Circular aromatic gamma peptides derived from phenol and methoxybenzene 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 inter

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

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

1.1GENERAL 1

1.2UNIMOLECULAR ION CHANNEL 4

1.3AGGREGATE ION CHANNELS 6

1.4OTHER TYPES OF ION CHANNELS 12

1.5APPLICATIONS 13

REFERENCES 15

CHAPTER TWO: SYNTHESIS AND STRUCTURAL INVESTIGATIONS OF CIRCULAR AROMATIC Γ-PEPTIDES DERIVED FROM PHENOL- AND METHOXYBENZENE-BASED BUILDING BLOCKS 18

2.1INTRODUCTION 18

2.2EXPERIMENTAL SECTION 20

2.2.1 Synthetic Scheme 20

2.2.2 General Methods 22

2.2.3 Synthetic Procedure 22

2.3THEORETICAL MODELING 33

2.3.1 Dimer 33

2.3.2 Higher Oligomers from Trimer to Pentamer 34

2.3.3 Cyclic Pentamers 35

2.4RESULTS AND DISCUSSION 36

2.4.1 Synthesis of Monomer, Higher Oligomers and Cyclic Pentamers 36

2.4.2 1D and 2D 1 H NMR Results 40

2.4.3 X-Ray Crystal Structure Analysis 41

2.5CONCLUSIONS 43

REFERENCES 44

CHAPTER THREE: SYNTHESIS AND STRUCTURAL INVESTIGATIONS OF METHOXYBENZENE-BASED CIRCULAR Γ-PEPTIDES 45

3.1INTRODUCTION 45

3.2EXPERIMENTAL SECTION 45

3.2.1 Synthetic Schemes 45

3.2.2 General Methods 47

3.2.3 Synthetic Procedure 48

3.3THEORETICAL MODELING 54

3.4RESULTS AND DISCUSSION 55

3.4.1 Synthesis of Oligomers and Circular Pentamer 55

3.4.2 X-Ray Crystal Structure Analysis 55

3.5CONCLUSIONS 56

REFERENCES 58

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,

demonstrating the rationality and validity of our design principle Further study of the function of the circular γ-peptides needs to be carried out

List of Tables

T ABLE 2.1EXPRIMENT CONDITIONS OF DEBENZYLATION THAT HAVE BEEN TRIED .39

List of Figures

F IGURE 1.1ION CHANNELS FORMED BY NATURAL COMOUNDS .2

F IGURE 1.2SCHEMATIC OF A VOLTAGE CLAMP EXPERIMENT .4

F IGURE 1.3SYNTHETIC CATION CHANNEL “HYDRAPHILES” CREATED BY GOKEL ET AL 5

F IGURE 1.4THE ION CHANNEL FORMED BY HYDRAPHILES 5

F IGURE 1.5SCHEME OF POST-MODIFICATION OF G-QUADRUPLEX 6

F IGURE 1.6AGGREGATION ION CHANNEL FORMED BY THE STACKING OF MACROCYCLES 7

F IGURE 1.7 CYCLO[-(TRP-D-LEU)3GLM-D-LEU-] AND THE ION CANNEL IT FORMED IN LIPID BILAYER 7

F IGURE 1.8MACROCYCLES THAT CAN STACK TO FORM TUBULAR ION CHANNELS .8

F IGURE 1.9STRUCTURE OF CUCURBITURIL 9

F IGURE 1.10 Β-BARREL ION CHANNEL WITH RIGID-ROD SCAFFOLD 9

F IGURE 1.11SCHEME OF PHOTOSYSTEM 1 PHOTO-SWITCHED INTO ION CHANNEL 2 10

F IGURE 1.12AGGREGATE ION CHANNELS FROM AMPHIPHILES 10

F IGURE 1.13A FEW EXAMPLES OF BOLAAMPHIPHILES 11

F IGURE 1.14MICELLE-LIKE CHANNEL FORMED BY SINGLE CHAIN AMPHIPHILES 12

F IGURE 1.15STRUCTURE OF SIMPLE COMPOUNDS THAT CAN FORM ION CHANNELS 12

F IGURE 1.16STRUCTURE OF ΑN AMINOXY ACID WHICH CAN FORM CHLORIDE CHANNELS 13

F IGURE 2.1CONCEPTUAL DEPICTION OF THE SYNTHETIC ION CHANNEL EMBEDDED WITHIN A LIPID BILAYER MEMBRANE 19

F IGURE 2.2THE STRUCTURE OF 1F PREDICTED BY AB INTIO CALCULATION 34

F IGURE 2.3THE STRUCTURE OF TRIMER 1H PREDICTED BY AB INTIO CALCULATION 34

F IGURE 2.4THE STRUCTURE OF TETRAMER 1J PREDICTED BY AB INTIO CALCULATION 35

F IGURE 2.5(A)TOP VIEW AND (B) SIDE VIEW OF THE STRUCTURE PREDICTED BY AB INTIO

CALCULATION OF CYCLIC PENTAMER 1 .35

F IGURE 2.6(A)TOP VIEW AND (B) SIDE VIEW OF THE STRUCTURE PREDICTED BY AB INTIO CALCULATION OF CYCLIC PENTAMER 1O 36

F IGURE 2.7TLC FOR CONDITIONS FROM ENTRY 1-10 38

F IGURE 2.81D1HNMR OF (A) PENTAMER 1 L,(B) TETRAMER 1 J,(C) TRIMER 1 H AND (D) DIMER

1 F IN CDCL3(298K,5 MM) .40

F IGURE 2.102DNOESY RESULT OF CIRCULAR PENTAMER 1(298K,500 MS,20 MM) 41

F IGURE 2.11CRYSTAL STRUCTURE OF DIMMER (COMPOUND 1 F) 42

F IGURE 2.12HYDROGEN BONDING IN DIMER 1 F IN (A) AB INTIO CALCULATED STRUCTURE AND

(B)X-RAY CRYSTAL STRUCTURE 42

F IGURE 3.1(A)TOP VIEW AND (B) SIDE VIEW OF THE STRUCTURE PREDICTED BY AB INTIO

CALCULATION OF CYCLIC PENTAMER 2 54

F IGURE 3.2(A)TOP VIEW AND (B) SIDE VIEW OF CRYSTAL STRUCTURE OF CIRCULAR PENTAMER

2(THE METHYL GROUPS ARE REMOVED FOR CLARITY) .56

F IGURE 3.3(A)TOP VIEW AND (B) SIDE VIEW OF THE CRYSTAL STRUCTURE OF 2 IN CPK

REPRESENTATIONS 56

DMSO Dimethyl sulfoxide

DMSO-d6 Deuterated Dimethyl Sulfoxide

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

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

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

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

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

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

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

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 107ions 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

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

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

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

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

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

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

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

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,

(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

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

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

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

O2N 0°C, 30 min reflux for 2d

1c R=Bn

2c R=Me

1d R=Bn 2d R=Me

reflux for 2h

OR

1e R=Bn 2e R=Me

1 NaOH, MeOH, reflux for 2h

H N OBn O

H N OBn O

O CH3reflux for 2h

H N OBn N H OBn

H N OBn N H OBn

O

O

O

CH3reflux for 2h

1b

OCH3COOCH3

O2N

2c

O 2 N OBn COOCH 3

1c

CH 3 I, K 2 CO 3 , DMF 60°C, 4h 82%

overall yield for 2steps: 28%

1e: 92%

2e: 92%

N OBn

O

O

O OCH 3

O

O

N OBn

O

O O

O

O

N OBn

O

OH O

O N

O N

O N

N O OBn

OBn

H 3 CO

BnO OBn

H H

H

H H BOP, DIEA, DCM, 40°C, 2h

1a

O N

O N

O N

O N

N O OH

OH

H 3 CO

HO OH

H H

H

H H

1

H 2 , Pd/C, cyclohexene, THF, EtOH, 40°C, 2h

H 2 N OBn O

N OBn

O

O

O OCH 3

O 2 N OCH 3 O

N OBn

O

O

N OBn

O

O O

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

purchased from Aldrich and used without further purification

2.2.3 Synthetic Procedure

Methyl 2-hydroxy-3-nitrobenzoate (1b)

Salicylic acid (10.0 g, 72.5 mmol) was dissolved in 200 mL of CH2Cl2, to which

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)

13C 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