Development of synthetic methodologies for ion channels

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ProQ uest Information and Leaming

300 North Z eeb Road. Ann Arbor, Ml 48106-1346 USA

800-521-0600

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by Chi-wei Hu

B.Sc. Chung-Yuan Christian University, Chung-Li, Taiwan, R.O.C. M.Sc. Florida Atlantic University, Boca Raton, Florida, USA

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree o f

DOCTOR OF PHILOSOPHY In the Department o f Chemistry

We accept this thesis as conforming to the required standard

s. Supervisor (bepartmei

Dr. T. M. Fyles, Supervisor (Department o f Chemistry)

Dr. R. H. Mitchell, Department Member (Department o f Chemistry)

Dr. R. G. Hicks, Department Member, (Department o f Chemistry)

de Membc

Dr. G. A. Beer, Outside Member (Department o f Physics and Astronomy)

___________________________________________________________________________________________________________________

Dr. N. R. Branda, External Examiner (Simon Fraser University) © Chi-wei Hu, 2002

University o f Victoria

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Abstract

The goal o f this research was to develop a synthetic strategy to synthesize voltage-gated (end-differentiated) ion channels with a minimal synthetic effort and this goal was ' pursued in two different ways with two different structural components: macrocycles and non-macrocycles (flexible acyclic components).

This thesis started with an end-differentiated macrocycle differentially protected with Boc and nitro as amine protecting groups. To test the m acrocycle’s suitability as a part o f an ion channel, a centrosymmetric ion channel candidate was synthesized. The synthesis started with the reduction o f the nitro to a free amine followed by a dimerization reaction with terephthaloyl dichloride to form a centrosymmetric diamide. The Boc protecting groups were removed to yield the target. Although the target compound showed ion channel activity, its very poor solubility made it almost impossible for a complete property investigation.

The second part o f this research was to examine the necessity o f macrocycles for ion channels. Some centrosymmetric non-macrocyclic bolaamphiphiles related to a known macrocyclic ion channel were synthesized and tested for their ion transport properties. An oligoester was prepared from 2-[2-(2-chloroethoxy)ethoxy]ethanol and dodecanedioyl

dichloride followed by estérification with the mono octyl ester o f maleic acid to yield a diene that reacted with mercaptoacetic acid to give the target compound. An oligoester homolog was also synthesized via the same chemistry.

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Other compounds with ester-amide and amide functionalities were also synthesized. The ester-amide compounds were synthesized from a mono Boc protected 1,8-diamino-

3,6-dioxaoctane followed by acylation with dodecanedioyl dichloride to yield a diamide that further reacted with the mono octyl ester o f fumaric acid to afford a diene. The diene reacted with mercaptoacetic acid to give the target ester-amide compound. The acyclic hexamide compound used the diamide intermediate above to react with the mono N- methyl-N-octyl amide o f maleic acid to afford a diene that reacted with mercaptoacetic acid to give the target compound. The other ester-amide compound started with 1,8- bis(methylamino)-3,6-dioxaoctane and followed the chemistry for the previous ester- amide to yield the target compound.

All the acyclic compounds were tested for their ion transport properties by pH-stat and carboxyfluorescein release experiments. Among the compounds tested, oligoesters showed clear ion channel activity, implying macrocycles are not necessary for ion channel formation. On the other hand, ester-amides were found to be active only in concentration ranges where they form large membrane defects. No ion transport activity was found for the hexamide compound.

The third part o f this thesis employed solid-phase synthesis to prepare some acyclic oligoesters as ion channel candidates. The synthesis provides end-differentiated compounds as required for voltage-gating applications. Building blocks with an acid functional group and THP or TBDMS protected alcohol were prepared for the solid- phase synthesis. Sequential coupling on Wang resin followed by cleavage and gel

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filtration gave products that showed the expected NMR spectra. The MALDI mass spectrum and G PC showed these samples contain deletion sequences due to incomplete conversion. The coupling efficiency was calculated be an average 93% for each step. The products formed active ion channels in a planar bilayer experiment, implying that this synthesis has achieved the goal o f the thesis.

Examiners:

Dr. T. M. Fyles. Supervisor (Department o f Chemistry)

Dr. R. H. Mitchell, Department Member (Department o f Chemistry)

Dr. R. G. Hicks, Department Member (Department o f Chemistry)

Dr. G. A. Beer, Outside Member (Department o f Physics and Astronomy)

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TITLE PAGE i ABSTRACT ii TABLE OF CONTENTS V LIST OF TABLES ix LIST OF FIGURES X LIST OF SCHEMES XV

LIST OF ABBREVIATIONS xvii

ACKNOWLEDGEMENTS xix

CHAPTER I INTRODUCTION 1

1.1 Ion transport phenomena 1

1.2 Examples o f small molecule ion channels from natural sources 5

1.2.1 Gramicidin A 5

1.2.2 Amphotericin B 6

1.2.3 Alamethicin 8

1.3 Synthetic ion channels 10

1.3.1 Bouquet ion channels 11

1.3.2 Ion channels formed by bis-macrocyclic bolaamphiphile 13

1.3.3 Multiple diaza-18-6 design 16

1.3.4 Steroid based transporters 18

1.3.5 Channels formed by ion pairs 22

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1.4 Goal o f the thesis 24

CHAPTER 2 DESIGN, SYNTHESIS, AND PROPERTIES OF BISCYCLOPHANE 28 BOLAAMPHIPHILES

2.1 Introduction 28

2.2 Synthesis 32

2.3 Ion transport activity studies 53

2.3.1 pH-stat titration investigation 54

2.3.2 Planar bilayer experiment 55

2.4 Conclusion 56

CHAPTER 3 DESIGN AND SYNTHESIS OF NON-MACROCYCLIC COMPOUNDS AND EVALUATION OF THEIR CHANNEL

FORMING PROPERTIES 58

3.1 Design 58

3.2 Synthesis 60

3.3 Ion transport studies 115

3.3.1 pH-stat titration 117

3.3.2 Carboxyfluorescein leakage assay 121

3.4 Conclusion 122

CHAPTER 4 SOLID-PHASE SYNTHESIS OF DEPSIDES FOR ION CHANNELS 124

4.1 Introduction 124

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4.3 Ion transport studies 169

CHAPTER 5 SUMMARY AND FUTURE PROSPECTS 172

CHAPTER 6 EXPERIMENTAL 178

6.1 Apparatus 178

6.2 Procedures 179

6.3 General procedures for solid-phase synthesis 201 6.3.1 Loading Wang resin with TBDMS protected hydroxyl dodecyl

glutarate 2 0 1

6.3.2 Deprotection o f TBDMS 202

6.3.3 Coupling o f THP protected hydroxyl acid to -O H on glutarate 202

6.3.4 Deprotection o f THP 202

6.3.5 Coupling o f THP protected hydroxyl acid to -O H on a hydroxyl

acid 203

6.3.6 Coupling o f TBDMS protected hydroxyl glutarate to -O H on a

hydroxyl acid 2 0 0

6.3.7 Cleaving from the solid support 203

6.4 Vesicle preparation and pH-stat titration 204

6.4.1 Stock solution 205

6.4.2 Egg lecithin vesicle preparation 205

6.5 Carboxyfluorescein leakage assay 207

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6.5.2 CF entrapped vesicle preparation 207 6.5.3 Percentage release o f CF after incubation with the surfactants 208

APPENDIX 209

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LIST O F TABLES

Table 3-1 Summary o f kinetic studies o f acyclic compounds 120 Table 4-1 Protocol o f solid-phase synthesis reactions 153 Table 4-2 Summary o f MALDI MS and HPLC results for

4-14

167 Table 4-3 Summary o f MALDI MS and HPLC results for

4-15

169

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Figure 1-1 Structure o f transport protein in bilayer membranes 1 Figure 1-2 Neurotransmitters bind to receptors to open ion channels 2

Figure 1-3 Planar bilayer experimental setup (left) 3

Typical current-time relationship (right)

Figure 1-4 pH-stat titration experimental setup and typical resulting curve 4

Figure 1-5 Structure o f gramicidin A 6

Figure 1-6 Amphotericin and proposed “barrel-stave” aggregation 7 Figure 1-7 Current-voltage relationship for alamethicin 8

Figure 1-8 Schematic representation of ion-transport mechanisms 1 0

Figure 1-9 One example o f (head-wall)n-core design 13

Figure 1-10 Tetrasteroid channel system 19

Figure 1-11 Nanotube formed by rigid rods 23

Figure 1-12 Schematic representation o f the synthetic strategy followed 25

Figure 2-1 'H NMR spectrum o f 2-14 in DMSO-c/e 33

Figure 2-2 NMR spectrum o f 2-14 in DMSG- d(, 35

Figure 2-3 *H NMR spectrum o f 2-17 in CDCI3 36

Figure 2-4 ‘H NMR spectrum o f 2-19 in CDiCl: 38

Figure 2-5 'H NMR spectrum o f 2-20 in DMSO-r/a at 150 “C 40 Figure 2-6 ’H NMR spectrum o f 2-20 in DMSG- c/e at 150 °C 42 Figure 2-7 *H NMR spectrum o f 2-22 in DMSG- c/e at 150 °C 44 Figure 2-8 '^C NMR spectrum o f 2-22 in DMSG- c/eat 150 °C 46

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Figure 2-10 '^C NMR spectrum o f 2-15 in CDCI3 49

Figure 2-11 'H NMR spectrum o f 2-23 in DMSO- d^at 120 °C 50 Figure 2-12 'H NMR spectrum o f 2-21 in DMSO- df. at 120 °C 52

Figure 2-13 pH-titration o f 2-15 54

Figure 2-14 Single ion channel conductance records o f 2-15 at an applied potential 56

Figure 3-1 ' H NMR spectrum o f 3-6 in CDCI3 61

Figure 3-2 ’^C NMR spectrum o f 3-6 in CDCI3 62

Figure 3-3 H NMR spectrum o f 3-7 in CDCI3 65

Figure 3-4 ‘H NMR spectrum o f 3-8 in CDCI3 6 6

Figure 3-6 'H NMR spectrum o f 3-9 in CDCI3 69

Figure 3-7 ‘^C NMR spectrum o f 3-9 in CDCI3 70

Figure 3-9 NMR spectrum o f 3-1 in CDCI3 73

Figure 3-13 NMR spectrum of3-15 in CDCI3 79

Figure 3-14 H NMR spectrum o f 3-16 in CDCI3 8 1

Figure 3-15 '^C NMR spectrum of3-16 in CDCI3 82

Figure 3-16 ‘H NMR spectrum o f 3-18 in CDCI3 84

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Figure 3-18 '^C NM R spectrum o f 3-19 in CDCI3 87

Figure 3-19 'H NMR spectrum o f 3-2 in CDCI3 8 8

Figure 3-20 ‘^C NM R spectrum o f 3-2 in CDCI3 90

Figure 3-21 ' H NM R spectrum o f 3 -2 1 in CDCI3 9 1

Figure 3-22 ‘^C NMR spectrum o f 3-2 1 in CDCI3 93

Figure 3-24 NM R spectrum o f 3-3 in CDCI3 96

Figure 3-25 DEPT NMR spectrum o f 3-3 in CDCI3 98

Figure 3-26 'H NMR spectrum o f 3-22 inC D C b 101

Figure 3-27 NM R spectrum o f 3-22 in CDCI3 102

Figure 3-30 NMR spectrum o f 3-23 in CDCI3 106

Figure 3-33 ‘^C NM R spectrum o f 3-24 in CDCI3 110

Figure 3-34 H NM R spectrum o f 3-25 in DMSO- 4 at 100 °C 111 Figure 3-35 '^C NMR spectrum o f 3-25 in DMSO- c4 at 100 °C 113 Figure 3-36 ‘H NM R spectrum o f 3-4 in DMSO- c/e at 100 °C 114 Figure 3-37 ‘^C NM R spectrum o f 3-4 in DMSO- ck at 100 T 116 Figure 3-38 A typical pH-stat titration curve o f 3-1 at 25 °C with K2SO4 117

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Figure 3-40 Initial rate o f transport as a function o f concentration o f

3-1

119 Figure 3-41 Carboxyfluorescein leakage assay results o f the acyclic compounds 122

Figure 4-1 ‘H NMR spectrum o f

4-5

in CDCh 130

Figure 4-2 '^C NMR spectrum o f

4-5

in CDCI3 131

4-6

in CDCI3 132

4-6

in CDCI3 133

Figure 4-5 'H NMR spectrum o f

4-9

in CDCI3 135

4-9

in CDCI3 137

4-11

in D3O 138

4-11

in D2O 140

4-12

in CDCI3 141

Figure 4-10 NMR spectrum o f

4-12

in CDCI3 142

Figure 4-11 H NMR spectrum o f

4-4

in CDCI3 144

4-4

in CDCI3 145

4-13

in CDCI3 147

Figure 4-14 ‘^C NMR spectrum o f

4-13

in CDCI3 148

Figure 4-15 Partial *H NM R spectrum o f the cleaved product following a single 152 cycle o f loading and coupling

Figure 4-16 H NMR spectrum o f

4-14

in CDCI3 155

4-15

in CDCI3 156

4-14

in CDCI3 157

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Figure 4-20 MALDI mass spectrum o f

4-14

160

Figure 4-21 MALDI mass spectrum o f

4-15

161

Figure 4-22 HPLC chromatogram o f

4-14

eluting with CHiCL and G PC Column 163 Figure 4-23 HPLC chromatogram o f

4-15

eluting with CHzCL and G PC Column 163 Figure 4-24 Current as a function o f time following addition o f

4-7

in to DiPhyPC

Membrane 170

Figure 4-25 Square tops observed for

4-14

170

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LIST O F SCHEM ES

Scheme I-I Some steps for synthesizing

1-1

12

Scheme 1-2 Macrocyclization reactions for compounds

1-4

and

1-5

15

Scheme 1-3 Partial synthesis o f

l-4a

16

Scheme 1-4 Partial steps to synthesize hydraphile channels 17

Scheme 1-5 Key syntheses for

1-1 la

and

1-llb

21

Scheme 1-6 Coupling o f phenyl rings for rigid rod synthesis 24

Scheme 2-1 Partial synthesis o f compound

l-4b

29

Scheme 2-2 Synthesis o f macrocycle

1-14

30

Scheme 2-3 Synthetic strategy to synthesize the corresponding cento-symmetric

diamide from

1-14

31

Scheme 2-4 Amines derived from

1-14

32

Scheme 2-5 Dimerization reaction o f

2-14

with glutaroyl dichloride 37 Scheme 2-6 Dimerization o f

1-14

with fumaroyl dichloride 39 Scheme 2-7 Dimerization reaction o f

2-14

with terephthaloyl chloride and

Reduction 41

Scheme 2-8 Dimerization o f

2-14

with pimeloyl dichloride 43 Scheme 2-9 Synthesis o f a centro-symmetric biscyclophane from

2-15

51 Scheme 3-1 Synthetic route to synthesize compound

3-1

and

3-5

63

Scheme 3-2 Proposed route for the synthesis o f

3-2

74

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Scheme 3-4 An alternative route to synthesize compound

3-2

3-3

3-4

99 Scheme 4-1 Solid-phase synthesis o f depsides according Riguera’s method 125 Scheme 4-2 Candidate substructures for the acid-ester-alcohol fragment 127 Scheme 4-3 Comparison o f the synthetic routes for potential aicd-ester-alcohol

fragments 127

Scheme 4-4 Synthetic route to synthesize

4-2

129

Scheme 4-5 Synthesis o f TBDMS protected building block

4-9

134

Scheme 4-6 Two synthetic routes that lead to

4-3

136

Scheme 4-7 Synthesis o f

4-4

and

4-13

146

Scheme 4-8 Reaction sequence to show colour change for montoring solid-phase

synthesis 151

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List of abbreviation

Boc /er/-butoxy carbonyl

Bz benzene

CF carboxyfluorescein

CBz carbobenzyloxy

DHP 3,4 dihydro-2 H-pyran DCC dicyclohexylcarbodiimide

DEPT distortionless enhancement by polarization transfer

DIG diisop rop ylcarb od iim id e

DiphyPC diphytanoyl phosphatidylcholine DMA dimethylacetamide

DMAP 4-dimethylaminopyridine

DMF dimethylformamide

DMSO dimethyl sulphoxide

Et ethyl

FCCP carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone HPLC high pressure liquid chromatography

IR infrared

LUV large unilamellar vesicle

LSIMS liquid secondary ion mass spectrum MALDI matrix assisted laser desorption ionization

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MS mass spectrum

NMR nuclear magnetic resonance mNBA /«- nitrobenzyl alcohol m/z mass to charge ratio

OMs methanesulfonyl

PA phosphatidic acid

PC phsophatidyl choline

PNBP p-nitrobenzylpyridine

TBAF tetrabutylammonium fluoride TBMDS /err-butyl dimethylsilyl

TEMPO 2,2,6,6-tetramethyl- 1 -piperidinyloxy

TFA trifluoroacetic acid

THF tetrahydrofuran

THP tetrahydropyran

TEC thin layer chromatography ;j-TsGH p-toluenesulfonic acid /7-TsC1 p-toluenesulfonyl chloride

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Acknowledgement

I would like to thank my supervisor Dr. Fyles for his guidance, patience and encouragement. Thanks are extended to Todd Sutherland for letting me use some o f his experimental results. I wish to thank X. Zhou for his friendly help at the beginning o f my graduate study here. I would also like to thank the chemistry department staff and faculty, especially Mrs. Christine Greenwood and Dr. David McGillivray. Thanks for the financial support from the department o f chemistry and University o f Victoria. And finally, I am grateful to my parents and my wife for their support throughout the long course.

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/. / Ion transport phenomenon

In biological systems, bilayer membranes physically separate the inside o f a living cell and the outside environment by restricting the free passage o f most polar species. However, in order to live, the cell must import necessary materials such as nutrients from outside o f the cell membrane and export the products generated in the cell such as hormones, enzymes, and toxins. Small molecules can directly diffuse through the cell membrane because they are hydrophobic.' On the other hand, polar species or ions can only be transported through the cell membrane via facilitated transport. Transport proteins play the most important role in facilitated transport in the biological world. Transport proteins span the cell membrane and offer a hydrophilic tunnel for passage o f polar molecules or ions. These proteins are embedded in the phospholipid bi layer membrane with their hydrophobic amino acids contacting the lipid and the hydrophilic

Amino I o n s terminus a helical section o f protein = Carboxyl term inus

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segment o f a transport protein is shown in Figure 1-1 where six a helices cluster together’. The cluster is roughly perpendicular to the membrane plane and the carboxyl and amino termini are on the opposite sides o f the membrane." The section o f the protein that functions as the transporter is only a small section o f the overall protein molecule. As shown in Figure 1-1, most o f the protein molecule is not embedded in membrane, only the section that acts as transporter. Other transport proteins bind to their passengers and physically deliver the passengers across the membrane.^ Transport proteins are also known to be selective. They only translocate specific species or closely related species across the membrane. For example, glucose in blood can be transported into liver cells rapidly via a specific transport protein but fructose, an isomer o f glucose, will be rejected by this transport protein.^

Transport proteins are also essential to the communication between neurons in the

S y n a p tic c le ft P r o te i n to n c h a n n e l ( o p e n ! N e u r o tr a n s in i tt e r tn o lc c u le n e u r o t r a n s m it te r __ m o le c u le b in tk t o a r e c e p to r - c h a n n e l o p e n s R e c e p to r P r o te i n io n c h a n n e l (c lo se d )

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are released from the presynaptic (transmitting) cell, they diffuse through the synaptic cleft to bind to receptors on the postsynaptic (receiving) cell membrane. These receptors control ion channels and the binding o f the neurotransmitters to the receptors opens the ion channels for specific ions. Ion transporters may be characterized by several common methods such as the planar bilayer experiment and the pH-stat titration method."*

Figure 1-3 depicts the planar bilayer experimental setup. In this picture, the two wells are essentially two separate vessels except for a small hole (-400 pm) on the smaller vessel. A lipid bi layer is formed on this hole and thus the two wells are insulated from one another. When an external potential is applied between the two wells, there is an ion flux through the membrane via ion channels. This ion flux can be observed by monitoring the

bilayers ch a n n e l Ag/AgCl IM KCl 4nm 4 0 0 nm C u r r e n t/ p A

Figure 1-3 Planar bilayer experimental setup (left) Typical current-time relationship (right)

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shown in Figure 1-3. In this figure, the lowest current step in the current-time plot is called the single-channel conductance which is the reciprocal o f the channel resistance. If the unit o f current is in amperes and the potential is in volts, the unit for conductance is given in Siemens. Multiple “square-tops” are observed channel openings. The planar bilayer experiment can observe the ion channel behavior o f single molecules, so the interpretation is complicated by the statistical distribution o f observations, yet the kinetic information observed is o f single molecule type.

Average or bulk kinetic data can be obtained by a different type o f experiment called the pH-stat titration experiment. The setup for this experiment is shown in Figure 1-4. The theory o f this experiment is that the pH o f the external solution is kept at 7.4 and the pH o f the internal buffer o f the vesicles is 6.6. The experiment records the amount o f base

consumed (or the amount o f protons released) as a function o f time. The pH o f the

A u to m a t ic t i t r a t o r E x t e r n a l so u tto n V ehicle u i t h a c h a n n e l p H e l e c t r o d e ose addition A d d itio n o f t r i t o n I A d d itio n o f tra n s p o rte r

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this pH gradient is the driving force for the release o f protons. Upon addition o f the transporter molecules, protons are released to the external solution, and in order to balance the charge, some cations such as Na*" will flow into the vesicles. As soon as the protons are in the external solution, the pH decreases and the base (titrant) will be added to maintain the pH at 7.4. As the experiment continues, a relationship of the volume o f base added and the time will develop as Figure 1-4 shows. At the end o f this phase o f channel activity, Triton (a detergent that releases all the protons from vesicles) is usually added to confirm the excess o f vesicles. From the plot, one can obtain the kinetic data such as kinetic order and initial proton transport rate. This technique is useful for determining averaged properties o f transporters.

1,2 Examples o f small molecule ion channels from natural sources

As introduced in the previous section, large transport proteins are the most common ion channels in nature; however, this thesis is only dedicated to the synthesis o f small molecule ion channels. This section serves to introduce some examples o f natural small molecule ion channels.

1.2.1 Gramicidin A

The bacterium Bacillus brevis produces a polypeptide called gramicidin. It consists o f 15 alternating l- and d- amino acid residues' (Figure 1-5) and usually exists as a mixture o f

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Ala-D-Val-L-Val-D-Val-L-Trp-D- Leu-L-T rp-D-Leu-L-T rp-o-Leu-L-

Trp-NHCHz-CHzOH

Figure 1-5 Structure o f gramicidin A

gramicidin forms a single ion channel that is a helical end-to-end dimer which spans a bilayer membrane. It is believed that the end-to-end dimer has both its N-termini in the middle o f the bilayer as Figure 1-5 indicates.^ The channel has a internal diameter o f 4 À and the P-helix has a length o f 26 Â^. Because o f its helical shape, the hydrophobic side chains o f the amino acids are on the outside o f the helix which helps the structure to insert into a bilayer membrane. On the other hand, the carbonyl oxygens are pointing towards the inside o f the helix to form a hydrophilic environment which is suitable for ion transport. Gramicidin shows selectivity towards alkali metal cations: Cs > Rb"> >Na > Li **. Although gramicidin A is an efficient ion channel towards monovalent cations, the channel can be blocked by divalent cations such as Ca" or Ba"'**. Unlike large transport protein molecules, gramicidin A is only a 15 amino acid peptide, and is small enough to be synthesized.

1.2.2 Amphotericin B

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hydrophobic

exterior

polar head

group

H HO O H C O jH OH Figure 1-6 Amphotericin and proposed "barre 1-stave” aggregation

structure contains a heptaene hydrocarbon chain on one side and a polyol region on the other (Figure 1-6). A mycosamine group and a carboxyl group at one end o f the molecule generate a zwitterion in neutral aqueous solutions, ft is believed that the active ion channel form o f amphotericin is an aggregate'” " and many involves additional sterols in the structure. The proposed form o f aggregation is called "barrel-stave model” which is shown in Figure 1-6. This hypothesis was based on conductance experiments which showed a high power dependence on amphotericin concentration'" The aggregate pore formed by amphotericin has a hydrophilic interior (polyol side o f the molecule) and a hydrophobic exterior (polyene side o f the molecule). The hydrophilic interior offers the polar environment for ions to pass through and the hydrophobic exterior helps the aggregate to be stabilized in the bilayer. It is believed that the aggregation number o f amphotericin is 8 ~ 12 and the pore size is about 4 À .'”" Other experiments, such as

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Extensive synthetic work has been made towards the synthesis o f amphotericin over the decades’"*''^ and the total synthesis o f amphotericin has been reported.17

1.2.3 Alamethicin

Alamethicin, produced from Trichoderma viride, is known as a transport antibiotic. It consists o f nineteen amino acids and one amino alcohol: Ac-Aib(a-amino-isobutyrate)- Pro-Aib-Ala-Aib-Ala-GIn-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Phe-OH. It is proposed that alamethicin forms membrane spanning aggregates which are also called a “barrel-stave.” The aggregation number o f alamethicin is very flexible; therefore, there is no saturation o f either current or voltage at high ionic concentration. This suggests that the aggregates formed by alamethicin can expand in size in response to membrane conditions. Another important property o f alamethicin is that its transmembrane current is voltage dependent'". As shown in Fig. 1-7, alamethicin is

1200 1000 000 ^ 600 2 0 0 >200 _tso 100 SO •50 •100 Voltage

Figure 1-7 Current-voltage relationship for alamethicin (DiPhvPC; 130pmol; IM KCl, PC/PA/ChoI)

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turned on only when the voltage is above +75 mV and shows no current below that. There have been an increasing number o f studies on the properties o f alamethicin in the

last 5 years. For example, the channels formed by alamethicin are mildly cation selective, but mildly anion-selective analogues have been synthesized by substituting a glutamine for a lysine residue at position 18 .“* Woolley and co-workers reported the synthesis o f an alamethicin dimer and other derivatives in structure-function relationship studies.'’* '*’ Bak and co-workers'" studied the conformation o f alamethicin in phospholipid bilayers by '^N-solid state NMR. Since alamethicin is almost a complete peptide, it can be synthesized with no difficulty by solid phase synthesis techniques; many derivatives have been synthesized for structure-property studies."* '"

The peptide melittin (from honey bee venom) is an example o f a membrane disrupter; it causes lysis o f blood cells by disrupting cell membranes. One characteristic o f membrane disrupters is that the “hole” they create is usually much larger than those created by the dimers or aggregates.

Typical ion transport mechanisms for these small molecule transporters are shown in Figure 1-8 as they apply within a phospholipid bilayer membrane. In this picture, A represents a carrier mechanism in which an ion can be carried across a membrane by a host molecule called a carrier. Carrier molecules usually have a hydrophilic interior to bind to ions and a hydrophobic exterior to enter into the bilayer. Ions can also be transported through a membrane by channel mechanisms. In the B case, a helical dimer such as that formed by gramicidin can transport ions using hydrophilic sites inside the

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B

Figure 1-8 Schematic representation o f ion-transport mechanisms

helix to stabilize the ion in transit. The hydrophobic outside stabilizes the channel within the bilayer membrane. In C, a number o f molecules spanning the bilayer membrane aggregate together to form a pore for ions to pass through. This structure is similar to the "barrel stave” formed by alamethicin and amphotericin. In D the mechanism is a less regulated phenomenon. Molecules insert into bilayers and disrupt the membrane structure: as a result, the membrane leaks and ions can pass through.

1.3 Synthetic ion channels

There are a great number o f synthetic ion channels with diverse structures reported in the literature; however, this introduction will only discuss a selected subset o f them. In order to go hand in hand with the compounds developed in this thesis, the emphasis will be on reported systems that form a family o f active channels or side-by-side aggregates. This thesis is focused on synthesis, so the synthetic challenges o f each system are important. Ion channels play an important role in biological world but their mechanisms are not very

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well understood. Unlike natural ion channels, synthetic ion channels can be simple molecules but still possess the desired functional groups and molecular dimensions for channel activity. Therefore, synthetic ion channels can be used as models to simplify the study o f ion channel phenomena. In addition, synthetic ion channels may find applications in drug delivery and separation. For these possible technologically goals, synthetic ion channels may have advantages over natural ion channels because they will function without the rest o f the cell components required in natural ion channels.

1.3.1 Bouquet ion channels

Lehn"' " reported two o f the first bilayer spanning ion channel models, compounds 1-1

and

1-2.

The molecules are generally referred to as "bouquet” channels because o f the

shape o f the molecules. The bouquets are based on either a tartaric acid crown ether or on P-cyclodextrin. In the crown ether case, the four bundles are linked to the core via amide bonds whereas in the cyclodextrin example

(1-2),

the 14 bundles are linked via ester

I O'^'R NaO" R= \ O N HN—^ o - / Bu 9 tt-cyclodextnn o4—( '—L_ 1 - 1 o \ y NaO^ / 1-2

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bonds. The proposed position for both bouquets in lipid bilayers is that the eight carboxylates (1-2. 14 carboxylates) act as head groups whereas the middle crown ethe core (P-cyclodextrin in 1-2) sits in the center o f the bilayer. As for the transport activity, ^Li and '^Na NM R were used to monitor the ion transfer rate and showed that the

OEt o a

CBz O OH

CBz O _NO,

Bu

Scheme l-l Some steps for synthesizing 1-1

transport rate was very low. The synthesis o f the bouquet 1-1 was straightforward and the overall yield was about 3.8% for 10 steps."" Since the crown ether involved only gave about 1 0% yield from commercially available materials, the overall yield for 1-1 is down

to 0.38%."" The synthetic route for 1-1 is shown in Scheme l - l . It started with alcohol a that was eventually converted to amine b in about 50% yield via OTs and N3. The amine

was then acylated with a carboxylic acid to form an amide c. The CBz protecting group was then removed to recover the free secondary amine which reacts with 5- nitroisophthaloyl chloride to give the aniline moiety by reduction. Compound 1-1 was synthesized by reacting 4 equivalents o f the amine d and the tartaric acid crown ether.

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1.3.2 Ion channels fo rm e d by bis-macrocyclic bolaamphiphiles

Fyles and co-workers have been interested in channels constructed from molecular components. Some early work done by Dr. T. James is shown in Figure 1-9. As the example shows, a core unit connects up to six equivalent walls forming a membrane spanning structure. On top o f each wall unit, there is a head group. In James’ research, a

head 0= ^ s, 0 o CO:R 'O 0 'CO;R R0,(

c:

CO,R core R= > wall V

Figure 1-9 One example o f (head-wall)n-core design

family o f 25 channel molecules was synthesized by using different core, wall, and head units. Experimental results showed that one molecule can form one active channel; no aggregation was required.'"* Later on, a newer design removed the middle crown ether core and used linkers such as shown in compound 1-3. This type o f molecule was mostly synthesized by Dr. M. Zojaji.'^ The fundamental difference o f this design is that the

OH

1-3

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active ion channels are not formed by just one molecule like the previous design; the molecules are supposed to aggregate to form a aqueous “pore” in between the molecules and thus allow ion passage. As opposed to a “channel” formed by a single molecule, molecules like this are called “pore formers”. Dr. X. Zhou'^~’ employed different linkers and head groups in a still later design o f ion channels such as compounds l-4a and l-4b.

- ^ 4

M b X = 0

The main difference o f the linkers is that the hydroxyl groups in 1-3 were removed in 1-4 making it more hydrophobic. This helps to stabilize the molecules in the bilayer membrane. Compound I-4b showed a typical single ion channel opening o f about 4 pA conductance for KCl. pH-stat vesicle experiments suggested that both l-4a and l-4b form aggregates in bilayer membranes. The specific conductance o f I-4a and l-4b is closely similar in vesicles indicating that ether oxygens are not essential for ion transport. Based on l-4b, compound 1-5 was synthesized with differentiated head groups. In 1-5, the head groups are 2-mercaptosuccinic acid and mercaptoacetic acid which have head

group charges o f -2 and -I respectively. Thus 1-5 is a candidate for a voltage-gated channel. Experimental results confirmed that it can show voltage-dependent transport due to an asymmetric distribution as 1-5 penetrates the bilayer membrane. Vesicle experiments showed a high concentration dependence suggesting the formation o f

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aggregates. Compound 1-5 also showed ion selectivity in the sequence Rb >Cs

One o f the possible ways to control the openings o f an ion channel is electrical potential. By applying specific electrical potentials across the membrane, a voltage-gated ion channel can be opened or closed. Therefore, it is o f interest to synthesize ion channels with such a property so the channel activity can be controlled by external means. To possess this property, an ion channel molecule usually requires differentiated head groups to allow an asymmetry when the channel inserts into the membrane.

The syntheses o f compounds l-4a and l-4b and 1-5 were challenging and unbearable.’*’ For example, the macrocycle needed for 1-4 or 1-5 was produced in only 14% or 6.5% yields’*’’** for the corresponding macrocyclization as Scheme 1-2 shows.

o II 1 . 8 -o c ta n c d io L ' " O " CHjSOjH ,0H H O ^ ^ n o r tr i e th y l c n e g ly c o l/ O CHjSOjH / - o 'o ' 6.5%

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Other challenging steps in the synthesis are shown in Scheme 1-3. Mono mesylation reaction o f the diene and the following coupling step both gave yields around 5%.'* Thus the very interesting functionality o f the system cannot be pursued further due to the limitations o f the synthesis.

l-4 a

Scheme 1-3 Partial synthesis o f l-4a

1.3.3 Multiple Jiaza-18-cro\vn-6 design

Gokel and co-workers first synthesized a compound based on diaza-18-crown-6'^ as shown (compound 1-6). The design strategy behind this “hydraphile” was that the three macrocycles are parallel to each other and perpendicular to the lipid axis. This hypothesis would give a “tunnel” conformation for ion passage. However, the experimental results showed the middle macrocycle is actually parallel to the lipid axis but the terminal ones are perpendicular to the lipid axis. Nevertheless, single ion channel experiments showed that the compounds o f the “hydraphile” family possess ion transport activities as much as

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R—N r u u —V / — O O - ^ y — u ü —V > / ^ \ ( > / \ < ) • N N — 4 CH2 # N N ( CH ; r* N N -( J \ “ / ÿ X 1 7 7 j \ — o o —/ N— o o — / O n — f N R N M e, n= 8. 10, 12 14, 16. _.so. etc 1-6 n=12, R=(CH2)i;CH3

40% o f that o f gramicidin. Many derivatives’’”'^" were synthesized by varying n ( 8 to 16)

and R mainly for the investigation o f the dimension required for maximum ion transport or the position o f the chain in bilayer membranes.

Because these derivatives are centrosymmetric, the syntheses o f these compounds are controlled by statistics; in many steps.'” the highest yield can be only 50% in theory. For example, as Scheme 1-4 shows, the alkylation o f one o f the amines in the diaza crown only gave 14% yield followed by another alkylation which gave 6 6% yield. These two

steps alone cut the overall yield down to 9%. The other key synthesis issue is the synthesis o f the diaza-18-crown-6 macrocycle which brings the overall yield to lower than 1%.29 NH HN L . . J +

r

NH

n

N—CH2(CH;),oCH3 ^ 14% B r(C U » ),.0 f H ]C(H ;C),oH 2C— N

r

3 N— CH2(CH2),.Br 6 6 % '—O O—'

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1.3.4 Steroid based transporters

One o f the characteristics o f steroids is that they incorporate into bilayer membranes easily. Regen""* took advantage o f this characteristic o f steroids to design an ion transporter. Compound 1-7 is an example o f the steroid strategy from the early 90 s. The idea for this transporter to work is that the steroid inserts into the bilayer with the result

l Æ HO .

1-7

that the two polyether chains are pulled into the bilayer and act as hydrophilic "hopping sites" for ions.^^ More recent designs o f transporters^'*’^' o f this type are single tail molecules such as compounds 1-8 and 1-9. The experimental results showed that the polyether 1-9 is only active for Na*^ but not for H /OH transport and the polyamine 1-8 is only active for H /O H transport but not for N a \ Regen also increased the number of

1-8 1-9

steroid moieties in the structure such as in compound 1-10 in Figure 1-10. The ion transport activity was studied with ‘^Na^ NMR at 35°C. The experimental results showed that the transport activity towards ‘^a*^ is second-order dependent on the concentration o f 1-10. This indicates that dimers are the active transporter species. However, cation selectivity and step conductance experiments were not reported.

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R= o

OH

MO

OH

Figure 1-10 Tetrasteroid channel system

The proposed structure for the active ion transporter is shown on the right o f Figure 1-10. The four steroid units o f the molecule line up in the lipid direction with the hydrophobic sides o f the steroids (shaded side o f the bar) in contact with the lipid. On the other side, the hydrophilic sides o f the steroids are all pointing toward the inside o f the bundle forming a hydrophilic interior for ions to pass through.

Compound 1-10, a tetra amide, was synthesized simply by coupling four cholic acids and a corresponding tetraamine with the assistance of DCC. The product was simply purified by column chromatography.

Kobuke and co-workers"^"’^ also employed a steroid moiety in their design of ion transporters. For example, compounds 1 -lla -d were synthesized and tested for their

R MeQ iMe MeO* 1 - I l a = R i = R2= C O O H l - l l b = R ,= R2= C H 2 N ^ (C H 3 )3 C 1 1 - I Ic= Ri= H 2 P 0 3 ; R 2 = C O O H l - l l d = R i = C O O H ; R 2 = C H 2 0 H

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transport properties.

The bilayer experiments showed that l - l la and l - l lb have similar ion transport properties. For example, l - l la and l - l lb showed ion selectivity towards cations:

K >Na » L i'^ and the life times o f openings are usually long (10 ms to 10 s). Also, both channels showed rectified current-voltage relationship and single channel conductance measurements gave results in the same range (5-20 pS for l - l la and 5-10 pS for l - l lb). Compounds I H e and l - l Id have differentiated terminals and are candidates for voltage-dependent ion channels. Experimental results showed that different amplitudes of currents were observed when opposite voltages o f same amplitude were applied. This observation suggested that oriented structures were produced, but that the aggregation size varied between experiments. The proposed mechanism for the ion transport activity observed is similar to that for compound I-IO because they all are based on the steroid moiety.

Using cholic acid as the starting material, the overall yields for l - l la and l - l lb are 2.7% and 0.2%^*'^’, respectively. The overall yields o f l - l l c and l - l Id could not be estimated because some steps were not fully reported; however, with all the known steps, the overall yield o f both l - l l c and l - l Id are less than 2.5%^’. Compounds l - l la and l - l lb were synthesized by converting the hydroxy groups in cholic acid to the desired methoxy groups (Scheme 1-5). However, this has to be done selectively because one hydroxyl group must be retained for a further linking reaction. Another key step for the formation o f l - l lb was the transformation from alcohol to ammonium head group which only gave

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2% yield. OMe OH HO' •OMe OMe HO' •OH ÇMe OCN. NCO •OMe OH (CH.I,N'(CllCll,COCI 1 -lla c t r i •OMe

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1.3.5 Channels form ed by ion pairs

Kobuke and co-workers^**'^^ synthesized some ion pairs such as compounds 1-12 and 1-13. The pair o f quaternary ammonium ion and carboxylate ion sometimes showed rectified current-voltage relationship one way and sometimes another. It also showed long-lived openings when inserted into bilayer membranes. The ammonium and phosphate pair also

1-12

1-13

showed a voltage-dependent property. The lipid bi layer experimental results showed that the short-lived single channel openings were only observed occasionally at ±50 mV. However, when the applied voltage across the bi layers is at ±70 mV or higher, the channel openings are frequent and longer-lived.^^ At ±100 mV o r higher, the channel is mostly open and only transient closings are observed. However, there was no cation selectivity for these ion pairs and the proposed mechanism for aggregation was only hypothetical,^'’ no direct evidence was found to support the mechanism. The major effort needed to synthesize the ion pairs was to synthesize the polyether part which gave an overall yield o f 7.6%^'’ from 1,4-butanediol.

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1.3.6 Nanotubes fo rm ed by self-assembly o f rigid rod ionophores

Matile and co-workers'*”"*^ designed tubular ionophores based on rigid rods formed by polyphenyl rings as Figure l- l 1 shows. The R groups on the rod can be short peptides or

.j

5

R= .OCU;CO-Lcu-Lys-NH;

= -OCMXO-Uu-Lys-Lcu-NII.

=

NHi-U-u-Lys-LcuCCXH^O-Figure 1-11 Nanotube formed by rigid rods

other functional groups ^”"*^connecting through ether bonds. The number o f phenyl rings can be varied as well, giving the variation o f length. The idea o f the tubular ionophore is based on the aggregation o f the rigid rods. They aggregate in a way that all the peptides stack on top o f each other and are held together by hydrogen bonding. Depending on the length of the peptides on the phenyl rings, the aggregation numbers can vary from 2 to 6;

as a result, the diameter o f the pore varies. It was reported"*" that when R= NfF-Leu-Lys-Leu-COCHiO-, the pore size is even big enough for carboxyfluorescein passage. In the same research, single ion channel conductance experiments showed long lasting openings (more than 1 min) and high conductance (~180pA).

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The original synthesis was very inefficient, however, after optimizing the syntheses the overall yield was increased up to 4.1%/'* A major step for the synthesis is to couple the biphenyl building blocks. The best yield for the coupling is about 10% as shown in

Scheme 1-6.

4.4.5.5-ietram cthy 1 J S t

dio.xaborulane. PdCU

Scheme 1-6 Coupling o f phenyl rings for rigid rod synthesis

1.4 Goal o f the thesis

It was the goal o f this research to design a synthetic strategy to synthesize voltage-gated ion channels with the least possible synthetic effort. At the beginning o f this research, we had learned that for a channel to be voltage-gated, it must have differentiated ends. Figure

1 - 1 2 shows the general synthetic strategy we adopted to synthesize voltage-gated ion

channels such as C. The functional groups X and Y represent alcohols, amines, and carboxylic acids etc. Once connected via the linking reactions, they will be amide or ester functional groups. The rectangles could be taken as either macrocyclic compounds, or as discussed below, non-macrocyclic units. The previous voltage-gated ion channels such as compounds in the 1-4 and 1-5 series were synthesized from a symmetric macrocyclic

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Precursors

X -

-X

X -

-Y

X -

-Y

X -

-X

Figure 1-12 Schematic representation o f the synthetic strategy followed

compound A to C via a dissymmetric compound B. Historically, the centrosymmetric compound A was designed for the synthesis o f the centrosymmetric dimer D and linking them in this system involves ester formation. Working from A to C requires conversion to B Unfortunately, this conversion causes a major loss o f overall synthetic efficiency; therefore, synthesizing an end-differentiated compound B directly from precursors is desired.

Cameron’**’ synthesized a cyclophane 1-14 that might be a suitable B (where X and Y in Figure 1-12 are protected amines) but never used this cyclophane as a part o f a channel. Therefore, the first goal o f this thesis was to investigate the suitability o f 1-14 as a part o f a channel compound. Although the general goal o f this thesis is to synthesize some

C

from some B, it is simpler in the case o f the available 1-14 to form a centrosymmetric dim er D via an amide connection. This will provide a quick answer about the suitability

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o f I-I4 as a precursor to voltage-gated channels. Although showing channel activity, the insolubility o f the corresponding D based on 1-14 makes it impossible to work with. As a result, the synthesis o f C based on 1-14 was not attempted.

1-14

Another weak point o f previous channel syntheses is the macrocyclization reactions to produce A or B. Although it was believed that macrocycles are essential for the functioning o f the aggregate type o f ion channels, this believed requirement had not been previously assessed. Therefore, we investigated the possibility to synthesize ion channels without macrocyclic units. By doing this, the rectangles in Figure 1-12 become some non-macrocyclic units such as a hydrocarbon chain (CH:)s or a polyether (CH:CH:0)n and the linkers remain as esters or amides. As will be discussed in Chapter 3, a non- macrocyclic B related to macrocyclic l-4a was synthesized to test if macrocyclic units are necessary for an active channel. As explained before, the symmetric dimer D is simpler to synthesize and therefore, a non-macrocyclic version o f it was prepared. This compound showed channel activity, meaning the macrocyclic units are not necessary. A series o f compounds with ester-amide, oligoamide and oligoester functionalities were synthesized and tested. Among the compounds tested, oligoesters show explicit channel activity and are pursued further for the possibility o f forming the acyclic version o f end- differentiated C (Figure 1-12).

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technique'*^ is used to prepare C from acyclic precursors B (X= OH, Y= COOH). We have synthesized several compounds with a C structure, and in the preliminary studies of the channel activity, they show ion channel activity and thus vindicate the success o f the new strategy. The principal goal o f a simple synthesis o f C-type channels has thus been achieved.

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Chapter 2 Design, synthesis, and properties of biscyclophane

bolaamphiphiles

2. /

Introduction

As discussed in the previous chapter, many examples o f unimolecular and aggregate types o f channel molecules have macrocyclic wall units. It was claimed that the macrocyclic wall units are essential to the pores formed;'^''*’ the more rigid the walls are, the better the channels will form. However, there are many disadvantages to include macrocycles in a potential ion channel molecule. For example, macrocyclization reactions are inherently low yielding reactions and a number o f isomers are produced by the reported syntheses. This chapter will address the issue o f isomer mixtures.

Scheme 2-1 shows a partial synthetic route to synthesize compound l-4b.'^ With the macrocycle 2-1, on one hand, the Michael addition was performed to the alkenes with mercaptoacetic acid and tetramethylpiperdine to yield the diacid 2-2; on the other hand.

Michael addition was performed selectively on one o f the two alkenes with mercaptoacetic acid to yield the alcohol 2-3 in low yield. Compound 2-3 was then converted to a mesylate 2-4 by reacting with mesyl chloride and triethylamine. The mesylated macrocycle 2-4 and the diacid 2-2 were then linked to form alkene acid 2-5 via the carboxylate alkylation reaction. This step also had a low yield since it too involves only one end o f symmetric structures. The last mercaptoacetic acid was added to the alkene via another Michael addition to afford l-4b. Both stereo and regio isomers arose in the synthesis o f l-4b. For example, there were 10 possible isomers formed in the

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R et1u.2hn Et.N CH,SO.CI CH.ci.irc : hnw 3‘r VIc,Nf)H DMSO w r t : Z5 hr> I2* -rS O H O H _Mrc l-4b

Scheme 2-1 Partial synthesis o f compound l-4b

formation o f 2-2, 4 for 2-3, 40 for 2-5, and eventually 160 for l-4b. Obviously, purifying any one isomer is not realistic. Fortunately, the isomer mixture showed channel activity and the isomers were apparently similarly active.

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2-6 X-Y=H 2-7 X-NO,. Y-CO.H

2-8 X' Y=H \ °

Y‘ NO,

processes leading to several macrocycles with differentiated ends. She found that macrocycles 2 - 6 and 2 - 8 are potentially suitable for ion channel molecules since they are

produced in reasonable yields. However, attempts to functionalize 2-6 differentially to make 2-7 were not successful. On the other hand, the attempt to differentially

OH DUPTHF rcrtitv 8 hr& 48* « 0 'o 2-9 Ei.NTHF rclliLX. 2 hrv 82“ n 2-10 _2-11 \ 9 ? I CH.CU rl' 3 Javs 8»":. 2-12 HCL McOH rf 1 hr, 70% OH NH- •a N O ; ■Cl 2-13

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functionalize 2-8 to 1-14 was successful. The synthesis o f macrocycle 1-14 is shown in Scheme 2-2. Starting with 1,8-octanediol, DHP was used to selectively protect one

alcohol functional group and gave compound 2-9. Compound 2-9 was then acylated with 5-nitro-iosphthaloyl dichloride to afford 2-10. The nitro group in 2-10 was reduced to the amine followed by protection with t-Boc and removal o f the THP protecting groups to give the diol 2-13. Macrocyclization o f 2-13 with 5-nitroisophthaloyl dichloride yielded macrocycle 1-14.

Recall the discussion at the end o f Chapter I: the goal o f the research based on the macrocycle 1-14 is to eventually synthesize a potential voltage-gated ion channel. However, it is necessary to know if the new macrocycle 1-14 is an appropriate wall unit for ion channels. Therefore, synthesizing and testing a centrosymmetric biscyclophane compound based on macrocycle 1-14 was carried out. As shown in Scheme 2-3, the macrocycle 1-14 could be converted to an amine that would react with diacyl chlorides to

1-14

P N

R - alkvl o r aryl

P is an amine protecting group

^

I y

D cprotcct P

,0—(

Y V

NH.

■0—\

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generate a centrosymmetric diamide for evaluation o f channel activity.

2.1 Synthesis

To convert 1-14 to a corresponding amine, there are two reasonable routes as shown in Scheme 2-4. One route is to remove the Boc protecting groups to yield compound 2-14 and the other route is to reduce the nitro in 1-14 to amine 2-15. Deprotecting Boc was chosen first and 2-14 synthesized by heating 1-14 in CH2CI2 with TFA at reflux overnight

as shown in Scheme 2-4. o=< ■NO; NH-1-14 NO; NH. NH ■NH, 2-14 2-15

Scheme 2-4 Amines derived from 1-14

The identification o f compound 2-14 was made by ’H NMR, ’■’C NMR and MS. The mass spectrum shows the molecular ion at m/z= 613.2 indicating the removal o f the Boc protecting group. In the 'H NMR spectrum (Figure 2-1), the two triplets at 4.2 and 4.4 ppm respectively are assigned to the methylene protons labeled a and b, which indicate

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2-14

r

f+s

/ j

JL

X

ppm

r

*

r

1

_ L

K J

V U U ;

— I—1—I I “ 1—I—I—r—f-'T—|“ T

5 . 0 4 . 0 3 . 0 PPM T I—|— 2 . 0 t I I - | 1 . 0

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the macrocycle is intact. A small peak at 5.6 ppm is assigned to the amine protons c. Peaks at 7.2 and 7.6 ppm are assigned to the d and e protons on the aniline ring. The multiplet at 8.7 ppm is the result o f the protons o f f and g on the nitrobenzene. The '"C NMR spectrum also provided evidence to support the structure. In Figure 2-2, the methylene carbons a and b adjacent to the esters are at 64 and 65.5 ppm respectively. The peaks at 116 and 118 ppm are assigned to the carbons labeled as c and d on the aniline ring. The peak at around 127 ppm is assigned to the nitro benzene ring carbon which is labeled as e. The assignment o f the next two peaks at 131 and 132 ppm is ambiguous; however, they are the f or g carbons on both rings. The peak at 134 ppm is a result o f the carbon labeled as h in nitro benzene ring. The two carbonyl carbons are at 163 and 165 ppm; the former should be assigned to the nitro benzene k and the latter should be assigned to the aniline I. The peaks at ~ 146 ppm labeled as i and j are the quartemary carbons directly connecting to both N atoms. Although the intensities are not high enough due to the solubility, these peaks can be reconfirmed from the spectra o f the precursors. Combining all the results obtained, it is clear that compound 2-14 can be made.

To link 2-14 to form the dim er indicated in Scheme 2-3, the first choice o f linker was glutaryl dichloride as shown in Scheme 2-5. Instead o f producing the desired 2-16, the cyclized product 2-17 is produced predominately. The strongest evidence for this cyclization is provided by the MS o f the product which shows the molecular ion o f 2-17 at m/z= 679.2 indicating the formation o f cyclized product 2-17. The 'H NMR spectrum o f compound 2-17 (Figure 2-3) shows a triplet labeled as a at 2.9 ppm. This is the peak which results from the a protons labeled as a. Also, the a peak has an integration o f half

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ppm

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2-17 b+c

H I

2 . 0 3 . 0 5 . 0 6 . 0

Figure 2-3 'H NMR spcclrum o f

2-17

in CDCI3

7 0 8 0 9 . 0 ppm u> 0 \

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HjN- NO; 2-14 Et.N/ T H F rellux/ 8 hr NO, 2-16 0% o o 2-17 70%

Scheme 2-5 Dimerization reaction o f 2-14 with glutaroyl dichloride

as much as all the methylene protons adjacent to the labeled b and c, indicating the formation o f 2-17 instead o f 2-16. The peak at -7 .9 ppm is assigned to the proton d and the peak at 8 . 6 ppm is assigned to proton e. On the nitro aromatic ring, the peak at 8 . 8

ppm is assigned to the f proton and the peak at 9.0 ppm is assigned to the protons labeled

8

Another linker, fumaryl dichloride, was chosen to avoid the intramolecular cyclization. The synthesis is illustrated in Scheme 2-6 and, surprisingly, the linker cyclized again to produce 2-19 instead o f 2-18. The MS shows molecular ion at m/z= 692.2, which is consistent with 2-19 rather than the expected product. The ‘H NMR spectrum o f 2-19 (Figure 2-4) shows two sets o f triplets assigned to the protons labeled a and b at 4.4 ppm. The peak at 6.9 ppm is a characteristic peak assigned to the alkene protons c in the imide.

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2-19

a+b

K A À I

U

1—r-T—T^—J— I— I— I— I—p-*T— I— I— I—I— I— r

9, ' o 8 . 0 7 . 0 6 . 0 ' 5 0 I I ' ' ' 4 . 0I P P M T—r T— I—I—I—r 3 . 0 T— r _I _r 2 0 ' T ' 1 o u> 00 ppm Figure 2-4 H NMR spectrum o f

2-19

in CD2CI2

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The integration o f this peak is about the same as peak d, the two protons on the benzene bearing the imide. The e proton is at ~8 . 6 ppm, which integrates about h alf as much as d

and c. O NO,

b 2-14

El,N THF/ rctluV S hrs •NO, NH •NH ,0 O, •NO,

2-19

70% Scheme 2-6 Dimerization o f

1-14

with fumaroyl dichloride

A third linker, terephthaloyl dichloride, was then chosen for its rigidity as shown in Scheme 2-7. As expected, the dimer

2-20

could be prepared but shows extremely low solubility. The success o f the formation o f

2-20

in Scheme 2-7 can be confirmed by the MS which shows the molecular ion peak at m/z= 1354.4 which is consistent with the structure o f the dimer,

2-20.

In the 'H NMR spectrum o f

2-20

(Figure 2-5), the methylene protons (a and b) adjacent to the ester are at 4.4 ppm as usual showing two sets o f triplets. The nitro benzene ring protons c and d are at 8.1 and 8.3 ppm. The integration (a+b)/(c+d) is 8:3 as expected for a dimer. The 4 equivalent protons in the terephthaloyl ring are assigned to the doublet at 8 . 6 ppm labeled e and the integration o f

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NO-HN 2-20 a+b B.t l ~ » ~ » 1 » I » I T' f ~ r ~ f ' T T Tf • ' * ' 1 I - ■ ! » * * ■ * ■ > * “ ■ I * • • * à 7 6 5 4 Figure 2-5 ' U NMR spcclrum o f 2-20 in D M S0-r4 at 150°C 1 0 ppm ê

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( J - N O , ’° ” o

2-14

Cl Cl E i,S THF/ rellux. 8 hrs.' ‘>0% D M .V l l 'C 3 d a y s / H ;. 35 p si/P lO j 2-21

Scheme 2-7 Dimerization reaction o f

2-14

with

terenhthalnvl ch lorid e and atfcm nted reduction

integration and the inset show; they are all clustered in the peaks assigned as f+g. This change in chemical shift is consistent with the amide formation. The '""C NMR spectrum

o f

2-20

(Figure 2-6) shows the carbons adjacent to the ester groups are at 64 and 65 ppm

assigned to a and b respectively. Peaks at 124 and 125 ppm are assigned to the c and d carbons on the amide ring. The peak at 126 ppm is unique because it indicates the 4 carbons in the terephthaloyl unit which are labeled as e. The peak at 127 ppm is observed at the precursor stage as the carbon f on the nitro benzene ring and it is not shifted. The other peaks carried over from the precursor are also observed here and are assigned to g or h, i, and I. The peak at 137 ppm is assigned to the carbon j in the terephthaloyl unit and carbon k is assigned to the carbon connecting to the amide N. As expected, for the structure in

2-20,

there are 3 carbonyl carbon peaks at 162, 164, and 164.5 ppm and they

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■NO; HN 2-20 g o r h gorh . 160 140 120 100 80 60 40 20 ppm Figure 2-6 'l l NMR spectrum o f 2-20 in DMS0-<4 at 150°C

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are assigned to

m, n,

and

o

carbons, respectively.

The last step to produce a channel compound is to reduce the nitro on 2-20 to amine 2-21. However, due to 2-20’s insolubility, the reaction did not go as expected. Many attempts were made to reduce 2 - 2 0 but no conditions were found to reduce the nitro without

fragmentation o f the esters. Typically, no reduction is observed even under prolonged exposure to hydrogen under pressure.

It was suspected that the terephthaloyl linker contributed the most to the extreme insolubility o f 2-2 0, therefore, another linker, pimeloyl dichloride, was used to replace

terephthaloyl linker as Scheme 2-8 shows. The product, 2-22, was synthesized as expected but shows extreme insolubility as well; as a result, the further hydrogenation reaction o f this compound was not attempted. In identifying the formation o f 2-2 2. the ’H

NMR spectrum (Figure 2-7) shows a characteristic triplet peak at 2.3 ppm assigned to the a protons in the linker. The two sets o f triplet peaks at 4.2 and 4.3 ppm are assigned to

■NOj HjN-2-14 T U F E t.N rctlu.x. 8 hrs NO. 2-22

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2-22 ; im f+g Jm

I

b+c - I 1 r - _{T "} 9 T " 8 “T6 “T9 I I 1 I 1--- '--- ' I ppm 4 3

(66)

ni g h i m m m m M — T - 129 m m * Il > ppni 160 140 120 100 60 60 40

(67)

2-15 b+c

/

J j

/ . /

( I t v I I \ OH Û ÜÛ I i\2 I I I I I I I I 64/ I > > > > ; > P l l l f F M T 7 > > > M F » >> 7 7 T ; M l I | l T H | T T T l | TT H | 1 1 1 1 1 n 1 I ^ > n 1 | I I 11 I I H l | I TT T p I ' M | M r » ! I M f | M I I I H T » | H f T | f 11 i p I I I 11 I 1 1 | I M 1 1 I I I T T ? I I I T T T f I f T r T l f M I I | > T H TTT I r i T l I M T~ so 8 5 80 7 5 70 65 60 55 50 45 40 35 30 25 20 1 5 1 0 05 00 ppm Figure 2-9 'H NMR spectrum o f

2-1

5 in THF-r/,»