Photolabeling of the Human Dopamine Transporter and the Reactivity of Aryl Azides with Amino Acid Analogs Open Access

Holmes Morris, Muhsinah Lateefah (2008)

Permanent URL: https://etd.library.emory.edu/concern/etds/qr46r101r?locale=en
Published

Abstract

This work explored the site of photolabeling of the human dopamine transporter
(hDAT) by the photoaffinity agent [125I]-DEEP, an aryl azide photoaffinity label, and also
the selectivity of aryl azides using phenyl azide and amino acid analogs. The site of
incorporation of [125I]-DEEP had been shown to incorporate within transmembrane
domains 1 and 2 of hDAT by Vaughan and Kuhar (1996). To further isolate the site of
incorporation, [125I]-DEEP was photolyzed with hDAT membranes, isolated by SDS-
PAGE, and digested with trypsin and cyanogen bromide (CNBr). The peptide,
N93GGAFLVPYLLFM106, which is located at the top portion of TMD2, was identified as
the possible site of [125I]-DEEP incorporation.

[125I]-DEEP contains a phenyl azide as the photoactive agent. In a followup study,
the photolabeling selectivity of phenyl azide with amino acids was examined using
analogs of amino acid side chains. Phenyl azide (100 μM) and amino acid analogs (1 M)
were irradiated at 254 nm for 4 minutes in polar and nonpolar conditions and the kinetics
of the reactions determined. 1-Octanethiol, butylamine, guanidine acetic acid, dimethyl
sulfide, ethylbenzene, butyric acid, imidazole, and phenol were used to model the side-
chains of cysteine, lysine, arginine, methionine, phenylalanine, glutamic acid, histidine,
and tyrosine, respectively. N-ethylacetamide was used as a model for the peptide
backbone.

The order of phenyl azide reactivity with analogs studied in polar conditions was
guanidine acetic acid ≈ imidazole > phenol. The order of phenyl azide reactivity with
analogs studied in nonpolar conditions was dimethyl sulfide > butyric acid ≈ butylamine >/=
N-ethylacetamide ≈ 1-octanethiol ≈ ethylbenzene. Product stability in basic and acidic
proteolytic conditions was also tested. Trypsin digestions were performed in 100mM
ammonium bicarbonate at pH 8.9, while CNBr cleavages were performed in 70%
trifluoroacetic acid at pH 1. Histidine, tyrosine and cysteine analogs were the most
stable, while all of the other analogs were not as stable, and decomposed by greater than
50% within 24 hours. Product stability in proteolytic conditions has implications for
photoaffinity labeling of proteins with aryl azides in which proteolysis is used for peptide
mapping.

Table of Contents

Table of Contents

Page

Chapter One

Cocaine Addiction and the Human Dopamine Transporter 1

Cocaine Addiction 2

Available Treatments for Addiction 5

Cocaine as an Inhibitor of the Dopamine Transporter 6

Other Inhibitors to the Dopamine Transporter 10

Structure and Function of the Dopamine Transporter 13

Characterization of Membrane Bound Proteins 18

Photoaffinity Labeling as a Method to Identify the Cocaine

Binding Site of the Human Dopamine Transporter 25

Photoaffinity Labeling of DAT 31

Photochemistry of Aromatic Nitrenes 36

Project Overview and Objectives 39

Chapter Two

Photoaffinity Labeling of the Human Dopamine Transporter

with [125I]-DEEP51

Introduction52

Methods56

Cell Culture and Photoaffinity Labeling 57

Cell Culture 57

Membrane Preparation 57

Photoaffinity Labeling 58

Purification and Isolation of Radiolabeled hDAT 61

Immobilized Metal Affinity Chromatography 61

Gel Electrophoresis 61

Autoradiography 62

Western Blotting 62

Digestion of Radiolabeled hDAT 64

Enzymatic Digestions of Labeled hDAT 64

Chemical Cleavage 64

Thin Layer Chromatography 65

HPLC 66

SDS-PAGE of Extracted hDAT Peptides 67

Solid-State Photolabeling of hDAT with Radioligands 67

General Protein/Mass Analysis for Windows (GPMAW) 67

Results70

Introduction to the Results 71

Suitability of Photoaffinity Labeling Experimental Conditions 74

Investigations into Localizing the Cocaine Binging Site with the

GBR Analog, [125I]-DEEP 78

Solid-State Photoreactions of [125I]-DEEP and Amino Acids 89

Effect of Photoaffinity Labeling Conditions on [125I]-DEEP 92

Preliminary Investigations of [125I]-JJC 3-24, a Rimcazole

Photolabel of DAT 96

Discussion103

Introduction to the Discussion 104

Investigation of Photoincorporation Site of [125I]-DEEP on hDAT 105

Suitability of Photoaffinity Labeling Experiment Conditions 105

WIN 35, 428 Protection Experiment 106

Analysis of [125I]-DEEP Labeled Peptides Resulting from

Proteolytic Digestions 107

Primary CNBr Digestion followed by Secondary

Trypsin Digestion 107

Primary Trypsin Digestion followed by Secondary

CNBr Cleavage: Gel and TLC Analysis 112

HPLC of [125I]-DEEP Labeled hDAT 112

Solid-state Photolabeling of TM2 Amino Acids 113

Effect of Photoaffinity Labeling Conditions on [125I]-DEEP 114

Analysis of Photolabeling of hDAT with [125I]-JJC 3-24 117

Future Studies 118

Chapter Three

Photoreactions of Phenyl Azide with Amino Acid Analogs120

Introduction121

Methods124

Photoreactions of Phenyl Azide/Para-azidoacetophenone

with Amino Acid Analogs125

General Notes 125

Synthesis of Phenyl Azide 125

Purification of Phenyl Azide 126

Analysis of the Irradiation of Phenyl Azide

with Amino Acid Analogs127

Sample Preparation 127

High Pressure Liquid Chromatography Analysis 127

Irradiation of Phenyl Azide and Amino Acid Analogs

for HPLC and Mass Spectral Analysis 129

Irradiation of para-azidoacetophenone (PAAP) and

Amino Acid Analogs for HPLC and Mass Spectral Analysis 129

Irradiation of Phenyl Azide and Amino Acid Analogs

for 1H Nuclear Magnetic Resonance (NMR) 130

Collection of the Product of the Reaction between

Phenyl Azide and the Amino Acid Analogs 130

High Resolution Mass Spectrometry Analysis 130

Stability of Products 130

Stability of the Products of Phenyl Azide and

the Amino Acid Analogs at pH 8.9 in

Ammonium Bicarbonate 130

Stability of the Products of Phenyl Azide and

the Amino Acid Analogs at 25oC in

Trifluoroacetic Acid (TFA), pH 1 131

Data Analysis 1 31

Results136

Introduction to the Results137

Photoreactions with Phenyl Azide and Butylamine as a Model for Lysine139

Phenyl Azide Photoreactivity with Butylamine as a Model for Lysine 139

Phenyl Azide Photoreactivity with Butylamine as a Model for Lysine

under NEAT conditions 148

The Identification of N-butyl-2H-azepin-7-amine by Electrospray

Ionization (ESI) Mass Spectrometry (MS) and Proton Nuclear

Magnetic Resonance (1H NMR) 154

Product Stability of Butylazepine under Proteolytic Conditions 159

Photoreactions with Phenyl Azide and 1-Octanethiol as a Model for Cysteine162

Phenyl Azide Photoreactivity with 1-Octanethiol as a Model for Cysteine 162

Phenyl Azide Photoreactivity with 1-Octanethiol as a Model for Cysteine

in NEAT Conditions 171

The Identification of 7-(octylthio)-2H-azepine by Atmospheric Pressure

Chemical Ionization (APCI) Mass Spectrometry (MS) 177

Product Stability of Octylthioazepine under Proteolytic Conditions 181

Single Exponential First-Order Rate Constants for Phenyl Azide

Irradiated with Other Amino Acid Analogs 184

Dimerization of Phenyl Azide195

Double Exponential Rate Constants Applied to the Photoreactions of Phenyl

Azide with Amino Acid Analogs203

Identification of Products by Mass Spectrometry for Aryl Azides Irradiated

with Amino Acid Analogs213

Products from the Irradiation of Phenyl Azide with Amino Acid Analogs218

Stability of Products in Proteolytic Conditions223

Discussion226

Introduction to the Discussion227

Reactivity of Phenyl Azide with Amino Acid Analogs

Interpretation of Results for Photoreactions of Phenyl Azide

with Amino Acid Analogs, Butylamine and 1-Octanethiol 229

Butylamine as a Model for Lysine 229

1-Octanethiol as a Model for Cysteine 230

Interpretation of Results for Photoreactions of Phenyl Azide with Amino

Acid Analogs in Nonpolar, Aqueous, and NEAT Conditions 233

Nonpolar Conditions 233

Aqueous Conditions 234

NEAT Conditions 234

Phenyl Azide Irradiated in Nonpolar and Aqueous Conditons 238

Interpretation of Phenyl Azide Reactivity with Amino Acid

Analogs 240

Discussion of the Product Formation of Azepines from the Irradiation

of Aryl Azides and Amino Acid Analogs245

Identification of Butylazepine

Analysis of Azepine Products from Phenyl Azide and Amino Acid

Analogs using Mass Spectrometry 247

Absorbance of Products from Photoreactions of Phenyl Azide and Amino

Acid Analogs248

Azepine Product Stability in Proteolytic Conditions252

Stability of Product in Basic Conditions 252

Stability of Product in Acidic Conditions 253

Correlation of Rate Constants vs. Product Stability in Proteolytic

Conditions 258

So What Gets Labeled? 260

References262

Appendices299


List of Figures

Chapter One

Cocaine Addiction and the Human Dopamine Transporter1

Figure 1-1. Forms of Cocaine 3

Figure 1-2. Dopaminergic Neurotransmission 8

Figure 1-3. DAT and VMAT Topology 9

Figure 1-4. DAT Inhibitors 12

Figure 1-5. 2-D Topology of the Human Dopamine Transporter (hDAT) 16

Figure 1-6. Proposed Mechanism for the Glutamate Transporter 20

Figure 1-7. The LeuTAa Topology 23

Figure 1-8. Structure of the LeuT-desipramine Complex 24

Figure 1-9. Steps for Photoaffinity Labeling 27

Figure 1-10. Typical Compounds used as Photoaffinity Labels 28

Figure 1-11. Photolysis of Photophores 29

Figure 1-12. GBR Analogs 34

Figure 1-13. Regions of Incorporation of PALs on hDAT 35

Figure 1-14. Pathways for Photolysis of Phenyl Azide 38

Figure 1-15. Structures of Cocaine and DAT Photolabels 41

Figure 1-16. Analogs of DAT Photolabels 42

Figure 1-17. Intermediates of Singlet and Triplet Nitrenes 49

Chapter Two

Photoaffinity Labeling of the Human Dopamine Transporter

with [125I]-DEEP 51

Figure 2-1. Structures of Cocaine and hDAT Inibitors 53

Figure 2-2. [125I]-DEEP as an Example of a DAT Photolabel 54

Figure 2-3. Western Blot of Purified DAT 75

Figure 2-4. [125I]-RTI-82 Labels Near the Cocaine Binding Site 76

Figure 2-5. Time-Dependent Irradiations with [125I]-RTI-82 77

Figure 2-6. [125I]-DEEP Labels Near the Cocaine Binding Site 80

Figure 2-7. Primary Proteolytic Digestions of [125I]-DEEP Labeled hDAT 81

Figure 2-8. HPLC of Primary Tryptic Digestion of [125I]-DEEP hDAT 82

Figure 2-9. TLC of Secondary Digestions of [125I]-DEEP hDAT 83

Figure 2-10. HPLC Analysis of Secondary CNBr Digestion of Tryptic Peptide,

T1 84

Figure 2-11. HPLC Analysis of Secondary CNBr Digestion of Tryptic Peptide,

T2 85

Figure 2-12. Primary Digestion of [125I]-DEEP Labeled hDAT with Trypsin

TPCK 87

Figure 2-13. HPLC Radioactive Trace of [125I]-DEEP with Methionine 89

Figure 2-14. HPLC Radioactive Trace of [125I]-DEEP with Leucine 90

Figure 2-15. HPLC Radioactive Trace of [125I]-DEEP with Tyrosine 91

Figure 2-16. Effect of Irradiation on [125I]-DEEP 92

Figure 2-17. Effect of Tryptic Conditions on [125I]-DEEP Labeled hDAT 93

Figure 2-18. Effect of CNBr/TFA Solution on the Photoaffinity Label,

[125I]-DEEP 94

Figure 2-19. TFA causes Decomposition of [125I]-DEEP Photolabel 95

Figure 2-20. High Concentration of [125I]-JJC 3-24 Labels hDAT 97

Figure 2-21. SDS-PAGE of Primary Tryptic Digestion of hDAT Labeled with

[125I]-DEEP and [125I]-JJC 3-24 98

Figure 2-22. Primary CNBr Cleavage of [125I]-JJC 3-24 Labeled hDAT 99

Figure 2-23. [125I]-JJC 3-24 Labels hDAT Peptide, PLFYM 100

Figure 2-24. Effect of Irradiation on [125I]-JJC 3-24 101

Figure 2-25. Effect of CNBr Conditions on [125I]-JJC 3-24 102

Figure 2-26. CNBr Cleavage Sites of hDAT 109

Figure 2-27. Primary CNBr and Secondary Tryptic Digestion Cleavage Sites 110

Figure 2-28. Primary Tryptic and Secondary CNBr Digestion Cleavage Sites 111

Figure 2-29. Effect of [125I]-DEEP on the Retention Time of Amino Acids 116

Chapter Three

Photoreactions of Phenyl Azide with Amino Acid Analogs120

Figure 3-1. Phenyl Azide and Butylamine in Cyclohexane Before Irradiation 140

Figure 3-2. Phenyl Azide and Butylamine in Cyclohexane After Irradiation 141

Figure 3-3. Phenyl Azide and Butylamine in Cyclohexane Before and After

Irradiation 142

Figure 3-4. Phenyl Azide and Butylamine in Cyclohexane Irradiated over Time 143

Figure 3-5. Single Exponential First-Order Decay Curve Fit for Phenyl Azide

and Butylamine in Cyclohexane 144

Figure 3-6. Double Exponential First-Order Decay Curve Fit for

Phenyl Azide and Butylamine in Cyclohexane 145

Figure 3-7. Phenyl Azide and Butylamine in Cyclohexane Product Formation 146

Figure 3-8. Phenyl Azide and Butylamine (NEAT) Before and After Irradiation 149

Figure 3-9. Phenyl Azide and Butylamine (NEAT) Irradiated over Time 150

Figure 3-10. Single First Order Exponential Decay Curve Fit for Phenyl Azide

and Butylamine (NEAT) 151

Figure 3-11. Phenyl Azide and Butylamine (NEAT) Product Formation 152

Figure 3-12. Mass Spectrometry of the Butylazepine Product 156

Figure 3-13. Experimental Proton NMR of N-butyl-3H-azepin-2-amine. 157

Figure 3-14. Theoretical Proton NMR of N-butyl-3H-azepin-2-amine 158

Figure 3-15. Stability of Butylazepine Product in Basic Conditions 160

Figure 3-16. Stability of Butylazepine in Acidic Conditions 161

Figure 3-17. Phenyl Azide and 1-Octanethiol in Cyclohexane Before Irradiation 163

Figure 3-18. Phenyl Azide and 1-Octanethiol in Cyclohexane After Irradiation 164

Figure 3-19. Phenyl Azide and 1-Octanethiol in Cyclohexane Before and After Irradiation 165

Figure 3-20. Phenyl Azide and 1-Octanethiol in Cyclohexane Irradiated

over Twenty Minutes 166

Figure 3-21. Single Exponential First-Order Decay Curve Fit for Phenyl Azide

and 1-Octanethiol in Cyclohexane 167

Figure 3-22. Double Exponential Decay Curve Fit for Phenyl Azide and

1-Octanethiol in Cyclohexane 169

Figure 3-23. Product Formation for Phenyl Azide and 1-Octanethiol

in Cyclohexane 170

Figure 3-24. Phenyl Azide and 1-Octanethiol (NEAT) Before and After

Irradiation 172

Figure 3-25. Phenyl Azide and 1-Octanethiol (NEAT) Irradiated over

Sixty Minutes 173

Figure 3-26. Phenyl Azide and 1-Octanethiol (NEAT) First-Order

Exponential Decay Curve Fit 174

Figure 3-27. Rate of Product Formation for Phenyl Azide and

1-Octanethiol (NEAT) 176

Figure 3-28. Mass Spectrometry of the Octylthioazepine Product 178

Figure 3-29. Stability of Octylthioazepine in Basic Conditions 182

Figure 3-30. Stability of Octylthioazepine in Acidic Conditions 183

Figure 3-31. Single Exponential First-Order Curve Fit for Phenyl Azide

and Imidazole 185

Figure 3-32. Single Exponential First-Order Curve Fit for Phenyl Azide

and Guanidine Acetic Acid 186

Figure 3-33. Single Exponential First-Order Curve Fit for Phenyl Azide

and Phenol 187

Figure 3-34. Single Exponential First-Order Curve Fit for Phenyl Azide

and N-ethylacetamide 188

Figure 3-35. Single Exponential First-Order Curve Fit for Phenyl Azide

and N-ethylacetamide (NEAT) 189

Figure 3-36. Single Exponential First-Order Curve Fit for Phenyl Azide

and Ethylbenzene 190

Figure 3-37. Single Exponential First-Order Curve Fit for Phenyl Azide

and Butyric Acid 191

Figure 3-38. Single Exponential First-Order Curve Fit for Phenyl Azide

and Dimethyl Sulfide First-Order Curve Fit 192

Figure 3-39. Loss of Phenyl Azide in Aqueous Conditions for 4 Minutes

of Irradiation 197

Figure 3-40. Dimerization of Phenyl Azide in Aqueous Conditions 198

Figure 3-41. Loss of Phenyl Azide in Cyclohexane for 4 Minutes of Irradiation 199

Figure 3-42. Dimerization of Phenyl Azide in Cyclohexane 200

Figure 3-43. Double Exponential First-Order Curve Fit for Phenyl Azide

and Guanidine Acetic Acid 204

Figure 3-44. Double Exponential First-Order Curve Fit for Phenyl Azide

and Imidazole 205

Figure 3-45. Double Exponential First-Order Curve Fit for Phenyl Azide

and Phenol 206

Figure 3-46. Double Exponential First-Order Curve Fit for Phenyl Azide

and Dimethyl Sulfide 208

Figure 3-47. Double Exponential First-Order Curve Fit for Phenyl Azide

and Butyric Acid 209

Figure 3-48. Double Exponential First-Order Curve Fit for Phenyl Azide

and N-ethylacetamide 210

Figure 3-49. Double Exponential First-Order Curve Fit for Phenyl Azide

and Ethylbenzene 211

Figure 3-50. Product Peak Heights for Cysteine, Lysine, and Peptide

Backbone Analogs 219

Figure 3-51. Product Peak Heights in Nonpolar Environment 220

Figure 3-52. Product Peak Heights in Aqueous Conditions 221

Figure 3-53. Product Peak Heights in NEAT Conditions 222

Figure 3-54. Stability of Products in Basic Proteolytic Conditions 224

Figure 3-55. Stability of Products in Acidic Proteolytic Conditions 225

Figure 3-56. UV Absorbance of Products in Different Solvent Conditions 250

Figure 3-57. Acid Hydrolysis of Azepine 259

Figure 3-58. Azepine becomes the Protonated Salt upon Addition of Acid 259


List of Tables

Chapter One

Cocaine Addiction and the Human Dopamine Transporter1

Table 1-1. Amino Acid Analogs 44

Chapter Two

Photoaffinity Labeling of the Human Dopamine Transporter

with [125I]-DEEP 51

Table 2-1. HPLC Gradient Description of Sara3 Method 66

Table 2-2. GPMAW Codes for Theoretical Proteolytic Digestions 68

Table 2-3. Summary of Peptides from Primary and Secondary Digestions of

[125I]-DEEP Labeled hDAT 86

Chapter Three

Photoreactions of Phenyl Azide with Amino Acid Analogs120

Table 3-1. HPLC Gradient Description for Method SHORT1 128

Table 3-2. HPLC Gradient Description for Method 16 128

Table 3-3. Single Exponential First-Order Rate Constants 193

Table 3-4. Single Exponential First-Order Rate Constants Divided

by Solvent Conditions 194

Table 3-5. Initial Reaction Rates of Phenyl Azide Reacting with

Amino Acid Analogs 201

Table 3-6. Single and Double Exponential Rate Constants for Amino

Acid Analogs in Aqueous Conditions 207

Table 3-7. Single and Double Exponential Rate Constants for Amino

Acid Analogs in Cyclohexane 212

Table 3-8. Mass Spectrometry Analysis of Amino Acid Analogs with

Phenyl Azide 214

Table 3-9. Mass Spectrometry Analysis of Amino Acid Analogs with

Para-azidoacetophenone (PAAP) 216

Table 3-10. Rate Constants of Amino Acids in Nonpolar Conditions 235

Table 3-11. Rate Constants of Amino Acids in Aqueous Conditions 237

Table 3-12. Partial List of Amino Acids Labeled by Azido Photolabels 242

Table 3-13. Arylnitrene Reactivity with Amino Acid Functional Groups 244

Table 3-14. Product Stability in Basic and Acidic Conditions 255


List of Schemes

Chapter One

Cocaine Addiction and the Human Dopamine Transporter1

Scheme 1-1. Photolysis of Phenyl Azide 48

Chapter Two

Photoaffinity Labeling of the Human Dopamine Transporter

with [125I]-DEEP 51

Scheme 2-1. Localization of Photoaffinity Labels 60

Chapter Three

Photoreactions of Phenyl Azide with Amino Acid Analogs120

Scheme 3-1. Irradiation of Phenyl Azide forms 3H-Azepine and Azobenzene 123

Scheme 3-2. Reaction Schemes for Butylamine and Phenyl Azide 154

Scheme 3-3. Reaction Scheme for 1-Octanethiol and Phenyl Azide 177

Scheme 3-4. Photolysis of Phenyl Azide in Aqueous and Organic Solvents 246

Scheme 3-5. Photoisomerization of 3H-Azepines 257

Scheme 3-6. Irradiation of N-butyl-3H-azepin-2-amine 257



List of Appendices

A. The Amino Acid Sequence of the Flag 6XHis Tagged Human

Dopamine Transporter299

B. Correlations of Physical Properties vs. Rate Constants302

Table of Physical Constants for the Amino Acid Analog

Functional Groups 302

Correlation Between Phenyl Azide Reactivity and Electronegativity 303

Correlation Between the Phenyl Azide Reactivity and Dielectric Constant 304

Correlation Between the Phenyl Azide Reactivity and Polarizability 305

C. ANOVA Data Tables for the Rate Constants306

One-Way ANOVA of Rate Constants in Cyclohexane 306

One-Way ANOVA of Rate Constants in Aqueous Conditions 308

One-Way ANOVA of Rate Constants in NEAT Conditions 309

D. Correlations of Rate Constants vs. Product Stability in

Proteolytic Conditions310

Correlation Between the Phenyl Azide Reactivity and Product Stability

in Acidic Conditions 310

Correlation Between the Phenyl Azide Reactivity and Product Stability

in Basic Conditions 311


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