CIRCULAR AROMATIC Γ-PEPTIDES DERIVED FROM PHENOL- AND METHOXYBENZENE-BASED BUILDING BLOCKS SHU YINGYING (B.Sc.), SICHUAN UNIVERSITY, CHINA A THESIS SUMBITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements I would like to express my sincere gratitude to my supervisor, Dr. Zeng Huaqiang, Ph. D., Assistant Professor, Department of Chemistry, National University of Singapore, for his valuable guidance, persistent help and encouragement throughout these years. He conveys a spirit of adventure in regard to research and devotes considerable amount of time to guide students in the projects, not only sharing his knowledge but also inspiring students to contribute to knowledge. I would like to express my sincere thanks to Dr. Qin Bo, Research Fellow, and Sun Chang for their valuable and kind help in my project. And I would also like to thank the other members in Dr. Zeng’s group, Yan Yan, Fang Xiao, Ong Wei Qiang, Ren Changliang, Yip Yeow Kwan, Hii Meng Ni, Liang Hui Fang and Pan Si Yan, for their collaboration and friendship. I would like to express my sincere gratitude to Department of Chemistry and National University of Singapore for the award of the research scholarship. In addition, I am so grateful for the moral support and warmest encouragement from my parents and friends to complete the project. Thank you all. Shu Yingying i Table of Contents ACKNOWLEDGEMENTS............................................................................................................ I TABLE OF CONTENTS...............................................................................................................II SUMMARY .................................................................................................................................... V LIST OF TABLES ...................................................................................................................... VII LIST OF FIGURES .................................................................................................................. VIII LIST OF ILLUSTRATIONS ........................................................................................................ X CHAPTER ONE: INTRODUCTION............................................................................................1 1.1 GENERAL .................................................................................................................................1 1.2 UNIMOLECULAR ION CHANNEL ................................................................................................4 1.3 AGGREGATE ION CHANNELS ....................................................................................................6 1.4 OTHER TYPES OF ION CHANNELS ...........................................................................................12 1.5 APPLICATIONS ........................................................................................................................13 REFERENCES ................................................................................................................................15 CHAPTER TWO: SYNTHESIS AND STRUCTURAL INVESTIGATIONS OF CIRCULAR AROMATIC Γ-PEPTIDES DERIVED FROM PHENOL- AND METHOXYBENZENE-BASED BUILDING BLOCKS............................................................18 2.1 INTRODUCTION ......................................................................................................................18 2.2 EXPERIMENTAL SECTION .......................................................................................................20 ii 2.2.1 Synthetic Scheme ...........................................................................................................20 2.2.2 General Methods ...........................................................................................................22 2.2.3 Synthetic Procedure .......................................................................................................22 2.3 THEORETICAL MODELING ......................................................................................................33 2.3.1 Dimer.............................................................................................................................33 2.3.2 Higher Oligomers from Trimer to Pentamer .................................................................34 2.3.3 Cyclic Pentamers...........................................................................................................35 2.4 RESULTS AND DISCUSSION .....................................................................................................36 2.4.1 Synthesis of Monomer, Higher Oligomers and Cyclic Pentamers .................................36 2.4.2 1D and 2D 1H NMR Results ..........................................................................................40 2.4.3 X-Ray Crystal Structure Analysis...................................................................................41 2.5 CONCLUSIONS ........................................................................................................................43 REFERENCES ................................................................................................................................44 CHAPTER THREE: SYNTHESIS AND STRUCTURAL INVESTIGATIONS OF METHOXYBENZENE-BASED CIRCULAR Γ-PEPTIDES....................................................45 3.1 INTRODUCTION ......................................................................................................................45 3.2 EXPERIMENTAL SECTION .......................................................................................................45 3.2.1 Synthetic Schemes..........................................................................................................45 3.2.2 General Methods ...........................................................................................................47 3.2.3 Synthetic Procedure .......................................................................................................48 3.3 THEORETICAL MODELING ......................................................................................................54 3.4 RESULTS AND DISCUSSION .....................................................................................................55 iii 3.4.1 Synthesis of Oligomers and Circular Pentamer.............................................................55 3.4.2 X-Ray Crystal Structure Analysis...................................................................................55 3.5 CONCLUSIONS ........................................................................................................................56 REFERENCES ................................................................................................................................58 iv Summary The aim of this project is to establish a new class of macrocyclic aromatic γ-peptides derived from methoxybenzene- and phenol-based building blocks. These circular γ-peptides with good ion-binding selectivities will be attached to some linear scaffolds, potentially leading to the formation of synthetic ion channels with tunable interior properties that may possess the function of selective ion transport in lipid bilayer membrane. According to the designed structure, the backbone involves the alternative arrangement of aromatic building blocks and amide functionalities in which the free rotation of amide bonds is restricted by hydrogen-bonding interactions. The utilization of bifurcated hydrogen bond rigidifies the molecule and enforces the molecule to stay in a crescent conformation, which is the key design principle of this project. The circular γ-peptides have five monomeric building blocks which are derived from methoxybenzene or phenol moieties. Therefore, the cavity formed is decorated by -OCH3 groups or –OH groups. The oxygen atoms in these groups are supposed to serve as anion donors so that the circular peptides may selectively bind cations and facilitate ion transport in the future study. Both experimental synthesis and theoretical modeling were carried out to testify the design. And results of X-ray crystallography and 2D NOESY collectively show the curved conformation of the oligomers or the circular γ-peptide in solid state and solution state, v demonstrating the rationality and validity of our design principle. Further study of the function of the circular γ-peptides needs to be carried out. vi List of Tables TABLE 2.1 EXPRIMENT CONDITIONS OF DEBENZYLATION THAT HAVE BEEN TRIED. ..........................39 vii List of Figures FIGURE 1.1 ION CHANNELS FORMED BY NATURAL COMOUNDS. ................................................2 FIGURE 1.2 SCHEMATIC OF A VOLTAGE CLAMP EXPERIMENT. ....................................................4 FIGURE 1.3 SYNTHETIC CATION CHANNEL “HYDRAPHILES” CREATED BY GOKEL ET AL .............5 FIGURE 1.4 THE ION CHANNEL FORMED BY HYDRAPHILES........................................................5 FIGURE 1.5 SCHEME OF POST-MODIFICATION OF G-QUADRUPLEX.............................................6 FIGURE 1.6 AGGREGATION ION CHANNEL FORMED BY THE STACKING OF MACROCYCLES .........7 FIGURE 1.7 CYCLO[-(TRP-D-LEU)3GLM-D-LEU-] AND THE ION CANNEL IT FORMED IN LIPID BILAYER ............................................................................................................................7 FIGURE 1.8 MACROCYCLES THAT CAN STACK TO FORM TUBULAR ION CHANNELS. ...................8 FIGURE 1.9 STRUCTURE OF CUCURBITURIL ..............................................................................9 FIGURE 1.10 Β-BARREL ION CHANNEL WITH RIGID-ROD SCAFFOLD ..........................................9 FIGURE 1.11 SCHEME OF PHOTOSYSTEM 1 PHOTO-SWITCHED INTO ION CHANNEL 2................10 FIGURE 1.12 AGGREGATE ION CHANNELS FROM AMPHIPHILES ...............................................10 FIGURE 1.13 A FEW EXAMPLES OF BOLAAMPHIPHILES ............................................................ 11 FIGURE 1.14 MICELLE-LIKE CHANNEL FORMED BY SINGLE CHAIN AMPHIPHILES ....................12 FIGURE 1.15 STRUCTURE OF SIMPLE COMPOUNDS THAT CAN FORM ION CHANNELS ................12 FIGURE 1.16 STRUCTURE OF ΑN AMINOXY ACID WHICH CAN FORM CHLORIDE CHANNELS .....13 FIGURE 2.1 CONCEPTUAL DEPICTION OF THE SYNTHETIC ION CHANNEL EMBEDDED WITHIN A LIPID BILAYER MEMBRANE ..............................................................................................19 FIGURE 2.2 THE STRUCTURE OF 1F PREDICTED BY AB INTIO CALCULATION .............................34 viii FIGURE 2.3 THE STRUCTURE OF TRIMER 1H PREDICTED BY AB INTIO CALCULATION ................34 FIGURE 2.4 THE STRUCTURE OF TETRAMER 1J PREDICTED BY AB INTIO CALCULATION ...........35 FIGURE 2.5 (A) TOP VIEW AND (B) SIDE VIEW OF THE STRUCTURE PREDICTED BY AB INTIO CALCULATION OF CYCLIC PENTAMER 1. ..........................................................................35 FIGURE 2.6 (A) TOP VIEW AND (B) SIDE VIEW OF THE STRUCTURE PREDICTED BY AB INTIO CALCULATION OF CYCLIC PENTAMER 1O .........................................................................36 FIGURE 2.7 TLC FOR CONDITIONS FROM ENTRY 1-10 .............................................................38 FIGURE 2.8 1D 1H NMR OF (A) PENTAMER 1L, (B) TETRAMER 1J, (C) TRIMER 1H AND (D) DIMER 1F IN CDCL3 (298 K, 5 MM). ..........................................................................................40 FIGURE 2.10 2D NOESY RESULT OF CIRCULAR PENTAMER 1 (298 K, 500 MS, 20 MM) ..........41 FIGURE 2.11 CRYSTAL STRUCTURE OF DIMMER (COMPOUND 1F) ............................................42 FIGURE 2.12 HYDROGEN BONDING IN DIMER 1F IN (A) AB INTIO CALCULATED STRUCTURE AND (B) X-RAY CRYSTAL STRUCTURE ....................................................................................42 FIGURE 3.1 (A) TOP VIEW AND (B) SIDE VIEW OF THE STRUCTURE PREDICTED BY AB INTIO CALCULATION OF CYCLIC PENTAMER 2...........................................................................54 FIGURE 3.2 (A) TOP VIEW AND (B) SIDE VIEW OF CRYSTAL STRUCTURE OF CIRCULAR PENTAMER 2 (THE METHYL GROUPS ARE REMOVED FOR CLARITY). ..................................................56 FIGURE 3.3 (A) TOP VIEW AND (B) SIDE VIEW OF THE CRYSTAL STRUCTURE OF 2 IN CPK REPRESENTATIONS..........................................................................................................56 ix List of Abbreviations CHCl3 Chloroform CDCl3 Deuterated Chloroform DCM Dichloromethane DIEA N, N-Diisopropylethylamine DMF N, N-Dimethylformamide DMSO Dimethyl sulfoxide DMSO-d6 Deuterated Dimethyl Sulfoxide EA Ethyl Acetate EtOH Ethanol MeOH Methanol NMM N-Methylmorpholine NMR Nuclear Magnetic Resonance Pd/C Palladium on Carbon THF Tetrahydrofuran 1D 1-Dimensional 2D 2-Dimensional x Chapter One: Introduction 1.1 General Ion transport through lipid bilayer membranes has always been a fascinating research topic among researchers, perticularly supramolecular chemists. In nature, ion transport occurs through ion carriers and ion channels. The former moves through the membrane together with ions; while the latter stays with the membrane and let ions flow through. Early efforts on mimicking the highly functionalized and sophisticated ion transporter were focused on ion carriers1. In spite of that, natural ion channels have also inspired supramolecular chemists for a long time. As long as 27 years ago, Tabushi2 and Nolte3 independently reported synthetic ion channels for the first time. After that, the first crystal structure of natural occurring ion channel--the potassium channel was revealed in 19984, which had profoundly enriched the understanding of ion channel transport mechanism. Thereafter, more and more synthetic ion channels have been created. Besides the hints given by natural ion channels, molecules which are membrane-active and functional as ion transporters inspired us substantially. For example, Gramicidin, a pentadecapeptide made up of alternating D- and L- amino acids, dimerize to form β-helix in lipid bilayer membrane. And amphotericin, a polyene antibiotic, aggregates end-to-end in lipid bilayer membrane to form a membrane-spanning channel (Figure 1.11). These two types of structure potently represent two major strategies for the design of synthetic ion channels, known as “unimolecular ion channel” and “aggregate ion channel”. Although the 1 ion channel formed by Gramicidin is the product of dimerization, here we regard it as a paradigm of unimolecular ion channels. Figure 1.1 Ion channels formed by natural compounds. Gramicidin forms β-helix in membrane. Amphotericin forms an aggregate channel in membrane. Inspiring from nature, various biomimetic ion channels have been created, either with well defined tubular structure or with the association of small components 1, 5, 6 . Although the strategies are fairly different, all of the synthetic ion channels need to meet certain design criteria for ion transport in lipid bilayer membranes. (1) Membrane-spanning structure with the length of 3-4 nm given that lipid bilayer membrane is about 4 nm thick and the hydrophobic core is about 3-3.5 nm. (2) Encompassment of a sufficient volume for the passage of the ion. (3) Stabilizing contacts for the transporting ion. (4) The ability to embed into a lipid bilayer membrane. 2 There are mainly two ways for the investigation of synthetic ion channels: vesicles (or liposomes) and planar bilayer membranes. When vesicles are used, pH-sensitive or ion-selective fluorescence dyes are employed to deduce the internal ionic composition. Sodium NMR spectroscopy can also be used in this case through a line-shape analysis method. A proper paramagnetic relaxation agent is able to produce a difference of chemical shift between internal sodium ions and outer sodium ions of the vesicle. And the addition of a membrane active channel can result in a corresponding change of the signal. Also the line width and peak shape will be altered. Through the analysis of signals, the exchange rate constant can be calculated. But it is better to use ion-selective electrodes via a pH-stat kinetic method to directly measure the exchange rate constant or the concentration. When planar bilayer membranes are used, the voltage clamp technique, which was initially used for natural ion channels, is adopted. During the voltage clamp experiment, a constant transmembrane potential is applied. Therefore the current changes are monitored as a function of time (Figure 1.2). Very little current of the membrane is observed. When the cannel opens, a current is produced due to the ionic flux. When the channel closes, the current falls back. 3 Figure 1.2 Schematic of a voltage clamp experiment. (a) cuvette;(b) electrolyte;(c) Agar salt-bridges;(d) reference electrolyte;(e) electrical contacts with reference electrolyte 1.2 Unimolecular ion channel A series of synthetic ion channels called hydraphiles, which consist of several crown ether units and side arms, are most typical among the unimolecular ion channels as shown in Figure 1.3. There are three aza-crown ethers in channel 1, in which the ones at two ends are act as headgroups to anchor the channel properly in the membrane. The three crowns were initially expected to stack co-facially to form a tubular channel so that ions could flow through the three circles. Experiments showed that the ion channel was active for cations. When the central macrocycle changed to smaller crown ethers, the channels were still active. This result showed that the cations did not pass through the central macrocycle. It was assumed that donor groups in the central macrocycle could stabilize the cation in transit. The hypothetic conformation of the channel is shown in Figure 1.4. It was later proved by fluorescence experiments using the dansyl derivative 1d7. Another observation was that channel 2 was more active than channel 1. It suggested that the increase of cation donors enhanced the activity. 4 Figure 1.3 Synthetic cation channel “hydraphiles” created by Gokel et al Figure 1.4 The ion channel formed by hydraphiles. The central macrocycle is along the direction which the ion flows through. To confirm the function of these channels, patch clamp technique was used and planar bilayer conductance measurements were carried out to test the transport of alkali-metal cation7. With Gramicidin as the control, the exchange rate of sodium ion through phospholipid bilayer of vesicles was detected by 23Na NMR. The result showed that it was concentration dependent. For channel 1, the exchange rate of sodium ion was about 27% of that of gramicidin. 5 In recent years, it was discovered that G-quartet might serve as a scaffold for building synthetic ion channels8. Even though the noncovalent assembly is thermodynamically stable, there is dynamic equilibrium between individual guanosine and its hexadecamer in solution. To fundamentally avoid the kinetic instability, post-assembly modification was carried out. Using Olefin metathesis to cross-link subunits turned the assembly into a unimolecular G-quadruplex (Figure 1.5) 9. According to the experiments of pH gradient assay and 23Na NMR, the unimolecular G-quadruplex obviously fulfiled the transport of Na+ ions across phospholipid bilayer membranes. Figure 1.5 Scheme of post-modification of G-quadruplex. The G-quadruplex 3 is obtained through metathesis. 1.3 Aggregate Ion Channels The inspiration of aggregate ion channels came from the channel formed by amphotericin. The design involving self-assembling structural units into a channel was a challenging task. There were a few strategies to achieve this goal. One strategy of the formation of aggregate ion channels was through the stacking of macrocycles with the help of H-bonding (Figure 1.6). One such example was the cyclic 6 peptides created by Gharidi et al10, 11 (Figure 1.7). The cyclic peptides, which adopt a flat conformation, are composed of alternating D- and L- amino acids. The cyclic peptides stack face-to-face when H-bonding is formed between the upper and the lower macrocycle and appear as a peptide nanotube. The cyclic peptide cyclo[-(Trp-D-Leu)3Glm-D-Leu-] could rapidly partition into the lipid bilayers and self-assemble into membrane channel structures when it was added to aqueous liposomal suspensions. The putative hydrogen-bonded tubular channel structure in the membrane was supported by Fourier transform-infrared spectroscopy. And the channel-mediated ion transport rate was 2.2 x 107 ions s-1 for K+ and 1.8 x 107 ions s-1 for Na+. It was nearly three times faster than that of gramicidin A in similar conditions11. Figure 1.6 Aggregation ion channel formed by the stacking of macrocycles Figure 1.7 cyclo[-(Trp-D-Leu)3Glm-D-Leu-] and the ion cannel it formed in lipid bilayer 7 The ureido-crown ether could stack in a similar manner in lipid bilayer membrane to form a self-assembly that could transport cations12 (Figure 1.8). One more recent example was aromatic oligoamide macrocycle made by Helsel et al13. Among the variants, 1d and 1e are membrane active. 23Na NMR technique was used to ensure the vesicles did not undergo lysis and also to test the ion transport function. Figure 1.8 Macrocycles that can stack to form tubular ion channels. (1) Ureido-crown ether (left). (2) Aromatic oligoamide macrocycle (right). As shown in Figure 1.9, there is another type of macrocycle that can form a channel in lipid bilayer membrane. The transport activity of the channel 1 formed by cucurbit[6]uril has an order of Li+ > Cs+ ≈ Rb+ > K+ > Na+, which is totally opposite to the binding affinity of cucurbit[6]uril toward alkali metal ions14. For channel 2 formed by cucurbit[5]uril, because the cavity size (diameter 2.4 Å) is not large enough, there is no transport of K+, Rb+, and Cs+ ions. However, the transport activity still follows the order of Li+ > Na+, which is also the same as channel 1 opposite to the binding affinity of itself. Therefore, it suggests that they selectively transport cations under a channel mechanism. Another representative paradigm of aggregate synthetic ion channels is rigid-rod β-barrels. 8 These synthetic ion channels all have rigid-rod p-oligophenyl scaffolds. Every phenyl unit connects with a side chain. The side chain can be peptide or other structure units. Figure 1.9 Structure of Cucurbituril Intercalating happens between several scaffolds and then the aggregate channel is formed. The scaffold with peptide chains can form an anti-parallel β-sheet. Due to the torsion angles at the inter-ring octiphenyl, a β-barrel is formed after the closure of β-sheets. And the side chain of the amino groups points to the opposite direction with that of alternative amino groups. In other words, the side chains either points outside or inside the β-sheet (Figure 1.105). This type of channel was named as synthetic multifunctional pore. It was found out that this type of channel was not only capable of translocation but also able to catalyze esterolysis15. Figure 1.10 β-Barrel ion channel with a rigid-rod scaffold. 9 One example of a different aggregate β-barrel channel is shown in Figure 1.11.16 Blue, red-fluorescent rigid-rod photosystem 1 was self-assembled with four p-octiphenyls as scaffolds through π-stacking of naphthalene diimide side chains. Multifunctionality was introduced when ligands 3 intercalate between the stacking layers of 1, which makes photosystem 1 transform into ion channel 2. Figure 1.11 Scheme of photosystem 1 photo-switched into ion channel 2 In another strategy, edge to edge aggregation is adopted. Bolaamphiphiles are especially typical in this case. The length of the bolaamphiphile is long enough to span across the whole lipid bilayer membrane. Figure 1.12 Aggregate ion channels from amphiphiles. 10 A series of this type of molecules are shown below. The central macrocycle in molecule 1 in Figure 1.13 was found out that it did not contribute much to the transport activity because when it changed to a bridging tartaric acid in 2 the activity stayed at the same level as 1. Modification was carried out. And until molecule 4 was made, the activity was largely enhanced1. Two to three units of them could aggregate to form a channel within the membrane. Voltage clamp studies showed that the monomers were not active in membrane. Only when channel was formed, it possessed activity. The aggregation process depended on the concentration of the monomer raised to a power that revealed stoichiometry (for example, 2 for dimers, and 3 for trimers.) Nevertheless, aggregates can not become too large if there is any specific stabilizing inter-molecular interaction that is missing, because it is unfavorable. The head groups of this type of molecule are deprotonated under the experimental condition. Therefore, impulsion is generated between the monomers. Figure 1.13 A few examples of bolaamphiphiles. 11 1.4 Other Types of Ion Channels Considering the design criteria of synthetic ion channels, amphiphility is expected. The amphiphility reminds us of detergents. Studies have already shown that many common detergents can perform like ion channels at concentrations below their critical micelle concentrations5. This type of ion channels is generally irregular, transient and hard to reproduce. But it has been proven that in voltage clamp experiments the simple compound 1-3 (Figure 1.15) can produce regular channel openings. Figure 1.14 Micelle-like channel formed by single chain amphiphiles. Figure 1.15 Structures of simple compounds that can form ion channels 12 Channels formed by ion pairs salts 1a, b are voltage dependent. But a small imbalance in the number of molecules on each side of the membrane could happen. And the activities of channels formed by compound 2 and 3 are surprisingly sensitive to the length of hydrocarbon chain even though the compounds themselves are not expected to span the whole lipid bilayer membrane17. For compound 3, even adding two more methylene groups results in completely inactive product. One recent synthetic ion channel is also based on small molecules as shown in Figure 1618. It is an unnatural analogue of α-amino acid. Fluorescence assay shows that the small molecule can facilitate the transport of chloride ion. Patch clamp experiments were carried out to prove that the transport of chloride ions was mediated by a channel mechanism instead of ion carrier mechanism. Experiments in living cells have also been carried out, showing its ability to facilitate chloride ion transport through lipid bilayer membrane in living cells. Figure 1.16 Structure of α-aminoxy acid which can form chloride channels 1.5 Applications With decades of effort, various synthetic ion channels have been created. Some of them have already shown that their function could exceed our expectation. For example, they can 13 be catalyst, detectors or sensors. And in the discipline of medicinal chemistry, synthetic ion channels are expected to contribute to the development of drug delivery vehicles and even become drugs that have antimicrobial activity in the future. 14 References 1. Fyles, T. M., Synthetic ion channels in bilayer membranes. Chem. Soc. Rev. 2007, 36, (2), 335-347. 2. Tabushi, I.; Kuroda, Y.; Yokota, K., A,B,D,F-tetrasubstituted [beta]-cyclodextrin as artificial channel compound. Tetrahedron Lett. 1982, 23, (44), 4601-4604. 3. van Beijnen, A. J. M.; Nolte, T. J. M.; Zwikker, J. W., A Molecular Cation Channel. Recl. Trav. Chim Pays-Bas 1982, 101, 409-410. 4. Doyle, D. A.; Cabral, J.; atilde; o, M.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J. M.; Cohen, S. L.; Chait, B. T.; MacKinnon, R., The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity. Science 1998, 280, (5360), 69-77. 5. Matile, S.; Som, A.; Sord, N., Recent synthetic ion channels and pores. Tetrahedron 2004, 60, (31), 6405-6435. 6. McNally, B. A.; Leevy, W. M.; Smith, B. D., Recent Advances in Synthetic Membrane Transporters. Supramolecular Chem. 2007, 19, (1), 29 - 37. 7. Gokel, G. W.; Ferdani, R.; Liu, J.; Pajewski, R.; Shabany, H.; Uetrecht, P., Hydraphile Channels: Models for Transmembrane, Cation-Conducting Transporters. Chem. Eur. J 2001, 7, (1), 33-39. 8. Forman, S. L.; Fettinger, J. C.; Pieraccini, S.; Gottarelli, G.; Davis, J. T., Toward Artificial Ion Channels: A Lipophilic G-Quadruplex. J. Am. Chem. Soc. 2000, 122, (17), 4060-4067. 9. Kaucher, M. S.; Harrell, W. A.; Davis, J. T., A Unimolecular G-Quadruplex that Functions as a Synthetic Transmembrane Na+ Transporter. J. Am. Chem. Soc. 2006, 128, 15 (1), 38-39. 10. Jorge, S.-Q.; Hui Sun, K.; Ghadiri, M. R., A Synthetic Pore-Mediated Transmembrane Transport of Glutamic Acid13. Angew. Chem. Int. Ed. 2001, 40, (13), 2503-2506. 11. Ghadiri, M. R.; Granja, J. R.; Buehler, L. K., Artificial transmembrane ion channels from self-assembling peptide nanotubes. Nature 1994, 369, (6478), 301-304. 12. Cazacu, A.; Tong, C.; van der Lee, A.; Fyles, T. M.; Barboiu, M., Columnar Self-Assembled Ureido Crown Ethers: An Example of Ion-Channel Organization in Lipid Bilayers. J. Am. Chem. Soc. 2006, 128, (29), 9541-9548. 13. Helsel, A. J.; Brown, A. L.; Yamato, K.; Feng, W.; Yuan, L.; Clements, A. J.; Harding, S. V.; Szabo, G.; Shao, Z.; Gong, B., Highly Conducting Transmembrane Pores Formed by Aromatic Oligoamide Macrocycles. J. Am. Chem. Soc. 2008, 130, (47), 15784-15785. 14. Jeon, Y. J.; Kim, H.; Jon, S.; Selvapalam, N.; Oh, D. H.; Seo, I.; Park, C.-S.; Jung, S. R.; Koh, D.-S.; Kim, K., Artificial Ion Channel Formed by Cucurbit[n]uril Derivatives with a Carbonyl Group Fringed Portal Reminiscent of the Selectivity Filter of K+ Channels. J. Am. Chem. Soc. 2004, 126, (49), 15944-15945. 15. Sakai, N.; Matile, S., Synthetic multifunctional pores: lessons from rigid-rod beta-barrels. Chem. Comm. 2003, (20), 2514-2523. 16. Bhosale, S.; Sisson, A. L.; Talukdar, P.; Furstenberg, A.; Banerji, N.; Vauthey, E.; Bollot, G.; Mareda, J.; Roger, C.; Wurthner, F.; Sakai, N.; Matile, S., Photoproduction of Proton Gradients with pi-Stacked Fluorophore Scaffolds in Lipid Bilayers. Science 2006, 313, (5783), 84-86. 17. Fyles, T. M.; Knoy, R.; Mullen, K.; Sieffert, M., Membrane Activity of Isophthalic 16 Acid Derivatives: Ion Channel Formation by a Low Molecular Weight Compound. Langmuir 2001, 17, (21), 6669-6674. 18. Li, X.; Shen, B.; Yao, X.-Q.; Yang, D., A Small Synthetic Molecule Forms Chloride Channels to Mediate Chloride Transport across Cell Membranes. J. Am. Chem. Soc. 2007, 129, (23), 7264-7265. 17 Chapter Two: Synthesis and Structural Investigations of Circular Aromatic γ-Peptides Derived from Phenol- and Methoxybenzene-Based Building Blocks 2.1 Introduction As mentioned in Chapter One, a variety of ion transport systems through lipid bilayer membranes have been created in the last three decades. Many of them have shown characters of ion channels. Enormous effort has been made to catch up with the creativity, high selectivity and high efficiency of nature. In this project, it was our aim to design and synthesize a new class of ion channels. The inspiration of the structure of the designed channels comes from foldamers. Foldamers, first named by Gellman1, are molecules with well-defined secondary structure enhanced by non-covalent bonds. In the past few years, a class of aromatic oligoamides with well-defined crescent backbones was reported2-4. The backbone of these oligoamides consists of benzene rings meta-linked by secondary amide groups. Three-center hydrogen bonds strongly bias the crescent conformation of the rigid aromatic amide backbone. Through changing linking position (meta- or para-) or the building blocks the cavity size of the folding oligomers can be tuned. Based on the well-defined crescent backbone and rigidity, the circular aromatic γ-peptide derived from phenol- and methoxybenzene-based building blocks is designed. It derived 18 from meta-linking benzene rings via amide linkages. And the circular peptide will be attached to a linear scaffold in the future hopefully to form a rigid synthetic ion channel as shown in Figure 2.1. Figure 2.1 Conceptual depiction of the synthetic ion channel embedded within a lipid bilayer membrane Ab initio calculations are used to predict the conformation of the circular γ-peptide. The calculation results are compared with the synthesized compound. And it is aimed to evaluate the conformation of the circular γ-peptide. It is hoped that the circular γ-peptide could be constructed as synthetic ion channels and obtain therapeutic properties that can be used as antimicrobials in the future. 19 2.2 Experimental Section 2.2.1 Synthetic Scheme HNO3 , H 2SO4, CH2 Cl2 COOH OH MeOH, H 2SO4 reflux for 2d 0°C, 30 min overall yield for 2steps: 28% O 2N COOCH3 OH 1b CH 3I, K2CO3, DMF 60°C, 4h O2 N O2 N COOCH 3 OCH 3 2c 82% COOCH 3 OH BnBr, K2CO3, DMF 60°C, 5h 1b O 2N COOCH3 OBn 1c 75% Fe, AcOH, EtOH reflux for 2h O2 N H2N COOCH 3 OR 1d R=Bn 2d R=Me O 2N COOH OR 1e R=Bn 2e R=Me COOCH 3 OR 1 NaOH, MeOH, reflux for 2h 2 HCl, H2 O 1c R=Bn 2c R=Me 1e : 92% 2e: 92% O2 N COOH O 2N 2 1d, DIEA, CH 2Cl2 OBn 1e O CH3 OBn O 74% 1f H N Fe, AcOH, EtOH H 2N reflux for 2h OBn O H N 1 NMM, ClCOOEt, CH 2Cl2 OBn O O CH3 OBn O 1g 1 (COCl)2 , DMF, CH2Cl2 O2 N COOH O2N 2 1g, DIEA, DMF OBn 1e OBn O N H OBn O 65% O OBn O 1h H N Fe, AcOH, EtOH, CH 2Cl2 reflux for 2h H N H2 N OBn O N H OBn O O CH3 OBn O 1i 20 CH 3 1 (COCl)2 , DMF, CH2 Cl2 2 1i, DIEA, DMF O2 N O2 N COOH 66% OBn OBn O H N N H OBn O 1e OBn O H N OCH 3 OBn O 1j H2 N reflux for 2h OBn O H N Fe, AcOH, EtOH, CH2Cl2 OBn O OBn O H N N H OCH 3 OBn O 1k 1 (COCl) 2, DMF, CH 2Cl2 2 1k, DIEA, DMF O2 N COOH OCH 3 OBn O H N O 2N 76% N H OCH3O 2e H N OBn O N H OBn O O CH 3 OBn O 1l Fe, AcOH, EtOH, CH2Cl2 reflux for 3h H N H 2N OBn O N H OCH 3O 95% OBn O H N N H OBn O O CH 3 OBn O 1m 1 KOH, MeOH H N H 2N 2 HCl, H2O OBn O N H OCH 3O OBn O H N N H OBn O OH OBn O 1n O N O N H BOP, DIEA, DCM, 40°C, 2h OBnH H 3CO OBn 16% N H O H N OBn O BnO H N O 1a O N O N H H2 , Pd/C, cyclohexene, THF, EtOH, 40°C, 2h OH H H3 CO OH 70% N H H N O HO H N OH O O 1 Scheme 1 Synthetic route for oligomers and cyclic pentamer 1. 21 2.2.2 General Methods All the reagents were obtained from commercial suppliers and used as received unless otherwise noted. Aqueous solutions were prepared from distilled water. The organic solutions from all liquid extractions were dried over anhydrous Na2SO4 for a minimum of 15 minutes before filtration. Reactions were monitored by thin-layer chromatography (TLC) on silica gel precoated glass plates (0.25 mm thickness, 60F-254, E. Merck). Flash column chromatography was performed using pre-coated 0.2 mm silica plates from Selecto Scientific. Chemical yields refer to pure isolated substances. 1H and 13C NMR spectra were recorded on either a Bruker ACF-300, AVF-500 or DRX-500 spectrometer. In addition, key compounds were characterized by 2D NOSEY and X-ray Diffraction. 1H NMR spectra were recorded on Bruker ACF500 (500 MHz) and DRX500 spectrometers (500 MHz). The solvent signal of CDCl3 was referenced at δ= 7.26 ppm and DMSO-d6 was referenced at δ= 2.50 ppm. Coupling constants (J values) are reported in Hertz (Hz). 1H NMR data are recorded in the order: chemical shift value, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), number of protons that gave rise to the signal and coupling constant, where applicable. 13C NMR spectra are proton-decoupled and recorded on Bruker ACF500 spectrometers (500 MHz). The solvent CDCl3 was referenced at δ= 77 ppm and DMSO-d6 was referenced at δ= 40 ppm. CDCl3 and DMSO-d6 (99.8%- Deuterated) was purchased from Aldrich and used without further purification. 2.2.3 Synthetic Procedure Methyl 2-hydroxy-3-nitrobenzoate (1b) 22 Salicylic acid (10.0 g, 72.5 mmol) was dissolved in 200 mL of CH2Cl2, to which concentrated HNO3 (69%, 6.05 mL, 94.2 mmol) was added with stirring at 0 °C. Concentrated H2SO4 (95%, 10.6 mL, 145 mmol) was then added dropwise to the reaction mixture. After 20 min, the reaction was quenched with 500mL of distilled water and the mixture was filtered. The crude product was dissolved in methanol (250 mL), and to the resultant solution was added concentrated H2SO4 (21.9 mL, 388 mmol). The mixture was heated under reflux for 48 hours. The solvent was then removed in vacuo and the residue was dissolved in CH2Cl2 (200 mL), washed successively with water (2 x 100 mL) and aq. NaHCO3 (100 mL), dried over anhydrous Na2SO4. Removal of CH2Cl2 gave a yellow solid which was purified by flash column chromatography on silica gel using hexane/CH2Cl2 (6:1) as the eluent to give pure product 1b (4.00 g, overall yield: 28%) as a bright yellow solid. 1H NMR (300 MHz, CDCl3) δ 11.99 (s, 1H), 8.15 (m, 2H), 7.01 (m, 1H), 4.02 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ 169.2, 155.6, 138.0, 135.7, 131.3, 118.3, 115.8, 53.1. Methyl 2-methoxy-3-nitrobenzoate (2c) Compound 1b (6.00 g, 30.4 mmol) was dissolved in DMF (125mL) to which anhydrous K2CO3 (15.6 g, 112.9 mmol) and iodomethane (6.98 mL, 112 mmol) were added to it. The mixture was heated at 60°C for 4 hours. The reaction mixture was then filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (100 mL), washed with water (2 x 50 mL), and dried over anhydrous Na2SO4. Removal of CH2Cl2 gave a pure light yellow solid 2c. Yield: 6.41g, 82%. 1H NMR (300 MHz, CDCl3) δ 8.01 (dd, 1H, J = 7.9, 1.8), 7.90 (dd, 1H, J = 8.1, 1.8), 7.26 (m, 1H), 3.99 (s, 3H), 3.95 (s, 3H). 13C NMR (75 23 MHz, CDCl3) δ 164.4, 152.9, 145.17, 135.3, 128.0, 127.1, 123.5, 63.9, 52.4. MS-ESI: calculated for [M]+ (C9H9NO5): m/z 211.0, found: m/z 211.1. 2-Methoxy-3-nitrobenzoic acid (2e) Compound 2c (4.00 g, 19.0 mmol) was dissolved in hot methanol (10 mL) to which 1M NaOH (40 mL, 40 mmol) was added. The mixture was heated under reflux for 2 hours and then quenched with water (100 mL). The aqueous layer was neutralized by addition of 1M HCl (80 mL) until the pH was at least 1. The precipitated crude product was collected by filtration, which was recrystallized from hot methanol to give a pure white solid 2e. Yield: 3.45 g, 92%. 1H NMR (300 MHz, CDCl3) δ 8.28 (dd, 1H, J = 7.9, 1.8), 8.03 (dd, 1H, J = 8.1, 1.8), 7.36 (t, 1H, J = 7.9), 4.08 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 165.0, 151.9, 144.5, 134.7, 127.3, 126.8, 122.8, 63.1. Methyl 2-(benzyloxy)-3-nitrobenzoate (1c) Compound 1b (3.94 g, 20.0 mmol) was dissolved in DMF (200 mL) to which anhydrous K2CO3 (11 g, 80 mmol) and benzyl bromide (2.6 mL, 22 mmol) were added to. The mixture was heated at 60°C for 5h. The reaction mixture was then filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2, washed with water and dried over anhydrous Na2SO4. Removal of CH2Cl2 and recrystallization from MeOH gave pure yellow solid 1c. Yield: 4.30 g, 75%. 1H NMR (500 MHz, CDCl3) δ 8.06 (dd, 1H, J=7.9, 1.3 ), 7.94 (dd, 1H, J=7.9, 1.9), 7.49 (d, 2H), 7.41-7.36 (m, 3H), 7.30 (t, 1H), 5.17 (s, 2H), 3.90 (s, 3H). 13 C NMR (125 MHz, CDCl3) δ 164.9, 151.5, 145.9, 135.7, 135.6, 128.7, 128.6, 128.5, 24 128.5, 128.1, 124.1, 78.6, 52.7. MS-ESI: calculated for [M+Na]+ (C15H13O5N123Na1): m/z 310.1, found: m/z 310.1. Methyl 3-amino-2-(benzyloxy) benzoate (1d) Compound 1c (2.87 g, 10.0 mmol) was dissolved in EtOH (50 mL). Iron powder (2.8 g, 50 mmol) and acetic acid (10 mL) was added to the solution. The mixture was heated under reflux for 2h. After cooling down, the reaction mixture was filtered and the solvent was removed in vacuo. Saturated NaHCO3 solution (100 mL) was added to the residue. Then the solution was extracted with CH2Cl2 and washed with water. Removal of CH2Cl2 gave a pure light yellow liquid 1d. Yield: 2.41 g, qualitative. 2-(Benzyloxy)-3-nitrobenzoic acid (1e) Compound 1c (5.74 g, 20.0 mmol) was dissolved in hot methanol (20 mL) to which 1M NaOH (40 mL, 40 mmol) was added. The mixture was heated under reflux for 2 hours and then quenched with water (100 mL). The aqueous layer was neutralized by addition of 1M HCl (60 mL, 60 mmol) until the pH was at least 1. The precipitated crude product was collected by filtration, which was recrystallized from hot methanol to give a pure white solid 1e. Yield: 5.02 g, 92%. 1H NMR (500 MHz, CDCl3) δ 8.28 (dd, 1H, J=8.2, 1.9), 8.05 (dd, 1H, J=8.2, 1.9), 7.48-7.47 (m, 2H), 7.40-7.37 (m, 4H). 13C NMR (125 MHz, CDCl3) δ 166.2, 151.7, 145.4, 136.9, 134.6, 129.9, 129.2, 129.1, 128.8, 126.3, 124.6, 79.4. MS-ESI: calculated for [M-H]+ (C14H10O5N1): m/z 272.1, found: m/z 272.1. 25 Methyl 2-(benzyloxy)-3-(2-(benzyloxy)-3-nitrobenzamido) benzoate (1f) Acid 1e (3.30 g, 12.1 mmol) was dissolved in CH2Cl2 (20 mL) to which NMM (1.90 mL, 17.3mmol) and ethyl chloroformate (1.70 mL, 18.0 mmol) was added at 0 °C. The reaction mixture was stirred for at least 15 min after which a solution of amine 1d (2.41 g, 9.38 mmol) dissolved in CH2Cl2 (50 mL) was added. The reaction mixture was allowed to stir continuously 6 hours at room temperature. The reaction mixture was washed with 1 M KHSO4 (100 mL), followed by saturated NaHCO3 (100 mL) and saturated NaCl (100 mL). Drying over Na2SO4 and removal of solvent in vacuo gave the crude product, which was recrystallized from methanol to give the pure product 1f as a pale yellow solid. Yield: 3.82 g, 74%. 1H NMR (500 MHz, CDCl3) δ 9.59 (s, 1H), 8.66 (dd, 1H, J=8.2, 1.9), 8.11 (dd, 1H, J=8.2, 1.9), 7.92 (dd, 1H, J=8.2, 1.9), 7.66 (dd, 1H, J=8.2, 1.9), 7.35-7.12 (m, 12H), 4.98 (s, 2H), 4.81 (s, 2H), 3.94 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 165.9, 161.6, 149.1, 148.2, 145.0, 136.1, 135.5, 133.7, 132.9, 131.1, 129.6, 129.1, 128.7, 128.5, 128.4, 128.0, 126.6, 124.6, 124.4, 124.3, 124.0, 79.6, 77.7, 52.3. MS-ESI: calculated for [M+Na]+ (C29H24O7N223Na1): m/z 535.1, found: m/z 535.1. Methyl 2-(benzyloxy)-3-(2-(benzyloxy)-3-(2-(benzyloxy)-3-nitrobenzamido) benzamido) benzoate (1h) Compound 1f (3.06 g, 5.98 mmol) was dissolved in CH2Cl2 (6.0 mL) and EtOH (60 mL). Iron powder (1.68 g, 30.0 mmol) and acetic acid (6.0 mL) was added to the solution. The mixture was heated under reflux for 2h. After cooling down, the reaction mixture was filtered and the solvent was removed in vacuo. Saturated NaHCO3 solution (100 mL) was 26 added to the residue. Then the solution was extracted with CH2Cl2 (3 x 50 mL) and washed with water (2 x 100 mL). Removal of CH2Cl2 gave a light yellow solid 1g, which was used directly in the next step without further purification. Acid 1e (2.36 g, 8.64 mmol) was placed in a very dry round bottom flask and saturated with nitrogen gas. Dry CH2Cl2 (15 mL) and DMF (0.40 mL) were added to the acid, followed by dropwise addition of oxayl chloride (0.99 mL, 1.17 mmol). The reaction mixture was allowed to stir for 2 hours. The solvent was then removed in vacuo and protected under nitrogen before addition of 10mL dry CH2Cl2. Amine 1g was dissolved in 15 mL dry CH2Cl2 and DIEA (2.1 mL, 12.0 mmol) before addition to the reaction mixture above. The reaction mixture was stirred at room temperature overnight and then was washed with aq NaHCO3 (100 mL) and water (200 mL). Drying over anhydrous Na2SO4 and removal of solvent in vacuo gave the crude product, which was purified by flash column chromatography (silica gel as the stationary phase) using Hexane/Ethyl acetate (from 10:1 to 3:1) as the eluent to give pure product 1h as a white solid. Yield: 2.87 g, 65%. 1H NMR (500 MHz, CDCl3) δ 9.73 (s, 1H), 9.04 (s, 1H), 8.78 (d, 1H, J=8.8), 8.56 (d, 1H, J=7.0), 8.03 (d, 1H, J=7.6), 7.97 (d, 1H, J=8.2), 7.77 (d, 1H, J=8.2), 7.66 (d, 1H, J=6.9), 7.37 (t, 1H, J=8.2), 7.31 (t, 1H, J=8.2), 7.24-7.10 (m, 10H), 6.99 (d, 1H, J=7.0), 6.93 (t, 2H, J=8.2), 6.83 (d, 1H, J=6.9). 13C NMR (125 MHz, CDCl3) δ 166.0, 163.3, 161.5, 149.0, 148.0, 145.8, 145.0, 136.2, 135.5, 134,3, 133.9, 133.2, 132.1, 131.1, 129.6, 129.2, 129.2, 128.8, 128.5, 128.5, 128.4, 128.3, 128.2, 128.2, 127.9, 127.5, 126.5, 126.4, 125.3, 124.8, 124.8, 124.4, 124.1, 79.9, 79.0, 77.3, 52.3. MS-ESI: calculated for [M+Na]+ (C43H35O9N323Na1): m/z 760.2, found: m/z 760.2. 27 Methyl 2-(benzyloxy)-3-(2-(benzyloxy)-3-(2-(benzyloxy)-3-(2-(benzyloxy)-3-nitro- benzamido) benzamido) benzamido) benzoate (1j) Compound 1h (2.88 g, 3.90 mmol) was dissolved in CH2Cl2 (6.0 mL) and EtOH (20 mL). Iron powder (1.09 g, 19.5 mmol) and acetic acid (4.0 mL) was added to the solution. The mixture was heated under reflux for 2h. After cooling down, the reaction mixture was filtered and the solvent was removed in vacuo. Saturated NaHCO3 solution (100 mL) was added to the residue. Then the solution was extracted with CH2Cl2 (3 x 50 mL) and washed with water (2 x 100 mL). Removal of CH2Cl2 gave a light yellow liquid 1i, which was used directly in the next step without further purification. Acid 1e (1.64 g, 6.01 mmol) was placed in a very dry round bottom flask and saturated with nitrogen gas. Dry CH2Cl2 (10 mL) and DMF (0.28 mL) were added to the acid, followed by dropwise addition of oxayl chloride (0.66 mL, 7.60 mmol). The reaction mixture was allowed to stir for 2 hours. The solvent was then removed in vacuo and protected under nitrogen before addition of 15mL dry CH2Cl2. Amine 1i was dissolved in 10 mL dry CH2Cl2 and DIEA (1.44 mL, 8.00 mmol) before addition to the reaction mixture above. The reaction mixture was stirred at room temperature overnight and then was washed with aq NaHCO3 (100 mL). Drying over anhydrous Na2SO4 and removal of solvent in vacuo gave the crude product, which was purified by flash column chromatography (silica gel as the stationary phase) using Hexane/CH2Cl2/Ethyl acetate (5:1:1) as the eluent to give pure product 1j as a white solid. Yield: 2.50 g, 66%. 1H NMR (500 MHz, CDCl3) δ 9.34 (s, 1H), 9.31 (s, 1H), 9.05 (s, 1H), 8.71-8.67 (m, 3H), 8.61 (dd, 1H, J=8.3, 1.3), 8.08 (dd, 1H, J=7.9, 1.9), 7.99 (dd, 1H, J=7.9, 1.9), 7.72-7.60 (m, 4H), 7.42-6.85 (m, 22H), 5.01 (s, 2H), 4.81 (s, 2H), 4.67 (s, 2H), 4.56 (s, 28 2H), 3.90 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 165.8, 163.4, 163.1, 161.6, 149.0, 148.1, 145.8, 145.4, 144.9, 136.1, 135.4, 134.2, 133.8, 133.0, 132.4, 132.0, 131.2, 129.4, 129.1, 129.0, 128.8, 128.8, 128.7, 128.7, 128.5, 128.5, 128.4, 128.4, 128.3, 128.2, 128.1, 128.1, 128.0, 127.9, 127.5, 126.4, 126.2, 125.6, 125.3, 125.2, 124.8, 124.5, 124.3, 124.2, 124.1, 123.9, 79.9, 78.8, 78.4, 77.5, 52.1. MS-ESI: calculated for [M+Na]+ (C57H46O11N423Na1): m/z 985.3, found: m/z 985.3. Methyl 2-(benzyloxy)-3-(2-(benzyloxy)-3-(2-(benzyloxy)-3-(2-(benzyloxy)-3- (2-methoxy-3- nitrobenzamido) benzamido) benzamido) benzamido) benzoate (1l) Compound 1j (2.50 g, 2.60 mmol) was dissolved in CH2Cl2 (4.0 mL) and EtOH (13 mL). Iron powder (0.72 g, 12.9 mmol) and acetic acid (2.6 mL) was added to the solution. The mixture was heated under reflux for 2h. After cooling down, the reaction mixture was filtered and the solvent was removed in vacuo. Saturated NaHCO3 solution (50 mL)was added to the residue. Then the solution was extracted with CH2Cl2 (3 x 20 mL) and washed with water (2 x 20 ml). Removal of CH2Cl2 gave a white solid 1k, which was used directly in the next step without further purification. Acid 2e (0.98 g, 4.98 mmol) was placed in a very dry round bottom flask and saturated with nitrogen gas. Dry CH2Cl2 (15 mL) and DMF (0.18 mL) were added to the acid, followed by dropwise addition of oxayl chloride (0.57 mL, 6.50 mmol). The reaction mixture was allowed to stir for 2 hours. The solvent was then removed in vacuo and protected under nitrogen before addition of 15mL dry CH2Cl2. Amine 1k was dissolved in 15 mL dry CH2Cl2 and DIEA (0.90 mL, 5.03 mmol) before addition to the reaction mixture above. The reaction mixture was stirred at room 29 temperature overnight and then was washed with aq NaHCO3 (100 mL). Drying over anhydrous Na2SO4 and removal of solvent in vacuo gave the crude product, which was purified by flash column chromatography (silica gel as the stationary phase) using CH2Cl2/Acetone (50:1) as the eluent to give pure product 1l as a white solid. Yield: 2.07 g, 76%. 1H NMR (500 MHz, CDCl3) δ 9.69 (s, 1H), 9.48 (s, 1H), 9.19 (s, 1H), 9.07 (s, 1H), 8.79-8.67 (m, 5H), 8.22 (d, 1H, J=7.6), 7.94 (d, 1H, J=7.6), 7.67-7.59 (m, 5H), 7.37-6.88 (m, 23H), 3.89 (s, 3H), 3.66 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 165.9, 163.7, 163.6, 163.4, 161.3, 151.0, 148.2, 145.7, 145.7, 145.6, 144.4, 136.3, 136.0, 134.8, 134.5, 134.4, 133.2, 132.5, 132.5, 132.4, 130.8, 129.4, 129.3, 129.1, 129.0, 129.0, 128.8, 128.7, 128.6, 128.6, 128.6, 128.5, 128.4, 128.3, 128.3, 128.0, 126.3, 126.0, 125.8, 125.6, 125.6, 125.4, 125.3, 124.7, 124.7, 124.4, 124.2, 124.1, 124.0, 79.2, 78.7, 78.5, 77.6, 64.1, 52.2. MS-ESI: calculated for [M+Na]+ (C65H53O13N523Na1): m/z 1134.4, found: m/z 1134.4. Methyl 3-(3-(3-(3-amino-2-(benzyloxy)benzamido)-2-(benzyloxy)benzamido)-2- (benzyloxy) benzamido)-2-(benzyloxy) benzoate (1m) Compound 1l (2.11 g, 1.90 mmol) was dissolved in CH2Cl2 (12 mL) and EtOH (25 mL). Iron powder (0.56 g, 10.0 mmol) and acetic acid (2 mL) was added to the solution. The mixture was heated under reflux for 3h. After cooling down, the reaction mixture was filtered and the solvent was removed in vacuo. Saturated NaHCO3 solution (50 mL) was added to the residue. Then the solution was extracted with CH2Cl2 (3 x 15 mL) and washed with water (2 x 20 mL). Removal of CH2Cl2 gave a white solid 1m. Yield: 1.95 g, 95%. 1H NMR (500 MHz, CDCl3) δ 10.04 (s, 1H), 9.40 (s, 1H), 9.13 (s, 1H), 9.11 (s, 1H), 8.83 (d, 30 1H, J=6.9), 8.69 (m, 3H), 7.66-7.57 (m, 4H), 7.44 (d, 1H, J=7.6), 7.36-6.88 (m, 19H), 4.92 (s, 2H), 4.85 (s, 2H), 4,70 (s, 2H), 4.67 (s, 2H), 3.91 (s, 3H), 3.57 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 165.9, 163.8, 163.8, 163.5, 148.2, 145.7, 145.4, 145.4, 144.8, 140.1, 136.1, 134.7, 134.6, 134.3, 133.2, 133.1, 132.5, 129.1, 129.0, 128.9, 128.8, 128.6, 128.4, 128.4, 128.3, 128.2, 28.0, 127.8, 126.7, 126.3, 125.7, 125.5, 125.4, 125.3, 125.1, 124.7, 124.4, 124.3, 124.1, 123.9, 120.6, 119.6, 78.6, 78.5, 78.5, 77.6, 60.6, 52.2. MS-ESI: calculated for [M+Na]+ (C65H55O11N523Na1): m/z 1104.4, found: m/z 1104.4. Circular Pentamer (1a) Compound 1m (0.97 g, 0.90 mmol) was dissolved in hot methanol (5 mL) to which 1M KOH (5 mL, 5 mmol) was added. The mixture was heated under reflux for 2 hours and then quenched with water (10 mL). The aqueous layer was neutralized with 1M HCl (6.0 mL) then extracted with CH2Cl2 (3 x 10 mL). Removal of CH2Cl2 gave compound 1n. Yield: 0.90 g, qualitative. Compound 1n (0.90 g, 0.84 mmol) and BOP (1.19 g, 2.70 mmol) were dissolved in CH2Cl2 (3.2 mL). DIEA (0.49 mL, 2.72 mmol) was added and the reaction mixture was stirred continuously at room temperature overnight. Removal of solvent in vacuo gave the crude product, which was purified by gradient flash column chromatography on silica gel using Hexane/CH2Cl2/THF (from 15:15:1 to 3:3:1) as the eluent and then recrystylization with MeOH to give a pure white product 1a. Yield: 0.16 g, 16%. 1H NMR (500 MHz, CDCl3) δ 10.40 (s, 1H), 10.28 (s, 1H), 9.94 (s, 1H), 9.78 (s, 1H), 9.76 (s, 1H), 8.86 (m, 3H), 8.65 (d, 1H, J=8.0), 8.54 (d, 1H, J=7.7), 7.91 (d, 1H, J=7.7), 7.83 (m, 2H), 7.74 (d, 1H, J=7.7), 7.61 (d, 1H, J=7.7), 7.44-7.29 (m, 8H), 7.05 (m, 15H), 31 5.29-4.83 (m, 11H), 3.87 (s, 1H). 13C NMR (125 MHz, CDCl3) δ 163.1, 162.6, 162.4, 162.3, 146.4, 144.8, 144.6, 144.4, 144.3, 133.5, 133.5, 133.3, 133.2, 133.1, 133.0, 132.9, 132.4, 129.7, 129.6, 129.6, 129.4, 129.4, 129.2, 129.2, 128.9, 128.6, 128.5, 128.5, 128.4, 128.4, 128.0, 127.9, 127.8, 127.0, 126.1, 126.1, 126.0, 125.9, 125.8, 125.7, 125.6, 124.0, 123.6, 123.4, 123.3, 79.6, 79.5, 79.4, 79.3, 63.6. MS-ESI: calculated for [M+Na]+ (C64H51O10N523Na1): m/z 1072.4, found: m/z 1072.4. Circular Pentamer (1) Compound 1a (0.024 g, 0.023 mmol) was going through hydrogenolysis at 40°C in cyclohexene (6.0 mL), THF (12 mL) and EtOH (18 mL) using Pd/C (0.048 g, 200 wt%) as the catalyst for 3h. The reaction mixture was then filtered and the solvent removed in vacuo to give the green solid. Add CH2Cl2 to the solid and stir for 1h then filter to get green solid. Recrystalize with MeOH and then purify the crude product by flash column chromatograpy using Acetone/CH2Cl2 (2:1) as eluent to get green solid 1n. Yield: 9.6 mg, 70%. 1 H NMR (500 MHz, DMSO-d6) δ 15.14 (s, 1H), 13.36 (s, 1H), 13.15 (s, 1H), 13.00 (s, 1H), 11.49 (s, 1H), 8.85 (d, 1H, J=8.0), 8.54 (d, 1H, J=7.3), 8.49 (s, 1H, J=7.3), 8.40 (m, 2H), 7.65 (d, 1H, J=8.0), 7.56 (d, 1H, J=7.9), 7.51 (d, 1H, J=7.9), 7.46 (m, 2H), 7.21 (m, 1H), 6.30-6.17 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ 167.7, 167.5, 166.7, 166.4, 165.8, 162.4, 161.7, 160.9, 160.4, 148.4, 135.9, 134.3, 134.1, 133.7, 132.3, 132.2, 132.1, 129.1, 127.5, 124.6, 124.5, 123.7, 123.3, 123.3, 123.2, 122.7, 120.8, 120.2, 119.7, 119.5, 119.4, 119.0, 117.2, 109.6, 109.2, 58.1. MS-ESI: calculated for [M-H]+ (C36H26O10N5): m/z 688.2, found: m/z 688.2. 32 2.3 Theoretical Modeling Ab initio calculations with Gaussian 98 (B3LYP/6-31G) were used to help design the aromatic γ-peptides to find out the preferred conformation possibly adopted by the molecules. 2.3.1 Dimer As Figure 2.2 shows, the aromatic amide backbone is rigid and planar while the protection groups for –OH, the two benzyl groups, are too bulky to stay on the same side of the plane. There are two possible reasons for the formation of the rigid and planar conformation. Firstly, the lone pair electrons of nitrogen atom in the amide bond are partially delocalized with the carbonyl group. As a result, the C-N bond in the amide bond is different from usual C-N bond as it has partial character of double bond. Therefore, it can no longer rotate freely like a single bond. Secondly, from the structure of 1f (Figure 2.2 a), it is probable that there is an intramolecular hydrogen bond. In the six-member hydrogen-bonded ring, the hydrogen bond length is 1.825 Å. At the five-member hydrogen bonded ring, the hydrogen bond length is 2.107 Å. These are supported by the X-Ray crystal structure, which will be discussed in the section 2.4. 33 Figure 2.2 (a) Top view and (b) side view of the structure of dimer 1f predicted by ab intio calculation. 2.3.2 Higher Oligomers from Trimer to Pentamer Ab initio calculation of trimer 1h and tetramer 1j is also carried out. But the structures gained are not as well-defined as dimer 1f. For trimer 1h, the backbone of the molecule cannot stay planar. Part of the molecule is constrained within a plane but the other part is twisted to another side probably due to the bulky benzyl groups as shown in Figure 2.3. For tetramer 1j, the whole backbone is twisted due to the existence of four benzyl groups. The backbone is no longer rigid and planar in order to avoid the strain caused by bulky groups (Figure 2.4). Figure 2.3 The structure of trimer 1h predicted by ab intio calculation. 34 Figure 2.4 The structure of tetramer 1j predicted by ab intio calculation. 2.3.3 Cyclic Pentamers Ab initio calculation of cyclic pentamers 1 and 1o is performed. Cyclic pentamer 1 (Figure 2.5) is almost a planar structure and the methyl group is out of the plane. The cavity size of the cyclic pentamer 1 is 5.5 Å at the narrowest and 6.4 Å at the widest. Cyclic pentamer 1o (Figure 2.6) also adopted an approximately planar conformation in the calculation. However, it is not as flat as expected, probably due to the strain. As a result, the cavity size is unexpectedly a little smaller than cyclic pentamer 1, which is 5.3 Å at the narrowest and 6.1 Å at the widest. Figure 2.5 (a) Top view and (b) side view of the structure predicted by ab intio calculation of cyclic pentamer 1. 35 Figure 2.6 (a) Top view and (b) side view of the structure predicted by ab intio calculation of cyclic pentamer 1o. 2.4 Results and Discussion 2.4.1 Synthesis of Monomer, Higher Oligomers and Cyclic Pentamers In scheme 1, dimer 1f was synthesized through active ester method using ethyl chloroformate and NMM with a yield of 74%. In the beginning, dimer 1f was synthesized through acyl chloride. Thionyl chloride was used to convert monomeric acid 1e into the corresponding acyl chloride. And then the acyl chloride is coupled with monomeric amine 1d in the presence of DIEA. And the yield was around 50%. Considering that active ester method was not so moisture-sensitive as the method using thionyl chloride, the active ester method was adopted. Trimer 1h was also synthesized using thionyl chloride at first to give the product in yield 58%. It was changed into using oxayl chloride. The acyl chloride was obtained in a yield of 65%. At the beginning, the target molecule was cyclic pentamer 1o. As shown in Scheme 2, pentamer 1p, with five benzyl groups, was synthesized. It went through reduction and hydrolysis as same as pentamer 1l. But it could not produce the expected compound 1s 36 1 (COCl)2 ,DMF,CH 2Cl2 2 1k, DIEA, DMF COOH O 2N O2 N OBn O H N N H OBn O OBn 1e OBn O H N O N H OBn O OBn O 1p Fe, AcOH, EtOH, CH2 Cl2 ref lux for 3h OBn O H N H 2N H N N H OBn O OBn O N H OBn O O OBn O 1q 1 KOH, MeOH 2 HCl, H2 O OBn O H N H 2N N H OBn O H N OBn O OBn O N H OH OBn O 1r O N O N H OBnH BnO OBn N H H N OBn O O BnO H N O 1s O N O N H OH H HO OH N H H N OH O O HO H N O 1o Scheme 2 A synthetic route designed for cyclic pentamer 1o 37 using BOP or any other coupling reagent such as HATU, PyBOP, and PyBrOP5. A probable reason is that the benzyl groups are too bulky for the acyclic pentamer to shape like a cycle. Due to the huge steric hindrance, the carboxylic acid terminal and the amino group terminal cannot react with each other to form a cyclized product. As a result, the fifth building block 1e in compound 1p was replaced by methoxy-containing compound 2e in order to reduce the steric hindrance during the cyclization process. This modification led to the successful cyclization from 1l to 1a albeit with a relatively low yield around 16%. Debenzylation was achieved by hydrogenolysis using Pd/C as catalyst. A few conditions have been tried as the table shown. Using Hexane/EA (1:1) as eluent to do TLC can find out whether the starting material 1a (sm for short) has been consumed completely and the debenzylated compound stays at the bottom line as shown in Figure 2.7. But TLC can hardly tell how many spots totally in the reaction mixture, because the spot of debenzylated compound has a long tail pointing to the frontier. ESI-MS was used to find out whether there was the expected product in the reaction mixture. Figure 2.7 TLC for conditions from entry 1-10 38 1 Volumetric Ratio of EtOH: THF: Cyclohexene 5:-:1 2 5:-:1 45 50 4 1, unknown compound 3 5:-:1 45 unknownb 5 1, P-1OH, P-3OH 4 5:-:1 45 unknown 10 1, P-2OH 5 4:1:1 45 unknown 4 6 4:1:1 45 unknown 6 1, P-1OH, P-2OH, P-3OH, P-4OH 1 7 4:1:1 45 unknown 10 1, unknown compound 8 2:3:1 45 unknown 6 1 9 2:3:1 45 unknown 3 P-3OH 10 3:2:1 45 unknown 4 1 Entry T/ºC P/psi t/h Major Peaks Shown in ESI-MS Results 25 50 4 1a, 1 (P-4OHa) a: P-nOH represents that there are n –OH groups in the circular pentamer after the debenzylation. b: For entry 3-10, exact pressure was not measured because balloon filled with hydrogen gas was used under these conditions. Table 2.1 Exprimental conditions of debenzylation that have been tried. The circular pentamer 1 has very poor solubility in dichloromethane, acetone, THF and chloroform. In methanol, ethanol and DMSO, the solubility is much better due to the hydrophilic –OH groups. Considering its poor solubility, CH2Cl2 was added to the crude product in order to dissolve the soluble impurities and then filtered off. The crude product was recrystallized with MeOH and then purified by flash column chromatography. However, there are still a small amount of impurities in the final product, which can be seen in the 1H NMR spectrum (see the next section). 39 2.4.2 1D and 2D 1H NMR Results Figure 2.8 1D 1H NMR of (a) pentamer 1l, (b) tetramer 1j, (c) trimer 1h and (d) dimer 1f in CDCl3 (500 MHz, 298 K, 5 mM). Figure 2.9 1D 1H NMR of circular pentamer 1 in DMSO-d6 (500 MHz, 298K, 20mM) Based on 1H NMR results of oligomers and circular pentamers, we can see that in circular 40 pentamer 1, the chemical shift of the protons in amide bonds moves from 9-11 ppm (in DMSO-d6) to lower field (11-15 ppm). It is assumed that the intramolecular hydrogen bonds are formed between the amide-bond proton and neighboring oxygen atoms in –OH/-OCH3. And it also suggests a crescent conformation. In addition, according to 2D NOESY result, strong NOE contacts between protons a and b or c were observed and there is no contact between proton a, d, e and f. And there is also no contact between the protons in amide bonds (a-f) and the prontons in benzene rings. Therefore, the 2D NOESY result shows the evidence for circular conformation of 1. Figure 2.10 2D NOESY result of circular pentamer 1 (298 K, 500 ms, 20 mM) 2.4.3 X-Ray Crystal Structure Analysis The single crystal structure of dimer 1f is shown in Figrure 2.11. The backbone is flat and ridid, though the two benzyl groups stretch out of the plane of the backbone. This structure is consistent with the ab intio calculation result. It shows that there is a bifurcated hydrogen bond formed (Fig 2.12). The hydrogen length in the five-membered hydrogen-bonded ring 41 is 2.169 Å, which is close to that of 2.107 Å calculated by ab initio calculation. Similarly, the hydrogen bond length in the six-membered hydrogen-bonded ring is 2.013 Å, which does not differ too much from that of 1.825 Å predicted by the calculation. It is assumed that this intramolecular hydrogen bond is one reason that makes the backbone rigid and maintains a planar and crescent conformation. a b Figure 2.11 (a) Top view and (b) side view of crystal structure of dimer 1f. Figure 2.12 Hydrogen bonding in dimer 1f in (a) ab intio calculated structure and (b) X-Ray crystal structure As discussed in the section 2.4.1, the circular pentamer 1 has poor solubility in many 42 different solvents, making difficult to identify a suitable solvent pair to grow single crystal.. And for other oligomers, crystal growth using the method of slow evaporation of mixed solvents failed. A possible reason is that the methylene groups in the benzyl groups can rotate freely together with the benzene rings. Therefore, the conformation is not favorable for packing. 2.5 Conclusions Phenol- and methoxybenzen-based aromatic circular γ-peptide 1 was designed and synthesized through oligomers, 1f, 1h, 1j, 1l, and circular γ-peptide 1a. Protection of phenol hydroxyl group with benzyl groups led to successful installation of hydroxyl group onto the circular pentamer after Pd/C-mediated deprotection of benzyl groups. Short oligomers can maintain crescent and rigid amide backbone with the forces of intramolecular hydrogen bonding. Introduction of the bulky benzyl groups shall not disrupt the rigid, crescent conformation. Ab intio calculations and X-Ray crystallography analysis were carried out to support the speculates made about the structures. 43 References 1. Gellman, S. H., Foldamers: A Manifesto. Acc. Chem. Res. 1998, 31, (4), 173-180. 2. Yuan, L.; Sanford, A. R.; Feng, W.; Zhang, A.; Zhu, J.; Zeng, H.; Yamato, K.; Li, M.; Ferguson, J. S.; Gong, B., Synthesis of Crescent Aromatic Oligoamides. J. Org. Chem. 2005, 70, (26), 10660-10669. 3. Gong, B., Crescent Oligoamides: From Acyclic ldquoMacrocyclesrdquo to Folding Nanotubes. Chem. Euro. J. 2001, 7, (20), 4336-4342. 4. Gong, B.; Zeng, H.; Zhu, J.; Yua, L.; Han, Y.; Cheng, S.; Furukawa, M.; Parra, R. D.; Kovalevsky, A. Y.; Mills, J. L.; Skrzypczak-Jankun, E.; Martinovic, S.; Smith, R. D.; Zheng, C.; Szyperski, T.; Zeng, X. C., Creating nanocavities of tunable sizes: Hollow helices. PNAS 2002, 99, (18), 11583-11588. 5. Han, S.-Y.; Kim, Y.-A., Recent development of peptide coupling reagents in organic synthesis. Tetrahedron 2004, 60, (11), 2447-2467. 44 Chapter Three: Synthesis and Structural Investigations of Methoxybenzene-Based Circular γ-Peptides 3.1 Introduction This project follows the same design principles mentioned in Chapter one and Chapter two. We aimed to design and synthesize a class of methoxybenzene-based circular aromatic γ-Peptides which hopefully become synthetic ion channels in the future. The building blocks are different with those described in Chapter two so that the size of the cavity finally formed is not the same. The pentameric backbone also consists of meta-linked benzene rings by secondary amide groups. The backbone is crescent and rigidified by three-center hydrogen bonds. In this project, Ab intio calculations have also been used to predict the optimized conformation. 3.2 Experimental Section 3.2.1 Synthetic Schemes O2N COOCH 3 OH 1b CH 3I, K2 CO 3, DMF 60°C, 4h 82% O2N H2, Pd/C, THF 40°C, 3h qualitative COOCH 3 OCH 3 1 NaOH, MeOH, reflux for 2h 2c 2 HCl, H2O 92% H 2N COOCH 3 OCH 3 2d O2 N COOH OCH 3 2e 45 O2N COOH OCH 3 O 2N 2 2d, DIEA, CH2Cl2 OCH 3O H N Fe, AcOH, EtOH H 2N reflux for 2h O OCH3O 2f 71% 2e OCH 3O H N 1 NMM, ClCOOEt, CH 2Cl2 O OCH 3O 2g O2N COOH O2 N 2 2d, DIEA, CH 2Cl2 OCH3 2e N H OCH3O O OCH 3O 2h 82% OCH3O H N Fe, AcOH, EtOH, CH2 Cl2 H2N reflux for 2h OCH3O H N 1 NMM, ClCOOEt, CH 2Cl2 O N H OCH3O OCH 3O 2i 1 (COCl)2, DMF, CH 2Cl2 2 2i, DIEA, DMF O2N COOH OCH 3 H N O2 N 61% OCH3O 2e OCH 3O OCH 3 OCH 3O 2j H N Fe, AcOH, EtOH, CH 2Cl2 ref lux for 2h N H OCH3O H N H2 N OCH3O OCH3O N H H N OCH 3O OCH 3 OCH 3O 2k 46 1 (COCl)2 , DMF, CH 2Cl2 2 2k, DIEA, DMF O2 N COOH OCH 3 H N O2N 60% OCH 3O N H OCH 3O 2e H N OCH3O N H OCH3O O OCH3O 2l Fe, AcOH, EtOH, CH 2 Cl2 H 2N reflux for 3h OCH 3O H N H N N H OCH 3O OCH3O N H OCH3O O OCH3O 2m 1 KOH, MeOH 2 HCl, H2 O H N H 2N OCH 3O H N N H OCH 3O OCH 3O OCH3O N H OH OCH3O 2n O N O N H BOP, DIEA, DCM, DMF, 40°C, 2h H O O O H N overall yield for 3 steps: 62% N H O O O H N O O 2 Scheme 3 Synthetic route for oligomers and circular pentamer 2 3.2.2 General Methods All the reagents were obtained from commercial suppliers and used as received unless otherwise noted. Aqueous solutions were prepared from distilled water. The organic solutions from all liquid extractions were dried over anhydrous Na2SO4 for a minimum of 15 minutes before filtration. Reactions were monitored by thin-layer chromatography (TLC) on silica gel precoated glass plates (0.25 mm thickness, 60F-254, E. Merck). Flash column chromatography was performed using pre-coated 0.2 mm silica plates from Selecto Scientific. Chemical yields refer to pure isolated substances. 1H and 13C NMR spectra were 47 recorded on either a Bruker ACF-300 or AVF-500 spectrometer. In addition, key compounds were characterized by 2D NOSEY and X-ray Diffraction. 1H NMR spectra were recorded on Bruker ACF300 (300 MHz) and ACF500 spectrometers (500 MHz). The solvent signal of CDCl3 was referenced at δ= 7.26. Coupling constants (J values) are reported in Hertz (Hz). 1H NMR data are recorded in the order: chemical shift value, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), number of protons that gave rise to the signal and coupling constant, where applicable. 13 C NMR spectra are proton-decoupled and recorded on Bruker ACF300 (300 MHz) and ACF500 spectrometers (500 MHz). The solvent, CDCl3, was referenced at δ= 77 ppm. CDCl3 (99.8%-Deuterated) was purchased from Aldrich and used without further purification. 3.2.3 Synthetic Procedure Methyl 2-methoxy-3-nitrobenzoate (2c) Compound 1b (6.00 g, 30.4 mmol) was dissolved in DMF (125mL) to which anhydrous K2CO3 (15.6 g, 113 mmol) and iodomethane (6.98 mL, 112 mmol) were added to it. The mixture was heated at 60°C for 4 hours. The reaction mixture was then filtered and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (100 mL), washed with water (2 x 50 mL), and dried over anhydrous Na2SO4. Removal of CH2Cl2 gave a pure light yellow solid 2c. Yield: 6.41g, 82%. 1H NMR (300 MHz, CDCl3) δ 8.01 (dd, 1H, J = 7.9, 1.8), 7.90 (dd, 1H, J = 8.1, 1.8), 7.26 (m, 1H), 3.99 (s, 3H), 3.95 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 164.4, 152.9, 145.2, 135.3, 128.0, 127.1, 123.5, 63.9, 52.4. MS-ESI: calculated for [M]+ (C9H9NO5): m/z 211.0, found: m/z 211.1. 48 Methyl 3-amino-2-methoxybenzoate (2d) Compound 2c (2.00 g, 9.48 mmol) was reduced by catalytic hydrogenation in THF (50 mL) at 40°C, using Pd/C (0.20g, 10%) as the catalyst for 3 hours. The reaction mixture was then filtered and the solvent removed in vacuo to give the pure brown liquid 2d. Yield: 1.72g, qualitative. 1H NMR (300MHz, CDCl3) δ 6.92-7.14 (m, 1H), 6.83-6.89 (m, 2H), 3.85 (s, 3H), 3.80 (s,3 H). 13C NMR (75 MHz, CDCl3) δ 166.5, 146.6, 124.1, 123.9, 120.1, 119.3, 60.5, 51.8. 2-Methoxy-3-nitrobenzoic acid (2e) Compound 2c (4.00 g, 19.0 mmol) was dissolved in hot methanol (10 mL) to which 1M NaOH (40 mL, 40 mmol) was added. The mixture was heated under reflux for 2 hours and then quenched with water (100 mL). The aqueous layer was neutralized by addition of 1M HCl (80 mL) until the pH was at least 1. The precipitated crude product was collected by filtration, which was recrystallized from hot methanol to give a pure white solid 2e. Yield: 3.45 g, 92.0%. 1H NMR (300 MHz, CDCl3) δ 8.28 (dd, 1H, J = 7.9, 1.8), 8.03 (dd, 1H, J = 8.1, 1.8), 7.36 (t, 1H, J = 7.9), 4.08 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 165.0, 151.9, 144.5, 134.7, 127.3, 126.8, 122.8, 63.1. Methyl 2-methoxy-3- (2-methoxy-3-nitrobenzamido)benzoate (2f) Acid 2e (3.00 g, 15.2 mmol) was dissolved in CH2Cl2 (30 mL) to which 4-methylmorpholine, NMM (2.2mL, 17.9mmol) and ethyl chloroformate (1.96 mL, 16.4 mmol) was added at 0 °C. The reaction mixture was stirred for at least 15 min then a 49 solution of amine 2d (2.70 g, 14.9 mmol) dissolved in CH2Cl2 (30 mL) was added. The reaction mixture was allowed to stir continuously 6 hours at room temperature. The reaction mixture was washed with 1M KHSO4 (100 mL), followed by saturated NaHCO3 (100 mL) and saturated NaCl (100 mL). Drying over Na2SO4 and removal of solvent in vacuo gave the crude product, which was recrystallized from methanol to give the pure product 2f as a white solid. Yield: 3.49 g, 71%. 1H NMR (300 MHz, CDCl3) δ 10.37 (s, 1H), 8.80 (dd, 1H, J = 8.2, 1.6), 8.45 (dd, 1H, J = 7.9, 1.8), 7.99 (dd, 1H, J = 8.1, 1.8), 7.63 (dd, 1H, J = 7.9, 1.6), 7.41 (t, 1H, J = 8.1), 7.24 (t, 1H, J = 8.1), 4.10 (s, 3H), 3.95 (s, 6H). 13C NMR (125 MHz, CDCl3) δ 165.9, 161.3, 151.5, 149.4, 136.5, 132.7, 128.8, 126.6, 124.6, 123.5, 64.5, 62.6. MS-ESI: calculated for [M]+ (C17H16N2O7): m/z 360.2, found: m/z 360.1. Methyl 2-methoxy-3-(2-methoxy-3-(2-methoxy-3-nitrobenzamido)benzamido) benzoate (2h) Compound 2f (3.46 g, 10.0 mmol) was reduced by catalytic hydrogenation in THF (50 mL) at 40 °C, using Pd-C (0.35 g, 10%) as the catalyst for 3 hours. The reaction mixture was then filtered and the solvent removed in vacuo to give the pure amine 2g. Yield: 3.16 g, qualitative. Acid 2e (2.06 g, 10.5 mmol) was dissolved in CH2Cl2 (50 mL) to which NMM (1.35mL, 12.4mmol) and ethyl chloroformate (1.08 mL, 11.3 mmol) was added at 0 °C. The reaction mixture was stirred for at least 15 min after which a solution of amine 2g (3.1 g, 9.04 mmol) dissolved in CH2Cl2 (50 mL) was added. The reaction mixture was allowed to stir continuously 6 hours at room temperature. The reaction mixture was washed with 1M KHSO4 (100 mL), followed by saturated NaHCO3 (100 mL) and saturated NaCl (100 50 mL). Drying over Na2SO4 and removal of solvent in vacuo gave the crude product, which was recrystallized from methanol to give the pure product 2h as a white solid. Yield: 3.64 g, 82%; mp 163-164 oC. 1H NMR (300 MHz, CDCl3) δ 10.23 (s, 1H), 10.22 (s, 1H), 8.85 (dd, 1H, J = 8.2, 1.5), 8.78 (dd, 1H, J = 8.2, 1.6), 8.47 (dd,, 1H, J = 7.9, 1.8), 8.03 (dd, 1H, J = 8.0, 1.8), 7.92 (dd, 1H, J = 7.9, 1.7), 7.62 (dd, 1H, J = 7.9, 1.7), 7.48-7.34 (m, 2H), 7.23-7.20 (m, 1H), 4.14 (s, 3H), 3.98 (s, 3H), 3.96 (s, H). 13C NMR (75 MHz, CDCl3) δ 165.9, 162.8, 161.3, 151.3, 149.2,147.3, 144.3, 136.5, 133.9, 132.0, 129.1, 128.9, 126.7, 126.2, 125.7, 125.0, 124.7, 124.6, 124.4, 123.5, 64.4, 63.2, 62.5, 53.4, 52.3. MS-ESI: calculated for [M]+ (C25H23N3O9): m/z 509.1, found: m/z 509.1. Methyl 2-methoxy-3-(2-methoxy-3-(2-methoxy-3-(2-methoxy-3-nitrobenzamido) benzamido) benzamido) benzoate (2j) Compound 2h (3.00 g, 6.09 mmol) was reduced by catalytic hydrogenation in THF (50 mL) at 40°C, using Pd-C (0.60 g, 20%) as the catalyst for 3 hours. The reaction mixture was then filtered and the solvent removed in vacuo to give the pure amine 2i. Yield: 2.82 g, qualitative. Acid 2e (1.79 g, 9.10 mmol) was placed in a very dry round bottom flask and saturated with nitrogen gas. Dry CH2Cl2 (50 mL) and DMF (0.42 mL) were added to the acid, followed by dropwise addition of oxayl chloride (1.05 mL, 7.27 mmol). The reaction mixture was allowed to stir for 2 hours. The solvent was then removed in vacuo and protected under nitrogen before addition of 15mL dry CH2Cl2. Amine 2i (2.81 g, 6.06 mmol) was dissolved in 35mL dry CH2Cl2 and triethylamine (1.86 mL, 12.1 mmol) before addition to the reaction mixture above. The reaction mixture was stirred at 40 0C for 2 hours 51 and then was washed with aq NaHCO3 (100 mL). Drying over anhydrous Na2SO4 and removal of solvent in vacuo gave the crude product, which was recrystallized from methanol to give the pure product 2j as a white solid. Yield: 2.34g, 61%. 1H NMR (500 MHz, CDCl3) δ 10.18 (s, 1H), 10.10 (s, 1H), 10.00 (s, 1H), 8.86-8.77 (m, 3H), 8.47-8.44 (m, 1H), 8.06-8.03 (m, 1H), 7.94-7.88 (m, 2H), 7.64-7.61 (m, 1H), 7.49-7.34 (m, 3H), 7.23-7.20 (m, 1H), 4.15 (s, 3H), 4.01 (s, 3H), 3.97 (s, 3H), 3.96 (s, 3H), 3.94 (s, 3H) . 13C NMR (75 MHz, CDCl3) δ 165.8, 162.9, 161.4, 151.2, 149.2, 147.2, 147.2, 144.3, 136.4, 133.0, 132.2, 131.9, 129.2, 128.9, 126.7, 126.6, 126.3, 126.1, 125.8, 125.7, 125.0, 124.9, 124.7, 124.6, 124.3, 123.4, 64.4, 63.1, 63.0, 62.4, 52.3. MS-ESI: calculated for [M]+ ( C33H30N4O11): m/z 658.2, found: m/z 658.2. Methyl 2-methoxy-3-(2-methoxy-3-(2-methoxy-3-(2-methoxy-3-(2-methoxy-3-nitrobenzamido) benzamido) benzamido) benzamido) benzoate (2l) Compound 2j (3.77g, 5.87 mmol) was reduced by catalytic hydrogenation in THF (50 mL) at 40°C, using Pd-C (0.75g, 20%) as the catalyst for 3 hours. The reaction mixture was then filtered and the solvent removed in vacuo to give the pure brown liquid 2k. Yield: 3.59 g, qualitative. Acid 2e (0.27 g, 1.36 mmol) was placed in a dry round bottom flask and saturated with nitrogen gas. Dry CH2Cl2 (5 mL) and DMF (60.0 μL) were added to the acid, followed by dropwise addition of oxayl chloride (142 μL, 1.12 mmol). The reaction mixture was allowed to stir for 2 hours. The solvent was then removed in vacuo and saturated with nitrogen gas before addition of 10 mL dry CH2Cl2. Amine 2k (0.57 g, 0.93 mmol) was dissolved in 25 mL dry CH2Cl2 and triethylamine, TEA (0.27 mL, 1.86 mmol) 52 before addition to the reaction mixture above. The reaction mixture was stirred at 40 °C for 2 hours and then was washed with aq NaHCO3 (50mL). Drying over anhydrous Na2SO4 and removal of solvent in vacuo gave the crude product which was recrystallised from methanol and further purified by flash column chromatography (silica gel as the stationary phase) using CH2Cl2/CH3CN (10:1) as the eluent to give pure product 2l as a white solid. Yield: 0.44 g, 60%. 1H NMR (500 MHz, CDCl3) δ 10.25 (d, 2H), 9.85 (s, 1H), 9.65 (s, 1H), 8.81 (m, 4H), 8.44 (m, 1H), 8.02 (m, 4H), 7.89 (m, 2H), 7.61 (m, 4H), 4.03 (s, 15H), 3.88 (m, 3H). 13C NMR (125 MHz, CDCl3) δ 165.6, 163.1, 162.8, 161.4, 151.3, 149.2, 147.2, 147.2, 144.1, 136.4, 132.2, 131.9, 128.8, 126.5, 125.7, 124.8, 123.3, 64.4, 63.1, 63.0, 52.1. MS-ESI: calculated for [M]+ (C41H37N5O13): m/z 807.2, found: m/z 807.3. Circular Pentamer (2) Compound 2l (0.44 g, 0.56 mmol) was reduced by catalytic hydrogenation in THF (50 mL) at 50°C, using Pd-C (0.75g, 20%) as the catalyst for 3 hours. The reaction mixture was then filtered and the solvent removed in vacuo to give a brown liquid 2m. Yield: 0.43g, quantitative. Compound 2m (0.43 g, 0.56 mmol) was dissolved in hot methanol (5 mL) to which 1M KOH (1.20 mL, 1.20 mmol) was added. The mixture was heated under reflux for 2 hours and then quenched with water (20 mL). The aqueous layer was neutralized with 1M KHSO4 (1.2 mL). The precipitated crude product 2n was collected by filtration. Compound 2n (0.76 g, 1.0 mmol) and BOP (0.88 g, 2.0 mmol) were dissolved in CH2Cl2 (3.2 ml) at 0°C. DIEA (0.50 ml, 3.0 mmol) was added and the reaction mixture was stirred continuously for 1 hr at 0°C, then stirred at room temperature for 2 hours. Removal of 53 solvent in vacuo gave the crude product, which was purified by flash column chromatography on silica gel using CH2Cl2/CH3CN (1:10) as the eluent to give a pure white product 2. Yield: 0.47 g, 62%. 1H NMR (500 MHz, CDCl3) δ 10.88 (s, 5H), 9.00 (dd, 5H, J = 8.2, 1.5), 8.02 (dd, 5H, J = 8.0, 1.5), 7.44 (t, 5H, J = 8.1), 4.09 (s, 15H). 13C NMR (125 MHz, CDCl3) δ 162.3, 146.5, 132.9, 126.6, 126.2, 125.6, 124.3, 63.3. HRMS-EI: exact mass calculated for [M]+ (C40H35N5O10): m/z 745.2384, found: m/z 745.2387. 3.3 Theoretical Modeling Ab initio calculations were initially used to design the circular γ-peptides with Gaussian 98 (B3LYP/6-31G) to find out what conformation the molecules we designed might adopt. As shown in Figure 3.1, Ab initio calculation of cyclic pentamers 2 is performed. Cyclic pentamer 2 is a planar structure and the methyl groups are out of the plane. The cavity size of the cyclic pentamer 1 is 5.4 Å at the narrowest and 7.8 Å at the widest. It appears that the cavity size is a little bigger than phenol-based circular pentamer 1 and 1o since the cycle itself is more planar. Figure 3.1 (a) Top view and (b) side view of the structure predicted by ab intio calculation of cyclic pentamer 2. 54 3.4 Results and Discussion 3.4.1 Synthesis of Oligomers and Circular Pentamer The synthesis of circular pentamer 2 is much easier than that of 1 due to the less sterically hindered methyl group compared with benzyl group. As we know, for coupling reaction, active ester is less reactive than chloride. The trimer 2h can be synthesized using active ester method at a yield of 82% while trimer 1h gains very poor yield when using active ester method. The work-up process is also much simpler than that of phenol-based oligomers. As discussed in Chapter two, except the dimer 1f can be directly recrystallized with MeOH, all the other oligomers and circular pentamers need to be purified through flash chromatography. However, all of the methoxybenzene-based oligomers and circular pentamers can be purified by recrystallization with MeOH, which makes the work-up process much more convenient. 3.4.2 X-Ray Crystal Structure Analysis X-ray crystal structure analysis of circular pentamer 2 is very consistent with theoretically modeled structure. The crystal was grown by Dr Qin Bo. The circular molecule is in an almost perfect planar conformation as shown in the figure below where the methyl groups are removed for clarity. 55 Figure 3.2 (a) Top view and (b) side view of crystal structure of circular pentamer 2 (the methyl groups are removed for clarity). The cavity size is 5.4 Å, which is large enough for binding certain cations. However, binding study of the circular pentamer, done by Dr Qin Bo, has shown poor activity towards alkali metal ions. The most probable reason is the steric hindrance caused by methyl groups. Although the cavity size is big enough, three methyl groups on one side of the plane and two on the other side block the entrance for the ions (Figure 3.3). Figure 3.3 (a) Top view and (b) side view of the crystal structure of 2 in CPK representations. 3.5 Conclusions Methoxybenzene-based aromatic noncircular peptides (2f-2n) and circular γ-peptide 2 were designed and synthesized. Ab intio calculations and X-Ray crystallography analysis were carried out. And the results of them are highly consistent with the fact that the circular 56 pentamer 2 maintains very planar and flat conformation. However, from crystal structure of the circular pentamer it can be seen that the methyl groups might block the designed binding site for ion transport. 57 References 1. Yuan, L.; Sanford, A. R.; Feng, W.; Zhang, A.; Zhu, J.; Zeng, H.; Yamato, K.; Li, M.; Ferguson, J. S.; Gong, B., Synthesis of Crescent Aromatic Oligoamides. J. Org. Chem. 2005, 70, (26), 10660-10669. 2. Gong, B.; Zeng, H.; Zhu, J.; Yua, L.; Han, Y.; Cheng, S.; Furukawa, M.; Parra, R. D.; Kovalevsky, A. Y.; Mills, J. L.; Skrzypczak-Jankun, E.; Martinovic, S.; Smith, R. D.; Zheng, C.; Szyperski, T.; Zeng, X. C., Creating nanocavities of tunable sizes: Hollow helices. PNAS 2002, 99, (18), 11583-11588. 3. Han, S.-Y.; Kim, Y.-A., Recent development of peptide coupling reagents in organic synthesis. Tetrahedron 2004, 60, (11), 2447-2467. 4. Zeng, H. Ph.D. Dissertation, University at Buffalo, 2002. 5. Qin, B.; Chen, X.; Fang, X.; Shu, Y; Yip, Y. K.; Yan, Y.; Pan, S.; Ong, W. Q.; Ren, C.; Su, H; Zeng, H., Crystallographic Evidence of an Unusual, Pentagon-Shaped Folding Pattern in a Circular Aromatic Pentamer. Org. Lett. 2008, 10 (22), 5127-5130 58 [...]... rigid aromatic amide backbone Through changing linking position (meta- or para-) or the building blocks the cavity size of the folding oligomers can be tuned Based on the well-defined crescent backbone and rigidity, the circular aromatic γ-peptide derived from phenol- and methoxybenzene- based building blocks is designed It derived 18 from meta-linking benzene rings via amide linkages And the circular. .. Forms Chloride Channels to Mediate Chloride Transport across Cell Membranes J Am Chem Soc 2007, 129, (23), 7264-7265 17 Chapter Two: Synthesis and Structural Investigations of Circular Aromatic γ -Peptides Derived from Phenol- and Methoxybenzene- Based Building Blocks 2.1 Introduction As mentioned in Chapter One, a variety of ion transport systems through lipid bilayer membranes have been created in the... cyclic 6 peptides created by Gharidi et al10, 11 (Figure 1.7) The cyclic peptides, which adopt a flat conformation, are composed of alternating D- and L- amino acids The cyclic peptides stack face-to-face when H-bonding is formed between the upper and the lower macrocycle and appear as a peptide nanotube The cyclic peptide cyclo[-(Trp-D-Leu)3Glm-D-Leu-] could rapidly partition into the lipid bilayers and. .. br, broad), number of protons that gave rise to the signal and coupling constant, where applicable 13C NMR spectra are proton-decoupled and recorded on Bruker ACF500 spectrometers (500 MHz) The solvent CDCl3 was referenced at δ= 77 ppm and DMSO-d6 was referenced at δ= 40 ppm CDCl3 and DMSO-d6 (99.8%- Deuterated) was purchased from Aldrich and used without further purification 2.2.3 Synthetic Procedure... bilayer membrane Ab initio calculations are used to predict the conformation of the circular γ-peptide The calculation results are compared with the synthesized compound And it is aimed to evaluate the conformation of the circular γ-peptide It is hoped that the circular γ-peptide could be constructed as synthetic ion channels and obtain therapeutic properties that can be used as antimicrobials in the future... 40°C, 2h OH H H3 CO OH 70% N H H N O HO H N OH O O 1 Scheme 1 Synthetic route for oligomers and cyclic pentamer 1 21 2.2.2 General Methods All the reagents were obtained from commercial suppliers and used as received unless otherwise noted Aqueous solutions were prepared from distilled water The organic solutions from all liquid extractions were dried over anhydrous Na2SO4 for a minimum of 15 minutes... from Selecto Scientific Chemical yields refer to pure isolated substances 1H and 13C NMR spectra were recorded on either a Bruker ACF-300, AVF-500 or DRX-500 spectrometer In addition, key compounds were characterized by 2D NOSEY and X-ray Diffraction 1H NMR spectra were recorded on Bruker ACF500 (500 MHz) and DRX500 spectrometers (500 MHz) The solvent signal of CDCl3 was referenced at δ= 7.26 ppm and. .. understanding of ion channel transport mechanism Thereafter, more and more synthetic ion channels have been created Besides the hints given by natural ion channels, molecules which are membrane-active and functional as ion transporters inspired us substantially For example, Gramicidin, a pentadecapeptide made up of alternating D- and L- amino acids, dimerize to form β-helix in lipid bilayer membrane And. .. DMF (200 mL) to which anhydrous K2CO3 (11 g, 80 mmol) and benzyl bromide (2.6 mL, 22 mmol) were added to The mixture was heated at 60°C for 5h The reaction mixture was then filtered and the solvent was removed in vacuo The residue was dissolved in CH2Cl2, washed with water and dried over anhydrous Na2SO4 Removal of CH2Cl2 and recrystallization from MeOH gave pure yellow solid 1c Yield: 4.30 g, 75%... (Figure 1.8) One more recent example was aromatic oligoamide macrocycle made by Helsel et al13 Among the variants, 1d and 1e are membrane active 23Na NMR technique was used to ensure the vesicles did not undergo lysis and also to test the ion transport function Figure 1.8 Macrocycles that can stack to form tubular ion channels (1) Ureido-crown ether (left) (2) Aromatic oligoamide macrocycle (right) As ... backbone and rigidity, the circular aromatic γ-peptide derived from phenol- and methoxybenzene-based building blocks is designed It derived 18 from meta-linking benzene rings via amide linkages And. .. (23), 7264-7265 17 Chapter Two: Synthesis and Structural Investigations of Circular Aromatic γ-Peptides Derived from Phenol- and Methoxybenzene-Based Building Blocks 2.1 Introduction As mentioned... 15 CHAPTER TWO: SYNTHESIS AND STRUCTURAL INVESTIGATIONS OF CIRCULAR AROMATIC Γ-PEPTIDES DERIVED FROM PHENOL- AND METHOXYBENZENE-BASED BUILDING BLOCKS 18 2.1 INTRODUCTION