell was titrated with Aspartic acid amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= No binding 120 Figure II-S31. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (100 ?M) in the cell was titrated with Tyrosine amide (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1.01 ? 106 M-1 121 Figure II-S32. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (100 ?M) in the cell was titrated with Isoleucine amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 9.43 ? 104 M-1 122 Figure II-S33. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (100 ?M) in the cell was titrated with Methionine amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 1.32 ? 105 M-1 123 Figure II-S34. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (100 ?M) in the cell was titrated with Lysine amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 7.46 ? 105 M-1 124 Figure II-S35. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (250 ?M) in the cell was titrated with Histidine amide (3.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 2.35 ? 104 M-1 125 Figure II-S36. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (100 ?M) in the cell was titrated with Tryptophan amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 2.56 ? 105 M-1 126 Figure II-S37. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (100 ?M) in the cell was titrated with Leucine amide (1.50 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka= 2.85 ? 105 M-1 127 Figure II-S38. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Acetyl-phenylalanine amide (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.99 ? 104 M-1 128 Figure II-S39. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (1.00 mM) in the cell was titrated with Acetyl-Lysine amide (20.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 8.06 ? 103 M-1 129 Figure II-S40. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with Acetyl-Tryptophan amide (2.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. K 4 -1 a = 3.56 ? 10 M 130 Figure II-S41. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Acetyl- Tyrosine amide (1.00 mM) in the cell was titrated with Motor 2 (20.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. K 3 -1 a = 3.06 ? 10 M 131 Figure II-S42. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (1.00 mM) in the cell was titrated with Acetyl-Arginine amide (20.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. K 3 -1 a = 7.87 ? 10 M 132 Figure II-S43. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (1.00 mM) in the cell was titrated with Acetyl-Lysine amide (20.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 9.80 ? 103 M-1 133 Figure II-S44. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (1.00 mM) in the cell was titrated with Acetyl-Tryptophan amide (10.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 8.40 ? 102 M-1 134 Figure II-S45. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Acetyl- Tyrosine amide (1.00 mM) in the cell was titrated with Motor 1 (10.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.26 ? 103 M-1 135 Figure II-S46. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (1.00 mM) in the cell was titrated with Acetyl-Phenylalanine amide (20.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 4.17 ? 103 M-1 136 Figure II-S47. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (1.00 mM) in the cell was titrated with Acetyl-Arginine amide (20.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 8.13 ? 103 M-1 137 Figure II-S48. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (5.00 mM) in the cell was titrated with Lysine (50.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 497.5 M?? 138 Figure II-S49. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (1.00 mM) in the cell was titrated with phenylalanine (15.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 9.61 ? 104 M-1 139 Figure II-S50. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (1.00 mM) in the cell was titrated with Tryptophan (10.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.14 ? 105 M-1 140 Figure II-S51. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Tyrosine (1.00 mM) in the cell was titrated with Motor 2 (20.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.03 ? 104 M-1 141 Figure II-S52. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (5.00 mM) in the cell was titrated with Arginine (50.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.39 ? 103 M-1 142 Figure II-S53. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (5.00 mM) in the cell was titrated with Lysine (40.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 333.3 M-1 143 Figure II-S54. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (1.00 mM) in the cell was titrated with Tryptophan (15.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.06 ? 104 M-1 144 Figure II-S55. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Tyrosine (1.00 mM) in the cell was titrated with Motor 1 (20.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.01 ? 104 M-1 145 Figure II-S56. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (1.00 mM) in the cell was titrated with Phenylalanine (10.0 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 7.87 ? 104 M-1 146 Figure II-S57. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 1 (2.00 mM) in the cell was titrated with Arginine (50.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.51 ? 103 M-1 147 Figure II-S58. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Gly-Ala-NH2 (100 ?M) in the cell was titrated with Motor 2 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.20 ? 105 M-1 148 Figure II-S59. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with H-Phe-Pro-Ala-NH2 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.06 ? 105 M-1 149 Figure II-S60. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Ile-Ala-NH2 (100 ?M) in the cell was titrated with Motor 2 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.62 ? 105 M-1 150 Figure II-S61. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Motor 2 (100 ?M) in the cell was titrated with H-Phe-Phe-Ala-NH2 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.22 ? 105 M-1 151 Figure II-S62. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Leu-Ala-NH2 (100 ?M) in the cell was titrated with Motor 2 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.59 ? 105 M-1 152 Figure II-S63. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Lys-Ala-NH2 (100 ?M) in the cell was titrated with Motor 2 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 5.32 ? 104 M-1 153 Figure II-S64. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Val-Ala-NH2 (100 ?M) in the cell was titrated with Motor 2 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 3.84 ? 105 M-1 154 Figure II-S65. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Gly-Ala-NH2 (100 ?M) in the cell was titrated with Motor 1 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.48 ? 106 M-1 155 Figure II-S66. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Pro-Ala-NH2 (100 ?M) in the cell was titrated with Motor 1 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 6.62 ? 104 M-1 156 Figure II-S67. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Phe-Ala-NH2 (100 ?M) in the cell was titrated with Motor 1 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.52 ? 105 M-1 157 Figure II-S68. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Leu-Ala-NH2 (100 ?M) in the cell was titrated with Motor 1 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.57 ? 105 M-1 158 Figure II-S69. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Ile-Ala-NH2 (100 ?M) in the cell was titrated with Motor 1 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.40 ? 105 M-1 159 Figure II-S70. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Lys-Ala-NH2 (100 ?M) in the cell was titrated with Motor 1 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.33 ? 105 M-1 160 Figure II-S71. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of H-Phe-Val-Ala-NH2 (100 ?M) in the cell was titrated with Motor 1 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 3.89 ? 105 M-1 161 Figure II-S72. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Insulin (200 ?M) in the cell was titrated with CB [7] (2.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4 containing 4 mM EDTA. Ka = 5.59 ? 105 M-1 162 Figure II-S73. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Insulin (200 ?M) in the cell was titrated with Motor 2 (2.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4 containing 4mM EDTA. Ka = 3.47 ? 105 M-1 163 Figure II-S74. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of Insulin (200 ?M) in the cell was titrated with Motor 1 (2.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4 containing 4mM EDTA. Ka = 1.32 ? 105 M-1 164 Figure II-S75. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Isoleucine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Isoleucine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Isoleucine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Isoleucine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Isoleucine amide (0.5 mM), f) Motor 1 (1mM). 165 Figure II-S76. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Lysine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Lysine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Lysine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Lysine amide (1 mM), e) a mixture Motor 1 (1 mM) and Lysine amide (0.5 mM), f) Motor 1 (1 mM). 166 Figure II-S77. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Proline amide (2 mM), b) a mixture of Motor 1 (1 mM) and Proline amide (2 mM), c) a mixture of Motor 1 (1 mM) and Proline amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Proline amide (1 mM), e) a mixture Motor 1 (1 mM) and Proline amide (0.5 mM), f) Motor 1 (1 mM). 167 Figure II-S78. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Alanine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Alanine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Alanine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Alanine amide (1 mM), e) a mixture Motor 1 (1 mM) and Alanine amide (0.5 mM), f) Motor 1 (1 mM). 168 Figure II-S79. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Serine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Serine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Serine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Serine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Serine amide (0.5 mM), f) Motor 1 (1 mM). 169 Figure II-S80. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Glutamic acid amide (2 mM), b) a mixture of Motor 1 (1 mM) and Glutamic acid amide (2 mM), c) a mixture of Motor 1 (1 mM) and Glutamic acid amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Glutamic acid amide (1 mM), e) a mixture of Motor 1 (1 mM) and Glutamic acid amide (0.5 mM), f) Motor 1 (1 mM). 170 Figure II-S81. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Glycine amide (3 mM), b) a mixture of Motor 1 (1 mM) and Glycine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Glycine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Glycine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Glycine amide (0.5 mM), f) Motor 1 (1 mM). 171 Figure II-S82. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Valine amide (10 mM), b) a mixture of Motor 1 (1 mM) and Valine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Valine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Valine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Valine amide (0.5 mM), f) Motor 1 (1 mM). 172 Figure II-S83. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Leucine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Leucine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Leucine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Leucine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Leucine amide (0.5 mM), f) Motor 1 (1 mM). 173 Figure II-S84. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Tryptophan amide (2 mM), b) a mixture of Motor 1 (1 mM) and Tryptophan amide (2 mM), c) a mixture of Motor 1 (1 mM) and Tryptophan amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Tryptophan amide (1 mM), e) a mixture of Motor 1 (1 mM) and Tryptophan amide (0.5 mM), f) Motor 1 (1 mM). 174 Figure II-S85. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Tyrosine amide (10 mM), b) a mixture of Motor 1 (1 mM) and Tyrosine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Tyrosine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Tyrosine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Tyrosine amide (0.5 mM), f) Motor 1 (1 mM). 175 Figure II-S86. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Glutamine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Glutamine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Glutamine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Glutamine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Glutamine amide (0.5 mM), f) Motor 1 (1 mM). 176 Figure II-S87. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Methionine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Methionine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Methionine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Methionine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Methionine amide (0.5 mM), f) Motor 1 (1 mM). 177 Figure II-S88. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Histidine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Histidine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Histidine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Histidine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Histidine amide (0.5 mM), f) Motor 1 (1 mM). 178 Figure II-S89. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Threonine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Threonine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Threonine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Threonine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Threonine amide (0.5 mM), f) Motor 1 (1 mM). 179 Figure II-S90. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Aarginine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Arginine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Arginine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Arginine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Arginine amide (0.5 mM), f) Motor 1 (1 mM). 180 Figure II-S91. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Aspartic acid amide (2 mM), b) a mixture of Motor 1 (1 mM) and Aspartic acid amide (2 mM), c) a mixture of Motor 1 (1 mM) and Arginine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Aspartic acid amide (1 mM), e) a mixture of Motor 1 (1 mM) and Aspartic acid amide (0.5 mM), f) Motor 1 (1 mM). 181 Figure II-S92. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Asparagine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Asparagine amide (2 mM), c) a mixture of Motor 1 (1 mM) and Asparagine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Asparagine amide (1 mM), e) a mixture of Motor 1 (1 mM) and Asparagine amide (0.5 mM), f) Motor 1 (1 mM). 182 Figure II-S93. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Tryptophan amide (2 mM), b) a mixture of Motor 2 (1 mM) and Tryptophan amide (2 mM), c) a mixture of Motor 2 (1 mM) and Tryptophan amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Tryptophan amide (1 mM), e) a mixture of Motor 2 (1 mM) and Tryptophan amide (0.5 mM), f) Motor 2 (5 mM). 183 Figure II-S94. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Valine amide (10 mM), b) a mixture of Motor 2 (1 mM) and Valine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Valine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Valine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Valine amide (0.5 mM), f) Motor 2 (5 mM). 184 Figure II-S95. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Asparagine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Asparagine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Asparagine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Asparagine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Asparagine amide (0.5 mM), f) Motor 2 (5 mM). 185 Figure II-S96. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Tyrosine amide (10 mM), b) a mixture of Motor 2 (1 mM) and Tyrosine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Tyrosine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Tyrosine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Tyrosine amide (0.5 mM), f) Motor 2 (5 mM). 186 Figure II-S97. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Glutamine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Glutamine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Glutamine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Glutamine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Glutamine amide (0.5 mM), f) Motor 2 (5 mM). 187 Figure II-S98. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Glutamic acid amide (2 mM), b) a mixture of Motor 2 (1 mM) and Glutamic acid amide (2 mM), c) a mixture of Motor 2 (1 mM) and Glutamic acid amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Glutamic acid amide (1 mM), e) a mixture of Motor 2 (1 mM) and Glutamic acid amide (0.5 mM), f) Motor 2 (5 mM). 188 Figure II-S99. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Alanine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Alanine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Alanine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Alanine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Alanine amide (0.5 mM), f) Motor 2 (5 mM). 189 Figure II-S100. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Aspartic acid amide (2 mM), b) a mixture of Motor 2 (1 mM) and Aspartic acid amide (2 mM), c) a mixture of Motor 2 (1 mM) and Aspartic acid amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Aspartic acid amide (1 mM), e) a mixture of Motor 2 (1 mM) and Aspartic acid amide (0.5 mM), f) Motor 2 (5 mM). 190 Figure II-S101. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Leucine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Leucine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Leucine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Leucine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Leucine amide (0.5 mM), f) Motor 2 (5 mM). 191 Figure II-S102. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Isoleucine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Isoleucine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Isoleucine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Isoleucine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Isoleucine amide (0.5 mM), f) Motor 2 (5 mM). 192 Figure II-S103. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Glycine amide (3 mM), b) a mixture of Motor 2 (1 mM) and Glycine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Glycine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Glycine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Glycine amide (0.5 mM), f) Motor 2 (5 mM). 193 Figure II-S104. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Serine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Serine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Serine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Serine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Serine amide (0.5 mM), f) Motor 2 (5 mM). 194 Figure II-S105. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Proline amide (2 mM), b) a mixture of Motor 2 (1 mM) and Proline amide (2 mM), c) a mixture of Motor 2 (1 mM) and Proline amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Proline amide (1 mM), e) a mixture of Motor 2 (1 mM) and Proline amide (0.5 mM), f) Motor 2 (5 mM). 195 Figure II-S106. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Threonine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Threonine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Threonine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Threonine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Threonine amide (0.5 mM), f) Motor 2 (5 mM). 196 Figure II-S107. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Methionine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Methionine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Methionine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Methionine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Methionine amide (0.5 mM), f) Motor 2 (5 mM). 197 Figure II-S108. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Lysine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Lysine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Lysine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Lysine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Lysine amide (0.5 mM), f) Motor 2 (5 mM). 198 Figure II-S109. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Histidine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Histidine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Histidine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Histidine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Histidine amide (0.5 mM), f) Motor 2 (5 mM). 199 Figure II-S110. 1H NMR spectra recorded (D2O, 400 MHz, RT) for: a) Arginine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Arginine amide (2 mM), c) a mixture of Motor 2 (1 mM) and Arginine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Arginine amide (1 mM), e) a mixture of Motor 2 (1 mM) and Arginine amide (0.5 mM), f) Motor 2 (5 mM). 200 Figure II-S111. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Acetyl phenylalanine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Acetyl phenylalanine amide (2 mM ), c) a mixture of Motor 1 (1 mM) and Acetyl phenylalanine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Acetyl phenylalanine amide (1 mM), e) a mixture of Motor 1 (1 mM ) and Acetyl phenylalanine amide (0.5 mM ), f) Motor 1 (1 mM ). 201 Figure II-S112. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Acetyl arginine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Acetyl arginine amide (2 mM ), c) a mixture of Motor 1 (1 mM) and Acetyl arginine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Acetyl arginine amide (1 mM), e) a mixture of Motor 1 (1 mM ) and Acetyl arginine amide (0.5 mM ), f) Motor 1 (1 mM ). 202 Figure II-S113. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Acetyl tryptophan amide (2 mM), b) a mixture of Motor 1 (1 mM) and Acetyl tryptophan amide (2 mM ), c) a mixture of Motor 1 (1 mM) and Acetyl tryptophan amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Acetyl tryptophan amide (1 mM), e) a mixture of Motor 1 (1 mM ) and Acetyl tryptophan amide (0.5 mM ), f) Motor 1 (1 mM ). 203 Figure II-S114. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Acetyl tyrosine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Acetyl tyrosine amide (2 mM ), c) a mixture of Motor 1 (1 mM) and Acetyl tyrosine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Acetyl tyrosine amide (1 mM), e) a mixture of Motor 1 (1 mM ) and Acetyl tyrosine amide (0.5 mM ), f) Motor 1 (1 mM ). 204 Figure II-S115. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Acetyl lysine amide (2 mM), b) a mixture of Motor 1 (1 mM) and Acetyl lysine amide (2 mM ), c) a mixture of Motor 1 (1 mM) and Acetyl lysine amide (1.5 mM), d) a mixture of Motor 1 (1 mM) and Acetyl lysine amide (1 mM), e) a mixture of Motor 1 (1 mM ) and Acetyl lysine amide (0.5 mM ), f) Motor 1 (1 mM ). 205 Figure II-S116. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Acetyl lysine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Acetyl lysine amide (2 mM ), c) a mixture of Motor 2 (1 mM) and Acetyl lysine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Acetyl lysine amide (1 mM), e) a mixture of Motor 2 (1 mM ) and Acetyl lysine amide (0.5 mM ), f) Motor 2 (5 mM ). 206 Figure II-S117. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Acetyl tyrosine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Acetyl tyrosine amide (2 mM ), c) a mixture of Motor 2 (1 mM) and Acetyl tyrosine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Acetyl tyrosine amide (1 mM), e) a mixture of Motor 2 (1 mM ) and Acetyl tyrosine amide (0.5 mM ), f) Motor 2 (5 mM ). 207 Figure II-S118. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Acetyl arginine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Acetyl arginine amide (2 mM ), c) a mixture of Motor 2 (1 mM) and Acetyl arginine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Acetyl arginine amide (1 mM), e) a mixture of Motor 2 (1 mM ) and Acetyl arginine amide (0.5 mM ), f) Motor 2 (5 mM ). 208 Figure II-S119. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Acetyl phenylalanine amide (2 mM), b) a mixture of Motor 2 (1 mM) and Acetyl phenylalanine amide (2 mM ), c) a mixture of Motor 2 (1 mM) and Acetyl phenylalanine amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Acetyl phenylalanine amide (1 mM), e) a mixture of Motor 2 (1 mM ) and Acetyl phenylalanine amide (0.5 mM ), f) Motor 2 (5 mM ). 209 Figure II-S120. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Acetyl tryptophan amide (2 mM), b) a mixture of Motor 2 (1 mM) and Acetyl tryptophan amide (2 mM ), c) a mixture of Motor 2 (1 mM) and Acetyl tryptophan amide (1.5 mM), d) a mixture of Motor 2 (1 mM) and Acetyl tryptophan amide (1 mM), e) a mixture of Motor 2 (1 mM ) and Acetyl tryptophan amide (0.5 mM ), f) Motor 2 (5 mM ). 210 Figure II-S121. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Phenylalanine (2 mM), b) a mixture of Motor 1 (1 mM) and phenylalanine (2 mM ), c) a mixture of Motor 1 (1 mM) and phenylalanine (1.5 mM), d) a mixture of Motor 1 (1 mM) and phenylalanine (1 mM), e) a mixture of Motor 1 (1 mM ) and phenylalanine (0.5 mM ), f) Motor 1 (1 mM ). 211 Figure II-S122. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Lysine (2 mM), b) a mixture of Motor 1 (1 mM) and lysine (2 mM ), c) a mixture of Motor 1 (1 mM) and lysine (1.5 mM), d) a mixture of Motor 1 (1 mM) and lysine (1 mM), e) a mixture of Motor 1 (1 mM ) and lysine (0.5 mM ), f) Motor 1 (1 mM ). 212 Figure II-S123. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Tryptophan (2 mM), b) a mixture of Motor 1 (1 mM) and tryptophan (2 mM ), c) a mixture of Motor 1 (1 mM) and tryptophan (1.5 mM), d) a mixture of Motor 1 (1 mM) and tryptophan (1 mM), e) a mixture of Motor 1 (1 mM ) and tryptophan (0.5 mM ), f) Motor 1 (1 mM ). 213 Figure II-S124. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Tyrosine (2 mM), b) a mixture of Motor 1 (1 mM) and tyrosine (2 mM ), c) a mixture of Motor 1 (1 mM) and tyrosine (1.5 mM), d) a mixture of Motor 1 (1 mM) and tyrosine (1 mM), e) a mixture of Motor 1 (1 mM ) and tyrosine (0.5 mM ), f) Motor 1 (1 mM ). 214 Figure II-S125. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Arginine (2 mM), b) a mixture of Motor 1 (1 mM) and arginine (2 mM ), c) a mixture of Motor 1 (1 mM) and arginine (1.5 mM), d) a mixture of Motor 1 (1 mM) and arginine (1 mM), e) a mixture of Motor 1 (1 mM ) and arginine (0.5 mM ), f) Motor 1 (1 mM ). 215 Figure II-S126. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Lysine (2 mM), b) a mixture of Motor 2 (1 mM) and lysine (2 mM ), c) a mixture of Motor 2 (1 mM) and lysine (1.5 mM), d) a mixture of Motor 2 (1 mM) and lysine (1 mM), e) a mixture of Motor 2 (1 mM ) and lysine (0.5 mM ), f) Motor 2 (5 mM ). 216 Figure II-S127. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Phenylalanine (2 mM), b) a mixture of Motor 2 (1 mM) and phenylalanine (2 mM ), c) a mixture of Motor 2 (1 mM) and phenylalanine (1.5 mM), d) a mixture of Motor 2 (1 mM) and phenylalanine (1 mM), e) a mixture of Motor 2 (1 mM ) and phenylalanine (0.5 mM ), f) Motor 2 (5 mM ). 217 Figure II-S128. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Tryptophan (2 mM), b) a mixture of Motor 2 (1 mM) and tryptophan (2 mM ), c) a mixture of Motor 2 (1 mM) and tryptophan (1.5 mM), d) a mixture of Motor 2 (1 mM) and tryptophan (1 mM), e) a mixture of Motor 2 (1 mM ) and tryptophan (0.5 mM ), f) Motor 2 (5 mM ). 218 Figure II-S129. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Arginine (2 mM), b) a mixture of Motor 2 (1 mM) and arginine (2 mM ), c) a mixture of Motor 2 (1 mM) and arginine (1.5 mM), d) a mixture of Motor 2 (1 mM) and arginine (1 mM), e) a mixture of Motor 2 (1 mM ) and arginine (0.5 mM ), f) Motor 2 (5 mM ). 219 Figure II-S130. 1H NMR spectra recorded (D2O , 400 MHz, RT) for: a) Tyrosine (2 mM), b) a mixture of Motor 2 (1 mM) and tyrosine (2 mM ), c) a mixture of Motor 2 (1 mM) and tyrosine (1.5 mM), d) a mixture of Motor 2 (1 mM) and tyrosine (1 mM), e) a mixture of Motor 2 (1 mM ) and tyrosine (0.5 mM ), f) Motor 2 (5 mM ). 220 Appendix II Triptycene Walled Glycoluril Trimer: Synthesis and Recognition Properties Sandra Zebaze Ndendjio,a Wenjin Liu,a,b Nicolas Yvanez,a,c Zhihui Meng,b,* Peter Y. Zavalij,a and Lyle Isaacsa,* Supporting Information a Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA. E-mail: lisaacs@umd.edu b School of Chemistry and Chemical Engineering, Beijing Institute of Technology, 5 South Zhongguancun Street, Beijing 100081, P. R. China c ?cole Nationale Sup?rieure de Chimie de Paris, 11 rue Pierre et Marie Curie, F75231 Paris cedex 05, France Table of Contents Pages ????????????????????????????????????????????????????????????????????? General experimental 222 1H and 13C NMR recorded for 1 223-225 Dilution experiment performed for 1 226-227 1H NMR stack plots for 1?guest complexes 228-251 ITC binding studies for 1?guest complexes 252-275 ESI-MS spectrum recorded for 1 276 ????????????????????????????????????????????????????????????????????? 221 General experimental Starting materials were purchased from commercial suppliers and used without further purification or were prepared by literature procedures. Melting points were measured on a Meltemp apparatus in open capillary tubes and are uncorrected. IR spectra were recorded on a JASCO FT/IR 4100 spectrometer and are reported in cm-1. NMR spectra were measured on Bruker spectrometers operating at 400 or 600 MHz for 1H and 100 or 125 MHz for 13C using D2O, or DMSO-d6 as solvents. Chemical shifts (?) are referenced relative to the residual resonances for HOD (4.80 ppm) and DMSO-d6 (2.50 ppm for 1H, 39.51 ppm for 13C). Mass spectrometry was performed using a JEOL AccuTOF electrospray instrument (ESI). ITC data was collected on a Malvern Microcal PEAQ-ITC instrument and analyzed using the software provided by the vendor. 222 1H and 13C NMR spectra of host 1 Figure III-S1. 1H NMR spectrum (D2O, 20 mM sodium phosphate, pD 7.4, 600 MHz, RT) recorded for 1. 223 Figure III-S2. 1H NMR spectrum (DMSO-d6, 600 MHz, RT) recorded for 1. 224 Figure III-S3. 13C NMR spectrum (D2O, 600 MHz, RT, 1,4-dioxane as internal reference) recorded for 1. 225 1H NMR Dilution Experiments Performed for host 1 Self-association Binding Model implemented in ScientistTM // Micromath Scientist Model File // self-association model for NMR IndVars: concTot DepVars: Deltaobs Params: Ka, Deltasat, Deltazero Ka = concBound/(concFree*concFree) concTot=concFree + concBound/2 Deltaobs = Deltazero + (Deltasat - Deltazero) * (1/2*concBound/concTot) //Constraints 0 < Ka 0 < concFree < concTot 0 < concBound < concTot *** 226 Figure III-S4. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for the dilution of host 1 (3 - 0.05 mM). Host 1 weakly self-associates in water (Ks = 480 ? 81 M-1). 1H NMR spectra of guests (4-26) with host 1 227 Figure III-S5. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 400 MHz, RT) for: a) 4 (5 mM), b) a mixture of 1 (125 ?M) and 4 (250 ?M), c) a mixture of 1 (125 ?M) and 4 (125 ?M), d) 1 (250 ?M). 228 Figure III-S6. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 5 (5 mM), b) a mixture of 1 (250 ?M) and 5 (500 ?M), c) a mixture of 1 (250 ?M) and 5 (250 ?M), d) 1 (250 ?M). 229 Figure III-S7. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 6 (5 mM), b) a mixture of 1 (250 ?M) and 6 (500 ?M), c) a mixture of 1 (250 ?M) and 6 (250 ?M), d) 1 (250 ?M). 230 Figure III-S8. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 6DQ?2Br- (6 mM), b) a mixture of 1 (250 ?M) and 6DQ?2Br- (500 ?M), c) a mixture of 1 (250 ?M) and 6DQ?2Br- (250 ?M), d) 1 (250 ?M). 231 Figure III-S9. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 400 MHz, RT) for: a) 6Q?Br- (4 mM), b) a mixture of 1 (125 ?M) and 6Q?Br- (250 ?M), c) a mixture of 1 (125 ?M) and 6Q?Br- (125 ?M), d) 1 (250 ?M). 232 Figure III-S10. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 7 (2 mM), b) a mixture of 1 (125 ?M) and 7 (250 ?M), c) a mixture of 1 (125 ?M) and 7 (125 ?M), d) 1 (250 ?M). 233 Figure III-S11. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 9 (2 mM), b) a mixture of 1 (125 ?M) and 9 (250 ?M), c) a mixture of 1 (125 ?M) and 9 (125 ?M), d) 1 (250 ?M). 234 Figure III-S12. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 10 (2 mM), b) a mixture of 1 (125 ?M) and 10 (250 ?M), c) a mixture of 1 (125 ?M) and 10 (125 ?M), d) 1 (250 ?M). 235 Figure III-S13. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 11 (5 mM), b) a mixture of 1 (250 ?M) and 11 (500 ?M), c) a mixture of 1 (250 ?M) and 11 (250 ?M), d) 1 (250 ?M). 236 Figure III-S14. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 400 MHz, RT) for: a) 12 (2 mM), b) a mixture of 1 (125 ?M) and 12 (250 ?M), c) a mixture of 1 (125 ?M) and 12 (125 ?M), d) 1 (250 ?M). 237 Figure III-S15. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 13 (2 mM), b) a mixture of 1 (125 ?M) and 13 (250 ?M), c) a mixture of 1 (125 ?M) and 13 (125 ?M), d) 1 (250 ?M). 238 Figure III-S16. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 14 (4 mM), b) a mixture of 1 (125 ?M) and 14 (250 ?M), c) a mixture of 1 (125 ?M) and 14 (125 ?M), d) 1 (250 ?M). 239 Figure III-S17. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 15 (4 mM), b) a mixture of 1 (250 ?M) and 15 (500 ?M), c) a mixture of 1 (250 ?M) and 15 (250 ?M), d) 1 (250 ?M). 240 Figure III-S18. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 16 (4 mM), b) a mixture of 1 (250 ?M) and 16 (500 ?M), c) a mixture of 1 (250 ?M) and 16 (250 ?M), d) 1 (250 ?M). 241 Figure III-S19. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 17 (2 mM), b) a mixture of 1 (250 ?M) and 17 (500 ?M), c) a mixture of 1 (250 ?M) and 17 (250 ?M), d) 1 (250 ?M). 242 Figure III-S20. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 18 (1 mM), b) a mixture of 1 (125 ?M) and 18 (250 ?M), c) a mixture of 1 (125 ?M) and 18 (125 ?M), d) 1 (250 ?M). 243 Figure III-S21. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 19 (4 mM), b) a mixture of 1 (250 ?M) and 19 (500 ?M), c) a mixture of 1 (250 ?M) and 19 (250 ?M), d) 1 (250 ?M). 244 Figure III-S22. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 20 (250 ?M), b) a mixture of 1 (125 ?M) and 20 (250 ?M), c) a mixture of 1 (125 ?M) and 20 (125 ?M), d) 1 (250 ?M). 245 Figure III-S23. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 21 (1 mM), b) a mixture of 1 (125 ?M) and 21 (250 ?M), c) a mixture of 1 (125 ?M) and 21 (125 ?M), d) 1 (250 ?M). 246 Figure III-S24. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 22 (2 mM), b) a mixture of 1 (125 ?M) and 22 (250 ?M), c) a mixture of 1 (125 ?M) and 22 (125 ?M), d) 1 (250 ?M). 247 Figure III-S25. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 23 (250 ?M), b) a mixture of 1 (125 ?M) and 23 (250 ?M), c) a mixture of 1 (125 ?M) and 23 (125 ?M), d) 1 (250 ?M). 248 Figure III-S26. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 400 MHz, RT) for: a) 24 (250 ?M), b) a mixture of 1 (125 ?M) and 24 (250 ?M), c) a mixture of 1 (125 ?M) and 24 (125 ?M), d) 1 (250 ?M). 249 Figure III-S27. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 25 (1 mM), b) a mixture of 1 (125 ?M) and 25 (250 ?M), c) a mixture of 1 (125 ?M) and 25 (125 ?M), d) 1 (250 ?M). 250 Figure III-S28. 1H NMR spectra recorded (D2O, 20 mM sodium phosphate, pD 7.40, 600 MHz, RT) for: a) 26 (250 mM), b) a mixture of 1 (62.5 ?M) and 26 (125 ?M), c) a mixture of 1 (125 ?M) and 26 (125 ?M), d) 1 (250 ?M). 251 Isotherm of guests (4-26) with host 1 Figure III-S29. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (100 ?M) in the cell was titrated with 4 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.92 ? 104 M-1 and ?H = -6.03 ? 0.260 kcal?mol-1 252 Figure III-S30. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 19 (500 ?M) in the cell was titrated with 6 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.30 ? 107 M-1 and H = -10.8 ? 0.044 kcal?mol-1. 253 Figure III-S31. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 19 (500 ?M) in the cell was titrated with 6DQ (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 5.00 ? 107 M-1 and ?H = -12.7 ? 0.028 kcal?mol-1 254 Figure III-S32. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (100 ?M) in the cell was titrated with 6Q (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.20 ? 106 M-1 and ?H = -8.54 ? 0.027 kcal?mol-1. 255 Figure III-S33. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 19 (500 ?M) in the cell was titrated with 7 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 7.24 ? 107 M-1 and ?H = -10.1 ? 0.036 kcal?mol-1. 256 Figure III-S34. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 5 (500 ?M) in the cell was titrated with 8 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.41 ? 108 M-1 and ?H = -11.5 ? 0.094 kcal?mol-1. 257 Figure III-S35. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 5 (500 ?M) in the cell was titrated with 9 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.42 ? 108 M-1 and ?H = -11.4 ? 0.062 kcal?mol-1. 258 Figure III-S36. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 5 (200 ?M) in the cell was titrated with 10 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.81 ? 108 M-1 and ?H = -11.3 ? 0.068 kcal?mol-1. 259 Figure III-S37. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (100 ?M) in the cell was titrated with 11 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 3.57 ? 105 M-1 and ?H = -4.83 ? 0.036 kcal?mol-1. 260 Figure III-S38. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 5 (500 ?M) in the cell was titrated with 12 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 4.55 ? 108 M-1 and ?H = -10.4 ? 0.064 kcal?mol-1. 261 Figure III-S39. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (10.0 ?M) in the cell was titrated with 13 (100 ?M) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. K = 1.13 ? 107 M-1 a and ?H = -10.1 ? 0.119 kcal?mol-1. 262 Figure III-S40. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (20.0 ?M) in the cell was titrated with 14 (200 ?M) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. K = 4.08 ? 106 M-1 a and ?H = -7.41 ? 0.084 kcal?mol-1. 263 Figure III-S41. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (100 ?M) in the cell was titrated with 15 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.11 ? 106 M-1 and ?H =-5.88 ? 0.049 kcal?mol-1. 264 Figure III-S42. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 19 (500 ?M) in the cell was titrated with 16 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 8.77 ? 106 M-1 and ?H = -10.5 ? 0.044 kcal?mol-1. 265 Figure III-S43. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 19 (500 ?M) in the cell was titrated with 17 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 5.81 ? 107 M-1 and ?H = -12.4 ? 0.045 kcal?mol-1. 266 Figure III-S44. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 5 (500 ?M) in the cell was titrated with 18 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 3.57 ? 108 M-1 and ?H = -13.7 ? 0.039 kcal?mol-1. 267 Figure III-S45. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (100 ?M) in the cell was titrated with 19 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 5.95 ? 104 M-1 and ?H = -6.61 ? 0.088 kcal?mol-1. 268 Figure III-S46. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 19 (500 ?M) in the cell was titrated with 20 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.32 ? 107 M-1 and ?H = -14.7 ? 0.036 kcal?mol-1. 269 Figure III-S47. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (100 ?M) in the cell was titrated with 21 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 9.80 ? 104 M-1 and ?H = -5.09 ? 0.042 kcal?mol-1 270 Figure III-S48. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (200 ?M) in the cell was titrated with 22 (5.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. K = 5.61 ? 104 M-1a and ?H = -3.98 ? 0.094 kcal?mol-1. 271 Figure III-S49. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (100 ?M) in the cell was titrated with 23 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. K = 8.47 ? 103 M-1a and ?H = -4.95 ? 2.30 kcal?mol-1. 272 Figure III-S50. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (100 ?M) in the cell was titrated with 24 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. K = 9.43 ? 105 M-1a and ?H =-9.63 ? 0.025 kcal?mol-1. 273 Figure III-S51. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (100 ?M) in the cell was titrated with 25 (2.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 3.70 ? 104 M-1 and ?H =-9.99 ? 0.129 kcal?mol-1. 274 Figure III-S52. Isothermal Titration Calorimetry (ITC) curve obtained when a solution of 1 (200 ?M) in the cell was titrated with 26 (2.40 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. K = 4.67 ? 103 M-1a and ?H = -8.92 ? 0.445 kcal?mol-1. 275 Figure III-S53. Electrospray mass spectrum for 1 recorded in the negative ion mode. The peak at 829.20204 corresponds to the [M + 1H ? 3Na]2- ion. 276 Appendix III Acyclic Cucurbit[n]uril-Type Receptors: Optimization of Electrostatic Interactions for Dicationic Guests By Xiaoyong Lu, Sandra A. Zebaze Ndendjio, Peter Y. Zavalij, Lyle Isaacs* Department of Chemistry and Biochemistry, University of Maryland, College Park, College Park, MD 20742, USA Supporting Information Table of Contents Pages General experimental ????????????????????.??? 278 Synthesis and characterization of compounds 1 and 4?????????...?. 279 1H NMR spectra of compound 1 and 4 ???????????????...... 280-285 Self-association study of compound 1 ????????????????... 286 1H NMR spectra of selected guests with host 1 ??????????............. 287-305 Isothermal Titration Calorimetry study of host 1 with guests?????...?...... 306-330 Single crystal X-ray data for host 1?7a and 1?7b?????????.??? 331-332 277 General experimental All chemicals and reagents were purchased from commercial suppliers and were used without further purification. Compound 2 was prepared as described previously.1 NMR spectra were measured at 400, 500 or 600 MHz for 1H and 100 and 125 MHz for 13C. The solvent for NMR experiments was deuterated water (D2O), deuterated chloroform (CDCl3), or deuterated dimethyl sulfoxide (DMSO-d6). Chemical shifts (?) are referenced relative to the residual resonances for HOD (4.80 ppm), CHCl3 (7.26 ppm for 1H, 77.16 ppm for 13C), DMSO-d6 (2.50 ppm for 1H, 39.51 ppm for 13C). Isothermal titration calorimetry was performed using a MicroCal PEAQ-ITC Isothermal Titration Calorimeter using 20 mM phosphate buffered water at pH = 7.4 at 298 K. Mass spectrometry was performed using a JEOL AccuTOF electrospray instrument. Melting points were measured by a Meltemp apparatus in open capillary tubes and are uncorrected. IR spectra were measured on a Thermo Nicolet NEXUS 670 FT/IR spectrometer by attenuated total reflectance (ATR) and are reported in cm - 1. 1. Ma, D.; Hettiarachchi, G.; Nguyen, D.; Zhang, B.; Wittenberg, J. B.; Zavalij, P. Y.; Briken, V.; Isaacs, L. Nature Chemistry 2012, 4, 503-510. 278 Synthesis and characterization of compounds 4 and 1. Container 4. To a mixture of glycoluril tetramer bisether 2 (17 g, 22.1 mmol) and hydroquinone (9.7 g, 88.2 mmol) was added trifluoroacetic acid (300 mL). The resulting heterogeneous mixture was stirred under N2 at 25 ?C for 16 hours. The reaction mixture was then poured into MeOH (600 mL) and stirred for 1 hour. The crude product was collected by filtration and subsequently washed with MeOH (400 mL), acetone (300 mL) and water (400 mL) to remove the unreacted hydroquinone. The residue was dried under the high vacuum to yield 2 as a pale-yellow solid (20.8 g, yield 99%). M.p. > 300 ?C, IR (ATR, cm-1): 3400w, 1713s, 1460s, 1226s, 1082m, 971w, 797s. 1H NMR (DMSO-d6, 600 MHz): ? 8.60 (s, 4H), 6.55 (s, 4H), 5.56 (d, J = 14.5 Hz, 2H), 5.49 (d, J = 15.1 Hz, 4H), 5.38 (d, J = 8.9 Hz, 2H), 5.27 (d, J = 8.9 Hz, 2H), 5.18 (d, J = 15.7 Hz, 4H), 4.09 ? 4.04 (m, 10H), 1.68 (s, 6H), 1.62 (s, 6H). 13C NMR (DMSO-d6, 125 MHz): ? 155.3, 154.2, 147.0, 126.1, 116.7, 77.4, 76.4, 70.7, 70.3, 48.1, 35.0, 17.1, 15.8 ppm (13 out of the 14 expected resonances were observed). HR-MS (ESI, positive, para-xylenediammonium dichloride as guest): m/z 551.22446 ([M + guest- 2Cl-]2+), C50H 2+58N18O12 calcd. for 551.22408. Container 1. To a mixture of compound 2 (0.80 g, 0.83 mmol) and pyridine sulfur trioxide complex (2.6 g, 16.3 mmol) was added dry pyridine (25 mL). The resulting mixture was stirred at 90 ?C under N2 for 18 hours. The reaction mixture was cooled to RT. The product precipitated out of the solution and was collected by filtration. The solid was slurried in water (1 mL), and the pH was adjusted to 8.4 by slow addition of saturated aqueous NaHCO3. After addition of EtOH (70 mL), the crude product was collected by centrifugation 7000 rpm ? 7 min. The precipitate was suspended in ethanol (50 mL ? 2), sonicated for 30 minutes, and solid collected by centrifugation. The crude solid was analyzed by NMR and process was repeated until the trapped pyridine was fully removed. Then the pH of the crude solution was adjusted to 7.0 by slow addition of 1 M HCl and the solvent was evaporated. The crude solid was treated with 30 mL of a mixture of CH3CN/ H2O (2: 1) and the heterogeneous mixture was centrifuged, the supernatant was collected and then evaporated to give a crude solid. The crude solid was redissolved in minimum amount of water and purified by size exclusion chromatography using Sephadex? G25 resin (30mm x 200mm) and eluted by water. Pure product was collected as the front fractions with violet fluorescent color under UV long wavelength (366 nm). After drying under high vacuum, the compound 1 was obtained as a light-yellow solid (0.50 g, 60% yield). M.p. > 300 ?C, IR (ATR, cm-1): 1720m, 1468s, 1228s, 1048s, 970m, 798s. 1H NMR (D2O, 500 MHz): ? 7.53 (s, 4H), 6.66 (d, J = 15.4 Hz, 2H), 5.57 (d, J = 15.8 Hz, 4H), 5.46 (d, J = 8.9 Hz, 2H), 5.41 (d, J = 8.9 Hz, 2H), 5.28 (d, J = 16.5 Hz, 4H), 4.41 (d, J = 16.5 Hz, 4H), 4.29 (d, J = 15.8 Hz, 4H), 4.16 (d, J = 15.4 Hz, 2H), 1.84 (s, 6H), 1.82 (s, 6H). 13C NMR (D2O, 125 MHz, dioxane as external reference): ? 156.5, 155.9, 146.0, 131.9, 123.0, 78.3, 77.3, 71.1, 70.9, 52.5, 48.1, 35.8, 15.6, 14.6 ppm (14 out of the 14 expected resonances were observed). HR-MS (ESI, positive, 6d as guest): m/z 787.1679 ([M + 6d-2Cl-]2+), C 2+54H70N18O24Na4 calcd. for 787.1642. 279 1H NMR spectra of compound 1 and 2 Figure IV-S1. 1H NMR spectra (600 MHz, D2O, RT) recorded for compound 1. 280 Figure IV-S2. 1H, 1H DQCOSY NMR spectra (600 MHz, D2O, RT) recorded for compound 1. 281 Figure IV-S3. 13C NMR spectra (600 MHz, D2O, RT) recorded for compound 1. 282 Figure IV-S4. 13C DEPT135 NMR spectra (600 MHz, D2O, RT) recorded for compound 1. 283 Figure IV-S5. 1H NMR spectra (600 MHz, DMSO-d6, RT) recorded for compound 2. 284 Figure IV-S6. 13C NMR spectra (125 MHz, DMSO-d6, RT) recorded for compound 2. 285 Figure IV-S7. 1H NMR spectra (600 MHz, D2O, RT) recorded for 1 as a function of concentration. 286 1H NMR spectra of selected guests with host 1 Figure IV-S8. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 5a (1.0 mM), c) host 1 (1.0 mM) and guest 5a (2.0 mM), d) guest 5a (1.0 mM). 287 Figure IV-S9. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 6Q (1.0 mM), c) host 1 (1.0 mM) and guest 6Q (2.0 mM), d) guest 6Q (1.0 mM). 288 Figure IV-S10. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 11d (1.0 mM), c) host 1 (1.0 mM) and guest 11d (2.0 mM), d) guest 11d (1.0 mM) 289 Figure IV-S11. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 11a (1.0 mM), c) host 1 (1.0 mM) and guest 11a (2.0 mM), d) 11a (1.0mM). 290 Figure IV-S12. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 6d (1.0 mM), c) host 1 (1.0 mM) and guest 6d (2.0 mM), d) guest 6d (1.0 mM). 291 Figure IV-S13. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 6c (1.0 mM), c) host 1 (1.0 mM) and guest 6c (2.0 mM), d) guest 6c (1.0 mM). 292 Figure IV-S14. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 6b (1.0 mM), c) host 1 (1.0 mM) and guest 6b (2.0 mM), d) guest 6b (1.0 mM). 293 Figure IV-S15. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 6a (1.0 mM), c) host 1 (1.0 mM) and guest 6a (2.0 mM), d) guest 6a (1.0 mM). 294 Figure IV-S16. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 7d (1.0 mM), c) host 1 (1.0 mM) and guest 7d (2.0 mM), d) guest 7d (1.0 mM). 295 Figure IV-S17. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 8d (1.0 mM), c) host 1 (1.0 mM) and guest 8d (2.0 mM), d) guest 8d (1.0 mM). 296 Figure IV-S18. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 9d (1.0 mM), c) host 1 (1.0 mM) and guest 9d (2.0 mM), d) guest 9d (1.0 mM). 297 Figure IV-S19. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 10d (1.0 mM), c) host 1 (1.0 mM) and guest 10d (2.0 mM), d) guest 10d (1.0 mM). 298 Figure IV-S20. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 12d (1.0 mM), c) host 1 (1.0 mM) and guest 12d (2.0 mM), d) guest 12d (1.0 mM). 299 Figure IV-S21. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 12a (1.0 mM), c) host 1 (1.0 mM) and guest 12a (2.0 mM), d) guest 12a (1.0 mM). 300 Figure IV-S22. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 13a (1.0 mM), c) host 1 (1.0 mM) and guest 13a (2.0 mM), d) guest 13a (1.0 mM). 301 Figure IV-S23. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (250 ?M) and guest 19 (250 ?M), c) host 1 (250 ?M) and guest 19 (500 ?M), d) guest 19 (500 ?M). 302 Figure IV-S24. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (250 ?M) and guest 20 (250 ?M), c) host 1 (250 ?M) and guest 20 (500 ?M), d) guest 20 (500 ?M). 303 Figure IV-S25. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 22 (1.0 mM), c) host 1 (1.0 mM) and guest 22 (2.0 mM), d) guest 22 (1.0 mM). 304 Figure IV-S26. 1H NMR spectra (600 MHz, D2O, RT) recorded for a) host 1 (1.0 mM), b) host 1 (1.0 mM) and guest 23 (1.0 mM), c) host 1 (1.0 mM) and guest 23 (2.0 mM), d) guest 23 (1.0 mM). 305 Isothermal Titration Calorimetry study of host 1 with selected guests Figure IV-S27. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding titration studies. A solution of 1 (100 ?M) in the cell was titrated with 5a (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.68 ? 106 M-1. 306 Figure IV-S28. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 5a (500 ?M) in the cell was titrated with 6a (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 3.70 ? 108 M-1 307 Figure IV-S29. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 13a (500 ?M) in the cell was titrated with 6b (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 5.26 ? 108 M-1 308 Figure IV-S30. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 13a (500 ?M) in the cell was titrated with 6c (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 5.74 ? 108 M-1 309 Figure IV-S31. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 13a (2000 ?M) in the cell was titrated with 6d (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 6.71 ? 109 M-1 310 Figure IV-S32. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding titration studies. A solution of 1 (10 ?M) in the cell was titrated with 6Q (100 ?M) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 7.57 ? 106 M-1 311 Figure IV-S33. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 13a (2000 ?M) in the cell was titrated with 7d (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 6.06 ? 109 M-1. 312 Figure IV-S34. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 13a (500 ?M) in the cell was titrated with 8d (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.75 ? 109 M-1. 313 Figure IV-S35. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 13a (500 ?M) in the cell was titrated with 9d (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 7.57 ? 108 M-1. 314 Figure IV-S36. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 13a (500 ?M) in the cell was titrated with 10d (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. K 8 -1a = 5.43 ? 10 M 315 Figure IV-S37. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding studies. A solution of 1 (100 ?M) in the cell was titrated with 12a (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 9.90 ? 105 M-1 316 Figure IV-S38. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (50 ?M) and 12d (1500 ?M) in the cell was titrated with 6d (500 ?M) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 6.66 ? 106 M-1 317 Figure IV-S39. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding studies. A solution of 1 (10.0 ?M) in the cell was titrated with 13a (100 ?M) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 3.41 ? 106 M-1. 318 Figure IV-S40. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 5a (500 ?M) in the cell was titrated with 11a (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 9.71 ? 108 M-1. 319 Figure IV-S41. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 5a (500 ?M) in the cell was titrated with 11d (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.05 ? 109 M-1. 320 Figure IV-S42. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding studies. A solution of 1 (100.0 ?M) in the cell was titrated with 23 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.31 ? 105 M-1. 321 Figure IV-S43. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding studies. A solution of 1 (100.0 ?M) in the cell was titrated with 22 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 2.41 ? 104 M-1 322 Figure IV-S44. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 5a (500 ?M) in the cell was titrated with 19 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 6.29 ? 108 M-1. 323 Figure IV-S45. Isothermal Titration Calorimetry (ITC) curve obtained through competition binding studies. A solution of 1 (100 ?M) and 13a (500 ?M) in the cell was titrated with 20 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.00 ? 109 M-1. 324 Figure IV-S46. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding studies. A solution of 1 (80.0 ?M) in the cell was titrated with 21 (500.0 ?M) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 5.32 ? 105 M-1. 325 Figure IV-S47. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding studies. A solution of 1 (30.0 ?M) in the cell was titrated with 14 (300.0 ?M) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 3.02 ? 106 M-1. 326 Figure IV-S48. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding studies. A solution of 1 (30.0 ?M) in the cell was titrated with 15 (300.0 ?M) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 3.64 ? 106 M-1. 327 Figure IV-S49. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding studies. A solution of 1 (100.0 ?M) in the cell was titrated with 17 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 7.69 ? 105 M-1. 328 Figure IV-S50. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding studies. A solution of 1 (100.0 ?M) in the cell was titrated with 16 (1.00 mM) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 1.89 ? 105 M-1. 329 Figure IV-S51. Isothermal Titration Calorimetry (ITC) curve obtained through direct binding studies. A solution of 1 (30.0 ?M) in the cell was titrated with 18 (300.00 ?M) in the syringe at 298.0 K in 20 mM sodium phosphate buffered water at pH 7.4. Ka = 4.85 ? 106 M-1. 330 Single crystal X-ray data for host 1 and guest 6a and 6d Figure IV-S52. 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