CLINICAL AND ANIMAL STUDIES OF LIPID-DERIVED PROTEIN MODIFICATIONS IN AUTISM, KIDNEY DIALYSIS, KERATITIS AND AGE-RELATED MACULAR DEGENERATION
by LIANG LU
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy
Thesis Advisor: Dr. Robert G. Salomon
Department of Chemistry CASE WESTERN RESERVE UNIVERSITY August 2007
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the dissertation of
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candidate for the Ph.D. degree *.
(signed)______(chair of the committee)
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*We also certify that written approval has been obtained for any proprietary material contained therein.
This thesis is dedicated to my parents, my husband, my daughter, and my sisters.
iii TABLE OF CONTENTS Table of Contents iv List of Schemes ix List of Tables xi List of Figures xiv Acknowledgements xxiv List of Abbreviations and Acronyms xxvi Abstract xxxiii
CLINICAL AND ANIMAL STUDIES OF LIPID-DERIVED PROTEIN MODIFICATIONS IN AUTISM, KIDNEY DIALYSIS, KERATITIS AND AGE-RELATED MACULAR DEGENERATION
Chapter 1. Introduction 1
1.1. Oxidative stress and aging 2
1.2. Lipid oxidation 4
1.3. 4-Hydroxy-2-nonenal and its protein adducts 6
1.4. Levuglandins, isolevuglandins and their protein adducts 7
1.5. Oxidatively truncated phospholipids and carboxyalkylpyrrole modifications
of proteins 10
1.6. Carboxyethylpyrroles (CEPs) and their potential clinical applications 11
1.7. References 17
Chapter 2. Syntheses and Characterization of Carboxyethylpyrroles 27 2.1. Background 28
2.2. Results and Discussion 30
2.2.1. Paal-Knoor synthesis using 4,7-dioxoheptanoic acid is ineffective
iv for the preparation of CEPs 30
2.2.2. Synthesis of a 9-fluorenylmethyl (Fm) ester of DOHA 31
2.2.3. Syntheses of CEP-peptide and CEP-protein adducts by Paal-Knoor
synthesis with DOHA-Fm 31
2.2.4. CEP linked to proteins with an ω-aminohexanoyl tether 34
2.2.5. Syntheses of biotinylated CEP derivatives 36
2.2.6. Syntheses of ethanolamine phospholipid CEP derivatives 39
2.2.7. Synthesis of an active pentafluorophenyl ester of a lysyl CEP 39
2.2.8. Characterization of CEP-modified protein 40
2.2.9. Tertiary protein structures of CEP modified HSA 41
2.3. Conclusions 43
2.4. Experimental Procedures 44
2.5. References 70
Chapter 3. Lipid Oxidative Modifications in Autistic Disease 74
3.1. Background 75
3.2. Materials and Methods 82
3.3. Results and Discussion 84
3.3.1. CEP and iso[4]LGE2 protein adducts immunoreactivity in human plasma 84
3.3.2. Presence of anti-CEP and anti-iso[4]LGE2 autoantibodies in
human plasma 87
3.3.3. Protein bound nitrotyrosine, chlorotyrosine and bromotyrosine levels
in ASD and healthy controls 90
3.3.4. Correlations among lipid oxidative protein adducts and nitrative proteins 92
v 3.3.5. Correlations between autoantibodies and lipid oxidation
immunoreactivities 94
3.3.6. Lipid oxidation products and birth events in ASD patients 96
3.4. Conclusions 99
3.5. Experimental Procedures 101
3.6. References 112
Chapter 4. Serum Vitamin E and Oxidative Protein Modification in
Hemodialysis 117
4.1. Background 118
4.2. Results 120
4.3. Discussion 128
4.4. Conclusions 133
4.5. Experimental Procedures 134
4.6. References 150
Chapter 5. Identification of Carboxyethylpyrrole
Phosphatidylethanolamine Adducts in vitro and in vivo 161
5.1. Background 162
5.2. Results and Discussion 168
5.2.1. Syntheses of authentic samples 168
5.2.2. Immunoreactivity of authentic CEP-PE 168
5.2.3. Identification of CEP-PE in oxidized lipid extracts 169
5.2.4. Identification of CEP-PE in lipid extracts that had not been
oxidized in vitro 170
vi 5.2.5. Quantification of CEP-PE in bovine retina 175
5.2.6. Identification of lysoCEP-PE in light-promoted oxidized lipid extracts 175
5.2.7. Identification of lysoCEP-PE in bovine retina 180
5.2.8. Identification of lysoCEP-PE in human plasma 180
5.2.9. Quantification of lysoCEP-PE in bovine retina and human plasma 183
5.3. Conclusions 185
5.4. Experimental Procedures 187
5.5. References 196
Chapter 6. Pilot Studies towards Identification of Levuglandin
Modified Proteins in Cornea 203
6.1. Introduction 204
6.1.1. LPS and inflammation 204
6.1.2. The cyclooxygenase and isoprostane pathways 204
6.1.3. LGE2 and iso[4]LGE2 bind avidly with proteins 207
6.1.4. Pyridoxamine, a potent trap that prevents protein modification by reactive
electrophiles 207
6.2. Results and Discussion 209
6.3. Experimental Procedures 212
6.4. References 214
Chapter 7. Syntheses of Isosteric Pyrazole Derivatives 221
7.1. Background 222
7.2. Results and Discussions 225
7.2.1. Synthesis of a 1,3-dione 225
vii 7.2.2. Synthesis and structure identification of 1-alkyl-5-pentylpyrazole-3-
carboxylic acid 225
7.2.3. Decarboxylation of a pyrazole carboxylic acid 227
7.2.4. Condensation of an alkylhydrazine with a β-ketoaldehyde 227
7.2.5. Attempted ozonolysis of an alkehylpyrazole 228
7.2.6. A strategy for conjugation of the isostere hapten with peptides 229
7.3. Experimental Procedures 230
7.4. References 236
Appendix 242
Bibliography 303
viii LIST of SCHEMES
Chapter 1
Scheme 1.1. Cyclooxygenase oxidation of AA generates PGs and LGs via rearrangement of PGH2. 8
Scheme 1.2. Free radical-induced oxidation of AA-PC produces isoLGs by rearrangement of isoP intermediates. 8
Scheme 1.3. Formation of LG-protein adducts, protein-protein and DNA-protein crosslinks. 10
Scheme 1.4. Oxidation of polyunsaturated fatty acids generates hydroxy-ω- oxoalkenoic acids, that react with proteins and form 2-(ω- carboxyalkyl)pyrroles. 12
Chapter 2
Scheme 2.1. DOHA exists in equilibrium with a spiroacylal hemiacetal. 30
Scheme 2.2. Synthesis of 9H-fluoren-9-ylmethyl ester 4,7-dioxo-heptanoic acid (DOHA-Fm, 2.4). 31
Scheme 2.3. Synthesis of CEP-dipeptide. 31
Scheme 2.4. Syntheses of CEP protein adducts. 33
Scheme 2.5. Syntheses of CEPs bound to proteins through a tether. 35
Scheme 2.6. Synthesis of biotinylated CEP derivative 2.21. 38
Scheme 2.7. Synthesis of biotinylated CEP derivative 2.25. 38
Scheme 2.8. Syntheses of ethanolamine phospholipid CEP derivatives. 39
Scheme 2.9. Synthesis of a lysyl CEP pentafluorophenyl derivative. 40
Chapter 5
Scheme 5.1. 4,5-(E)-Epoxy-2-(E)-heptenal reacts with phosphatidylethanolamine to produce pyrrole derivatives. 166
Scheme 5.2. Synthesis of CEP-PE and lysoCEP-PE. 168
ix Chapter 6
Scheme 6.1. Representative pathways showing enzyme and free radical induced generation of LGE2 and iso[4]LGE2 from AA-PC. 206
Scheme 6.2. Possible mechanism of pyrrole formation between pyridoxamine and 1,4-dicarbonyls. 208
Chapter 7
Scheme 7.1. Synthesis of 1-alkyl-5-pentylpyrazole through a decarboxylation route. 225
Scheme 7.2. General scheme of Barton decarboxylation. 227
Scheme 7.3. An alternative route for synthesis of pyrazole 7.5. 228
Scheme 7.4. Reductive amination of proteins with pyrazole aldehyde 7.6 that is to be generated by ozonolysis of alkene 7.5. 229
x LIST OF TABLES
Chapter 2
Table 2.1. Pyrrole concentration (μM) generated in 1 mg/mL CEPH-BSA prepared by coupling various initial protein concentrations with various CEPFmSu/Lys ratios. 36
Table 2.2. ELISA data for CEPH-BSA of Figure 2.1. 69
Table 2.3. ELISA data for CEP-HSA of Figure 2.1. 69
Chapter 3
Table 3.1. ELISA data for iso[4]LGE2-HSA standard for Figure 3.6. 103
Table 3.2. ELISA data for ASD patient (patient code A27) for Figure 3.6. 103
Table 3.3. ELISA data for control (normal code N11) for Figure 3.6. 104
Table 3.4. Data for CEP, iso[4]LGE2 immunoractivites and CEP, iso[4]LGE2 autoantibodies in ASD patients. 105
Table 3.5. Data for CEP, iso[4]LGE2 immunoractivites and CEP, iso[4]LGE2 autoantibodies in normal controls. 106
Table 3.6. Nitrotyrosine, chlorotyrosine and bromotyrosine levels in autistic patients. 109
Table 3.7. Nitrotyrosine, chlorotyrosine and bromotyrosine levels in normal controls. 110 Chapter 4
Table 4.1. Baseline clinical characteristics of patients. 120
Table 4.2. Plasma levels of alpha and gamma tocopherol in placebo and vitamin E treated patients. 122
Table 4.3. Plasma glycoxidation and protein-lipid oxidation products in placebo and vitamin E treated patients. 124
Table 4.4. Linear regression of glycoxidation and protein-lipid oxidation products with alpha or gamma tocopherol at baseline. 126
Table 4.5. Linear regression of iso[4]LGE2 with alpha or gamma tocopherol during placebo treatment at 3 months, 6 months and 9 months. 126
xi
Table 4.6. Linear regression of iso[4]LGE2 with alpha or gamma tocopherol during during treatment with α-tocopherol at 3 months, 6 months and 9 months. 127
Table 4.7. Linear regression matrix for glycoxidation and protein-lipid oxidation products at baseline. 127
Table 4.8. α-Tocopherol levels (μg/mL) in plasma from patients treated with vitamin E at baseline, three months, six months, nine months and twelve months. 136
Table 4.9. α-Tocopherol levels (μg/mL) in plasma from HD patients treated with placebo at baseline, three months, six months, nine months and twelve months. 137
Table 4.10. γ-Tocopherol levels (μg/mL) in plasma from patients treated with vitamin E at baseline, three months, six months, nine months and twelve months. 138
Table 4.11. γ-Tocopherol levels (μg/mL) in plasma from HD patients treated with placebo at baseline, three months, six months, nine months and twelve months. 138
Table 4.12. Pentosidine levels (pmol/mg protein) in plasma from HD patients treated with vitamin E supplementation at baseline, three months, six months, nine months and twelve months. 140
Table 4.13. Pentosidine levels (pmol/mg protein) in plasma from HD patients treated with placebo at baseline, three months, six months, nine months and twelve months. 141
Table 4.14. HNE-derived pentylpyrrole immunoreactivities in plasma from patients treated with vitamin E at baseline, 3 months, 6 months, 9 months and 12 months. 144
Table 4.15. HNE-derived pentylpyrrole immunoreactivities (nmol/mL) in plasma from patients treated with placebo at baseline, 3 months, 6 months, 9 months and 12 months. 145
Table 4.16. Iso[4]LGE2-protein immunoreactivities (nmol/mL) in plasma from patients treated with vitamin E at baseline, 3 months, 6 months, 9 months and 12 months. 146
xii Table 4.17. Iso[4]LGE2-protein immunoreactivities (nmol/mL) in plasma from patients treated with placebo at baseline, 3 months, 6 months, 9 months and 12 months. 147
Table 4.18. ONE-protein immunoreactivities (pmol/mg protein) in plasma from patients treated with vitamin E at baseline, 3 months, 6 months and 9 months. 148
Table 4.19. ONE-protein immunoreactivities (pmol/mg protein) in plasma from patients treated with placebo at baseline, 3 months, 6 months and 9 months. 149
Chapter 5
Table 5.1. Distribution of the fatty acids of PE and PC in human ROS (as mol% of total fatty acid). 162
Table 5.2. ELISA data for CEP-PE in Figure 5.1. 188
Table 5.3. ELISA data for CEP-CEO in Figure 5.1. 188
Chapter 7
Table 7.1. HMQC and HMBC data for 7.4 (in parts per million). 226
Appendix
Table S1. Total iron levels of patients treated with vitamin Eα or placebo at baseline, three months, six months and nine months. 292
Table S2. Erythropoietin administrated in patients treated with vitamin Eα or placebo at baseline, three months, six months and nine months. 293
Table S3. Ferritin levels of patients treated with vitamin Eα or placebo at baseline, three months, six months and nine months. 294
xiii LIST OF FIGURES
Chapter 1
Figure 1.1. The sources and cellular responses to reactive oxygen species (ROS). 3
Figure 1.2. CEP-HSA as well as dipeptide (Ac-gly-lys-OH) induces angiogenesis in a rat corneal micropocket assay. (A) HSA (1 μg), (B) CEP-HSA (1 μg), (C) dipeptide (41 ng), (D) CEP-dipeptide (37 ng). 13
Figure 1.3. Outer retinal pathology present in mice immunized with CEP-MSA (A-J). Large arrows indicate several inflammatory cells immediately adjacent to the RPE or in the IPM. Large empty vacuoles are also evident (C-I). Some of which appear to be intracellular (I). In (J) the RPE has degenerated (asterisks). Bar in lower right of (J) represent 25 μm. 14
Chapter 2
Figure 2.1. Inhibition curves showing cross-reactivity of the anti-CEP-KLH antibody for CEP-HSA (▲) and CEPH-BSA (●) against CEP-HSA as coating agent. 36
Figure 2.2. A ball-and-stick model of 3D structure for human serum albumin. All the lysines are shown as space-filling structures in the left unit. Only lysines that become CEP modified are shown in the right unit. The lysine in the sequence which is common to HSA, BSA and MSA is marked. 42
Chapter 3
Figure 3.1. Anti-CEP staining of brain cortical regions from 3 of the control cases aged 5, 6 and 11 years (A, B, C) and 3 autism cases aged 9, 7, and 5 years (D, E, F). 80
Figure 3.2. CEP immunoreactivity detected in human plasma from (◆) ASD and (■) normal controls. The figure also shows mean levels detected (O). The error bars indicate the standard deviation (S. D.) for each data set. 84
Figure 3.3. Levels of individual CEPs in plasma from 27 patients with diagnosed ASD and 11 healthy controls. 85
Figure 3.4. Iso[4]LGE2 immunoreactivity detected in human plasma from (◆) ASD and (■) normal controls. The figure also shows mean levels
xiv detected (O). The error bars indicate the standard deviation (S. D.) for each data set. 85
Figure 3.5. Levels of individual iso[4]LGE2-protein adducts in plasma from 27 patients with diagnosed ASD and 11 healthy controls. 86
Figure 3.6. Inhibition curves for binding of anti-iso[4]LGE2-KLH to iso[4]LGE2- BSA by iso[4]LGE2-HSA (●), plasma from an ASD patient (■), and a normal control (▲). 86
Figure 3.7. CEP autoantibody titer detected in human plasma from (◆) ASD and (■) normal controls. The figure also shows mean levels detected (O). The error bars indicate the standard deviation (S. D.) for each data set. 88
Figure 3.8. Levels of individual individual CEP autoantibody titer in plasma from 26 patients with diagnosed ASD and 5 healthy controls. 88
Figure 3.9. Iso[4]LGE2 autoantibody titer detected in human plasma from (◆) ASD and (■) normal controls. The figure also shows mean levels detected (O). The error bars indicate the standard deviation (S. D.) for each data set. 89
Figure 3.10. Levels of individual iso[4]LGE2 autoantibody titer in plasma from 27 patients with diagnosed ASD and 11 healthy controls. 89
Figure 3.11. Nitrotyr/Tyr ratio detected in human plasma from (◆) ASD and (▲) normal controls. The figure also shows mean levels detected (O). The error bars indicate the standard deviation (S. D.) for each data set. 91
Figure 3.12. Cltyr/Tyr ratio detected in human plasma from (◆) ASD and (■) normal controls. The figure also shows mean levels detected (O). The error bars indicate the standard deviation (S. D.) for each data set. 91
Figure 3.13. Brtyr/Tyr ratio detected in human plasma from (◆) ASD and (■) normal controls. The figure also shows mean levels detected (O). The error bars indicate the standard deviation (S. D.) for each data set. 92
Figure 3.14. Correlation between CEP immunoreactivity and iso[4]LGE2 immunoreactivity in human plasma from (●) ASD and (∆) normal controls. 93
Figure 3.15. Correlation between iso[4]LGE2 and CEP immunoreactivities in human plasma from (◆) ASD and (■) normal controls. The horizontal and vertical dashed lines indicate the mean values of CEP
xv and iso[4]LGE2 immunoreactivities, respectively. Out of 27 ASD patients, 6 (22%) are in region IV while 1 out of 11 normal controls (9%) is in region IV. ASD patients account for 86% (6/7) of the data in region IV. 93
Figure 3.16. Correlation between CEP immunoreactivity and nitrotyro/Tyr ratio in human plasma from (●) ASD and (∆) normal controls. 94
Figure 3.17. Correlation between iso[4]LGE2 immunoreactivity and nitrotyro/Tyr ratio in human plasma from (●) ASD and (∆) normal controls. 94
Figure 3.18. Correlation between CEP autoantibody titer and iso[4]LGE2 autoantibody titer in human plasma from (●) ASD and (∆) normal controls. 95
Figure 3.19. Correlation between CEP immunoreactivity and CEP autoantibody titer in human plasma from (●) ASD and (∆) normal controls. 95
Figure 3.20. Correlation between iso[4]LGE2 immunoreactivity and iso[4]LGE2 autoantibody titer in human plasma from (●) ASD and (∆) normal controls. 96
Figure 3.21. CEP immunoreactivity grouped according to birth events of ASD patients with no events, born premature or other events birth events. 97
Figure 3.22. CEP autoantibody titer grouped according to birth events of ASD patients with no events, born premature or other events birth events. 97
Figure 3.23. Iso[4]LGE2 immunoreactivity grouped according to birth events of ASD patients with no events, born premature or other events birth events. 98
Figure 3.24. Iso[4]LGE2 autoantibody titer grouped according to birth events of ASD patients with no events, born premature or other events birth events. 98
Chapter 4
Figure 4.1. Relationship between plasma levels of Vitamin E alpha and gamma before and after treatment. A. Baseline R = 0.46, p = 0.0055. B. The positive correlation between vitamin E alpha and gamma is present in the placebo group, left regression line (R = 0.42, p = 0.05). In the patients treated with vitamin E, there is a negative correlation between the two metabolites, right regression line (R = 0.11, “not significant”). Gray symbols, placebo. Black symbols, Vitamin E treatment. Open circles represent baseline; squares, 3 month values. 123
xvi
Figure 4.2. Levels of HNE-derived pentylpyrrole over the course of study. 125
Figure 4.3. Levels of iso[4]LGE2-protein adduct over the course of study. 125
Figure 4.4. Fenton and Fenton-like reactions. 131
Figure 4.5. Reduction of transition metal ions by tocopherols. 131
Chapter 5
Figure 5.1. Inhibition curves showing crossreactivity of the anti-CEP-KLH antibody for CEP-CEO (y) and CEP-PE () against CEP-CEO as coating agent. 169
Figure 5.2. Proposed MRM fragments of CEP-PE and corresponding m/z. 171
Figure 5.3. Daughter scan of CEP-PE. 171
Figure 5.4. MRM chromatograms of CEP-PE. 172
Figure 5.5. MRM of lipid extracts from bovine retina after UV light promoted oxidation. A single peak with a retention time that is identical to that of authentic standard CEP-PE was present in the MRM chromatogram of oxidative lipid at each parent/daughter ion pair channel. 173
Figure 5.6. MRM of bovine retinal extracts that had not been oxidized in vitro. A single peak with a retention time that is identical to that of authentic CEP-PE is present in the MRM chromatogram monitoring the 838.3 → 255.4 and 838.3 → 391.2 transitions. 174
Figure 5.7. Standard curve and calibration equation for CEP-PE. 175
Figure 5.8. Proposed fragments of lysoCEP-PE and corresponding m/z. 177
Figure 5.9. Daughter scan of lysoCEP-PE. 177
Figure 5.10. MRM chromatograms of lysoCEP-PE. Pure lysoCEP-PE (10 ng/μL in methanol) was injected onto a Shimadzu LC-10AD HPLC system, eluting with 1 mM ammonium acetate in 20% acetonitrile and using a linear gradient to 100% acetonitrile over 3 minutes, followed by 100% acetonitrile for 10 minutes. 178
xvii Figure 5.11. MRM of bovine retinal lipids after UV-promoted oxidation. Lipid extracts (50 ng/μL in methanol) was injected onto a Shimadzu LC- 10AD HPLC system as described in Figure 5.10. A single peak with a retention time that is identical to that of authentic lysoCEP-PE was present in the MRM chromatogram of oxidized lipid in each parent/daughter ion pair channel. 179
Figure 5.12. MRM chromatograms of authentic lysoCEP-PE. Pure lysoCEP-PE (50 ng) was injected onto a Waters 2790 HPLC system, eluting with 1 mM ammonium acetate in water and using a linear gradient to 100% methanol over 4 minutes, followed by 100% methanol for 9 minutes. Note: This is a different system than Figures 5.10-5.11 and retention times are, therefore, different. 181
Figure 5.13. MRM chromatograms of lipid extracts from a plasma sample of a normal control. Lipid extracts (150 μg in 20 μL methanol) was injected onto a Waters 2790 HPLC system, eluting with 1 mM ammonium acetate in water and using a linear gradient to 100% methanol over 4 minutes, followed by 100% methanol for 9 minutes. 182
Figure 5.14. MRM chromatograms of lipid extracts from a plasma sample of an AMD patient. Lipid extracts (100 μg in 20 μL methanol) was injected onto a Waters 2790 HPLC system, eluting with 1 mM ammonium acetate in water and using a linear gradient to 100% methanol over 4 minutes, followed by 100% methanol for 9 minutes. 183
Figure 5.15. Standard curve and calibration equation of authentic lysoCEP-PE. 184
Chapter 6
Figure 6.1. Eicosanoid synthesis. 206
Figure 6.2. Cornea of C57BL/6 mouse was treated in vivo with LPS or PBS for 24 h by injection or scratch. Sections (5 μm) of cornea were stained with LGE2 and iso[4]LGE2 pAb. A, B, C, J, K, L – Untreated; D, E, F – PBS injection; G, H, I – LPS injection; M, N, O – PBS treated (scratch); P, Q, R – LPS treated (scratch). Panels treated with preimmune, LGE2 and iso[4]LGE2 pAbs are indicated in the Figure. 210
Chapter 7
Figure 7.1. Pyrroles cross-link with proteins. 224
Figure 7.2. Structural description of C – H HMBC coupling for both 3-carboxyl- 5-pentyl pyrazole and 3-pentyl-5-carboxyl pyrazole. 226
xviii
Appendix
1 Figure S1. H NMR (200 MHz, CDCl3) spectrum of methyl 6-(2,5-dioxolanyl) -4-oxohexanoate (2.1). 243
13 Figure S2. C NMR (50 MHz, CDCl3) spectrum of methyl 6-(2,5-dioxolanyl) -4-oxohexanoate (2.1). 243
1 Figure S3. H NMR (200 MHz, CDCl3) spectrum of 6-(2,5-dioxolanyl)-4- oxohexanoic acid (2.2). 244
13 Figure S4. C NMR (100 MHz, CDCl3) spectrum of 6-(2,5-dioxolanyl)-4- oxohexanoic acid (2.2). 244
1 Figure S5. H NMR (200 MHz, CDCl3) spectrum of (2.3). 245
13 Figure S6. C NMR (50 MHz, CDCl3) spectrum of (2.3). C, CH2 (-); CH, CH3 (+). 245
1 Figure S7. H NMR (200 MHz, CDCl3) spectrum of DOHAFm (2.4). 246
13 Figure S8. C NMR (100 MHz, CDCl3) spectrum of DOHAFm (2.4). 246
1 Figure S9. H NMR (400 MHz, CDCl3) spectrum of (2.5). 247
13 Figure S10. C NMR (100 MHz, CDCl3) spectrum of (2.5). 247
1 Figure S11. H NMR (400 MHz, CDCl3) spectrum of (2.6). 248
13 Figure S12. C NMR (150 MHz, CDCl3) spectrum of (2.6). 248
1 Figure S13. H NMR (400 MHz, CDCl3) spectrum of (2.9). 249
13 Figure S14. C NMR (100 MHz, CDCl3) spectrum of (2.9). 249
1 Figure S15. H NMR (400 MHz, CDCl3) spectrum of CEPFmSu (2.10). 250
13 Figure S16. C NMR (100 MHz, CDCl3) spectrum of CEPFmSu (2.10). 250
1 Figure S17. H NMR (400 MHz, CD3OD) spectrum of (2.18). 251
1 Figure S18. H NMR (200 MHz, CD3OD) spectrum of (2.19). 251
1 Figure S19. H NMR (200 MHz, CDCl3) spectrum of (2.20). 252
xix 1 Figure S20. H NMR (400 MHz, CD3OD) spectrum of (2.21). 252
1 Figure S21. H NMR (200 MHz, CDCl3) spectrum of (2.22). 253
1 Figure S22. H NMR (400 MHz, CD3OD) spectrum of (2.23). 253
13 Figure S23. C NMR (100 MHz, CD3OD) spectrum of (2.23). 254
1 Figure S24. H NMR (400 MHz, CD3OD) spectrum of (2.24). 254
1 Figure S25. H NMR (400 MHz, CD3OD) spectrum of (2.25). 255
13 Figure S26. C NMR (100 MHz, CD3OD) spectrum of (2.25). 255
1 Figure S27. H NMR (400 MHz, CDCl3 : CD3OD = 1 : 1) spectrum of (2.26). 256
13 Figure S28. C NMR (50 MHz, CDCl3:CD3OD = 1:1) spectrum of (2.26). 256
1 Figure S29. H NMR (400 MHz, CDCl3 : CD3OD = 1 : 1) spectrum of (2.27). 257
13 Figure S30. C NMR (100 MHz, CDCl3:CD3OD = 1:1) spectrum of (2.27). 257
1 Figure S31. H NMR (400 MHz, CDCl3 : CD3OD = 1 : 1) spectrum of (2.28). 258
13 Figure S32. C NMR (400 MHz, CDCl3 : CD3OD = 1 : 1) spectrum of (2.28). 258
1 Figure S33. H NMR (400 MHz, CDCl3) spectrum of (2.29). 259
13 Figure S34. C NMR (100 MHz, CDCl3 :CD3OD:D2O = 50:50:1) spectrum of (2.29). 259
1 Figure S35. H NMR (400 MHz, CDCl3) spectrum of (2.30). 260
13 Figure S36. C NMR (100 MHz, CDCl3) spectrum of (2.30). 260
1 Figure S37. H NMR (400 MHz, CDCl3) spectrum of (2.31). 261
13 Figure S38. C NMR (100 MHz, CDCl3) spectrum of (2.31). 261
Figure S39. Tandem MS characterization of CEP modified HSA shows series of fragment ions sufficient to identify CEP modifications on lysyl residues of the HSA peptides. (A). MS/MS spectrum of the doubly charged ion m/z 589.266. (B). MS/MS spectrum of the doubly charged ion m/z 674.762. (C). MS/MS spectrum of the doubly charged ion m/z 881.401. (D). MS/MS spectrum of the doubly charged ion m/z 687.784. (E). MS/MS spectrum of the doubly
xx charged ion m/z 636.252. Asterisks denote fragment ions with the modified lysyl residue. 263
Figure S40. Tandem MS characterization of CEP modified MSA. (A). MS/MS spectrum of the doubly charged ion m/z 650.3296. (B). MS/MS spectrum of the doubly charged ion m/z 1047.0802. (C). MS/MS spectrum of the doubly charged ion m/z 769.4069. (D). MS/MS spectrum of the doubly charged ion m/z 612.8407. (E). MS/MS spectrum of the doubly charged ion m/z 872.9611. (F). MS/MS spectrum of the triplet charged ion m/z 849.4302. (G). MS/MS spectrum of the triplet charged ion m/z 857.4633. (H). MS/MS spectrum of the triplet charged ion m/z 694.6888. Asterisks denote fragment ions with the modified lysyl residue. 266
Figure S41. Tandem MS characterization of CEP modified CEO. (A). MS/MS spectrum of the doubly charged ion m/z 540.2886. (B). MS/MS spectrum of the triplet charged ion m/z 801.7021. (C). MS/MS spectrum of the doubly charged ion m/z 839.3332. (D). MS/MS spectrum of the triplet charged ion m/z 808.0259. (E). MS/MS spectrum of the doubly charged ion m/z 610.2589. (F). MS/MS spectrum of the doubly charged ion m/z 630.8334. (G). MS/MS spectrum of the doubly charged ion m/z 459.1887. Asterisks denote fragment ions with the modified lysyl residue. 269
Figure S42. Tandem MS characterization of CEP modified myoglobin. (A). MS/MS spectrum of the doubly charged ion m/z 671.2667. (B). MS/MS spectrum of the doubly charged ion m/z 604.9635. (C). MS/MS spectrum of the doubly charged ion m/z 815.2087. (D). MS/MS spectrum of the quartet charged ion m/z 557.4288. (E). MS/MS spectrum of the doubly charged ion m/z 785.6061. (F). MS/MS spectrum of the doubly charged ion m/z 899.6666. Asterisks denote fragment ions with the modified lysyl residue. 272
Figure S43. Tandem MS characterization of CEP modified GPDH. (A). MS/MS spectrum of the doubly charged ion m/z 655.3343. (B). MS/MS spectrum of the doubly charged ion m/z 669.3289. (C). MS/MS spectrum of the doubly charged ion m/z 923.9847. (D). MS/MS spectrum of the doubly charged ion m/z 639.3413. (E). MS/MS spectrum of the doubly charged ion m/z 740.8362. Asterisks denote fragment ions with the modified lysyl residue. 274
Figure S44. Tandem MS characterization of CEPH modified BSA. (A). MS/MS spectrum of the triplet charged ion m/z 594.9934. (B). MS/MS spectrum of the doubly charged ion m/z 541.8060. (C). MS/MS spectrum of the doubly charged ion m/z 937.9577. (D). MS/MS spectrum of the doubly charged ion m/z 526.7835. (E). MS/MS
xxi spectrum of the doubly charged ion m/z 612.3604. (F). MS/MS spectrum of the doubly charged ion m/z 689.4319. (G). MS/MS spectrum of the doubly charged ion m/z 765.3984. (H). MS/MS spectrum of the doubly charged ion m/z 964.5088. Asterisks denote fragment ions with the modified lysyl residue. 278
Figure S45. Tandem MS characterization of CEPH modified BSA. (A). MS/MS spectrum of the doubly charged ion m/z 512.2829. (B). MS/MS spectrum of the doubly charged ion m/z 627.8604. (C). MS/MS spectrum of the doubly charged ion m/z 608.8177. (D). MS/MS spectrum of the doubly charged ion m/z 732.3707. Asterisks denote fragment ions with the modified lysyl residue. 280
Figure S46. Tandem MS characterization of the doubly charged ion m/z 596.8715 from a MS scan of tryptic digested CEPH modified CEO. Asterisks denote fragment ions with the modified lysyl residue. 281
Figure S47. Tandem MS characterization of CEPH modified GPDH. (A). MS/MS spectrum of the doubly charged ion m/z 635.0367. (B). MS/MS spectrum of the doubly charged ion m/z 712.5500. (C). MS/MS spectrum of the doubly charged ion m/z 919.7650. (D). MS/MS spectrum of the doubly charged ion m/z 572.9763. (E). MS/MS spectrum of the triplet charged ion m/z 899.6923. (F). MS/MS spectrum of the doubly charged ion m/z 726.0422. (G). MS/MS spectrum of the doubly charged ion m/z 980.7797. (H). MS/MS spectrum of the doubly charged ion m/z 731.1005. (I). MS/MS spectrum of the doubly charged ion m/z 941.7422. (J). MS/MS spectrum of the triplet charged ion m/z 579.9560. (K). MS/MS spectrum of the doubly charged ion m/z 508.9000. (L). MS/MS spectrum of the doubly charged ion m/z 696.1018. (M). MS/MS spectrum of the doubly charged ion m/z 797.5823. Asterisks denote fragment ions with the modified lysyl residue. 286
Figure S48. Tandem MS characterization of CEPFmHSA revealed five CEP modifications. (A). MS/MS spectrum of the doubly charged ion m/z 636.248. (B). MS/MS spectrum of the doubly charged ion m/z 881.408. (C). MS/MS spectrum of the doubly charged ion m/z 571.262. (D). MS/MS spectrum of the doubly charged ion m/z 687.787. (E). MS/MS spectrum of the doubly charged ion m/z 674.765. 289
Figure S49. Tandem MS characterization of CEPFmHSA revealed five CEPFm modifications. (A). MS/MS spectrum of the doubly charged ion m/z 725.281. (B). MS/MS spectrum of the doubly charged ion m/z 660.289. (C). MS/MS spectrum of the doubly charged ion m/z 776.820. 291
xxii
1 Figure S50. H NMR (300 MHz, CDCl3) spectrum of (7.1). 295
13 Figure S51. C NMR (75 MHz, CDCl3) spectrum of (7.1). 295
1 Figure S52. H NMR (300 MHz, CDCl3) spectrum of (7.2). 296
13 Figure S53. C NMR (75 MHz, CDCl3) spectrum of (7.2). 296
1 Figure S54. H NMR (300 MHz, CDCl3) spectrum of (7.3). 297
1 Figure S55. H NMR (300 MHz, CDCl3) spectrum of (7.4). 297
13 Figure S56. C NMR (75 MHz, CDCl3) spectrum of (7.4). 298
1 Figure S57. H NMR (400 MHz, CDCl3) spectrum of (7.5). 298
13 Figure S58. C NMR (100 MHz, CDCl3) spectrum of (7.5). 299
Figure S59. HMQC of 3-carboxyl-5-pentyl pyrazole (7.4). 299
Figure S60. HMBC of 3-carboxyl-5-pentyl pyrazole (7.4). 300
1 Figure S61. H NMR (300 MHz, CDCl3) spectrum of (7.7). 300
1 Figure S62. H NMR (300 MHz, CDCl3) spectrum of (7.8). 301
1 Figure S63. H NMR (300 MHz, CDCl3) spectrum of (7.9). 301
1 Figure S64. H NMR (400 MHz, CDCl3) spectrum of (7.10). 302
xxiii Acknowledgements
First of all, I would like to express my deepest sense of gratitude and everlasting
respect to my research advisor Dr. Robert G. Salomon for his invaluable guidance,
fruitful discussions, strictly scientific attitude, and humane understanding throughout my
graduate studies. He provided me with the best education that an advisor can bring to his
students. He encouraged me to never accept anything short of my personal best in my
research. I am also deeply moved by his noble personality. His intelligence, his kindness,
and his generosity make me very thankful to be one of his students. I wish I could stay
longer to experience more of his guidance, both academically and personally.
I am very grateful to my committee members: Dr. Pearson, Dr. Zagorski, and Dr.
Burda for their precious time, efforts, suggestions, and support in my candidate
qualification and thesis. Special thanks to Dr. Weiss for being on my committee, and for
her efforts, intelligence and time on our paper and my thesis.
It is my pleasure to acknowledge Dr. John W. Crabb, Dr. Joe Hollyfield, Dr.
Victor Perez, and their groups, at the Cole Eye Institute, Cleveland Clinic Foundation, for
their collaboration. Special thanks are due to Dr. Xiaorong Gu for her valuable help and
sisterly care.
I would like to thank Dr. Lian Shan, at Frantz Biomarkers, for all the help and instruction on mass spectrometry.
I would like to thank Dr. Eric Pearlman and his group at the Center for Global
Health & Diseases, for their training on immunostaining techniques.
I would like to thank Dr. Salomon’s group, former and current members: Dr.
Bogdan Gugiu, Dr. Suresh Annangudi, Dr. James Laird, and Ken, Wujuan, Jawoo, Xi, xxiv Wei, Xiaodong, Yunfeng, Li, and Hua for many thoughtful discussions and the overall friendly atmosphere. Special thanks to Jim, for all his assistance in my effort to improve my writing and pronunciation.
I would also like to thank Dr. Dale Ray and Dr. Jim Faulk and other staff members in Department of Chemistry in Case Western Reserve University for their help on numerous occasions.
I greatly appreciate Jun Li, Ming Zhang, Liqing Ma and all my friends for all the beautiful memories we had together. Their friendship made my stay at U.S.A. pleasant, enjoyable and deeply cherished.
Finally, I would like to express my deepest gratitude to my parents, my husband, my sisters and their family, and my sweety heart--my daughter-- for their endless love, support, and countless sacrifices.
Liang Lu
xxv LIST OF ABBREVIATIONS AND ACRONYMS
Abbreviations and Acronyms Equivalent
AA Arachidonyl acid
AA-PC Arachidonic phosphatidylcholine
AGE Advanced glycation end product
ALPE Advanced lipid peroxidation end product
AMD Age-related macular degeneration
AOPP Advanced oxidative protein product
APT Attached proton test
AS Atherosclerosis
ASD Autism spectrum disorders
BCA Bicinchoninic acid
BrTyr Bromotyrosine
BSA Bovine serum albumin
CAT Catalase
CDCl3 Deutrated chloroform
CD3OD Deutrated methanol
γ-CEHC 2,7,8-Trimethyl-2-(β-carboxyethyl)-6-
hydroxychroman
CEO Chicken egg ovulbumin
CEP 2-(ω-Carboxyethyl)pyrrole
xxvi CEPH Carboxyethylpyrrole derivatives of 6-
aminohexanoic acid
CEP-PE Carboxyethylpyrrole
phosphatidylethanolamine adducts
CHAOS Cambridge Heart Antioxidant Study
CHP 2-(ω-Carboxyheptyl)pyrrole
ClTyr Chlorotyrosine
CME Carboxymethyl-PE
CPP 2-(ω-Carboxypropyl)pyrrole
COX Cyclooxygenase
DBF Dibenzofulvene
DBU 1,8-Diazabicyclo[5.4.0] undec-7-ene
DCC Dicyclohexylcarbodiimide
DHA Docosahexaenoic acid
DAPI 4,6-Diamidino- -2-phenylindole
DMAP 4-N,N-Dimethylaminopyridine
DMF Dimethyl formide
DNA Desoxyribonucleic acid
D2O Deutrated water
DOHA 4,7-Dioxoheptanoic acid
DOHA-Fm 9H-fluoren-9-ylmethyl ester of DOHA
DPCs DNA-protein cross-links
xxvii DSM Diagnostic and Statistical Manual of Mental
Disorders
EDTA Ethylenediaminetetraacetic acid
EI Electron impact
ELISA Enzyme-linked immunosorbent assay
ESRD End stage renal disease
FAB Fast atom bombardment
FCS Fetal calf serum
Fm 9-Fluorenylmethyl
GISSI Gruppo Italiano per lo Studio della
Sopravvivenza nell’Infarto miocardio
GPDH Glycerol-3-phosphate dehydrogenase
GPx Glutathione peroxidase
GSH Reduced glutathione
GSSH Glutathione-glutathione disulfide
GTP Guanosine triphosphate pHA p-Hydroxyphenylacetaldehyde
HD Hemodialysis
HDDE 4-Hydroxydodeca-(2E,6Z)-dienal
HEPE N-(Hexanoyl)phosphatidylethanolamine HHE 4-Hydroxy-2E- hexenal HMBC Heteronuclear multiple bond coherence
HMQC Heteronuclear multiple quantum coherence
xxviii HNE 4-Hydroxy-2-nonenal
HODA 9-Hydroxy-12-oxo-10(E)-dodecenoic acid
HOHA 4-Hydroxy-7-oxohept-5-enoic acid
HODA-PC 1-Palmitoyl-2-(9-hydroxy-12-oxododec-10- enoate)-sn-glycero-3-phosphatidylcholine
HOOA 5-Hydrox-8-oxo-6-octenoic acid
HOPE Heart Outcomes Prevention Evaluation
HPLC High performance liquid chromatography
HRMS High resolution mass spectrometry
HSA Human serum albumin
IFN-γ Interferon-γ
IL-2 Interleukin-2
IPM Interphotoreceptor matrix iso[4]LGE2 Iso[4]levuglandin E2 isoLGE2 Isolevuglandin E2 isoP Isoprostane
KLH Keyhole limpet hemocyanin
LA Linoleic acid
LC Liquid chromatography
LGD2 Levuglandin D2
LGE2 Levuglandin E2
LDL Low density lipoprotein
LF Lipofuscin
xxix LG Levuglandin
LOOH Lipid hydroperoxide
LPS Lipopolysaccharide
Lyso-PC 1-Palmityl-2-hydroxy-sn-glycero-3- phosphatidylcholine
Lyso-PE 1-Palmityl-2-hydroxy-sn-glycero-3-phospho- - ethanolamine MALDI-TOF Matrix assisted laser desorption ionization- time of flight m-CPBA m-Chloroperbenzoic acid
MCP-1 Monocyte chemoattractant protein-1
MDA Malondialdehyde
MHC Major histocompatibility complex
MHz Megahertz
MMR Measles-mumps-rubella
M-PE 1-Myristyl-2-hydroxy-sn-glycero-3-
phosphatidylethanolamine
MPM Mouse peritoneal macrophage
MPO Myeloperoxidase
MRM Multiple reaction monitoring
MS Mass spectrometry
MSA Mouse serum albumin
MS/MS Tandem mass spectrometry
MT Metallothionein
NMR Nuclear magnetic resonance
xxx NO Nitric oxide
NOS Nitric oxide synthase
ONE 4-Oxo-2-nonenal
ON 9-Oxononanyl
OV 2-Oxovaleryl oxLDL Oxidized low density lipoprotein oxPC Oxidized phosphatidylcholines oxPL Oxidized phospholipids
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffered saline
PC Phosphatidylcholine
PDD Pervasive developmental disorders
PE Phosphatidylethanolamine
PG Prostaglandin
PGD2 Prostaglandin endoperoxide D2
PGE2 Prostaglandin endoperoxide E2
PGH2 Prostaglandin endoperoxide H2
PL Phospholipids
POPE 1-Palmityl-2-oleyl-sn-glycero-3- phosphoethanolamine PP 2-Pentylpyrrole ppm Parts per million
PTCA Percutaneous transluminal coronary
angioplasty
xxxi PUFA Polyunsaturated fatty acid
QTOF Quadrupole time-of-flight
Rf Retention factor
ROS Rod outer segment
ROxS Reactive oxygen species
RPE Retina pigment epithilium
S.D. Standard deviation
SDS/PAGE Sodium dodecyl sulfate/ polyacrylamide gel electrophoresis
SH Sulfhydryl
SO Superoxide
SOD Superoxide dismutase
Su N-Hydroxysuccinimide
TCA Trichloroacetic acid
TLC Thin layer chromatography
TNF Tumor necrosis factor
Tyr Tyrosine
α-TTP α-Tocopherol transfer protein
UV Ultraviolet
xxxii Clinical and Animal Studies of Lipid-Derived Protein Modifications in Autism, Kidney Dialysis, Keratitis and Age-Related Macular Degeneration
Abstract
By
LIANG LU
Lipid oxidation in diverse biological systems contributes to normal physiological
processes and is increasingly believed to be associated with age-related diseases. Lipid
oxidation products, e.g., isolevuglandins and γ-hydroxyalkenals, and their protein adducts
are produced during and may even contribute to disease pathogenesis. This thesis reports
clinical studies on the occurrence of 2-pentylpyrrole, iso[4]LGE2-protein adducts and
carboxyethylpyrrole (CEP) protein modifications in brain and blood from autistic
individuals, blood from hemodialysis patients, as well as cornea from a mouse model of
keratitis. Similar levels of these modifications and autoantibodies against them were
detected in blood from both autistic individuals and normal controls. Autistic patients born prematurely showed significantly elevated iso[4]LGE2-protein adduct levels. Oral
supplementation with α-tocopherol had no significant effect on levels of oxidative
protein modifications in patients treated by hemodialysis, and thus, does not result in an
antioxidant benefit in hemodialysis. We detected the formation of iso[4]LGE2- and
LGE2-protein adducts upon injection of lipopolysaccharide into mouse cornea, a model of
keratitis.
xxxiii We anticipated that docosahexaenoic acid-derived oxidatively truncated
phospholipids containing reactive electrophilic 4-hydroxy-7-oxohept-5-enoates (HOHA)
would convert the primary amino group of PEs into carboxyethylpyrrole phosphatidylethanolamine adducts (CEP-PEs). I detected a low level of CEP-PE in a bovine retinal lipid extract and quantified the generation of CEP-PE adduct and a CEP- modified lysophospholipid, lysoCEP-PE, upon UV light-promoted oxidation of the retinal lipid extract. Similar levels, about 1.2 ng/mL, of lysoCEP-PE were detected in plasma samples from a normal control and a patient with age-related macular degeneration (AMD).
A reliable source of CEP-modified proteins was needed to provide: (1) reagents for immunoassays that measure levels of CEPs or anti-CEP autoantibodies as biomarkers
for clinical prognosis of AMD, and (2) antigens to test the hypothesis that immune responses against CEP-protein adducts generated in the retina contribute to the pathogenesis of AMD, (3) for studies on the stimulation of angiogenesis by CEPs and its inhibition, and (4) for the production of CEP-PEs as standards for analyses of these potential markers of oxidative injury. An efficient, reliable synthesis of CEPs was
developed that exploits a flourenylmethyl ester derivative of HOHA as a key reagent.
xxxiv
Chapter 1
Introduction
1 1.1 Oxidative stress and ageing.
Living in an oxygenated environment has required the evolution of effective cellular
strategies to detect and detoxify metabolites of molecular oxygen known as reactive
oxygen species. The appropriate and inappropriate production of oxidants, together with the ability of organisms to respond to oxidative stress, is intricately connected to ageing and life span.
------Toren Finkel and Nikki J. Holbrook
Aging has been characterized as “the progressive accumulation of changes with time
that are associated with or responsible for the ever-increasing susceptibility to disease and
death which accompanies advancing age.”1 In 1956, Denham Harman proposed his
famous free radical theory of aging which speculated that free oxygen radicals produced
during aerobic respiration cause cumulative oxidative damage, resulting in aging and
death.2 Results from disparate experimental systems have recently shown that reactive
oxygen species (ROxS) production provides the strongest correlation with overall
longevity: the greater the production, the shorter the life span.3-6 Whether or not they
determine life span, it is becoming apparent that oxygen radicals are certainly important
players in aging’s pathophysiology.
ROxS include a variety of diverse chemical species such as superoxide anions,
hydroxyl radicals and hydrogen peroxide. These radical species can either be generated
2 exogenously or produced intracellularly from several different sources (Figure 1.1).
Figure 1.1. The sources and cellular responses to reactive oxygen species (ROxS).7
Although free radicals may certainly cause harm to biological processes, there is increasing evidence that products of free radical-induced lipid oxidation serve as specific signaling molecules under both physiological and pathophysiological conditions.8-11 For example, ROxS produced in response to stimulation by growth factors participate in regulating the proliferative response.12 The immune system utilizes some of these
3 products to mark foreign invaders or damaged tissue, and thus indicate which tissues
need to be removed from the body.13, 14 As a result, an elevation in intracellular oxidant
levels has two potentially important effects: damage to various cell components and triggering of the activation of specific signaling pathways.7 Both of these effects are
related to numerous cellular processes linked to aging and the development of age-related diseases.
An intricate enzymatic and non-enzymatic antioxidant defence system encompassing catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx), as well as small antioxidant molecules such as tocopherols, flavonoids, ascorbic acids, and carotenoids, restrains and regulates overall ROxS levels to maintain physiological homeostasis.15, 16 Perturbation of the physiological role of oxidants in cellular
proliferation and host defence might be caused by lower ROxS levels than the
homeostatic set point. Meanwhile, high ROxS levels may result in cell death or accelerate
aging and age-related diseases.7 This is traditionally referred to as oxidative stress.
Consequences of this stress include modification to cellular proteins, lipids and DNA.
1.2. Lipid oxidation.
Oxygen-dependent deterioration of lipids has been observed since antiquity as a big
problem in the storage of oils (mainly olive oil). The first studies of this lipid oxidation
problem were those of the Swiss chemist Nicolas-Theodore de Saussure who noticed around 1800 that oil became viscous and had a bad smell when exposed to air.17
4 Systematic studies of lipid autoxidation may be regarded to have initiated around the
1940s since Criegee et al.18 proposed that hydroperoxides are the primary products of
hydrocarbon oxidation. In 1961, Bolland19 documented that the primary autoxidation
products of linoleic acid are hydroperoxides (on carbon atom 9 or 13) containing
conjugated dienes. The free radical-induced oxidation of all-cis 8,11,14-eicosatrienoate and arachidonate was discovered in the 60s.20 Detailed studies of the autoxidation of
polyunsaturated fatty acids were initiated in the 70s by several research groups,
disclosing more complex mixtures21-23 than those previously proposed. The further
demonstration of oxidation of membrane phospholipids24 (PL) led to a new fruitful era
with a continuous flow of considerable research devoted to chemistry, biochemistry and
medicine.
Lipid oxidation in diverse biological systems contributes to normal physiological
processes. Hydroperoxides derived from arachidonic acid serve as modulators of the
enzymes involved in the biosynthesis of prostaglandins and leukotrienes.25-28 Oxidized
phospholipids (oxPL) are suspected in the pathogenesis of atherosclerosis29-33 and several
chronic inflammatory diseases such as antiphospholipid antibody syndrome34,
rheumatoid arthritis35, inflammatory bowel disease36, and multiple sclerosis.37 Lipid
oxidation may also be involved in the immune response38 or apoptosis (programmed cell
death) 39, 40, and is increasingly believed to be associated with age-related diseases.41-43
Research on the chemistry of lipid oxidation and the reactions of lipid oxidation products led to the identification of some new biological effects such as the covalent adduction
5 that modifies DNA or protein function as well as the receptor-mediated responses
triggered by oxPL or lipid-based protein modifications that act as “oxidative
messengers”.39, 44 However, our understanding of the intrinsic role that lipid oxidation
plays in both physiological and pathological processes is still in its infancy. Research in
Dr. Salomon’s group aims to understand the biologically important chemistry of oxidized
lipids and to determine the extent and consequences of such chemistry in pathogenesis of human diseases.
1.3. 4-Hydroxy-2-nonenal and its protein adducts. An important effect of lipid
peroxidation is decreased membrane fluidity, which alters membrane integrity and can
significantly disrupt membrane-bound proteins. Moreover, the peroxidation of lipids
generates reactive aldehydes, that may act as mutagens45 or inactivate enzymes,46-48 or react with proteins and nucleic acids to form heterogeneous cross-links.49 One of these aldehydes, 4-hydroxy-2(E)-nonenal (HNE), has been investigated extensively since the report that it inhibits glucose 6-phosphatase and cytochrome P-450.47, 50 HNE is a product of oxidative fragmentation of arachidonic acid (AA) or linoleic acid (LA) phospholipid esters. It is highly cytotoxic to Ehrlich ascites tumour cells.51 It can cause lysis of
erythrocytes50 and arouse chemiluminescence and pentane production in isolated
hepatocytes.52
HNE is highly reactive towards the nucleophilic residues of proteins such as lysine,
histidine and cysteine and generates Michael and Schiff base adducts as well as a pyrrole
adduct.53, 54 HNE can also mediate the formation of lysine-lysine crosslinks.55
6 Among the numerous adducts that HNE forms with proteins, 2-pentylpyrrole (PP) adducts, that incorporate the ε-amino group of protein lysyl residues, have been cited as one example of an “advanced lipid peroxidation end product” (ALPE).54 Mean PP levels
in plasma from individuals with atherosclerosis are double those found in healthy
controls.56 HNE and its protein adducts are therefore considered as established markers
for oxidative stress.57
1.4. Levuglandins, isolevuglandins and their protein adducts. The discovery of
levuglandins was the culmination of a ten year effort to elucidate the chemistry of the
prostaglandin endoperoxide PGH2. PGH2 is the cyclooxygenase metabolite of
58 arachidonic acid (AA). Spontaneous rearrangements of PGH2 generate PGD2 and PGE2.
In addition, rearrangement of PGH2 in aqueous solution produces levulinaldehyde
23 derivatives with prostanoid side chains, namely levuglandins (LGs). Two LGs, LGD2
and LGE2, are generated (Scheme 1.1). Non-enzymatic (free radical-induced) oxidation
of arachidonyl phospholipids (AA-PL) gives rise to four structurally isomeric families of
phospholipid endoperoxide stereoisomers, that have been named isoprostanes (isoPs).
Rearrangement of these endoperoxides produces four LGE2 structural isomers, i. e.,
isoLGE2, iso[4]LGE2, iso[10]LGE2, iso[7]LGE2 and four corresponding LGD2 structural
isomers (Scheme 1.2).59
Levuglandins and isolevuglandins are biologically active. Guanosine triphosphate
(GTP)-induced assembly of bovine microtubule protein is inhibited by LGs and
60, 61 chymotrypsin-like proteasomal activity is effectively restrained by isoLGE2.
7 (CH ) COOH HO 2 3 C5H11 O (CH ) COOH AA 2 3 (CH2)3COOH
C H O2 cyclooxygenase 5 11 C5H11 O OH PGD 2 O OH LGD2 (CH2)3COOH + O O C H O 5 11 O (CH2)3COOH PGH OH 2 (CH2)3COOH
C5H11 C5H11 HO OH PGE2 O OH LGE2
Scheme 1.1. Cyclooxygenase oxidation of AA generates PGs and LGs via rearrangement of PGH2.
O 10 7 O O C H Arachidonyl O O 15 31 phosphatidylcholine C H O P N(CH3)3 ester 13 5 11 O O -7 H -10 H -10 H -13 H
OH OH (CH2)3CO2R 8 O (CH2)3CO2R O (CH2)3CO2R O (CH2)3CO2R O O O C5H11 O C5H11 O C5H11 9 OH iso[7]PGH2-PC iso[10]PGH2-PC OH iso[4]PGH2-PC isoPGH2-PC
O OH O O OH O
(CH2)3CO2R 8 (CH2)3CO2 (CH2)3CO2R (CH2)3CO2R R 12 C5H11 C5H11 C5H11 9 O O OH O O OH
iso[7]LGE2-PC iso[4]LGE2-PC iso[10]LGE2-PC isoLGE2-PC ++ + +
O OH O O OH O
(CH2)3CO2R 8 (CH2)3CO2 (CH2)3CO2R (CH2)3CO2R R 12 C5H11 C5H11 C5H11 9 O O OH O O OH
iso[7]LGD2-PC iso[4]LGD2-PC iso[10]LGD2-PC isoLGD2-PC Scheme 1.2. Free radical-induced oxidation of AA-PC produces isoLGs by rearrangement of isoP intermediates.
8 LGs and isoLGs covalently bind avidly with proteins within minutes due to their
reactive electrophilic γ-ketoaldehyde functionality.62 Moreover, protein-protein
cross-links and protein polymerization can occur in conjunction with the binding, and this
is believed to be associated with some pathological process, e. g., Alzheimer’s disease
(AD). Levels of levulglandinyl-lysine lactam adducts of proteins are elevated in AD hippocampi compared to normal.63
A Schiff base, formed initially, eventually is transformed into a lactam through an
intermediate pyrrole. The lactam is the primary end product form of LGE2-protein
adducts.64 The LG-protein Schiff base adduct can act as a reactive electrophile. It binds
with a primary amino group of another protein leading to a protein-protein crosslink. LGs
also cause DNA-protein cross-links (DPCs) which are repair-resistant and were shown to
be relevant to cell killing (Scheme 1.3).65
Levuglandin-protein adducts are detectable in vivo using polyclonal rabbit antibodies
and are useful as unambiguous markers of oxidative injury. The mean levels of
iso[4]LGE2 and iso[7]LGE2-protein adducts, as well as LGE2-protein adducts, are
significantly elevated in plasma from individuals with atherosclerosis and renal disease
compared to normal individuals.66, 67 In addition, oxidative stress induced by Candida
infection in mice leads to a 3.5-fold elevation in plasma levels of iso[4]LGE2-protein
adducts.68 In this thesis, more pathological diseases are explored to monitor the levels of
iso[4]LGE2-protein adducts in plasma and thereby to further understand the biological
significance of levulglandin chemistry.
9 bis-pyrrole adduct O R1 R1 protein N
levuglandin R2 H C R2 2 R1 O protein N
protein NH2 R2
H2O R1 R O 1 protein N protein N R2 R2 Schiff Base LG-pyrrole protein NH NH2 DNA 2 O2, . ROO
OH R1 R1 O O R1 N protein N protein protein N R2 R2 H H R2 NH NH O protein DNA hydroxylactam +
OH OH R R1 R1 1 protein protein N protein N N R R2 R2 2 protein NH DNA NH O lactam
protein-protein crosslink DNA-protein crosslink
Scheme 1.3. Formation of LG-protein adducts, protein-protein and DNA-protein
crosslinks.
1.5. Oxidatively truncated phospholipids and carboxyalkylpyrrole modifications of
proteins. Free radical induced oxidation of arachidonate (C20) and linoleate (C18) phospholipids generates truncated C5 or C9 ω-oxoalkanoate phospholipids, 2-oxovaleryl
(OV) or 9-oxononanoyl (ON) phospholipids, respectively, as well as C8 or C12
γ–hydroxyalkenal phospholipids, 5-hydroxy-8-oxo-6-octenoic acid (HOOA) or
10 9-hydroxy-12-oxododec-10-enoic acid (HODA), which are precursors of
carboxyalkylpyrrole derivatives with proteins (Scheme 1.4).69 Both OV
phosphatidylcholine (OV-PC), and HOOA-PC induce monocyte binding to endothelial
cells but inhibit lipopolysaccharide (LPS) induced E-selectin expression which is a major
endothelial adhesion molecule involved in neutrophil binding.70 Moreover, HOOA-PC
and HODA-PC and their protein modification products, 2-(ω-carboxypropyl) pyrroles
(CPPs) and 2-(ω-carboxyheptyl) pyrroles (CHPs), have been confirmed to be present in
human atherosclerotic lesions. HOOA-PC and HODA-PC also impair the proteolytic
degradation of internalized macromolecules by mouse peritoneal macrophages (MPMs)
and inhibit the activity of the lysosomal protease, cathepsin B, as well as block the
maturation of the fusion protein Rab5a.71 All these results implicate that the oxidatively truncated phospholipids may contribute to chronic inflammation such as atherosclerotic plaques associated with the accumulation of cholesterol esters and the formation of foam cells.
1.6. Carboxyethylpyrroles (CEPs) and their potential clinical applications. Oxidative cleavage of phospholipids containing docosahexaenoic acid (DHA) produces reactive electrophilic 4-hydroxy-7-oxohept-5-enoic acid (HOHA)-PL that convert the primary
amino group of protein lysyl residues into 2-(ω-carboxyethyl)pyrrole (CEP) derivatives
(Scheme 1.4). DHA is most abundant in the brain72 and retina, and is concentrated in the light-sensitive photoreceptor rod outer segment (ROS) membranes and the retinal pigment epithelium (RPE).73 The extremely high oxygen levels of the environment, the
11 exquisite sensitivity to being oxidized due to its six double bonds, make DHA especially
important in the pathological processes associated with retinal diseases.
Polyunsatuated Fatty Acid (PUFAs) Hydroxy-ω-oxoalkenoic Acid Carboxyalkylpyrrole
Protein OH C5H11 (CH2)7COOH O2 Protein linoleic acid (LA) O N(CH2)7COOH (CH2)7COOH C H (CH ) COOH 2 5 2 7 HODA CHP linolenic acid
C5H11 (CH2)3COOH γ-linolenic acid Protein OH O Protein 2 O N(CH2)3COOH C5H11 (CH2)3COOH 2 (CH2)3COOH arachidonic acid (AA) HOOA CPP
C2H5 (CH2)3COOH 3 eicosapentaenoic acid Protein OH O2 Protein O N(CH2)2COOH C2H5 (CH2)2COOH 4 (CH2)2COOH docosahexaenoic acid (DHA) HOHA CEP Scheme 1.4. Oxidation of polyunsaturated fatty acids generates hydroxyl-ω-oxoalkenoic acids, that react with proteins and form 2-(ω-carboxyalkyl)pyrroles.
There are promising results that implicate CEPs as clinically useful markers of
age-related macular degeneration (AMD). CEPs and autoantibodies against CEPs are
elevated in the blood of individuals with AMD.42 Western blot analysis of PAGE gels of
protein extracts from the choroid/RPE/ROS complex of AMD retinas revealed markedly
elevated levels of CEP immunoreactivity. Proteomic characterization of drusen, deposits
that are a risk factor for AMD and accumulate below the RPE on Bruch’s membrane, also showed the presence of CEPs.41 A recent report announced that CEPs could induce
angiogenesis by promoting sprouting of new vessels into the retina (choroidal
neovascularization) (Figure 1.2), and damaging the photoreceptor cells.43 CEP levels are
12 elevated by a factor of four in the retinas of rats exposed to intense light compared to those raised in the dark (unpublished data from Renganathan, K.).
A HSA B CEP-HSA
C Dipeptide D CEP-Dipeptide
Figure 1.2. CEP-HSA as well as dipeptide (Ac-gly-lys-OH) induces angiogenesis in a rat corneal micropocket assay. (A) HSA (1 μg), (B) CEP-HSA (1 μg), (C) dipeptide (41 ng),
(D) CEP-dipeptide (37 ng).
We recently conducted mouse model studies that showed retina degeneration, holes forming inside the retina which looks exactly like AMD symptoms, after one year of
CEP-mouse serum albumin (MSA) injection into mice (Figure 1.3).74 Moreover, a significant number of CEP-MSA specific interferon-γ (IFN-γ) and interleukin-2 (IL-2) producing T cells in lymph nodes and spleen of immunized mice as well as an increase of anti-CEP autoantibody titers were detected in immunized mice treated with CEP-MSA.75
13 These observations demonstrate that immune responses to a CEP adducted self-protein in
the retina can lead to the generation of an adaptive immune response that results in retinal
damage and possibly AMD. It is also suggested that an in vivo chemical modification
induced by oxidative stress in a specific organ can lead to autoimmunity.
Figure 1.3. Outer retinal pathology present in mice immunized with CEP-MSA (A-J).
Large arrows indicate several inflammatory cells immediately adjacent to the retinal pigment epithelium (RPE) or in the interphotoreceptor matrix (IPM). Large empty vacuoles are also evident (C-I). Some of which appear to be intracellular (I). In (J) the
RPE has degenerated (asterisks). Bar in lower right of (J) represent 25 μm.
In view of the abundance of DHA in brain tissues, it is presumed that CEP locates in the brain and its level might be related to neurological diseases. Immunohistochemical staining of cortical tissue from four autistic brains and five age-matched normal brains
14 using anti-CEP polyclonal antibody exhibited stained filaments in all of the autistic brains
while no staining in the control brains.76
In summary, there is increasing evidence that lipid oxidation products and lipid-derived
protein adducts are related to specific pathological processes. This thesis focuses on
understanding the role of oxidative injury in various diseases and clinically characterizing the disease-related levels of specific lipid oxidation products in tissues and plasma. These studies provide a foundation for the development of diagnostic methods and therapeutic interventions for diseases associated with oxidative injury.
Chapter 2 describes the development of an efficient synthesis of CEP derivatives such
as CEP-proteins, CEP-phosphatidylethanolamine (PE) and CEP biotinylated derivatives.
The synthesis of CEP-MSA enables the establishment of a mouse animal model to study
immune mechanisms that could lead to the development of AMD and could be used to
develop new therapies. The characterization of CEP-proteins was also accomplished
using tryptic digestion in conjunction with quadrupole time-of-flight (Q-TOF) mass
spectrometry and tandem mass spectrometric analysis.
Chapter 3 explores the contribution of oxidative injury to autism by monitoring levels
of iso[4]LGE2-protein adducts, CEP and corresponding autoantibody titers as well as nitrotyrosine in plasma from autistic patients and normal controls.
Chapter 4 assesses the effects of oral vitamin Eα supplementation on the levels of
pentosidine, iso[4]LGE2-, 2-pentylpyrrole-, and (E)-4-oxo-2-nonenal (ONE)-protein
adducts in hemodialysis patients.
15 Chapter 5 establishes methodology that was used to quantify CEP-PEs in lipid extracts
from bovine retina as well as from plasma samples of an AMD patient and a normal
control by using authentic standards. The formation of these adducts in vivo is predicted
to be associated with the pathogenesis of AMD.
Chapter 6 describes a pilot study of LPS-induced generation of LGE2 and iso[4]LGE2 protein modifications in mouse cornea model of keratitis.
Chapter 7 describes the total synthesis of a pyrazole isosteres of 2-pentylpyrroles.
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26
Chapter 2
Syntheses and Characterization of Carboxyethylpyrroles
27 2.1. Background
Although scarce in most human tissues, docosahexaenoic acid (DHA) is essential for
the growth, functional development and maintenance of the brain.1 Phospholipids
containing DHA are major structural elements of photoreceptor cell membranes in the
retina.2, 3 Owing to the presence of the six homoconjugated C=C bonds in DHA, it is exquisitely sensitive to oxidative damage.4 Oxidative cleavage of retinal phospholipids
containing DHA produces reactive electrophilic 4-hydroxy-7-oxohept-5-enoates that
convert the primary amino group of protein lysyl residues into 2-(ω-carboxyethyl)pyrrole
(CEP) derivatives. CEPs are especially abundant in the retinas of individuals with age-
related macular degeneration (AMD), a slow, progressive disease of the eye5 that is the
major cause of untreatable loss of vision among the elderly in developed countries.6, 7
Roughly 11 % of people in the United States have AMD, and owing to increases in
human life span, AMD is expected to nearly double in the next 25 years.8, 9 Proteomic
characterization of drusen, deposits that accumulate in the back of the retina, revealed
that CEP adducts are associated with drusen proteins.10 CEPs and autoantibodies against
CEPs are elevated in the blood of individuals with AMD.11 CEPs are not simply benign
markers of oxidative damage. We recently reported that CEPs promote the growth of
capillaries into the retina (choroidal neovascularization), and destruction of the
photoreceptor cells.12 Such neovascularization is responsible for 90 % of the loss of
vision associated with AMD.
This chapter describes the development of an efficient synthesis of CEPs that will
enable (1) their use as biomarkers for clinical prognosis of AMD, (2) their use as antigens
to test the hypothesis that immune responses against CEP-protein adducts generated in
the retina contribute to the pathogenesis of AMD through the activation of B- and T-cells,
28 (3) their use as “bait” to capture human ScFv antibodies from a phage display library, and
(4) for studies on the stimulation of angiogenesis by CEPs and its inhibition. The key
finding of this study is that a 9-fluorenylmethyl ester of 4,7-dioxoheptanoic acid reacts
with primary amines to provide esters of CEPs that can be deprotected with DBU without
causing protein denaturation. The introduction of multiple CEPs into proteins is readily
achieved using this strategy. In addition, the preparation of CEPs tethered to proteins
through an ω-amino hexanoate linker and their strong binding with anti-CEP antibodies is described. Characterization of the CEP adducts generated on the lysyl primary amino groups of proteins was achieved by quadrupole time-of-flight (Q-TOF) mass spectrometry and MS/MS analysis.
29 2.2. Results and Discussion
2.2.1. Paal-Knoor synthesis using 4,7-dioxoheptanoic acid is ineffective for the
preparation of CEPs. The reaction of γ-ketoaldehydes with primary amines, the Paal-
Knoor synthesis, is generally an efficient method for the preparation of pyrroles.13 This reaction was successfully applied to the generation of carboxyheptylpyrrole and carboxypropylpyrrole derivatives through the reactions of 9,12-dioxododecanoic or 5,8- dioxooctanoic acid with proteins. However, attempts at preparing the corresponding carboxyethylpyrrole derivatives of proteins by treatment with 4,7-dioxoheptanoic acid
(DOHA) generally caused precipitation, and in the few instances in which precipitation did not occur, the ratio of pyrrole to protein, e.g. 1.6:1 for human serum albumin,11 was
much lower than were obtained previously for the longer chain carboxyalkylpyrroles.14
Another distinguishing feature of DOHA was the nearly complete absence of a signal for the aldehydic hydrogen in its 1H NMR spectrum. We postulated that the unusual 1H
NMR spectrum and abberant reactivity of DOHA is a consequence of the proximity of the carboxyl group to the γ-ketoaldehyde array, and that DOHA exists in equilibrium with a spiroacylal hemiacetal (Scheme 2.1). To obviate complications engendered by the carboxyl group, we sought a masked derivative that could be deprotected under conditions that would not lead to denaturation and consequent precipitation of proteins.
This excluded acidic conditions. Therefore, we opted for a 9-fluorenylmethyl ester.
OH O N O O OH Protein CEP O DOHA NH HO 2 O Protein O O
Scheme 2.1. DOHA exists in equilibrium with a spiroacylal hemiacetal.
30 2.2.2. Synthesis of a 9-fluorenylmethyl (Fm) ester of DOHA. Coupling of 3-
carbomethoxypropionyl chloride with the Grignard reagent derived from 2-(2-
bromoethyl)-1,3-dioxolane provides access to the methyl ester 2.1 (Scheme 2.2). The
desired 9H-fluoren-9-ylmethyl ester 4,7-dioxo-heptanoic acid (DOHA-Fm, 2.4) was then obtained through saponification to afford the carboxylic acid 2.2, and esterificaton with
9-fluorenylmethanol, followed by hydrolysis of ethylene ketal in 2.3.
O O O Br OR 1) Mg, THF O O 2) ClCO(CH ) COOMe O 2 2 2.1, R = CH (60%) THF, -78 °C 3 NaOH, H O/MeOH/THF 2.2, R = H (90%) 2 FmOH, DCC, DMAP, CH Cl 2.3, R = Fm (95%) 2 2
O 88% O O HOAc:H2O (3:1, v/v) O 50 °C DOHA-Fm (2.4)
Scheme 2.2. Synthesis of 9H-fluoren-9-ylmethyl ester 4,7-dioxo-heptanoic acid (DOHA-
Fm, 2.4).
2.2.3. Syntheses of CEP-peptide and CEP-protein adducts by Paal-Knoor synthesis
with DOHA-Fm. Paal-Knoor synthesis of the Fm ester 2.5 of a CEP dipeptide was
O O O O H H N N N OMe N OMe H H O 80% O DOHA-Fm methanol
NH2 N COOR
Ac-Gly-Lys-OMe 2.5, R = Fm DBU,THF 2.6, R = H 86%
Scheme 2.3. Synthesis of CEP-dipeptide.
31 readily achieved in 80 % yield by the condensation of methyl 6-amino-2-((2-
acetylamino)acetyl)amino)hexanoate (Ac-Gly-Lys-OMe) with 1 equivalent of DOHA-
Fm (Scheme 2.3). For peptide synthesis, the deprotection of 9-fluorenylmethyl esters is
normally accomplished with piperidine in DMF. Piperidine serves both as a base to
fragment the Fm group and as a scavenger to trap the dibenzofulvene (DBF) released.15
The removal of an Fm group from an ester bound to a protein has not been reported.
Piperidine was unsatisfactory, vide infra. This was readily determined by the persistence of a UV absorbtion at 265 nm that is characteristic of the flourene group. Therefore, we examined the efficacy of a stronger base. 1,8-Diazabicyclo[5.4.0] undec-7-ene (DBU) successfully removed all Fm groups from protein adducts, vide infra, and this reagent was also applied to deprotection of the dipeptide Fm ester 2.5. Removal of the Fm protecting group by treatment of 2.5 with DBU in THF solution delivered the CEP dipeptide 2-(2-acetylamino-acetylamino)-6-[2-(2-carboxy-ethyl)-pyrrol-1-yl]-hexanoic acid methyl ester (2.6) in 86 % yield.
CEP-modified proteins are needed for immunoassays that measure levels of CEPs or anti-CEP autoantibodies in vivo. We also required CEP-protein adducts as antigens to immunize animals as models to test the hypothesis that immune responses against CEP- protein adducts generated in the retina contribute to the pathogenesis of AMD. We now find that the preparation of CEP-protein adducts can be readily accomplished by incubation of DOHA-Fm (2.4) with protein in 30 % DMF/phosphate-buffered saline
(PBS) solution for 5 days at 37 ˚C followed by deprotection by addition of DBU to the reaction mixture and stirring for an additional 9 h. One equivalent of DOHA-Fm was used for each lysine group present in a particular protein (Scheme 2.4). Proteins used for making CEP adducts were: human serum albumin (HSA), mouse serum albumin (MSA),
32 chicken egg albumin (CEO), rabbit myoglobin and glycerol-3-phosphate dehydrogenase
(GPDH). Low molecular weight contaminants were removed by dialysis (Mr cutoff
14000) of the reaction mixture against 20% DMF in 10 mM PBS. An especially
important feature of the use of Fm esters of DOHA is the ease with which residual Fm
groups can be detected and their complete removal assured by UV spectroscopy, i. e.,
absorption at 265 nm. The final protein concentration was determined by the Pierce
bicinchoninic acid (BCA) protein assay16 or Bio-rad protein assay.17 The pyrrole concentration was determined by the generation of a characteristic chromophore through reaction with 4-(dimethylamino)benzaldehyde, the Ehrlich reagent,18 using the CEP
dipeptide 2.6 as a quantitative standard. In contrast with the preparation of CEP-HSA by direct treatment with DOHA, that delivered a pyrrole to protein ratio of 1.6:1 for CEP-
HSA,11 the new synthetic method using DOHA-Fm provided CEP-HSA (2.7) with a
much higher pyrrole to protein ratio, 7.6 ± 1.1 to 1, and provided CEP-MSA (2.8) with a
pyrrole to protein ratio of 5.2 ± 1.0 to 1. Further characterization of the CEP adducts
generated on the primary amino groups of protein lysyl residues was achieved by QTOF
mass spectrometry and MS/MS analysis.
NH2 O N protein DOHA-Fm (2.4) O DMF, PBS protein protein = HSA protein = MSA protein = CEO protein = myoglobin 2.7, protein = HSA protein = GPDH COOH 2.8, protein = MSA N DBU,DMF, PBS 2.9, protein = CEO protein 2.10, protein = myoglobin 2.11, protein = GPDH
Scheme 2.4. Syntheses of CEP protein adducts.
33 2.2.4. CEP linked to proteins with an ω-aminohexanoyl tether. Direct coupling of
DOHA-Fm to proteins results in a high yield of CEP modifications of lysyl residues. As an alternative approach for anchoring CEP haptens to proteins for use as coating agents to capture anti-CEP antibodies, we examined the utility of CEPs anchored to proteins through hexanoyl amides of protein lysyl residues. An Fm masked 2-carboxyethylpyrrole
2.12 was generated through the reaction of DOHA-Fm with 6-aminocaproic acid. After purification, 2.12 was activated by conversion into an N-hydroxysuccinimide ester 2.13
(CEPFmSu). Incubation of the active ester 2.13 with bovine serum albumin (BSA)
( CEPFmSu : Lys = 1.5 :1, mol/mol), followed by deprotection in situ by addition of
DBU to the reaction mixture, delivered a 6-(2-carboxyethyl-1-pyrrolyl)hexanoyl amide derivative of BSA, CEPH-BSA (2.14, Scheme 2.5). Low molecular weight impurities were readily removed by dialysis (Mr cutoff 14000) with 20% DMF in 10 mM PBS and then with 10 mM PBS. The protein concentration was determined by a modified Lowry protein assay19 using the Lowry protein assay reagent and Folin-Ciocalteu reagent. The pyrrole concentration was determined using an Ehrlich assay. The pyrrole to BSA ratio in
CEPH-BSA (2.14) was 5.4 ± 0.9 to 1. CEP linked to Mouse albumin, chick albumin and
GPDH with this tether was also prepared.
34 O HOOC(CH ) NH 90% 2 5 2 HOOC(CH2)5N 80% N OOC(CH2)5N + HOSu H O 2 DCC DOHA-Fm (2.4) MeOH O 2.12 COO-Fm 2.13 CH2Cl2 COO-Fm DMF, PBS O O NH2 HN (CH ) N HN (CH2)5N 2 5 DBU protein protein protein DMF protein = BSA PBS COOH protein = MSA COO-Fm protein = CEO 2.14, CEPH-BSA, protein = BSA, protein = GPDH 2.15, CEPH-MSA, protein = MSA 2.16, CEPH-CEO, protein = CEO 2.17, CEPH-GPDH, protein = GPDH Scheme 2.5. Syntheses of CEPs bound to proteins through a tether.
The antibody binding affinity of CEPH-BSA (2.14) was determined by competitive
enzyme-linked immunosorbent assay (ELISA)20 using an anti-CEP-KLH polyclonal
antibody (Figure 2.1). CEP-HSA was used as a coating agent and standard whose binding was inhibited by CEPH-BSA. The IC50 of CEPH-BSA (1.93 pmol/mL) is lower than the
IC50 of CEP-HSA (3.02 pmol/mL) indicating that this preparation of CEPH-BSA has a slightly higher affinity than CEP-HSA for binding anti-CEP-KLH antibody.
To optimize the coupling reaction conditions, we used different protein concentrations
(1 mg/mL, 2.6 mg/mL, 3 mg/mL, 4 mg/mL, and 10 mg/mL), and coupled the protein with different CEPFmSu:Lys mole ratios (0.5:1, 1:1, 1.5:1). The pyrrole concentration
(relative to protein) of different coupling reactions was compared after normalizing the final protein concentration to 1 mg/mL (Table 2.1). The best coupling was achieved by incubating 3 mg/mL BSA with CEPFmSu:Lys 1.5:1 in 15% DMF.
35 120
100
80
60
IC50 = 3.02 40
20 IC50 = 1.93 Absorbance (%max)
0
0.01 0.1 1 10 100 1000 Inhibitor (pmol/ml)
Figure 2.1. Inhibition curves showing cross-reactivity of the anti-CEP-KLH antibody for CEP-HSA (▲) and CEPH-BSA (●) against CEP-HSA as coating agent.
Table 2.1. Pyrrole concentration (μM) generated in 1 mg/mL CEPH-BSA prepared by coupling various initial protein concentrations with various CEPFmSu/Lys ratios.
Protein Conc. 1 mg/ml 2.6 mg/ml 3 mg/ml 4 mg/mL 10 mg/mL CEPFmSu:Lys 0.5:1 20 25 35 N/A N/A
1:1 28 N/A 40 31 N/A
1.5:1 30 34 42 33 precipitate
2.2.5. Syntheses of biotinylated CEP derivatives. We want to explore the utility of monoclonal scFv antibodies (vide infra) as competitive inhibitors of the choroidal neovascularization that is promoted by CEPs. Immunoglobins and B-cells bind divalently through their variable (Fv) regions with haptens such as CEP. This can cause aggregation
36 of proteins and/or cells displaying CEP modifications, and can activate B-cells.
Immunoglobins also possess a constant (Fc) effector region that activates immune responses. In contrast, monoclonal scFv antibodies are monovalent constructs that contain only a single Fv binding region and no constant Fc region, and are therefore not expected to cause protein aggregation and deposition. Furthermore, monoclonal scFv antibodies are likely to favor penetration of the internal limiting membrane and access the subretinal space when injected intravitreously because of their relatively low molecular weight.21 Therefore, we anticipate that a monoclonal anti-CEP scFv antibody will be useful as a competitive inhibitor of CEP induced neovascularization in the eye.
Specific human scFv antibodies can be selected from engineered libraries of phage that display the antibody protein on their exterior. Selection is accomplished by using the corresponding hapten as “bait” to catch phage that produce (with the help of E. coli) and display antigen specific scFv on their exterior. One approach involves anchoring the hapten, through a biotin linker, to streptavidin-coated magnetic beads. For this purpose we prepared the biotinylated CEP Fm ester (2.20) from DOHA-Fm and 4-amino butylbiotin (Scheme 2.6). Deprotection of the intermediate 3-(1-[4-[5-(2-Oxo-hexahydro- thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-butyl]-1H-pyrrol-2-yl)-propionic acid 9H- fluoren-9-ylmethyl ester 2.20 by treatment with DBU in THF generated 3-(1-[4-[5-(2-
Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-butyl]-1H-pyrrol-2-yl)- propionic acid (2.21).
37 O O H N O-t-Bu NH NH 80% NH NH H2N(CH2)4 O O O H (CH2)4 N O-t-Bu S (CH2)4 OPhNO S (CH2)4 N 2 H 2.18 O O O 90% 84% ROOC NH NH NH NH CF3COOH O DOHA-Fm MeOH O CH2Cl2 (CH ) NH (CH ) N 2 4 2 (CH2)4 N S 2 4 S (CH2)4 N H H 2.19 2.20, R = Fm DBU, THF 2.21, R = H 85% Scheme 2.6. Synthesis of biotinylated CEP derivative 2.21.
All of the functionality in biotinylated CEP derivative 2.21 survived treatment with
sodium hydroxide ethanol solution. This observation suggested the feasibility of simpler
synthesis for preparing CEP derivatives of substrates that are stable to strong base. Thus,
we prepared 3-(1-[4-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino] -
hexyl]-1H-pyrrol-2-yl)-propionic acid (2.25) by reacting 4,7-dioxo-heptanoic acid methyl
ester (DOHA-Me, 2.22) with biotinyl-1,6-diaminohexane (2.23) followed by hydrolysis
of the methyl ester 2.24 with ethanolic sodium hydroxide (Scheme 2.7).
O O OMe O O 87% OMe H O O 2.1 O 2 HOAc 2.22 O O O 90% NH NH O NH NH O H2N(CH2)6NH2 OC H NO pyridine 6 4 2 NH(CH2)6NH2 S H2O S O 2.23
NH NH 2.22 75% O + MeOH N 2.23 NH(CH2)6 S O 2.24, R = CH 3 NaOH, EtOH 2.25, R = H OR 84%
Scheme 2.7. Synthesis of biotinylated CEP derivative 2.25.
38 2.2.6. Syntheses of ethanolamine phospholipid CEP derivatives.
Phosphatidylethanolamines (PEs) are major components of certain membranes in brain
cells and in photoreceptor cells of the retina. Because levels of PEs are strictly regulated,
they are believed to have unique functional importance.22 In view of the reactivity of the
primary amino group of PEs and the abundance of DHA in brain and retina, we anticipate
that DHA-derived oxidatively truncated phospholipids containing reactive electrophilic
4-hydroxy-7-oxohept-5-enoates convert the primary amino group of PEs into CEP-PE
derivatives. We synthesized CEP-PEs to facilitate their detection and identification in
vivo. Reaction of DOHA-Fm with 1-palmityl-2-oleyl-sn-glycero-3-phosphoethanolamine
(POPE) or 1-palmityl-2-hydroxy-sn-glycero-3-phosphoethanolamine (lysoPE) followed
by deprotection of the intermediate Fm esters 2.26 or 2.28 with DBU (Scheme 2.8) delivered the CEP-PE 2.27 and lysoCEP-PE 2.29. Using these authentic standards, we
identified CEP-PEs in lipids extracted from retina. Details of those studies are reported in
Chapter 5.
O CO POPE 76% R O P O O Fm O 2.4 + or OH N lysoPE TEA, CHCl3 O C H 14 29 2.26, R = oleyl O 2.28, R = H
O CO R O P O OH 90% O OH N DBU, CHCl 3 O C H 14 29 2.27, R = oleyl O 2.29,R=H
Scheme 2.8. Syntheses of ethanolamine phospholipid CEP derivatives.
2.2.7. Synthesis of an active pentafluorophenyl ester of a lysyl CEP.
Pentafluorophenyl esters of protected amino acids are widely used in peptide synthesis.
We are interested in testing the ability of CEP modified peptides bound to major
39 histocompatibility proteins to elicit an immune response to antigen specific T-cells.
Furthermore, complexes of CEP modified peptides bound to constructs called “dimer X”,
that have major histocompatibility proteins fused to immunoglobin Fc constant regions,
can be used to fluorescently label antigen specific T-cells, and consequently enable their
quantitation by fluorescence activated cell sorting. The pentafluorophenyl ester 2.31 of a
CEP-Fm modified lysine was synthesized as a building block for construction of CEP
modified peptides. Reaction of DOHA-Fm with 6-amino-2-(9H-fluoren-9-
ylmethoxycarbonylamino)-hexanoic acid (Fmoc-Lys-OH) delivered 2.30. The latter was
then coupled with pentafluorophenol using the traditional DCC, DMAP method (Scheme
2.9).
FmO O H COOH H COOH F F Fmoc N Fmoc N O 75% O O 90% F N DOHA-Fm OFm C F OH, DCC 6 5 F F HN MeOH DMAP, CH Cl HOAC N 2 2 NH2 2.30 Fmoc 2.31 Scheme 2.9. Synthesis of a lysyl CEP pentafluorophenyl derivative.
2.2.8. Characterization of CEP-modified protein. To characterize the CEP adducts in
protein following modification with DOHAFm or DOHAFmSu and DBU deprotection,
the proteins were digested with trypsin (Protein:trypsin = 50:1, w/w) and analyzed by
liquid chromatography tandem MS (LC MS/MS) with a CapLC system (Micromass,
Beverly, MA) and a quadrupole time-of-flight mass spectrometer (QTOF2,
Micromass).23 Peptides were separated on a 75 μm x 5 cm Biobasic C18 column (New
Objective, Cambridge, MA) by using aqueous formic acid/ acetonitrile solvents, a flow
rate of 250 nL/min, and a gradient of 5-40% acetonitrile over 57 min followed by 80%
acetonitrile for 2 min. Protein identifications from MS/MS data was accomplished with
MASSLYNX 3.5 software (Waters), the Mascot search engine (Matrix Science) and the
40 Swiss-protein and National Center for Biotechnology information protein sequence
databases.24, 25 MS/MS analysis of modifications at specific m/z revealed peptide
sequences with unambiguous CEP or CEPH adducts on lysyl residues of the CEP-
proteins or CEPH-proteins. No CEPFm or CEPHFm modifications were found after
deprotection. We also analyzed CEPFm modified HSA before deprotection. The LC-MS
revealed 3 peptides with CEPFm modifications at m/z 725.281, 660.289 and 776.820 on
lysyl residues of the HSA peptides 25DAHKSEVAHR34, 234AFKAWAVAR242, and
247FPKAEFAEVSK257in CEPFmHSA (Appendix Figure S49). In addition, owing to spontaneous loss of the Fm group, 5 peptides with CEP modifications were found at m/z
636.248, 881.408, 571.262, 687.787 and 674.765 on lysyl residues of the HSA peptides
25DAHKSEVAHR34, 438KVPQVSTPTLVEVSR452, 234AFKAWAVAR242,
247FPKAEFAEVSK257, and 35FKDLGEENFK44 respectively in CEPFmHSA (Appendix
Figure S48).
2.2.9. 3 D protein structures of CEP modified HSA. LC-MS/MS revealed six CEP
modifications at m/z 589.266, 571.260, 674.762, 881.401, 687.784, 636.252 on lysyl
residues of the HSA peptides 161KYLYEIAR 168, 234AFKAWAVAR242,
35FKDLGEENFK44, 438KVPQVSTPTLVEVSR452, 247FPKAEFAEVSK257, and
25DAHKSEVAHR34 respectively. Notably, the sequence 234AFKAWAVAR242 is very
similar to the sequence 233ALKAWSVAR241 in BSA and identical with
234AFKAWAVAR242 in MSA. MS/MS analysis revealed modification on ALKAWSVAR
sequence of CEPH-BSA in preparations generated with different CEP ratios, implying
that this sequence is readily accessed and modified. Figure 2.2 represents a ball-and-stick
model of 3D visualization of HSA using PyMOL v0.99. X- ray crystallography of HSA
reveals a dimer structure with two identical units. We show all lysines as space-filling
41 structures in the left unit while only the lysines that become modified by CEP are shown
as space-filling structures in the right unit. The lysine residue in the 234AFKAWAVAR242
sequence is marked. The 3D structure shows that this lysine is located at the surface of
HSA and protrudes from the surface of the molecule. In addition, this sequence
incorporates nonpolar amino acid residues on both ends. The physical and chemical
characteristics of this specific sequence probably facilitate access by DOHA-Fm. Further
research is underway to synthesize this CEP modified peptide bound to major
histocompatibility complex (MHC) and to test its ability to elicit an immune response to
antigen specific T-cells.
K236
Figure 2.2. A ball-and-stick model of 3D structure for human serum albumin. All the lysines are shown as space-filling structures in the left unit. Only lysines that become
CEP modified are shown in the right unit. The lysine in the sequence which is common to
HSA, BSA and MSA is marked.
42 2.3. Conclusions.
The introduction of multiple CEP modifications of lysyl residues is readily achieved
through reaction of proteins with DOHA-Fm, a 9-fluorenylmethyl ester of 4,7- dioxoheptanoic acid, followed by deprotection of intermediate Fm esters of CEPs, without causing protein denaturation, by treatment with DBU. CEP-proteins and peptides, that are now readily available through the new methodology, are being evaluated as biomarkers for clinical prognosis of AMD, and in studies of their role in promoting AMD through the activation of B- and T-cells. The DOHA-Fm reagent is also useful for the preparation of CEPs linked to biotin as ligands to capture human scFv antibodies from phage display libraries, and to generate CEP modified ethanolamine phospholipids as standards to confirm their presence in retina. The characterization of CEP modified proteins was done by tandem mass spectrometry. LC-MS/MS analysis of modifications revealed peptide sequences with unambiguous CEP or CEPH adducts on lysyl residues of
HSA, BSA, MSA, CEO, myoglobin and GPDH. CEP peptides for HSA were described above, CEP and CEPH modified peptides from the remaining proteins are detailed in the experimental section.
43 2.4. Experimental Procedures
General Methods.
1H NMR spectra were recorded on a Varian Gemini spectrometer operating at 200, 300
MHz, or on a Varian Inova spectrometer operating at 400 MHz using CDCl3 (δ 7.24) or
CD3OD (δ 3.30) as internal standard. All chemical shifts are reported in parts per million
(ppm) on the δ scale relative to the internal standard used. 1H NMR spectral data are
tabulated in terms of multiplicity of proton absorption (s, singlet; d, doublet; dd, doublet
of doublets; dt, doublet of triplets; t, triplet; m, multiplet), coupling constants (Hz),
number of protons. 13C NMR spectra were recorded on a Varian Gemini spectrometer
operating at 75 MHz or on a Varian Inova spectrometer operating at 100 MHz using
CDCl3 (δ 77.0), or CD3OD (δ 49.0) as internal standard. High-resolution mass spectra
were recorded on a Kratos AEI MS25 RFA high-resolution mass spectrometer at 20 eV.
Chromatography was performed with ACS grade solvents (ethyl acetate, hexane,
chloroform, or methanol). Thin-layer chromatography (TLC) was performed on glass
plates precoated with silica gel (Kieselgel 60 F254, E. Merck, Darmstadt, West Germany).
Rf values are quoted for plates of thickness 0.25 mm. The plates were visualized by
viewing the developed plates under short-wavelength UV light, iodine, or a phospholipid
spray (Molybdenum spray).26 Flash column chromatography was performed on 230-400
mesh silica gel supplied by E. Merck. For all reactions performed in an inert atmosphere,
argon was used unless otherwise specified.
Bovine serum albumin (BSA, Cat. A2153), human serum albumin (HSA, Cat. A1887)
and chicken egg albumin (CEO, Cat. A5503) as well as myoglobin (Cat. M1882) and
glycerol-3-phosphate dehydrogenase (GPDH, Cat. G6880) were purchased from Sigma.
44 6-[1,3]Dioxolan-2-yl-4-oxo-hexanoic acid methyl ester (2.1). A solution of 2-(2-
bromoethyl)-1,3-dioxolane (1 g, 5.5 mmol) in anhydrous THF (2 mL) was added
dropwise to a flame dried 100 mL flask with Mg turnings (150 mg, 6.25 mmol) and THF
(4 mL) and a small piece of I2 were added under argon at room temperature to initiate the reaction. After adding a few drops, the reaction started as evidence by disappearance of the red-brown I2 color. Then THF (5 mL) was added to the flask. After completion of the
addition, more THF (15 mL) was added. The reaction mixture was stirred for another 1 h
and then cooled to -78 °C followed by slow addition of 3-carbomethoxypropionyl
chloride (710 mg, 4.7 mmol) dissolved in dry THF (2.5 mL). The resulting mixture was
stirred for another 40 min, then quenched with 30 mL of a saturated aqueous solution of
NH4Cl, and extracted with EtOAc (4 x 15 mL). The combined organic phase was washed
with brine, dried with MgSO4, and evaporated to obtain the crude product. The crude
product was purified by silica gel chromatography (30 % ethyl acetate in hexane, TLC: Rf
1 = 0.3) to give 714 mg (60 %) of pure 2.1. H NMR (CDCl3, 200 MHz) δ 4.85 (t, J = 4.3
Hz, 1H), 3.7-3.90 (4H), 3.62 (s, 3H), 2.67 (t, J = 7.48 Hz, 2H), 2.56 (t, J = 7.48 Hz, 4H),
13 1.93 (dt, J = 4.3, 7.48 Hz, 2H). C NMR (CDCl3, 50 MHz, APT) δ 207.81 (+) (CO),
173.09 (+) (COO), 103.06 (-) (CH), 64.83 (+) (CH2), 51.61 (-) (CH3), 36.87 (+) (CH2),
36.30 (+) (CH2), 27.62 (+) (CH2), 27.41 (+) (CH2). HRMS (FAB) (m/z) calcd for
+ C10H17O5 (MH ) 217.1076, found 217.1081.
6-[1,3]Dioxolan-2-yl-4-oxo-hexanoic acid (2.2). Ester 2.1 (390 mg, 1.8 mmol) in 10
mL of H2O/MeOH/THF (2:5:3, v/v/v) was stirred for 3 h with NaOH (367 mg, 9.2 mmol)
at room temperature. The reaction mixture was then acidified with 3 N HCl to pH 3.0 and
extracted with EtOAc (3 x 15 mL). The combined organic phase was washed with brine,
1 dried with MgSO4, and evaporated to give acid acetal 2.2 (360 mg, 90%). H NMR
45 (CDCl3, 200 MHz), δ 4.85 (t, J = 4.3 Hz, 1H), 3.7-3.90 (4H), 2.5-2.64 (6H), 1.93 (dt, J =
13 4.3, 7.48 Hz, 2H). C NMR (CDCl3, 100 MHz,) δ 207.81 (CO), 178.03 (COOH), 103.17
(CH), 64.97 (CH2), 36.76 (CH2), 36.36 (CH2), 27.71 (CH2), 27.51 (CH2). HRMS (FAB)
+ (m/z) calcd for C9H15O5 (MH ) 203.0919, found 203.0917.
6-[1,3]Dioxolan-2-yl-4-oxo-hexanoic acid 9H-fluoren-9-ylmethyl ester (2.3). (9H-
Fluoren-9-yl)-methanol (373 mg, 1.9 mmol) in 3 mL dry CH2Cl2 was slowly added to the
solution of dicyclohexylcarbodiimide (DCC, 295 mg, 1.425 mmol), dimethlamino
pyridine (DMAP, 58 mg, 0.475 mmol) and the acid 2.2 (96 mg, 0.475 mmol) in 5 mL dry
CH2Cl2. The resulting mixture was stirred for 72 h at room temperature. The solvent was
removed. Flash chromatography of the residue (30 % ethyl acetate in hexane, TLC: Rf =
1 0.3) gave the ester 2.3 (153 mg, 95 %). H NMR (CDCl3, 200 MHz) δ 7.77 (d, J = 6.74
Hz, 2H), 7.60 (d, J = 7.7 Hz, 2H), 7.42 (dd, J = 6.74, 7.7 Hz, 2H), 7.29 (dd, J = 6.74, 7.7
Hz, 2H), 4.90 (t, J = 4.3 Hz, 1H), 4.40 (d, J = 7.38 Hz, 2H), 4.21 (t, J = 7.38 Hz, 1H),
3.80-3.96 (4H), 2.62-2.73 (4H), 2.56 (t, J = 7.3 Hz, 2H), 1.98 (td, J = 7.3, 4.3 Hz, 2H).
13 C NMR (CDCl3, 50 MHz, APT), δ 207.76 (+) (CO), 172.62 (+) (COO), 143.69 (+) (C),
141.19 (+) (C), 127.69 (-) (CH), 127.04 (-) (CH), 125.00 (-) (CH), 119.92 (-) (CH),
103.12 (-) (CH), 66.45 (+) (CH2), 64.90 (+) (CH2), 46.67 (-) (CH), 36.89 (+) (CH2), 36.35
+ (+) (CH2), 27.90 (+) (CH2), 27.48 (+) (CH2). HRMS (FAB) (m/z) calcd for C23H24O5 (M )
+ 380.1624, found 380.1614; calcd for C23H25O5 (MH ) 381.1702, found 381.1711.
4,7-Dioxo-heptanoic acid 9H-fluoren-9-ylmethyl ester (DOHA-Fm, 2.4). Ester 2.3
(94 mg, 0.25 mmol) in 10 mL of AcOH/H2O (3:1, v/v) was stirred at 50 °C for 5 h. TLC
(5 % CHCl3 in MeOH): Rf = 0.5 showed the completion of the reaction.The solvent was
removed by rotary evaporation. Flash chromatography of the residue (25 % ethyl acetate
1 in hexane) gave 2.4 (73 mg, 88 %). H NMR (CDCl3, 400 MHz), δ 9.78 (s, 1H), 7.77 (d,
46 J = 6.74 Hz, 2H), 7.60 (d, J = 7.7 Hz, 2H), 7.42 (dd, J = 6.74, 7.7 Hz, 2H), 7.30 (dd, J =
6.74, 7.7 Hz, 2H), 4.39(d, J = 7.38 Hz, 2H), 4.20 (t, J = 7.38 Hz, 1H), 2.67-2.79 (8H). 13C
NMR (CDCl3, 100 MHz), δ 206.56 (CO), 200.27 (CHO), 172.53 (COO), 143.70 (C),
141.23 (C), 127.73 (CH), 127.07 (CH), 124.98 (CH), 119.96 (CH), 66.47 (CH2), 46.70
(CH), 37.42 (CH2), 36.87 (CH2), 34.53 (CH2), 27.93 (CH2). HRMS (FAB) (m/z) calcd for
+ C21H20O4 (M ) 336.1362, found 336.1361.
2-(2-Acetylamino-acetylamino)-6-[2-[2-(9H-fluoren-9-ylmethoxycarbonyl)-ethyl]- pyrrol-1-yl]-hexanoic acid methyl ester (2.5). Methyl 6-amino-2-((2- acetylamino)acetyl) amino) hexanoate (Ac-Gly-Lys-OMe, 25.6 mg, 0.08 mmol) in 1 mL methanol was added dropwise to DOHAFm (27 mg, 0.08 mmol) in 1.5 mL methanol.
The solution was stirred for 9 h at room temperature under argon. The solvent was removed by rotary evaporation. TLC (4 % methanol in chloroform): Rf = 0.34. The crude
compound was purified by silica gel chromatography (4 % methanol in chloroform ) to
1 give 35.8 mg (80%) of pure 2.5. H NMR (CDCl3, 400 MHz), δ 7.77 (d, J = 7.6 Hz, 2H),
7.60 (d, J = 7.6 Hz, 2H), 7.36-7.32 (dd, J = 7.6, 7.2 Hz, 2H), 7.27 (dd, J = 7.6, 7.2 Hz,
2H), 6.60 (dd, J = 2.4, 1.6 Hz, 1H), 6.54 (m, 1H), 6.28 (s, 1H), 6.09 (dd, J = 3.6, 2.4 Hz,
1H), 5.90 (m, 1H), 4.56-4.61 (m, 1H), 4.40 (d, J = 7.2 Hz, 2H), 4.21 (t, J = 7.2 Hz, 1H),
3.91 (dABq, J = 5.2, 14 Hz, 2H), 3.82 (t, J = 7.2 Hz, 2H), 3.76 (s, 3H), 2.65-2.86 (4H),
13 2.01 (s, 3H), 1.86 (2H), 1.66 (2H), 1.36 (2H). C NMR (CDCl3, 100 MHz), δ 172.98
(CO), 172.33 (CO), 170.58 (CO), 168.66 (CO), 143.74 (C), 141.31 (C), 130.73(C),
127.80 (CH), 127.12 (CH), 125.01 (CH), 120.37 (CH), 120.04 (CH), 106.99 (CH),
105.26 (CH), 66.47 (CH2), 52.53 (CH), 51.86 (CH3O), 46.78 (CH), 46.09 (CH2), 43.23
(CH2), 33.22 (CH2), 31.89 (CH2), 30.67 (CH2), 22.92 (CH2), 22.40 (CH2), 21.41 (CH3).
+ + HRMS (FAB) (m/z) calcd for C32H38N3O6 (MH ) 560.2755, found 560.2747.
47 2-(2-Acetylamino-acetylamino)-6-[2-(2-carboxy-ethyl)-pyrrol-1-yl]-hexanoic acid
methyl ester (CEP-dipep, 2.6). DBU (75 μL) was added to 2.5 (27 mg, 0.047 mmol) in
2.5 mL THF. The system was stirred for 6 h under argon. After the removal of solvent,
the crude compound was purified by silica gel chromatography (6 % methanol in
chloroform) to give 21 mg (86 %) of CEP-dipep. TLC (10 % methanol in chloroform): Rf
1 = 0.3; H NMR (CDCl3, 400 MHz), δ 7.18 (d, J = 8 Hz, 1H), 6.81 (m, 1H), 6.53 (m, 1H),
6.03 (dd, J = 3.2, 3.2 Hz, 1H), 5.87 (m, 1H), 4.53 (dt, J = 1H), 3.95 (dABq, J = 5.2, 13.6
Hz, 2H), 3.81 (t, J = 6.8 Hz, 2H), 3.70 (s, 3H), 2.65-2.86 (4H), 2.01 (s, 3H), 1.8-1.62
13 (4H), 1.36 (m, 2H). C NMR (CDCl3, 100 MHz), δ 176.01 (COOH), 172.40 (COO),
171.35 (CO), 169.28 (CO), 131.40 (C), 119.92 (CH), 107.25 (CH), 105.00 (CH), 52.50
(CH), 52.00 (CH3O), 45.66 (CH2), 43.19 (CH2), 32.98 (CH2), 31.44 (CH2), 30.90 (CH2),
+ + 22.94 (CH2), 22.07 (CH2), 21.37 (CH3). HRMS (FAB) (m/z) calcd for C18H28N3O6 (MH )
382.1978, found 382.1986.
Tryptic Digestion. CEP modified proteins were dissolved in NH4HCO3 buffer (8 M
urea/2M NH4HCO3) to make a final concentration 2-3 pmol/μL. Trypsin solution (0.1
μg/μL) was made by suspending sequencing grade modified porcine trypsin (20 μg)
(Promega, Cat. V511A) in trypsin resuspension buffer (Promega, Cat. V511A) (200 μL).
This trypsin solution was then added to protein solution in a ratio of 1:50 (w/w) enzyme/protein. After incubation at 37 °C for 24 h, the solution was centrifuged at 3000 rpm for 5 mins. The upper layer (10 μL) was subjected to LC-MS analysis.27
Carboxyethyl pyrrole human albumin adduct (CEP-HSA, 2.7). A solution of
DOHA-Fm (2 mg, 0.006 mmol) in DMF (750 μL) was added to HSA solution (0.08 mM,
1.5 mL) in PBS. The mixture was stirred under argon for 4 days. DBU (200 μL) was
48 added and the resulting mixture was stirred overnight under argon followed by two
successive 12 h dialyses (Mr cutoff 14000) against 500 mL 20 % DMF in 10 mM PBS
(pH 7.4) and two additional dialyses (12 h each) against 500 mL 10 mM PBS (pH 7.4) at
4 °C. The final protein concentration (1.80 mg/mL) was determined by the Pierce bicinchoninic acid (BCA) protein assay. The pyrrole concentration (187 μM) was determined by Ehrlich assay. LC-MS/MS revealed six CEP modifications at m/z 589.266,
571.260, 674.762, 881.401, 687.784, 636.252 on lysyl residues of the HSA peptides
161KYLYEIAR168, 234AFKAWAVAR242, 35FKDLGEENFK44, 438KVPQVSTPTLVEVS-
R452, 247FPKAEFAEVSK257, 25DAHKSEVAHR34 respectively (Appendix Figure S39).
No CEPFm modifications were found after deprotection. Figure 2.3 shows the tandem
MS spectrum of the doubly charged ion m/z 571.260 from a MS scan of tryptic digested
CEP-HSA. The spectrum shows a series of fragment ions (b ions and y ions) sufficient to
identify a CEP modification on Lys236 from HSA residues 234AFKAWAVAR242. This
particular sequence is very similar with the sequence 233ALKAWSVAR241 in BSA and is
exactly the same as in MSA. MS/MS analysis revealed unambiguous modification on the
ALKAWSVAR sequence of CEP-BSA and the AFKAWAVAR sequence of MSA.
Presumably, this sequence is readily accessed and modified.
49 ) Relative Abundance (%
Figure 2.3. Tandem MS characterization of the doubly charged ion m/z 571.260 from
a MS scan of tryptic digested CEP modified HSA shows series of fragment ions
sufficient to unambiguously identify a CEP modification on the lysyl residue (K236).
Asterisks denote fragment ions with the modified lysyl residue.
Carboxyethyl pyrrole mouse albumin adduct (CEP-MSA, 2.8). A solution of
DOHAFm (18.5 mg, 0.055 mmol) in DMF (8 mL)was added slowly to the solution of mouse serum albumin (100 mg) in 18 mL 10 mM PBS (pH 7.4). The mixture was stirred under argon for 4 days. DBU (360 μL) was added and the resulting mixture was stirred overnight under argon followed by two successive 24 h dialyses (Mr cutoff 14000) against 1 L 20 % DMF in 10 mM PBS (pH 7.4) and two additional dialyses (24 h each) against 1 L 10 mM PBS (pH 7.4) at 4 °C. The final protein concentration (2.43 mg/mL) was determined by the Pierce bicinchoninic acid (BCA) protein assay. The pyrrole concentration (210 μM) was determined by Ehrlich assay. LC-MS/MS revealed 15 CEP modifications at m/z 650.3296, 872.9611, 571.3492, 849.4302, 612.8407, 769.4069,
50 694.6888, 1047.0802, 857.4633 on lysyl residues of the MSA peptides 25EAHKSEI
AHR34, 206LDGVKEKALVSSVR219 (2 CEPs), 234AFKAWAVAR242, 243LSQTFPNADF
AEITKLATDLTK264, 258LATDLTKVNK267, 373LAKKYEATLEK383 (2 CEPs),
435YTQKAPQVSTPTLVEAAR452, 544EKQIKKQTALAELVK558 (3 CEPs), 550QTA
LAELVKHKPKATAEQLK 569 (3 CEPs) respectively (Appendix Figure S40). The sequence coverage was 35%. No CEPFm modifications were found after deprotection.
The tandem MS spectrum of AFKAWAVAR was shown in Figure 2.4. Relative Abundance (%)
Figure 2.4. Tandem MS characterization of the doubly charged ion m/z 571.3492 from
a MS scan of tryptic digested CEP modified MSA shows series of fragment ions
sufficient to unambiguously identify a CEP modification on the lysyl residue (K236).
Asterisks denote fragment ions with the modified lysyl residue.
Carboxyethyl pyrrole chicken egg albumin adduct (CEP-CEO, 2.9). A solution of
DOHAFm (18 mg, 0.054 mmol) in DMF (3.2 mL) was added slowly to the solution of
CEO (76 mg) in pH 7.4 PBS (10 mM, 7 mL). The mixture was stirred under argon for 4
days. DBU (140 μL) was added and the resulting mixture was stirred overnight under
51 argon followed by two successive 24 h dialyses (Mr cutoff 3500) against 1 L 20% DMF
in 10 mM pH 7.4 PBS and two more dialyses (24 h each) against 1 L 10 mM pH 7.4 PBS
at 4 °C. The final protein concentration (2.94 mg/mL) was determined by the Pierce
bicinchoninic acid (BCA) protein assay. The pyrrole concentration (93 μM) was determined by Ehrlich assay. LC-MS/MS revealed eight CEP modifications at m/z
540.2886, 801.7021, 839.3332, 808.0259, 610.2589, 630.8334, 459.1887 on lysyl residues of the CEO peptides 51TQINKVVR58, 85DILNQITKPNDVYSFSLASR104,
187AFKDEDTQAMPFR199, 200VTEQESKPVQMMYQIGLFR218, 219VASMASEKMK228,
277KIKVYLPR284, 285MKMEEK290 respectively (Appendix Figure S41). The sequence
coverage was 46%. No CEPFm modifications were found after deprotection.
Carboxyethyl pyrrole myoglobin adduct (CEP-myoglobin, 2.10). A solution of
DOHAFm (12 mg, 0.036 mmol) in DMF (3.6 mL) was added slowly to the solution of myoglobin (32.38 mg) in pH 7.4 PBS (10 mM, 5 mL). The mixture was stirred under argon for 4 days. DBU (140 μL) was added and the resulting mixture was stirred overnight under argon followed by two successive 24 h dialyses (Mr cutoff 3500) against
500 mL 20% DMF in 10 mM pH 7.4 PBS and two more dialyses (24 h each) against 500 mL 10 mM pH 7.4 PBS at 4 °C. The final protein concentration (0.65 mg/mL) was determined by Bio-Rad protein assay. The pyrrole concentration (91 μM) was determined by Ehrlich assay. LC-MS/MS revealed CEP modifications at m/z 671.2667, 604.9635,
815.2087, 557.4288, 785.6061, 899.6666, on lysyl residue of the myoglobin peptides
32LFTGHPETLEKFDKF KHLK50 (3 CEP modifications), 48HLKTEAEMK56, 63KHGT
VVLTALGGILK77, 79KGHHEAELKPLAQSHATK96, 119HPGDFGADAQGAMTKALE
LFR139, 140NDIAAKYKELGFQG153 (2 CEP modifications) (Appendix Figure S42). The
52 peptide sequence coverage achieved 91%. No CEPFm modifications were found after
deprotection.
Carboxyethyl pyrrole GPDH adduct (CEP-GPDH, 2.11). GPDH (30 mg) was added in 10 mL of 10 mM PBS (pH 7.4). After vortexing for 5 mins and centrifugation (4 °C) at 3000 rpm for 10 mins (GPDH does not dissolve well in PBS), 4 mL of clear solution was taken and DOHAFm (5.8 mg, 0.017 mmol) in DMF (4 mL) was added slowly to it.
The mixture was stirred under argon for 4 days. DBU (120 μL) was then added and the resulting mixture was stirred overnight under argon followed by two successive 24 h dialyses (Mr cutoff 3500) against 500 mL 20% DMF in 10 mM pH 7.4 PBS and two more dialyses (24 h each) against 500 mL 10 mM pH 7.4 PBS at 4 °C. The final protein concentration (0.29 mg/mL) was determined by the Bio-Rad protein assay. The pyrrole concentration (65 μM) was determined by Ehrlich assay. LC-MS/MS revealed six CEP modifications at m/z 655.3343, 669.3289, 923.9847, 639.3413, 740.8362 on lysyl residues of the GPDH peptides 105AGAHLKGGAKR115, 184TVDGPSGKLWR194,
198GAAQNIIPASTGAAKAVGK216, 258VVKQASEGPLK268, 321VVDLMVHMASKE332
respectively (Appendix Figure S43). The peptide sequence coverage achieved 45%. No
CEPFm modifications were found after deprotection.
6-[2-[2-(9H-fluoren-9-ylmethoxycarbonyl)-ethyl]-pyrrol-1-yl]-hexanoic acid (2.12).
6-Aminocaproic acid (10.8 mg, 0.082 mmol) in water (400 μL) was slowly added to
DOHA-Fm (21 mg, 0.0625 mmol) in methanol (600 μL). The solution became cloudy
with the addition. The heterogeneous system was stirred for 48 h under argon at room
temperature during which became homogenous. The solution was extracted with CH2Cl2,
washed with brine, dried with Na2SO4 and evaporated. The yellowish residue was loaded
onto silica gel in a sintered-glass funnel with chloroform and washed with chloroform (15
53 mL), 10 % ethyl acetate in hexane (15 mL), 20 % ethyl acetate in hexane (15 mL) and 50
% ethyl acetate in hexane (60 mL) successively. The fractions eluted with 50 % ethyl
acetate/hexane were collected and dried to give 21.8 mg (81 %) acid 2.12. TLC (ethyl
1 acetate/hexane, 2:3 v/v): Rf = 0.22. H NMR (CDCl3, 400 MHz), δ 7.77 (d, J = 6.74 Hz,
2H), 7.58 (d, J = 7.7 Hz, 2H), 7.42 (dd, J = 7.7, 7.2 Hz, 2H), 7.30 (dd, J = 6.74, 7.2 Hz,
2H), 6.58 (dd, J = 3.2, 3.6 Hz, 1H), 6.06 (dd, J = 3.2, 3.2 Hz, 1H), 5.87 (m, 1H), 4.42 (d,
J = 7.2 Hz, 2H), 4.22 (t, J = 7.2 Hz, 1H), 3.79 (t, J = 7.2 Hz, 2H), 2.72-2.84 (4H). 2.34 (t,
13 J = 7.2 Hz, 2H), 1.75-1.34 (6H). C NMR (CDCl3, 100 MHz), δ 177.40 (COOH), 172.90
(COO), 143.73 (C), 141.20 (C), 130.66 (C), 127.78 (CH), 127.10 (CH), 125.00 (CH),
120.34 (CH), 120.04 (CH), 106.88 (CH), 105.20 (CH), 66.40 (CH2), 46.79 (CH2), 46.28
(CH), 33.45 (CH2), 33.20 (CH2), 31.01 (CH2), 26.23 (CH2), 24.26 (CH2), 21.44 (CH2).
+ HRMS (FAB) (m/z) calcd for C27H30NO4 (MH ) 432.2175, found 432.2190.
6-[2-[2-(9H-fluoren-9-ylmethoxycarbonyl)-ethyl]-pyrrol-1-yl]-hexanoic acid 2,5-
dioxo-pyrrolidin-1-yl ester (CEPFmSu, 2.13). Acid 2.12 (15 mg, 0.035 mmol) and N-
hydroxysuccinimide (4.5 mg, 0.039 mmol), DCC (7.5 mg, 0.036 mmol) were dissolved in
dry CH2Cl2 (7.5 mL) under Argon. The clear solution became cloudy after 15 minutes.
The reaction mixture was stirred for 3.5 h. Solvent was then removed by evaporation.
The crude product was purified by silica gel chromatography with ethyl acetate/hexane
(2:3, v/v) to deliver 16.6 mg (90 %) active ester 2.13. TLC (ethyl acetate/hexane, 2:3): Rf
1 = 0.25. H NMR (CDCl3, 400 MHz), δ 7.77 (d, J = 6.74 Hz, 2H), 7.58 (d, J = 7.7 Hz, 2H),
7.42 (dd, J = 7.7, 7.2 Hz, 2H), 7.30 (dd, J = 6.74, 7.2 Hz, 2H), 6.58 (dd, J = 3.2, 3.6 Hz,
1H), 6.06 (dd, J = 3.2, 3.2 Hz, 1H), 5.87 (m, 1H), 4.42 (d, J = 7.2 Hz, 2H), 4.22 (t, J = 7.2
Hz, 1H), 3.79 (t, J = 7.2 Hz, 2H), 2.72-2.86 (8H), 2.60 (t, J = 7.2 Hz, 2H), 1.81-1.70 (4H),
13 1.48-1.40 (m, 2H). C NMR (CDCl3 100 MHz), δ 172.79 (COO), 169.10 (COO), 168.39
54 (CO), 143.75 (C), 141.29 (C), 130.69 (C), 127.77 (CH), 127.10 (CH), 125.01 (CH),
120.34 (CH), 120.01 (CH), 106.92 (CH), 105.24 (CH), 66.36 (CH2), 46.79 (CH2), 46.21
(CH), 33.31 (CH2), 30.86 (CH2), 30.72 (CH2), 25.87 (CH2), 25.55 (CH2), 24.25 (CH2),
+ 21.44 (CH2). HRMS (FAB) (m/z) calcd for C31H33N2O6 (MH ) 529.2338, found 529.2340.
6-(2-carboxyethyl-1-pyrrolyl)-hexanoyl BSA amide (CEPH-BSA, 2.14) A solution of CEPFmSu (1.4 mg) in DMF (150 μL) was added to BSA (3 mg/mL, 1 mL) in PBS (10 mM, pH 7.4) solution. The cloudy solution became homogenous with overnight stirring.
After 2 days, DBU (25 μL) was added and the resulting mixture was stirred overnight under argon followed by two successive 12 h dialyses (Mr cutoff 14000) against 500 mL
20 % DMF in PBS (10 mM, pH 7.4) and two additional dialyses (12 h each) against 500 mL PBS (10 mM, pH 7.4) at 4 °C. The final protein concentration (1.34 mg/mL) was determined by modified Lowry protein assay. The pyrrole concentration (134 μM) was determined by Ehrlich assay. LC-MS/MS revealed nine CEPH modifications at m/z
594.9934, 618.8188, 541.8060, 937.9577, 526.7835, 612.3604, 689.4319, 765.3984,
964.5088 corresponding to the BSA peptides 400LKHLVDEPQNLIK412,
233ALKAWSVAR 241, 242LSQKFPK248, 437KVPQVSTPTLVEVSR451, 452SLGKVGTR459,
490TPVSEKVTK498, 548KQTALVELLK557, 246FPKAEFVEVTK256, 249AEFVEVTKLV
TDLTK263 respectively and unambiguously confirmed CEPH adducts on lysyl residues
(Appendix Figure S44). No CEPHFm modifications were found after deprotection. The tandem MS spectrum of ALKAWSVAR is shown in Figure 2.5.
55 Relative Abundance (%)
Figure 2.5. Tandem MS characterization of the doubly charged ion m/z 618.8188 from a
MS scan of tryptic digested CEPH-BSA shows series of fragment ions sufficient to
unambiguously identify a CEP modification on lysyl residue (K235). Asterisks denote
fragment ions with the modified lysyl residue.
Preparation of carboxyethyl pyrrole hexanoic acid 2,5-dioxo-pyrrolidin-1-yl
mouse serum albumin (CEPH-MSA, 2.15). A solution of CEPFmSu (630 μg) in DMF
(150 μL) was added in MSA (2 mg/mL, 1 mL) in PBS (10 mM, pH 7.4). The cloudy
solution became clear with overnight stirring. After 2 days, DBU (25 μL) was added, and
the resulting mixture was stirred overnight under argon followed by two successive 12 h
dialyses (Mr cutoff 3500) against 500 mL 20% DMF in PBS (10 mM, pH 7.4) and two
more dialyses (12 h each) against 500 mL PBS (10 mM, pH 7.4) at 4 °C. The final
protein concentration (2.06 mg/mL) was determined by modified Lowry protein assay.
The pyrrole concentration (69.5 μM) was determined by Ehrlich assay. LC-MS/MS
revealed four CEPH modifications at m/z 512.2829, 627.8604, 608.8177, 732.3707 on
56 lysyl residues of the MSA peptides 206LDGVKEK 212, 234AFKAWAVAR242, 376KYEATL
EK383, 435YTQKAPQVSTPTLVEAAR452, respectively (Appendix Figure S45). No
CEPHFm modifications were found after deprotection.
Preparation of carboxyethyl pyrrole hexanoic acid 2,5-dioxo-pyrrolidin-1-yl
chicken egg albumin (CEPH-CEO, 2.16). A solution of CEPFmSu (1.0 mg) in DMF
(150 μL) was added in CEO (4 mg/mL, 1 mL) in PBS (10 mM, pH 7.4). After the
coupling underwent for 2 days, the solution was still not clear until the addition of DBU
(25 μL). The system was stirred overnight under argon followed by two successive 12 h
dialyses (Mr cutoff 14000) against 500 mL 20% DMF in PBS (10 mM, pH 7.4) and two
more dialyses (12 h each) against 500 mL PBS (10 mM, pH 7.4) at 4 °C. The final
protein concentration (3.46 mg/mL) was determined by modified Lowry protein assay.
The pyrrole concentration (85.4 μM) was determined by Ehrlich assay. LC-MS/MS revealed a CEPH modification at m/z 596.8715 on a lysyl residue of the CEO peptide
51TQINKVVR 58 (Appendix Figure S46). No CEPHFm modifications were found after
deprotection.
Preparation of carboxyethyl pyrrole hexanoic acid 2,5-dioxo-pyrrolidin-1-yl
GPDH adduct (CEPH-GPDH, 2.17). GPDH (10 mg) was added in PBS (10 mM, 10
mL). After vortexing for 5 min and centrifugation (4 °C) at 3000 rpm for 10 mins, the
upper clear solution (2 ml) was separated and CEPFmSu (770 μg) in DMF (300 μL) was
added. The cloudy solution became clear after 2 days. Then DBU (25 μL) was added and
the resulting mixture was stirred overnight under argon followed by two successive 12 h
dialyses (Mr cutoff 3500) against 500 mL 20% DMF in PBS (10 mM, pH 7.4) and two more dialyses (12 h each) against 500 mL PBS (10 mM, pH 7.4) at 4 °C. The final protein concentration (0.32 mg/mL) was determined by modified Lowry protein assay.
57 The pyrrole concentration (32.5 μM) was determined by Ehrlich assay. LC-MS/MS revealed CEPH modifications at m/z 635.0367, 712.5500, 919.7650, 572.9763, 899.6923,
726.0422, 980.7797, 731.1005, 941.7422, 579.9560, 508.9000, 696.1018, 797.5823 on lysyl residues of the GPDH peptides 1VKVGVNGFGR10, 53FHGTVKAENGK63,
64LVINGKAITIFQER77, 105AGAHLKGGAK114, 160VIHDHFGIVEGLMTTVHAITA
TQKTVDGPSGK191, 184TVDGPSGKLWR194, 198GAAQNIIPASTGAAKAVGK216,
213AVGKVIPELNGK224, 217VIPELNGKLTGMAFR231, 249AAKYDDIK256,
252YDDIKK257, 258VVKQASEGPLK268, and 321VVDLMVHMASKE332 (Appendix
Figure S47). No CEPHFm modifications were found after deprotection. The peptide
sequence coverage achieved 68%.
[4-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-butyl]-
carbamic acid tert-butyl ester (2.18). N-Boc-1,4-butanediamine (20 mg, 0.106 mmol)
in CH2Cl2 (2 mL) was added in d-biotin p-nitrophenyl ester (38 mg, 0.10 mmol) in
CH2Cl2 (2 mL). The mixture was stirred for 10 h. TLC analysis (30 % ethyl acetate in hexane) showed the completion of the reaction with a new spot at Rf = 0.25 generated.
After removal of the solvent by rotary evaporation, the crude compound was purified by flash chromatography (30 % ethyl acetate in hexane) to give 28 mg (80 %) 2.18. 1H
NMR (CD3OD, 400 MHz), δ 4.47-4.50 (m, 1H), 4.28-4.31 (m, 1H), 3.2 (m, 1H), 3.16 (t,
J = 7.2 Hz, 2H), 3.03 (t, J = 6.8 Hz, 2H), 2.92 (dd, J = 12.4, 4.8 Hz, 1H), 2.68-2.71 (d, J
= 12.8 Hz, 1H), 2.19 (t, J = 7.6 Hz, 2H), 1.44-1.7 (10H), 1.42 (s, 9H).
5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic acid (4-amino-butyl)- amide (2.19). Trifluoroacetic acid (100 μL) was added to 2.18 (6 mg) in CH2Cl2 (400
μL). After stirring for 3 h, TLC (20% methanol in chloroform, Rf = 0.1) showed the
reaction was completed. The solvent was removed by rotary evaporation. The yellowish
58 residue was basified with 1N NaOH to pH 8.5. After removal of the solvent, the residue
was triturated with methanol and the methanol extract was concentrated and dried to give
1 4 mg (90%) 2.19. H NMR (CD3OD, 400 MHz), δ 4.46-4.51 (m, 1H), 4.28-4.31 (m, 1H),
3.2 (m, 1H), 3.16 (t, J = 7.2 Hz, 2H), 2.9 (dd, J = 12.4, 4.8 Hz, 1H), 2.68-2.71 (d, J = 12.4
Hz, 1H), 2.65 (t, J = 7.2 Hz, 2H), 2.19 (t, J = 7.6 Hz, 2H), 1.34-1.7 (10H).
3-(1-[4-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-butyl]-
1H-pyrrol-2-yl)-propionic acid 9H-fluoren-9-yl-methyl ester (2.20). To amine 2.19 (4 mg, 0.012 mmol) in methanol (150 μL), was added DOHA-Fm (4 mg, 0.0119 mmol) in methanol (150 μL). The solution was stirred for 9 h at room temperature under argon.
The solvent was then removed by rotary evaporation. The crude compound was purified by silica gel chromatography (5 % methanol in chloroform, TLC: Rf = 0.2) to give 6 mg
1 (84 %) of pure 2.20. H NMR (CDCl3, 400 MHz), δ 7.70 (d, J = 7.6 Hz, 2H), 7.50 (dd, J
= 7.6, 1.2 Hz, 2H), 7.34 (dd, J = 7.6, 7.2 Hz, 2H), 7.27-7.23 (ddd, J = 7.6, 7.2, 1.2 Hz,
2H), 6.51 (dd, J = 1.6, 2.8 Hz, 1H), 5.98 (dd, J = 2.8, 3.6 Hz, 1H), 5.79 (m, 1H), 5.74 (m,
1H), 5.58 (s, 1H), 4.82 (s, 1H), 4.40 (m, 1H), 4.36 (d, J = 7.2 Hz, 2H), 4.20 (m, 1H), 4.15
(t, J = 7.2 Hz, 1H), 3.76 (t, J = 7.2 Hz, 2H), 3.15 (m, 2H), 3.05 (m, 1H), 2.84-2.79 (dd, J
= 4.8, 12.8 Hz, 1H), 2.78-2.76 (m, 2H), 2.71-2.66 (m, 2H), 2.64-2.61 (d, J = 12.8 Hz, 1H),
2.06-2.10 (dt, J = 3.6, 7.2 Hz, 2H), 1.72-1.36 (10H). HRMS (FAB) (m/z) calcd for
+ + C35H43N4O4S (MH ) 615.3005, found 615.2995.
3-(1-[4-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-butyl]-
1H-pyrrol-2-yl)-propionic acid (2.21). DBU (5 μL) was added to 2.20 (6 mg, 0.01
mmol) in THF (500 μL) and the resulting mixture was stirred for 2 h. TLC (10 % methanol in chloroform, Rf = 0.3) showed the completion of the reaction by
59 disappearance of the UV active starting spot. The corresponding acid was purified by
flash chromatography (7 % methanol in chloroform) to provide 3.7 mg (85 %) acid 2.21.
1 H NMR (CD3OD, 400 MHz), δ 6.57 (dd, J = 2.8, 1.6 Hz, 1H), 5.92 (dd, J = 3.2, 2.8 Hz,
1H), 5.79 (m, 1H), 4.60 (3H), 4.48 (m, 1H), 4.27 (m, 1H), 3.86 (t, J = 7.2 Hz, 2H), 3.19-
3.15 (3H), 2.94-2.89 (dd, J = 4.8, 12.8 Hz, 1H), 2.86-2.82 (m, 2H), 2.71-2.67 (d, J = 12.8
Hz, 1H), 2.60-2.56 (m, 2H), 2.20-2.17 (t, J = 7.2 Hz, 2H), 1.72-1.36 (10H). HRMS (FAB)
+ + + (m/z) calcd for C21H33N4O4S (MH ) 437.2222, found 437.2193; calcd for C21H31N4O3S
(M+-OH) 419.2117, found 419.2102.
4,7-Dioxo-heptanoic acid methyl ester (DOHA-Me, 2.22). Ester 2.1 (22.5 mg, 0.104 mmol) in 5 mL of AcOH/H2O (3:1, v/v) was stirred at 50 °C for 5 h. TLC (3% MeOH in
CHCl3, Rf = 0.4) showed the completion of the reaction. The solvent was removed by
rotary evaporation. Flash chromatography of the residue (25 % ethyl acetate in hexane)
1 gave 2.22 (15.6 mg, 87 %). H NMR (CDCl3, 200 MHz), δ 9.78 (s, 1H), 3.67 (s, 3H),
+ + 2.56-2.81 (8H); HRMS (FAB) (m/z) calcd for C8H11O4 (M -H) 171.0658, found
+ + 171.0656; calcd for C8H13O5 (M + OH) 189.0763, found 189.0783.
5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic acid (6-amino-hexyl)- amide (2.23). 1,6-Diaminohexane (270 mg, 2.324 mmol) in 10 mL pyridine-H2O (9:1, v/v) was added slowly to d-biotin p-nitrophenyl ester (Sigma) (100 mg, 0.274 mmol) in
28 20 mL pyridine-H2O (9:1,v/v). The clear yellow solution was stirred for 24 h at room
temperature. The solvent was removed by rotary evaporation. TLC (methanol (saturated
with NH3): CHCl3, 3:7, v/v), Rf = 0.24. The crude product was purified by silica gel
chromatography (30 % methanol saturated with NH3 in chloroform) to give 84 mg (90 %)
1 of pure 2.23. H NMR (CD3OD, 400 MHz), δ 4.46-4.51 (m, 1H), 4.28-4.31 (m, 1H),
3.20 (m, 1H), 3.16 (t, J = 7.2 Hz, 2H), 2.9 (dd, J = 12.4, 4.8 Hz, 1H), 2.68-2.71 (d, J =
60 12.4 Hz, 1H), 2.65 (t, J = 7.2 Hz, 2H), 2.19 (t, J = 7.6 Hz, 2H), 1.34-1.7 (14H). 13C NMR
(CD3OD, 100 MHz), δ 175.97 (CO), 166.10 (CO), 63.36 (CH), 61.60 (CH), 57.01 (CH),
42.3 (CH2), 42.0 (CH2), 40.2 (CH2), 36.8 (CH2), 33.2 (CH2), 30.50 (CH2), 29.8 (CH2),
29.5 (CH2), 28.8 (CH2), 28.6 (CH2), 28.0 (CH2). HRMS (FAB) (m/z) calcd for
+ + C16H31N4O2S (MH ) 343.2168, found 343.2165.
3-(1-[6-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexyl]-
1H-pyrrol-2-yl)-propionic acid methyl ester (2.24). Amine 2.23 (45 mg, 0.13 mmol) in
1 mL methanol was added to DOHA-Me (23 mg, 0.13 mmol) in 1.5 mL methanol. The
solution was stirred for 9 h at room temperature under argon. The solvent was removed
by rotary evaporation. The crude product was purified by silica gel chromatography (5 %
1 methanol in chloroform, TLC: Rf = 0.18) to give 47 mg (75 %) of pure 2.24. H NMR
(CD3OD, 400 MHz), δ 6.52 (dd, J = 2.0, 2.8 Hz, 1H), 5.9 (dd, J = 3.2, 2.8 Hz, 1H), 5.75
(m, 1H), 4.46-4.49 (dd, J = 8.0, 4.0 Hz, 1H), 4.29 (dd, J = 8.0, 4.8 Hz, 1H), 4.28-4.3 (m,
1H), 3.82 (t, J = 7.6 Hz, 2H), 3.65 (s, 3H), 3.1-3.2 (3H), 2.9 (dd, J = 12.4, 4.8 Hz, 1H),
2.85 (t, J = 7.2 Hz, 2H), 2.68-2.71 (d, J = 12.4 Hz, 1H), 2.65 (t, J = 7.2 Hz, 2H), 2.19 (t, J
= 7.6 Hz, 2H), 1.34-1.7 (14H).
3-(1-[4-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexyl]-
1H-pyrrol-2-yl)-propionic acid (2.25). Sodium hydroxide (16 mg, 0.4 mmol) was added to 2.24 (47 mg, 0.1 mmol) in 1 mL absolute ethanol and the resulting mixture was stirred for 4 h at room temperature. TLC (10 % methanol in chloroform, Rf = 0.3) showed the
completion of the reaction by disappearance of the starting spot. After removal of the
solvent, the residue was acidified with 3 N HCl to pH = 3 and then extracted with ethyl
acetate. The ethyl acetate extract was washed with brine, dried with MgSO4, and
1 concentrated to afford 38 mg (84 %) 2.25. H NMR (CD3OD, 400 MHz), δ 6.57 (dd, J =
61 2.0, 2.4 Hz, 1H), 5.93 (dd, J = 2.4, 3.6 Hz, 1H), 5.80 (m, 1H), 4.46 (dd, J = 7.6, 5.2 Hz,
1H), 4.29 (dd, J = 4.8, 7.6 Hz, 1H), 3.84 (t, J = 7.6 Hz, 2H), 3.1-3.2 (3H), 2.9 (dd, J =
12.4, 4.8 Hz, 1H), 2.85 (t, J = 7.2 Hz, 2H), 2.68-2.71 (d, J = 12.4 Hz, 1H), 2.61 (t, J = 7.2
13 Hz, 2H), 2.19 (t, J = 7.2 Hz, 2H), 1.34-1.7 (14H). C NMR (CD3OD, 100 MHz),
δ 176.82 (CO), 175.97 (CO), 166.10 (CO), 132.00 (C), 121.22 (CH), 107.52 (CH),
106.08 (CH), 63.36 (CH), 61.60 (CH), 57.01 (CH), 47.19 (CH2), 41.03 (CH2), 40.22
(CH2), 36.82 (CH2), 34.48 (CH2), 32.54 (CH2), 30.30 (CH2), 29.78 (CH2), 29.51 (CH2),
27.62 (CH2), 27.44 (CH2), 26.95 (CH2), 22.55 (CH2). HRMS (FAB) (m/z) calcd for
+ + C23H37N4O4S (MH ) 465.2530, found 465.2524.
Octadec-9-enoic acid 2-[(2-[2-[2-(9H-fluoren-9-ylmethoxycarbonyl)-ethyl]-pyrrol-
1-yl]-ethoxy)-hydroxy-phosphoryloxy]-1-hexadecanoyloxymethyl-ethyl ester (2.26).
Triethylamine (TEA) (25 μL, 0.247 mmol) was added to POPE (50 mg, 0.065 mmol) in
500 μL CHCl3, then DOHAFm (22 mg, 0.065 mmol) in 500 μL CHCl3 was added. The
resulting mixture was stirred 20 h under Argon. After evaporation of solvent, the crude
product was purified by silica gel chromatography (10 % methanol in chloroform, TLC:
1 Rf = 0.2) to give 50 mg (76 %) of pure 2.26. H NMR (CD3OD:CDCl3 = 1:1, 400 MHz),
δ 7.74 (d, J = 7.6 Hz, 2H), 7.53 (d, J = 7.6 Hz, 2H), 7.36 (dd, J = 7.6, 7.6 Hz, 2H), 7.27
(dd, J = 7.6, 7.6 Hz, 2H), 6.63 (dd, J = 2.8, 1.6 Hz, 1H), 5.94 (dd, J = 3.2, 2.8 Hz, 1H),
5.77 (m, 1H), 5.28 (m, 2H), 4.36 (d, J = 6.8 Hz, 2H), 4.28 (m, 1H), 4.17 (t, J = 6.8 Hz,
1H), 4.05-3.98 (6H), 3.68 (t, J = 6.4 Hz, 2H), 2.86-2.82 (t, J = 7.2 Hz, 2H), 2.73-2.69 (t, J
= 7.2 Hz, 2H), 2.25-2.21 (4H), 2.0-1.94 (4H), 1.57 (4H), 1.21 (44H), 0.84 (t, J = 6.4 Hz,
13 6H). C NMR (CD3OD:CDCl3 = 1:1, 50 MHz, APT), δ 175.21 (+) (CO), 174.84 (+)
(CO), 174.81 (+) (CO), 145.08 (+) (C), 142.68 (+) (C), 132.30 (+) (C), 131.24 (-) (CH),
130.95 (-) (CH), 129.13 (-) (CH), 128.53 (-) (CH), 126.31 (-) (CH), 121.30 (-) (CH),
62 121.22 (-) (CH), 108.49 (-) (CH), 106.77 (-) (CH), 71.86 (-) (CH), 67.96 (+) (CH2), 66.62
(+) (CH2), 64.60 (+) (CH2), 64.05 (+) (CH2), 48.10 (+) (CH2), 47.82 (-) (CH), 35.47 (+)
(CH2), 35.34 (+) (CH2), 34.66 (+) (CH2), 33.27 (+) (CH2), 31.03 (+) (CH2), 30.85 (+)
(CH2), 30.70 (+) (CH2), 30.66 (+) (CH2), 30.61 (+) (CH2), 30.49 (+) (CH2), 28.49 (+)
(CH2), 26.24 (+) (CH2), 26.20 (+) (CH2), 23.98 (+) (CH2), 22.64 (+) (CH2), 15.14 (-)
+ + (CH3). HRMS (FAB) (m/z) calcd for C60H91NNa2O10P [(M-H)Na2 ] 1062.6177, found
1062.6.
Octadec-9-enoic acid 2-([2-[2-(2-carboxy-ethyl]-pyrrol-1-yl]-ethoxy)-hydroxy-
phosphoryloxy]-1-hexadecanoyloxymethyl-ethyl ester (CEP-PE, 2.27). DBU (20 μL,
0.13 mmol) was added to 2.26 (24 mg, 0.025 mmol) in 500 μL CHCl3. After 3 h, the
reaction was completed and the system was diluted by 1.5 mL CHCl3. The solution was
washed with pH 5.5 phosphate buffer (2 mL). The organic phase was washed with brine,
dried over magnesium sulfate, then vacuum-filtered, concentrated, and purified by flash
chromatography (15 % methanol in chloroform, TLC: Rf = 0.18) to yield 19 mg (90 %) of
1 pure 2.27. H NMR (CD3OD:CDCl3 = 1:1, 400 MHz), δ 6.56 (m, 1H), 5.96 (m, 1H),
5.83 (m, 1H), 5.30 (m, 2H), 5.15 (m, 1H), 4.33 (m, 1H), 4.05-3.98 (6H), 3.81 (m, 2H),
2.83 (m, 2H), 2.58 (m, 2H), 2.27 (t, J = 7.2 Hz, 4H), 2.0-1.94 (4H), 1.57 (4H), 1.21 (44H),
13 0.84 (t, J = 6.4 Hz, 6H). C NMR (CD3OD:CDCl3 = 1:1, 100 MHz), δ 180.36 (COOH),
174.49 (CO), 174.08 (CO), 133.54 (C), 130.46 (CH), 130.14 (CH), 120.98 (CH), 107.26
(CH), 105.05 (CH), 71.00 (CH), 65.84 (CH2), 64.03 (CH2), 63.11 (CH2), 47.29 (CH2),
34.70 (CH2), 34.56 (CH2), 32.40 (CH2), 32.42 (CH2), 30.23 (CH2), 30.17 (CH2), 30.00
(CH2), 29.80 (CH2), 29.63 (CH2), 27.66 (CH2), 25.40 (CH2), 23.13 (CH2), 14.31 (CH3).
+ + HRMS (FAB) (m/z) calcd for C46H82NNaO10P (MNa ) 862.5574, found 862.5550; calcd
+ + for C46H81NNa2O10P [(M-H)Na2 ] 884.5394, found 884.5291.
63 Hexadecanoic acid 3-[(2-[2-[2-(9H-fluoren-9-ylmethoxycarbonyl)-ethyl]-pyrrol-1-
yl]-ethoxy)-hydroxy-phosphoryloxy]-2-hydroxy-propyl ester (2.28). Triethylamine
(TEA) (12 μL, 0.116 mmol) was added to lyso-PE (42 mg, 0.093 mmol) in 500 μL
CHCl3, then DOHAFm (26 mg, 0.077 mmol) in 500 μL CHCl3 was added to the mixture.
The cloudy system was stirred 20 h under Ar. After evaporation of solvent, the crude
product was purified by silica gel chromatography (10 % methanol in chloroform, TLC:
1 Rf = 0.2) to give 40 mg (69 %) of pure 2.28. H NMR (CDCl3, 400 MHz), δ 7.70 (d, J =
7.6 Hz, 2H), 7.52 (d, J = 7.6 Hz, 2H), 7.35 (dd, J = 7.6, 7.6 Hz, 2H), 7.25 (dd, J = 7.6, 7.6
Hz, 2H), 6.58 (m, 1H), 5.94 (m, 1H), 5.74 (m, 1H), 5.28 (m, 2H), 4.31 (d, J = 6.8 Hz, 2H),
4.14 (t, J = 6.8 Hz, 1H), 4.03-3.60 (9H), 2.82-2.78 (t, J = 7.2 Hz, 2H), 2.69-2.65 (t, J =
7.2 Hz, 2H), 2.21-2.17 (t, J = 7.2 Hz, 4H), 1.54-1.45 (m, 2H), 1.23-1.19 (24H), 0.86 (t, J
13 = 6.8 Hz, 3H). C NMR (CDCl3, 100 MHz), δ 173.69 (CO), 173.42 (CO), 143.69 (C),
141.22 (C), 131.07 (CH), 127.77 (CH), 127.09 (CH), 125.02 (CH), 120.97 (CH), 119.98
(CH), 107.20 (CH), 105.34 (CH), 77.20 (CH) 69.07 (CH2), 66.66 (CH2), 65.37 (CH2),
64.30 (CH2), 46.65 (CH2), 46.35 (CH), 33.97 (CH2), 33.11 (CH2), 31.92 (CH2), 29.73
(CH2), 29.67 (CH2), 29.58 (CH2), 29.37 (CH2), 29.23 (CH2), 24.83 (CH2), 22.69 (CH2),
+ + 21.23 (CH2), 14.11 (CH3). HRMS (FAB) (m/z) calcd for C41H58NNaO9P (MNa )
+ + 776.3904, found 776.3939; calc. for C41H57NNa2O9P (MNa2 ) 798.3724, found
798.3717.
Hexadecanoic acid 3-([2-[2-(2-carboxy-ethyl)-pyrrol-1-yl]-ethoxy]-hydroxy-
phosphoryloxy)-2-hydroxy-propyl ester (lysoCEP-PE, 2.29). DBU (20 μL, 0.13 mmol) was added to 2.28 (24 mg, 0.03 mmol) in 500 μL CHCl3. After 3 h, the reaction was
completed and the mixture was diluted by 1.5 mL CHCl3. The solution was washed with
64 pH 5.5 phosphate buffer (2 mL). The organic layer was washed with brine, dried over
magnesium sulfate and vacuum-filtered, concentrated, and purified by flash
chromatography (CHCl3:MeOH:H2O = 65:25:4, v/v, TLC: Rf = 0.18) to yield 16 mg (95
1 %) of pure lysoCEP-PE. H NMR (CD3OD:CDCl3 = 1:1, 400 MHz), δ 6.60 (m, 1H), 5.96
(m, 1H), 5.83 (m, 1H), 4.10-4.0 (5H), 3.74-3.6 (m, 2H), 3.6-3.5 (m, 2H), 2.8-2.9 (m, 2H),
2.68-2.65 (m, 2H), 2.32 (t, J = 6.8 Hz, 2H), 1.57 (m, 2H), 1.23 (24H), 0.85 (t, J = 7.2 Hz,
13 3H). C NMR (CD3OD:CDCl3:D2O = 50:50:1, 100 MHz), δ 175.20 (CO), 133.00 (C),
121.23 (CH), 107.36 (CH), 105.56 (CH), 70.80 (CH), 66.05 (CH2), 65.63 (CH2), 47.17
(CH2), 34.61 (CH2), 33.29 (CH2), 32.48 (CH2), 30.22 (CH2), 30.05 (CH2), 29.90 (CH2),
29.88 (CH2), 29.72 (CH2), 27.04 (CH2), 25.42 (CH2), 23.21 (CH2), 14.38 (CH3). HRMS
+ + (FAB) (m/z) calcd for C27H48NNaO9P (MNa ) 598.3121, found 598.3053.
2-(9H-Fluoren-9-ylmethoxycarbonylamino)-6-[2-[2-(9H-fluoren-9-ylmethoxy
carbonyl)-ethyl]-pyrrol-1-yl]-hexanoic acid (2.30) 6-Amino-2-(9H-fluoren-9-ylmeth-
oxycarbonylamino)-hexanoic acid (40 mg, 0.1 mmol) was suspended in 10 mL methanol
with DOHAFm (30 mg, 0.089 mmol). Then 20 μL of acetic acid was added. The
suspension dissolved gradually and a light yellow oil was generated at the bottom of the
flask as the reaction proceeded. The mixture was stirred 24 h under argon. After the
removal of solvent, the crude compound was purified by silica gel chromatography (4 %
methanol in chloroform) to give 45 mg (75 %) of a light yellow oil 2.30. TLC (4 %
1 methanol in chloroform): Rf = 0.15; H NMR (CDCl3, 400 MHz), δ 7.75 (d, J = 7.2 Hz,
4H), 7.55 (d, J = 7.6 Hz, 4H), 7.39 (dd, J = 7.2, 7.6 Hz, 4H), 7.30 (dd, J = 7.2, 7.6 Hz,
4H), 6.57 (m, 1H), 6.06 (dd, J = 3.2, 3.2 Hz, 1H), 5.87 (m, 1H), 5.4 (m, 1H), 4.41 (d, J =
6.8 Hz, 2H), 4.39 (m, 1H), 4.20 (t, J = 6.8 Hz, 2H), 3.78 (t, J = 6.8 Hz, 2H), 2.85-2.73
65 13 (4H), 1.91-1.39 (6H). C NMR (CDCl3, 100 MHz), δ 176.58 (COOH), 173.08 (COO),
156.04 (CO), 143.75 (C), 143.66 (C), 141.25 (C), 130.58 (C), 127.75 (CH), 127.69 (CH),
127.07 (CH), 127.03 (CH), 125.01 (CH), 124.95 (CH), 119.99 (CH), 106.99 (CH),
105.23 (CH), 67.04 (CH2), 66.46 (CH2), 53.49 (CH), 47.07 (CH), 46.71 (CH), 46.04
(CH2), 33.18 (CH2), 31.84 (CH2), 30.73 (CH2), 22.40 (CH2), 21.37 (CH2). HRMS (FAB)
+ + (m/z) calcd for C42H41N2O6 (MH ) 669.2959, found 669.2949.
2-(9H-Fluoren-9-ylmethoxycarbonylamino)-6-[2-[2-(9H-fluoren-9-ylmethoxy
carbonyl)-ethyl]-pyrrol-1-yl]-hexanoic acid pentafluorophenyl ester (2.31). Freshly
distilled CH2Cl2 (10 mL) was added to a mixture of pentafluorophenol (50 mg,
0.27mmol), dicyclohexylcarbodiimide (DCC, 41.5 mg, 0.201 mmol), dimethlamino pyridine (DMAP, 8 mg, 0.067 mmol) and acid 2.30 (45 mg, 0.067 mmol). The resulting mixture was stirred for 72 h at room temperature. The solvent was then removed. Flash chromatography of the residue (15 % ethyl acetate in hexane, TLC: Rf = 0.2) gave a low
1 melting white solid 2.31 (50 mg, 90 %). H NMR (CDCl3, 200 MHz) δ 7.75 (dd, J = 6.8,
6.8 Hz, 4H), 7.56 (dd, J = 7.2, 7.2 Hz, 4H), 7.38 (dd, J = 7.2, 7.6 Hz, 4H), 7.29 (4H), 6.58
(dd, J = 3.2, 1.6 Hz, 1H), 6.06 (dd, J = 3.2, 3.2 Hz, 1H), 5.87 (m, 1H), 5.38 (d, J = 8.2 Hz,
1H), 4.39-4.45 (5H), 4.17-4.22 (m, 2H), 3.84 (t, J = 7.2 Hz, 2H), 2.86 (t, J = 6.8 Hz, 2H),
13 2.77 (t, J = 6.8 Hz, 2H), 2.0-1.46 (6H). C NMR (CDCl3, 100 MHz), δ 173.14 (COO),
168.96 (COO), 156.09 (COO), 143.93 (C), 143.82 (C), 141.57 (C), 141.51 (C), 130.94
(C), 128.03 (CH), 127.32 (CH), 125.22 (CH), 120.57 (CH), 120.25 (CH), 107.41 (CH),
105.62 (CH), 67.46 (CH2), 66.73 (CH2), 53.83 (CH), 47.32 (CH), 46.99 (CH), 46.31
(CH2), 33.40 (CH2), 32.18 (CH2), 30.99 (CH2), 22.72 (CH2), 21.66 (CH2). HRMS (FAB)
+ + (m/z) calcd for C48H40F5N2O6 (MH ) 835.2801, found 835.2813.
66 Competitive ELISA for inhibition of anti-CEP antibody by CEPH-BSA. CEP-
HSA was used as a coating agent and a standard, CEPH-BSA was used as an inhibitor. A
blank, a positive control containing no inhibitor, and up to eight serial dilutions of the
inhibitor and eight serial dilutions of the CEP-HSA standard were run. Each well of a 96
well ELISA plate (Cat. 9018, Corning Inc.) was coated with CEP-HSA solution (100 μL),
prepared by diluting a solution containing 187.14 nmol/mL HSA-bound CEP in PBS to
187 pmol/mL with pH 7.4 PBS (10 mM). The plate was incubated at 37 °C for 1 h, then
washed with 10 mM PBS (3 x 300 μL), and then blocked by incubating 1 h at 37 °C with
300 μL of 1 % chicken ovalbuman (CEO) in 10 mM PBS. The plate was then rinsed with
0.1 % CEO in 10 mM PBS (300 μL). Eight serial dilutions of CEPH-BSA inhibitor or
CEP-HSA standard (120 μL each with a dilution factor of 0.2) were preincubated at 37
°C for 1 h with anti-CEP-KLH antibody solution (120 μL) that was prepared by adding 5
μL of protein G column-purified antibody (1.8 mg/mL) in PBS to 10 mL of 0.2% CEO in
10 mM PBS. The initial inhibitor and standard concentrations were 1162 pmol/mL and
935 pmol/mL, respectively. These were prepared by diluting a CEPH-BSA solution
(116.2 nmol/mL) or CEP-HSA solution (187.14 nmol/mL) with 10 mM PBS,
respectively. Blank wells were filled with 0.1 % CEO (100 μL). Positive control wells
were filled with the diluted antibody solution (50 μL) and PBS (50 μL). The antibody- antigen complex solutions (100 μL) were then added in duplicate to their respective halves of the plate, which was then incubated at room temperature with gentle agitation on a shaker for 1 h. After the supernatant was discarded, the wells were washed with 0.1
% CEO (3 x 300 μL), and then goat anti-rabbit IgG-alkaline phosphatase solution
(Boehringer-Mannheim, Indianapolis, Indiana) (100 μL) which was prepared by adding
67 10 μL of the commercial enzyme-linked secondary antibody in 10 mL of 1 % CEO was
added. The plate was then incubated at room temperature with gentle agitation for 1 h
and washed with 0.1 % CEO (3 x 300 μL). A solution of of p-nitrophenyl phosphate in
0.2 M Tris buffer (100 μL, 1.0 mg/mL, Cat. N1891, Sigma) was added. The plate was then incubated at room temperature for 20 min until the maximum absorbance reached
0.6-0.8. The development was terminated by adding 3 N NaOH (50 μL) to each well before measuring the final absorbance values. The absorbance in each well was measured with a dual-wavelength Bio-Rad 450 microplate reader with detection at 405 nm relative to 655 nm. Absorbance values for duplicate assays were averaged and scaled to make the maximum curve fit value close to 100 percent. The averaged and scaled percent absorbance values were plotted against the log of concentration. Theoretical curves for each plot were fit to the absorbance data with a four parameter logistic function, f(x) =
(a-d)/[1+(x/c)^b]+d using SigmaPlot 9.0 (Jandel Scientific Software, San Rafael, CA).
Parameter a = the asymptotic maximum absorbance, b = slope at the inflection point, c = the inhibitor concentration at the 50 % absorbance value (IC50), and d = the asymptotic minimum absorbance.
68 Table 2.2. ELISA data for CEPH-BSA of Figure 2.1.
Pmol/mL Absorbance % Absorbance Parameters Curve Fit
1162.0000 5.0000e-3 0.9634 a = 99.4463 2.6770
232.0000 0.0230 4.4316 b = 0.8436 3.9224
46.5000 0.0570 10.9827 c = 1.9337 8.4609
9.3000 0.1090 21.0019 d = 2.2389 22.6527
1.8600 0.2670 51.4451 51.6393
0.3720 0.4270 82.2736 80.0698
0.0744 0.4860 93.6416 93.5954
0.0149 0.5190 100.0000 97.8689
Table 2.3. ELISA data for CEP-HSA of Figure 2.1.
Pmol/mL Absorbance % Absorbance Parameters Curve Fit
935.0000 0.0270 3.9187 a = 102.7627 4.7109
187.0000 0.0560 8.1277 b = 0.6935 8.2860
37.4000 0.1320 19.1582 c = 3.0304 17.7498
7.4800 0.2640 38.3164 d = 2.8697 37.6602
1.4960 0.4220 61.2482 64.8032
0.2990 0.6240 90.5660 86.0684
0.0598 0.6560 95.2104 96.6024
0.0119 0.6890 100.0000 100.6648
69 2.5. References
1. Skinner, E. R.; Watt, C.; Besson, J. A.; Best, P. V., Differences in the fatty acid
composition of the grey and white matter of different regions of the brains of patients
with Alzheimer's disease and control subjects. Brain 1993, 116 ( Pt 3), 717-25.
2. Wang, N.; Anderson, R. E., Enrichment of polyunsaturated fatty acids from rat
retinal pigment epithelium to rod outer segments. Curr. Eye Res. 1992, 11, (8), 783-91.
3. Alvarez, R. A.; Aguirre, G. D.; Acland, G. M.; Anderson, R. E., Docosapentaenoic
acid is converted to docosahexaenoic acid in the retinas of normal and prcd-affected
miniature poodle dogs. Invest. Ophthalmol. Vis. Sci. 1994, 35, (2), 402-8.
4. Farnsworth, C. C.; Dratz, E. A., Oxidative damage of retinal rod outer segment
membranes and the role of vitamin E. Biochim. Biophy. Acta. 1976, 443, (3), 556-70.
5. Bok, D., New insights and new approaches toward the study of age-related macular
degeneration. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 14619-14621.
6. Ferris, F. L., 3rd Am. J. Epidem. 1983, 118, 132-151.
7. Klein, R.; Wang, Q.; Klein, B. E.; Moss, S. E.; Meuer, S. M., The relationship of
age-related maculopathy, cataract, and glaucoma to visual acuity. Invest. Ophthalmol. Vis.
Sci. 1995, 36, (1), 182-91.
8. Stone, E. M.; Sheffield, V. C.; Hageman, G. S., Molecular genetics of age-related macular degeneration. Human Molecular Genetics 2001, 10, (20), 2285-2292.
9. Williams, R. A.; Brody, B. L.; Thomas, R. G.; Kaplan, R. M.; Brown, S. I., The psychosocial impact of macular degeneration. Arch. Ophthalmol. 1998, 116, (4), 514-20.
10. Crabb, J. W.; Miyagi, M.; Gu, X. R.; Shadrach, K.; West, K. A.; Sakaguchi, H.;
Kamei, M.; Hasan, A.; Yan, L.; Rayborn, M. E.; Salomon, R. G.; Hollyfield, J. G.,
70 Drusen proteome analysis: An approach to the etiology of age-related macular
degeneration. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, (23), 14682-14687.
11. Gu, X.; Meer, S. G.; Miyagi, M.; Rayborn, M. E.; Hollyfield, J. G.; Crabb, J. W.;
Salomon, R. G., Carboxyethylpyrrole protein adducts and autoantibodies, biomarkers for
age-related macular degeneration. J. Biol. Chem. 2003, 278, (43), 42027-42035.
12. Ebrahem, Q.; Renganathan, K.; Sears, J.; Vasanji, A.; Gu, X. R.; Lu, L.; Salomon,
R. G.; Crabb, J. W.; Anand-Apte, B., Carboxyethylpyrrole oxidative protein modifications stimulate neovascularization: Implications for age-related macular degeneration. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, (36), 13480-13484.
13. Jackson, A. H., Pyrroles. . Comprehensive Organic Chemistry (Barton, D., and
Ollis, D. W., Eds.) 1980, 4, pp 275-320, peergamon, New York.
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1396.
15. Sheppeck, J. E.; Kar, H.; Hong, H., A convenient and scaleable procedure for
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17. Bio-rad Protein Assay Instruction Manual. www.bio-
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Quantitation of Imines, Pyrroles, and Stable Nonpyrrole Adducts in 2,5-Hexanedione-
Treated Protein. Mol. Pharmacol. 1987, 32, (4), 542-548.
71 19. Instructions of modified Lowry protein assay kit. www.piercenet.com.
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Salomon, R. G., Immunochemical evidence supporting 2-pentylpyrrole formation on
proteins exposed to 4-hydroxy-2-nonenal. Chem. Res. Toxicol. 1996, 9, (7), 1194-1201.
21. Krzystolik, M. G.; Afshari, M. A.; Adamis, A. P.; Gaudreault, J.; Gragoudas, E. S.;
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experimental choroidal neovascularization with intravitreal anti-vascular endothelial
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22. Kruijff, B., Nature 1997, 386, 129-130.
23. Miyagi, M. S., H.; Darrow, R. M.; Yan, L.; West, K. A.; Aulak, K. S.; Stuehr, D. J.;
Hollyfield, J. G.; Organisciak, D. T. & Crabb, J. W., Mol. Cell. Proteomics 2002, 1, 293-
303.
24. Aulak, K. S.; Miyagi, M.; Yan, L.; West, K. A.; Massillon, D.; Crabb, J. W.; Stuehr,
D. J., Proteomic method identifies proteins nitrated in vivo during inflammatory
challenge. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, (21), 12056-61.
25. West, K. A.; Yan, L.; Miyagi, M.; Crabb, J. S.; Marmorstein, A. D.; Marmorstein,
L.; Crabb, J. W., Proteome survey of proliferating and differentiating rat RPE-J cells. Exp.
Eye Res. 2001, 73, (4), 479-91.
26. Stillway, L. W.; Harmon, S. J., A procedure for detecting phosphonolipids on thin-
layer chromatograms. J. Lipid Res. 1980, 21, (8), 1141-3.
27. Crabb, J. W.; Nie, Z.; Chen, Y.; Hulmes, J. D.; West, K. A.; Kapron, J. T.; Ruuska,
S. E.; Noy, N.; Saari, J. C., Cellular retinaldehyde-binding protein ligand interactions.
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20712-20.
72 28. Uebel, R.; Jaarsveld, P. v.; Hawtrey, A. O., Preparation of biotinylated spermine and the improved synthesis of some related amino derivatives. Med. Sci. Res. 1990, 18,
777-778.
73
Chapter 3
Lipid Oxidative Modifications in Autistic Disease
74 3.1. Background1, 2
In 1943, Dr. Leo Kanner, a psychiatrist at Johns Hopkins Hospital, studied a group of
11 children and applied the term ‘autism’ to children who were self-absorbed and who
had severe social, communication, and behavioral problems.3 Autism appears to affect an
estimated 34 out of every 10,000 children ages 3-10 and is three times more likely to
affect males than females.4 Autism and closely related pervasive developmental disorders
(PDDs), Asperger’s syndrome, fragile X syndrome, Rett’s syndrome and childhood
disintegrative disorder are referred to as autism spectrum disorders (ASD) that
corresponds to what the Diagnostic and Statistical Manual of Mental Disorders (DSM)-
IV refers to collectively as PDD.
Characteristics which are quite common in autism are impairments or delays in social
interaction, a lack of skills necessary for imaginative play and fantasy, and restricted,
repetitive patterns of behavior.4, 5 Autistic children do not follow the typical patterns of
child development. Some do not babble, point or make meaningful gestures by 1 year of
age. Some do not speak one word by 16 months. Some do not smile. Most children with
autism seem to have tremendous difficulty learning to participate in everyday human
interaction. They are also slower in learning to interpret what others are thinking and
feeling. Autistic patients usually exhibit odd, intensely repetitive motions although they
are physically normal and have good muscle control.
Although there is no known unique cause of autism, a number of factors have been
implicated in the pathogenesis of autism, including genetic, environmental, immunological and neurological. There is some indication of genetic influences such as
high concordance rates in siblings of autistic probands, as well as in monozygotic twins.6
75 Recently, many researchers speculate that three to five genes are likely associated with
autism.6, 7
Viruses associated with vaccinations, such as the measles component of measles-
mumps-rubella (MMR) vaccine and the pertussis component of the DPT shot were also
suspected being linked to autism.8 In the past few years, there was a public concern that the use of thimerosal (a mercury based preservative used in the MMR that is no longer used in vaccinations) linked to autism. However, a thorough review concluded that there is no causal relationship between thimerosal and autism.9, 10
Research associated with ASD
Zinc deficiency and copper excess are associated with ASD. In analyzing 503 patients
presenting diagnosis of ASD, Bill Walsh and colleagues reported 428 (85%) patients
exhibited Cu/Zn ratios in blood (1.63 ± 0.54) which were severely elevated ( p < 0.001) compared to 25 healthy controls (1.15 ± 0.24).11 Walsh proposes that copper:zinc
imbalance leads to emotional instability, attention deficit and hyperactivity,
neurotransmitter imbalances, and impairment of hippocampus and amygdala function.
The average autistic unbound (by ceruloplasmin) serum copper level (40 μg/dL) is 4
times higher than that of normal controls. An impaired metallothionein (MT) system is
postulated to be a pervasive factor in ASD. The absence of Cu and Zn homeostasis and
severe Zn deficiency are suggestive of an MT disorder. MTs are a family of proteins that
naturally chelate and buffer Zn as well as Cu and other redox active metals due to the
many sulfhydryl (-SH) groups they contain which give them extraordinary metal-binding
capability.12 MT synthesis and functioning involves (1) induction of thionein (apoMT), (2)
“preloading” with Zn ions, and (3) replacement of Zn by other metals, e. g., Cu. MT
76 plays a significant role in regulating the Cu levels throughout the body. It serves as a site
for temporary storage and detoxification of excess amounts of intracellular Cu.13, 14
Low plasma GSH levels are found in ASD patients. A primary mechanism for zinc loading and metal binding is the glutathione-glutathione disulfide (GSSH) redox couple.
Reduced glutathione (GSH) efficiently transfers zinc into apo-MT. Once MT is loaded with zinc, GSSH oxidizes MT causing the release of zinc in exchange for another ion, e. g., copper. Concentrations of total GSH at 4.1 ± 0.5 μmol/L were reported for autistics compared with 7.6 ± 1.4 μmol/L for healthy controls, p < 0.001.15 These abnormally low
levels of plasma GSH could contribute to the defective Cu and Zn metabolism common
in ASD patients. Furthermore, low levels of GSH also favor oxidative stress because
GSH is a cofactor for glutathione peroxidase (GPx), an antioxidant enzyme.
Sulfur metabolism defects are associated with autism. In a study of 232 children with
ASD matched with 68 control children, blood sulphate levels were found to be greatly
reduced in autism, with mean values of 0.55 nmol/mg protein compared with 4.9
nmol/mg protein for age-matched controls. In addition, urinary excretion of sulphate was
significantly elevated in autism (6819.0 ± 6712.3 nmol/mL) compared with 3030.8 ±
1461.0 nmol/mL for controls.16 Loss of sulphate in urine may partly explain low blood
sulphate levels. Sulphates in the blood help rid the body of waste peoducts by making them water soluble and therefore easily excreted. Low levels of sulphate may lead to retention of toxins, which can lead to biochemical effects on the central nervous system.
Glutathione peroxidase deficiency and superoxide dismutase excess are associated
with ASD. A major cause of damage to cells results from reactive oxygen species (ROS)
-induced alteration of macromolecules such as polyunsaturated fatty acids in membrane
77 lipids, essential proteins, and DNA. Oxidative stress and ROS have been implicated in
disease states, such as Alzheimer's disease, Parkinson’s disease, cancer, and aging. Under
normal conditions, ROS are cleared from the cell by the action of superoxide dismutase
(SOD), catalase, or glutathione peroxidase (GPx). SOD, MnSOD in the mitochondria and
CuZnSOD in the cytoplasm, removes superoxide anion by converting it into hydrogen
peroxide (H2O2). Catalase and GPx reduce H2O2 to water. In the presence of unbound Cu,
under certain conditions, SOD can promote oxidative injury owing to a Haber-Weiss
reaction of H2O2 that is catalyzed by Cu, that generates ●OH, a potent ROS. Statistically significant elevations in ZnCuSOD were documented in erythrocytes (+16.6%) and in platelets (+23.7%) of autistic individuals compared with controls, as well as severely depressed levels of GPx (-44.4%) in erythrocytes of autistic individuals compared with
17 controls. As a result, the H2O2 formed by the action of SOD would not be efficiently
removed owing to diminished levels of GPx. The imbalance is exacerbated by the low
plasma GSH levels found in ASD patients, because GSH is a cosubstrate needed for the
GPx-promoted reduction of H2O2.
Abnormalities of the immune system have been described in ASD patients. Immune
abnormalities in autism include depressed cell-mediated immunity and decreased natural
killer-cell activity;18 elevated levels of serum IgE;19 decreased proportions of interleukin-
2 (IL-2) and interferon-γ (IFN-γ) synthesizing CD4+ (T-helper-1) T cells and an increased production of IL-4 synthesizing CD4+ (T-helper-2) T cells.20 Although it is
unlikely that an immune disorder is the only or primary factor in ASD symptom
pathogenesis, the indications have led investigators to consider new multifactorial models
for the pathogenesis of autism.
78 Antibrain autoantibodies are present in the blood of ASD patients. Autoimmunity, an
abnormal immune reaction in which the immune system becomes primed to react against
body organs, can result from (1) defective “tolerance”, i. e., the absence of an immune
response to self antigens. (2) cross reactivity of self antigens with antibodies engendered
by foreign proteins. (3) an immune response against altered self proteins, e. g.., modified
by adduction of lipid-derived electrophiles generated by oxidative injury. IgG anti-brain
autoantibodies were present in 27% of sera from children with ASD compared with 2%
from healthy children. IgM autoantibodies were present in 36% of sera from children
with ASD compared with 0% of control sera.21 In another study, sera from 40 healthy
subjects and 40 autistic children were analyzed for the presence of IgG, IgM, and IgA
antibodies against nine neuron-specific antigens and three encephalitogenic and cross-
reactive proteins. Only 7.5-10% of controls had IgA, IgG or IgM antibodies against
neurofilaments compared to 37.5%, 50% and 57.5% of autistic subjects.22
The possible operation of an immune response against altered self proteins, has not been examined. Research in our group has demonstrated significantly elevated levels carboxyethylpyrrole (CEP) epitopes – specific docosahexaenoate (DHA)-derived oxidative protein modifications – and the corresponding autoantibodies in the blood of
AMD patients compared with healthy age and sex matched controls.23 Since DHA
containing lipid concentrations are uniquely high not only in retina but also in brain, it
seemed reasonable to anticipate that the detection and quantification of CEP epitopes and
autoantibodies in ASD patients will be a valuable tool for assessing oxidative injury in
ASD brain as it appears to be for assessing oxidative injury in AMD retina.
79 CEP and iso[4]LGE2-protein modifications in autistic brain. Immunohistochemical analysis of brain tissues such as cerebellum, hippocampus and neocortical regions has shown significant CEP-staining in the white matter in every autism cases but is absent in any of the control cases (age-matched or older) examined, suggesting elevated oxidative damage in these brain regions in autism (Figure 3.1).24 There is also prominent staining
24 of iso[4]LGE2-protein adduct in autistic brain but is not present in normal brain. An increased understanding of the extent and mechanisms of oxidative stress within the brain is needed, and is likely to lead to improvements in the treatment of autism. In this chapter, in order to determine whether the oxidative injury detected immunocytochemically in autistic brain was also present in blood, we examined the oxidative modifications of plasma proteins.
A B C
D E F
Figure 3.1. Anti-CEP staining of brain cortical regions from 3 of the control cases aged
5, 6 and 11 years (A, B, C) and 3 autism cases aged 9, 7, and 5 years (D, E, F).
80 In the present study, levels of CEP and iso[4]LGE2 adducts in plasma from patients
with documented ASD were compared with age-matched healthy controls. Anti-CEP,
anti-iso[4]LGE2, autoantibodies were also measured. In addition, since recent studies
proposed a possible participation of enhanced nitrative stress in AMD pathologies,25 levels of protein bound nitrotyrosine in plasma from ASD and normal controls were compared in this study by stable isotope dilution tandem mass spectrometry. No significant elevations in the levels of any of these markers of oxidative injury were detected in ASD plasma. However, an unanticipated strong elevation of iso[4]LGE2 immunoreactivity levels were found in ASD patients born prematurely compared with
ASD patients with no birth events or other birth events.
81 3.2. Materials and Methods
Human plasma
Blood samples were obtained by phlebotomists at the Institute’s Pfeiffer Treatment
Center. Eligibility for the study was based on a diagnosis of ASD, as defined by DSM-IV.
Subjects with questionable diagnoses and patients with comorbidity for seizures, and
birth anoxia were excluded. In total, 27 autistic subjects and 11 healthy control subjects
were recruited for the study.
ELISA
A competitive enzyme-linked immunosorbent assay (ELISA) of plasma from ASD and
healthy controls was performed as described previously for CEP and iso[4]LGE2
26 23 immunoreactivities. A direct ELISA was performed for anti-CEP and anti-iso[4]LGE2
autoantibodies. Absorbance values were measured on a Bio Rad Microplate Reader using
dual wavelength (405 nm to read the plate and 650 nm as a reference).
Mass spectrometric analysis of nitrotyrosine
Protein-bound nitrotyrosine in plasma from ASD and healthy controls was quantified
by stable isotope dilution LC/MS/MS as described27 on a triple quadrupole mass
spectrometer (API 365, Applied Biosystems, Foster City, CA) with Ionic EP 10+ upgrade
(Concord, Ontario, CA) interfaced to a Cohesive Technologies Aria LX Series HPLC
multiplexing system (Franklin, MA). Samples were delipidated, hydrolyzed, passed over mini solid-phase C18 extraction columns (Supelclean LC-C18-SPE minicolumn, 3 mL,
Supelco, Inc., Bellefone, PA) and subjected to mass spectrometric analysis. Synthetic
13 [ C6]-labeled 3-nitrotyrosine was used as internal standard for quantification of natural abundance 3-nitrotyrosine. Simultaneously, a universal labeled precursor amino acid,
82 13 15 [ C9, N1]tyrosine, was added, permitting potential intrapreparative formation of
13 15 nitro[ C9, N1]tyrosine to be routinely monitored and shown to be negligible (i.e., <<
5% of the level of the natural abundance product observed). Results are normalized to the content of the precursor amino acid tyrosine (i.e., nitrotyrosine/tyrosine, micromoles/ moles), which was monitored within the same injection.
Statistical analyses
ASD and normal controls were compared using CEP, iso[4]LGE2 protein adducts, anti-
CEP, anti-iso[4]LGE2 antibodies and nitroTyr/Tyr ratio. P-values were calculated by
independent t-test and analysis of variance (ANOVA) using Microsoft Excel 2003 for
Windows. The significance of group comparisons was corrected for the effect of repeated measures. Correlations between measured parameters were ascertained using pair-wise
comparisons by linear regression and were performed using JMP 5.12 (SAS Institute,
Cary NC). Data were expressed as mean ± SD. Statistical significance was defined as p less than 0.05.
83 3.3. Results and Discussion
3.3.1. CEP and iso[4]LGE2 protein adducts immunoreactivity in human plasma.
Levels of CEP and iso[4]LGE2-protein adducts in human blood were measured by
competitive ELISA using polyclonal anti-CEP and anti-iso[4]LGE2-KLH antibodies
respectively. We examined plasma from 27 patients with diagnosed ASD and 11 healthy
controls with matched ages. Comparison of levels of CEP-protein adducts in ASD and
controls is presented in Figure 3.2. Levels of individual CEP-protein adducts are shown
in Figure 3.3.
CEP Immunoreactivity in ASD and Control 350
p = 0.45 300
250
200
150 125 ± 58 110 ± 30 100
CEP Immunoreactivity (pmol/mL) 50
0 Autism Control (n = 27) (n = 11)
Figure 3.2. CEP immunoreactivity detected in human plasma from (◆) ASD and (■) normal controls. The figure also shows mean levels detected (O). The error bars indicate the standard deviation (S. D.) for each data set.
84 CEP Immunoreactivity of ASD and Controls
350
300
250 ASD Control 200
150
100
50 CEP Im m unoreactivity (pm ol/m L)
0
2 4 6 7 9 8 3 1 4 6 9 A1 A A3 A A5 A A A8 A 10 11 12 14 16 17 19 21 22 24 26 27 N N2 N3 N N5 N N7 N8 N 11 A A A A13 A A15 A A A1 A A20 A A A2 A A25 A A N10 N Sample ID Figure 3.3. Levels of individual CEPs in plasma from 27 patients with diagnosed ASD and 11 healthy controls.
Comparison of levels of iso[4]LGE2-protein adducts is presented in Figure 3.4. Levels
of individual iso[4]LGE2-protein adducts are shown in Figure 3.5.
Iso[4]LGE2 Immunoreactivity in ASD and Control 40 p = 0.09 35
30
25
20 16.7 ± 5.8 15 Immunoreactivity (nmol/mL)
2 13.4 ± 3.4
10
iso[4]LGE 5 Autism Control (n = 27) (n = 11)
Figure 3.4. Iso[4]LGE2 immunoreactivity detected in human plasma from (◆) ASD and
(■) normal controls. The figure also shows mean levels detected (O). The error bars
indicate the standard deviation (S. D.) for each data set.
85 Iso[4]LGE2 Immunoreactivity of ASD and Control
35
30 ASD Control 25
20
15 Immunoreactivity 2 (nmol/mL) 10
5 Iso[4]LGE
0
3 4 5 6 7 2 3 4 5 2 3 4 5 6 7 8 9 A1 A2 A A A A A A8 A9 1 1 1 1 16 2 2 2 2 N1 N2 N3 N4 N5 N N N N A10A11A A A A A A17A18A19A20A21A A A A A26A27 N10N11 Sample ID
Figure 3.5. Levels of individual iso[4]LGE2-protein adducts in plasma from 27 patients
with diagnosed ASD and 11 healthy controls.
A typical inhibition curve for binding of iso[4]LGE2-KLH with a naturally occurring
epitope in plasma from a patient with documented ASD and a normal control is shown in
Figure 3.6.
120
100
80
60
40
Absorbance (% max) Absorbance (% 20
0
10-3 10-2 10-1 100 101 102 103 104 Inhibitor (nmol/mL)
Figure 3.6. Inhibition curves for binding of anti-iso[4]LGE2-KLH to iso[4]LGE2-BSA by
iso[4]LGE2-HSA (●), plasma from an ASD patient (■), and a normal control (▲).
86 There were no significant differences in the mean levels of CEP adduct between the
plasma of ASD, 124.5 ± 57.9 pmol/mL, and the plasma of age-matched healthy
controls, 110.4 ± 30.3 pmol/mL, p = 0.45. Nor were there statistically significant
differences in the mean levels of iso[4]LGE2 protein adduct between ASD, 16.7 ± 5.8
nmol/mL, and controls, 13.4 ± 3.4 nmol/mL, p = 0.088.
3.3.2. Presence of anti-CEP and anti-iso[4]LGE2 autoantibodies in human plasma.
ASD seems to arise from environmental factors interacting with a genetic predisposition. It often occurs in conjunction with a family history of autoimmune
diseases.28-30 As mentioned above, autistic patients often have immune abnormalities.
Antibrain antibodies IgG, IgM, IgE and IgA are present in ASD patients.21 Inflammatory
over activation is suggested by the observation that many autistic children appear to
produce excessive amounts of TNFα and other pro-inflammatory cytokines.31 In this
study, we were interested in probing the association between ASD and lipid oxidation.
Anti-CEP autoantibody titer in plasma from 26 ASD and 5 healthy controls were
measured by ELISA. Data obtained from triplicate assays are summarized in Figures 3.7
and 3.8. Anti-iso[4]LGE2 autoantibody titer in plasma of 27 ASD and 11 healthy
controls are summarized in Figures 3.9 and 3.10.
87 CEP Autoantibody Titer in ASD and Control 4 p = 0.9 ) 0
3
2
1.2 ± 0.7 1.2 ± 0.3 1 CEP Autoantibody Titer (A/A Titer CEP Autoantibody
0 Autism Control (n = 26) (n = 5)
Figure 3.7. CEP autoantibody titer detected in human plasma from (◆) ASD and (■)
normal controls. The figure also shows mean levels detected (O). The error bars indicate
the standard deviation (S. D.) for each data set.
CEP Autoantibody Titre of ASD and Control
4.0 3.5 3.0 ASD Control 2.5 2.0 1.5 1.0 0.5
CEP Autoantibody Titre(A/A0 0.0
1 4 6 9 0 5 0 2 5 2 3 5 A A2 A3 A A5 A A7 A8 A 13 14 19 24 N1 N N N4 N A1 A11A12A A A1 A16A17A18A A2 A21A2 A23A A2 A26 Sample ID
Figure 3.8. Levels of individual individual CEP autoantibody titer in plasma from 26 patients with diagnosed ASD and 5 healthy controls.
88 Iso[4]LGE2 Autoantibody Titer in ASD and Control
8
) p = 0.6 7
6
5
4
3
Autoantibody Titer (A/A0 Autoantibody 2 2 1.3 ± 1.6 1 1.0 ± 0.9
0 Iso[4]LGE
-1 Autism Control (n = 27) (n = 11)
Figure 3.9. Iso[4]LGE2 autoantibody titer detected in human plasma from (◆) ASD and
(■) normal controls. The figure also shows mean levels detected (O). The error bars
indicate the standard deviation (S. D.) for each data set.
Iso[4]LGE2 Autoantibody Titer 9
8
7 ASD Control 6
5 4
3 Autoantibody Titer (A/A0) Titer Autoantibody 2 2 1
Iso[4]LGE 0
3 6 9 2 6 9 3 6 3 4 6 7 A1 A2 A A4 A5 A A7 A8 A 10 1 13 1 17 1 20 2 24 2 27 N1 N2 N N N5 N N N8 N9 10 A A11A A A14A15A A A18A A A21A22A A A25A A N N11 Sample ID
Figure 3.10. Levels of individual iso[4]LGE2 autoantibody titer in plasma from 27
patients with diagnosed ASD and 11 healthy controls.
89 There were no significant differences in the mean levels of anti-CEP autoantibody
titer between the plasma of ASD, 1.2 ± 0.7, and the plasma of age-matched healthy
controls, 1.2 ± 0.3, p = 0.9. Nor were there statistically significant differences in the
mean levels of anti-iso[4]LGE2 autoantibody titer between ASD, 1.3 ± 1.6, and
controls, 1.0 ± 0.9, p = 0.6.
3.3.3. Protein bound nitrotyrosine, chlorotyrosine and bromotyrosine levels in ASD and healthy controls.
A number of inflammatory and neurodegenerative disorders have been linked with protein modification by reactive nitrogen species including ocular inflammation, atherosclerosis, retinal ischemia, lung infection, cancer, Parkinson’s disease, and
32 Alzheimer’s disease. Measurement of NO2Tyr, a posttranslational modification of proteins generated by reactive nitrogen species, serves as a quantitative index of nitrative stress in vivo.
In this study, there were no differences between the ratio of NO2Tyr/Tyr in plasma
from 27 ASD patients, 7.81 E -06 ± 3.29 E -06, and that from 11 healthy controls, 7.87
E-06 ± 1.62 E -06. The data are shown in Figure 3.11.
We also measured chlorotyrosine and bromotyrosine levels in autistic and normal plasma.
There were no differences between the ratio of ClTyr/Tyr in plasma from 27 ASD
patients, 1.37 E -05 ± 1.03 E -05, and that from 11 healthy controls, 1.55 E -05 ± 1.14
E-05. The data are shown in Figure 3.12. Nor were there any significant differences
between the ratio of BrTyr/Tyr in plasma from ASD patients, 4.17 E -05 ± 5.69 E -06,
and that from healthy controls, 3.77 E -05 ± 7.41 E -06. The data are shown in Figure
3.13.
90 NitroTyr/Tyr in Autism and Control Plasma 2.00E-05 p = 0.95 1.80E-05
1.60E-05
1.40E-05
1.20E-05
1.00E-05
8.00E-06 7.81 ± 3.29 7.87 ± 1.62 6.00E-06
4.00E-06
2.00E-06 NitroTyr/Tyr Conc. Ratio (mol/mol) Ratio Conc. NitroTyr/Tyr 0.00E+00 Autism Control (n = 27) (n = 11)
Figure 3.11. Nitrotyr/Tyr ratio detected in human plasma from (◆) ASD and (▲) normal controls. The figure also shows mean levels detected (O). The error bars indicate the standard deviation (S. D.) for each data set.
ClTyr/Tyr in Autism and Control Plasma
4.50E-05 p = 0.59 4.00E-05
3.50E-05
3.00E-05
2.50E-05
2.00E-05
1.50E-05 1.55 ± 1.14 1.37 ± 1.03 1.00E-05 ClTyr/Tyr Conc. Ratio (mol/mol) Ratio Conc. ClTyr/Tyr 5.00E-06
0.00E+00 Autism Control (n = 27) (n = 11)
Figure 3.12. Cltyr/Tyr ratio detected in human plasma from (◆) ASD and (■) normal controls. The figure also shows mean levels detected (O). The error bars indicate the standard deviation (S. D.) for each data set.
91 BrTyr/Tyr in Autism and Control Plasma
7.00E-05 p = 0.08 6.50E-05
6.00E-05
5.50E-05
5.00E-05
4.50E-05 4.17 ± 0.57 4.00E-05 3.77 ± 0.74 3.50E-05 BrTyr/Tyr Conc. Ratio (mol/mol) Ratio Conc. BrTyr/Tyr 3.00E-05
2.50E-05 Autism Control (n = 27) (n = 11)
Figure 3.13. Brtyr/Tyr ratio detected in human plasma from (◆) ASD and (■) normal controls. The figure also shows mean levels detected (O). The error bars indicate the standard deviation (S. D.) for each data set.
3.3.4. Correlations among lipid oxidative protein adducts and nitrative proteins.
Linear regression analyses of CEP, iso[4]LGE2 immunoreactivities and nitroTyr/Tyr
were conducted (Figures 3.14-3.17). Out of 27 ASD patients, 6 (22%) showed elevated
CEP and iso[4]LGE2 immunoreactivities while 1 out of 11 normal controls (9%) showed
both elevated CEP and iso[4]LGE2 immunoreactivities (Figure 3.15 ).
A significant linear correlation, R = 0.34, p = 0.036, was found between the levels
of CEP and iso[4]LGE2 immunoreactivities (Figure 3.14, non-parametric Spearman’s
correlation was used). There were no significant linear correlations between CEP and
nitroTyr/Tyr (Figure 3.16), R = 0.09, p = 0.56, or iso[4]LGE2 immunoreactivity and
nitroTyr/Tyr (Figure 3.17), R = 0.1, p = 0.5.
92 300
250
200 ● ASD (n = 27) ▲ Control (n = 11) 150 R = 0.34, p = 0.036
100
50 CEP immunoreactivity (pmol/mL) 10 15 20 25 30
Iso[4]LGE2 immunoreactivity (nmol/mL)
Figure 3.14. Correlation between CEP immunoreactivity and iso[4]LGE2
immunoreactivity in human plasma from (●) ASD and (∆) normal controls.
Correlation of Iso[4]LGE2 and CEP Immunoreactivity 350.0
III 300.0 IV ASD (n = 27) 250.0 Control (n = 11) c Ave. 200.0
eactivity (pmol/mL) 150.0
100.0 II 50.0 I CEP Immunor CEP
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0
Iso[4]LGE2 Immunoreactivity (nmol/mL)
Figure 3.15. Correlation between iso[4]LGE2 and CEP immunoreactivities in human plasma from (◆) ASD and (■) normal controls. The horizontal and vertical dashed lines
indicate the mean values of CEP and iso[4]LGE2 immunoreactivities, respectively. Out of
27 ASD patients, 6 (22%) are in region IV while 1 out of 11 normal controls (9%) is in region IV. ASD patients account for 86% (6/7) of the data in region IV.
93 300
250
200 ● ASD (n = 27) ▲ Control (n = 11) CEP 150 R = 0.09, p = 0.56
immunoreactivity 100
50
0 5 10 15 20 Nitrotyro/Tyr
Figure 3.16. Correlation between CEP immunoreactivity and nitrotyro/Tyr ratio in
human plasma from (●) ASD and (∆) normal controls.
30
25 ● ASD (n = 27) y
▲ Control (n = 11) 20 R = 0.1, p = 0.5 immunoreactivity 2
(nmol/mL) 15
10 Iso[4]LGE
0 5 10 15 20 Nitrotyro/Tyr
Figure 3.17. Correlation between iso[4]LGE2 immunoreactivity and nitrotyro/Tyr ratio in human plasma from (●) ASD and (∆) normal controls.
3.3.5. Correlations between autoantibodies and lipid oxidation immunoreactivities.
Although CEP autoantibody titer was only monitored in plasma from 26 ASD and 5
controls, a strong correlation was seen in CEP autoantibody titer and iso[4]LGE2
autoantibody titer (Figure 3.18), R = 0.76, p < 0.001.
94 No correlation was found between CEP imunoreactivity and CEP autoantibody levels
(Figure 3.19), R = 0.28, p = 0.6. Nor was there any correlation between iso[4]LGE2 immunoreactivity and iso[4]LGE2 autoantibody levels (Figure 3.20), R = 0.04, p = 0.8.
3.5
3
2.5 ● ASD (n = 26) ▲ Control (n = 5) 2
titer R = 0.76, p < 0.001
1.5 CEP autoantibody
1
0.5 0 1 2 3 4 5 6 7 8
Iso[4]LGE autoantibody titer 2
Figure 3.18. Correlation between CEP autoantibody titer and iso[4]LGE2 autoantibody
titer in human plasma from (●) ASD and (∆) normal controls.
300
250
200 ● ASD (n = 26) ▲ Control (n = 5) CEP 150 R = 0.28, p = 0.6
immunoreactivity 100
50
.5 1 1.5 2 2.5 3 3.5 CEP autoantibody titer
Figure 3.19. Correlation between CEP immunoreactivity and CEP autoantibody titer in
human plasma from (●) ASD and (∆) normal controls.
95 30
25
)
20 ● ASD (n = 27) ▲ Control (n = 11)
immunoreactivity R = 0.04, p = 0.8 2 nmol/mL ( 15
Iso[4]LGE 10
0 1 2 3 4 5 6 7 8
Iso[4]LGE autoantibody titer 2
Figure 3.20. Correlation between iso[4]LGE2 immunoreactivity and iso[4]LGE2 autoantibody titer in human plasma from (●) ASD and (∆) normal controls.
3.3.6. Lipid oxidation products and birth events in ASD patients.
Figures 3.21-24 show differences in levels of lipid oxidation protein adducts and autoantibody titers, grouped according to birth events of ASD patients with no events, born premature or other events such as C-section, cord wrapped and low birth weight.
The “born premature" group exhibits a significantly elevated mean level of
iso[4]LGE2 immunoreactivity, compared to “no birth events” group (p = 0.008) and
the “other events” group (p = 0.016). However, neither CEP immunoreactivity level
nor CEP or iso[4]LGE2 autoantibody titers showed any significant differences.
It must be noted that all of the “born premature” group were also a cohort of the
autistic group. Thus, we had no “born premature” individuals in the control group. So it is
not possible to distinguish between a general characteristic of premature birth and a
characteristic of a subgroup of autistic patients.
96 Birth Events and CEP Immunoreactivity 255
p = 0.88 p = 0.23 205
155
128 ± 34 124 ± 59 105 102 ± 32
55
5 CEP Immunoreactivity (pmol/mL) Immunoreactivity CEP No Significant Born Other Events Birth Events Premature (n=5) (n=16) (n=6)
Figure 3.21. CEP immunoreactivity grouped according to birth events of ASD patients with no events, born premature or other birth events.
Birth Events and CEP Autoantibody Titer 4.0 p = 0.8 p = 0.9 3.5
3.0
2.5
2.0
1.5 1.2 ± 0.8 1.1 ± 0.7 1.2 ± 0.7 1.0
0.5 CEP autoantibody titer (A/A0) titer CEP autoantibody
0.0 No Significant Born Other Events Birth Events Premature (n=5) (n=16) (n=5)
Figure 3.22. CEP autoantibody titer grouped according to birth events of ASD patients with no events, born premature or other birth events.
97 Birth events and iso[4]LGE2 immunoreactivity 35 p = 0.008 p = 0.016
30
25 22.7 ± 5.9 20
15.3 ± 5.0 15 14.0 ± 3.0 immunoreactivity (nmol/mL) 2
10
Iso[4]LGE 5 No Significant Born Other Events Birth Events Premature (n=5) (n=16) (n=6)
Figure 3.23. Iso[4]LGE2 immunoreactivity grouped according to birth events of ASD patients with no events, born premature or other birth events.
Birth Events and iso[4]LGE Autoantibody Titer 8.5 2
7.5 p = 0.8 p = 0.98 6.5
5.5
4.5
3.5
2.5 autoantibody titer (A/A0) 2 1.5 1.4 ± 1.6 1.5 ± 1.3 1.2 ± 1.8 0.5 iso[4]LGE -0.5 No Significant Born Other Events Birth Events Premature (n=5) (n=16) (n=6)
Figure 3.24. Iso[4]LGE2 autoantibody titer grouped according to birth events of ASD patients with no events, born premature or other birth events.
98 3.4. Conclusions
In view of the high omega-3 poly unsaturated fatty acid content of the brain, it is likely
that these fats participate in brain biochemistry, physiology and functioning; and may
play a role in some neuropsychiatric diseases and ageing. By using polyclonal anti-
iso[4]LGE2 and anti-CEP antibodies, we measured levels of the lipid oxidation protein adducts iso[4]LGE2 and CEP in blood plasma from 27 autistic patients and 11 normal controls. Autoantibodies against iso[4]LGE2 and CEPs were also monitored by direct
ELISA. Similar levels of these protein modifications and autoantibodies against them
were detected in blood from both autistic individuals and normal controls.
Immunohistochemically, it was discovered that modifications of brain protein by
products of free radical-induced lipid oxidation, i.e., CEP and iso[4]LGE2-protein adducts,
are uniquely present in autistic brain but not in normal brain. Taken together, our
observations suggest that the oxidative protein modifications associated with autistic
brain are highly localized and do not cross the blood/brain barrier.
Unexpectedly, a subpopulation consisting of autistic patients born prematurely showed significantly elevated iso[4]LGE2-protein adduct immunoreactivity levels compared with patients with no birth events or other birth events. No control subjects were born
prematurely in this study, so it can not be concluded whether or not this phenomenon is associated with autism. The possibly general association of the phenomenon of elevated plasma iso[4]LGE2-protein adduct levels does warrant further investigation as a
heretofore unrecognized consequence of premature birth or postnatal treatment protocols.
Other observations, that are not associated with autism, are strong correlations between
CEP and iso[4]LGE2-protein adduct immunoreactivity levels in plasma from 27 ASD
99 patients and 11 normal controls, as well as between anti-CEP and anti-iso[4]LGE2- protein adduct autoantibody titers in plasma from 26 ASD patients and 5 normal controls.
The positive correlation between the two lipid oxidation protein modifications suggests that oxidative damage to docosahexaenoate and arachidonate are equally likely. Also, the proclivity of an individual to develop an immune response to altered self protein is similar for a diverse structural array of immunogenic modifications, e. g., CEPs and isoLGs.
Tandem mass spectrometry was used to monitor the levels of protein nitrative products
(nitrotyrosine) in plasma from 27 ASD and 11 normal controls. We found no significant differences between ASD and controls.
100 3.5. Experimental Procedures
Human plasma
Plasma samples were treated according to following protocol:
1. Collect 5 mL of blood in 7 mL lavender top vacutainer tube with EDTA (chilled tube
is not necessary). Gently invert the tube 5-6 times to distribute anticoagulant.
2. Spin in refrigerated centrifuge (4 °C) at 3000 rpm for 15 minutes.
3. Transfer plasma (2.0 mL, be sure to avoid the buffy coat) to a 15 mL Falcon tube.
4. Add 10 μL of BHT/ethanol solution (5 μL/mL plasma) and 20 μL of protease inhibitor
cocktail kit (10 μL/mL plasma, Sigma, Cat. P8340) to plasma. Mix gently 5-6 times by
inversion. Do not vortex. The BHT/ethanol solution is made by combining BHT (44 mg)
with ethanol (10 mL). If refrigerated, the BHT solution is good for 3 weeks.
5. Aliquot 250 μL into 8 prelabeled screw-top microfuge tubes. Gently flush the vials
with argon and seal with screw caps.
6. Quench freeze in liquid nitrogen by placing the 8 vials into a Scienceware Round
Bubble Rack (Fisher, Cat. 14-792-14 Bel-Art, fishersci.com) and then immersing the
bottom of the rack and the tubes into liquid nitrogen in Nalgene polyethylene Dewar flask
for 1 minute.
7. Store vials in fiberboard storage boxes (Fisher, Cat. 11-678-24A, fishersci.com) with fiberboard box dividers (Fisher, Cat. 11-678-24C, fishersci.com) in dry ice for transport
or at -80 °C.
101 Biomarkers
CEP-BSA and CEP-HSA were prepared as described in Chapter 2. Iso[4]LGE2-BSA and iso[4]LGE2-HSA were prepared as follows: BSA (20.6 mg) in pH = 7.4 PBS solution
(10 mM, 2 mL) was incubated with iso[4]LGE2 (3 mg) at 37 °C for 20 h under Argon.
The light yellow clear solution changed to green-yellow clear solution as the reaction
going. The consequent iso[4]LGE2-BSA was diluted with PBS (10 mM, 3 mL) and
transferred to a dialysis membrane tube ( Mr cutoff 14000). Two successive dialyses (12
h each) against 1 L 10 mM PBS (pH 7.4) at 4 °C were performed. The final protein
concentration (3.745 mg/mL) was determined by the Pierce bicinchoninic acid (BCA)
protein assay.33 The pyrrole concentration (1.094 mM) was determined by Ehrlich
34 assay. The pyrrole : protein ratio was found to be 11 : 1. Iso[4]LGE2-HSA was
synthesized similarly except that it was diluted with 4 mL PBS (10 mM). Protein concentration was 2.82 mg/mL by the Pierce bicinchoninic acid (BCA) protein assay33,
and pyrrole concentration was 0.842 mM by Ehrlich assay.34 The pyrrole : protein ratio
was also found to be 11 : 1. Anti-CEP-KLH, anti-iso[4]LGE2-KLH polycolonal
antibodies were purified as described previously.23, 26
Competitive ELISA for CEP and iso[4]LGE2 immunoreactivity. ELISA of plasma
from ASD and healthy controls was performed as described in Chapter 2. For CEP immunoreactivity, CEP-BSA was used as a coating agent and CEP-HSA was used as a standard. The initial concentration of CEP-HSA was 16.7 nmol/mL. For iso[4]LGE2 immunoreactivity, iso[4]LGE2-BSA was used as a coating agent and iso[4]LGE2-HSA was used as a standard. The initial concentration of iso[4]LGE2-HSA was 810 nmol/mL.
A dilution factor of 0.2 was employed for standard and samples. Eight serial dilutions for
102 standard, five serial dilutions of samples were performed. Data for Figure 3.6 are presented in Tables 3.1-3.3. Levels of CEP and iso[4]LGE2 immunoreactivities in plasma
of ASD patients versus age matched healthy control are summarized in Table 3.4-3.5.
Table 3.1. ELISA data for iso[4]LGE2-HSA standard for Figure 3.6.
nmol/mL Absorbance % Absorbance Parameters Curve fit
810.0000 0.0100 1.4493 98.9827 1.6297
162.0000 0.0200 2.8986 1.2176 2.4593
32.4000 0.0540 7.8261 3.6946 7.9634
6.4800 0.2330 33.7681 1.4923 34.1853
1.3000 0.5440 78.8406 77.6367
0.2590 0.6350 92.0290 95.2946
0.0520 0.6920 100.2899 98.4430
0.0100 0.6860 99.4203 98.9098
Table 3.2. ELISA data for ASD patient (patient code A27) for Figure 3.6.
nmol/mL Absorbance % Absorbance Parameters Curve fit
810.0000 0.1350 17.9045 98.4569 19.3619
162.0000 0.4080 54.1114 0.9558 52.4454
32.4000 0.6180 81.9629 185.7748 82.8472
6.4800 0.6960 92.3077 1.7911e-7 94.6278
1.3000 0.7540 100.0000 97.6062
103 Table 3.3. ELISA data for control (normal code N11) for Figure 3.6.
nmol/mL Absorbance % Absorbance Parameters Curve fit
810.0000 0.1250 17.3130 99.2831 17.9040
162.0000 0.4790 66.3435 1.3534 65.5440
32.4000 0.6640 91.9668 264.6144 93.8141
6.4800 0.7160 99.1690 1.5995e-7 98.6319
1.3000 0.7220 100.0000 99.2086
CEP and iso[4]LGE2 autoantibodies in human plasma.
Direct ELISAs were performed to determine the antibody titer. The plates were coated with CEP-BSA or iso[4]LGE2-BSA (100 μL/well). BSA (2 %) in 10 mM PBS (100 μL) was added to the corresponding blank wells. The plates were incubated for 1 h at 37 °C, washed with PBS (10 mM, 300 μL) 3 times, and blocked with 1% chicken egg ovalbumin (CEO, 300 μL) for 1 h at 37 °C. After washing once with 0.1 % CEO plus
0.05 % Tween 20 (300 μL), the plates were loaded with plasma, diluted 20 times in 0.2 %
CEO plus 0.05 % Tween 20, and then incubated for 1 h at room temperature. The plates were then washed 3 times with 0.1 % CEO plus 0.05 % Tween 20 (300 μL), and then incubated 1 h at room temperature with alkaline phosphatase conjugated goat anti-human
IgG (Sigma-Aldrich, Milwaukee, WI, Cat. Sigma A-8542), which was diluted 1:2000 with 1% CEO plus 0.05 % Tween 20 (100 μL). After washing with 0.1% CEO (3 × 300
µL), a solution of p-nitrophenyl phosphate in 0.2 M Tris buffer (1.0 mg/mL, 100 µL,
Sigma-Aldrich, Milwaukee, WI, Cat. Sigma N1891) was added. The absorbance was read at 405 nm with reference at 650 nm after incubation at room temperature for 30 min. The
104 titer was defined as the ratio of plasma binding to antigen (A) vs. binding to BSA blank
(A0). The data for CEP and iso[4]LGE2 autoantibodies are list in Tables 3.4 and 3.5.
Table 3.4. Data for CEP, iso[4]LGE2 immunoractivites and CEP, iso[4]LGE2 autoantibodies in ASD patients.
CEP iso[4]LGE2 CEP iso[4]LGE2
Patient code immunoreactivity immunoreactivity autoantibody autoantibody
(pmol/mL) (nmol/mL) (A/A0) (A/A0)
A1 60.8 8.8 0.9 1.1
A2 105 14.4 1.1 0.6
A3 143.2 15.2 0.9 0.4
A4 215.6 14 3.4 2.1
A5 55.2 14 2.8 7.7
A6 87.1 12.8 2.2 3.6
A7 185 20 0.7 0.3
A8 102.5 24.8 1.1 1.4
A9 167.5 9.6 0.7 0.5
A10 172 21.6 0.9 0.8
A11 287.5 23.2 1.5 1
A12 237.5 13.6 1.1 1.2
A13 93.2 12 1.6 1.8
A14 135.7 29.6 2.4 4.5
A15 158.8 22.4 0.6 0.5
A16 79.4 22.8 0.8 0.6
105 A17 104 12 0.7 0.9
A18 91.5 14.8 0.9 0.7
A19 154.6 19.2 0.7 0.3
A20 72.8 14 0.6 0.7
A21 41.7 9.6 1.2 1.3
A22 84.3 13.6 0.7 0.2
A23 112.5 16 0.8 0.4
A24 100 29.6 1 0.8
A25 115.3 10.4 0.9 0.4
A26 87.6 16.8 0.7 0.5
A27 111.2 16.2 . 1.5
Table 3.5. Data for CEP, iso[4]LGE2 immunoractivites and CEP, iso[4]LGE2 autoantibodies in normal controls.
CEP iso[4]LGE2 CEP iso[4]LGE2
Normal code immunoreactivit immunoreactivity autoantibody autoantibody
y (pmol/mL) (nmol/mL) (A/A0) (A/A0)
N1 102.1 10 1.6 0.4
N2 130.1 9.6 1.4 1.1
N3 182.5 19.2 0.8 0.3
N4 70.5 9.6 1.1 0.6
N5 98.6 16.8 1.2 0.4
N6 111.9 13.1 3.2
106 N7 83.6 13.1 0.5
N8 91.8 10.5 1.5
N9 97.4 16.9 1.7
N10 111.6 16.2 0.4
N11 134.5 12.8 1.2
Detection of nitrotyrosine in plasma of ASD and control. This work was done
collaboratively with Xiaoming Fu and Zeneng Wang.35
Protein Hydrolysis—Protein was delipidated and desalted using two sequential
extractions with a single phase mixture of H2O/methanol/H2O-saturated diethyl ether
13 13 (1:3:8 v/v/v). Oxidized tyrosine standards, C6-ortho-tyrosine (o-Tyr), C6-meta-
13 13 13 tyrosine (m-Tyr), C6-nitrotyrosine (Nitro-Tyr), C6-chlorotyrosine (Cl-Tyr), C6-
13 15 bromotyrosine (Br-Tyr) (2 pmol each) and universal labeled tyrosine, C9- N-tyrosine
(2 nmol) were added to protein pellets. Samples were hydrolyzed in degassed 4M
methane sulfonic acid (500 μL) supplemented with 1% phenol for 24 h at 110 °C under
argon atmosphere. Amino acid hydrolysates were resuspended in 0.1% trifluoroacetic acid (2 mL) and applied to mini solid-phase C18 extraction columns (Supelclean LC-C18
SPE mini-column, 3 ml; Supelco, Inc., Bellefone, PA) pre-equilibrated with 0.1% trifluoroacetic acid. Following sequential washes with trifluoroacetic acid (2 ml, 0.1%), oxidized tyrosines and tyrosine were eluted with 2 ml of 30% methanol in 0.1% trifluoroacetic acid, dried under vacuum, and then analyzed by mass spectrometry as described below.
107 Mass Spectrometry (MS)—Nitrotyrosine in lysates was analyzed by HPLC with on-line
electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) using stable isotope
dilution methodology on a triple quadrupole mass spectrometer (API 365; Applied
Biosystems, Foster City, CA) with Ionics EP 10+ upgrade (Ionics, Concord, Ontario, CA)
interfaced to a Cohesive Technologies (Franklin, MA) Aria LX Series HPLC
multiplexing system. Samples were suspended in equilibration solvent (H2O with 0.1%
formic acid) and injected onto an Ultrasphere C18 column (Phenominex, 5 μm, 2.0 × 150
mm). L-Tyrosine and its oxidation products were eluted at a flow rate of 0.2 mL/min
using a linear gradient generated against 0.1% formic acid in acetonitrile pH 2.5, as the
second mobile phase. Analytes were monitored in positive ion mode with full scan
product ion MS/MS at unit resolution. Response was optimized with a spray voltage
setting of 5 kV. The heated capillary voltage was set at 10 V and the temperature to 350
°C. Nitrogen was used both as sheath and auxiliary gas, at a flow rate of 12 and 8 psi,
respectively. The precursor ion isolation width was 1.0 and 3.0 for nitrotyrosine and
tyrosine, respectively. The analyte abundance was evaluated by measuring the
chromatographic peak areas of selected product ions extracted from the full scan total ion
chromatogram, according to the corresponding ion trap product ion spectra. The ions
12 monitored for each analyte were: 3-nitro[ C6]tyrosine (mass-to-charge-ratio (m/z)
13 13 15 227→181); 3-nitro[ C6]tyrosine (m/z 233→187); 3-nitro[ C9 N1]tyrosine (m/z
35 12 35 13 237→190); chloro-[ C6]tyrosine (m/z 216→170); chloro-[ C6]tyrosine (m/z
35 13 15 12 222→176); chloro-[ C9- N]tyrosine (m/z 226→179); bromo-[ C6]tyrosine (m/z
13 13 15 260→214); bromo -[ C6]tyrosine (m/z 266→220); bromo -[ C9- N]tyrosine (m/z
12 13 15 270→223); [ C6]tyrosine (m/z 182→136); and [ C9 N1]tyrosine (m/z 192→145). The
108 maximum ion injection time was 100 ms; a scan rate was used that permitted a minimum sampling rate of at least 9 points/chromatographic peak. For all analyses, results were normalized to the content of the precursor amino acid L-tyrosine, which was monitored within the same injection of each oxidized amino acid. The results are listed in Tables 3.6 and 3.7.
Table 3.6. Nitrotyrosine, chlorotyrosine and bromotyrosine levels in autistic patients.
nitroTyr/Tyr ClTyr/Tyr BrTyr/Tyr Patient code (mol/mol) (mol/mol) (mol/mol)
A1 3.7300e-6 1.0700e-6 4.8871e-5
A2 6.9951e-6 5.8656e-6 3.4013e-5
A3 7.5331e-6 8.5004e-6 4.6604e-5
A4 6.4280e-6 1.0165e-5 3.5161e-5
A5 8.6893e-6 6.3993e-6 3.8037e-5
A6 6.2144e-6 6.4514e-6 3.1739e-5
A7 8.7463e-6 1.2837e-5 4.8275e-5
A8 5.8117e-6 9.2790e-6 3.7834e-5
A9 5.4147e-6 7.2821e-6 3.9738e-5
A10 2.0685e-6 1.4240e-5 3.9119e-5
A11 1.2195e-5 7.3088e-6 4.0735e-5
A12 7.3582e-6 6.2857e-6 3.6257e-5
A13 1.0113e-5 3.3894e-5 3.9214e-5
A14 4.8539e-6 2.7447e-5 5.1203e-5
A15 1.0264e-5 4.1406e-5 4.3703e-5
109 A16 1.1858e-5 2.7301e-5 4.7188e-5
A17 8.7110e-6 1.4430e-5 4.3352e-5
A18 1.2641e-5 1.3353e-5 3.9286e-5
A19 3.4437e-6 5.4245e-6 3.9175e-5
A20 1.7499e-5 2.0681e-5 4.7605e-5
A21 6.9135e-6 3.7879e-6 5.5540e-5
A22 9.3421e-6 1.9860e-5 3.6565e-5
A23 1.0140e-5 3.2724e-5 3.7768e-5
A24 5.1831e-6 6.1468e-6 4.1817e-5
A25 6.2409e-6 8.8281e-6 3.9079e-5
A26 6.3528e-6 4.4593e-6 3.9699e-5
A27 6.0950e-6 5.1037e-6 4.7011e-5
Average 7.8087e-6 1.3710e-5 4.1651e-5
STDEV 3.2943e-6 1.0338e-5 5.6909e-6
Table 3.7. Nitrotyrosine, chlorotyrosine and bromotyrosine levels in normal controls.
nitroTyr/Tyr ClTyr/Tyr BrTyr/Tyr Normal code (mol/mol) (mol/mol) (mol/mol)
N1 9.5160e-6 6.8757e-6 3.1430e-5
N2 8.0010e-6 1.0466e-5 3.1475e-5
N3 6.3955e-6 2.5877e-5 4.2179e-5
N4 8.5125e-6 1.7408e-5 3.9366e-5
N5 6.4621e-6 9.1273e-6 4.0811e-5
110 N6 6.0418e-6 2.7017e-6 2.8182e-5
N7 1.1408e-5 2.7262e-5 4.9403e-5
N8 7.2531e-6 3.3149e-5 2.9346e-5
N9 6.3484e-6 3.1250e-6 4.6537e-5
N10 8.4462e-6 2.8620e-5 4.3595e-5
N11 8.2132e-6 5.5500e-6 3.2361e-5
Average 7.87e-6 1.55e-5 3.77e-5
STDEV 1.62e-6 1.14e-5 7.41e-6
111 3.6. References
1. Kidd, P. M., Autism, an extreme challenge to integrative medicine. Part 1: the
knowledge base. Alternative Medicine Review. 2002,
http://www.findarticles.com/p/articles/mi_m0FDN/is_4_7/ai_91155402.
2. Edelson, S. M., Overview of autism. http://www.autism.org/overview.html.
3. Kanner, L., Autistic disturbances of affective contact. Nervous Child 1943, 2, 217-
250.
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116
Chapter 4
Serum Vitamin E and Oxidative Protein Modifications in Hemodialysis
117 4.1. Background
In the early 1990’s epidemiologic studies established an inverse relationship between
taking supplemental vitamin E and cardiovascular disease1, 2 or cancer.3 By 2000, an estimated 23 million Americans took supplemental vitamin E.4 However large
prospective randomized controlled trials of treatment failed to show improved
cardiovascular or other health outcomes.5 Thus, proponents of antioxidant therapy argued
that its greatest utility might be found in the setting of abnormal levels of oxidative stress, such as in patients with end stage renal disease (ESRD) treated by dialysis.6
Excess oxidant production and decreased antioxidant defense7 have been recognized
characteristics of the uremic state. In end stage renal disease (ESRD), oral vitamin E
supplementation has been shown to lower the susceptibility of low-density lipoprotein
(LDL) to oxidation,8, 9 and to prevent the oxidative stress associated with intravenous iron
administration for treatment of anemia.10 The SPACE trial showed a significant decrease in cardiovascular events in 196 dialysis patients with pre-existing cardiovascular disease taking oral vitamin E supplements.11 These hopeful results emphasized the importance of
understanding basic mechanisms of action of antioxidants in the uremic milieu.
The clinical observation that vitamin E was protective against morbid events in some
settings,11, 12 but not in all, has been named the antioxidant paradox.6 Factors that lead to
oxidative stress in living systems are complex. For example, the kinetics of reaction
between vitamin E and oxidants are slower than the rate at which free radical species
inactivate nitric oxide.13 Therefore vitamin E would not be expected to correct
118 abnormalities in flow-mediated vasodilation14-16 despite effectively decreasing the
oxidation of LDL.8, 9 In addition, vitamin E has been shown to act as a pro-oxidant, particularly in the absence of vitamin C.17, 18
Patients with ESRD have increased quantities of oxidatively modified circulating and
tissue proteins – including advanced glycation end products (AGEs),19, 20 advanced
oxidative protein products (AOPP),21, 22 oxidized low density lipoprotein (oxLDL),23
(E)-4-hydroxy-2- nonenal (HNE)-derived-2-pentylpyrroles (PP) and isolevuglandin adducts24, 25 – as well as oxidized lipids including isoprostanes26-29 and oxysterols.30 The goal of this project was to discover the effect of supplemental vitamin E on levels of oxidative protein modifications. We measured pentosidine, a glycoxidation-derived protein modification, as well as protein modifications derived from the lipid peroxidation products, iso[4]levuglandin (iso[4]LGE2), HNE, and (E)-4-oxo-2-nonenal (ONE), in a highly oxidative clinical state – ESRD treated by hemodialysis. We explored the relationships between the levels of these products at baseline and over the course of
treatment, and examined the relationship between levels of oxidation products and factors
known to influence oxidation in the clinical setting.
119 4.2. Results
The demographic characteristics of the 34 study subjects are summarized in Table 4.1.
Table 4.1. Baseline clinical characteristics of patients.
Placebo Group Treatment Group P value ∗
Number of Patients 17 17
Gender (male/female) 8/9 6/11 .48
Race (African American/Caucasian) 13/4 16/1 .13
Diabetes (no/yes) 7/10 6/11 .72
Age (years) 54.7 56.7 .67 ±13.5 ±14.1
Time on dialysis at start of study 47 44 .83 (M) ±49 ±34 Creatinine (mg/dL) 9.9 9.6 .79 ±3.4 ±2.2 Urea (md/dL) 51 44 .24 ±19 ±15
Standardized Kt/V (dimensionless 1.42 1.43 .92 measure of dialysis adequacy) ±.30 ±.34 Body Mass Index (kg/m2) 34.4 30.1 .18 ±9.7 ±8.1
∗ For significant difference in mean values for treatment group versus controls.
Individual participation in the study ranged between 2.1-20.6 months (mean follow up
9.8 ± 4.7). During the course of the study 17 subjects developed thrombotic events
120 involving their hemodialysis vascular accesses, as previously described.31 There were no differences in numbers of events between placebo and vitamin E treatment groups. Nor were there differences in numbers of events between patients with diabetes and without diabetes. During the course of the study 1 patient received a transplant, 1 transferred out of the city, 4 patients had documented myocardial infarctions, and there were 5 deaths.
Clinical laboratory measurements of total iron, iron binding protein and ferritin, were not found to influence levels of glycoxidation or lipid peroxidation products. Nor was there a dose effect related to the quantity of exogenous intravenous iron and erythropoietin administered as part of the routine treatment of anemia in patients on dialysis (data are shown in Appendix Tables S1-S3).
α-Tocopherol levels increased two fold in treated patients (Table 4.2). In contrast
γ-tocopherol levels dropped to half of baseline in patients treated with oral vitamin E alpha (α-tocopherol) (Table 4.2). At baseline there was a positive correlation between levels of α- and γ-tocopherol, Fig 4.1 A. A positive correlation continued in the placebo group. After treatment with vitamin E alpha, the correlation between plasma levels of α- and γ-tocopherol became negative, with the highest levels of α-tocopherol associated with the lowest levels of γ-tocopherol (Table 4.2), and shown at the 3 month follow up time point (Fig 4.1B).
121 Table 4.2. Plasma levels of alpha and gamma tocopherol in placebo and vitamin E treated patients.
Time point Baseline 3 months 6 months 9 months
Alpha Tocopherol Time p
Placebo 12.15 12.44 12.76 13.79 .74
SD 3.48 3.44 4.00 5.31
N 17 14 13 12
Treatment 12.78 26.02 27.27 30.19 .0042
SD 4.52 16.95 14.31 16.40
N 17 16 14 11
Placebo vs Treatment p .65 .006 .0017 .0035
Gamma Tocopherol Time p
Placebo 3.68 3.47 3.51 3.97 .69
SD 2.10 1.69 1.62 2.05
N 17 14 13 12
Treatment 3.92 2.30 1.45 1.49 .0001
SD 1.54 1.47 1.05 1.08
N 17 16 14 11
Placebo vs Treatment p .70 .051 .0006 .0017
122 A B
10 10
8 8
6 6
4 4
2 2
gamma tocopherol (µg/mL) 0 0 0 10 20 30 0 10 20 30 40 50 60 70
alpha tocopherol (µg/mL) Figure 4.1. Relationship between plasma levels of Vitamin E alpha and gamma
before and after treatment. A. Baseline R = 0.46, p = 0.0055. B. The positive
correlation between vitamin E alpha and gamma is present in the placebo group, left
regression line (R = 0.42, p = 0.05). In the patients treated with vitamin E, there is a
negative correlation between the two metabolites, right regression line (R = 0.11,
“not significant”). Gray symbols, placebo. Black symbols, Vitamin E treatment.
Open circles represent baseline; squares, 3 month values.
There were no significant changes in circulating levels of oxidative protein modifications over the course of the study (Table 4.3 and Figures 4.2-4.3). The data set displayed wide standard deviations for pentosidine and ONE-protein adduct. Despite blinded randomization, mean values at baseline were not balanced. In addition an apparent increase in pentosidine and ONE-protein adduct in patients treated by vitamin E alpha was not statistically significant at baseline or at any follow up time point. At baseline, the regression relationships between the lipid peroxidation-derived oxidative
123 protein modifications and both α- and γ-tocopherol were positive, with the highest correlation coefficients for iso[4]levuglandin-protein adducts (Table 4.4).
Table 4.3. Plasma glycoxidation and protein-lipid oxidation products in placebo and vitamin E treated patients.
Time point Baseline 3 months 6 months 9 months pentosidine (pmol/mg protein) Time p Placebo 14.76 15.73 15.65 15.66 .99 SD 9.99 11.18 12.28 15.04 Treatment 18.41 18.93 21.33 23.07 .63 SD 8.65 9.88 9.04 14.01 Placebo vs Treatment p .26 .41 .18 .27 iso[4]LGE2 (nmol/ml) Time p Placebo 8.43 8.84 8.31 8.39 .94 SD 2.68 2.32 2.55 1.69 Treatment 8.26 8.39 8.46 9.90 .29 SD 1.99 2.56 2.37 1.86 Placebo vs Treatment p .85 .64 .87 .06 HNE (nmol/ml) Time p Placebo 0.46 0.49 0.51 0.49 .78 SD 0.13 0.11 0.11 0.12 Treatment 0.48 0.45 0.51 0.51 .43 SD 0.11 0.09 0.08 0.11 Placebo vs Treatment p .72 .37 .89 .63 ONE (pmol/mg protein) Time p Placebo 189 189 189 194 .91 SD 60 65 44 56
124 Treatment 236 204 227 243 .54 SD 68 61 72 102 Placebo vs Treatment p .07 .52 .12 .17
0.55
HNE (nmol/mL) 0.5 placebo 0.45 vitamin E alpha 0.4 treated
0.35
0.3 Baseline 3months 6months 9months
Figure 4.2. Levels of HNE-derived pentylpyrrole over the course of study.
12 Iso[4]LGE 11
10
2 placebo (nmol/mL) 9 vitamin E alpha 8 treatment 7
6
5 Baseline 3months 6months 9months
Figure 4.3. Levels of iso[4]LGE2-protein adduct over the course of study.
Iso[4]-levuglandin-protein adduct level was correlated positively with both α- and
γ-tocopherol levels at baseline, during placebo treatment and during treatment with
125 α-tocopherol (Tables 4.5 and 4.6). HNE-protein adduct level was correlated positively with α-tocopherol at baseline (Table 4.4). In addition at baseline, the correlations among the lipid oxidation products were highly significant (Table 4.7). In contrast, there was no correlation between the glycoxidation product pentosidine and any of the lipid peroxidation protein adducts (Table 4.7), and the regression relationship between the glycoxidation product pentosidine and γ-tocopherol was negative (Table 4.4, p = 0.08).
Table 4.4. Linear regression of glycoxidation and protein-lipid oxidation products with alpha or gamma tocopherol at baseline.
pentosidine HNE iso[4]LGE2 ONE
alpha tocopherol -.02 .46 .42 .29 R .92 .01 .03 .12 p gamma tocopherol -.30 .01 .44 .25 R .08 .95 .02 .20 p
Table 4.5. Linear regression of iso[4]LGE2 with alpha or gamma tocopherol during placebo treatment at 3 months, 6 months and 9 months.
3 months 6 months 9 months alpha tocopherol .1 .07 .2 R .29 .42 .12 p gamma tocopherol .01 .1 .4 R .71 .27 .02 p
126 Table 4.6. Linear regression of iso[4]LGE2 with alpha or gamma tocopherol during
treatment with α-tocopherol at 3 months, 6 months and 9 months.
3 months 6 months 9 months alpha tocopherol .07 .53 .13 R .4 .003 . 3 p gamma tocopherol .03 .006 .25 R .58 .79 .12 p
Table 4.7. Linear regression matrix for glycoxidation and protein-lipid oxidation products at baseline.
pentosidine HNE iso[4]LGE2
HNE .01 R .96 p
iso[4]LGE2 -.05 .39 R .80 .04 p
ONE .04 .41 .37 R .85 .03 .05 p
127 4.3. Discussion
The term “vitamin E” refers to a group of eight naturally occurring chromanols,
including four tocopherols (α, β, γ, δ) and four tocotrienols (α, β, γ, δ). In addition, there
are several stereoisomers of each form.32 Although the naturally occurring RRR-
stereoisomer of α-tocopherol is marketed as “natural vitamin E”, normal dietary
quantities of γ-tocopherol are greater than alpha tocopherol.33 Nevertheless, α-tocopherol
represents the majority of plasma vitamin E.34 This is because α- tocopherol has the
greatest binding affinity to α-tocopherol transfer protein (α-TTP).35 The highly specific
α-TTP preferentially transports α-tocopherol into the blood stream.36 Notably, oral
supplementation with α-tocopherol decreases circulating and tissue levels of γ-tocopherol in normals,37, 38 the elderly,39 and patients with ESRD.28, 32 Presumably, supplementation
with α-tocopherol competitively inhibits α-TPP mediated absorption of dietary
γ-tocopherol.
α-Tocopherol levels are in the normal range in patients with ESRD,32 whereas
γ-tocopherol levels may be normal (as in this study),28 decreased40 or increased.32 Patients in this study received 800 IU of natural RRR-α-tocopherol per day, a dose and form reported to be effective in preventing secondary coronary events in patients with ESRD,11
and in preventing restenosis after percutaneous transluminal coronary angioplasty
(PTCA).41 The power of this study was too small to show an effect of treatment on
cardiovascular outcome. However we have reported previously on an association
128 between lower baseline levels of α- and γ-tocopherol and subsequent thrombotic events
in the hemodialysis (HD) vascular access.31
One important activity of the E vitamins is their ability to function as lipid soluble chain-breaking antioxidants in biological membranes. Vitamin E is hypothesized to protect against atherosclerosis through limiting LDL oxidation in the arterial wall.
Unfortunately there is little evidence that vitamin E inhibits LDL oxidation in healthy humans lacking signs of increased oxidative stress.42 Thus, when α-tocopherol is given
for primary prevention, there is no benefit against atherosclerosis or cancer as shown in
clinical trials such as the Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto
miocardio (GISSI prevention),5 and the Heart Outcomes Prevention Evaluation
(HOPE).43, 44 In secondary prevention, the Cambridge Heart Antioxidant Study (CHAOS)
did show benefit of α-tocopherol supplementation – markedly reducing non-fatal
myocardial infarction in patients with documented coronary artery disease.12 In contrast,
in the HOPE study, there was evidence of harm associated with α-tocopherol
supplementation, with significant increases in heart failure and trends towards increased ischemic complications of atherosclerosis.43, 45, 46
α-Tocopherol is considered the “most potent” non-specific lipid-soluble antioxidant in
the body.47 However, only small differences are expected in abilities of the various
tocopherols and tocotrienols to neutralize free radicals by hydrogen atom transfer. For example, the rate constants for H-atom transfer to a peroxyl radical from α−tocopherol
and γ-tocopherol are 23.5, 15.9 x 105 M-1S-1, respectively.48 Therefore, all of the forms of
129 vitamin E are expected to be similarly effective as chain-breaking antioxidants for inhibiting lipid peroxidation. If the dominant activity of E vitamins was to prevent lipid peroxidation by neutralizing free radicals through hydrogen atom transfer, levels of lipid peroxidation should decrease as those of the E vitamins increase. Two observations of the present study are not in accord with this expectation: (1) there are striking positive correlations between circulating levels of lipid peroxidation products and circulating levels of both α- or γ-tocopherol at baseline and (2) the huge increase in the circulating level of α-tocopherol accomplished by supplementation is not accompanied by a significant decrease in levels of lipid peroxidation products.
The striking positive correlations seen in the present study, between lipid peroxidation products and those of tocopherols at baseline, suggest that in the chronically oxidizing environment of ESRD, the E vitamins are exerting prooxidative effects. Tocopherols may promote lipid autoxidation by recycling catalytic amounts of transition metal ions to low-valent metals which can initiate free radical oxidative chain reactions by inducing
Fenton and Fenton-like reactions, as shown in Figure 4.4. Tocopheryl quinone is produced by oxidation of α-tocopherol with concomitant reduction of transition metal ions (Figure 4.5) that then mediate reductive cleavage of hydroperoxides to alkoxy radicals.49 Thus, tocopherols can promote the generation of alkoxy radicals from lipid hydroperoxides, and thereby initiate free radical chain reactions that generate more lipid hydroperoxides through autoxidation of lipids.50
130 n+ (n+1)+ Fenton Reacton H2O2 + M OH + OH + M
LOOH + M n+ LO + OH + M (n+1)+ Fenton-like Reaction Figure 4.4. Fenton and Fenton-like reactions.
O O O H C H C H C 31 15 M+(n+1) 31 15 M+n 31 15 H OH OH O
α-tocopherol cation radical radical
O O H C H C H2O HO 31 15 M+(n+1) 31 15 M+n O O H O O
C15H31 α radical cation -tocopherylquinone
Figure 4.5. Reduction of transition metal ions by tocopherols.
Another, more subtle, prooxidant effect of supplementation with α-tocopherol may result from the consequent reduction in levels of γ-tocopherol (Figure 4.1).
Myeloperoxidase (MPO), which represents up to 5% of cell protein in inflammatory phagocytes, functions as a major catalyst for lipid peroxidation.51 We previously showed
that MPO promotes the generation of iso[4]LGE2-protein adducts in vivo in a mouse
model of chronic inflammation.52 α-Tocopherol is ineffective against MPO-mediated
52 oxidation. In contrast γ-tocopherol may have unique specificity for protection against
reactive nitrogen species.53
Further complexity arises from other biological activities of vitamin E. In addition to its antioxidant functions, vitamin E is involved in cell signaling, gene expression,
immune response and apoptosis.54 γ-Tocopherol is metabolized largely to
131 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (γ -CEHC).55 Both γ -tocopherol
and γ-CEHC, but not α-tocopherol, inhibit cyclooxygenase activity and are, thus, anti-inflammatory.56 γ-Tocopherol is more potent than α-tocopherol in modulating
inflammatory pathways.56, 57 Oral supplementation with α-tocopherol results in a
decrease in circulating levels of γ-tocopherol with a resultant increase in inflammation in
unstable atherosclerotic lesions.58
In ESRD chronic inflammation is a major source of increased oxidative stress.7, 59
Untreated hemodialysis membranes activate complement and the immune system during
dialysis.60 In contrast with the present study, dialysis membranes coated with
α-tocopherol decrease the peroxidation of red blood cell and plasma lipids.61 In addition,
hemodialysis with α-tocopherol coated dialysis membranes normalizes endothelial response62 and corrects the over-production of cytokines usually associated with dialysis, normalizing immune function.63, 64 Dialysis with α-tocopherol coated dialyzers decreased
levels of AGEs,65, 66 and of free circulating HNE.67 In the present study, circulating levels
of AGEs, i.e., pentosidine, were inversely related to levels of γ-tocopherol, particularly in
the multivariate model, suggesting that inflammatory pathways that are modulated
normally by γ-tocopherol influence pentosidine formation. Evidence that α-tocopherol
supplementation can increase inflammation is demonstrated in hemodialysis patients who took a 14 day course of this vitamin. Interleukin-6 levels increased in response to treatment, but c-reactive protein levels decreased when the patients received supplements rich in γ-tocopherol.32 Our data emphasize an important contrast between the action of
132 α-tocopherol localized to the site of oxidative and inflammatory activation on the
hemodialysis membrane, versus the dietary interaction of α-tocopherol as an oral
supplement.58
4.4. Conclusions
In summary, in the clinical setting of oxidative stress experienced by patients with
ESRD treated by hemodialysis, the balance of oxidant/antioxidant systems is highly
complex. Both chemical pathways and inflammatory components appear to be involved
in interactions that lead to the formation of glycoxidation and protein-bound lipid
oxidation products. These data demonstrate that intervention by oral supplementation
with the commercially prevalent “natural vitamin E”, i.e., α-tocopherol, does not result in predictable antioxidant benefit.
133 4.5. Experimental Procedures
Methods
Patients
Thirty-four stable patients treated by HD at one of the Centers for Dialysis Care,
Cleveland were recruited for the study. Informed consent was obtained in compliance
with the Institutional Review Board of University Hospitals of Cleveland. Some patients left the study due to death, transplantation, or the initiation of anticoagulant therapy.
Study Design
Patients were provided with Vitamin E (α-tocopherol, 800 IU) or identical placebo capsules to be taken daily. Investigators and patients were blinded as to treatment.
“Natural vitamin E” (RRR-alpha-tocopherol also known as d-alpha-tocopherol) was the kind gift of Cognis Inc (LaGrange, IL). Clinical data was collected by interview, and chart review. Clinical parameters and routine clinical chemistry and hematology results used in standard care and management were obtained in a single reference clinical laboratory. Results were recorded by chart review every three months throughout the study. The total administered dosages of erythropoietin and intravenous iron were recorded for each 3 month time period (Appendix Tables S1-S3), as were significant clinical events, including hospitalizations and surgical interventions.
Assays
At each time point blood was collected in EDTA-metal free tubes for plasma or plain glass tubes for serum. Samples for plasma were placed on ice, and spun in the cold at
134 3000 rpm for 15 minutes. Serum was allowed to clot before centrifugation. Plasma for
analysis of alpha and gamma tocopherol was protected from light at each step. Plasma for
enzyme-linked immunosorbent assay (ELISA) was stabilized prior to storage by addition
of butylated hydroxytoluene (BHT, 10 mM final concentration) and a protease inhibitor
cocktail (Sigma-Aldrich, Milwaukee, WI, Cat. Sigma P8340) (10 μL/mL plasma), as
described.24 After processing, serum and plasma aliquots were quench-frozen in liquid
nitrogen and transported to the laboratory where they were layered with argon and stored
at -80 °C.
Tocopherols were measured by Penny Erhard using a modification of the method of
Sommerburg.68 Briefly, in plasma (200 μL) was added ethanol (200 μL, 200 proof,
Pharmacia) containing alpha tocopherol acetate (32 μg/mL). Hexane (500 μL) was added and the system was allowed to sit in the dark for 5 minutes, vortexed, and then centrifuged at 2000 RPM for 5 mins at 15 °C. The upper hexane layer was saved and the extraction was repeated twice. The hexane extract was dried under nitrogen and reconstituted in methanol. Then the sample (80 μL) was injected into an HPLC system with a Waters UV detector at 292 nm using a reverse phase C18 μBondapack column (3 micron, 15 cm, Waters). The sample was eluted using an isocratic elution with 95% methanol and 5% water at a flow rate of 1 mL/min for 20 mins. Standards for α- and
γ-tocopherol were purchased from Sigma, and prepared freshly for each assay. The intra-assay coefficient of variation was 0.039 for α-tocopherol and 0.041 for γ-tocopherol, and the inter-assay coefficients were 0.078 for both α- and γ-tocopherol . The normal
135 range for α-tocopherol was 12.50 ± 0.23 µg/ml; the normal range for γ-tocopherol was
2.41 ± 0.26 µg/ml. α-Tocopherol levels in plasma from HD patients treated with vitamin
E and placebo at baseline, three months, six months, nine months and twelve months are listed in tables 4.8 and 4.9 respectively. γ-Tocopherol levels in plasma from HD patients treated with vitamin E and placebo at baseline, three months, six months, nine months and twelve months are listed in tables 4.10 and 4.11 respectively.
Table 4.8. α-Tocopherol levels (μg/mL) in plasma from patients treated with vitamin E. Patient Code Baseline 3 months 6 months 9 months 12 months T1 16.04 23.18 56.63 60.81 55.49 T2 15.21 66.53 40.28 T3 8.69 15.97 14.95 14.91 13.24 T4 12.15 17.95 18.95 T5 7.02 15.62 12.69 16.60 14.94 T6 10.93 16.67 28.31 31.06 30.21 T7 14.36 9.19 15.49 16.76 20.55 T8 6.99 61.88 42.63 43.68 37.57 T9 17.65 32.26 25.49 49.29 33.13 T10 15.89 28.02 15.39 13.39 T11 17.80 42.87 48.32 42.26 T12 16.69 14.62 28.46 17.70 T13 12.23 17.83 20.66 25.61 T14 8.46 15.69 13.49 T15 13.94 Average 12.78 26.02 27.27 30.19 29.30 STDEV 4.52 16.95 14.31 16.40 14.78
136 Table 4.9. α-Tocopherol levels (μg/mL) in plasma from HD patients treated with placebo
at baseline, three months, six months, nine months and twelve months.
Patient Code Baseline 3 months 6 months 9 months 12 months P1 16.94 16.80 16.22 18.97 18.17 P2 15.27 16.71 11.81 13.91 12.44 P3 13.50 13.86 14.82 23.87 P4 14.54 15.18 11.54 11.61 15.36 P5 8.95 9.07 7.70 7.70 8.25 P6 20.16 15.52 21.16 19.46 19.01 P7 11.14 12.19 12.77 13.30 13.76 P8 13.34 15.79 17.58 15.76 22.34 P9 10.43 8.92 9.04 10.18 8.68 P10 14.94 13.79 13.56 15.14 P11 11.47 11.56 12.76 10.71 P12 6.43 6.63 7.51 P13 7.66 10.37 9.36 Average 12.15 12.44 12.76 13.79 16.14 STDEV 3.48 3.44 4.00 5.31 7.15
Table 4.10. γ-Tocopherol levels (μg/mL) in plasma from patients treated with vitamin E at baseline, three months, six months, nine months and twelve months.
Patient Code Baseline 3 months 6 months 9 months 12 months T1 6.04 4.98 1.51 2.14 T2 6.19 2.56 0.91 T3 3.49 2.81 2.79 2.72 T4 2.96 0.66 0.41 T5 2.58 4.71 4.16 3.93 1.27
137 T6 3.88 3.67 1.25 0.72 5.41 T7 4.16 1.17 1.23 1.39 T8 2.19 1.15 0.97 0.69 1.51 T9 5.14 2.37 2.19 0.63 2.51 T10 4.35 1.65 2.01 1.72 T11 6.95 0.99 0.77 1.05 T12 5.02 2.71 0.47 0.96 T13 2.69 0.61 0.36 0.39 T14 2.74 0.89 1.24 T15 2.49 Average 3.92 2.30 1.45 1.49 2.68 STDEV 1.54 1.47 1.05 1.08 1.90
Table 4.11. γ-Tocopherol levels (μg/mL) in plasma from HD patients treated with placebo at baseline, three months, six months, nine months and twelve months.
Patient Code Baseline 3 months 6 months 9 months 12 months P1 6.91 5.73 5.25 8.40 2.05 P2 4.38 6.20 3.62 4.23 P3 0.61 0.75 1.38 1.39 2.00 P4 3.01 2.23 2.68 2.85 3.07 P5 5.82 5.65 5.42 4.76 4.72 P6 9.12 4.45 6.46 6.61 P7 2.69 2.90 3.16 2.43 5.20 P8 4.08 5.21 4.91 5.49 0.76 P9 2.08 1.61 1.37 2.98 P10 2.56 2.47 3.64 3.33 P11 2.17 3.34 2.52 3.37
138 P12 3.27 2.66 3.49 P13 2.04 2.94 1.69 Average 3.68 3.47 3.51 3.97 2.97 STDEV 2.10 1.69 1.62 2.05 1.72
Pentosidine was measured by HPLC using a modification of the method of Odetti as
previously published.69, 70 Plasma (500 μL) was subjected to precipitation with 10% trichloroacetic acid (TCA) (500 μL) on ice followed by centrifugation. The pellets were washed twice with 5% cold TCA and acid hydrolyzed in 6 N HCl (1 mL) for 16 h at 110
°C in borosilicate tubes with Teflon coated screw caps. Acid was then removed by vacuum centrifugation. The hydrolyzed pellet was dissolved in 250 μL of water/0.01 M heptafluorobutyric acid. The equivalent of 0.8 mg of plasma protein was injected onto an
HPLC system (Waters). A Vydac type 218 TP C18 column (25 × 0.46 cm, 10 micron)
(Separations Group, Hesperia, CA) was used. The HPLC was programmed with a linear gradient from 1 to 35 min of 10-17% acetonitrile in water and 0.1% heptafluorobutyric acid as a counter ion. Pentosidine eluted at 30 min as monitored by fluorescence excitation at 335 nm and emission at 385 nm. Pentosidine prepared according to a published procedure71 was used as a standard. Results were calculated per milligram
added protein. This work was done by Penny Erhard. The results are listed in tables 4.12
and 4.13 respectively. In previous studies the range for pentosidine in healthy control
subjects was 1.35 ± 0.6 pmol/mg protein; the usual range in patients on HD was 22.9 ±
10.8 pmol/mg protein.72, 73
139 Table 4.12. Pentosidine levels (pmol/mg protein) in plasma from HD patients treated with vitamin E supplementation at baseline, three months, six months, nine months and twelve months.
Patient Code Baseline 3 months 6 months 9 months 12 months
T1 14.68 19.78 31.18 37.20 27.19
T2 18.42 16.90 19.01
T3 28.62 35.41 35.60 43.40 40.43
T4 37.71 18.38 17.36
T5 20.61 18.04 17.50 20.57 23.00
T6 21.92 13.60 17.21 15.30 18.08
T7 5.37 17.27 13.06 19.08 19.85
T8 13.82 9.12 9.02 9.17 9.48
T9 8.36 13.40 13.31 11.52 11.94
T10 10.31 31.36 30.95 22.74
T11 22.91 7.52 8.93 6.80
T12 10.98 37.68 32.54 44.96
T13 26.78 24.58 25.73
T14 19.32 26.47 27.17
T15 28.06
Average 18.41 18.93 21.33 23.07 21.42
STDEV 8.65 9.88 9.04 14.01 10.35
140 Table 4.13. Pentosidine levels (pmol/mg protein) in plasma from HD patients treated with placebo at baseline, three months, six months, nine months and twelve months.
Patient Code Baseline 3 months 6 months 9 months 12 months
P1 10.53 13.80 13.15 13.48 12.11
P2 8.78 7.55 6.73 7.46 6.85
P3 48.09 50.70 52.04 56.82
P4 16.26 15.63 12.13 11.87 10.91
P5 13.62 16.06 15.19 11.36 11.57
P6 8.41 8.55 8.59 9.79 11.07
P7 4.36 5.05 4.91 5.13 6.20
P8 21.91 16.42 17.90 17.86 20.16
P9 5.85 8.69 5.37 6.39
P10 13.82 17.36 16.39 16.46
P11 23.69 23.58 23.10
P12 8.97 9.16 8.94
P13 12.04 13.03 18.99
Average 14.76 15.73 15.65 15.66 11.27
STDEV 9.99 11.18 12.28 15.04 4.57
141 Protein adducts of iso[4]LGE2, HNE derived pentylpyrrole and ONE were measured
by competitive ELISA as described above (Chapter 2). Iso[4]LGE2-BSA and iso[4]LGE2-HSA were synthesized as described in Chapter 3. ON-BSA and ON-HSA
were synthesized as described previously24, and specific antibodies were produced,
purified and characterized.74 For pentylpyrrole immunoreactivity, ON-BSA (final
concentration was 187 pmol/mL) was used as a coating agent. ON-HSA was used as a standard. The initial concentration of ON-HSA was 13800 pmol/mL. A dilution factor of
0.3 was employed for standard and samples. The normal range for HNE–protein adduct was 436 ± 77 pmol/mL; the usual range in patients on HD was 543 ± 190 pmol/mL. The intra-assay and inter-assay coefficient of variation were 5% and 6% respectively. The pentylpyrrole immunoreactivities in plasma from patients treated with vitamin E and placebo were listed in tables 4.14 and 4.15 respectively. For iso[4]LGE2 immunoreactivity, iso[4]LGE2-BSA (final concentration was 22.8 nmol/mL) was used as a coating agent and iso[4]LGE2-HSA was used as a standard. The initial concentration of
iso[4]LGE2-HSA was 810 nmol/mL. A dilution factor of 0.2 was employed for standard and samples. Eight serial dilutions for standard, five serial dilutions of samples were
performed. The normal range for iso[4]LGE2-protein adduct was 4087 ± 47.9 pmol/mL;
the usual range in patients on HD was 8344 ± 2296 pmol/mL.74 The intra-assay and
inter-assay coefficient of variation for iso[4]LGE2-protein adduct were 4.65% and 7.11%
respectively. Iso[4]LGE2 immunoreactivities in plasma from patients treated with vitamin
E and placebo are listed in tables 4.16 and 4.17. ONE -BSA and anti-ONE-RNase
142 antibody were kind gifts of Dr. Lawrence Sayre. ONE-BSA was used as a coating agent
(final concentration was 1170 pmol/mg) and a standard (initial concentration was 11700
pmol/mg). The normal range for ONE-RNase was 114.72 ± 28.73 pmol/mg BSA; the
usual range in patients on HD was 152.58 ± 33.68 pmol/mg BSA. The intra-assay coefficient of variation was 3.36%, and the inter-assay coefficient was 6.92%.
ONE-RNase immunoreactivities in plasma from patients treated with vitamin E and placebo are listed in tables 4.18 and 4.19 respectively.
Statistical analyses
Treatment and placebo groups were compared with respect to demographic and baseline characteristics using paired comparisons for parametric data, and chi square analysis for
descriptive variables. Longitudinal effects of treatment were compared using analysis of
variance (ANOVA) with subsequent pair-wise analysis using a 2-tailed t test. The
significance of group comparisons was corrected for the effect of repeated measures.
Correlations between measured parameters were ascertained using pair-wise comparisons
by linear regression. Models of interactions were constructed using multiple regression analysis and the least squares model of fit. Data were expressed as mean ± SD. Statistical significance was defined as P less than 0.05. All analyses were performed using JMP
5.12 (SAS Institute, Cary NC).
143 Table 4.14. HNE-derived pentylpyrrole immunoreactivities in plasma from patients treated with vitamin E at baseline, 3 months, 6 months, 9 months and 12 months.
Patient Code Baseline 3 months 6 months 9 months 12 months
T1 0.47 0.45 0.46 0.43 0.38
T2 0.53 0.54 0.42
T3 0.50 0.23 0.55 0.49 0.49
T4 0.39 0.43 0.40
T5 0.47 0.41 0.46 0.47 0.47
T6 0.60 0.39 0.70 0.74 0.80
T7 0.50 0.55 0.53 0.56 0.53
T8 0.55 0.55 0.55 0.56 0.58
T9 0.45 0.43 0.50 0.47 0.49
T10 0.56 0.46 0.52 0.44
T11 0.72 0.47 0.61 0.67
T12 0.51 0.61 0.54 0.50
T13 0.38 0.34 0.38 0.35
T14 0.30 0.44 0.47
Average 0.48 0.45 0.51 0.51 0.53
STDEV 0.11 0.09 0.08 0.11 0.13
144 Table 4.15. HNE-derived pentylpyrrole immunoreactivities (nmol/mL) in plasma from patients treated with placebo at baseline, 3 months, 6 months, 9 months and 12 months.
Patient Code Baseline 3 months 6 months 9 months 12 months
P1 0.47 0.40 0.44 0.39 0.43
P2 0.53 0.50 0.61 0.65 0.75
P3 0.53 0.47 0.59 0.60
P4 0.66 0.62 0.67 0.66 0.73
P5 0.30 0.37 0.36 0.29 0.38
P6 0.54 0.51 0.55 0.53 0.58
P7 0.54 0.53 0.56 0.54 0.51
P8 0.22 0.39 0.42 0.42 0.32
P9 0.54 0.66
P10 0.30 0.32 0.36 0.38 0.23
P11 0.49 0.58 0.61 0.39
P12 0.51 0.56 0.55 0.52
P13 0.57 0.55 0.54 0.56
P14 0.31 0.37 0.37
Average 0.46 0.49 0.51 0.49 0.49
STDEV 0.13 0.11 0.11 0.12 0.19
145 Table 4.16. Iso[4]LGE2-protein immunoreactivities (nmol/mL) in plasma from patients treated with vitamin E at baseline, 3 months, 6 months, 9 months and 12 months.
Patient Code Baseline 3 months 6 months 9 months 12 months
T1 12.96 12.21 15.51 13.61 11.71
T2 9.13 9.32 8.22
T3 8.74 4.46 9.60 8.50 9.96
T4 9.29 8.86 7.68
T5 10.10 9.78 8.50 12.00 11.85
T6 9.54 2.85 9.04 11.17 12.56
T7 8.00 8.49 6.60 8.90 8.83
T8 7.37 9.38 9.22 8.76 8.79
T9 8.33 8.32 7.54 8.85 8.86
T10 7.74 6.88 5.66
T11 8.72 8.69 9.19 9.89
T12 7.50 9.79 8.79 9.92
T13 6.09 6.38 6.46 7.43
T14 6.22 7.88 6.52
Average 8.26 8.39 8.46 9.90 11.71
STDEV 1.99 2.56 2.37 1.86 1.64
146 Table 4.17. Iso[4]LGE2-protein immunoreactivities (nmol/mL) in plasma from patients treated with placebo at baseline, 3 months, 6 months, 9 months and 12 months.
Patient Code Baseline 3 months 6 months 9 months 12 months
P1 16.26 15.31 14.94 12.88 14.35
P2 8.46 7.51 8.10 6.68 8.00
P3 7.51 9.03 8.28 8.06
P4 8.79 7.70 8.40 7.74 8.36
P5 7.20 6.93 6.84 6.86 9.70
P6 9.91 8.61 7.64 10.06 8.94
P7 6.36 7.42 8.07 7.72 6.11
P8 5.34 7.14 8.97 7.69 7.43
P9 5.98 8.03 7.62 8.88 4.15
P10 8.61 11.60 8.84 8.73
P11 8.44 9.50 7.73 7.01
P12 8.85 8.85 10.81 8.11
P13 7.92 7.29 5.81
P14
Average 8.43 8.84 8.31 8.39 8.38
STDEV 2.68 2.32 2.55 1.69 2.97
147 Table 4.18. ONE-protein immunoreactivities (pmol/mg protein) in plasma from patients treated with vitamin E at baseline, 3 months, 6 months and 9 months.
Patient Code Baseline 3 months 6 months 9 months
T1 160.5 187.5 118.7 84.6
T2 242.6 234.9 210.4
T3 360.9 151.6 N/A 341.7
T4 244.2 194.1 184.3
T5 252.1 268.9 315.6 437.9
T6 252.1 110.3 280.2 252.1
T7 216.4 211.6 298.2 231.6
T8 221.1 212.8 211.2 208.0
T9 302.5 297.5 335.5 307.7
T10 266.9 202.3 216.4 158.1
T11 339.7 325.5 305.8 325.0
T12 159.7 176.6 143.4 156.9
T13 158.3 128.8 144.7 175.0
T14 130.0 165.7 193.8
Average 236.2 204.9 227.5 243.5
STDEV 68.9 61.1 72.3 102.2
148 Table 4.19. ONE-protein immunoreactivities (pmol/mg protein) in plasma from patients treated with placebo at baseline, 3 months, 6 months and 9 months.
Patient Code Baseline 3 months 6 months 9 months
P1 299.5 224.0 208.8 171.7
P2 168.0 133.7 150.6 165.0
P3 225.8 180.3 203.0 252.1
P4 100.3 92.2 130.1 90.4
P5 151.6 176.4 171.3 146.3
P6 238.5 220.7 236.6 200.0
P7 186.7 157.7 157.7 169.9
P8 99.3 207.5 236.9 276.6
P9 222.4 280.2
P10 223.0 335.5 242.9 280.7
P11 266.9 219.7 202.3 233.6
P12 143.4 134.7 135.9 176.6
P13 192.0 167.2 246.2 175.0
P14 132.3 123.2 138.3
Average 189.3 189.5 189.3 194.8
STDEV 60.4 64.9 43.9 56.3
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160
Chapter 5
Identification of Carboxyethylpyrrole Phosphatidylethanolamine
Adducts in vitro and in vivo
161 5.1. Background
Retinal membrane contains the most highly unsaturated fatty acids found in vertebrate tissues. Over 50% of the total retinal fatty acids are unsaturated.1 Of all subcellular components of retina, the rod outer segment (ROS) is the most enriched in polyunsaturated fatty acid (PUFA) phospholipid esters, among which docosahexaenoic acid (DHA) phospholipids predominate (see Table 5.1). The major phospholipids in ROS are phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs), which comprise
37.6% and 32.5% of total lipids respectively.
Table 5.1. Distribution of the fatty acids of PE and PC in human ROS (as mol% of total fatty acid).
Phosphatidylethanolamine Phosphatidylcholine (PC)
(PE)
Palmitic acid (16:0) 13.3 32.5
Linoleic acid (18:2, ω6) 0.3 1.1
Arachidonic acid (20:4, 3.5 4.7
ω6)
Docosahenaenoic acid 34.2 19.5
(22:6, ω3)
% saturation 52.3 48.9
% unsaturation 43.1 27.3
162 In retina, light and oxygen are essential for vision. However, the formation of reactive
oxygen species, which leads to photochemical damage, is also favored by light and
oxygen.2 Retina photoreceptor rod outer segments (ROS) consist of a stack of hundreds of densely packed membrane disks that are renewed continually. New disks are generated
in the rod inner segment and migrate to the base of the rod outer segment, and old disks
are detached from the end of the rod outer segment where they are deformed, engulfed,
and displaced within the retinal pigment epithelium (RPE).3, 4 This process is believed to be related to oxidative modifications induced by photogenerated radicals.5 The high
levels of oxygen consumption and irradiation received by retina is conceivably
responsible for the generation of reactive oxygen species through photosensitizers.2, 6 A
large number of chromophores may serve as potential sensitizers such as the visual
pigments (11-cis-retinal-protein complexes), melanin and lipofuscin, flavins and
flavoproteins, and the macular pigment.2 Lipofuscin (LF) is an ill-defined conglomerate
of lipids, metals, organic molecules, and biomolecules that commonly fluoresce at 360 to
470 nm. Lipofuscin-like substances were observed by a reaction of lipids containing
primary amines, with either an aldehyde or a ketone, in the presence of a peroxide.7, 8
Lipofuscin serves to facilitate generation of reactive oxygen species that contribute to
age-related decline of retinal pigment epithelium function and blue light damage.9 It has
been confirmed that age-related macular degeneration (AMD) is significantly correlated
(P = 0.01) with lipofuscin pigment accumulation in the basal lamina and Bruch’s
membrane cells of the retina.10
163 The structure of fluorophores in lipofuscin granules remained unknown until the documentation of A2E.11 The name A2E stemmed from the characterization that this unusual bis-retinoid is derived from 2 molecules of retinal and one molecule of ethanolamine.12 A2E is biosynthesized in ROS from all-trans-retinal and PEs in the form of a phospholipid bisretinoid adduct (A2-PE), which is subject to hydrolytic cleavage by phospholipase D to deliver A2E.13 A2E accumulates in the RPE as the consequence of the accumulation of large amounts of all-trans-retinal which failed to be efficiently reduced to all-trans-retinol.14 Upon irradiation (430 nm), A2E acts as a photosensitizer to release singlet oxygen and other reactive oxygen species, which could trigger the epoxidation and peroxidation of polyunsaturated fatty acids and therefore contribute to photooxidative damage such as modifications of DNA and protein.15-18 It is also conceivable that A2E could induce cell apoptosis after exposure to blue light (480 nm), and exert a detergent-like perturbation of cell membranes.19-21 A2E was previously suggested to exercise toxic effects on RPE cells by directly damaging lysosomal function and structure.22 Extralysosomal RPE cell structures were also proposed to be severely harmed by A2E during the pathogenesis of AMD. The identification of A2E was a breakthrough that exemplified the pathological potential of lipid-modified PEs.
The primary amino groups of PEs, as well as proteins, are potential targets of covalent modifications by carbohydrates and lipid peroxidation products. In 1993, Bucala et al.23 first proposed that glucose would modify the amino group of PEs. Subsequent in vitro studies24, 25 confirmed the formation of glucose-modified PE through an unstable Schiff
164 base that rearranges to a PE-linked α-aminoketone (Amadori) product. The existence of
glycated aminophospholipids in human red blood cells and plasma was further
documented by Ravandi et al.26 Another glycation adduct, carboxymethyl-PE (CME) was identified in red blood cell membranes and fasting urine, although the amount of CME did not show statistically significant differences for control versus diabetic subjects.27
PEs modified by lipid peroxidation products were also reported in recent years.
Malonaldehyde, an aldehydic degradation product from lipid peroxidation, was proposed to react with amino groups in phosphatidylethanolamine in a model study to generate fluorescent Schiff’s bases with a 1-amino-3-imino-propene structure.28 It was further
documented by Esterbauer, et al., that the reaction between 4-hydroxynonenal and
phosphatidylethanolamine led to the formation of a fluorescent chromophore identical
with the fluorophore formed in peroxidizing microsomes and mitochondria. Tsuji et al.29
characterized the formation of N-(hexanoyl)phosphatidylethanolamine (HEPE), in the
reaction of egg PE with 13-hydroperoxyoctadecadienoic acid. A Michael adduct,
generated by covalent binding of 4-HNE to PEs, was elucidated to be a poor substrate of
30 secreted phospholipase A2 and phospholipase D. In an in vitro reaction, phosphatidyl
ethanolamine-derived pyrroles 5.3 and 5.4 were identified as products from incubation of phosphatidylethanolamine with 4,5-(E)-epoxy-2-(E)-heptenal (5.1) followed by GC/MS or HPLC/MS (Scheme 5.1).31 By comparing the ability of 4-HNE, 4-hydroxydodeca-
(2E,6Z)-dienal (4-HDDE), and 4-hydroxy-2E- hexenal (4-HHE) from n-3 fatty acids to
covalently modify various ethanolamine phospholipids (PEs), chosen for their biological
165 relevance, it was concluded that aldehydes that are more hydrophobic generate more
adducts with the PEs.32 Moreover, the aldehydes exhibited stronger reactivity toward
32 PEs having unsaturated fatty acids compared with saturated ones. Levuglandin E2 stereoisomers, generated by the free radical induced peroxidation of arachidonic acid via the isoprostane pathway, were also shown to covalently modify PEs in vitro to deliver pyrrole and Schiff base adducts.33
O O R-NH2 O Et Et + Et H NR N N R R OH 5.1 5.2 5.3 5.4
Scheme 5.1. 4,5-(E)-Epoxy-2-(E)-heptenal reacts with phosphatidylethanolamine
(R-NH2) to produce pyrrole derivatives.
Modified PEs were shown to have some biological activities. Oxidative modifications
of the LDL-associated PEs stimulate a pronounced prothrombotic response by increasing
the activity of the platelet prothrombinase complex.34 The concentration of
p-hydroxyphenylacetaldehyde (pHA)-modified phospholipid, that is generated from this
product of L-tyrosine oxidation and the amino group of PEs, is strikingly elevated in
LDL isolated from human atherosclerotic lesions.35 Synthetically prepared
Amadori-glycated PE triggered lipid peroxidation when this Amadori-PE was incubated
with linoleic acid in the presence of Fe3+ in a micellar system.36
Previously, research in our group has shown that oxidative cleavage of retinal
phospholipids containing DHA produces reactive electrophilic 4-hydroxy-7-oxohept
166 -5-enoates that convert the primary amino group of protein lysyl residues into
2-(ω-carboxyethyl)pyrrole (CEP) derivatives.37 Evidence is accumulating showing that
CEPs are closely associated with age-related macular degeneration (AMD).38, 39 In view
of the reactivity of the primary amino group of PEs and the abundance of DHA in brain
and retina, we anticipate that DHA-derived oxidatively truncated phospholipids containing reactive electrophilic 4-hydroxy-7-oxohept-5-enoates convert the primary amino group of PEs into carboxyethylpyrrole phosphatidylethanolamine adducts
(CEP-PEs). In this chapter, we identified and quantified the CEP-PEs in lipid extracts from light promoted oxidation of bovine retina. Pilot studies on plasma from an age-related macular degeneration (AMD) patient and a normal control show that carboxyethylpyrrole phosphatidylethanolamine adducts are endogenously generated in vivo. The formation of these adducts in vivo could be involved in the pathogenesis of
AMD and other diseases associated with oxidative injury.
167 5.2. Results and Discussion
5.2.1. Syntheses of authentic samples. Authentic samples of CEP-PE and lysoCEP-PE
were synthesized by reaction of DOHA-Fm with 1-palmityl-2-oleyl-sn-glycero-3-
phosphoethanolamine (POPE) or 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-
-ethanolamine (lysoPE) followed by deprotection of the intermediate Fm esters with
DBU (Scheme 5.2). The synthesis of authentic standards and their characterization by
high resolution mass spectrometry (HRMS) and multinuclear NMR were described in
Chapter 2.
O CO POPE 76% R O P O O DOHAFm + or O lysoPE OH N TEA, CHCl3 O C14H29 CEPFm-PE, R = oleoyl O lysoCEPFm-PE, R = H O CO R O P O OH 90% O OH N DBU, CHCl 3 O C14H29 CEP-PE, R = oleoyl O lysoCEP-PE,R= H Scheme 5.2. Synthesis of CEP-PE and lysoCEP-PE.
5.2.2. Immunoreactivity of authentic CEP-PE. Like CEP-dipeptide, CEP-PE has a
CEP epitope, that is expected to be recognized by anti-CEP antibody. A competitive
ELISA was performed to characterize the competitive binding of CEP-PE to anti-CEP-KLH polyclonal antibody. CEP-CEO (chicken egg ovalbumin) was used as a coating agent and a standard, CEP-PE was used as an inhibitor. As shown in Figure 5.1,
CEP-PE is cross-reactive with anti-CEP antibody, although the binding of CEP-PE to
168 anti-CEP antibody is not as strong as that of CEP-CEO, perhaps due to the low molecular
weight of the former.
100
80
60
40 Absorbance (%max) 20
0
10-1 100 101 102 103 104 Inhibitor (pmol/mL)
Figure 5.1. Inhibition curves showing crossreactivity of the anti-CEP-KLH antibody for
CEP-CEO (y) and CEP-PE () against CEP-CEO as coating agent.
5.2.3. Identification of CEP-PE in oxidized lipid extracts. It has been reported that light damage promotes the peroxidation of DHA in retina.40 We postulated that irradiation of a lipid extract from retina would produce CEP modified PEs through oxidation of DHA to HOHA, that would, in turn, covalently bind with the primary amino group of PEs. Oxidation of lipid vesicles containing 50% retinal lipid extract and 50%
POPC was promoted with UV light of 350 nm. Butylated hydroxytoluene (BHT) was added after 33 h to prevent further autoxidation of the lipid samples. Lipids were extracted using the Bligh and Dyer method,41 dried under a nitrogen stream at room
169 temperature, sealed under argon, and stored in the dark at -80°C until they were analyzed.
The extracts were analyzed by LC/MS, and preliminary identification of products was achieved by comparing retention times with those of authentic samples. Negative ion
ESI-MS/MS analysis of CEP-PE yielded both parent and daughter ions which were used as the specific mass transition ion pairs in LC-MS/MS analysis. The multiple reaction monitoring (MRM) transitions used to identify the CEP-PE were the mass-to-charge ratios (m/z) for the molecular ion [M-H]- and its daughter ions, which correspond to the fragments listed in Figure 5.2. A daughter scan spectrum of CEP-PE is presented in
Figure 5.3. MRM chromatograms of CEP-PE are presented in Figure 5.4. Oxidized lipid extracts from bovine retina produced the expected CEP-PE. A single peak with a retention time that is identical to that of standard was present in the MRM chromatogram of oxidized lipid in each parent/daughter ion pair channel (Figure 5.5).
5.2.4. Identification of CEP-PE in lipid extracts that had not been oxidized in vitro.
Lipid extracts from bovine retina were dissolved in methanol and analyzed by LC-MS
using the negative ion MRM mode. The specific parent-daughter ion pairs were
monitored and a single peak with a retention time that is identical to that of the standard
is present in the MRM chromatogram monitoring the 838.3 → 255.4 and 838.3 → 391.2
transitions (Figure 5.6).
170 COO Parent ion O O O : 838.5 P N O O OH C8H17 O C14H29 O
O O O P O OH O Daughter ion: 673.6 C8H17 O C14H29 O O O O P Daughter ion: 417.2 O O OH
C8H17 O O P O OH Daughter ion: 391.2 C15H31 O O O
O Daughter ion: 281.2
C8H17
O Daughter ion: 255.2 O O O HO P Daughter ion: 152.9 O OH
Figure 5.2. Proposed MRM fragments of CEP-PE and corresponding m/z.
Relative Intensity (%) m/z Figure 5.3. Daughter scan of CEP-PE.
171 Relative Intensity (%) Relative Intensity
Figure 5.4. MRM chromatograms of CEP-PE.
172 Relative Intensity (%) Relative Intensity
Figure 5.5. MRM of lipid extracts from bovine retina after UV light-promoted oxidation.
A single peak with a retention time that is identical to that of authentic standard CEP-PE was present in the MRM chromatogram of oxidative lipid at each parent/daughter ion pair channel.
173
Relative Intensity (%) Relative Intensity
Figure 5.6. MRM of bovine retinal extracts that had not been oxidized in vitro. A single peak with a retention time that is identical to that of authentic CEP-PE is present in the
MRM chromatogram monitoring the 838.3 → 255.4 and 838.3 → 391.2 transitions.
174 5.2.5. Quantification of CEP-PE in bovine retina. Calibration curves for quantitative
analyses of CEP-PE were constructed by adding a fixed amount of internal standard
1-myristyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine (M-PE) into various amounts of authentic CEP-PE samples. LC-MS analysis was performed and peak area ratio vs analyte weight ratio was plotted. Calibration curves of CEP-PE and equations describing the calibration curves are presented in Figure 5.7. Samples containing lipid extracts and a fixed amount of internal standard (M-PE), that were or were not exposed to light-promoted oxidation, were analyzed by LC-MS. There was 60 pg CEP-PE in 1.0 μg
of lipid extract with light-promoted oxidation and 0.7 pg CEP-PE in 1.0 μg of lipid extract without oxidation.
8 y = 1.2168x - 0.1121
E 7 R2 = 0.9986 6 5 4 3 2 1 Peak AreaRatio (Analyte/MP 0 01234567 Analyte (w)/MPE (w)
Figure 5.7. Standard curve and calibration equation for CEP-PE.
5.2.6. Identification of lysoCEP-PE in light-promoted oxidized lipid extracts. In glycerophospholipids, the sn-1 position is usually occupied with saturated C16 or C18
175 fatty acids, while the sn-2 position may have a variety of saturated or unsaturated fatty acyls. Phospholipase A2 (PLA2) enzymes catalyze hydrolysis of sn-2 acyl chains from membrane phospholipids in human tissue and cells to liberate free fatty acids and lysophospholipids.42 To simplify the analysis of oxidative phospholipid modifications, we explored monitoring of lysoCEP-PE in retina samples. A complex mixture of CEP modified PEs can be converted into a simpler mixture by the action of PLA2. An authentic standard sample of lysoCEP-PE was obtained by reaction of DOHA-Fm with lysoPE followed by deprotection of the intermediate Fm ester with DBU. We also converted authentic CEP-PE to lysoCEP-PE by treating it with PLA2. This sample of lysoCEP-PE was characterized by HRMS and NMR. Negative ion ESI-MS/MS analysis of lysoCEP-PE showed parent and daughter ions that were used as the specific mass transition ion pairs for LC-MS/MS analysis. The MRM transitions used to detect the lysoCEP-PE were the mass-to-charge ratios (m/z) for the molecular ion [M-H]- and its daughter ions, which correspond to the fragments listed in Figure 5.8. A daughter scan spectrum of lysoCEP-PE is presented in Figure 5.9. It should be noted that the fragment that represents m/z = 317.98 has the specific carboxyethylpyrrole group in it. The detection of this fragment thus provides presumptive evidence that irradiated lipid extracts from bovine retina contain lysoCEP-PE (vide infra). Chromatograms of authentic standard lysoCEP-PE are presented in Figure 5.10. A lipid extract from bovine retina was converted into vesicles and irradiated with UV light (350 nm) for 33 h, and then extracted and analyzed by LC/MS as described above. A single peak with a retention time that is
176 identical to that of authentic standard is present in the MRM chromatogram of oxidized lipid in each parent/daughter ion pair channel (Figure 5.11).
COO
O O P N Parent ion: 574.07 HO O OH
O C14H29 O O P O HO O OH Daughter ion: 409.29
O C14H29 O COO
O P O Daughter ion: 317.98 N HO O OH
O Daughter ion: 255.2 O O O P Daughter ion: 152.9 O OH HO Figure 5.8. Proposed fragments of lysoCEP-PE and corresponding m/z. Relative Intensity (%)
m/z Figure 5.9. Daughter scan of lysoCEP-PE.
177 5.15 100 574.1 > 409.3
0 5.15 100 574.1 > 317.98
0
100 5.16 574.1 > 255.2 Relative Intensity (%) Relative Intensity
0
100 5.16 574.1 > 152.89
0 0 Time (minute) 14 Figure 5.10. MRM chromatograms of lysoCEP-PE. Pure lysoCEP-PE (10 ng/μL in methanol) was injected onto a Shimadzu LC-10AD HPLC system, eluting with 1 mM ammonium acetate in 20% acetonitrile and using a linear gradient to 100% acetonitrile over 3 minutes, followed by 100% acetonitrile for 10 minutes.
178 574.07 > 409.29 100 5.16
0 5.16 100 574.07 > 317.98
0 5.16 574.07 > 255.22
Relative Intensity (%) Relative Intensity 100
0 5.16 100 574.07 > 152.89
0 14 0 Time (minute) Figure 5.11. MRM of bovine retinal lipids after UV-promoted oxidation. Lipid extracts
(50 ng/μL in methanol) was injected onto a Shimadzu LC-10AD HPLC system as described in Figure 5.10. A single peak with a retention time that is identical to that of authentic lysoCEP-PE was present in the MRM chromatogram of oxidized lipid in each parent/daughter ion pair channel.
179 5.2.7. Identification of lysoCEP-PE in bovine retina. Bovine retinal lipid that had not been modified by UV-promoted oxidation was also analyzed through LC-MS/MS. Its
MRM chromatogram did not display these characteristic peaks. We also treated these
lipid extracts with PLA2 to convert a mixture of CEP-PEs into a simpler mixture of
lysoCEP-PEs and monitored lysoCEP-PE using the MRM mode by LC-MS/MS.
However, the results did not show any characteristic peaks for lysoCEP-PE in the MRM
chromatogram. This may be because lipids from a different retina than that described
above (P.170) were used and they had no CEP-PE. Alternatively, the presence of EDTA
may have interfered with the action of PLA2. This issue should be resolved by further
studies.
5.2.8. Identification of lysoCEP-PE in human plasma. Lipid extracts from plasma
samples of an AMD patient and a normal control were analyzed by LC-MS/MS. A single
peak with a retention time that is identical to authentic lysoCEP-PE is present in the
MRM chromatogram in each parent/daughter ion pair channel (Figures 5.12-5.14).
180 100 8.80 574.07 > 409.29
0
100 8.80 574.07 > 317.98
0 8.84
Relative Intensity (%) Relative Intensity 100 574.07 > 255.22
0 8.84 100 574.07 > 152.89
0 0 15 Time (minute)
Figure 5.12. MRM chromatograms of authentic lysoCEP-PE. Pure lysoCEP-PE (50 ng) was injected onto a Waters 2790 HPLC system, eluting with 1 mM ammonium acetate in water and using a linear gradient to 100% methanol over 4 minutes, followed by 100% methanol for 9 minutes. Note: This is a different system than Figures 5.10-5.11 and retention times are, therefore, different.
181 100 8.84 574.07 > 409.29
0
100 8.88 574.07 > 317.98
0
Relative Intensity (%) Relative Intensity 8.88 100 574.07 > 255.22
0
100 8.88 574.07 > 152.89
0
0 Time (minute) 18
Figure 5.13. MRM chromatograms of lipid extracts from a plasma sample of a normal control. Lipid extracts (150 μg in 20 μL methanol) was injected onto a Waters 2790
HPLC system, eluting with 1 mM ammonium acetate in water and using a linear gradient to 100% methanol over 4 minutes, followed by 100% methanol for 9 minutes.
182 100 8.77 574.07 > 409.29
0 100 8.81 574.07 > 317.98
0
Relative Intensity (%) Relative Intensity 100 8.77 574.07 > 255.22
0 8.77 100 574.07 > 152.89
0 0 Time (minute) 18 Figure 5.14. MRM chromatograms of lipid extracts from a plasma sample of an AMD patient. Lipid extracts (100 μg in 20 μL methanol) was injected onto a Waters 2790
HPLC system, eluting with 1 mM ammonium acetate in water and using a linear gradient to 100% methanol over 4 minutes, followed by 100% methanol for 9 minutes.
183 5.2.9. Quantification of lysoCEP-PE in bovine retina and human plasma. Calibration
curves for quantitative analyses of lysoCEP-PE were constructed by adding a fixed
amount of internal standard 1-myristyl-2-hydroxy-sn-glycero-3-phosphatidyl
ethanolamine (M-PE) into various amounts of authentic lysoCEP-PE samples. LC-MS
analysis was performed and peak area ratio vs analyte weight ratio was plotted.
Calibration curves of lysoCEP-PE and equations describing the calibration curves are
presented in Figure 5.15. Lipid extracts samples containing a fixed amount of internal
standard (M-PE) were analyzed by LC-MS. There was 260 pg lysoCEP-PE in 1.0 μg of
lipid extract with light-promoted oxidation. No lysoCEP-PE was detected in lipid extract
that had not been subjected to light-promoted oxidation. There was 1.2 ng lysoCEP-PE in
1 mL of plasma from a normal control and 1.3 ng lysoCEP-PE in 1 mL of plasma from
an AMD patient. These levels are virtually identical.
7 E 6 y = 2.7858x R2 = 0.9995 5 4 3 2 1 Peak Area Ratio (Analyte/MP 0 00.511.522.5 Analyte (w)/MPE (w)
Figure 5.15. Standard curve and calibration equation of authentic lysoCEP-PE.
184 5.3. Conclusions
Recent studies have strongly implicated reactive aldehydes derived from lipid peroxidation could be key mediators of oxidant injury in view of their potency to covalently modify proteins, lipids, and DNA.34, 43 Modification of aminophospholipids in
plasma membrane might disturb the phospholipid membrane distribution, alter membrane
fluidity, and damage membrane organization and function.44 HNE and isolevuglandins
have been documented to react with PEs. In view of the striking abundance of DHA and
PEs in retinal photoreceptor outer segment membranes, we explored the possibility that
CEP-PE adducts are generated in retina, presumably through oxidation of
DHA-containing phospholipids to HOHA-containing phospholipids, followed by
covalent binding to the primary amine of PEs and partial phospholipolysis. Using an
authentic standard CEP-PE, 60 pg CEP-PE in 1.0 μg lipid extract and 0.7 pg CEP-PE in
1.0 μg of lipid extract were detected and quantified in bovine retinal extracts that had and
had not been modified by UV light-promoted oxidation respectively. In addition, 260 pg of a CEP-modified lysophospholipid, lysoCEP-PE was identified and quantified in 1.0 μg
of bovine retinal extracts that had been modified by UV light-promoted oxidation. In a pilot study, similar levels, about 1.2 ng/mL, of lysoCEP-PE were detected in plasma
samples from a normal control and an AMD patient. While further studies on a large
number of individuals may reveal a significant difference between AMD and normal
individuals, it seems unlikely that a large difference will be found.
To simplify the analysis of oxidative phospholipid modifications, I treated the lipid
185 extract with PLA2. When I extracted lipid from bovine retina, I added Na2EDTA and
2+ BHT to the lipid extract to prevent oxidation. In view of the fact that PLA2 requires Ca
as a cofactor, I added extra Ca2+ solution to the system. Nevertheless, I did not detect
lysoCEP-PE in lipid extracts from non-oxidized bovine retina before or after treatment
with PLA2. There are two possible issues: 1, In the presence of Na2EDTA, the activity of
PLA2 was blocked and therefore no CEP-PE was converted to lysoCEP-PE. 2. There was
no HODA-PCs present in the retina sample used for this experiment. The following
experiments may clarify the issues. Add authentic CEP-PE to lipid extracts, then treat the
2+ mixture with PLA2 plus extra Ca solution to see if CEP-PE converts to lysoCEP-PE
under these conditions. Another possible solution may be to replace Na2EDTA with
CaEDTA that is expected to chelate Fe2+ or Cu1+ by exchange for Ca2+ to prevent oxidation of lipid extracts. Then we can treat real samples containing CaEDTA with PLA2 and analyze through LC-MS to check for the generation of lysoCEP-PE. As a control, we will treat CEP-PE with PLA2 under these same conditions.
186 5.4. Experimental Procedures
Materials and Methods
1-Palmityl-2-oleyl-sn-glycero-3-phosphatidylethanolamine (PO-PE),
1-palmityl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine (Lyso-PE) and
1-myristyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine (M-PE) were obtained
from Avanti Polar Lipids (Alabaster, AL).
Phospholipase A2 from porcine pancreas was obtained from Sigma (Cat. P0861).
ELISA Characterization of competitive binding of CEP-PE to anti-CEP antibody.
To test the immunoreactivity of authentic CEP-PE to anti-CEP-KLH polyclonal antibody,
a competitive ELISA was performed. CEP-PE vesicles were prepared by mixing CEP-PE
(50 μg) and PO-PC (450 μg) in PBS buffer (80 μL, 10 mM, pH 7.4). The mixture was
sonicated for 5 min and made into unilamellar vesicles by extrusion (4 passes) using an
Avanti Mini-Extruder (Avanti Polar Lipids, Inc.).45 CEP-CEO was used as a coating
agent and a standard. The CEP-CEO was synthesized as described in Chapter 2. The
standard was prepared by diluting a 251.4 μmol/L CEP-CEO solution to 2514 nmol/L
with PBS (10 mM, pH 7.4). A serial dilution factor of 0.2 was used, and up to eight serial dilutions of CEP-PE vesicles and CEP-CEO standard were run. CEP-CEO (100 µL,
125.7 nmol/L) solution was coated onto each well of the plate. The ELISA was carried out as described in Chapter 2. The results are summarized in Table 5.2 and Table 5.3 and shown graphically in Figure 5.1.
187 Table 5.2. ELISA data for CEP-PE in Figure 5.1.
Pmol/mL Absorbance % Absorbance Parameters Curve Fit
5200. 0.1840 23.2911 a = 100.00 25.5699
1040. 0.2280 28.8608 b = 0.8649 27.0222
208. 0.2400 30.3797 c = 1169.1756 31.2217
41.6 0.3770 47.7215 d = 25.5178 44.1505
8.32 0.5020 63.5443 68.1990
1.664 0.7190 91.0127 88.3608
0.3328 0.7880 99.7468 96.7218
0.0666 0.7940 100.5063 99.1573
Table 5.3. ELISA data for CEP-CEO in Figure 5.1.
Pmol/mL Absorbance % Absorbance Parameters Curve Fit
2514. 4.0000e-3 0.3035 a = 100.00 0.3105
502.8 0.0200 1.5175 b = 1.1669 0.6099
100.56 0.0360 2.7314 c = 4.0079 2.5249
20.112 0.1520 11.5326 d = 0.2563 13.4347
4.0224 0.6780 51.4416 50.0232
0.8045 1.1330 85.9636 86.7251
0.1609 1.2540 95.1442 97.7126
0.0322 1.3180 100.0000 99.6434
188 Extraction of Phospholipids from Retina. Bovine retinas were harvested from 2 normal cows. To preclude contamination by blood and to prevent in vitro oxidation, the retinas were rinsed with saline antioxidant cocktail [saline PBS, pH 7.4, containing 2 mM
ethylenediaminotetraacetic acid (Na2EDTA) and 100 μM butylated hydroxytoluene
(BHT)]. Lipid extraction was performed using the following procedure. Methanol (1 mL)
and 0.5 M HCl (5 μL) were added to the retina. The tissue was immediately homogenized
manually using a stainless steel pestle coated with Teflon. Then chloroform (1 mL) was
added. The mixture was vortexed, centrifuged at 3000 rm, 4 °C for 3 minutes. The lower
organic layer was carefully collected into a brown vial. The aqueous layer was extracted
with chloroform (2 x 1 mL) and the organic layer was combined and dried under a stream
of argon and stored under argon in an amber vial at -80 °C before being analyzed by
LC-MS/MS.
Light-promoted lipid oxidation in vitro. Unilamellar vesicles were prepared as follows.
Lipid extract (1 mg) and PO-PC (1 mg) were suspended in PBS buffer (1 mL, 10 mM,
pH 7.4). The mixture was vortexed for 1 minute, sonicated for 5 minutes and passed
through an extruder. The vesicles were kept in a quartz tube placed in the center of a
Rayonet photochemical reactor with three 80 W low-pressure mercury UV (350 nm)
Rayonet lamps (Southern New England Ultraviolet, Midtown, CT) at room temperature
for 33 h. Lipid extraction was then performed according to Bligh and Dyer41, and the
samples were dried, stored, and sealed, in vials, under argon, at -80 ºC.
189 Bligh and Dyer method for lipid extraction. Chloroform and methanol (3.75 mL,
CHCl3/MeOH = 1:2, v/v) was added to lipid vesicles in PBS buffer (1 mL). The mixture was vortexed vigorously. Chloroform (1.25 mL) was added and the mixture was vortexed vigorously again. Finally H2O (1.25 mL) was added and the system was vortexed well.
The resulting mixture was centrifuged at 1000 RPM for 5 minutes at room temperature to give two phases. The bottom organic phase was withdrawn very carefully through a
Pasteur pipette to avoid disturbing the interface or upper phase. The lipid was concentrated by rotary evaporation and the residue dried fully under a stream of argon.
Synthesis of lysoCEP-PE by treating authentic CEP-PE with PLA2. CEP-PE (14 mg,
0.017 mmol) was dissolved in THF (50 μL). CaCl2 (5 mM, 70 μL) in pH 7.4 PBS solution (10 mM, 1 mL) was added. The system was sonicated for 2 min followed by the
addition of PLA2 (10,000 unit/mL, 10 μL) and then incubated in 37 °C for 5 h. TLC
(CHCl3 : MeOH : H2O = 65 : 25 : 4, Rf = 0.3) showed the reaction was completed. The mixture was extracted with CHCl3/MeOH (2 : 1, v/v, 1 mL x 3) and the organic layer was concentrated to afford crude product. Flash chromatography (CHCl3 : MeOH : H2O = 65 :
1 25 : 4) provided pure lysoCEP-PE (6 mg, 65%). H NMR (CD3OD : CDCl3 = 1 : 1, 400
MHz), δ 6.60 (m, 1H), 5.96 (m, 1H), 5.83 (m, 1H), 4.10-4.0 (5H), 3.74-3.6 (m, 2H),
3.6-3.5 (m, 2H), 2.8-2.9 (m, 2H), 2.68-2.65 (m, 2H), 2.32 (t, J = 6.8 Hz, 2H), 1.57 (m,
2H), 1.23 (24H), 0.85 (t, J = 7.2 Hz, 3H).
LC/MS/MS analysis and quantification of CEP-PE in lipids from bovine retina.
A. Analysis of standard. LC-MS analysis was performed on a Quattro Ultima mass
190 spectrometer (Micromass, Wythenshawe, United Kingdom) equipped with an
electrospray ionization (ESI) probe interfaced with a Shimadzu LC-10AD (Kyoto, Japan)
HPLC system. A standard curve for CEP-PE was generated by incorporating a fixed
amount of internal standard (M-PE) and varying levels of pure CEP-PE, and plotting
peak area ratio vs analyte weight ratio. M-PE (10 μL, 1 ng/μL) was added to 90 μL each of various concentrations (15 pg/μL, 30 pg/μL, 60 pg/μL, 120 pg/μL, 240 pg/μL, 480 pg/μL) of CEP-PE in methanol. Each analyte (30 μL) mixed with internal standard was chromatographed on a BetaBasic C18 column (20 x 2.1 mm, 5 μ, Thermo Electron
Corporation, Part NO. 71505-022150) at a flow rate of 0.2 mL/min, eluting with 1 mM ammonium acetate in water and using a linear gradient to 100% methanol over 10 min, followed by 100% methanol for 10 min. Mass spectrometric analysis was performed online using an ESI tandem mass spectrometer in the negative ion multiple reaction monitoring (MRM) mode (cone energy, 60 V; collision energy, 40 eV). The total ion current was measured in the mass range of m/z 200-1000 in the negative ion mode. A 3.1 kilovolt potential was applied to the electrospray capillary. The optimal collision energy to obtain the most intense daughters was determined to be 40 eV. The MRM transitions used to detect the CEP-PE were the mass-to-charge ratios (m/z) for the molecular ion
[M-H]- and its daughter ions, which correspond to the fragments listed in Figure 5.2. The
following mass transitions, m/z 838.3 → 391.2, 838.3 → 281.4, 838.3 → 255.4, 838.3 →
153 were monitored simultaneously. Chromatograms are displayed in Figure 5.4.
B. Analysis of bovine lipids. Bovine retinal lipids that had been oxidized (promoted by
191 UV irradiation) in vitro (500 μg) were dissolved in chloroform : methanol (1 : 1, 1 mL).
Bovine retinal lipids that had not been oxidized in vitro (500 μg)was dissolved in chloroform : methanol (1 : 1, 100 μL). To these solutions (90 μL) was added M-PE (10
μL, 1 ng/μL in methanol). The resulting mixture (30 μL) was analyzed by LC-MS/MS using the same HPLC gradient and MS/MS conditions as described above for pure
CEP-PE. The mass transitions, m/z 838.3 → 391.2, 838.3 → 281.4, 838.3 → 255.4,
838.3 → 153 were monitored simultaneously. Chromatograms of lipid extracts with and without oxidation are displayed in Figures 5.5 and 5.6, respectively. The amount of
CEP-PE was 60 pg in 1 μg lipid extracts from oxidized bovine retina and 0.7 pg in 1 μg lipid extracts from bovine retina without light-promoted oxidation.
LC/MS/MS analysis and quantification of lysoCEP-PE generated in light-promoted oxidation of bovine retina. A standard curve for lysoCEP-PE was generated by incorporating a fixed amount of internal standard (M-PE) and varying levels of pure lysoCEP-PE, and plotting peak area ratio vs analyte weight ratio. M-PE (10 μL, 1 ng/μL in methanol) was added to 90 μL each of various concentrations (5 pg/μL, 10 pg/μL, 20 pg/μL, 40 pg/μL, 80 pg/μL, 250 pg/μL) of lysoCEP-PE in methanol. Each analyte (30 μL) mixed with internal standard was chromatographed on a BetaBasic C18 column (20 x 2.1 mm, 5 μ, Thermo Electron Corporation, Part NO. 71505-022150) at a flow rate of 0.2 mL/min, eluting with 1 mM ammonium acetate in 20% acetonitrile and using a linear gradient to 100% acetonitrile in 3 min, followed by 100% acetonitrile for 10 min. Mass spectrometric analysis was performed online using an ESI tandem mass spectrometer in
192 the negative ion multiple reaction monitoring (MRM) mode (cone energy, 45 V; collision
energy, 24 eV). The total ion current was measured in the mass range of m/z 200-1000 in
the negative ion mode. A 3.77 kilovolt potential was applied to the electrospray capillary.
The optimal collision energy to obtain the most intense daughters was determined to be
24 eV. The following mass transitions, m/z 574.07 → 409.29, 574.07 → 317.98, 574.07
→ 255.22, 574.07 → 152.89 were monitored simultaneously. A calibration curve and chromatograms are presented in Figures 5.12 and 5.13 respectively.
Oxidized lipid extract (500 μg) was dissolved in chloroform : methanol (1 : 1, 1 mL), and an aliquot was diluted 1 : 10 (v/v) in methanol. To this solution (90 μL) was added the M-PE standard (10 μL, 1 ng/μL in methanol), and the resulting mixture (10 μL) was analyzed by LC-MS/MS using the same HPLC gradient and MS/MS conditions as described above for authentic lysoPE-CEP. The mass transitions, m/z 574.07 → 409.29,
574.07 → 317.98, 574.07 → 255.22, 574.07 → 152.89 were monitored simultaneously.
Chromatograms are shown in Figure 5.11. The absolute amount of lysoCEP-PE was 260 pg in 1 μg lipid extract from UV-promoted oxidized bovine retina. No lysoCEP-PE was detected in lipid extracts from unoxidized bovine retina.
LC/MS/MS analysis and quantification of lysoCEP-PE in bovine retina treated with
PLA2. Lipid extract (1 mg) and PO-PC (1 mg) were suspended in PBS buffer (1 mL, 10
mM, pH 7.4). The mixture was vortexed for 1 min, sonicated for 5 min and passed
through an extruder. CaCl2 (5 mM, 70 μL) in pH 7.4 PBS solution (10 mM, 1 mL) was added. The system was sonicated for 2 min followed by the addition of PLA2 (10,000
193 unit/mL, 10 μL) and then incubated in 37 °C for 5 h. Lipid was then extracted using
Bligh and Dyer method. Lipid extract was dried under a stream of argon. Lipid (400 μg)
was dissolved in methanol (400 μL). To this solution (90 μL) was added the M-PE standard (10 μL, 1 ng/μL in MeOH), and the lipid mixture (10 μL) was analyzed by
LC-MS/MS using the same HPLC gradient and MS/MS conditions as described above for pure lysoPE-CEP. The mass transitions, m/z 574.07 → 409.29, 574.07 → 317.98,
574.07 → 255.22, 574.07 → 152.89 were monitored simultaneously. No peaks expected for lysoCEP-PE were detected.
LC/MS/MS analysis and quantification of lysoCEP-PE in human plasma.
LysoCEP-PE (50 ng in 20 μL methanol) was injected onto a Waters 2790 HPLC system, eluting with 1 mM ammonium acetate in water and using a linear gradient to 100% methanol over 4 minutes, followed by 100% methanol for 9 minutes. Mass spectrometric analysis was performed online using an ESI tandem mass spectrometer in the negative ion multiple reaction monitoring (MRM) mode (cone energy, 45 V; collision energy, 24 eV). The total ion current was measured in the mass range of m/z 200-1000 in the negative ion mode. A 3.77 kilovolt potential was applied to the electrospray capillary. The optimal collision energy to obtain the most intense daughters was determined to be 24 eV.
The following mass transitions, m/z 574.07 → 409.29, 574.07 → 317.98, 574.07 →
255.22, 574.07 → 152.89 were monitored simultaneously. The chromatograms are presented in Figure 5.12.
Lipids were extracted from plasma samples of a normal control and an AMD patient
194 using the Bligh and Dyer method as described above. Lipid extracts (300 μg) from an
AMD plasma sample or (420 μg) from a normal plasma sample were dissolved in methanol (60 μL). Then the M-PE standard (10 μL, 1 ng/μL in methanol) was added, and the resulting mixture (20 μL) was analyzed by LC-MS/MS using the same HPLC gradient and MS/MS conditions as described above for authentic lysoPE-CEP. The mass transitions, m/z 574.07 → 409.29, 574.07 → 317.98, 574.07 → 255.22, 574.07 → 152.89 were monitored simultaneously. Chromatograms of normal and AMD samples are shown in Figures 5.13 and 5.14. There was 1.2 ng lysoCEP-PE in 1 mL plasma from a normal control and 1.3 ng lysoCEP-PE in 1 mL plasma from an AMD patient.
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Lett. 2000, 481, (1), 26-30.
200 37. Gu, X.; Sun, M.; Gugiu, B.; Hazen, S.; Crabb, J. W.; Salomon, R. G., Oxidatively
truncated docosahexaenoate phospholipids: total synthesis, generation, and Peptide
adduction chemistry. J. Org. Chem. 2003, 68, (10), 3749-61.
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M.; Hasan, A.; Yan, L.; Rayborn, M. E.; Salomon, R. G.; Hollyfield, J. G., Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc.
Natl. Acad. Sci. U. S. A. 2002, 99, (23), 14682-7.
39. Ebrahem, Q.; Renganathan, K.; Sears, J.; Vasanji, A.; Gu, X.; Lu, L.; Salomon, R. G.;
Crabb, J. W.; Anand-Apte, B., Carboxyethylpyrrole oxidative protein modifications stimulate neovascularization: Implications for age-related macular degeneration. Proc.
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40. Bicknell, I. R.; Darrow, R.; Barsalou, L.; Fliesler, S. J.; Organisciak, D. T., Mol. Vis.
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42. Dennis, E. A., Diversity of group types, regulation, and function of phospholipase A2.
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43. Murthi, K. K.; Friedman, L. R.; Oleinick, N. L.; Salomon, R. G., Formation of
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10016-23.
202
Chapter 6
Pilot Studies towards Identification of Levuglandin Modified Proteins in
Cornea
203 6.1. Introduction
6.1.1. LPS and inflammation
Inflammation involves the interaction of multiple cell types at the target site releasing
eicosanoids (prostaglandins, prostacyclins, thromboxanes and leukotrienes), proteolytic
enzymes, cytokines, and other inflammatory mediators.1 Insults to the endothelial layer of
the blood vessels can stimulate the production of leukocyte chemo-attractant molecules
and the expression of adhesion molecules that bind to the monocytes and make them
adhere to the endothelial layer through integrins. The activation of chemotactic factors,
e.g., monocyte chemoattractant protein-1 (MCP-1), usually in response to inflammatory
cytokines such as interleukin-1 (IL-1), interleukin-4 (IL-4), tumor necrosis factor α
(TNF-α), and interferon-γ (IFN-γ), recruits more phagocytic and immune cells to the site
of inflammation.2-5 It is well established that lipopolysaccharide (LPS) stimulates a
variety of inflammatory responses including activation of the alternate complement
cascade and the production of cytokines.6 LPS, a structural element of Gram-negative
bacterial cell wall consisting of lipids and polymers of carbohydrates, serves as a ligand
for receptors that can activate proinflammatory cytokines in many cell types.6-8 Acute
exposure to LPS in serum concentrations as low at 1 ng/mg could cause death.9 Released cytokines, such as tumor necrosis factor and interleukin-1, can activate endothelial cells
to upregulate receptors for various immune cells.10, 11
6.1.2. The cyclooxygenase and isoprostane pathways. Cyclooxygenase (COX), also
known as prostaglandin endoperoxide (PGH2) synthase, is the first enzyme of the
204 pathway by which arachidonic acid (AA) is highly stereoselectively cyclooxygenated to a
12 single, enantiomerically pure prostaglandin endoperoxide PGH2. The existence of two
cyclooxygenases in human endothelial cells has been proven. COX-1 is considered a
constitutive enzyme, being found in most mammalian cells. More recently it has been
shown to be upregulated in several carcinomas and to have a central role in tumorigenesis.
COX-2 is undetectable in most normal tissues and is induced in a number of cell types
including endothelial cells,13 neutrophils,13, 14 macrophages15, 16 and monocytes16, 17 by a
variety of inflammatory mediators, such as IL-1, IL-2 and LPS1, 18-20 at sites of
inflammation.
PGH2, which is generated enzymatically through the action of COX from arachidonic
acid (AA), is a branch point in the biosynthesis of numerous hormone-like mediators
21, 22 (PGD2, PGE2, PGI2, PGF2, and thromboxane TXA2) of normal physiological
processes (Figure 6.1). Non-enzymatical rearrangement of PGH2 in protic solvents
produces two levulinaldehyde derivatives with prostanoid side chains which were named
23 levuglandin (LG) D2 and E2. In addition, free radical-induced cyclooxygenation of AA
and its phospholipid esters through the isoprostane pathway generates isomers of PGs
which were coined isoprostanes (isoPs).24, 25 Non-enzymatic rearrangement of isoP endoperoxides gives rise to both stereoisomers and structural isomers of levuglandins,
26 which were defined as isoLGs and iso[n]LGs respectively. LGE2, one of the stereoisomers designated collectively as isoLGE2, is a product of both the COX and
isoprostane pathways whereas iso[4]LGE2 derives only from isoprostane pathway
205 (Scheme 6.1).
Figure 6.1. Eicosanoid synthesis.
(CH2)3COOPC (CH ) COOH Phospholipase A2 2 3
C5H11 C5H11 AA-PC AA
Free radical induced Cyclooxygenase
(CH2)3COOPC O O (CH2)3COOH O O C5H11 C5H11 OH OH
isoPGH2-PC PGH2
O O (CH2)3COOPC (CH2)3COOH
OHC C5H11 OHC C H 5 11 OH OH Levuglandin E2 Iso[4]levuglandin E2-PC
Scheme 6.1. Representative pathways showing enzyme and free radical induced generation of LGE2 and iso[4]LGE2 from AA-PC.
206 6.1.3. LGE2 and iso[4]LGE2 bind avidly with proteins. LGE2 and iso[4]LGE2 have exceptional reactivity to biological nucleophiles such as proteins and DNA.27-29 It was
proposed that levuglandins are the reactive electrophiles responsible for much (if not all)
of the COX-dependent binding of AA metabolites.30 Upon incubation of bovine serum
3 albumin (BSA) with an excess of 5,6- H-LGE2, 10 molecules of LGE2 irreversibly bind
30 to BSA within 1 min. LGE2 and iso[4]LGE2 protein adducts were found in human
plasma by immunoassays using antibodies raised against corresponding LGE2 and
31 iso[4]LGE2 keyhole limpet hemocyanin (KLH) adducts. More importantly, the levels of
both LGE2 and iso[4]LGE2 protein adduct immunoreactivity were significantly elevated
in plasma from end-stage renal disease (RD) and atherosclerosis (AS) than in plasma from healthy controls.32 These clinical findings provide unambiguous evidence that lipid
oxidation is correlated to pathological disease.
6.1.4. Pyridoxamine, a potent trap that prevents protein modification by reactive
electrophiles. In view of the damage caused by protein modification by products of lipid
oxidation, many approaches have been explored to protect against the assault of lipid
oxidation products on amino groups of proteins.33 Pyridoxamine (vitamine B6), an
essentially nontoxic amine, was first proposed by Hudson and colleagues as an inhibitor
of the formation of advanced glycoxidation end products (AGEs).34, 35 Recently,
pyridoxamine was documented to be an extremely potent scavenger of 1,4-dicarbonyl
compounds. Pyridoxamine reacted with 1,4-dicarbonyls at rates 2300 times greater than
to α–N-AcLys in the formation of pyrroles and it strongly reduced adduction to lysine by
207 a γ-ketoaldehyde when present at equimolar concentrations to α–N-AcLys in vitro.36 The
acidic phenol group on pyridoxamine was proposed to be responsible for its extraordinary
reactivity. This hydroxyl protonates the carbonyl in a hemiaminal intermediate and holds
it into a conformation that favors intramolecular attack of the amine, facilitating
cyclization to a pyrrolidine diol (Scheme 6.2).36 In animal model studies, pyridoxamine,
administrated in drinking water at 1 g/l, inhibited AGE formation and retarded the
development of early renal disease. It also reduced the development of acellular
capillaries which is one of the earliest hallmarks of diabetic retinopathy in
streptozotocin-induced diabetic rats.37-40 All these experiments encourage us to examine
the ability of pyridoxamine to inhibit the accumulation of LG modifications.
In this chapter, a pilot study was constructed on the LPS induced formation of LGE2 and iso[4]LGE2 protein adducts in a mouse cornea model. Further studies are planned to test the ability of pyridoxamine to prevent LPS-promoted haze development using mouse models.
R2 R R H O HO 2 2 H H OH R2 O N O O NH2 O N N H R1 + OH R1 N OH N HO R1 O OH N N OH OH R1 pyridoxamine hemiaminal pyrrolidine diol pyrrole
Scheme 6.2. Possible mechanism of pyrrole formation between pyridoxamine and
1,4-dicarbonyls.
208 6.2. Results and Discussion
Cornea of C57BL/6 mouse was treated in vivo with ultrapure LPS (2 μg) in water (2
μL) or PBS (2 μL) by intrastromal injection. Alternatively, the cornea was scratched three times and then LPS in water (2 μL) or PBS alone (2 μL) was dropped onto the scratches.
After 24 h, the cornea was excised. To localize the LGE2 and iso[4]LGE2 modified
proteins in the cornea and to determine the effect of LPS on the generation of LG- or
isoLG-protein adducts, corneal sections were stained with the corresponding antibodies.
The slides were then incubated with a fluorescent second antibody and analyzed with a
fluorescence microscope. The results are presented in Figure 6.2. The green fluorescence
represents the staining of LGE2-protein or iso[4]LGE2-protein adducts, while the blue
represents nuclear staining by 4,6-diamidino- -2-phenylindole (DAPI). The LPS-injected
cornea showed visibly elevated green fluorescence of both LGE2-protein and
iso[4]LGE2-protein adducts compared to the PBS-injected cornea. However, cornea
scratched and treated with LPS did not seem to show expected differences compared with
PBS treatment.
In the eye, corneal inflammation caused by microbial keratitis or bacterial products, e.
g., LPS, leads to a significant loss of vision.41 The translucent nature of the cornea allows in vivo visualization of inflammatory events in a noninvasive manner.42 Intrastromal
injection of LPS has been reported to induce COX-2 expression.43-45 As mentioned above,
COX-2 catalyzes the synthesis of PGH2, which undergoes non-enzymatic rearrangement
to give rise to LGE2. Iso[4]LGE2 is a unique product from free-radical induced
209 Untreated cyclooxygenation.A The resultsB show that LPS injection C
D E F PBS LPS
G H I
K J L Untreated
M N O PBS
P Q R LPS
Preimmune LGE2 Iso[4]LGE2
Figure 6.2. Cornea of C57BL/6 mouse was treated in vivo with LPS or PBS for 24 h by injection or scratch. Sections (5 μm) of cornea were stained with LGE2 and iso[4]LGE2 pAb. A, B, C, J, K, L – Untreated; D, E, F – PBS injection; G, H, I – LPS injection; M, N, O – PBS treated (scratch); P, Q, R – LPS treated (scratch).
Panels treated with preimmune, LGE2 and iso[4]LGE2 pAbs are indicated in the Figure.
210 increased both LGE2-protein and iso[4]LGE2-protein adducts. The formation of
iso[4]LGE2-protein adducts confirms that free radical mediated lipid oxidation is promoted by LPS. However, the formation of LGE2-protein adducts does not show that
LPS induces COX pathway because these adducts are also expected to be generated
through free radical-induced lipid oxidation, i. e., the isoprostane pathway.
Further experiments in continuation of this pilot project should be focused on
examining the efficacy of pyridoxamine for scavenging endogenous γ-ketoaldehydes and
retarding the accumulation of LGE2-protein or iso[4]LGE2-protein modifications, and
thereby preventing haze development in keratitis.
211 6.3. Experimental Procedures.
General methods. The following commercially available materials were used as
received: fetal calf serum (FCS, Invitrogen, Carlsbad, CA); phosphate buffered saline
(PBS, Sigma, St. Louis, MO); goat anti-rabbit IgG conjugated with Alexa 1000 and
Vectashield mounted with media containing 4,6-diamidino-2-phenylindole (DAPI) were
from Vector Laboratory Inc, Burlingam, CA; Anti-LGE2-KLH and anti-iso[4]LGE2-KLH polyclonal antibodies were purified as described previously.26
Immunohistochemical analysis. Eyes were enucleated 24 hours after intrastromal
injection or scratch, and snap frozen in optimum temperature cutting compound
(Tissue-Tek; Miles Scientific, Napierville, IL). Five-micrometer frozen sections of the
central cornea were cut and fixed on slides for immunohistochemical analysis. The slides
were fixed by submersion 4% paraformaldehyde solution for 30 min, rinsed with PBS
and then blocked with 2% fetal calf serum (FCS) (200 μL/slide) for 30 min at room
temperature. Subsequently, the slides were incubated with appropriate first antibody
solutions (200 μL/slide) for 2 h at room temperature. The antibody solution was prepared
by adding 25 μL of concentrated antibody (1.94 mg/mL polyclonal rabbit anti-LGE2 or
1.14 mg/mL polyclonal rabbit anti-iso[4]LGE2) into 2.5 mL 1% FCS in PBS containing
3% dry milk. The slides were then washed with 1% FCS in PBS (3 x 5 min) and
subsequently incubated with goat anti-rabbit IgG conjugated with Alexa 1000 (Vector
Laboratory Inc, Burlingam, CA) (10 ng/slide) for 1 h at room temperature. Slides were
washed with 1% FCS in PBS three times and with PBS two times. Slides were dried
212 carefully with kimwipes, sealed with Vectashield, mounted with media containing DAPI
(blue) (Vector Laboratory Inc, Burlingam, CA), and analyzed with a Nikon EFD-3 fluorescence microscope attached to a CCD camera.
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220
Chapter 7
Syntheses of Isosteric Pyrazole Derivatives
221 7.1. Background
Numerous biological processes are proposed to be mediated through pyrrole formation
on biological molecules, and pyrrole adducts on protein and DNA have been associated
with pathological diseases.1-4 Humans or experimental animals can develop a distal
neuropathy, which is an accumulation of neurofilaments in large myelinated axons
(myelin is an insulating lipid layer formed around nerves), or even lose distal sensory and
motor functions when exposed to hexane. Pyrrole formation, which stems from the
hexane oxidation product 2,5-hexanedione that reacts with lysyl ε-amino groups of
proteins, is believed to be the first step in the induction of neuropathy.3, 4 Peroxidation
products from fatty acids also are known to form pyrroles upon reaction with lysyl
ε-amino groups. For example, 4-hydroxy-2-nonenal (HNE), which has been touted as the
“most cytotoxic aldehyde”5, 6 released during peroxidation of linoleate and arachidonate
fatty esters, has been confirmed to adversely modify proteins and irreversibly generate a
2-pentylpyrrole adduct as well as Michael and Shiff-base adducts.7-10 This HNE-derived
pyrrole has been called an example of an “advanced lipid peroxidation end product”
(ALPE) based on the multistep nature of the condensation which leads to a pyrrole.7
Levuglandin E2 (LGE2), a nonenzymatic rearrangement seco prostanoid product derived from the prostaglandin endoperoxide PGH2 during metabolism of arachidonic acid (AA),
covalently binds to proteins and generates pyrrole derivatives as major primary
11-16 products. Both 2-pentylpyrrole and LGE2 protein adducts can be detected in human
plasma, and are more prevalent in plasma from patients with renal disease or
222 atherosclerosis than in plasma from normal controls,17, 18 making them indelible markers of oxidative injury associated degenerative diseases.
The pyrrole ring is an electron-rich heterocycle, and its derivatives readily undergo
oxidation under photochemical and electrochemical conditions, or in the presence of
molecular oxygen.19, 20 The oxidations are normally sophisticated and generally result in
various products in different distribution.1, 21-23 It has been established that proteins carrying pyrrole rings tend to covalently cross-link under oxidizing conditions. Either
pyrrole-pyrrole dimerization24 or reaction of a putative electrophilic autoxidized pyrrole
moiety with nucleophilic protein side chains such as lysyl ε-amino groups or cysteine
thiols are possible contributions to cross-linking under physiological conditions (Figure
7.1).1, 25, 26 The limited stability of pyrroles detracts from their utility as standards in
immunosorbent assays. When the pyrroles are used as standards, they must be freshly
prepared. For example, HNE-derived 2-pentylpyrroles degrade over a period of one year.
Since each batch contains different amounts of pyrroles and there is always the
possibility that various pyrrole-Nu crosslinks exist simultaneously, it is not reliable to
compare levels of pyrrole determined with the use of different batches of standard.
Therefore, we opted for a more reliable approach that exploits the high crossreactivity anticipated between pyrroles and their stable pyrazole isosteres. The pyrazoles are
electron deficient, and consequently, are unlikely to undergo electrophilic aromatic substitution or oxidation.27
223 R2 R 2 protein N R2 X protein O2 N protein R1 + R2 CH2 N protein R1 HX protein R1 CH3 N protein CH R1 3 X = N, S
Figure 7.1. Pyrroles cross-link with proteins.
Since 2-pentylpyrrole generated by condensation of HNE with the ε–amino groups of
protein lysyl residues are appended to the protein by an n-alkyl tether to the pyrrole ring nitrogen, an isosteric pyrazole hapten was designed that would be linked to a carrier protein by an n-alkyl tether to the 1-position of the pyrazole ring. An aldehyde group was chosen to allow coupling by reductive alkylation with peptides or proteins.
In this chapter, the synthesis of pyrazole derivatives, that are analogues of
2-pentylpyrrole, is reported. These are potential standards applicable in immunosorbent assays.
224 7.2. Results and Discussions
7.2.1. Synthesis of a 1,3-dione. Claisen condensation provides a general route to
1,3-dicarbonyl compounds.28-30 An enol 7.1 was obtained by Claisen condensation, preferentially at the methyl position of 2-heptanone, with diethyl oxalate in 64 % yield
(Scheme 7.1). We found no evidence for condensation at the methylene position since
TLC of the crude product showed only one major spot, our target product. Acidification of this sodium salt generated a dione that exists mainly as the enol 7.1.
O 1, Na, Ethanol O OH 2, HCl + (COOC H ) C H C CO Et C5H11 CH3 2 5 2 5 11 2 64% H 7.1
CO2Et 1, I-(CH2)4CH=CH2 (7.3), COOH NH2-NH2H2O NaH, Toluene EtOH, HOAc N N C H 2, NaOH, EtOH 5 11 N C5H11 N 84% H 50% (CH2)4CH=CH2 7.2 7.4
170 oC, 9hr N C5H11 N 45% (CH2)4CH=CH2 7.5 acetone, reflux Cl-(CH2)4CH=CH2 + NaI I-(CH2)4CH=CH2 73% 7.3
Scheme 7.1. Synthesis of 1-alkyl-5-pentylpyrazole through a decarboxylation route.
7.2.2. Synthesis and structure identification of 1-alkyl-5-pentylpyrazole-3-carboxylic acid. Condensation of hydrazine with 1,3-dicarbonyl compounds provides an expeditious construction of pyrazoles.31, 32 An N-substituted pyrazole carboxylic acid 7.4 was
225 prepared from the pyrazole ester 7.2 and iodoalkene 7.3 followed by hydrolysis of the
ester with alcoholic NaOH.
Heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond
coherence (HMBC) were used to identify the pyrazole structure. Notably, in the HMBC
α β γ spectrum, H , Η and H all correlate with the same C*. This confirmed that this product
is the 3-carboxyl-5-pentylpyrazole and not the 3-pentyl-5-carboxylpyrazole in which
β γ H and H would not correlate with the same C* (Figure 7.2). The HMQC and HMBC
data are listed in Table 7.1, and the spectra are displayed in Appendix Figures S59 and
S60.
α γ α H H COOH H γ CH C4H9 H * C N *C N CH N HOOC N C4H9 βH CH βH CH (CH2)3CH=CH2 (CH2)3CH=CH2 3-carboxyl-5-pentyl pyrazole 3-pentyl-5-carboxyl pyrazole Figure 7.2. Structural description of C – H HMBC coupling for both 3-carboxyl-5-pentyl pyrazole and 3-pentyl-5-carboxyl pyrazole.
Table 7.1. HMQC and HMBC data for 7.4 (in parts per million).a
HMQC HMBC α α β γ 6.63 (H )↔ 107.37 144.68 (C*)↔ 6.63 (H ), 4.11 (H ), 2.59 (H ) 4.99 ↔ 114.89 141.69 ↔ 6.63 β 4.11 (H )↔ 49.52 137.98 ↔ 2.10 γ 2.59 (H )↔ 25.70 114.89 ↔ 2.10 0.92 ↔ 13.85 107.37 ↔ 2.59
a The symbol ↔ denotes the C-H correlation; solvent: CDCl3.
226 7.2.3. Decarboxylation of a pyrazole carboxylic acid.33 The melting point of pyrazole
carboxylic acid is 145 °C. Melting point was measured on a Thomas Hoover Unimelt
capillary melting point apparatus and is uncorrected. Copper chromite catalyzes decarboxylation of indole 2-carboxylic acid.34 We heated the acid 7.4 with copper
chromite at 200 - 250 °C in a vacuum, but failed to obtain the decarboxylation product.
Barton decarboxylation35, 36 is a potential route from 7.4 to 7.5. However, this route is
lengthy and involves noxious reagents, and was therefore not pursued. It was reported
that heating such pyrazole carboxylic acids at a temperature a little bit higher than their
melting points causes decarboxylation.31 This thermal decarboxylation proved to be effective when the acid 7.4 was heated at 170 °C for 9 h with no catalyst or solvent.
Although the high temperature resulted in a dark, tar-like residue, the desired product 7.5 was easily purified by flash chromatography to deliver pure decarboxylation product in
45% yield.
O O N S S Cl C C Cl ONa O RCOOH RCOCl DMAP N O C R
S SnBu3 HSnBu3 N + CO2 + RH AIBN, heat Scheme 7.2. General scheme of Barton decarboxylation.
7.2.4. Condensation of an alkylhydrazine with a β-ketoaldehyde. An alternative was
explored to generate the requisite N-substituted pyrazole by condensation of an
alkylhydrazine with a β-ketoaldehyde (Scheme 7.3). However, a mixture of 3- and
227 5-substituted isomers usually occurs. The failure to isolate the individual isomers led
early workers to the conclusion that these two forms are indistinguishable and that the 3-
and 5-positions are equivalent.37 Actually, the isomers are separatable using normal flash chromatography. The problem with this route consists of two factors: (1) 2-heptanone condenses with ethyl formate at both the methyl and the methylene positions to provide a mixture of sodium salts of hydroxymethylene ketones (7.7 and the isomer);30, 38 and (2)
7.7 readily undergoes self-condensation to form a symmetrical 1,3,5-triacylbenzene.39-41
As a consequence of the above factors, the yield of 7.5 through this route was low (10%).
O O OH O OH 1. Na, ether. 2. HCl + HCOOC H + C H CH 2 5 C5H11 C H H C C H 5 11 3 crude: 45% H 3 C H 7.7 4 9 C5H11 O OH NH2-NH-(CH2)4CH=CH2 N + N C5H11 N N C5H11 C H ethanol H (CH2)4CH=CH2 (CH2)4CH=CH2 7.5 (10%) 7.10 (11%) ether + 1/3 PBr Br-(CH ) CH=CH HO-(CH2)4CH=CH2 3 -78 °C, 50% 2 4 2 7.8 MeOH, 50 hr NH2-NH2 · H2O + Br-(CH2)4CH=CH2 NH2-NH-(CH2)4CH=CH2 60% 7.9 Scheme 7.3. An alternative route for synthesis of pyrazole 7.5.
7.2.5. Attempted ozonolysis off an alkenylpyrazole. Oxidation of an alkene to an
aldehyde is generally accomplished by ozonolysis. However, the ozonolysis of pyrazole
7.5 encountered problems. Apparently, the pyrazole ring is also oxidized. After trying the ozonolysis three times, I failed to get the expected pyrazole aldehyde 7.6 (Scheme 7.4).
Since the double bond on the side chain is supposed to be more readily oxidized than the
228 double bonds on the aromatic ring, it may be possible to control consumption of ozone by
careful TLC monitoring of the reaction.
7.2.6. A strategy for conjugation of the isostere hapten with peptides. Sodium
cyanoborohydride is generally useful for imine reduction and reductive amination of
proteins in aqueous solution at pH 6 - 8.42 It is anticipated that the aldehyde 7.6 can be coupled with the dipeptide N-acetyl- gly-lys-OMe or bovine serum albumin (BSA) by reductive alkylation in methanol (Scheme 7.4).
N (1),ozone, MeOH N NaBH CN, HOAc N N 3 C5H11 C5H11 N C5H11 N (CH ) CH=CH -78 °C, (2), Me S (CH ) CHO protein protein 2 4 2 2 2 4 NH2 (CH2)5NH 7.5 7.6 protein=Actyl-Gly-Lys-OMe or BSA
Scheme 7.4. Reductive amination of proteins with pyrazole aldehyde 7.6 that is to be generated by ozonolysis of alkene 7.5.
229 7.3. Experimental Procedures.
Preparation of 2-hydroxy-4-oxo-non-2-enoic acid ethyl ester (7.1). To a suspension of
metallic sodium (1.06 g, 46.08 mmol ) in freshly distilled ethanol (50 mL) was slowly
added dropwise a mixture of 2-heptanone (4.18 g, 36.6 mmol) and diethyl oxalate (6.4 g,
43.8 mmol). After being stirred at 60 °C for 9 h, the mixture was treated with 3 N HCl to
adjust the pH to 5. The cloudy mixture was extracted with ethyl acetate (3 x 15 mL). The
red-brown organic layer was washed with brine, dried with sodium sulphate and
concentrated to obtain the crude product. The crude compound was purified by silica gel
chromatography (15 % ethyl acetate in hexane, TLC: Rf = 0.3) to deliver 5.02 g pure 7.1
1 as a red-brown oil. H NMR (CDCl3, 300 MHz) δ 6.36 (s, 1H), 4.36 (q, J = 7.14 Hz, 2H),
2.48 (t, J = 7.4 Hz, 2H), 1.65 (m, 2H), 1.37 (t, J = 7.14 Hz, 3H), 1.34-1.29 (4H), 0.90 (t, J
13 = 6.87 Hz, 3H). C NMR (CDCl3, 50 MHz, APT) δ 203.42 (+) (CO), 166.69 (+) (COH),
162.22 (+) (COO), 101.66 (-) (CH), 62.53 (+) (CH2), 40.94 (+) (CH2), 31.33 (+) (CH2),
24.62 (+) (CH2), 22.42 (+) (CH2), 14.10 (-) (CH3), 13.93 (-) (CH3).
5-Pentyl-1H-pyrazole-3-carboxylic acid ethyl ester (7.2). Hydrazine hydrate (1.65 mL,
0.033 mol) was slowly added to a solution of 7.1 (4.61 g, 0.0215 mol) in ethanol (10 mL)
with acetic acid (4 mL) under ice-cooling. The mixture was then heated for 24 h under
reflux, then cooled, neutralized with sat. NaHCO3 solution, and extracted with ethyl
acetate. The ethyl acetate extract was washed with saturated NaHCO3 solution, dried with
Na2SO4, and evaporated to dryness. The crude residue was purified by silica gel chromatography (25 % ethyl acetate in hexane, TLC: Rf = 0.3) to give 3.8 g (84 %) of
230 1 pure 7.2. H NMR (CDCl3, 300 MHz) δ 6.60 (s, 1H), 4.36 (q, J = 7.1 Hz, 2H), 2.68 (t, J =
11.2 Hz, 2H), 1.65 (m, 2H), 1.36 (t, J = 7.1 Hz, 3H), 1.32-1.27 (4H), 0.90 (t, J = 10.6 Hz,
13 3H). C NMR (CDCl3, 50 MHz) δ 162.25 (COO), 146.73 (C), 142.07 (C), 105.68 (CH),
60.39 (CH2), 31.04 (CH2), 28.52 (CH2), 25.38 (CH2), 22.08 (CH2), 13.94 (CH3), 13.66
+ (CH3). HRMS (EI) (m/z) calcd for C11H18N2O2 (M ) 210.1368, found 210.1371; calcd for
+ C10H15N2O2 (M -CH3) 195.1134, found 195.1132.
Preparation of 6-iodohexene (7.3). 6-Chlorohexene (500 mg, 4.22 mmol) and sodium
iodide (3.8 g, 25.3 mmol) were dissolved in acetone (28 mL). The solution was stirred for
9 h under reflux. Insoluble sodium chloride was generated as the reaction proceeded. The
mixture was then cooled, evaporated to remove acetone. Water (10 mL) was added to
dissolve the salt. The mixture was extracted with ethyl acetate (3 x 15 mL). The brown
extract was washed with Na2S2O3 and brine, dried with Na2SO4, and evaporated to give
1 650 mg (3.1 mmol, 73 %) 7.3 without further purification. H NMR (CDCl3, 300 MHz) δ
5.77 (m, 1H), 5.0 (m, 2H), 3.2 (t, J = 5.1 Hz, 2H), 2.08 (m, 2H), 1.84 (m, 2H), 1.52 (m,
2H).
Preparation of 1-hex-5-enyl-5-pentyl-1H-pyrazole-3-carboxylic acid (7.4). Pyrazole
7.2 (565 mg, 2.69 mmol) in toluene (5 mL) was added dropwise to a stirred suspension of
NaH (60 % in oil) (143 mg, 3.58 mmol) in toluene (10 mL) at room temperature. The resulting mixture was stirred at 60 °C for 1 h, then 6-iodohexene (650 mg, 3.1 mmol) was added and the resulting mixture was heated at 100 °C overnight. Solvent was removed by evaporation, and water was added to the residue. The mixture was extracted with EtOAc
231 (3 x 10 mL). The organic layer was washed with brine, dried with Na2SO4, and evaporated to give crude ethyl ester (593 mg). The ester (593 mg) was then dissolved in a solution of NaOH (1.5 N, 6 mL) in ethanol (10 mL), and refluxed for 3 h. After removal of the solvent, the residue was acidified with 3 N HCl to pH = 4.0, and then extracted with EtOAc (3 x 10 mL). The organic layer was washed with brine, dried with Na2SO4, and concentrated. The crude acid was purified by silica gel chromatography (1 % acetic
acid in ethyl acetate, TLC: Rf = 0.23) to give 353 mg (50 %) of pure 7.4 as light yellow
1 crystals (melting point: 145°C). H NMR (CDCl3, 300 MHz) δ 6.63 (s, 1H), 5.77 (m, 1H),
4.99 (m, 2H), 4.11 (t, J = 11.1 Hz, 2H), 2.59 (t, J = 11.0 Hz, 2H), 2.10 (m, 2H), 1.86 (m,
13 2H), 1.67 (m, 2H), 1.45-1.3 (6H), 0.92 (t, J = 10.4 Hz, 3H). C NMR (CDCl3, 50 MHz,
APT) δ 166.00 (+) (COO), 144.68 (+) (C), 141.69 (+) (C), 137.98 (-) (CH), 114.89 (+)
(CH2), 107.37 (-) (CH), 49.52 (+) (CH2), 33.15 (+) (CH2), 31.25 (+) (CH2), 29.65 (+)
(CH2), 27.93 (+) (CH2), 25.70 (+) (CH2), 25.28 (+) (CH2), 22.33 (+) (CH2), 13.85 (-)
(CH3).
Preparation of 1-hex-5-enyl-5-pentyl-1H-pyrazole (7.5). Acid 7.4 (140 mg, 0.53 mmol)
was put into an evacuated flask and heated under 170 °C for 9 h using a heating mantle
filled with sand. The dark residue was purified by silica gel chromatography (10 % ethyl
1 acetate in hexane, TLC: Rf = 0.2) to afford (53 mg, 45%) of pure 7.5. H NMR (CDCl3,
400 MHz) δ 7.38 (d, J = 1.6 Hz, 1H), 5.98 (d, J = 1.6 Hz, 1H), 5.76 (m, 1H), 5.01-4.93
(m, 2H), 4.01 (t, J = 7.2 Hz, 2H), 2.56 (t, J = 7.6 Hz, 2H), 2.07 (m, 2H), 1.82 (m, 2H),
13 1.63 (m, 2H), 1.44-1.34 (6H), 0.92 (t, J = 7.2 Hz, 3H). C NMR (CDCl3, 100 MHz) δ
232 142.52 (C), 138.21 (CH), 138.05 (CH), 114.81 (C), 103.65 (CH), 48.62 (CH2), 33.24
(CH2), 31.44 (CH2), 29.73 (CH2), 28.29 (CH2), 25.90 (CH2), 25.36 (CH2), 22.37 (CH2),
+ 13.92 (CH3). HRMS (FAB) (m/z) calcd for C14H25N2 (MH ) 221.2018, found 221.2027.
Ozonolysis. Alkene 7.5 (17 mg, 0.078 mmol) was dissolved in methanol (10 mL) and the
solution was cooled to -78 °C. Ozone, generated from a cylinder of oxygen with a
Welsbach ozonizer operated with an O2 pressure of 6 lb per square inch and a voltage
setting of 58 v, was bubbled through the methanol solution at -78 °C until a blue color
appeared and persisted for 5 minutes. The excess of ozone was then flushed away with
nitrogen. While still at -78 °C, Me2S (1 mL) was added and the solution was allowed to warm gradually to room temperature. The solvent was removed by rotary evaporation. A
1H NMR spectrum of the mixture showed the disappearance of protons on the pyrazole
double bonds. Thus, the desired aldehyde pyrazole was not obtained.
Preparation of 1-hydroxyoct-1-en-3-one (7.7). To a suspension of metallic sodium (201
mg, 8.75 mmol ) in freshly distilled ether (25 mL) was slowly added dropwise a mixture
of 2-heptanone (1 g, 8.75 mmol) and ethyl formate (648 mg, 8.75 mmol) with ice bath
cooling. The mixture became a slurry after being stirred for 9 h at room temperature. The slurry was poured into cold water (10 mL). The mixture was separated. The aqueous
layer was washed with ether twice and then acidified with 3 N HCl to pH = 5. The
resulting mixture was extracted with ethyl acetate (3 x 10 mL). The light yellow organic
layer was washed with brine, dried with sodium sulphate and concentrated to obtain 561
mg of the crude product 7.7 (45 %) which was used without further purification. 1H NMR
233 (CDCl3, 300 MHz) δ 7.9 (d, J = 4.8 Hz, 1H), 5.52 (d, J = 4.8 Hz, 1H), 2.35 (t, J = 7.4 Hz,
2H), 1.6 (m, 2H), 1.4-1.2 (4H), 0.90 (t, J = 6.87 Hz, 3H).
Preparation of 6-bromohexene (7.8).43 To a solution of 5-hexen-1-ol (3.41 g, 34.1 mmol) in freshly distilled ether (25 mL) under argon at -78 °C was added dropwise a solution of phosphorus tribromide (3.54 g, 13.7 mmol) in ether (5 mL). The reaction mixture was slowly allowed to warm to room temperature overnight, whereupon water (5 mL) and ether (15 mL) were added. The mixture was washed with an aqueous solution of saturated potassium carbonate (10 mL). After drying and removal of the solvents, the residue was distilled in vacuo to afford 2.78 g (50 %) of the hexenyl bromide, bp 34-36
1 °C (0.05 mm). H NMR (CDCl3, 300 MHz) δ 5.77 (m, 1H), 5.0 (m, 2H), 3.4 (t, J = 7.4
Hz, 2H), 2.1 (m, 2H), 1.88 (m, 2H), 1.55 (m, 2H).
Preparation of 5-hexen-1-yl-hydrazine (7.9). Hydrazine hydrate (10 mL, 0.2 mol) was added to 6-bromohexene (2.78 g, 17.05 mmol) in methanol (10 mL). The cloudy system
was flushed with argon and stirred for 50 h at room temperature. The solution was then
extracted three times with ether (10 mL). The combined extracts were dried over Na2SO4.
After removal of solvent by evaporation, the crude residue was purified by silica gel chromatography (20 % ethyl acetate in hexane, TLC: Rf = 0.2) to afford (1.1 g, 60%) of
1 pure 7.9. H NMR (CDCl3, 300 MHz) δ 5.77 (m, 1H), 5.0 (m, 2H), 2.9 (b, s, 3H), 2.77 (t,
J = 7.4 Hz, 2H), 2.05 (m, 2H), 1.6-1.4 (4H).
Preparation of 1-hex-5-enyl-5-pentyl-1H-pyrazole (7.5) and 1-hex-5-enyl-3-pentyl
-1H-pyrazole (7.10). Hydrazine 7.9 (495 mg, 4.35 mmol) was slowly added to a solution
234 of 7.7 (561 mg, 3.95 mmol) in ethanol (10 mL) and acetic acid (4 mL) under ice-cooling.
The mixture was heated for 24 h under reflux, then cooled, neutralized with saturated
NaHCO3 solution, and extracted with ethyl acetate. The ethyl acetate extract was washed with saturated NaHCO3 solution, dried with Na2SO4, and evaporated to dryness. The
crude residue was purified by silica gel chromatography (10 % ethyl acetate in hexane) to
1 give pure 7.5 (84 mg, Rf = 0.3, 10%) and pure 7.10 (98 mg, Rf = 0.35, 11 %). H NMR for
1 7.5 is displayed above. H NMR for 7.10 (CDCl3, 400 MHz) δ 7.18 (d, J = 2.4 Hz, 1H),
5.93 (d, J = 2.4 Hz, 1H), 5.71 (m, 1H), 4.95-4.85 (m, 2H), 3.98 (t, J = 7.2 Hz, 2H), 2.54 (t,
J = 7.6 Hz, 2H), 2.00 (m, 2H), 1.78 (m, 2H), 1.56 (m, 2H), 1.35-1.24 (6H), 0.82 (t, J =
7.2 Hz, 3H).
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241
Appendix
242 O O OMe
O O
1 Figure S1. H NMR (200 MHz, CDCl3) spectrum of methyl 6-(2,5-dioxolanyl)-4 -oxohexanoate (2.1).
O O OMe
O O
13 Figure S2. C NMR (50 MHz, CDCl3) spectrum of methyl 6-(2,5-dioxolanyl)- -4-oxohexanoate (2.1).
243 O O OH
O O
1 Figure S3. H NMR (200 MHz, CDCl3) spectrum of 6-(2,5-dioxolanyl)-4 -oxohexanoic acid (2.2).
O O OH
O O
13 Figure S4. C NMR (100 MHz, CDCl3) spectrum of 6-(2,5-dioxolanyl)-4- oxohexanoic acid (2.2).
244
O O O
O O
1 Figure S5. H NMR (200 MHz, CDCl3) spectrum of (2.3).
O O O O O
13 Figure S6. C NMR (50 MHz, CDCl3) spectrum of (2.3). C, CH2 (-); CH, CH3 (+).
245 O O O
O
1 Figure S7. H NMR (200 MHz, CDCl3) spectrum of DOHAFm (2.4).
O O O
O
13 Figure S8. C NMR (100 MHz, CDCl3) spectrum of DOHAFm (2.4).
246
H CO O 3 O H N N N H O O
O
1 Figure S9. H NMR (400 MHz, CDCl3) spectrum of (2.5).
H3CO O O H N N N H O O
O
13 Figure S10. C NMR (100 MHz, CDCl3) spectrum of (2.5).
247 O H H3CO O N N O N H
O O H
1 Figure S11. H NMR (400 MHz, CDCl3) spectrum of (2.6).
O H H3CO O N
N O N H
O O H
13 Figure S12. C NMR (150 MHz, CDCl3) spectrum of (2.6).
248 O N O 3
O OH
1 Figure S13. H NMR (400 MHz, CDCl3) spectrum of (2.9).
O N O 3
O OH
13 Figure S14. C NMR (100 MHz, CDCl3) spectrum of (2.9).
249 N O O
3 O O O N O
1 Figure S15. H NMR (400 MHz, CDCl3) spectrum of CEPFmSu (2.10).
N O O
3 O O O N O
13 Figure S16. C NMR (100 MHz, CDCl3) spectrum of CEPFmSu (2.10).
250
O
NH NH O H N O C N C S H O
1 Figure S17. H NMR (400 MHz, CD3OD) spectrum of (2.18).
O
NH NH O NH2 N S H
1 Figure S18. H NMR (200 MHz, CD3OD) spectrum of (2.19).
251 O
NH NH O N O
N O S H
1 Figure S19. H NMR (200 MHz, CDCl3) spectrum of (2.20).
O
NH NH O N N S H O
OH
1 Figure S20. H NMR (400 MHz, CD3OD) spectrum of (2.21).
252 O O OMe
O
1 Figure S21. H NMR (200 MHz, CDCl3) spectrum of (2.22).
O
NH NH O
N 4 NH2 S H
1 Figure S22. H NMR (400 MHz, CD3OD) spectrum of (2.23).
253 O
NH NH O
N 4 NH2 S H
13 Figure S23. C NMR (100 MHz, CD3OD) spectrum of (2.23).
O
NH NH O N N 4 S H O
OMe
1 Figure S24. H NMR (400 MHz, CD3OD) spectrum of (2.24).
254
O
NH NH O N N 4 S H O
OH
1 Figure S25. H NMR (400 MHz, CD3OD) spectrum of (2.25).
O
NH NH O N N 4 S H O
OH
13 Figure S26. C NMR (100 MHz, CD3OD) spectrum of (2.25).
255 O O CO O P O O 7 O C8H17 O N O C14H29 O
1 Figure S27. H NMR (400 MHz, CDCl3 : CD3OD = 1 : 1) spectrum of (2.26).
O O CO O P O O 7 O C8H17 O N O C14H29 O
13 Figure S28. C NMR (50 MHz, CDCl3 : CD3OD = 1 : 1) spectrum of (2.26).
256 O O CO O P O OH 7 O C8H17 O N O C14H29 O
1 Figure S29. H NMR (400 MHz, CDCl3 : CD3OD = 1 : 1) spectrum of (2.27).
O O CO O P O OH 7 O C8H17 O N O C14H29 O
13 Figure S30. C NMR (100 MHz, CDCl3:CD3OD = 1:1) spectrum of (2.27).
257 COO
O O P N HO O OH
O C14H29 O
1 Figure S31. H NMR (400 MHz, CDCl3 : CD3OD = 1 : 1) spectrum of (2.28).
COO
O O P N HO O OH
O C14H29 O
13 Figure S32. C NMR (400 MHz, CDCl3 : CD3OD = 1 : 1) spectrum of (2.28).
258 COOH
O O P N HO O OH
O C14H29 O
1 Figure S33. H NMR (400 MHz, CDCl3) spectrum of (2.29).
COOH
O O P N HO O OH
O C14H29 O
13 Figure S34. C NMR (100 MHz, CDCl3 :CD3OD:D2O = 50:50:1) spectrum of (2.29).
259
HO H O N O O O O N
1 Figure S35. H NMR (400 MHz, CDCl3) spectrum of (2.30).
HO H O N O O O O N
13 Figure S36. C NMR (100 MHz, CDCl3) spectrum of (2.30).
260
F F F F O F H O N O O O O N
1 Figure S37. H NMR (400 MHz, CDCl3) spectrum of (2.31).
F F
F F
O F H O N O O O O N
13 Figure S38. C NMR (100 MHz, CDCl3) spectrum of (2.31).
261 A
B
C
262 D
E
Figure S39. Tandem MS characterization of CEP modified HSA shows series of
fragment ions sufficient to identify CEP modifications on lysyl residues of the HSA peptides. (A). MS/MS spectrum of the doubly charged ion m/z 589.266. (B). MS/MS spectrum of the doubly charged ion m/z 674.762. (C). MS/MS spectrum of the doubly charged ion m/z 881.401. (D). MS/MS spectrum of the doubly charged ion m/z 687.784.
(E). MS/MS spectrum of the doubly charged ion m/z 636.252. Asterisks denote fragment ions with the modified lysyl residue.
263 A
B
C
264 D
E
F
265 G
H
Figure S40. Tandem MS characterization of CEP modified MSA. (A). MS/MS spectrum of the doubly charged ion m/z 650.3296. (B). MS/MS spectrum of the doubly charged ion m/z 1047.0802. (C). MS/MS spectrum of the doubly charged ion m/z 769.4069. (D).
MS/MS spectrum of the doubly charged ion m/z 612.8407. (E). MS/MS spectrum of the doubly charged ion m/z 872.9611. (F). MS/MS spectrum of the triplet charged ion m/z
849.4302. (G). MS/MS spectrum of the triplet charged ion m/z 857.4633. (H). MS/MS spectrum of the triplet charged ion m/z 694.6888. Asterisks denote fragment ions with the modified lysyl residue.
266 A
B
C
267 D
E
F
268 G
Figure S41. Tandem MS characterization of CEP modified CEO. (A). MS/MS spectrum
of the doubly charged ion m/z 540.2886. (B). MS/MS spectrum of the triplet charged ion
m/z 801.7021. (C). MS/MS spectrum of the doubly charged ion m/z 839.3332. (D).
MS/MS spectrum of the triplet charged ion m/z 808.0259. (E). MS/MS spectrum of the doubly charged ion m/z 610.2589. (F). MS/MS spectrum of the doubly charged ion m/z
630.8334. (G). MS/MS spectrum of the doubly charged ion m/z 459.1887. Asterisks
denote fragment ions with the modified lysyl residue.
269 A
B
C
270 D
E
271 F
Figure S42. Tandem MS characterization of CEP modified myoglobin. (A). MS/MS spectrum of the doubly charged ion m/z 671.2667. (B). MS/MS spectrum of the doubly charged ion m/z 604.9635. (C). MS/MS spectrum of the doubly charged ion m/z
815.2087. (D). MS/MS spectrum of the quartet charged ion m/z 557.4288. (E). MS/MS spectrum of the doubly charged ion m/z 785.6061. (F). MS/MS spectrum of the doubly charged ion m/z 899.6666. Asterisks denote fragment ions with the modified lysyl residue.
272 A
B
C
273 D
E
Figure S43. Tandem MS characterization of CEP modified GPDH. (A). MS/MS spectrum of the doubly charged ion m/z 655.3343. (B). MS/MS spectrum of the doubly charged ion m/z 669.3289. (C). MS/MS spectrum of the doubly charged ion m/z
923.9847. (D). MS/MS spectrum of the doubly charged ion m/z 639.3413. (E). MS/MS spectrum of the doubly charged ion m/z 740.8362. Asterisks denote fragment ions with the modified lysyl residue.
274 A
B
275 C
D
276 E
F
G
277 H
Figure S44. Tandem MS characterization of CEPH modified BSA. (A). MS/MS spectrum of the triplet charged ion m/z 594.9934. (B). MS/MS spectrum of the doubly charged ion m/z 541.8060. (C). MS/MS spectrum of the doubly charged ion m/z
937.9577. (D). MS/MS spectrum of the doubly charged ion m/z 526.7835. (E). MS/MS spectrum of the doubly charged ion m/z 612.3604. (F). MS/MS spectrum of the doubly charged ion m/z 689.4319. (G). MS/MS spectrum of the doubly charged ion m/z
765.3984. (H). MS/MS spectrum of the doubly charged ion m/z 964.5088. Asterisks denote fragment ions with the modified lysyl residue.
278 A
B
C
279 D
Figure S45. Tandem MS characterization of CEPH modified BSA. (A). MS/MS spectrum of the doubly charged ion m/z 512.2829. (B). MS/MS spectrum of the doubly charged ion m/z 627.8604. (C). MS/MS spectrum of the doubly charged ion m/z
608.8177. (D). MS/MS spectrum of the doubly charged ion m/z 732.3707. Asterisks denote fragment ions with the modified lysyl residue.
280
Figure S46. Tandem MS characterization of the doubly charged ion m/z 596.8715 from a
MS scan of tryptic digested CEPH modified CEO. Asterisks denote fragment ions with the modified lysyl residue.
281 A
B
C
282 D
E
F
283 G
H
I
284 J
K
L
285 M
Figure S47. Tandem MS characterization of CEPH modified GPDH. (A). MS/MS spectrum of the doubly charged ion m/z 635.0367. (B). MS/MS spectrum of the doubly charged ion m/z 712.5500. (C). MS/MS spectrum of the doubly charged ion m/z
919.7650. (D). MS/MS spectrum of the doubly charged ion m/z 572.9763. (E). MS/MS spectrum of the triplet charged ion m/z 899.6923. (F). MS/MS spectrum of the doubly charged ion m/z 726.0422. (G). MS/MS spectrum of the doubly charged ion m/z
980.7797. (H). MS/MS spectrum of the doubly charged ion m/z 731.1005. (I). MS/MS spectrum of the doubly charged ion m/z 941.7422. (J). MS/MS spectrum of the triplet charged ion m/z 579.9560. (K). MS/MS spectrum of the doubly charged ion m/z
508.9000. (L). MS/MS spectrum of the doubly charged ion m/z 696.1018. (M). MS/MS spectrum of the doubly charged ion m/z 797.5823. Asterisks denote fragment ions with the modified lysyl residue.
286 A
9 8 7 6 5 4 3 1 y
D A H K S E V A H R
b 3 4 6 7
B
14 11 10 9 8 y
K V P Q V S T P T L V E V S R
b 2 5
287 C 8 7 6 5 4 y
A F K A W A V A R b 3
10 9 8 7 y D F P K A E F A E V S K
b 3 5 8
288 E 10 9 8 7 y F K D L G E E N F K
b 3
Figure S48. Tandem MS characterization of CEPFmHSA revealed five CEP modifications. (A). MS/MS spectrum of the doubly charged ion m/z 636.248. (B).
MS/MS spectrum of the doubly charged ion m/z 881.408. (C). MS/MS spectrum of the doubly charged ion m/z 571.262. (D). MS/MS spectrum of the doubly charged ion m/z
687.787. (E). MS/MS spectrum of the doubly charged ion m/z 674.765.
289
A
9 8 7 6 5 4 1 y
D A H K S E V A H R
b 3 4 5 6 7
B
8 6 5 4 3 2 y
A F K A W A V A R
b 4
290
C 10 9 8 7 6 5 3 y
F P K A E F A E V S K
b 2
Figure S49. Tandem MS characterization of CEPFmHSA revealed five CEPFm modifications. (A). MS/MS spectrum of the doubly charged ion m/z 725.281. (B).
MS/MS spectrum of the doubly charged ion m/z 660.289. (C). MS/MS spectrum of the doubly charged ion m/z 776.820.
291 Table S1. Total iron levels of patients treated with vitamin Eα or placebo at baseline, three months, six months and nine months. Patient code VE treatment/ Baseline 3 months 6 months 9 months placebo P1 placebo 67 54 57 53 T1 treatment 57 48 41 60 T2 treatment 43 60 34 41 P2 placebo 50 42 62 75 P3 placebo 43 39 65 P4 placebo 42 39 41 61 T3 treatment 48 54 69 98 T4 treatment 60 46 62 P5 placebo 62 112 74 69 T5 treatment 49 48 53 25 T6 treatment 37 64 71 27 P6 placebo 52 58 62 27 P7 placebo 69 64 74 93 P8 placebo 70 64 57 56 T7 treatment 72 107 98 T8 treatment 58 89 56 46 T9 treatment 47 57 42 65 P9 placebo 41 46 45 42 P10 placebo 69 110 55 91 T10 treatment 49 32 38 28 T11 treatment 95 81 45 68 T12 treatment 50 60 53 60 P11 placebo 70 52 66 65 P12 placebo 59 68 83 86 T13 treatment 60 61 52 52 T14 treatment 106 52 128 P14 placebo 46 122 131
292 Table S2. Erythropoietin administrated in patients treated with vitamin Eα or placebo at baseline, three months, six months and nine months. Patient VE treatment Baseline 3 months 6 months 9 months code /placebo P1 placebo 37200 16800 23200 33600 T1 treatment 345600 294000 198000 241200 T2 treatment 51600 44400 42000 96000 P2 placebo 252000 271200 324000 295200 P3 placebo 100800 51600 64800 49200 P4 placebo 284400 622800 752400 924000 T3 treatment 19600 151800 78100 278400 T4 treatment 702000 628200 399600 P5 placebo 644400 577200 432000 505200 T5 treatment 276000 115200 124800 192000 T6 treatment 142800 110400 90000 120500 P6 placebo 160800 120000 120000 177600 P7 placebo 104400 37200 32400 27600 P8 placebo 175200 87600 45600 36000 T7 treatment T8 treatment 165600 147600 60000 68400 T9 treatment 250800 306000 385200 397200 P9 placebo 468000 518400 367200 368400 P10 placebo T10 treatment 993600 993600 993600 1009200 T11 treatment 159600 241200 406800 334800 T12 treatment 86400 68400 97200 112800 P11 placebo 18000 8400 17200 75600 P12 placebo 50400 25200 8000 22000 T13 treatment 109200 90000 90000 79200 T14 treatment 20400 60000 26100 P14 placebo 98400 139800 91200
293 Table S3. Ferritin levels of patients treated with vitamin Eα or placebo at baseline, three months, six months and nine months. Patient VE treatment Baseline 3 months 6 months 9 months code /placebo P1 placebo 405 506 469 345 T1 treatment 92 105 205 107 T2 treatment 809 525 386 2271 P2 placebo 523 94 159 280 P3 placebo 414 265 481 P4 placebo 245 189 92 124 T3 treatment 837 287 837 306 T4 treatment 748 610 793 P5 placebo 493 729 793 212 T5 treatment 1092 1543 617 536 T6 treatment 738 1131 536 841 P6 placebo 563 553 774 379 P7 placebo 256 406 661 794 P8 placebo 1268 487 408 273 T7 treatment 215 403 377 T8 treatment 517 1077 424 289 T9 treatment 417 299 165 558 P9 placebo 70 190 171 191 P10 placebo 464 693 877 822 T10 treatment 820 742 706 1029 T11 treatment 740 587 310 580 T12 treatment 850 303 314 633 P11 placebo 598 694 741 745 P12 placebo 212 240 435 396 T13 treatment 500 407 360 462 T14 treatment 796 789 737 P14 placebo 167 459 653
294
O OH
C5H11 CO2Et
1 Figure S50. H NMR (300 MHz, CDCl3) spectrum of (7.1).
O OH
C5H11 CO2Et
13 Figure S51. C NMR (75 MHz, CDCl3) spectrum of (7.1).
295 CO2Et
N C5H11 N H
1 Figure S52. H NMR (300 MHz, CDCl3) spectrum of (7.2).
CO2Et
N C5H11 N H
13 Figure S53. C NMR (75 MHz, CDCl3) spectrum of (7.2).
296 I-(CH2)4CH=CH2
1 Figure S54. H NMR (300 MHz, CDCl3) spectrum of (7.3).
COOH
N C5H11 N (CH2)4CH=CH2
1 Figure S55. H NMR (300 MHz, CDCl3) spectrum of (7.4).
297
COOH
N C5H11 N (CH2)4CH=CH2
13 Figure S56. C NMR (75 MHz, CDCl3) spectrum of (7.4).
N C5H11 N (CH2)4CH=CH2
1 Figure S57. H NMR (400 MHz, CDCl3) spectrum of (7.5).
298 N C5H11 N (CH2)4CH=CH2
13 Figure S58. C NMR (100 MHz, CDCl3) spectrum of (7.5).
COOH
N C5H11 N (CH2)4CH=CH2
Figure S59. HMQC of 3-carboxyl-5-pentyl pyrazole (7.4).
299 COOH
N C5H11 N (CH2)4CH=CH2
Figure S60. HMBC of 3-carboxyl-5-pentyl pyrazole (7.4). O OH
C H C H 5 11 H
1 Figure S61. H NMR (300 MHz, CDCl3) spectrum of (7.7).
300 Br-(CH2)4CH=CH2
1 Figure S62. H NMR (300 MHz, CDCl3) spectrum of (7.8).
NH2-NH-(CH2)4CH=CH2
1 Figure S63. H NMR (300 MHz, CDCl3) spectrum of (7.9).
301 C5H11
N N
(CH2)4CH=CH2
1 Figure S64. H NMR (400 MHz, CDCl3) spectrum of (7.10).
302
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