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Photooxidation Chemistry and Photodynamic Therapy: Pointsource Delivery of Singlet Oxygen, Sensitizer and Nitrosamine Drugs
Ashwini Anil Ghogare Graduate Center, City University of New York
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PHOTOOXIDATION CHEMISTRY AND PHOTODYNAMIC THERAPY: POINTSOURCE DELIVERY OF SINGLET OXYGEN, SENSITIZER AND NITROSAMINE DRUGS
by
ASHWINI ANIL GHOGARE
A dissertation submitted to the Graduate Faculty in Chemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The City University of New York
2016
ii
2016
ASHWINI ANIL GHOGARE
All Rights Reserved
iii
This manuscript has been read and accepted for the Graduate Faculty in Chemistry in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy.
PROFESSOR ALEXANDER GREER
Date Chair of Examining Committee
April 7, 2016 PROFESSOR BRIAN R. GIBNEY
Date Executive Officer
PROFESSOR WAYNE W. HARDING
Supervision Committee
PROFESSOR DAVID R. MOOTOO
Supervision Committee
THE CITY UNIVERSITY OF NEW YORK
iv
ABSTRACT
Photooxidation Chemistry and Photodynamic Therapy: Pointsource Delivery of
Singlet Oxygen, Sensitizer and Nitrosamine Drugs
by
Ashwini Anil Ghogare
Advisor: Professor Alexander Greer
Eradication of residual tumor cells that are directly adjacent to vital tissue is a daunting challenge to surgeons. Because the field needs advances in intraoperative settings and a means for high-precision delivery of singlet oxygen for photodynamic therapy (PDT) of cancers, this dissertation outlines the development and application of a “pointsource” fiber optic device. The device offers highly localized and simultaneous delivery of sensitizer drug, light, and oxygen
(components necessary for PDT) for cancer cell eradication in-vitro and in-vivo. The following chapters describe (a) the photokilling activity and precision of pointsource PDT in monolayer ovarian and brain cancer cells in-vitro by creating a halo of singlet oxygen, (b) minimal biomaterial fouling on the pace of sensitizer drug photorelease into in-vivo tumors of the head and neck cancer, and (c) synthesis of PEGylated pheophorbide sensitizers to enhance their bio-availability and uptake in cancer cells. (d) With the view of delivering nitrosamine drugs alongside singlet oxygen for dual chemo-photodynamic therapy, the photooxidation mechanism in N-nitrosamines was studied. 18O-isotopic labelling, photochemistry, tandem mass spectrometry and DFT calculations were utilized and an 18O label scrambling into aromatic but not aliphatic N-nitrosamine drugs from v
18 molecular O2, was discovered. The oxygen atom exchange mechanism was proposed to occur by nitrosoperoxy intermediates and might provide a clue to new factors significant in nitrosamine
1 phototoxicity. (e) Lastly, a review of the literature is presented on using singlet oxygen ( O2) to synthesize natural products and drugs that intends to draw a logical link between flow and batch
1 reactions in the current state of O2 in synthesis.
vi
DEDICATION
This dissertation is dedicated to my loving parents, Sheela and Anil Ghogare, my beloved sisters,
Prachi and Manasi Ghogare, and my husband, Mahendran Adaickapillai. I am truly blessed to
have all of you in my life.
vii
ACKNOWLEDGMENTS
This doctoral dissertation was made possible with the contribution, mentorship and support from many people. First and foremost, I want to express my immense appreciation and gratitude to my advisor, Dr. Alexander Greer. He has been a good friend and a great mentor throughout the course of my doctoral research. His strong belief in my potential and his constant encouragement, motivated me to achieve my goals. He introduced me to the field of photochemistry and photobiology and gave me the freedom to explore my scientific curiosity which were key in shaping me as a scientific researcher. I have been inspired by his perseverance and passion for exploring new scientific ideas and I want to thank him for giving me the opportunity to work on many interdisciplinary research projects under his guidance.
I want to thank members of my thesis committee, Dr. Wayne W. Harding and Dr. David
R. Mootoo for their guidance and insightful comments during the committee meetings which helped to widen the scope of my research. I am grateful to many friends and colleagues, who made my time enjoyable at Brooklyn College and the Graduate Center. The past and present members of the Greer group and other groups in the Chemistry Department have contributed immensely to my personal and professional growth and have been a great source of support throughout my doctoral research.
I was fortunate to work with some great collaborators during the course of my dissertation.
I want to thank Dr. Imran Rizvi and Dr. Tayyaba Hasan for giving me the opportunity to work in their laboratory at the Wellman Center for Photomedicine, Massachusetts General Hospital,
Boston. It was a very enriching experience for me as a doctoral student in organic chemistry to learn new techniques in cancer biology and photodynamic therapy. viii
I would like to thank Joann M. Miller, Dr. Keith A. Cengel and Dr. Theresa M. Busch from
Department of Radiation Oncology, University of Pennsylvania, PA for their efforts in developing head and neck cancer model in mice and conducting all the in-vivo procedures. I would also like to thank Dr. Bikash Mondal and Dr. Alan M. Lyons from the Department of Chemistry, College of Staten Island, CUNY for their efforts with XPS analysis of proteins on silica surface.
I would like to thank Dr. Marilene Silva Oliveira, Fernanda Manso Prado and Dr. Paolo Di
Mascio from Departamento de Bioquímica, Instituto de Química, Universidade de Sao Paulo, Sao
Paulo for their collaborative efforts in the HPLC-MS/MS and HRMS analysis, and Dr. Edyta M.
Greer from Department of Natural Sciences, Baruch College, CUNY for the computational calculations, on nitrosamine compounds.
I gratefully acknowledge the generous financial support through the CUNY Science
Scholarship 2010-2015 and Doctoral Dissertation Year Award 2015-2016 from the Graduate
Center of the City University of New York. This dissertation was also supported by the grants from National Institute of Health (NIH) and National Science Foundation (NSF).
Finally I want to thank the most important people in my life, my family. They have supported me and encouraged me during the challenges of graduate school and throughout my life.
Their unwavering faith and confidence in my abilities have inspired me to strive towards fulfilling my dreams. Especially my dad, who motivated me to pursue doctoral studies and Dr. Mahendran
Adaickapillai who helped me to complete this endeavour in innumerous ways. Thank you very much!
ix
TABLE OF CONTENTS
Page
TITLE i
APPROVAL PAGE iii
ABSTRACT iv
DEDICATION vi
ACKNOWLEDGEMENT vii
TABLE OF CONTENTS ix
LIST OF SYMBOLS AND ABBREVIATIONS xv
LIST OF FIGURES xix
LIST OF SCHEMES xxiv
LIST OF TABLES xxix
Chapter 1. Introduction
1.1 Photooxidation Chemistry and Photodynamic Therapy (PDT) 1
1.2 Sections in the Thesis 3
1.3 References 5
Chapter 2. “Pointsource” Delivery of a Photosensitizer Drug and Singlet
Oxygen: Eradication of Glioma Cells In-vitro
2.1 Introduction 7
2.2 Results and Discussion 9
2.2.1 Cell Phototoxicity and Sensitizer Uptake 9 x
2.2.2 Course of the Sensitizer Drug Photorelease in Phosphate 11
Buffered Saline (PBS)
2.2.3 Fiber Tip-Guided Sensitizer Delivery for Cell Killing in 12
Discrete Locations
2.2.4 “Lengthening” the Toxic Radius of Singlet Oxygen 13
2.2.5 Mechanism 15
2.3 Conclusion 16
2.4 Experimental Section 17
2.4.1 Materials and Methods 17
2.4.2 Device Fabrication and Instruments 18
2.4.3 Sensitizer Photorelease in Phosphate Buffered Saline (PBS) 19
2.4.4 Cellular Uptake in U87 MG Cell Monolayer 20
2.4.5 Phototoxicity of Pyropheoporbide-a in U87 MG Cell 21
Monolayer
2.4.6 Treatment Procedure 21
2.4.7 Sources of Error 22
2.5 References 23
Chapter 3. Fluorinated PDT Device Tips and Their Resistance to Fouling for In-
vivo Sensitizer Release
3.1 Introduction 25
3.2 Results 27
3.2.1 Effects of Biofouling on Device Tip Sensitizer Release 28
3.2.2 Effect of Adsorption of Cellular Material 31 xi
3.3 Discussion 34
3.4 Conclusion 36
3.5 Experimental Section 36
3.5.1 Materials and Methods 36
3.5.2 Sensitizer Photorelease Studies 37
3.5.3 BCA Studies 38
3.5.4 XPS Studies 39
3.6 References 39
Chapter 4. Synthesis of an Poly(ethylene glycol) Galloyl Sensitizer Tip for a
Pointsource Photodynamic Device
4.1 Introduction 42
4.2 Results and Discussion 44
4.2.1 Sensitizer Design and Solubility 44
4.2.2 Synthesis 45
4.2.3 Structural Assignments 47
4.2.4 Comparative Analysis 50
4.3 Conclusion 52
4.4 Experimental Section 53
4.4.1 Computations 53
4.4.2 Materials and Methods 53
4.4.3 Synthesis of TriPEG-galloyl (8) 54
4.4.4 Synthesis of TriPEG-galloyl-ether-31-pyropheophorbide Methyl 55
Ester (10) xii
4.4.5 Synthesis of TriPEG-galloyl-ether-31-pyropheophorbide 56
Carboxylic Acid (11)
4.4.6 Synthesis of TriPEG-galloyl-ether-31-pyropheophorbide-(Z)- 57
alkene Ester (13)
4.4.7 TriPEG-galloyl Pheophorbide Modified Fluorinated Silica (1) 58
4.4.8 Hydrolytic Stability 58
4.5 References 85
Chapter 5. Mechanism of Photochemical O-Atom Exchange in Nitrosamines
with Molecular Oxygen
5.1 Introduction 88
5.2 Results and Discussion 91
5.2.1 Direct Excitation of Aromatic and Aliphatic Nitrosamines in the 92
18 Presence of Isotopically Labeled Molecular Oxygen ( O2)
5.2.2 Nitrosamine Photolytic Instability 94
5.2.3 Effects of Added Radical Scavenger Butylated Hydroxytoluene 95
18 5.2.4 Photo-oxygen Exchange by Adventitious H2 O is Ruled Out 96
18 18 5.2.5 Experiments with O-Labeled Singlet Oxygen ( O2) 96
5.2.6 DFT Computed Bond Dissociation Energies 97
5.2.7 Proposed Mechanism 98
5.3 Conclusion 100
5.4 Experimental Section 101
5.4.1 Materials and Methods 101
5.4.2 18O-Photoexchange Reactions 102 xiii
5.4.3 UV Photolysis of N-nitrosodiphenylaniline (1) 102
5.4.4 UV Photolysis of N-nitroso-N-methylaniline (2) 103
5.4.5 UV Photolysis of N-butyl-N-(4-hydroxybutyl)nitrosamine (3) 103
and Nitrosodiethylamine (4)
5.4.6 Photochemical and Chemical Generation of Singlet Oxygen 103
18 1 [ ( O2)]
5.4.7 Computational Methods 104
5.5 References 122
Chapter 6. Literature Review of the Use of Singlet Oxygen to Synthesize Natural
Products and Drugs
6.1 Introduction and Background 125
6.2 Scope of the Review 128
6.3 Singlet Oxygen in Synthesis 129
6.3.1 Background 129
6.3.2 Endoperoxides 131
6.3.3 Carbasugars 135
6.3.4 Epoxides 140
6.3.5 Tropones and Tropolones 145
6.3.6 Polycyclic Ethers and Polyols 149
6.3.7 Sterols 154
6.3.8 Opioids 157
6.3.9 Ring-Fused Examples 159
6.3.10 Phenols 163 xiv
6.3.11 Self-Sensitized Examples 171
6.3.12 Indoles 173
6.3.13 Lactams and Related Examples 178
6.4 Singlet Oxygen in Flow Synthesis 184
6.4.1 Background Information 184
6.4.2 Singlet Oxygen Microphotoreactors 194
6.4.2.1 Microphotoreactors with Solution-Phase Sensitizer 194
6.4.2.2 Microphotoreactors with Immobilized Sensitizers 196
6.4.3 Singlet Oxygen Macrophotoreactors 200
6.4.3.1 Macrophotoreactors with Solution-Phase Sensitizer 200
1 6.4.4 Supercritical Carbon Dioxide O2 Photoreactor 203
6.4.4.1 Supercritical Carbon Dioxide Photoreactors Using 204
Dissolved Sensitizer
6.4.4.2 Supercritical Carbon Dioxide Photoreactor Using an 204
Immobilized Sensitizer
6.4.5 Bubbling Photoreactors 205
6.4.5.1 Bubbling Photoreactor Using a Dissolved Sensitizer 206
6.4.5.2 Bubbling Photoreactor Using a “Shielded” 206
Heterogeneous Sensitizer
6.5 Prospectives 207
1 6.5.1 State of O2 Synthetic Science 207
6.6 Summary and Outlook 208
6.7 References 209 xv
LIST OF SYMBOLS AND ABBREVIATIONS
13C NMR carbon-13 nuclear magnetic resonance
1H NMR proton nuclear magnetic resonance
1 1 1 O2 excited state O2 ( ∆g)
3 3 3 — O2 ground-state O2 ( Σg )
Å angstrom
Ac acetyl
Ac2O acetic anhydride
API active pharmaceutical ingredient
B3LYP/6-31G (d) Becke-style-Parameter Density Functional Theory
BANT bisacenapthalenethiophene
BMP p-biphenylmethyl
Bz benzyl
Boc t-butyloxycarbonyl
BTIB [bis(trifuloroacetoxy)iodo]-benzene calcd calculated d doublet
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
DCM Dichlorometane
DFT Density Functional Theory
DIPEA diisopropylethylamine
DMAP 4-(dimethylamino)pyridine xvi
DMF N,N-dimethylformamide
DPBF 1,3-diphenylisobenzofuran
DTBN di-t-butyl hyponitrite
Et2O diethyl ether
EtOAc ethyl acetate
FEP fluorinated ethylene propylene
GC gas chromatography
GCMS gas chromatography mass spectroscopy h hour
HPLC high pressure liquid chromatography
Hz hertz i.d. inner diameter
J coupling constant kcal kilocalorie
L liter
LAH lithium aluminum hydride
LDA lithium diisopropylamide, m multiplet m-CPBA m-chlorobenzoic acid
MB methylene blue
MCF microcapillary film
MeOH methanol mg milligram xvii min minute mL milliliter mmol millimole
MOM methoxymethyl
Ms methanesulfonyl
MTAD N-methyltriazolinedione
NBO natural bond order o.d. outer diameter oC degree Celsius
OLED organic light emitting diode p-TsOH p-toluenesulfonic acid
Pc phthalocyanine
PCC pyridinium chlorochromate
PDC pyridinium dichromate
PDMS polydimethylsiloxane
PDT photodynamic therapy pH potential of hydrogen pKa ionization constant
PNB p-nitrobenzylester ppm parts per million
PPTS pyridinium p-toluenesulfonate
PTAD N-phenyltriazolinedione
PTFE polytetrafluoroethylene xviii
Py pyridine q quartet
RB rose bengal rt room temperature s singlet scCO2 supercritical CO2
STY space-time yield t triplet
TBAF tetra-n-butylammonium fluoride
TBDPS t-butyldiphenylsilyl
TBS t-butyldimethylsilyl
TDCPP tetra-2,6-chlorophenyl porphyrin
TES triethylsilyl
TFA trifuloroacetic acid
TG Tentagel
THF tetrahydrofuran
TIPS triisopropylsilyl ether
TMEDA tetramethyl ethylenediamine
TMSCN trimethylsilyl cyanide
TMSI trimethylsilyl iodide
TPP tetraphenylporphyrin
TS transition state
chemical shift in ppm xix
LIST OF FIGURES
Chapter 1
Figure Page
1. Schematic presentation of the “pointsource” fiberoptic PDT device 3
and functionalized device tips 4-7.
Chapter 2
Figure Page
1. Pointsource device for targeted singlet oxygen delivery. 8
2. Comparison of sensitizer uptake efficiency in the dark after 1 h (light red bars) 10
with phototoxicity effects on U87 cells treated by a bare-tipped device devoid
of sensitizer, but sparging O2 with red light irradiation with externally added
pheophorbide (dark blue bars).
3. A time-sequence analysis of pheophorbide photoreleased free from the probe 11
tip of the device in PBS at 25 °C (solid circles) and fluence from the tip (solid
triangles).
4. Time-sequence analysis of phototoxicity effects on U87 cells in 14 mm 13
diameter microwell experiments treated with the device tip (solid circles) and
fluence from the tip (solid squares).
5. Device tip treatment of a U87 cell monolayer (154 mm2 area) revealed a radius 14
of photokilling as a function of time.
xx
Chapter 3
Figure Page
1. Schematic presentation of the pointsource PDT technique with various device 26
tips.
2. A schematic of the cylindrical device tip held vertically, through an incision 27
revealing the SQ20B tumor on the flank of a nude mouse.
3. The percent of sensitizer 4 photoreleased free from device tip 1 (black lines) 30
and tip 2 (grey lines) in 1 mL n-butanol.
4. A plot of remaining sensitizer 4 photoreleased into n-butanol vs time for tip 1 31
(■) and tip 5 (●).
5. A plot of blood cells adsorbed to tip 6 (■) and tip 7 (●) vs time when 32
immersed in blood.
6. Time profile for XPS peak area ratio changes of C 1s/Si 2p (▲), N 1s/Si 2p 33
(●), and C 1s/N 1s (■) of tip 6 immersed in whole blood.
7. XPS spectra of clean silica 6 and silica 6 contaminated by whole blood. 34
Chapter 4
Figure Page
1. Chemical structures of the new triPEG-galloyl pheophorbide silica 1, and 44
previously reported triPEG-chlorin silica 2 and triPEG-chlorin Teflon/PVA
nanocomposite 3.
2. 1H NMR spectra for the pheophorbide methylester 9 and triPEG-galloyl 49
pheophorbide methylester 10.
3. 1H NMR spectrum of the triPEG-galloyl pheophorbide alkene ester 13. 49 xxi
4. Scheme of pointsource PDT device. 52
1 5. H NMR spectrum of compound 8 in CDCl3. 59
13 6. C NMR spectrum of compound 8 in CDCl3. 60
1 7. H NMR spectrum of compound 10 in CDCl3. 61
13 8. C NMR spectrum of compound 10 in CDCl3. 62
9. HSQC spectra of compound 10 in CDCl3. 63
10. HMBC spectra of compound 10 in CDCl3. 64
11. Expanded HMBC spectra of compound 10 in CDCl3. 65
12. COSY spectra of compound 10 in CDCl3. 66
13. HRMS of compound 10. 67
14. HPLC spectrum of compound 10 in 5% (v/v) water in acetonitrile. 68
1 15. H NMR spectrum of compound 11 in CDCl3. 69
13 16. C NMR spectrum of compound 11 in CDCl3. 70
17. HSQC spectra of compound 11 in CDCl3. 71
18. HMBC spectra of compound 11 in CDCl3. 72
19. COSY spectra of compound 11 in CDCl3. 73
20. HRMS of compound 11. 74
21. HPLC spectrum of compound 11 in 5% (v/v) water in acetonitrile. 75
1 22. H NMR spectrum of compound 13 in CDCl3. 76
13 23. C NMR spectrum of compound 13 in CDCl3. 77
24. HSQC spectra of compound 13 in CDCl3. 78
25. HMBC spectra of compound 13 in CDCl3. 79
26. Expanded HMBC spectra of compound 13 in CDCl3. 80 xxii
27. COSY spectra of compound 13 in CDCl3. 81
28. HRMS of compound 13 in CDCl3. 82
29. HPLC spectrum of compound 13 in 5% (v/v) water in acetonitrile. 83
30. UV-Vis spectra of compounds 9, 10, 11 and 13 (20 µM) in chloroform. 84
Chapter 5
Figure Page
1. HPLC-MS/MS analysis of (A) 1 prior to photolysis, and of (B) 1 with the 18O- 94
label photochemically introduced.
2. HPLC/MS isotopic abundances of nitrosamine reagent [M+H]+ (▲) and 18O- 95
labeled nitrosamine product [(M+2)+H]+ (■) as a function of photolysis time
18 of 1 (A) and 2 (B) with O2 in acetonitrile-d3.
18 3. HPLC/MS analysis of the photoreactions of 1-4 with O2. 106
18 4. HPLC-MS/MS analysis of 1 before and after photolysis with O2. 107
18 5. HPLC-MS/MS analysis of the photoreaction of 1 with O2. 108
18 6. HPLC/MS analysis of 1 before and after photolysis with O2. 109
18 7. HRMS analysis of the photoreaction of 1 with O2. 110
18 8. HPLC-MS/MS analysis of the thermal reaction of 1 with DHPN O2. 111
18 9. HPLC-MS/MS analysis of 2 before and after photolysis with O2. 112
18 10. HPLC-MS/MS analysis of the photoreaction of 2 with O2. 113
18 11. HPLC/MS analysis of 2 before and after photolysis with O2. 114
18 12. HRMS analysis of the photoreaction of 2 with O2. 115
18 13. HPLC-MS/MS analysis of the photoreaction of 3 with O2. 116
18 14. HPLC/MS analysis of 3 before and after photolysis with O2. 117 xxiii
18 15. HPLC-MS/MS analysis of the photoreaction of 3 with O2 with BHT. 118
18 16. HPLC-MS/MS analysis of the photoreaction of 4 with O2. 119
18 17. HPLC/MS analysis of 4 before and after photolysis with O2. 120
18. Absorption spectra of 1-4. 121
Chapter 6
Figure Page
1 1. A microflow system capable of generating O2 in micrometer sized channels. 192
1 2. A macroflow system capable of generating O2 in a millimeter sized channel. 192
1 3. Murray’s gaseous O2 photoreactor device. 193
xxiv
LIST OF SCHEMES
Chapter 2
Scheme Page
1 1. Sensitizer drug and O2 delivery and glioblastoma cell killing mechanism 16
Chapter 4
Scheme Page
1. Self-sensitization, singlet oxygen formation and dye cleavage 43
2. Synthesis of the triPEG galloyl compound 8 46
3. Synthesis of triPEG-galloyl pheophorbide silica 1 47
Chapter 5
Scheme Page
1. Speculated intermediates in a O-atom exchange reaction between singlet 88
1 oxygen ( O2) and N-nitrosamine
2. Photo 18O exchange process and generation of 18O-labeled nitrosamines 91
3. Proposed 18O exchange mechanism for generating the 18O-labeled 92
nitrosamine
18 18 1 4. Thermal decomposition of DHPN O2 to generate ( O2) 97
Chapter 6
Scheme Page
1 1. Synthetic utility of O2 for generating oxygenated hydrocarbons 127
2. Generation of singlet oxygen 128
3. Schematic of type I/II photooxidation reactions 130 xxv
4. Synthesis of marine sponge endoperoxide isomers 132
5. Synthesis of (±)-6-epiplakortolide E 133
6. Synthesis of (±)-chondrillin, (±)-plakorin and a stannyl alkenyl 134
hydroperoxide
7. Synthesis of DL-talo-quercitol and DL-vibo-quercitol 135
8. Synthesis of DL-proto- and DL-gala-quercitol and a gala-aminoquercitol 136
derivative
9. Synthesis of a DL-tetrol and a DL-pentaol 137
10. Synthesis of isomeric heptols 138
11. Synthesis of 5a-carba-6-deoxy-α-DL-galacto-heptopyranose and 5a-carba-6- 139
deoxy-α-DL-gulo-heptopyranose
12. Synthesis of DL-bis-homoinositol 140
13. Synthesis of elysiapyrone A and B 141
14. Synthesis of (±)-limonin 142
15. Synthesis of fusicogigantepoxides and fusicogigantones 143
16. Synthesis of a triptolide derivative 144
17. Photooxidation of isocolchicine atropisomers 146
18. Photooxidation of (-)-colchicine 147
19. Synthesis of tropolone compounds 148
20. Synthesis of (±)-clavularin A and B 149
21. A singlet oxygen reaction in the synthesis of brevetoxin A 150
22. Synthesis of polycyclic ethers 151
23. Synthesis of milbemycin E 152 xxvi
24. Synthesis of the C29-C46 subunit of oasomycin A 153
25. Synthesis of 5,6-dihydro-glaucogenin C 155
26. Synthesis of withanolide A 156
27. Synthesis of imidazoline steroid mimic 157
28. Photooxidation of thebaine 158
29. Synthesis of (R)-methylnaltrexone 159
30. Synthesis of a tashironin-like compound 160
31. Synthesis of (±)-phomactin A 161
32. Synthesis of epoxyxanthatin endoperoxides 162
33. Synthesis of (±)-oxytridachiahydropyrone 162
34. Synthesis of tigliane and daphnane-type compounds 164
35. Synthesis of (-)-adunctin E 165
36. Synthesis of the BCE ring structure of ryanodine 166
37. Synthesis of moracin M 167
38. Synthesis of vanillin, dehydroisoeugenol and derivatives 168
39. Synthesis of kuwanons I and J 169
40. Photooxidation of prenylated coumarins and xanthones 170
41. Synthesis of (-)-balanol 171
42. Synthesis of vineomycinone B2 methyl ester 172
43. Synthesis of oxindoles 173
44. Synthesis of the natural product (-)-CJ-12662 175
45. Synthesis of (+)-okaramine N 176
46. Synthesis of (-)-melohenine B 177 xxvii
47. Synthesis of Antibiotic (±)-PS-5 178
48. Synthesis of pandamarine 179
49. Synthesis of pandamarilactone-1 180
50. Synthesis of PI-091 181
51. Synthesis of a tetracyclic compound similar to the natural product 182
erysotramidine
52. Synthesis DL and meso isochrysohermidin 183
53. Synthesis of a 1,5-dihydro-pyrrol-2-one 183
54. Flow reactor photooxidation of α-terpinene 185
55. Flow reactor photooxidation of α-pinene 185
56. Flow reactor photooxidation of (-)-β-citronellol 185
57. Flow reactor photooxidation of cyclopentadiene 186
58. Flow reactor photooxidation of alkenes 186
59. Flow reactor photooxidation of 1,5-dihydroxynaphthalene and phenol 187
60. Flow reactor photooxidation of 2-(3-methoxyphenyl)-3-methyl-1-benzofuran 187
61. Flow reactor photooxidation of 9,10-dimethylanthracene and 9,10- 188
anthracene dipropionate dianion
62. Flow reactor photooxidation of cholesterol 189
63. Flow reactor photooxidation of methionine derivatives and an organic 189
sulfide
64. Flow reactor photooxidation of dihydroartemisinic acid 190
65. Flow reactor photooxidation of amines 190
66. Flow reactor photooxidation of furan derivatives 191 xxviii
67. Structure of the glass-attached porphyrin 197
68. Structure of a silica-attached porphyrin 198
69. Structures of the tentagel-supported 199
70. Synthesis of paracaseolide A 203
71. Structures of the immobilized sensitizers in flow devices 205 xxix
LIST OF TABLES
Chapter 3
Table Page
1. Tumor- or blood-contact dependence of the photorelease of sensitizer 4 from 29
device tips 1 and 5 into n-butanol
2. Blood and tumor cell adsorption to fluorinated silica 6 and native silica 7 32
surfaces
Chapter 4
Table Page
1. Computed octanol-water partition coefficients (log P) using the ACD 45
program
2. Comparative analysis of synthetic, stability and materials data 51
Chapter 5
Table Page
1. HPLC/MS data of the nitrosamine percent 18O exchange and decomposition 93
18 in the presence of O2 in CHCl3
2. Energetics and parameters of nitrosamines 1-4 with DFT and TD-DFT 98
calculations
3. Experimental conditions and percent abundances of isotopes of 18O 105
photoexchanged into nitrosamines 1-4 1
Chapter 1. Introduction
1.1 Photooxidation Chemistry and Photodynamic Therapy (PDT)
Radiation, chemotherapy and surgery have been the mainstays of treatment for cancer, 1-3 but the eradication of residual tumor cells near vital organs remains a major challenge to
4-6 1 surgeons. Photodynamic therapy (PDT), where singlet oxygen ( O2) is a key cytotoxic agent, is a successful treatment regimen. But treatment of tumors by conventional PDT is a two-step process that involves the systemic delivery of the photosensitizer followed by local illumination with a sensitizer-exciting wavelength of light.7-9 We have used basic concepts in organic and photochemistry to develop a one-step process for local delivery of sensitizer drug, oxygen and illumination in an effort to simplify the application of PDT. Adjuvant chemo-photodynamic therapy has also been known to enhance the overall effect of treatment of tumors.10-12 For example chemotherapeutic nitrosamine drugs13-15 such as carmustine and lomustine are given in combination with PDT to improve the efficiency in treating several types of brain cancer.16-18
The field is in need of a device that could potentially (i) offer precise delivery of sufficient sensitizer (with minimal loss), light and oxygen to generate adequate concentrations of cytotoxic singlet oxygen at the target site, (ii) limit near-neighbor effects in tumors that are close to vital tissue, and (iii) eliminate tumor hypoxia ensuring high local oxygen concentrations, sufficient to sustain PDT.
In this thesis we have taken basic (Chapter 5) and applied (Chapters 2-4) research approach, using organic synthesis and photochemistry as well as in-vitro and some preliminary in- vivo studies, that are mutually dependent. In view of the utility in PDT,19-21 a “pointsource” fiber optic device capable of delivering the components necessary for PDT in a highly localized and 2 controllable fashion, was developed in our lab.22-26 This device offers high precision simultaneous delivery of sensitizer drug, light, and oxygen to produce singlet oxygen for photodynamic action in-vitro and in-vivo.
Figure 1A shows the “pointsource” PDT device where red diode laser light and O2 gas travel through a hollow fiber optic and emerge from a silica probe tip attached at the distal end of the fiber. The surface of the silica probe tip is fluorinated to make it more Teflon like (hydrophobic) and loaded with sensitizer drug via photolabile alkene linker. Singlet oxygen produced by the attached sensitizer helps to cleave the linker27-29 via dioxetane intermediate (1) and photorelease the sensitizer drug (3) in the surrounding medium. The photoreleased sensitizer traverses a
1 relatively long 0.25 mm distance in media and sensitizes the production of O2 away from the probe via light and O2 gas delivered from probe tip.
Fluorination of the surface helps to reduced physical quenching of singlet oxygen,25 solves the problem of sensitizer re-adsorption onto the surface and improves localized concentration of oxygen around the probe which helps to maintain the efficiency of the probe even under hypoxic conditions. Thus far, four different kinds of silica probe tips have been synthesized, fluorinated- sensitizer coated tip 4, only sensitizer coated tip 5, only fluorinated tip 6 and uncoated bare tip 7
(Figure 1B). The “pointsource” device with the variety of probe tips were utilized in the eradication of glioma U87 and OVCAR-5 cancer cells in-vitro30,31 and SQ20B tumors of head and neck in mice in-vivo32 and in understanding the mechanism of phototoxicity in nitrosamine drugs, which has been elaborated in this thesis.
3
A B
Figure 1B. Schematic presentation of various functionalized device tips 4-7 (inset on the right) pyropheophorbide sensitizer structure. The 5 × 8 mm2 cylindrical shape probe tips are made of porous Vycor glass.
Figure 1A. Schematic presentation of the “pointsource” fiberoptic PDT device with fluorinated probe tip 4 and mechanism of sensitizer release after scission of dioxetane intermediate that arose from a [2 + 2] addition of singlet oxygen to the ethene bond. A cutaway view of the hollow fiber core, delivery of visible light and O2 through the fiber to the tip causing the photorelease of the sensitizer molecules, is shown.
1.2 Sections in the Thesis
The chapters to follow describe in detail, (a) Eradication of glioma cells in-vitro - the precision of pointsource PDT in the eradication of glioma (U-87) and ovarian cancer (OV5) cells 4 in-vitro by creating a halo of singlet oxygen. The efficiency of delivering sensitizer drug and singlet oxygen was assisted by an autocatalytic mechanism as can been seen in Chapter 2.
(b) Biomaterial fouling in-vivo - the resistance to biomaterial fouling on the surface of the fluorinated PDT device tips probe tips its effect on the pace of sensitizer photorelease in-vivo.
Biomaterial (e.g., proteins, cells, etc.) from SQ20B head and neck tumors and whole blood was used for an assessment of fouling of the silica tips by adsorption. Amount of biomaterial on the probe surface was quantified by bicinchoninic acid assay (BCA) and X-ray photoelectron spectroscopy (XPS) measurements and data summarized in Chapter 3.
(c) New triPEG-galloyl pheophorbide sensitizer - the efficient synthesis of a specialized silica tip for an optical fiber device capable of delivering oxygen, light and a cleavable tripolyethylene glycol (PEG)-galloyl pheophorbide sensitizer, all components necessary for photodynamic therapy. The hydrolytic stability of the attached PEGs and the extent to which the
PEG groups enhance solubility will also be discussed in Chapter 4. The new triPEG-galloyl sensitizer has the potential for use in intraoperative pointsource photodynamic therapy which aims for precision treatment of residual disease.
(d) Photooxidation in nitrosamines - the photooxidation mechanism in N-nitrosamines was studied as described in chapter 5. Integrative approaches such as 18O-isotopic labelling chemistry,33-35 photochemistry, tandem mass spectrometry (HPLC-MS, HPLC-MS/MS) and density functional theory (DFT) calculations, were exploited to reveal the products of an oxygen exchange route in nitrosamine photochemistry. This may provide a clue to new factors significant in nitrosamine phototoxicity.
1 (e) A review - of the literature on using photogenerated singlet oxygen ( O2) in the synthesis
1 of natural products and drugs. The visible-light sensitized production of O2 is not only useful for 5 synthesis, it is extremely common in nature. Chapter 6 intends to draw a logical link between flow
1 and batch reactions—a combination that leads to the current state of O2 in synthesis.
1.3 References
1. Bonadonna. G, Valagussa. P. N. Engl. J. Med. 1981, 304, 10. 2. Boland, C. R.; Goel, A. Gastroenterology, 2010, 138, 2073. 3. Scott, J.; Martin, I.; Redhead, D.; Hammond, P.; Garden, O. J. Clin. Radiol. 2000, 55, 187. 4. Peddu, P.; Quaglia, A.; Kana, P. A.; Karani, J. B. Crit. Rev. Oncol. Hematol. 2009, 70, 12. 5. Pass, H. I. J. Natl. Cancer. Inst. 1993, 85, 443. 6. Hopper, C. Lancet. Oncol. 2000, 1, 212. 7. Kessel, D.; Foster, T. H. Photochem. Photobiol. 2007, 83, 995. 8. Singlet Oxygen: Applications in Biosciences and Nanosciences (Edited by S. Nonell and C. Flors), pp. 1-450. Royal Society of Chemistry (RSC), Abingdon, Oxfordshire, UK. 9. Li, B.; Wilson, B. C. J. Innov. Opt. Health Sci. 2015, 8, 1502001. 10. Huang, H.-C.; Hasan, T. Austin J Nanomed Nanotechnol. 2014, 2, 1020. 11. Zhang, C.-J.; Hu, Q.; Feng, G.; Zhang, R.; Yuan, Y.; Lu, X.; Liu, B Chem. Sci. 2015, 6, 4580. 12. Khdair, A.; Handa, H.; Mao, G.; Panyam, J. Eur. J. Pharm. Biopharm. 2009, 71, 214. 13. Affronti, M. L.; Heery, C. R.; Herndon, J. E.; Rich, J. N.; Reardon, D. A.; Desjardins, A.; Vredenburgh, J. J.; Friedman, A. H.; Bigner, D. D.; Friedman, H. S. Cancer 2009, 115, 3501. 14. Garside, R.; Pitt, M.; Anderson, R.; Rogers, G.; Dyer, M. Health Technol Assess 2007, 11, 242. 15. Brandes, A. A.; Tosoni, A.; Basso, U.; Reni, M.; Valduga, F.; Monfardini, S.; Amistà, P.; Nicolardi, L.; Sotti, G.; Ermani, M. J. Clin. Oncol. 2004, 22, 4779. 16. Chen, L. F.; Ke, Y. Q.; Yang, Z. L.; Wang, S. Q.; Xu, R. X. Di Yi Jun Yi Da Xue Xue Bao. 2005, 1, 116. 17. Quirk, B. J.; Brandal, G.; Donlon, S.; Vera, J. C.; Mang, T. S.; Foy, A. B.; Lew, S. M.; Girotti, A. W.; Jogal, S.; LaViolette, P. S.; Connelly, J. M.; Whelan, H. T. Photodiagn. Photodyn. Ther. 2015, 12, 530. 18. Failla, V.; Wauters, O.; Caucanas, M.; Nikkels-Tassoudji, N.; Nikkels, A. F. Rare Tumors 2010, 2, e34. 19. Philipp, C. M.; Ziolkowska, M.; Mueller, U.; Urban, P.; Berlien, H.-P. Optik & Photonik 2013, 1, 42. 20. Berlien, H.-P.; Philipp, C. M. Photon. Lasers Med. 2015, 4, 352. 21. Lucky, S. S.; Soo, K. C.; Zhang, Y. Chem. Rev. 2015, 115, 1990. 22. Zamadar, M.; Ghosh, G.; Mahendran, A.; Minnis, M.; Kruft, B. I.; Ghogare, A.; Aebisher, D.; Greer, A. J. Am. Chem. Soc.2011, 133, 7882. 23. Mahendran, A.; Kopkalli, Y.; Ghosh, G.; Ghogare, A.; Minnis, M.; Kruft, B. I.; Zamadar, M.; Aebisher, D.; Davenport, L.; Greer, A. Photochem. Photobiol. 2011, 87, 1330. 24. Bartusik, D.; Aebisher, D.; Ghosh, G.; Minnis, M.; Greer, A. J. Org. Chem. 2012, 77, 4557. 25. Bartusik, D.; Minnis, M.; Ghosh, G.; Greer, A. J. Org. Chem. 2013, 78, 8537. 26. Ghosh, G.; Minnis, M.; Ghogare, A. A.; Abramova, I.; Cengel, K. A.; Busch, T. M.; Greer, A. J. Phys. Chem. B. 2015, 119, 4155. 6
27. Klan, P.; Solomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Chem Rev. 2013, 113, 119. 28. Arian, D.; Kovbasyuk, L.; Mokhir, A. J. Am. Chem. Soc.2011, 133, 3972. 29. Hossion, A. M.; Bio, M.; Nkepang, G.; Awuah, S. G.; You, Y. ACS Med. Chem. Lett. 2013, 4, 124. 30. Bartusik, D.; Aebisher, D.; Ghogare, A.; Ghosh, G.; Abramova, I.; Hasan, T.; Greer, A. Photochem. Photobiol. 2013, 89, 936. 31. Ghogare, A. A.; Rizvi, I.; Hasan, T.; Greer, A. Photochem. Photobiol. 2014, 90, 1119. 32. Ghogare, A. A.; Miller, J. M.; Mondal, B.; Lyons, A. M.; Cengel, K. A.; Busch, T. M.; Greer, A. Photochem. Photobiol. 2016, 92, 166. 33. Wasserman, H. H.; Vinick, F. J. J. Org. Chem. 1973, 38, 2407. 34. Sawwan, N.; Greer, A. Chem. Rev. 2007, 107, 3247. 35. Ishikawa, S.; Nojima, T.; Sawaki, Y. J. Chem. Soc., Perkin Trans. 2 1996, 1, 127.
7
Chapter 2. “Pointsource” Delivery of a Photosensitizer Drug and Singlet Oxygen:
Eradication of Glioma Cells In-vitro
2.1 Introduction
Conventional photodynamic therapy (PDT) techniques1 to treat tumors, systemically administer the sensitizer relying on homing to the appropriate site. There are examples of sensitizers conjugated to compounds or nanoparticles that lead to improved photodynamic therapy
(PDT) results,2-7 sensitizer conjugation to, for example, metal particles or silica have been seen to increase PDT efficiency8,9 and singlet oxygen production.10-14 As we mentioned in chapter 1, have developed a “pointsource” device that simultaneously delivers all the components essential for
PDT in highly localized regions.
In this chapter, we describe the application of our pointsource micro-optic device (Figure
1) in killing glioblastoma cells in monolayer in-vitro. Briefly, pheophorbide molecules readily
15 1 cleave off of the silica probe tip by oxidation of the ethene linker (Figure 1b and c) and emits O2 which is used to kill glioblastoma cells. Such oxidation processes resulting in bond cleavage16 based on singlet oxygen chemistry17-20 have been of interest to us.
Our interests are also in adsorption reactions that participate on silica surfaces. In recent work, we developed surface coatings (designed as a repellent fluorosilane probe surface) for enhanced pheophorbide photorelease of up to 99% of the ethane bonds broken in toluene.21 This fluorinated silica surface was also used for sensitizer drug release and photokilling of ovarian cancer (OVCAR-5) cells—providing initial estimates the device may function as a PDT implement.22 8
Figure 1. Pointsource device for targeted singlet oxygen delivery: (a) Red laser light and oxygen gas traveled through the hollow fiber optic and emerged from the probe tip. We used 200 mW output from a 669 nm laser and an O2 gas flow rate of ~0.2 ppm/min through the probe tip. The probe tip was held vertically in a perpendicular orientation above the cells so as not to kill them by mechanical action. (b) The fiber is equipped with a 5 × 10 mm2 (d × l) pheophorbide modified silica tip with a photocleavable ethene linker. The probe design includes a covalently bound nonafluorosilane to improve sensitizer photorelease into the surrounding medium. (c) A view of the singlet oxygen-generating probe tip is shown with sensitizer photorelease and factors that relate to the glioma cell killing mechanism. The sensitizer traverses a relatively long 0.25 mm distance, 1 which stands in contrast to the short ~150 nm diffusion distance of O2 in H2O. The sensitized 1 production of O2 also occurs away from the probe tip through diffusion of the pheophorbide via light and O2 delivered from the probe tip.
Based on recent work21-22, we sought to answer chemical and biological questions in the drug photorelease process. For example, can we explain mechanisms of sensitizer uptake into the cells and the precision of killing when the probe tip is placed in U87 monolayers? Our reasons for pursuing this work are (i) to address significant challenges in the clinic in removing cancers to minimize damage to normal tissue,23-24 and (ii) to identify potential benefits of the pointsource 9 approach over conventional systemic photosensitizer delivery, particularly for PDT in the brain, where sensitizer delivery is problematic due to the blood brain barrier. Here, we describe detailed studies of the pointsource PDT technique, using principles of organic chemistry and photobiology for a basic understanding of interfacial phenomena for sensitizer-photorelease control and cell- killing precision. In one respect, our work bears similarity to an optical fiber system developed by
Kandler et al.25 which is ideal for cultured neurons and brain slices containing caged reagents for photo-uncaging reactions where the light spot is focused.
2.2 Results and Discussion
2.2.1 Cell Phototoxicity and Sensitizer Uptake
Initially, we carried out control experiments to find conditions for efficient cell killing.
Figure 2 (light red bars) shows the percent U87 cell viability, but with a device probe tip that was devoid of any sensitizer molecules. Here, pyropheophorbide-a spiked into U87 cell samples followed by light and oxygen from the device tip was used as a control. The U87 cell viabilities were analyzed 24 h post-treatment with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. These controls demonstrate that greater than 90% cell killing is achievable with 0.5 µM pheophorbide after 1 h. The cell viability decreases as we add higher
1 pyropheophorbide-a concentrations due to greater concentrations of O2. Our control experiments also demonstrate that the pyropheophorbide-a toxicity in the dark is minimal (i.e. 0-3% for 0.025-
1.0 µM pheophorbide and 4% for 2.5 µM pheophorbide). Evidence for a photosensitized oxidation process is that in the absence of sensitizer, light, or O2 the cell viability was ~97%. In the absence of sensitizer, but the presence of light and O2 for 1 h the cell viability was 92-94%. 10
Figure 2. Comparison of sensitizer uptake efficiency in the dark after 1 h (light red bars) with phototoxicity effects on U87 cells treated by a bare-tipped device devoid of sensitizer, but sparging O2 with red light irradiation with externally added pheophorbide (dark blue bars). Cells were digested for the uptake measurements. Cell viability was assessed by MTT assay and results are shown as percent relative to control cells. Each value represents an average of two or more experiments.
Figure 2 (dark blue bars) shows the concentration of pheophorbide that was taken up by
U87 cells in MEM media. The dose of pheophorbide added to the cells ranged from 0.025 to 2.5
µM, which corresponded to cellular uptake ranging from 0.01 µM to 0.138 µM. Despite the difference in the amounts of pheophorbide introduced, over 1 h the quantity of pheophorbide taken up was relatively fixed 15-24%, where the uptake appeared to be a linear relationship over this concentration range (slope = 0.152; R2 = 0.9977).
The data from Figure 2 taken together point to a 92% cytotoxic response for the delivery of 0.5 µM and uptake of 0.08 µM uptake of pheophorbide after 1 h. Although the porous silica probe tip readily loads different amounts of pheophorbide, the information in Figure 2 helped us identify release quantities that achieve good-level killing.
11
2.2.2 Course of the Sensitizer Drug Photorelease in Phosphate Buffered Saline (PBS)
We have used a probe tip with 7-nmol pheophorbide covalently-bound to the working front face. Figure 3 shows the percent of sensitizer photoreleased from the probe tip into 0.20 mL PBS.
The plot shows a sigmoidal photorelease behavior. From 0 to 30 min, the photorelease was slow and was then followed by rapid photorelease. After 1.5 h, there was a deceleration. After 2 h, we found that ~10% of the sensitizer had departed from the probe tip, although most of the ethene bonds were consumed (92-95%). The amount of sensitizer adsorbed on the tip was 82±3% based on solvent washes with DMSO and n-butanol. This can be understood in terms of limited solubility of the sensitizer in PBS, thereby increasing its tendency to adsorb to the fluorinated silica probe surface.
Figure 3. A time-sequence analysis of pheophorbide photoreleased free from the probe tip of the device in PBS at 25 °C (solid circles) and fluence from the tip (solid triangles). The concentration of pheophorbide was measured by fluorescence spectroscopy at the indicated times. The values shown here are an average of 3 or more measurements.
We previously showed that externally irradiated silica-conjugated sensitizer samples also produce a sigmoidal photorelease in n-butanol.26 There was a kinetic likeness in the photorelease 12 although the sigmoid was 7.7-fold steeper in the n-butanol than the PBS. Silica surface properties29-34 and factors including greater solubility in n-butanol than PBS play key roles in the extent of the pheophorbide retention on the silica tip surface. The results were similar from internal irradiation (described here) vs external irradiation26 of the sensitizer solid, where increasing light intensity in the former was a correlation with, but not causation of the sigmoidal behavior.
As might have been expected, the light intensity emerging through the tip increased over the course of the PBS experiment. Power meter measurements have correlated the amount of light delivered through the tip with the fraction of detached sensitizer. Figure 3 shows that after 2 h, there is a net 10% sensitizer photorelease, which only yielded a 23% increase in the light intensity through the probe tip. With the increased light delivery, the affect may be one of increasing the
1 O2 concentration available at the end of the fiber adjacent to the probe tip. But, we did not attribute the increasing light intensity as playing a significant role in causing the sigmoidal photorelease behavior.
Guided by the results of the micro-tipped device for sensitizer photorelease in PBS, we proceeded to investigate the efficiency of the pointsource device for killing glioma cells in-vitro.
2.2.3 Fiber Tip-Guided Sensitizer Delivery for Cell Killing in Discrete Locations
We have used the device to demonstrate sensitizer photorelease and global phototoxicity to U87 cells in MEM-media. The device contained pheophorbide-attached probe tips, and cell viability was measured 24 h after device treatment by live/dead assay and fluorescence microscopy. Here, the phototoxicity was evaluated in 14 mm diameter microwell experiments.
Figure 4 shows a sigmoidal behavior for the photokilling, analogous to the photorelease behavior in solution (Figure 3). From 0 to 30 min, the cell killing was slow which was followed 13 by an acceleration and then deceleration at 1.5 h. The increasing sensitizer release and light intensity emerging through the tip over the course of the experiment was roughly proportional to the photokilling (inversely proportional to the normalized viability by live/dead assay). The phototoxicity reached a maximum of 79% after 2 hours (Figure 4). We did not observe 100% killing under our experimental conditions.
Figure 4. Time-sequence analysis of phototoxicity effects on U87 cells in 14 mm diameter microwell experiments treated with the device tip (solid circles) and fluence from the tip (solid squares). Cell viability was assessed by live/dead assay and results are shown as percent relative to control cells. Each value represents an average of two experiments.
2.2.4 “Lengthening” the Toxic Radius of Singlet Oxygen
1 35,36 Although the diffusion distance of O2 is short with toxicity that does not extend much beyond ~100 nm37-39 sensitizer release from our device gives a diffusible photocatalyst that effectively increases it. This was shown in our examination of the cell killing radius, where we placed the device tip 0.25 mm above U87 cells spread into a monolayer on a 200-µL microwell plate (diameter = 14 mm). In-vitro toxicity experiments and imaging were done under the 14 supervision of Dr. Imran Rizvi. Figure 5 shows the photokilling radius as a function of time. The non-viable, propidium iodide-stained detached cells were aspirated as part of the standard protocol for the live/dead assay. Consequently the images in Figure 5 show viable green attached cells. The radius of cell killing increases and proceeds from 0.1 to 2.9 mm for treatment times of 0.5 to 2.0 h, respectively. Compare the insets for “control” and treatment time = 0 h (Figure 5), which shows that cell viability with only light and O2 for 1 h is ~95% indicating that the sensitized formation of
1 O2 is required for the cell photokilling. A limited number of peripheral cell deaths were observed based on the fluorescence intensity of the attached cells from the no treatment image to those lying outside of the treatment zones.
Figure 5. Device tip treatment of a U87 cell monolayer (154 mm2 area) revealed a radius of photokilling as a function of time. Regions of the confocal fluorescence images show the probe tip radius in a white dashed line, the radius of photokilling, and that of the plate edge. The cells were stained with calcein AM (green/live) for 30 min. Magnification 10×. (In-vitro imaging were done under the supervision of Dr. Imran Rizvi)
15
2.2.5 Mechanism
Our data support the mechanism depicted in Scheme 1. Visible light and oxygen gas
1 emerge from the pointsource tip. The ethene group reacts with O2 for sensitizer release, following dioxetane cleavage, availing the sensitizer’s phototoxic activity to the U87 cells.
Notice there was an increased photokilling rate from 0.5 to 1.0 h (270 cells/min) relative to earlier 0 to 0.5 h (173 cells/min) or later in the reaction 1.0 to 2.0 h (182 cells/min). Thus, it follows the radius of cell killing increases in the mid-point of the reaction. The notion was the probe tip is most lethal at 0.5 to 1.0 h when the photorelease rate was at its fastest. We know as the result of work with native silica that using fluorinated silica increases the photorelease.26 In
1 addition to a Teflon-like repellent surface, evidence is for reduced physical quenching of O2 with the fluorinated silica compared to native silica21, which is an important factor in the self- accelerated sensitizer release.
1 We view O2 as a key species initiating events causing phototoxicity. Red-light irradiation of pheophorbides mainly proceeds by a Type-II (singlet oxygen) photosensitized mechanism40,41 rather than a Type-I mechanism involving superoxide, hydroxyl radical and related species.42,43 It was evident from Figure 2 that of the sensitizer delivered, ~20% diffused into the cells, but we did
1 not discriminate whether cell death depends more on extracellular or intracellular O2. Although an interconnection for the extracellular route could, in principle, be made with a membrane-
44 1 impermeable sensitizer. The diffusion coefficients of O2 or of pheophorbide were not estimated due to the heterogeneity of the system. 16
1 Scheme 1. Sensitizer drug and O2 delivery and glioblastoma cell killing mechanism
2.3 Conclusion
We describe a micro-optic device, which combines a diode laser, a hollow fiber optic, and a porous silica probe tip, to deliver a pheophorbide (sensitizer) and singlet oxygen. The sensitizer photocleaves away from the probe tip and diffuses through media until it reaches the glioma cells.
A rapid photorelease function was identified about midway through the reaction. This builds on the previous work that was published in 201326, which found an autocatalytic-assisted photorelease of a sensitizer bound to a fluorosilane-coated silica surface into butanol and octanol solutions. 17
Development of the device for cancer eradication applications requires good precision in cell killing. Precision is important in treatment of cancers like glioma to minimize damage to critical normal nearby tissue.23,24 Additional experiments will grow from these initial experiments including optimization sensitizer release, sensitizer cell uptake, light dose rates, and also probe tip shape and surface conditioning to further enhance the cell killing. Our basic message is the pointsource approach has potential benefits compared to conventional systemic photosensitizer delivery for PDT. Its significance may include the treatment of brain tumors, e.g. glioblastoma
3 multiforme. That the device tip delivers O2 is an essential (and somewhat indispensable) feature of the technique. The device can, in principle, connect to fiber optic methods to improve cell- killing precision in oxygen-poor sites during PDT.
2.4 Experimental Section
2.4.1 Materials and Methods
Sterile DMSO, chloroform, hydrofluoric acid, and propidium iodide solution (1 mg/mL in water, dead cell stain) were purchased from Sigma Aldrich (St. Louis, MO). Calcein AM (live cell stain), fetal bovine serum (FBS), and the MTT reagent were purchased from Life Technologies
(Carlsbad, CA). Pyropheophorbide-a was purchased from Frontier Scientific (Logan, UT).
Aqueous-based tissue solubilizer solution was purchased from PerkinElmer (Waltham, MA).
Pierce® BCA protein assay kit was purc hased from Thermo Scientific (Rockford, IL). Minimum
Essential Medium Eagle (Mod.) 1× (MEM), Dulbecco's Phosphate-buffered salt solution (PBS) and 5,000 I.U./mL penicillin/streptomycin and 50-mg/mL streptomycin were purchased from
Mediatech (Herndon, VA). U87 MG ATCC® HTB-14™ cells were purchased from ATCC
(Manassas, VA). To make complete MEM growth media, 1% (v/v) 5,000 I.U./mL 18 penicillin/streptomycin and 50-mg/mL streptomycin and 10% (v/v) FBS was added to a 500 mL bottle of MEM media. Cell culture glass bottom dishes (29 mm glass well size and 14 mm microwell) and #1 cover glass (0.13-0.16 mm) were purchased from In-Vitro Scientific
(Sunnyvale, CA). Falcon 35 mm cell culture dishes and 24-well cell culture plates were purchased from Becton Dickinson Labware (Franklin Lakes, NJ). Corning’s code 7930 porous Vycor glass
(PVG) was purchased from Advanced Glass and Ceramics (Holden, MA).
2.4.2 Device Fabrication and Instruments
We have used this device previously.22 A 3-ft long fiber optic was purchased from
Fiberoptic Systems, Inc. (Simi Valley, CA). It had an internal 1.1-mm diameter Teflon gas flow tube from the distal end to a T-valve that was surrounded by excitation fibers, as well as a 1.4-mm diameter black polyvinyl chloride jacket. The Vycor was shaped into cylindrical pieces 5 × 10 mm2 (d × l) with a Buehler IsoMet Low Speed Saw (Model 11-1280-160), a Buehler ultrasonic disk cutter (Model 170), and a Buehler variable speed grinder-polisher. A hole 1.5 × 7.0 mm2 (d × l) was bored into the glass pieces with a dremel drill (Model 200) to fit to the fiber optic and was glued in place with ethyl cyanoacrylate. The synthesis of the pheophorbide-modified probe tips was carried out using a procedure as previously described.15,26 The amount of sensitizer covalently bonded to the probe tips was obtained to be 70 nmol. The diameter of the sensitizer molecule is
~20 Å and pore sizes in the silica are ~40 Å. The penetration depth of the sensitizer is 0.08 mm along the outer faces of the probe tip. Light was delivered from a 669 nm CW diode laser (model
7404, 700 mW, 4.1 A output, Intense Inc., North Brunswick, NJ) that was connected to the optical fiber and the power (1 W/cm2, spot size 0.196 cm2) was determined with a VEGA Laser Power
Energy Meter (Ophir Laser Measurement Group, LLC, North Logan, UT). With the optical fiber 19 pinned to a translation stage (OptoSigma Corp., Santa Ana, CA) for ±0.1 mm precision movement, the diode laser was connected to its proximal end through an SMA connector. Based on a previous
27 report , the O2 gas flow rate through the probe tip was ~0.2 ppm/min as measured by a Clarke- type oxygen electrode. The amount of sensitizer covalently attached was determined by monitoring its Soret absorption (λ = 415 nm) after liberation from unused probe tips on dissolution with 40%
(v/v) aqueous hydrofluoric acid and extraction with chloroform. The total amount of sensitizer remaining on the probe tip after photorelease was measured in terms of mV using a Labsphere integrating sphere (North Sutton, NH) attached to a Fluke 79 Series II Digital Multimeter (John
Fluke Mfg. Co., Vail, AZ). The multimeter was calibrated prior to use and the amount of covalently bound and adsorbed sensitizer were obtained from calibration curves. Fluorescence measurements were made with a SpectraMax M5 Multi-Mode Microplate Reader from Molecular Devices
(Sunnyvale, CA). Absorbance measurements were made with an Evolution 300 UV-Vis
Spectrophotometer (Thermo Fisher Scientific, Franklin, MA).
2.4.3 Sensitizer Photorelease in Phosphate Buffered Saline (PBS)
Oxygen gas and 669 nm excitation light, intensity of 1 W/cm2 (measured with fiber, without cap) were delivered for 2 h, through the fiber optic to probe tip loaded with covalently bound sensitizer dipped in a 200-µL PBS solution. From the photocleaved pheophorbide PBS solution 50 µL was sampled out at 0.5, 1, 1.5 and 2 h periods and diluted with 50 µL DMSO to measure fluorescence of the sensitizer. The photocleavage of sensitizer away from the probe tip was followed by fluorescence in solution (λex = 405 nm, λem = 675 nm) using the plate reader. The concentration of photosensitizer was obtained from the pre-constructed calibration curves of pheophorbide in (1:1) (v/v) DMSO:PBS solution. The amount of pheophorbide photoreleased was 20 calculated as follows: % photorelease = [(photorelease/loading per area)] × 100. A 1/10th portion of the cap was dipped in 200-µL PBS solution and the quantity of dye that photocleaved was based on the amount of sensitizer covalently attached (7 nmol) to the front face of the cap (20 mm2 area).
The amount of sensitizer adsorbed was measured by soaking the cap in 1 mL n-butanol solution for 24 h followed by fluorescence measurements using the plate reader.
2.4.4 Cellular Uptake in U87 MG Cell Monolayer
Human brain carcinoma cells (U87 MG) were maintained in complete MEM growth media at 37 °C in a 5% CO2 incubator. U87 cells (100,000 per well) were seeded in a 24-well cell culture treated plate and maintained at 37 °C in the 5% CO2 incubator. Twenty-four hours later, 0.20 mL volume of MEM media (without phenol red) containing concentrations of pyropheophorebide-a ranging from 0.025 to 2.5 μM in 1% (v/v) DMSO were added to the cells under subdued light conditions. The cells were incubated with the pyropheophorebide-a containing media for times ranging from 0.5 to 2 h. At each time point, the 0.20 mL of the supernatant media was removed from each well and diluted with 0.20 mL DMSO to determine the amount of pheophorbide remaining in the media by fluorescence using a microplate reader. The pheophorbide taken up and associated with the membrane of the U87 MG cells was extracted in 200-µL of the tissue solubilizer solution by digesting the cells for 30 min and the concentration of pheophorbide in the cell lysates was measured by fluorescence. Intracellular and bound concentrations of pheophorbide were quantified from pre-constructed calibration curves of known concentration range of pheophorbide in cell lysates. The total protein content of the cell lysates was determined using the
BCA protein assay kit, and calibration curves prepared from known concentrations of BSA in the tissue solubilizer solution. 21
2.4.5 Phototoxicity of Pyropheoporbide-a in U87 MG Cell Monolayer
U87 cells (210,000 per well) were plated in a 35-mm cell culture dish in complete MEM media and maintained at 37 °C in a 5% CO2 incubator for 24 h. A concentration range of pyropheophorbide-a in 1.0 mL MEM media (0.025-2.5 µM) was added to U87 cell monolayer.
Immediately after addition of the media containing pheophorbide, the cells were treated with the device fitted with a “bare” tip at 669 nm laser, 150 mW/cm2 intensity and continuous oxygen sparging for 0.5 or 1.0 h. Control conditions such as ‘no treatment’, ‘light only’ and ‘dark control’ containing 2.5 µM pheophorbide were also done to ensure the reliability of the data. Post-device treatment, the media containing pheophorbide was aspirated and fresh complete growth MEM media was added to each treatment and control dish, and maintained at 37 °C in a 5% CO2 incubator. Next day, cell viability was measured by assay28 and the surviving fraction was normalized to the ‘no treatment’ controls.
2.4.6 Treatment Procedure
U87 cells (170,000 per well) were plated in 29 mm glass bottom cell culture dish with 14 mm glass bottom micro-well insert, in complete MEM media and maintained at 37 °C in a 5%
CO2 incubator. Before treatment, media were aspirated and 0.20 mL complete growth MEM media with 1% (v/v) DMSO were added to the cell culture dish. The probe tip loaded with covalently- bound sensitizer was placed 0.25 mm above the cells. Device treatment was carried out for 0.5 to
2 h periods with 669-nm light with an intensity of 1 W/cm2 (measured with fiber, without cap) with continuous oxygen sparging, as ‘treatment’ groups. Controls such as ‘no treatment’ control,
‘light only’ control with the bare tip and pheophorbide-loaded ‘tip only’ control were done to 22 evaluate the toxicity coming from only light or 1% (v/v) DMSO in MEM media. Post-device treatment, DMSO-containing MEM media were removed, complete growth MEM media were replenished in their place and cells were maintained at 37 °C in 5% CO2 incubator. To determine phototoxicity after 24 h, the media were removed from the cells, the cells were washed with PBS solution and then incubated for 30 min in a live/dead assay made of 0.001% (v/v) Calcein AM
(live cell stain) and 0.002% (v/v) propidium iodide (dead cell stain) in PBS. The confocal fluorescence images of the stained cells were taken on the Olympus FV-1000 confocal using a 10× objective for the entire 14 mm glass microwell. The 488 nm line from an Argon ion laser with paired with a FITC filter set and a 559 nm diode source paired with a TRITC filter set were used to detect cleaved Calcein to label live cells. Cell viability was quantified from the fluorescence images using Image J software, where the surviving fraction was normalized to ‘no treatment’ controls. Importantly, the red fluorescence was lowered due the loss of detached cells during treatment and washing steps prior to imaging. However, this did not impact the analysis because cell viability was measured using Calcein AM (green fluorescence).
2.4.7 Sources of Error
Errors arise from the following sources: (i) volumes were recorded by drawing the media up into a 250-µL Hamilton syringe (±5 µL resolution; equates to 2.5% error). (ii) Media evaporation took place (e.g., tens of microliters could be lost over the course of the experiment).
However, media were added every 30 min to account for this to maintain the volume at 200 µL.
(iii) The concentration of the pheophorbide in the PBS was based on its fluorescence via its extinction coefficient (accuracy ±0.1 µM). (iv) The diameter of the light spot that emerged from the tip was measured with a ruler. It was 0.196 cm2 viewed by eye (accuracy ~20%). The 23 measurement of the fluence (mW/cm2) of light had an error of ~1%. (v) The normalized cell viability was calculated as the relative absorbance of the MTT reagent using the plate reader. The procedure of incubation with MTT reagent and extraction with DMSO, pipetting out 200-µL sample for absorbance introduces error of 2-5% in the cell viability calculation. (vi) The radius of photoxicity was measured using the same protocol described in the ‘Treatment Procedure’ section, using the Image J software “circle tool” option which introduced a ~1% error.
2.5 References
1. Röder, B. In Encyclopedia of Analytical Chemistry; John Wiley & Sons, Ltd: 2006, 302-320 2. Srivatsan, A.; Ethirajan, M.; Pandey, S. K.; Dubey, S.; Zheng, X.; Liu, T. H.; Shibata, M.; Missert, J.; Morgan, J.; Pandey, R. K. Mol. Pharm. 2011, 8, 1186. 3. Zhou, A.; Wei, Y.; Wu, B.; Chen, Q.; Xing, D. Mol. Pharm. 2012, 9, 1580. 4. Lim, C. K.; Shin, J.; Kwon, I. C.; Jeong, S. Y.; Kim, S. Bioconjugate Chem.2012, 23, 1022. 5. Cui, Y.; Wu, Y.; Liu, Y.; Yang, G.; Liu, L.; Fu, H.; Li, Z.; Wang, S.; Wang, Z.; Chen, Y. Dyes Pigments 2013, 97, 129. 6. Zhao, J.; Fei, J.; Du, C.; Cui, W.; Ma, H.; Li, J. Chem. Comm. 2013, 49, 10733. 7. Dosselli, R.; Tampieri, C.; Ruiz-González, R.; De Munari, S.; Ragàs, X.; Sánchez-García, D.; Agut, M.; Nonell, S.; Reddi, E.; Gobbo, M. J. Med. Chem.2013, 56, 1052. 8. Cheng, Y.; Meyers, J. D.; Broome, A.-M.; Kenney, M. E.; Basilion, J. P.; Burda, C. J. Am. Chem. Soc.2011, 133, 2583. 9. Cheng, Y.; Samia, A. C.; Meyers, J. D.; Panagopoulos, I.; Fei, B.; Burda, C. J. Am. Chem. Soc.2008, 130, 10643. 10. Wang, S.; Gao, R.; Zhou, F.; Selke, M. J. Mater. Chem.2004, 14, 487. 11. Lacombe, S.; Soumillion, J. P.; El Kadib, A.; Pigot, T.; Blanc, S.; Brown, R.; Oliveros, E.; Cantau, C.; Saint-Cricq, P. Langmuir 2009, 25, 11168. 12. Panagiotou, G. D.; Tzirakis, M. D.; Vakros, J.; Loukatzikou, L.; Orfanopoulos, M.; Kordulis, C.; Lycourghiotis, A. Appl. Catal.A: 2010, 372, 16. 13. Ragàs, X.; Gallardo, A.; Zhang, Y.; Massad, W.; Geddes, C. D.; Nonell, S. J. Phys. Chem. C 2011, 115, 16275. 14. Mooi, S. M.; Heyne, B.Photochem. Photobiol. 2014, 90, 85. 15. Zamadar, M.; Ghosh, G.; Mahendran, A.; Minnis, M.; Kruft, B. I.; Ghogare, A.; Aebisher, D.; Greer, A. J. Am. Chem. Soc.2011, 133, 7882. 16. Klan, P.; Solomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Che.m Rev. 2013, 113, 119. 17. Arian, D.; Kovbasyuk, L.; Mokhir, A. J. Am. Chem. Soc.2011, 133, 3972. 18. Hossion, A. M.; Bio, M.; Nkepang, G.; Awuah, S. G.; You, Y. ACS Med. Chem. Lett. 2013, 4, 124. 24
19. Bio, M.; Rajaputra, P.; Nkepang, G.; Awuah, S. G.; Hossion, A. M.; You, Y. J. Med. Chem. 2013, 56, 3936. 20. Komeda, C.; Ikeda, A.; Kikuchi, J.; Ishida-Kitagawa, N.; Tatebe, H.; Shiozaki, K.; Akiyama, M. Org. Biomol. Chem. 2013, 11, 2567. 21. Bartusik, D.; Aebisher, D.; Ghosh, G.; Minnis, M.; Greer, A. J. Org. Chem. 2012, 77, 4557. 22. Bartusik, D.; Aebisher, D.; Ghogare, A.; Ghosh, G.; Abramova, I.; Hasan, T.; Greer, A. Photochem. Photobiol. 2013, 89, 936. 23. Mandel, S.; Glass, J. Pract. Neurol. 2009, 16. 24. Eljamel, S. Photodiagnosis Photodyn. Ther. 2010, 7, 76. 25. Kandler, K.; Nguyen, T.; Noh, J.; Givens, R. S. Cold Spring Harb. Protoc. 2013, 2013, 118. 26. Bartusik, D.; Minnis, M.; Ghosh, G.; Greer, A. J. Org. Chem.2013, 78, 8537. 27. Mahendran, A.; Kopkalli, Y.; Ghosh, G.; Ghogare, A.; Minnis, M.; Kruft, B. I.; Zamadar, M.; Aebisher, D.; Davenport, L.; Greer, A. Photochem. Photobiol.2011, 87, 1330. 28. Merlin, J. L.; Azzi, S.; Lignon, D.; Ramacci, C.; Zeghari, N.; Guillemin, F. Eur. J. Cancer 1992, 28a, 1452. 29. De Mayo, P. Pure & Appl. Chem. 1982, 54, 1623 30. Bauer, R. K.; Borenstein, R.; De Mayo, P.; Okada, K.; Rafalska, M.; Ware, W. R.; Wu, K. C. J. Am. Chem. Soc. 1982, 104, 4635. 31. Turro, N. J. Tetrahedron 1987, 43, 1589. 32. Ottaviani, M. F.; Turro, N. J.; Jockusch, S.; Tomalia, D. A. J. Phys. Chem. B 2003, 107, 2046. 33. Jockusch, S.; Sivaguru, J.; Turro, N. J.; Ramamurthy, V. Photochem. Photobiol. Sci. 2005, 4, 403. 34. Ramasamy, E.; Jayaraj, N.; Porel, M.; Ramamurthy, V. Langmuir 2012, 28, 10. 35. Natarajan, A.; Kaanumalle, L. S.; Jockusch, S.; Gibb, C. L. D.; Gibb, B. C.; Turro, N. J.; Ramamurthy, V. J. Am. Chem. Soc.2007, 129, 4132. 36. Greer, A. Nature 2007, 447, 273. 37. Moan, J. J. Photochem. Photobiol. B 1990, 6, 343. 38. Feigenbaum, J. J.; Choubal, M. D.; Crumrine, D. S.; Kanofsky, J. R. Peptides 1996, 17, 1213. 39. Hatz, S.; Poulsen, L.; Ogilby, P. R. Photochem. Photobiol. 2008, 84, 1284. 40. Roeder, B.; Naether, D.; Lewald, T.; Braune, M.; Nowak, C.; Freyer, W. Biophys. Chem. 1990, 35, 303. 41. Kuwabara, M.; Yamamoto, T.; Inanam, O.; Sato, F.Photochem. Photobiol. 1989, 49, 37. 42. Sauvaigo, S.; Douki, T.; Odin, F.; Caillat, S.; Ravanat, J. L.; Cadet, J. Photochem. Photobiol. 2001, 73, 230. 43. Di Mascio, P.; Teixeira, P. C.; Onuki, J.; Medeiros, M. H.; Dornemann, D.; Douki, T.; Cadet, J. Arch. Biochem. Biophys. 2000, 373, 368. 44. Pedersen, B. W.; Sinks, L. E.; Breitenbach, T.; Schack, N. B.; Vinogradov, S. A.; Ogilby, P. R. Photochem. Photobiol. 2011, 87, 1077. 25
Chapter 3. Fluorinated PDT Device Tips and Their Resistance to Fouling for In-vivo
Sensitizer Release
3.1 Introduction
As we discussed in Chapter 2, our pointsource PDT device produced a highly defined killing radius of glioma U871 and OVCAR-5 cancer cells in-vitro2. This observation raises key questions about limitations of pointsource PDT that have not yet been addressed. Does the device tip biofoul? Would biofouling be expected to arise during the time course of a typical PDT session?
Do cells adhere to the tip and impede sensitizer photorelease? Does fluorination of the tip increase biofouling resistance in the pointsource PDT technique?
Proteins, cells or microorganisms, in this manuscript called “biomaterial”, are known to adhere to surfaces, such as hydrophilic silica3,4, but less to hydrophobic fluorinated silica5. Indeed, polymers have been developed that have antibiofouling coatings, such as fluoropolymers and poly(ethylene glycol) (PEG) polymers.6-8 Some surfaces offer added biofouling protection by the production of reactive oxygen species (ROS). A vanadium pentoxide nanoparticle surface as a haloperoxidase mimic is known to produce singlet oxygen and resist biofouling.9 A porphyrin-
1 10 modified film that produced O2 was found to have antibacterial activity and anti-biofouling activity11. Similarly, silica/polydopamine/silver nanoparticle12, copper iodide nylon13 and electrochemical surfaces14, which produced ROS were found to resist biofouling. A self-cleaning superhydrophobic surface containing TiO2 nanoparticles was also found to photooxidize bovine serum albumin15.
Thus, our hypothesis was that inhibition of sensitizer release in pointsource PDT device
(Figure 1) will scale with biomaterial adsorption on the device tip. We had also tested the 26 hypothesis that sensitizer turnout levels will be better maintained based on the presence of a fluorosilane coating (tip 1) compared to the native silica surface (tip 5). We have analyzed whether surface biomaterial fouling limits tip output of sensitizer in pointsource PDT.
Figure 1. Schematic presentation of the pointsource PDT technique with various device tips. (a) Diode laser light and O2 gas are passed through a hollow fiber optic and emerge through the silica tip that is functionalized by a sensitizersilane and/or fluorosilane outer layer. An inset on the left shows the sensitizer structure. (b) The proposed mechanism is shown summarizing the steps in the sensitizer release system of tip 1. Much of the laser light is distributed out of the end of the tip. The device tip leaves behind the sensitizer upon conversion of the ethene to a dioxetane 3 and 1 additional O2 is generated away from the tip. (c) Shown is a photograph of four device tips [sized 5 × 10 mm2 (d × l)] and chemical drawings of the bottom of tips with the anchored groups or silanol.
In this chapter, we report on the biofouling of pointsource PDT device tips tested after placing the tip in contact with the surface of a surgically-exposed flank tumor (SQ20B tumors in nude mice). Figure 2 shows an image of the device tip placed on the exposed tumor surface. In 27 other experiments, device tips were soaked in whole blood as a phantom body fluid. Overlapping chromophores make the delivery of sensitizer into the tumor or blood samples difficult to quantitate. For this reason, fouling effects of the device tips were quantitated by a sensitizer photorelease inhibition analysis. Data were also collected with a bicinchoninic acid assay (BCA) and X-ray photoelectron spectroscopy (XPS) to quantitate the amount of biomaterial (e.g., proteins, cells, etc.) adsorbed on “dummy” tips 6 and 7, which contained no sensitizer. The results not only show that tip 1 biofouls by ~8%, based on experiments in the presence and absence of biomaterial, but retains a rapid sigmoidal release feature indicative of an autocatalytic mechanism.
Figure 2. A schematic of the cylindrical device tip held vertically, through an incision revealing the SQ20B tumor on the flank of a nude mouse. Red laser light and oxygen gas travel through the 3 1 hollow fiber optic. Sensitizer, O2 and O2 emerge out the device tip. (All in-vivo procedures were performed by Joann M. Miller)
3.2 Results
Studies were performed on device tips that were biofouled through exposure to one of two types of media (mouse flank tumor and rat whole blood). The effects of biofouling on sensitizer photorelease were evaluated. After biofouling effects are established, repellent materials could be further developed16-21 as more effective pointsource PDT device tips.
28
3.2.1 Effects of Biofouling on Device Tip Sensitizer Release
Initially, we conducted experiments to quantitate biofouling of the device tip by contact with SQ20B tumors. Sensitizer photorelease inhibition—which can occur from the adhering of tumor biomaterial—was used as evidence for device tip biofouling. Development of head and neck cancer model in mice and all the in-vivo procedures were conducted by Joann M. Miller in Dr.
Theresa M. Busch’s laboratory.
Table 1 shows that exposing the tip to SQ20B tumor affects the yield of sensitizer photorelease. When exposed to tumor for 2 h in the dark, Table 1 (entries 1 and 5, and 2 and 6) show an 8% reduction in tip 1, and an 18% reduction in tip 5. The inhibition of sensitizer 4 photoreleased was lower for fluorinated tip 1 compared to the native tip 5 (~10%). We found that the reduction in amount of sensitizer released was similar for tips biofouled with tumor and with blood (Table 1, compare entries 1 with 3, and 2 with 4). The tips were then dissolved by hydrofluoric acid and show that 5-8% of sensitizer remained bound to the surface, which indicates that the tips were comparable since all were near depleted of sensitizer.
29
Table 1. Tumor- or blood-contact dependence of the photorelease of sensitizer 4 from device tips 1 and 5 into n-butanol a
% covalently- test % photoreleased % adsorbed entry device tip bound sensitizer medium sensitizer 4 sensitizer on tip remaining on tip b 1 1 77±2 17±5 6±2 tumor 2 5 40±2 53±5 7±3 3 1 79±2 16±5 5±2 blood 4 5 38±3 55±5 7±4 5 1 85±2 9±3 6±5 none 6 5 58±3 34±5 8±4 a The device tip was placed in contact with the surface of a surgically-exposed flank tumor or presoaked in blood or n-butanol for 1 h under subdued light. The tip was then affixed to the hollow 2 optical fiber, delivering O2 and 669 nm laser light through the tip (irradiance = 51 to 550 mW/cm , time = 0 to 2 h) and the amount of 4 released determined after 1 h. After the 5 mm diameter operating front face of the device tip was exposed to tumor or blood, sensitizer photorelease was monitored in n-butanol. The amount of 4 adsorbed on the tip was determined by Soxhlet extraction with methanol at ~70 °C for 24 h. b The last remaining covalently-bound sensitizer was quantified by removal with HF and analysis of the Q-band (λ = 663 nm) of the sensitizer by UV-Vis spectroscopy judged against a prior constructed calibration curve of the sensitizer. Experiments were carried out 3 or more times.
Figure 3 shows the time-course of sensitizer photorelease from device tips 1 and 5 after exposure to the tumor or whole blood. That is, the photorelease in n-butanol was carried out after the tip had been in contact with biological media. Fluorinated tip 1 gave a sigmoidal photorelease, whereas the native silica tip 5 gave a slower (pseudolinear) photorelease. Furthermore, the amount of sensitizer adsorbed to tip 1 was three times less compared to tip 5 as revealed by Soxhlet extraction with methanol at 68-70 °C for 24 h, to detach any adsorbed sensitizer. After the covalent ethene bridge bonding the sensitizer to the surface is broken, the amount of sensitizer 4 adsorbed to tip 1 was ~17% (35 nmol) and to tip 5 was ~54% (11 nmol). The tip fouling experiments conducted with the tumors and blood are consistent with each other and complementary. 30
Figure 3. The percent of sensitizer 4 photoreleased free from device tip 1 (black lines) and tip 2 (grey lines) in 1 mL n-butanol. The tips were pre-exposed for 1 h to: (a) mouse flank tumor through an incision (●), (b) whole blood (▲), and (c) n-butanol (■). The concentration of sensitizer 4 was measured by UV−VIS following the sensitizer Q-band at λ = 663 nm.
Similar to Figure 3, Figure 4 also shows the time-course of sensitizer photorelease from device tips 1 and 5. The difference between Figure 3 and 4 is that the latter was collected with the tip placed on the SQ20B tumor with light and oxygen purging through it for 1 h (amount of sensitizer delivered to tumor: 15% for tip 1 and 3% for tip 5). Once placed in n-butanol, Figure 4 shows the fluorinated tip 1 release significantly more sensitizer (35%) than the native tip 5 (12%).
That is, sigmoidal release was observed since the recording of sensitizer departure started at time
= 1 h, not at time = 0 h. 31
Figure 4. A plot of remaining sensitizer 4 photoreleased into n-butanol vs time for tip 1 (■) and tip 5 (●). These points were collected after the tips already used for 4 photorelease in a mouse flank tumor for 1 h.
3.2.2 Effect of Adsorption of Cellular Material
The data show that biomaterial (e.g. proteins, cells, etc.) from the SQ20B tumors and rat blood adsorb onto the tip surfaces. The amount of this biofouling on both the fluorinated silica 6 and native silica 7 surfaces was determined based on a BCA assay and XPS measurements. XPS analysis was performed by Dr. Bikash Mondal in Dr. Alan M. Lyons’s laboratoty.
Table 2 and Figure 5 show the BCA assay results and the amount of biomaterial residue adhering to tips 6 and 7. A ~15% higher adsorption was observed on the native silica 7 compared to the fluorinated silica 6. In the first 5 min, there is a rapid adsorption (85 µg for 6; 102 µg for 7).
After the biofouling increases sharply during the first 5 min, it slows to 2 h and continues for 10 h. The rate of biomaterial adsorption onto the silica surfaces remains constant from ~1 h to 10 h. 32
Figure 5. A plot of blood cells adsorbed to tip 6 (■) and tip 7 (●) vs time when immersed in blood. The quantity of protein was determined by a BCA assay after stripping with a SolvableTM solution.
Table 2. Blood and tumor cell adsorption to fluorinated silica 6 and native silica 7 surfaces a
cell quantities adsorbed on device tips (µg) time whole blood b SQ20B tumor b fluorinated tip 6 native tip 7 fluorinated tip 6 native tip 7 6 min 85±4 102±5 - - 15 min 90±5 105±3 - - 0.5 h 100±3 115±3 - - 1 h 105±3 125±8 35±8 58±7 3 h 127±3 140±5 - - 10 h 198±8 201±5 - - a Device tips were pre-exposed to rat blood (100 µL) for 1 h. Error bounds were obtained from 2 or more measurements. b Adsorbed tumor or blood cells were stripped off by SolvableTM and quantitated by a BCA assay.
33
Figure 6 shows an XPS analysis of the adsorption of biomaterial from blood on tip 6. The ratio of the N 1s to Si 2p and C 2s to Si 2p peaks were used to determine the relative amount of protein and other biological materials on the surface. During the first hour of immersion, the amount of biomaterial adsorbed on the surface increases rapidly with time. The C 1s to N 1s ratio remains constant throughout this period, indicating that C and N adsorption rates are similar. This rapid initial adsorption of biomaterials is consistent with the adsorption isotherm of protein studied on various surfaces22.
Figure 6. Time profile for XPS peak area ratio changes of C 1s/Si 2p (▲), N 1s/Si 2p (●), and C 1s/N 1s (■) of tip 6 immersed in whole blood.
After 1 hour of immersion, however, the N 1s/Si 2p ratio remains relatively constant
(Figure 6). This apparent stability may be due to the formation of a complete biomaterial coating on the silica tip after 1 h of immersion. This layer is sufficiently thick to completely cover the underlying silica, preventing detection of Si 2p peaks in the XPS spectrum, as shown in Figure 7.
The N 1s (~400 eV) signal is characteristic of adsorbed biomaterial at the surface.23,24 Peak area ratio of N 1s/ Si 2p, C 1s/N 1s and C 1s/Si 2p were compared to eliminate any variation between 34 different XPS samples.25,26 Accumulation of biomaterial does continue, as shown in Figure 5 for the BCA assay results. However thicker layers of biomaterial would not be distinguished by XPS due to the limited penetration depth (5-30 nm) of the escaping electrons. Thus, once a sufficiently thick layer of biomaterial is deposited on the silica surface to obscure the Si 2p peak, XPS cannot be used to detect further biomaterial accumulation.
Figure 7. XPS spectra of clean silica 6 and silica 6 contaminated by whole blood. (XPS analysis was performed by Dr. Bikash Mondal)
3.3 Discussion
Some details are now available on how pointsource PDT device tips 1 and 5 are fouled.
Tip fouling experiments were carried out where SQ20B tumors and whole blood showed sensitizer release inhibition of ~6% for 1 and ~10% for 5 after 1 h. Thus, the hydrophobicity of the fluorinated tip provides some protection against biofouling. Figure 3 shows sigmoidal release behavior of 4 for tip 1 that is attributed to an autocatalytic process, where surface fouling does not significantly inhibit the release of sensitizer.
We now know that tip fouling was minimal because of the sensitizer turnout levels that were maintained. Thus, fouling is not expected to be problematic over the time course of a typical 35
PDT session; furthermore, the tips are intended to be replaced after each treatment. This is an important criterion to have met due to the sensitizer delivery feature of the pointsource PDT strategy.1 We demonstrate that ~15% less biomaterial adheres to the fluorinated silica than to native silica. The BCA assay shows a constant increase of biomaterial from 1 hour to 10 h while the XPS shows that the level after 1 h remains constant. This is because once a complete and sufficiently thick layer of biomaterial forms on the silica surface, the underlying Si can no longer be seen by the XPS instrument. It makes sense that when silica is treated with nonafluorosilane the fraction of silicon observable on the surface by XPS at time = 0 (before protein adsorption) is smaller than native silica. The fluorinated silica surface does adhere proteins and cells—just less than the native silica surface due to the residual charges on the untreated silica. Bacteria could be present as a foulant, although there are more cells present than bacteria. Our work did not examine whether the adsorption of biomaterial is due to a hydrophobic and electrostatic interactions27-29, or other mechanisms.
Lastly, we now know there is a complimentary effect where the fluorinated tip 1 not only
1 30-32 repels biofoulants better, it also suppresses surface O2 physical quenching for a more efficient sensitizer photorelease. It could be argued that added biofouling protection results from the
1 production of O2 at the surface of tips 1 and 5 as has been observed for other surfaces which
1 9-15 1 produce O2 or ROS. We believe that biomaterial on (or near) the tip where O2 is generated will retard and/or prevent fouling on that surface. The magnitude of this effect will depend on
1 several factors. If sufficient O2 is generated in an environment with low amounts of proteins, cells, microorganisms, etc. then biofouling might, indeed, be prevented. However, in an environment
1 rich in proteins, cells, etc., then the O2 production rate would need to be sufficiently high to
1 overcome the loading of biomaterial that could react with O2. Each cell, bacterium or protein 36
1 33-35 1 could consume many O2 molecules. Other studies have examined the reaction of O2 with biological media that produce peroxides, which can decompose and/or chemiluminesce36-50.
3.4 Conclusion
There is still much research to be done before pointsource PDT can be used clinically. Data obtained from the tumor and blood fouling studies described here will contribute to the ongoing development of pointsource PDT.1 The pointsource PDT device tip was modified with nonafluorosilane to improve its protection against biofouling. The fluorinated tip led to improved biofouling resistance based on sensitizer photorelease performance.
Future studies could continue to resolve outstanding questions concerning a one-step PDT process (i.e. simultaneous delivery of sensitizer, oxygen and light) in order to simplify the application of PDT. Other device configurations could be benefitial. Advantages may exist for micropillar roughened device tips, such as 3D-printed superhydrophobic surfaces, which reduce the contact between the tip and tissue.41,42 Device tips could also be designed with different sensitizer types43-49 to customize delivery based on tumor type.50-52 Finally, research efforts could seek advantages for intraoperative use of pointsource PDT for precision treatment of residual disease. Research efforts are in progress in these directions.
3.5 Experimental Section 3.5.1 Materials and Methods
(i) Device fabrication. A fiber-optic device with silica device tips 1 and 5 was used as described previously.1,30 Briefly, pieces of silica were fluorinated by soaking in 1×10-3 M
3,3,4,4,5,5,6,6,6 nonafluorohexyltrimethoxysilane and then refluxed in toluene for 24 h. Any 37 nonafluorosilane that was not covalently attached to the silica surface was washed away by Soxhlet extraction in methanol for 24 h.
(ii) Tumor model. SQ20B head and neck squamous carcinoma cells (ATCC, Manassas,
VA) were cultured in DMEM medium (ATCC) supplemented with 10% fetal bovine serum
(Gibco, Carlsbad, CA), 2 mM L-glutamine (Gibco), 100 units/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco) and maintained in a humidified atmosphere with 5% CO2 at 37 °C. Cells in log phase were harvested, and resuspended at 2×107 cells/mL in a 1:1 solution of phenol red-free matrigel basement membrane matrix (BD Biosciences, San Jose, CA) and normal saline53-55. Each
8-9–week old female athymic nude mouse (Charles River Laboratories in Frederick, MD) was inoculated with 1×106 SQ20B cells intradermally on the right and left flank of the mouse. The tumor suspension was in 50% matrigel. Animals were used for experiments two weeks after tumor inoculation when the intradermal tumors reached volumes of ~50-150 mm³. The mice were anesthetized by inhalation of isoflurane in medical air delivered through a nosecone (VetEquip anesthesia machine, Pleasanton, CA). An incision was made adjacent to the flank tumor and a skin flap was created in order to expose the tumor. A device tip (1 or 5-7) was placed on the tumor surface for 1 h. Once the device tip was in contact with the tumor, two drops of saline solution were applied to the exposed area to prevent dehydration of the tissue and its adhesion. After treatment, the device tips were removed and preserved in dry ice for further analysis.
3.5.2 Sensitizer Photorelease Studies
The bottom face of tips 1, 5-7 were placed (i) on the surgically-exposed surface of a SQ20B tumor grown intradermally on the flank of athymic nude mouse, or (ii) in 100 µL whole blood obtained from Sprague Dawley rats at 37 °C. The tips were placed for 1 h on the tumor or in blood 38 either (i) in the dark or (ii) with 669-nm laser irradiation via hollow fiber optic coupling with oxygen flowing through the tip. The tips were then rinsed with 5 mL saline and analyzed for subsequent sensitizer photorelease (attached to the device) from the tips into 1 mL n-butanol, quantified by monitoring the Q-band (λ = 663 nm) with UV−VIS spectroscopy (6). After photorelease in n-butanol, the device tips were subjected to Soxhlet extraction with methanol at
68-70 °C for 24 h, thereby removing any adsorbed sensitizer, and then subjected to a previously described hydrofluoric acid stripping method (5) in order to determine the amount of sensitizer bound to the surface. Treatments were at a fluence rate of 51 mW/cm2 to 550 mW/cm2 over a period of 0 to 2 h.
3.5.3 BCA Studies
After exposing the front face of device tips to the SQ20B tumor or the Sprague Dawley rat blood, the tips were washed with 5 mL saline and the adsorbed biomaterial was stripped off with
200 µL 20% (v/v) SolvableTM in 1 mL 1% SDS solution. The amount of biomaterial adsorbed on the device tips, as µg, was determined using BCA assay in comparison to the calibration curve of standard protein albumin.56,57 Experiments were carried out 2 or more times. From the plot, 10 µL blood has 876±20 µg protein. The compatibility of BCA assay in 20% (v/v) SolvableTM in 1 mL
1% SDS solution for detection of proteins in blood was validated by using different volumes of blood.
39
3.5.4 XPS Studies
The flat front faces of tips were placed in 100 µL whole blood obtained from Sprague
Dawley rats for various lengths of time at 37 °C. After exposing the device tips to the blood, the tips were washed with 5 mL saline and dried using a high vacuum freeze dryer lyophilizer. The amount of protein adsorbed on glass samples was analyzed with XPS using an Omicron
Nanotechnology system equipped with an Al Kα X-ray source (1486.6 eV). A hemispherical analyzer (EA-125) was operated in constant analyzer energy (CAE) mode and equipped with one channel electron multiplier to measure the binding energies of the emitted photoelectrons. XPS spectra were collected under ultra-high vacuum (<1×10-8 torr) with high resolution scans (0.5 eV step size) over the range of binding energy (from 600 to 0 eV) to cover N 1s (~400 eV), O 1s
(~532.8 eV) and C 1s-2p (~285-290 eV), for signals typical of adsorbed protein57. Surface areas with a diameter less than 1.5 mm were analyzed and referenced by setting C 1s peak to 284.8 eV to compensate for residual charging. Finally, the peak area ratios of N 1s/Si 2p, C 1s/N 1s and C
1s/Si 2p were calculated to compare the amount of adsorbed protein on the silica surfaces58.
3.6 References
1. Ghogare, A. A.; Rizvi, I.; Hasan, T.; Greer, A. Photochem. Photobiol.2014, 90, 1119. 2. Bartusik, D.; Aebisher, D.; Ghogare, A.; Ghosh, G.; Abramova, I.; Hasan, T.; Greer, A. Photochem. Photobiol.2013, 89, 936. 3. Pohl, J.; Saltsman, I.; Mahammed, A.; Gross, Z.; Roder, B. J. Appl. Microbiol. 2015, 118, 305. 4. Roy, I.; Kumar, P.; Kumar, R.; Ohulchanskyy, T. Y.; Yong, K.-T.; Prasad, P. N. RSC Adv. 2014, 4, 53498. 5. Costacurta, S.; Falcaro, P.; Malfatti, L.; Marongiu, D.; Marmiroli, B.; Cacho-Nerin, F.; Amenitsch, H.; Kirkby, N.; Innocenzi, P. Langmuir, 27, 3898. 6. Ionov, L.; Synytska, A.; Kaul, E.; Diez, S. Biomacromolecules 2010, 11, 233. 7. Imbesi, P. M.; Gohad, N. V.; Eller, M. J.; Orihuela, B.; Rittschof, D.; Schweikert, E. A.; Mount, A. S.; Wooley, K. L. ACS Nano 2012, 6, 1503. 8. Chiag, Y.-C.; Chang, Y.; Chen, W.-Y.; Ruaan, R.-C. Langmuir 2012, 28, 1399. 9. Natalio, F.; Andre, R.; Hartog, A. F.; Stoll, B.; Jochum, K. P.; Wever, R.; Tremel, W. Nat. 40
Nanotechnol. 2012, 7, 530. 10. Krouit, M.; Granet, R.; Krausz, P. Bioorg. Med. Chem. 2008, 16, 10091. 11. Li, J.; Yin, L.; Qiu, G.; Li, X.; Liu, Q.; Xie, J. J. Mater. Chem. A 2015, 3, 6781. 12. Zhangwei, G.; Junzeng, X.; Tao, L.; Xiao, S.; Yuanyuan, S.; Huixian, W. Micro Nano Lett. 2014, 9, 210. 13. Sato, T.; Fujimori, Y.; Nakayama, T.; Gotoh, Y.; Sunaga, Y.; Nemoto, M.; Matsunaga, T.; Tanaka, T. Appl. Microbiol. Biotechnol. 2012, 95, 1043. 14. Perez-Roa, R. E.; Anderson, M. A.; Rittschof, D.; Hunt, C. G.; Noguera, D. R. Biofouling 2009, 25, 563. 15. Zhao, Y.; Liu, Y.; Xu, Q.; Barahman, M.; Lyons, A. M. ACS Appl. Mater. Interfaces 2015, 7, 2632. 16. Bennett, S. M.; Finlay, J. A.; Gunari, N.; Wells, D. D.; Meyer, A. E.; Walker, G. C.; Callow, M. E.; Callow, J. A.; Bright, F. V.; Detty, M. R. Biofouling 2010, 26, 235. 17. Sokolova, A.; Bailey, J. J.; Waltz, G. T.; Brewer, L. H.; Finlay, J. A.; Fornalik, J.; Wendt, D. E.; Callow, M. E.; Callow, J. A.; Bright, F. V.; Detty, M. R. Biofouling 2012, 28, 143. 18. Hou, X.; Hu, Y.; Grinthal, A.; Khan, M.; Aizenberg, J. Nature 2015, 519, 70. 19. MacCallum, N.; Howell, C.; Kim, P.; Sun, D.; Friedlander, R.; Ranisau, J.; Ahanotu, O.; Lin, J. J.; Vena, A.; Hatton, B.; Wong, T.-S.; Aizenberg, J. ACS Biomater. Sci. Eng. 2015, 1, 43. 20. Rosenhahn, A.; Schilp, S.; Kreuzer, H. J.; Grunze, M. Phys. Chem. Chem. Phys.2010, 12, 4275. 21. Xiao, L.; Li, J.; Mieszkin, S.; Di Fino, A.; Clare, A. S.; Callow, M. E.; Callow, J. A.; Grunze, M.; Rosenhahn, A.; Levkin, P. A. ACS Appl. Mater. Interfaces 2013, 5, 10074. 22. Xu, Q. F.; Liu, Y.; Lin, F.-J.; Mondal, B.; Lyons, A. M. ACS Appl. Mater. Interfaces 2013, 5, 8915. 23. Malmsten, M. Colloids and Surf. B Biointerfaces 1995, 3, 297. 24. Iucci, G.; Polzonetti, G.; Infante, G.; Rossi, L. Surf. Interface Anal. 2004, 36, 724. 25. Gruian, C.; Vanea, E.; Simon, S.; Simon, V. Biochim. Biophys. Acta 2012, 1824, 873. 26. Vanea, E.; Simon, V. Appl. Surf. Sci. 2011, 257, 2346. 27. Zander, N. E.; Orlicki, J. A.; Rawlett, A. M.; Beebe, T. P., Jr. ACS Appl. Mater. Interfaces 2012, 4, 2074. 28. McUmber, A. C.; Randolph, T. W.; Schwartz, D. K. J. Phys. Chem. Lett. 2015, 6, 2583. 29. Moerz, S. T.; Huber, P. Langmuir 2014, 30, 2729. 30. Bartusik, D.; Aebisher, D.; Ghosh, G.; Minnis, M.; Greer, A. J. Org. Chem. 2012, 77, 4557. 31. Bartusik, D.; Minnis, M.; Ghosh, G.; Greer, A. J. Org. Chem. 2013, 78, 8537. 32. Ghosh, G.; Minnis, M.; Ghogare, A. A.; Abramova, I.; Cengel, K. A.; Busch, T. M.; Greer, A. J. Phys. Chem. B.2015, 119, 4155. 33. Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. 34. Xu, N.; Yao, M.; Farinelli, W.; Hajjarian, Z.; Wang, Y.; Redmond, R. W.; Kochevar, I. E. Lasers Surg. Med. 2015, 47, 17. 35. Yao, M.; Yaroslavsky, A.; Henry, F. P.; Redmond, R. W.; Kochevar, I. E. Lasers Surg. Med. 2010, 42, 123. 36. Takemura, T.; Ohta, N.; Nakajima, S.; Sakata, I. Photochem. Photobiol. 1992, 55, 137. 37. Alarcon, E.; Edwards, A. M.; Garcia, A. M.; Munoz, M.; Aspee, A.; Borsarelli, C. D.; Lissi, E. A. Photochem. Photobiol. Sci. 2009, 8, 255. 38. Ferraz, R. C. M. C.; Fontana, C. R.; Ribeiro, A. P. d.; Trindade, F. Z.; Bartoloni, F. H.; Baader, J. W.; Lins, E. C.; Bagnato, V. S.; Kurachi, C. J. Photochem. Photobiol. 2011, 103, 41
87. 39. Mano, C. M.; Prado, F. M.; Massari, J.; Ronsein, G. E.; Martinez, G. R.; Miyamoto, S.; Cadet, J.; Sies, H.; Medeiros, M. H. G.; Bechara, E. J. H.; Di Mascio, P. Sci. Rep. 2014, 4, 5938. 40. Miyamoto, S.; Martinez, G. R.; Medeiros, M. H.; Di Mascio, P. J. Photochem. Photobiol. B 2014, 139, 24. 41. Choi, C.-H.; Kim, C.-J. C. Langmuir 2009, 25, 7561. 42. Lima, A. C.; Mano, J. F. Nanomedicine 2015, 10, 271. 43. Kuthanapillil, J., Avirah, R. R.; Ramaiah, D. ARKIVOC 2007, 8, 296. 44. Dror, S. B.; Bronshtein, I.; Garini, Y.; O'Neal, W. G.; Jacobi, P. A.; Ehrenberg, B. Photochem. Photobiol. Sci. 2009, 8, 354. 45. Ben-Dror, S.; Bronshtein, I.; Wiehe, A.; Roder, B.; Senge, M. O.; Ehrenberg, B. Photochem. Photobiol. 2006, 82, 695. 46. Dosselli, R.; Ruiz-González, R.; Moret, F.; Agnolon, V.; Compagnin, C.; Mognato, M.; Sella, V.; Agut, M.; Nonell, S.; Gobbo, M.; Reddi, E. J. Med. Chem. 2014, 57, 1403. 47. Jux, N. and B. Röder (2010) Targeting Strategies for Tetrapyrrole-based Photodynamic Therapy. In Handbook of Porphyrin Science, Vol. 4 (Edited by K. M. Kadish, K. M. Smith and R. Guilard), pp. 325-401. World Scientific Publishing Co. Pte. Ltd., Singapore. 48. Majumdar, P.; Nomula, R.; Zhao, J. J. Mater. Chem. C 2014, 2, 5982. 49. Zhao, J.; Wu, W.; Sun, J.; Guo, S. Chem. Soc. Rev. 2013, 42, 5323. 50. Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D.; Korbelik, M.; Moan, J.; Mroz, P.; Nowis, D.; Piette, J.; Wilson, B. C.; Golab, J. CA Cancer J. Clin. 2011, 61, 250. 51. St. Denis, T. G., Y.-Y. Huang and M. R. Hamblin (2014) Cyclic Tetrapyrroles In Photodynamic Therapy: The Chemistry of Porphyrins and Related Compounds in Medicine. In Handbook of Porphyrin Science, Vol. 27, K. M. Kadish, K. M. Smith and R. Guilard, Eds., 27, pp. 255-301. World Scientific Publishing Co. Pte. Ltd., Singapore. 52. Dabrowski, J. M.; Arnaut, L. G. Photochem. Photobiol. Sci., 2015, 14, 1765. 53. Busch, T.; Cengel, K. A.; Finlay, J. Cancer Biol. Therapy 2009, 8, 540. 54. Grossman, C. E.; Pickup, S.; Durham, A.; Wileyto, E. P.; Putt, M. E.; Busch, T. M. Lasers Surg. Med. 2011, 43, 663. 55. Cerniglia, G. J.; Dey, S.; Gallagher-Colombo, S. M.; Daurio, N. A.; Tuttle, S.; Busch, T. M.; Lin, A.; Sun, R.; Esipova, T. V.; Vinogradov, S. A.; Denko, N.; Koumenis, C.; Maity, A. Mol. Cancer Ther. 2015, 14, 1928. 56. Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76. 57. Wyatt, A. R.; Wilson, M. R. J. Biol. Chem. 2010, 285, 3532. McArthur, S. L. Surf. Interface Anal. 2006, 38, 1380. 58. Vanea, E.; Simon, V. Appl. Surf. Sci. 2013, 280, 144.
42
Chapter 4. Synthesis of an Poly(ethylene glycol) Galloyl Sensitizer Tip for a Pointsource
Photodynamic Device
4.1. Introduction
Thus far, we have decribed a pointsource PDT device (Chapter 1) that channels O2 and red diode laser light through a silica probe tip and it can be implanted at the cancer cells in-vitro
(Chapter 2) and at tumor site (Chapter 3). Briefly, the pointsource device generates singlet oxygen
1 ( O2) which serves dual role, (a) causes the release of sensitizer through cleavage of a dioxetane intermediate (Scheme 1), and (b) leads to cell death. However, there is a need for further progress in device tip design and synthesis suited to releasing sensitizer molecules.
Other research groups have reported on pheophorbide1-12 and chlorin derivatives13-22 for efficient photosensitized eradication of tumor cells by alleviating solubility and aggregation problems. We23,24 and others25-32 have observed that polyethylene glycol (PEG) groups bonded to chlorophyll sensitizers can increase their solubility in solution, cell uptake, and phototoxicity in
23 cancer cells. But previous work with chlorin e6 had required chemically bonding each PEG group individually with the third PEG adding in low yield (triPEG-chlorin silica 2 and triPEG-chlorin
Teflon/PVA nanocomposite 3, Figure 1). This led us to pose the question: Can three PEG groups be installed in one step by the use of a galloyl group at its connecting point in tip fabrication (1,
Figure 1)?
43
Scheme 1. Self-sensitization, singlet oxygen formation and dye cleavage
Thus, the aims of the present work were to (i) design a PEG sensitizer based on computed solubility via octanol/water calculations, (ii) fabricate triPEG-galloyl pheophorbide silica 1 as a new device tip material, and (iii) develop a synthetic approach with fewer steps to reach 1 and with increased stability of the PEG-sensitizer bond compared to the previously reported 2 and 323. As we will see, 1 is synthesized in fewer steps, and in higher overall yield and stability compared to
2 and 3. Other aspects such as the efficacy for scaling up the production of the optical fiber device tips will also be discussed.
44
Figure 1. Chemical structures of the new triPEG-galloyl pheophorbide silica 1, and previously reported triPEG-chlorin silica 2 and triPEG-chlorin Teflon/PVA nanocomposite 3.23
4.2. Results and Discussion
4.2.1 Sensitizer Design and Solubility (Table 1)
Short chain PEG groups, such as [–O(CH2CH2O)3CH3] when bonded to sensitizers can reduce self-aggregation33-38, and retain some propensity to localize in cell membranes compared to unsubstituted sensitizers24,39,40. Therefore, we surmised that a galloyl unit carrying three PEG groups could be bonded to pheophorbide silica end-tips of a pointsource PDT device. We were unsure whether the triPEG-galloyl pheophorbide would ameliorate the low water solubility problem to the same extent as the triPEG chlorin. Experimental log P measurements were desirable in octanol and water, but the sensitizer quantity is sparse in water. Thus, computed log P values were obtained with the ACD algorithm, which has performed well in predicting the log P values of drugs41-45. 45
Table 1 shows the computed log P values. TriPEG-galloyl pheophorbide and triPEG- chlorin (entries 2 and 4) led to a lipophilic deamplification by 1.9 log units compared to unsubstituted sensitizers, respectively (entries 1 and 3). Lower log P values of PEGylated compounds would be expected, where their curling onto the porphyrin ring inhibits aggregation due to π-π stacking forces46-52. The reduced log P of the PEGylated pheophorbide versus native hydrophobic pheophorbide-a will make the former more accessible and less prone to aggregating, to potentially address the challenge sterilization of surgical margins. Next, the synthesis of triPEG- galloyl pheophorbide silica 1 was carried out.
Table 1. Computed octanol-water partition coefficients (log P) with three [–O(CH2CH2O)3CH3] PEG groups appended to chlorin e6 and pyropheophorbide-a using the ACD program
entry compound number of PEG groups computed log P
a 1 chlorin e6 0 6.6 ± 1.6
a 2 triPEG-chlorin e6 3 4.7 ± 1.7
3 pyropheophorbide-a 0 7.2 ± 1.6
4 triPEG-galloyl pheophorbide 11 3 5.1 ± 1.6 a Reference 24.
4.2.2 Synthesis (Schemes 2 and 3)
Scheme 2 shows that triethyleneglycol monomethylether (PEG) 4 reacted with 4- toluenesulfonyl chloride (TsCl) in presence of sodium hydroxide in THF to form PEG-tosylate 5.
Similar to literature reports53-55, a potassium carbonate-mediated PEGylation of methyl gallate 6 in acetone led to 7 which was followed by reduction with LiAlH4 in THF resulting in triPEG- galloyl alcohol 8 in 58% yield after 3 steps. Scheme 3 shows that the pheophorbide methylester 9 reacted with HBr in acetic acid to brominate the exocyclic alkene at the carbon 31 position and 46 subsequent nucleophilic substitution reaction with 8 in dichloromethane led to the triPEG-galloyl pheophorbide methylester 10 in 40% yield in 2 steps. The methyl ester 10 was hydrolysed by a reaction with potassium carbonate to form triPEG-galloyl pheophorbide acid 11. A condensation reaction of acid 11 with alkene 12 was carried out with EDC and DMAP to form triPEG-galloyl pheophorbide alkene ester 13 in 50% yield. Next, the fabrication of the photoactive surface 1 is described.
Fluorinated silica 11 with loading ratio of 1:60 for Si–OH:C–F was prepared by reaction of silica with nonafluorotrimethoxysilane in toluene under reflux. The triPEG-galloyl pheophorbide alkene ester 13 was reacted with (3-iodopropyl)trimethoxysilane in NaH and THF and added to the pieces of fluorinated silica 11 [each piece was 0.33 g and sized ∼5 mm × ∼8 mm
(d × l)] in refluxing toluene to reach 1 with loading of 115 nmol per g of silica (ratio of
1860:111,400:1 for Si–OH:C–F:sensitizer). Unreacted nonafluorotrimethoxysilane and 13 were washed off of the silica by Soxhlet extraction in methanol.
Scheme 2. Synthesis of the triPEG galloyl compound 8
47
Scheme 3. Synthesis of triPEG-galloyl pheophorbide silica 1
4.2.3 Structural Assignments (Figures 2 and 3)
There are literature examples of synthetic modifications of the exocyclic alkene of pheophorbide to form an ether bond56,57. In our case, NMR spectroscopy58 was particularly useful in assigning structures. Figure 2 shows NMR data of 9 and 10 indicate the shift of the 31-proton 48 from 7.9 to 6.0 ppm, disappearance of the 32-alkene protons and appearance of 2 aromatic protons
(Ar-H) at 6.55 ppm in the 1H NMR spectra for 10.
COSY spectra for 10 showed correlation between the newly formed peak for 32-proton at
2.19 ppm and 31-protons at 6.01 ppm. HSQC spectra for 10 showed that C peak at 107.6 belongs to Ar-H, 71.2 ppm belongs to Bz-H and 71.46 ppm belongs to 31-H. The regioselective attachment of triPEG-galloyl group at carbon 31 position of 10 was shown by HMBC spectra where correlation existed between (a) Ar-H (6.55 ppm) and Bz-C (71.2 ppm) of triPEG-galloyl group, (b) 31-H (6.01 ppm) of porphyrin ring and Bz-C (71.2 ppm) of triPEG-galloyl group, and (c) Bz-H (4.6 ppm) and
31-C (71.46 ppm) (Figure 11). Formation of 11 is evident by HRMS (+ESI) observed [M]+ =
+ 1128.5882 corresponded well with the calculated C61H84N4O16 [M] = 1128.5875.
Figure 3 shows 1H NMR data of 13 indicating the appearance of 2 alkene protons at 6.08 ppm and 8 aromatic protons between 6.90-7.32 ppm from the alkene linker. COSY spectra for 13 showed correlation between peaks for the 17-proton at 4.32 ppm and 171-protons at 2.61 ppm and
172-proton at 2.73 ppm. HSQC spectra of 13 showed that C peak at 29.96 belongs to Bz-H of alkene, 31.0 ppm belongs to 172-H, 29.8 ppm belongs to 171-H and 172.0 ppm belongs to 173 ester
C=O. The attachment of alkene group by ester bond to the oxygen attached to carbon 173 position was confirmed by HMBC spectra of 13 where correlation was found between (a) Bz-H (4.97 ppm) of alkene linker and 173-C (172.0 ppm), (b) 172-H (2.73 ppm) and 173-C (172.0 ppm) and (c) 171-
H (2.61 ppm) and 173-C (172.0 ppm) (Figure 25).
49
Figure 2. 1H NMR spectra for the pheophorbide methylester 9 and triPEG-galloyl pheophorbide methylester 10.
Figure 3. 1H NMR spectrum of the triPEG-galloyl pheophorbide alkene ester 13.
50
4.2.4 Comparative Analysis (Table 2)
Synthetic, stability and other device tip data are compared. We found that (i) the triPEG- galloyl pheophorbide silica 1 was synthesized in higher yield compared to triPEG-chlorin silica 2 and triPEG-chlorin Teflon/PVA nanocomposite 3. The number of steps to reach 1 was decreased and more efficient compared to 2 and 3 due to the ease of introduction of three PEGs through a galloyl unit. (ii) There was a greater hydrolytic stability of the PEG-sensitizer conjugate 1 (ether bond) compared to the labile PEG-sensitizer conjugates of 2 and 3 (ester bonds). Previously, researchers have examined PEG-drug conjugates through irreversible ether bonds and reversible
59-63 ester bonds . (iii) Figure 4 shows the silica tip with CF3CF2CF2CF2 groups where facile sensitizer release is expected. For example, PEGylated sensitizer molecules would otherwise adhere to non-fluorinated silica23. Thus, the fluorinated device tip yields repellent characteristics to discharge the sensitizer and also yields high oxygen concentrations consequently producing highly focused quantities of singlet oxygen. (iv) Lastly, synthetic efficiency is one parameter that influences the feasibility of scale-up. Thus, making batches of tip 1 is more economical that tips 2 and 3.
51
Table 2. Comparative analysis of synthetic, stability and materials data
triPEG-chlorin triPEG-galloyl triPEG-chlorin silica Teflon/PVA pheophorbide silica 1 2 nanocomposite 3 % yield of alkene 18% (over 4 steps) 3% (over 6 steps) 3% (over 6 steps) ester precursor total number of 6 8 7 synthetic steps sensitizer loading 115 nmol 90 nmol 23 nmol quantity (per g) stability of PEG ~0% decomposition ≥ 80% decomposition ≥ 80% decomposition (after 1 h) at pH 2 and 8 b at pH 2 and 8 b at pH 2 and 8 b surface C– 111,000:1 142,000:1 750,000:1 F:sensitizer ratio
O2 concentration in n-butanol in the 0.522 mM 0.522 mM 0.559 mM presence of 0.2 g of 1-3 at 25 °C Number of probe tips fabricated from 100 4 2 5 mg sensitizer a Total number of person hours required 60 150 130 to fabricate a batch of 12 probe tips (h) a Commercially available pyropheophorebide-a and chlorin e6. b The decomposition of the triPEG-galloyl pheophorbide 10 and triPEG-chlorin24 were measured in homogeneous solution the absence of the silica support.
52
Figure 4. Scheme of pointsource PDT device: red laser light and oxygen gas emerge through the device tip and triggers the sensitizer release for singlet oxygen production away but in the near vicinity of the device tip. The red circles represent oxygen atoms, and the green circles represent fluorine atoms.
4.3. Conclusions
Because of the need to further advance the pointsource PDT technique64,65, a photocleavable triPEG-galloyl pheophorbide silica 1 was designed and synthesized. Unlike 2 and
3 that required each of the three PEG groups to be introduced one at a time, 1 had the three PEG 53 groups introduced in one-step with a galloyl substituent. This is an advantage from the fabrication and scale-up perspective of a pointsource PDT device.
With the completion of the synthesis of 1, next we will be able to examine whether a cylindrical, hemispherical, or rectilinear probehead is best for the fiber to get the right amount of sensitizer and oxygen delivered to kill cells directionally, e.g., in specific quadrants of a culture plate. Future work may also focus on 3D-geometric shaped TMOS/MeOH/H2O xerogels cast with flanges and ridges and with matrix porosities that can be increased66-69. To this end, pointsource
PDT can augment the current tools available for intraoperative use with improved control for sterilization of residual cells.
4.4 Experimental Section
4.4.1 Computations
Octanol/water partition coefficient calculations were conducted with the Advanced
Chemistry Development Inc. (ACD) log P algorithm (version 10).
4.4.2 Materials and Methods
Methanol, dichloromethane, hydrofluoric acid (HF), tetrahydrofuran (THF), chloroform- d, chloroform, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N,N- dimethyl-4-aminopyridine (DMAP), tri(ethylene glycol) monomethyl ether (MW = 164.20), (3- iodopropyl)trimethoxysilane, 3,3,4,4,5,5,6,6,6-nonafluorohexyltrimethoxysilane, methyl 3,4,5- trihydroxybenzoate (methyl gallate), and pyropheophorbide-a methylester were used as received from Sigma Aldrich. Synthesized products were purified by column chromatography using flash silica gel 200-400 mesh particle size. 1D and 2D NMR data were acquired on Bruker DPX400 54
MHz NMR instrument. HPLC data were obtained on a PerkinElmer 200 series instrument equipped with a bondclone 10 C18 column at 254 nm. UV-vis spectra were collected on a Hitachi
U-2001 instrument. High-resolution mass spectrometry (HRMS) data were collected on an Agilent iFunnel 6550 Q-ToF LC/MS system.
4.4.3 Synthesis of TriPEG-galloyl (8)
Compound 8 was synthesized from diethyleneglycol monomethylether and methyl gallate in 3 steps following a modified procedure from literature2. Briefly, triethyleneglycol monomethylether (PEG) 4 was reacted with TsCl in presence of NaOH in THF to form PEG- tosylate 5 which was further reacted with methyl gallate 6 in the presence of K2CO3 in acetone to form PEGyled methyl gallate of 7. The ester group in 7 was reduced with LiAlH4 in THF to form
1 triPEG-galloyl alcohol 8 in 58% yield. H NMR (400 MHz, CDCl3) δ (ppm): 6.62 (s, 2H), 4.57 (s,
2H), 4.08-4.01 (m, 6H), 3.73 (ddd, J = 18.5, 5.8, 4.3 Hz, 6H), 3.64-3.57 (m, 6H), 3.56-3.50 (m,
13 12H), 3.50-3.42 (m, 6H), 3.29 (s, 9H), 2.80 (t, J = 6.5, 5.8 Hz, 1H). C NMR (101 MHz, CDCl3)
δ (ppm): 152.50, 137.38, 137.02, 106.20, 72.18, 71.85, 71.82, 70.67, 70.59, 70.45, 70.40, 69.71,
68.71, 64.69, 58.88.
55
4.4.4 Synthesis of TriPEG-galloyl-ether-31-pyropheophorbide Methyl Ester (10)
Pyropheophorbide-a methylester (9) (50 mg, 0.091 mmol) and 30% HBr/AcOH (Aldrich,
1.0 mL) were added to a 5 mL round-bottom flask and stirred at room temperature for 2 h. Acetic acid was removed under high vacuum (bath temperature was maintained at 30-40 °C) and the resulting concentrate was dissolved in anhydrous dichloromethane (10 mL). Galloyl-triPEG 8 (540 mg, 0.91 mmol) and K2CO3 (102 mg, 0.76 mmol) were added, and the reaction mixture was stirred at room temperature for 12 h under N2 atmosphere. The reaction was quenched with water (5 mL), and extracted with dichloromethane (15 mL). The dichloromethane layer was washed with sat.
NaCl solution (10 mL) and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue obtained was chromatographed on a silica column, eluted with 2% (v/v) methanol in dichloromethane to obtain 10 in 42% yield (43.0 mg, 0.038 mmol) and over 99% purity. HPLC: tR = 10.33 and 11.00 min (2 diastereomers of 10) 5% (v/v) water in acetonitrile
1 solvent system. H NMR (400 MHz, CDCl3) δ (ppm): 9.71 (s, 1H), 9.53 (s, 1H), 8.57 (s, 1H), 6.52
(s, 2H), 5.99 (q, J = 6.7 Hz, 1H), 5.36-5.03 (m, 2H), 4.65 (d, J = 11.7 Hz, 1H), 4.50 (dq, J = 11.8,
6.0, 4.8 Hz, 2H), 4.32 (dt, J = 8.9, 2.6 Hz, 1H), 4.12 (t, J = 5.2 Hz, 2H), 4.01 – 3.26 (m, 55H), 3.17
(d, J = 1.7 Hz, 3H), 2.81 – 2.49 (m, 2H), 2.29 (ddt, J = 14.6, 9.3, 4.8 Hz, 2H), 2.16 (dd, J = 6.6,
2.2 Hz, 3H), 1.93-1.78 (m, 3H), 1.71 (t, J = 7.6 Hz, 3H), 1.29 (s, 1H), -1.73 (s, 1H). 13C NMR (101
MHz, CDCl3) δ (ppm): 196.20, 173.48, 171.42, 160.37, 155.05, 152.63, 150.91, 148.98, 145.01,
141.25, 141.22, 138.73, 137.85, 137.82, 136.32, 135.47, 135.39, 133.74, 132.79, 132.76, 130.54,
128.47, 107.58, 107.55, 106.06, 104.12, 97.91, 92.77, 72.30, 71.95, 71.84, 71.45, 71.21, 71.17,
70.69, 70.64, 70.55, 70.53, 70.50, 70.44, 69.58, 68.62, 59.03, 58.97, 51.71, 51.68, 50.02, 48.05,
30.93, 30.91, 29.91, 29.89, 29.72, 24.57, 24.55, 23.21, 23.19, 19.50, 17.53, 12.11, 11.29, 11.20. 56
+ HRMS (+ESI): m/z calculated for C62H86N4O16 [M] 1142.6034, found: 1142.6039. UV−vis
(CHCl3): λmax = 411 nm and 664 nm.
4.4.5 Synthesis of TriPEG-galloyl-ether-31-pyropheophorbide Carboxylic Acid (11)
TriPEG-galloyl pheophorbide 10 (43.0 mg, 0.038 mmol) was dissolved in tetrahydrofuran
(2.0 mL) and methanol (4 mL). K2CO3 (10.2 mg, 0.076 mmol) dissolved in distilled water (1.6 mL) was added and the reaction mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC. After the completion of the reaction it was diluted with dichloromethane
(20 mL) and washed with 2% acetic acid (10 mL), again with distilled water till the pH of the aqueous solution was neutral. The dichloromethane layer was separated and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue obtained was chromatographed on a silica column, eluted with 10% (v/v) methanol in dichloromethane to obtain
11 in 95% yield (41.0 mg, 0.036 mmol) and over 99% purity. HPLC: tR = 7.49 and 8.35 min (2
1 diastereomers of 11) 5% (v/v) water in acetonitrile solvent system. H NMR (400 MHz, CDCl3) δ
(ppm): 9.69 (d, J = 22.2 Hz, 1H), 9.48 (s, 1H), 8.55 (d, J = 4.7 Hz, 1H), 6.48 (d, J = 5.0 Hz, 2H),
6.09 – 5.85 (m, 1H), 5.37 – 5.00 (m, 2H), 4.64 (dd, J = 11.8, 5.1 Hz, 1H), 4.56 – 4.42 (m, 2H),
4.38 – 4.26 (m, 1H), 4.07 (dt, J = 8.0, 5.2 Hz, 2H), 3.95 – 3.23 (m, 52H), 3.16 (d, J = 3.5 Hz, 3H),
2.78 – 2.48 (m, 2H), 2.36 – 2.22 (m, 2H), 2.15 (t, J = 6.3 Hz, 3H), 1.81 (dd, J = 7.4, 3.1 Hz, 3H),
13 1.69 (t, J = 7.6 Hz, 3H), 1.21 (s, 1H), -1.72 (s, 1H). C NMR (101 MHz, CDCl3) δ (ppm): 196.38,
171.48, 160.35, 160.31, 155.13, 155.09, 152.58, 150.91, 149.00, 145.00, 141.29, 141.25, 138.82,
138.75, 137.81, 136.32, 136.30, 135.56, 135.43, 133.70, 132.87, 132.73, 130.45, 128.44, 107.64,
106.08, 104.10, 98.05, 97.73, 92.78, 72.26, 71.94, 71.81, 71.46, 71.39, 71.28, 70.67, 70.58, 70.50,
70.47, 70.39, 70.37, 69.53, 68.58, 68.56, 59.01, 58.94, 51.56, 50.01, 48.00, 30.55, 29.72, 29.63, 57
24.61, 24.46, 23.20, 23.18, 19.48, 17.50, 12.08, 11.27, 11.24, 11.12. HRMS (+ESI): m/z calculated
+ for C61H84N4O16 [M] 1128.5875, found: 1128.5882. UV−vis (CHCl3): λmax = 411 nm and 664 nm.
4.4.6 Synthesis of TriPEG-galloyl-ether-31-pyropheophorbide-(Z)-alkene Ester (13)
To 41.0 mg (0.036 mmol) of 11 in 10 mL anhydrous dichloromethane were added 30.7 mg
(0.108 mmol) of alkene 12, 9.16 mg (0.072 mmol) of DMAP and 20.2 mg (0.108 mmol) of EDC under N2 atmosphere. The reaction was stirred overnight at room temperature. Upon completion of the reaction, solvent was removed under vacuum and the residue obtained was chromatographed on a silica column, eluted with 5% (v/v) methanol in dichloromethane to obtain 13 in 50% yield
(25.0 mg, 0.018 mmol) and 93% purity. HPLC: tR = 8.72 and 9.59 min (2 diastereomers of 13) in
1 5% (v/v) water in acetonitrile solvent system. H NMR (400 MHz, CDCl3) δ (ppm): 9.77 (s, 1H),
9.58 (s, 1H), 8.59 (s, 1H), 7.32 – 7.23 (m, 2H), 7.15 (dd, J = 8.7, 2.5 Hz, 2H), 7.06 – 6.98 (m, 2H),
6.98 – 6.90 (m, 2H), 6.52 (s, 2H), 6.08 (ddt, J = 16.6, 3.4, 0.8 Hz, 2H), 5.98 (q, J = 6.6 Hz, 1H),
5.33 – 5.03 (m, 2H), 5.03 – 4.86 (m, 2H), 4.70 – 4.56 (m, 3H), 4.48 (dd, J = 11.7, 7.4 Hz, 2H),
4.31 (d, J = 8.5 Hz, 1H), 4.18 – 4.06 (m, 2H), 4.01 – 3.25 (m, 52H), 3.18 (d, J = 2.0 Hz, 3H), 2.79
– 2.49 (m, 2H), 2.42 – 2.22 (m, 2H), 2.15 (dd, J = 6.7, 1.9 Hz, 3H), 1.80 (dd, J = 7.3, 1.8 Hz, 3H),
1.71 (t, J = 7.6 Hz, 3H), 1.26 (s, 1H embedded under grease peak 3H), -1.76 (s, 1H). 13C NMR
(101 MHz, CDCl3) δ (ppm): 196.15, 172.84, 171.45, 160.38, 157.26, 156.80, 152.64, 149.00,
144.98, 141.27, 138.76, 137.85, 136.28, 135.51, 133.73, 132.83, 130.58, 130.10, 130.05, 128.68,
128.55, 128.50, 128.01, 116.24, 116.12, 107.58, 106.17, 104.11, 97.90, 92.86, 72.31, 71.96, 71.84,
71.47, 71.21, 70.71, 70.66, 70.56, 70.53, 70.45, 69.60, 68.65, 65.93, 64.76, 59.05, 58.98, 51.62,
50.00, 48.05, 31.07, 29.81, 29.72, 24.57, 23.19, 19.51, 17.51, 12.12, 11.29, 11.21. HRMS (+ESI): 58
+ m/z calculated for C77H98N4O19 [M] 1382.6841, found: 1382.6825. UV−vis (CHCl3): λmax = 411 nm and 664 nm.
4.4.7 TriPEG-galloyl Pheophorbide Modified Fluorinated Silica (1)
Fluorinated silica 14 was prepared following previously established procedure70. Briefly,
Vycor pieces were added to the nonafluorotrimethoxysilane in 0.07% (wt/wt) in toluene under reflux for 24 h under N2 atmosphere to obtain fluorinated silica 14 with loading ratio of 1:60 for
Si–OH:C–F. Unreacted nonafluorotrimethoxysilane was removed by Soxhlet extraction in methanol for 24 h. The triPEG-galloyl pheophorbide alkene ester 13 was reacted with (3- iodopropyl)trimethoxysilane in NaH and THF and added to the pieces of fluorinated silica 14
[each piece was 0.33 g and sized ∼5 mm × ∼8 mm (d × l)] in toluene under reflux for 24 h to reach
1 with loading of 115 nmol/g of silica (ratio of 1860:111,400:1 for Si–OH:C–F:sensitizer).
Unreacted adsorbed sensitizer was washed off of the silica surface by Soxhlet extraction in methanol for 24 h and washings with CH2Cl2 and THF. Amount of sensitizer loading was determined after dissolution of silica 1 in HF, extraction with chloroform, and monitoring the UV- vis absorbance of the chloroform solution at λmax = 411 nm and 664 nm.
4.4.8 Hydrolytic Stability
The hydrolytic stabilities of triPEG-galloyl pheophorbide 10 and triPEG-chlorin24 were measured in 10% (v/v) water in methanol where the pH was adjusted to 2 or 8 with formic acid or ammonium hydroxide, respectively. After 1 h, the samples were injected and analyzed by LC−MS.
59
. 3 CDCl
in
8
compound
of
spectrum
NMR
H 1
: 5
Figure 60
. 3 CDCl
in
8
compound
of
spectrum
NMR
C 13
: 6
Figure
61
. 3 CDCl
in
10
compound
of
spectrum
NMR
H 1
: 7
Figure
62
. 3 in CDCl in 10 C NMR spectrum of NMR compound C 13 Figure 8:
63
. 3 in CDCl in 10 HSQC spectra of compound spectra HSQC Figure 9:
64
. 3 in CDCl in 10 HMBC spectra of compound HMBC spectra Figure 10:
65
. 10 Expanded HMBC spectra of compound of spectra compound HMBC Expanded Figure 11: 66
. 3 in CDCl in 10 COSY spectra of spectra compound COSY Figure 12:
67
. 10 in 5% (v/v) water in acetonitrile. acetonitrile. (v/v) water 5% in in 10 HRMS of compound of compound HRMS Figure 13: HPLC spectra of compound HPLC spectra Figure 14:
68
n5 vv ae naeoirl. 5% acetonitrile. in (v/v) in water 10 HPLC spectrum of compound HPLC spectrum Figure 14: 69
. 3 in CDCl in 11 H NMR spectrumH of NMR compound 1
Figure 15:
70
. 3 in CDCl in 11 C NMR spectrum NMR C of compound 13 Figure 16:
71
. 3 in CDCl in 11 HSQC spectra of spectra compound HSQC Figure 17: 72
. 3 in CDCl in 11 HMBC spectra of compound HMBC spectra Figure 18: 73
. 3 in CDCl in 11 COSY spectra of spectra compound COSY Figure 19: 74
. 11 HRMS of compound of compound HRMS Figure 20:
75
in 5% (v/v) water in acetonitrile. 5% acetonitrile. in (v/v) in water 11 HPLC spectrum of compound HPLC spectrum Figure 21: 76
. 3 in CDCl in 13 H NMR spectrumH of NMR compound 1
Figure 22:
77
. 3 in CDCl in 13 C NMR spectrum NMR C of compound 13 Figure 23:
78
. 3 in CDCl in 13 HSQC spectra of spectra compound HSQC Figure 24:
79
. 3 in CDCl in 13 HMBC spectra of compound HMBC spectra Figure 25: 80
. 3 in CDCl in 13 Expanded HMBC spectra of compound of spectra compound HMBC Expanded Figure 26: 81
. 3 in CDCl in 13 COSY spectra of spectra compound COSY Figure 27: 82
13. HRMS of compound of compound HRMS Figure 28: 83
n5 vv ae naeoirl. 5% acetonitrile. in (v/v) in water 13 HPLC spectrum of compound HPLC spectrum Figure 29: 84
(20 µM) in chloroform. chloroform. (20 in µM) 13 and 11 , 10 , 9 UV-Vis spectra of spectra compounds UV-Vis Figure 30:
85
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88
Chapter 5. Mechanism of Photochemical O-Atom Exchange in Nitrosamines with
Molecular Oxygen
5.1 Introduction
Despite the decades-long interest in N-nitrosamine organic chemistry and toxicity,1,2 no photochemical O-atom exchange process with molecular oxygen has been reported. One paper,3
1 speculated that an O-atom exchange between singlet oxygen ( O2) and N-nitrosamine would proceed by a 1,2,3,4-trioxazetidine intermediate (cycloNO3 species) (Scheme 1). In related papers,4-9 experimental and theoretical evidence have pointed to the intermediacy of a 1,2,3-
1 dioxazetidine (cycloCNO2 species) in the reaction of O2 with hydrazones, which cleaves the hydrazone C=N bond yielding carbonyls and N-nitrosamines.
1 Scheme 1. Speculated intermediates in a O-atom exchange reaction between singlet oxygen ( O2) and N-nitrosamine.
Little is known of the intermediates involved in the direct photolysis of nitrosamines in the
10 presence of O2 and the formation of peroxy intermediates. Early studies of Tanikaga and Chow
11 et al. were done in air-saturated solutions. Formed aminium radical cations (R2NH+∙), from protonation of aminyl radicals (R2N∙), were thought not react to react with O2 due to unaffected
-3 12 absorption signals at 10 M O2 concentrations. Past studies had focused on nitrosamine excited states, NO∙ release, and the formation of amine and imine, not on photooxygen atom exchange 89
11-16 chemistry. Indeed, nitrosamine photolysis studies mainly focused on N2-degassed solutions.
In this paragraph we review nitrosamine photochemistry to set the stage for our work.
Using N2-degassed solutions, the direct photolysis of nitrosamines can lead to the homolysis of
17,18 19-21 the N–N bond via excitation to the S1 and S2 states. For diarylnitrosamines, photodissociation of NO∙ occurs from the singlet excited state.15,22,23 The triplet state of diarylnitrosamine has a low propensity to dissociate NO∙.15 Later we will show that photoexpulsion of NO∙ from 1 and 2 is minor pointing to the importance of a triplet reaction in our system. For N-
1 dimethylnitrosamine, the first excited singlet state (1 A’’) contains npO π *NO character, while
1 20 16 the second excited singlet state (2 A’) contains npN π *NO character. Chow et al. also found a direct excitation route [S0 T1 (n π*) transition] for N-dimethylnitrosamine at 453 nm. The photolysis of alkyl nitrosamines leads to NO∙ release and aminyl radicals or aminium radical
20,21 13 cations after protonation in the presence of acid. On the S1 excited surface, CASSCF
20,24 calculations show that N-dimethylnitrosamine can lose NO∙ by loss of R2N–N=O planarity, where subsequent reactions were stymied due to high-energy barriers so that recombination of NO∙ and R2N∙ is favored and regenerates ground-state nitrosamine. When originating from the S1 state, aminyl radicals can have lifetimes (τ) upward of 0.5 s.16 Consistent with a long lifetime, Geiger et al. found that UV photolysis of N-dimethylnitrosamine led to isotopic exchange for 15NO∙ from
25,26 16 the S0 S1 transition but not the S0 S2 transition. Chow et al. also found that excitation
N-dimethylnitrosamine to S2 led to NO∙ release and products such as imines (CH2=NHCH3 and
11,16 CH2=NH) and methyl radical (CH3∙). Photolysis of N-nitrosopiperidine also yielded imines.
On the other hand, UV photolysis of 1 started with NO∙ release and led to Ph2NH, and after extended photolysis, carbazole as a secondary product. Photorelease of NO∙ has been recognized in other compounds, such as diazeniumdiolates.27-30 With time-dependent DFT calculations, an 90
O2-dependent process is known for n π * excitation of CH3CH2NHN=O with isomerization and
31 H-abstraction to reach CH3CH=NH.
Today, the potential for photochemical O-atom exchange in nitrosamines with O2 remains unexplored. We thought that an oxygen isotope labeling study would be useful since its use in rearrangement and cleavage reactions have helped deduce mechanisms in the past. For example,
18O isotope labeling has helped deduce the mechanism of the Robinson-Gabriel oxazole synthesis32 and has proved useful in tracking peroxy-nitrogen and -sulfur species in reactions,33,34 and other oxygen-transfer reactions.35-39 One example of 18O labeling described phenyl
40-47 dioxaziridine (cycloNO2 species) arising in the photooxidation of phenyl azide and accompanying calculations.48,49
Here, we describe a scrambling of O atoms in the photolysis of nitrosamines in the presence
18 of O2. We report experiments and calculations that suggest a new oxygen atom exchange scheme.
One feature is the presence of an intermediate, trioxazetidine (cycloNO3 species) as playing an important role in the reaction mechanism. HPLC/MS and HPLC-MS/MS were used to examine the photochemistry of four nitrosamines (N-nitrosodiphenylaniline 1, N-nitroso-N-methylaniline
2, N-butyl-N-(4-hydroxybutyl)nitrosamine 3, and N-nitrosodiethylamine, 4). These nitrosamines,
1-4, were chosen to vary in their number of phenyl and alkyl groups. The intention was to uncover a possible photoexchange process, and evaluate a substituent dependence for the process. DFT and
TD-DFT calculations were also carried out. Experimental and theoretical evidence that supports the reaction in Scheme 2 is described next.
91
Scheme 2. Photo 18O exchange process and generation of 18O-labeled nitrosamines
5.2 Results and Discussion
We thought that the research should focus on the photooxygen atom exchange mechanism of nitrosamines 1-4 because observations of the intermediates in O2-saturated solution have not yet been sought. Experiments and theoretical calculations have been conducted for determining
18 the likely intermediates produced upon excitation of nitrosamine in the presence of O2. The proposed reaction scheme is shown in Scheme 3. The experimental photoexchange results will be discussed first, followed by product analyses and theoretical calculations. HPLC-MS/MS and
HRMS analysis was performed by Dr. Marilene Silva Oliveira and Fernanda Manso Prado in Dr.
Paolo Di Mascio’s laboratory. Theoretical calculations were conducted by Dr. Edyta M. Greer.
92
Scheme 3. Proposed 18O exchange mechanism for generating the 18O-labeled nitrosamine
R1 * R1 R1 R1 hn 18O 18 N N 2 N O 18 N R2 N R2 N R2 N O R2 N 18O direct via triplet cyclization O irradiation O surface O O 18O nitrooxide A trioxazetidine B 1-4 via singlet head-to-tail surface dimerization
18 1 O R 1 18 2 R O O R 1 1 N R R R2 N N N N 18 N N NO R2 O 18 O R1 R2 N 18O + R2 N 18O O O 18O hexaoxadiazocane C O 18O cage release, a-CH H-abstraction abstraction greater amine formation 1 and 2 R (not 3 and 4) in presence of BHT R1
2 N N R H R 5 6
5.2.1 Direct Excitation of Aromatic and Aliphatic Nitrosamines in the Presence of
18 Isotopically Labeled Molecular Oxygen ( O2)
18 Photolyses of 1-4 (5 mM) in O2-saturated CHCl3 solutions were carried out at room temperature by irradiation with a metal halide or tungsten light source. Table 1 shows that the 18O- label was exchanged into nitrosamines 1 and 2, but not 3 and 4, as it will be discussed below.
Figure 1 shows mass spectral data for 1 prior to and after photolysis for 3 h. The base peak at m/z
= 199.08 is unlabeled 1 (M + H)+ and the peak with a +2 Da mass increase ([(M + 2) + H)]+, m/z
= 201.08) is 18O-labeled 1. Peaks with m/z = 200 and 201 correspond to natural abundance of isotopomers containing 1.1% 13C and 0.21% 18O, respectively. The small peak at m/z = 202 is due to the isotopomer with the 18O label and the natural abundance of 13C (1.1%). Figure 9 shows mass spectral data for 2 prior to and after photolysis where a peak with a +2 Da mass increase is found for 18O-labeled 2. Table 1 shows that the 18O-label is photoexchanged with limited efficiency, which led us to analyze decomposition yields and by-product formation. 93
Table 1. HPLC/MS Data of the Nitrosamine Percent 18O Exchange and Decomposition in the 18 Presence of O2 in CHCl3
a relative abundance of isotopes (%) percent irradiation nitrosamine decomposition time (h) b [M+H]+ [(M+1)+H] + [(M+2)+H] + (%)
0 100±0 17±2 1±0 0
3 65±4 11±2 8±2 35±4 (m/z = 199)
0 100±0 8±1 0±0 0
3 50±2 4±0 12±3 50±2
(m/z = 137)
0 100±0 12±1 1±0 0
1 30±3 4±1 0±0 70±3
(m/z = 175)
0 100±2 6±0 1±1 0
(m/z = 103) 0.5 48±2 3±0 0±0 52±2 a Relative to the sum of area of the [M+H]+, [(M+1)+H]+, and [(M+2)+H]+ peaks. b Based on the amount of reactant remaining after photolysis.
94
Figure 1. HPLC-MS/MS analysis of (A) 1 prior to photolysis, and of (B) 1 with the 18O-label photochemically introduced. (HPLC-MS/MS analysis was done by Marilene Silva Oliveira)
5.2.2 Nitrosamine Photolytic Instability
HPLC-MS/MS was used to determine the percent yields of products in the photolysis of 1-
4 (Figures 6, 11, 14 and 17). It is clear that nitrosamine photodecomposition rates are increased as the number of alkyl groups are increased. The di- and monophenyl nitrosamines 1 and 2 only photodecomposed by 35±4% and 50±2% photodegradation after 3 h, respectively. In contrast, the amount of 3 and 4 remaining was far less. After 1 h photolysis, the amount of starting material remaining of 1 was 89%, of 2 was 77%, of 3 was 30%, and of 4 was 4%. Figure 2 shows the rate of appearance of 18O-labeled 2 is 1.4-fold greater than 18O-labeled 1, but that the rate of the photodegradation of 2 is greater by 1.5 fold than 1. Clearly, the dialkyl substituted nitrosamines decompose rapidly. The time to fully photodegrade 3 was 3 h and 4 was 2 h. The photolysis of nitrosamines 1 and 2 for 3 h led to the detection of amines, namely N,N-diphenylamine [m/z = 170 95
(M + H)+] and N-methylaniline [m/z = 108 (M + H)+], in 17% and 28% yields, respectively. Amines and/or imines were detected upon the photolysis of nitrosamines 3 and 4.
A B
Figure 2. HPLC/MS isotopic abundances of nitrosamine reagent [M+H]+ (▲) and 18O-labeled + 18 nitrosamine product [(M+2)+H] (■) as a function of photolysis time of 1 (A) and 2 (B) with O2 + in acetonitrile-d3. The sum of the isotopic abundance of the 2 peaks and [(M+1)+H] was normalized to 100%.
5.2.3 Effects of Added Radical Scavenger Butylated Hydroxytoluene
Because Crumrine et al.15 found that the photolysis of nitrosamines leads to NO∙ and aminyl radicals where amine product formation was increased in protic solvents, we hypothesized that
BHT scavenging of the aminyl radical by H-atom transfer agent will decrease the α-C–H
18 abstraction reaction to imine 6. This is indeed what we find. In the presence of BHT and O2, UV photolysis of 3 was carried out for 1 h and followed by HPLC/MS. The formation of amine 5 increased from 42% to 60% and the formation of imine 6 decreased from 24% to 2%. The result suggests that the aminyl radical arises following NO∙ release and rapidly abstracts a hydrogen atom in the presence of BHT. Because of the possible instability due to moisture, we next examined
18 whether the source of the O in photolyzed 1 and 2 was due to adventitious trapping of H2O. 96
18 5.2.4 Photo-oxygen Exchange by Adventitious H2 O is Ruled Out
16 When the photolysis reaction was carried out in an O2-saturated atmosphere in CD3CN
18 18 in the presence of <1% w/v H2 O for 3 h, the O atom was found not to exchange into
18 18 nitrosamines 1 or 2. Similarly, the O atom exchange did not occur with H2 O addition in N2
18 18 degassed photolysis experiments in the absence of O2. Thus, the involvement of H2 O in the exchange of 18O can be ruled out. To provide further insight to the formation of these 18O labeled nitrosamines, we have conducted a study to explore whether the exchange process derived from singlet oxygen.
18 18 5.2.5 Experiments with O-Labeled Singlet Oxygen ( O2)
18O-Labeled singlet oxygen was generated by photochemical or chemical methods.
Visible-light irradiation [metal-halide lamp with a cutoff filter (λ < 500 nm) as the light source] of
18 0.2 mM SiPcCl2 or methylene blue in the presence of 1-4 (5 mM) was used in O2-saturated CHCl3 solutions. We found no evidence for the exchange of an 18O-atom in the nitrosamines. Scheme 4
18 1 18 18 shows that ( O2) was generated from a chemical source [ O– O labeled naphthalene
18 50 endoperoxide of N,N’-di(2,3-hydroxypropyl)-1,4-naphthalene dipropanamide (DHPN O2)],
18 18 which also failed to exchange an O atom into 1. The thermal decomposition of DHPN O2 was carried out in the presence of 1 in CH3CN/D2O buffer phosphate (2:1) at pD 7.4 and also in a 2- phase CHCl3/D2O buffer phosphate (2:1) system at pH 7.4 with stirring for 1 h at 37 °C. Figure S6
18 18 (Supporting Information) shows the HPLC/MS of O-labeled endoperoxide of DHPN O2 before and after its thermal decomposition. Theoretical evidence that supports the reaction in Scheme 3 is described next. 97
18 18 1 Scheme 4. Thermal decomposition of DHPN O2 to generate ( O2)
5.2.6 DFT Computed Bond Dissociation Energies
DFT calculations were conducted to predict bond dissociation energies (BDE), excited state energetics, and geometries and energetics of the reagents and intermediates in the nitrosamine/O2 photoreaction.
First, we discuss the N–N BDEs that have been computed. Table 2 shows that the BDEs are about 14-18 kcal/mol lower for 1 and 2 compared to 3 and 4. This is a telling result because the lower N–N BDEs of 1 and 2 do not result in greater loss of NO∙, but instead a greater propensity for 18O-photoexchange. Our BDE values are similar to previous theoretical studies for N–N BDEs of ~35 kcal/mol (when X is an aromatic substituent) and X–NH–NO ranging from 48 kcal/mol
(when X is an alkyl substituent).51 Due to resonance, the aminyl radicals derived from the nitrosamines 1 and 2 (i.e. Ph2N∙ and Ph(Me)N∙) are stabilized and account for the lower the N–N
BDE relative to the aliphatic aminyl radicals derived from 3 and 4. Nitrosamines 1 and 2 also have longer N–N bond lengths compared to 3 and 4 as anticipated for weaker N–N bonds. Table 2 and
Figure 3 show our TD-DFT computed vertical electronic excitation energy from the ground state
(S0) to the S1 and T1 states of nitrosamines 1-4 and 7. Our values are in fairly good agreement with previously reported experimental values.16 For example, for N-dimethylnitrosamine, the experimental reported excited singlet state is 72-73 kcal/mol and the excited triplet state is 58-59 kcal/mol.16 98
Table 2. Energetics and parameters of nitrosamines 1-4 with DFT and TD-DFT calculations
a N–N bond c c nitrosamine BDE (kcal/mol) S1 (kcal/mol) T1 (kcal/mol) distance (Å) b 1 28.6 1.35 71.9 48.4
2 32.6 1.34 76.6 53.0
3 47.8 1.32 79.3 55.6
4 46.4 1.32 79.1 55.5 a DFT computed enthalpies with UωB97XD/6-31+G(d,p) where BDE = [(R2N∙ + NO∙) - b c R2NN=O]. ωB97XD/6-31+G(d,p) optimized geometries. TD-DFT computed enthalpies with B3LYP/6-311+G(d,p). (DFT calculation were done by Edyta M. Greer)
Scheme 5
5.2.7 Proposed Mechanism
To assess the factors that underlie nitrosamine photochemistry in the presence of O2, three mechanistic aspects were considered. One emanates from the 18O exchange reaction of nitrosamines with O2 via hexaoxadiazocane and trioxazetidine intermediates that cleave apart, the second from unimolecular NO∙ release and formation of amine and/or imine products, and the last is the viability of the peroxy intermediates.
18 (i) O-Atom Exchange. That the photoexchange occurs with O2 is viewed as evidence for a reaction with triplet nitrosamine. Assuming nitrooxide A is sufficiently long-lived, subsequent dimerization of the nitrooxide to yield the hexaoxadiazocane C with loss of O=18O can lead to the exchange of the oxygen atom label. Alternatively, but higher in energy, is the conversion of 99 nitrooxide A to trioxazetidine B, which is followed by the unimolecular collapse of the trioxazetidine to nitrosamine and oxygen.
(ii) Nitrosamine Photodecomposition. The aliphatic substituents on nitrosamine play a key role in facilitating the photodecomposition. α-C–H groups are labile to hydrogen abstraction by radicals and offer an explanation to the rapid photodecomposition of 3 and 4, and the slightly faster decomposition of 2 compared to 1 in our series. Oxygen-derived radicals would be expected to react with the nitrosamines and contribute to their decomposition. For example, nitrosamines16
(and amines52) bearing α-C–H groups can more easily photodecompose than aromatic nitrosamines (and amines). Furthermore, dialkylaminium radical can react with an aliphatic nitrosamine via α-C–H abstraction leading to an alkylidenimine product.53 There are reports of nitrosamines losing NO∙ where the exciting wavelength modulates the NO∙ donor activity, where
14,16 the aminyl radical can persist for longer periods from the S1 state. That NO∙ formation in 1 and
2 is minor also argues for a triplet process. Expulsion of NO∙ occurs but is not the main reaction route of the aryl nitrosamines 1 and 2. It was interesting and unexpected that lower nitrosamine
N–N BDEs correlate with the ease of O-atom photoexchange since the exchange nets an identity reaction not a N–N bond broken compound.
(iii) Reactive Intermediates in the 18O Exchange Process. The mechanism in Scheme 3 is tentative and is based on of nitrosamines 1 and 2 and the dimerization of their corresponding nitrooxides in preference to the unimolecular cyclization of nitrooxides. Computations indicate high-energy barriers for the nitrooxide dimerization to hexaoxadiazocane C and for the conversion of nitrooxide A to trioxazetidine B. Thus, photochemical interconversions would be necessary to overcome the high barriers. We note there are similarities of our work to the previous literature.
That is, nitrooxide A bears similarity to the nitrosooxide, an intermediate formed in the reaction 100
3 40-49,54,55 of nitrenes with O2. Relatedly, the trioxazetidine B is reminiscent of the 1,2,3,4- dioxadiazete, which along with its radical cation (cycloN2O2 species) have been postulated in gas- phase ion–molecule reactions.56 The dimerization of nitrooxide A to hexaoxadiazocane C is reminiscent of the coupling of a carbonyl oxide yielding a dimeric benzophenone peroxide57 and also nitrosooxide yielding a tetraoxadiazine.27 Furthermore, the photocyclization of A and B is similar to that reported for the photocyclization of nitrosooxide to dioxaziridine,35,42,58 where 18O labeling also showed aryl dioxaziridines arise in the photooxidation of aryl azides (λ > 350 nm)
18 40,41 using O2 gas by photocyclization of the aryl nitrosooxide.
In summary as described above, a photochemical process that transposes an 18O label from
18 18 molecular oxygen O2 was seen for nitrosamines 1 and 2, but not for 3 and 4. The O atom source
18 was found not to be from moisture based on control photolysis experiments with O-labeled H2O.
18 18 Thermal or visible-light sensitized production of singlet O2 to give O-labeled nitrosamine was
1 3 not observed. In these reactions, physical quenching of O2 to O2 is likely a key pathway. An
1 ordinary [2 + 2] cycloaddition of O2 to the nitrosamine N=O bond is not operating as a means to reach the trioxazetidine and scramble the 16O and 18O atoms.
5.3 Conclusion
Four nitrosamines 1-4 were irradiated in the presence of 18O-labeled molecular oxygen gas to examine a substituent dependence in the O-atom exchange process. Di- and monophenyl nitrosamines 1 and 2 are more photostable than the dialkyl nitrosamines 3 and 4. Nitrosamines are
23,59 generally recognized not to be good sources of NO∙, even under N2 atmosphere, due to the formation of by-products. Whether triplet sensitization of nitrosamines is also a viable strategy to reach the nitrooxide is a question that we are also exploring. The formation of non-nitrosamine 101 products, such as nitroamine in photooxidation reactions is currently being explored. Another question yet to be addressed is related to possible chemiluminescence from the trioxazetidine (by analogy to 1,2-dioxetanes).60,61 Time-resolved IR methods62-65 would also provide insight into the mechanism. Lastly, the discovery of an oxygen exchange route in nitrosamine photochemistry and the formation of peroxy intermediates derived from this reaction is described here and may provide a clue to new factors significant in nitrosamine phototoxicity.
5.4 Experimental Section
5.4.1 Materials and Methods
Diphenylamine, N-methyl-N-(p-tolyl) amine, silicon phthalocyanine dichloride (SiPcCl2),
18 methylene blue, butylated hydroxytoluene (BHT), K2Cr2O7, CH3CN, CHCl3, CDCl3, H2 O
18 18 (96.9%), O2 gas (99% O) from a gas cylinder, and N-nitrosodiphenylaniline 1, N-nitroso-N- methylaniline 2, N-butyl-N-(4-hydroxybutyl)nitrosamine 3, N-nitrosodiethylamine 4 were purchased commercially. (Caution: nitrosamines are carcinogenic).
Positive ion mode electrospray ionization mass spectrometry data were collected as previously described.66 The analysis was done by direct injection to a mass spectrometer with a Z- spray atmospheric pressure ionization source. Samples were dried and reconstituted in acetonitrile prior to injection into the mass spectrometer. HPLC-MS/MS refers to liquid chromatography coupled to an electrospray ionization tandem mass spectrometry instrument67 that was used.
HPLC/MS data were collected as has been described in our previous work.68 NMR data were recorded on an instrument operating at 400 MHz for 1H NMR and 100.6 MHz for 13C NMR. UV- visible and GC/MS data were also collected. Micromass software was also used to generate theoretical spectra of nitrosamines to calculate natural abundance of isotopomers. 102
5.4.2 18O-Photoexchange Reactions
18 Photooxidations were carried out in a 3-mL sealed glass vial at 27 °C with periodic O2 bubbling and irradiation with two 400-W metal halide lights or two 500-W tungsten lights.
Experiments were conducted in 1-mL CHCl3 or CH3CN solutions of 1 mM or 5 mM 1-4. Prior to
18 photolysis, one of two methods was used to introduce O2 gas: (i) CHCl3 solutions were frozen
18 and thawed in liquid N2 and kept under vacuum, then the system was connected to an O2 gas cylinder, and the system was kept closed during the photolysis; or (ii) CH3CN solutions were
18 sparged with N2 for 15 min and then O2 gas for 5 min. During the photolysis, at 30-min intervals,
18 samples were sparged with O2 gas for 3-min periods. CHCl3 contains trace ethanol as a stabilizer, which was not removed. The results were reproducible and nearly identical in either CHCl3 or
CH3CN solutions. Reaction mixtures were analyzed by HPLC/MS and HPLC-MS/MS. For the
HPLC/MS, the column used for 1-4 was a 2.1 mm × 30 mm, 3.5 μm SB-C18 column. For the
HPLC-MS/MS, the column used for 1 and 2 was a 25 cm × 2.1 mm, 5 µm Supelcosil LC18-S column, and for 3 and 4 was a 25 cm × 4.6 mm, 5 µm C18 Gemini column.
5.4.3 UV Photolysis of N-nitrosodiphenylaniline (1)
18 HPLC/MS: tR = 5.48 min; HRMS (+ESI) calcd for labeled C12H11N2 O = 201.0938, found
201.0958; calcd unlabeled C12H11N2O = 199.0871, found 199.0872. When followed by HPLC-
MS/MS, peaks were observed for 1 (m/z = 199) and 18O-exchanged 1 (m/z = 201) at 24.69 min, and diphenylamine (m/z = 170) at 26.51 min. Spiking a commercial sample of diphenylamine led to an increase in the m/z = 170 peak.
103
5.4.4 UV Photolysis of N-nitroso-N-methylaniline (2)
18 HPLC/MS: tR = 3.91 min; HRMS (+ESI) calcd for labeled C7H9N2 O = 139.0757, found
139.0755; calcd unlabeled C7H9N2O = 137.0715, found 137.0724. When followed by HPLC-
MS/MS, peaks were observed for N-methylaniline (m/z = 108) at 4.81 min, 2 (m/z = 137) and 18O- exchanged 2 (m/z = 139) at 12.69 min.
5.4.5 UV Photolysis of N-butyl-N-(4-hydroxybutyl)nitrosamine (3) and nitrosodiethylamine
(4)
When followed by HPLC-MS/MS, peaks were observed for parent 3 and 4, but not 18O- exchanged 3 and 4. The photolysis of 3 led to an imine 4-((1-hydroxybutyl)imino)butan-1-ol or isomer [m/z = 144 (M + H)+] at 5.87 min and 4-(butylamino)butan-1-ol [m/z = 146 (M + H)+] at
7.26 min. The photolysis of 4 led to diethylamine (m/z = 74) at 3.92 min.
18 1 5.4.6 Photochemical and Chemical Generation of Singlet Oxygen [ ( O2)]
Experiments were conducted in 1-mL CDCl3 or CH3CN solutions of 1 mM or 5 mM 1-4.
18 1 The experiments for a photochemical source of ( O2) used a photosensitizer, 0.1 mM SiPcCl2 or methylene blue, in which samples were irradiated through a cutoff filter (λ < 500 nm) solution of
18 1 18 0.2 M K2Cr2O7 in 0.5% v/v H2SO4. The experiments for a chemical source of ( O2) used an O-
18O labeled endoperoxide [N,N’-di(2,3-dihydroxypropyl)-1,4-naphthalene dipropanamide
7 (DHPN18O2)] that was synthesized as described previously.
5.4.7 Computational Methods
Calculations were performed with Gaussian 09 (revision D.01)69 and visualized with 104
Gaussview 5.0.70 Geometries were optimized with unrestricted ωB97X-D, which includes empirical dispersion71 along with the 6-311+G(d,p) basis set. These calculations yielded results in
1 72 reasonably good agreement with CCSD(T) based on the reaction of ethene with O2. The energetics are reported as the thermal enthalpies. BDEs were determined by UB97X-D/6-
311+G(d,p) calculations of optimized geometries of 1-4 and the corresponding aminyl radicals and
NO∙ with the formula: BDE = [(R2N∙ + NO∙) - R2NN=O].
105
Table 3. Experimental conditions and percent abundances of isotopes of 18O photoexchanged into nitrosamines 1-4
nitros- irradiation irradiation mass found in spectra c sensitizer b solvent gas MW amine wavelength a time (h) [M+H]+ [(M+2)+H]+ 16 none chloroform O2 199 - 18 none chloroform O2 199 201 18 none acetonitrile O2 199 201 1 UV and vis 3 16 198 none acetonitrile O2 199 - d 16 none acetonitrile O2 199 - d none acetonitrile N2 199 - 16 UV and vis SiPcCl2 chloroform O2 137 - 16 visible SiPcCl2 chloroform O2 137 - 18 visible SiPcCl2 chloroform O2 137 - methylene 18 visible chloroform O2 137 - blue 2 3 18 136 UV and vis none chloroform O2 137 139 18 UV and vis none acetonitrile O2 137 139 16 UV and vis none acetonitrile O2 137 - d 16 UV and vis none acetonitrile O2 137 - d UV and vis none acetonitrile N2 137 - 18 none chloroform O2 175 - 3 UV and vis 3 18 174 none acetonitrile O2 175 - 18 none chloroform O2 103 - 4 UV and vis 3 18 102 none acetonitrile O2 103 - a Irradiation of 1-4 with a metal halide light source or visible light (λ < 500 nm by use of a 0.2 M K2Cr2O7 solution filter in 0.05% (v/v) H2SO4). [1-4] = 1 mM in CH3CN and 5 mM in CHCl3. The HPLC/MS and HPLC-MS/MS analyses used a CH3CN/H2O mobile phase b c d 18 system. [Sensitizer] = 0.1 µM in CH3CN and 0.2 mM in CHCl3. Data from >100 experiments. H2 O = <1% w/v 106
Figure 3. HPLC/MS data of relative mass isotopic abundance of [M+H]+ (grey), [(M+1)+H]+ (light grey), and [(M+2)+H]+ (dark grey) peaks prior to and after photolysis of nitrosamines 1-4 18 with O2 in acetonitrile. The sum of the isotopic abundance of the three peaks was normalized to 100%. 107
18 Figure 4. HPLC-MS/MS analysis of nitrosamine 1 before (A) and after (B) photolysis with O2 in chloroform. The data show unlabeled 1 ([M+H]+, m/z = 199) and a +2 Da mass increase due to the exchange of an 18O atom ([(M+2)+H]+, m/z = 201). A peak corresponding to an acetonitrile adduct is seen at m/z = 240.
108
18 Figure 5. HPLC-MS/MS of products formed after 3 h photolysis of nitrosamine 1 with O2 in chloroform. Ion selection analysis for (A) nitrosamine 1 (m/z = 199) and (B) N,N-diphenylamine (m/z = 170). MS/MS fragmentation of peak for (C) 18O-labeled nitrosamine 1 at 24.69 min and (D) N,N-diphenylamine at 26.51 min. 109
18 Figure 6. HPLC/MS analysis of nitrosamine 1 before (A) and after (B) photolysis with O2 in acetonitrile after 3 h. UV-visible detection is shown at 280 nm. For spectrum B, the peak at tR = 5.48 min corresponds to nitrosamine 1 and peak at tR = 5.71 min corresponds to N,N- diphenylamine. The other peaks in spectrum B are unidentified products. 110
18 Figure 7. HRMS data of 1 after photolysis with O2 in chloroform (A), showing fragment ions of 199 m/z (B), and 201 m/z (C). 111
Figure 8. HPLC-MS/MS of 18O-labeled N,N´-di(2,3-hydroxypropyl)-1,4-naphthalene 18 dipropanamide endoperoxide (DHPN O2) before (A) and after (B) thermal decomposition to 18 1 chemically generate ( O2) and N,N´-di(2,3-dihydroxypropyl)-1,4-naphthalene dipropanamide 18 18 and (C) of nitrosamine 1 upon exposure to DHPN O2. ESI+/MS of DHPN O2 before (D) and 18 1 after (E) thermal decomposition to chemically generate ( O2) and of (F) nitrosamine 1 upon 18 18 18 exposure to DHPN O2. DHPN O2 did not introduce an O atom into nitrosamine 1. 112
18 Figure 9. HPLC-MS/MS analysis of nitrosamine 2 before (A) and after (B) photolysis with O2 in chloroform. The data show unlabeled 2 ([M+H]+, m/z = 137) and a +2 Da mass increase due to the exchange of an 18O atom ([(M+2)+H]+, m/z = 139). A peak corresponding to an acetonitrile adduct is seen at m/z = 178. 113
18 Figure10. HPLC-MS/MS of products formed after 3 h photolysis of nitrosamine 2 with O2 in chloroform. Ion selection analysis for (A) nitrosamine 2 (m/z = 137) and (B) N-methylaniline (m/z = 108). MS/MS fragmentation of peak for (C) 18O-labeled nitrosamine 2 at 12.69 min and (D) N-methylaniline at 4.81 min. 114
18 Figure 11. HPLC/MS analysis of nitrosamine 2 before (A) and after (B) photolysis with O2 in acetonitrile after 3 h. UV-visible detection is shown at 280 nm. For spectrum B, the peak at tR = 3.92 min corresponds to nitrosamine 2 and peak at tR = 4.51 min corresponds to N-methylaniline.
115
18 Figure 12. HRMS data of 2 after photolysis with O2 in chloroform (A), showing fragment ions of 137 m/z (B), and 139 m/z (C). 116
18 Figure 13. HPLC-MS/MS of products formed after 1 h photolysis of nitrosamine 3 with O2 in chloroform. Ion selection analysis for (A) nitrosamine 3 (m/z = 175), (B) 4-(butylamino)butanol (m/z = 146), and (C) a tentative assignment of 4-(butylimino)butanol (m/z = 144). MS/MS scan of peaks found (D) nitrosamine 3 at 16.46 min, (E) 4-(butylamino)butanol at 7.26 min, and (F) 4- (butylimino)butanol or isomer at 5.87 min. The peak at 20.60 min in spectrum A and peak at 16.31 min in spectrum C, are unidentified products. MS/MS data show unlabeled 3 ([M+H]+, m/z = 175). A +2 Da mass increase for 18O-labeled 3 was not seen ([(M+2)+H]+, m/z = 177).
117
18 Figure 14. HPLC/MS analysis of 3 before (A) and after (B) 30 min photolysis with O2 in 18 acetonitrile. HPLC trace of 3 before (C) and after (D) 30 min photolysis with O2 in acetonitrile. UV-visible detection was conducted at 280 nm. The MS data show unlabeled 3 ([M+H]+, m/z = 175). A +2 Da mass increase for 18O-labeled 3 was not seen ([(M+2)+H]+, m/z = 177).
118
18 Figure 15. HPLC-MS/MS of products formed after 1 h photolysis of nitrosamine 3 with O2 in chloroform in the presence of BHT. Ion selection analysis for (A) nitrosamine 3 (m/z = 175), (B) 4-(butylamino)butanol (m/z = 146), and (C) a tentative assignment of 4-(butylimino)butanol (m/z = 144). MS/MS scan of peaks found (D) 3 at 16.39 min, (E) 4-(butylamino)butanol at 7.20 min, (F) unidentified compound at 16.39 min. The peak at 6.72 min in (B) is an unidentified compound.
119
18 Figure 16. HPLC-MS/MS of products formed after 1 h photolysis of nitrosamine 4 with O2 in chloroform. Ion selection analysis for (A) nitrosamine 4 (m/z = 103), (B) diethylamine (m/z = 74). MS/MS scan of peaks found (C) nitrosamine 4 at 13.42 min, and (D) diethylamine at 3.92 min. MS/MS data show unlabeled 4 ([M+H]+, m/z = 103). A +2 Da mass increase for 18O-labeled 4 was not seen ([(M+2)+H]+, m/z = 105).
120
18 Figure 17. HPLC/MS analysis of 4 before (A) and after (B) 30 min photolysis with O2 in 18 acetonitrile. HPLC trace of 4 before (C) and after (D) 30 min photolysis with O2 in acetonitrile. UV-visible detection is shown at 280 nm. The MS data show unlabeled 4 ([M+H]+, m/z = 103). A +2 Da mass increase for 18O-labeled 4 was not seen ([(M+2)+H]+, m/z = 105).
121
Figure 18. Absorption spectra of nitrosamines 1 (solid black), 2 (solid grey), 3 (dotted black), and 4 (dotted grey) in acetonitrile. Concentration = 50 µM.
122
5.5 References
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59. Karaki, F.; Kabasawa, Y.; Yanagimoto, T.; Umeda, N.; Urano, Y.; Nagano, T.; Otani, Y.; Ohwada, T. Chem. Eur. J. 2012, 18, 1127-1141. 60. Baader, W. J.; Stevani, C. V.; Bastos, E. L. Chemiluminescence of Organic Peroxides; In The Chemistry of Peroxides; Rappoport, Z., Ed.; Wiley: Chichester, 2006, Vol. 2, pp. 1211-1278. 61. Baumstark, A. L.; Moghaddari, M.; Chamblee, M. L. Heteroatom Chem. 1990, 1, 175-180. 62. Torres-Alacan, J.; Sander, W. J. Org. Chem. 2008, 73, 7118-7123. 63. Evans, A. S.; Sundaram, G. S. M.; Sassmannshausen, J.; Toscano, J. P.; Tippmann, E. M. Org. Lett. 2010, 12, 4616-4619. 64. Burdzinski, G.; Kubicki, J.; Sliwa, M.; Réhault, J.; Zhang, Y.; Vyas, S.; Luk, H. L.; Hadad, C. M.; Platz, M. S. J. Org. Chem. 2013, 78, 2026-2032. 65. Ranaweera, R. A. A.; Upul; S. T.; Li, Q.; Rajam, S.; Duncan, A.; Li, R.; Evans, A.; Bohne, C.; Toscano, J. P.; Ault, S.; Gudmundsdottir, A. D. J. Phys. Chem. A 2014, 118, 10433- 10447. 66. Oliveira, M. S.; Severino, D.; Prado, F. M.; Angeli, J. P. F.; Baptista, M. S.; Medeiros, M. H. G.; Di Mascio, P. Photochem. Photobiol. Sci., 2011, 10, 1546-1555. 67. Queiroz, R. F.; Paviani, V.; Coelho, F. R.; Marques, E. F.; Di Mascio, P.; Augusto, O. Biochem. J. 2013, 455, 37-46. 68. Mahendran, A.; Vuong, A.; Aebisher, D.; Gong, Y.; Bittman, R.; Arthur, G.; Kawamura, A.; Greer, A. J. Org. Chem. 2010, 75, 5549-5557. 69. Gaussian 09, Revision D.01, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, M. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian, Inc., Wallingford CT, 2009. 70. Dennington, R.; Keith, T.; Millam, J. Gaussview. Semichem Inc., Shawnee Mission KS, 2009. 71. Chai, J.-D.; Head-Gordon, M. Phys. Chem. Chem. Phys. 2008, 10, 6615-6620. 72. Saito, T.; Nishihara, S.; Kataoka, Y.; Nakanishi, Y.; Kitagawa, Y.; Kawakami, T.; Yamanaka, S.; Okumura, M.; Yamaguchi, K. J. Phys. Chem. A 2010, 114, 7967-7974.
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Chapter 6. Literature Review of the Use of Singlet Oxygen to Synthesize Natural Products and Drugs
6.1 Introduction and Background
1 This Review discusses photogenerated singlet oxygen ( O2) in synthetic organic chemistry.
Even though singlet oxygen is a short-lived metastable excited state of molecular oxygen, it is a practical reagent for compound oxidation and can form carbon-oxygen and heteroatom-oxygen bonds. The motivation for the Review is to encourage its further use in the synthesis of natural products and drugs.
1 Historically, O2 showed diffusible properties with photosensitization as a convenient
1,2 1 method for its production in the Foote-Wexler reaction 50 years ago. Since this time, O2 is established for its ability to oxidize organic and biological compounds,3-11 or be used in synthesis.
In the 1980s, reports emerged on the use of singlet oxygen in the synthesis of natural
1 12-14 products. The use of O2 in organic synthesis was pioneered and routes to natural products by
1 O2 began to be classified as biomimetic. While singlet oxygen’s frequency in the synthesis of natural products has increased in the past 10 years, it is still a relative newcomer.
1 Scheme 1 shows synthetically useful O2 reactions for generating oxygenated compounds, including the formation of endoperoxides from Diels-Alder reactions, dioxetanes from [2 + 2] cycloadditions, hydroperoxides from alkenes and phenols, sulfoxides from sulfides, and phosphine
15-22 1 oxides from phosphines. Not only is the chemical trapping of O2 easy (Scheme 1), its generation is also straight forward (Scheme 2).
1 Scheme 2 shows two methods that give rise to O2, although the Review focuses exclusively on the second. The first route is a thermal process, i.e. dark singlet oxygenation
126
23 24-27 (Scheme 2A), such as H2O2 with NaOCl or the use of peroxides [arene endoperoxides,
28-31 32,33 34-36 hydrotrioxides, dihydroperoxides, potassium monoperoxysulfate (KHSO5), dimethyldioxirane,37 ozone/heterocycle adducts with pyrroles, oxazoles, and imidazoles,38 or
39,40 triphenylphosphite ozonide, cyclo-(PhO)3PO3]. Such peroxides are oxidants themselves that can react with the substrates directly or have poor functional group tolerance, which are synthetic concerns. But these peroxide reagents can be eliminated in the second route.
1 41 The second route is the photosensitized production of O2 (Scheme 2B). This oxygen- dependent photosensitization reaction is not only useful in organic synthesis, it is extremely common in nature. The route is appealing since it only requires visible light, oxygen and a
1 sensitizer to produce O2. Light excites the sensitizer and not the substrate. Thus, the process is wavelength-selective, where narrow and broad-band light sources can be used.42 Furthermore, fabricated capillary reactors that run on miniature light-emitting diodes (LEDs) have advantages to batch reactors. Small reactors with low-energy long wavelengths are more appealing than large reactors with high-energy short wavelengths, as we will see.
127
1 Scheme 1. Synthetic utility of O2 for generating oxygenated hydrocarbons
128
Scheme 2. Generation of singlet oxygen
6.2 Scope of the Review
The Review is intended to be of interest to synthetic organic chemists. Only modest information is known for singlet oxygen’s success in organic synthesis applied to complex targets
1 1 or the use of O2 in flow synthesis. No comprehensive coverage exists for O2 in the organic synthesis of natural products and drugs. The Review has been organized into two sections
(Sections 6.3 and 6.4).
In Section 6.3, natural products or related compounds are discussed where singlet oxygenation is a key step in their biomimetic synthesis. Because the synthesis of butenolides43-47
48-59 1 and trioxane antimalarial drugs using O2 chemistry has been reviewed, our description of
1 these topics will be confined to Section 5. Similarly, because there are reviews on O2 in physical- organic chemistry,60-63 this literature will not be explicitly covered. Physical and environmental
1 64-67 chemistry aspects of O2 will also not be covered due to preexisting reviews.
1 In Section 6.4, O2 flow photoreactors and their success in synthesis will be summarized in an effort to help validate them. Previous reviews on flow photoreactors that have selected
1 68-70 coverage of O2 in flow synthesis have been published. Our Review is up-to-date where
129
1 1 Section 5 exclusively covers O2 flow photoreactors in synthesis. “Flowing” O2 generators such
71-75 as the chemical oxygen-iodine laser (COIL) with supersonic I2/O2 mixing will not be covered because there is no overlap with organic synthesis. Extensive studies have been carried out with direct substrate photolysis in flow reactions,76-83 which will also not be covered. Lastly, the Review will emphasize the interplay between basic (Section 6.3) and applied research (Section 6.4) to
1 further establish O2 in the synthesis of targets with industrial scale syntheses in mind.
6.3 Singlet Oxygen in Synthesis
6.3.1 Background
1 In this section, we provide accounts of O2 in batch reactions for the synthesis of natural products and drugs. Schemes 4-53 show the extent of complex targets synthesized to date using
1 1 84-96 O2. There are reports of tandem additions of O2 molecules in the literature. Many studies formed peroxide cycloadducts as intermediates, which often rearranged or were reduced to more stable groups. Common reducing agents used are thiourea, dimethylsulfide and triphenylphosphine. As we will see, tetraphenylporphyrin (TPP), rose bengal (RB) and methylene blue (MB) are the most common sensitizers (Sens) in homogeneous solutions.97-101 Irradiation of
Sens0 causes Sens* to efficiently produce singlet oxygen (type II) and trace amounts of oxygen radicals (type I) (Scheme 3), the latter is responsible for photobleaching over long irradiation times. Heterogeneous sensitizers are diphasic with two appeals (pun intended), they are generally less prone to photobleaching compared to homogeneous sensitizers102-106 and they are easy to separate from solution after reaction; examples include, polymer-supported RB, secoporphyrazine and fullerenes.107-116 Self-sensitized photooxygenation reactions are also known,117-120 where the
130
1 substrate absorbs visible light to generate O2 which reacts on itself; three examples are described in Section 4.11 in the context of natural products and drug synthesis.
Scheme 3. Schematic of type I/II photooxidation reactions
In the context of synthesis, there are caveats unique to singlet oxygen chemsitry: (i) An
1 inverse relationship exists between temperature and rate, where O2 often adds more rapidly at low
1 121-124 temperature (many O2 reactions are under entropy-control). (ii) Peroxide product stabilitites vary and low-temperature NMR spectroscopy is beneficial for detection in crude reaction mixtures.
1 Furthermore, deuterated NMR solvents show duplicity with O2 in synthesis for in situ monitoring
1 and faster reaction rates. (iii) The rate of O2 reactions is faster in deuterated solvents due to longer
1 O2 lifetimes (τ ∆); for example, compare toluene-d8 (τ ∆ = 280 µs) vs toluene (τ∆ = 29 µs), and
125,126 CDCl3 (τ ∆ = 7.0 ms) vs CHCl3 (τ ∆ = 230 µs). In contrast, protic solvents produce the shortest
1 3 O2 lifetimes due to facile deactivation by O–H vibrational quenching to ground state O2. (iv)
1 1 Some O2 is wasted through substrate physical quenching. The total rate constants (kT) of O2 of a compound is known to be the sum of the chemical quenching rate constant (kr) and the physical quenching rate constant (kq), where reactions can be efficient (kr ≈ kT) or inefficient (kq ≈ kT). The
1 7 8 latter case is observed with amines, which deactivate O2 by charge-transfer quenching (10 to 10
-1 -1 127-130 1 M s ). Additionally, amines can diverge the chemistry from type II ( O2) to type I,
131
131 1 generating oxygen radicals. (v) In terms of selectivity, O2 reacts preferentially with compounds
132-134 of higher nucleophilicity owing to singlet oxygen’s electrophilicity. For example, the kT of a tetrasubstituted diene (2,5-dimethyl-2,4-hexadiene)135 is ~100 fold greater than a disubstituted diene [(E,Z)-2,4-hexadiene] so the former site is selectively reacted as is relevant in the first example in Section 6.3.2, the synthesis of endoperoxide natural products.
6.3.2 Endoperoxides
We have located 3 studies; first, a 2002 report describes the RB-sensitized photooxygenation of a trisubstituted diene selectively in the presence of a trisubstituted alkene to furnish a mixture of diastereomeric endoperoxides. The photooxidation of triene 1 was followed by a reaction with diazomethane leading to endoperoxides 2 and 3 in 40% yield (Scheme 4).136 To reach 2 and 3, the synthesis was 18 steps in length with a total yield of 2.8%. Based on the assignment of diastereomeric (3R,6S,8R,10R)-2 and (3S,6R,8R,10R)-3, the natural product 4 is tentatively assigned as 3S,6R,8S,10R. Carboxylic acid derivatives of 2 and 3 are known to have potent cytotoxic and anti-fungal activity.137
132
Scheme 4. Synthesis of marine sponge endoperoxide isomers
Second, a 2002 report describes the RB-sensitized photooxidation of a 1.8:1.0 ratio of
(3E,5E):(3Z,5E) dienes 5 (Scheme 5),138 to form diastereomeric endoperoxides 6 and 7 (also in a ratio of 1.8:1.0) and a 45% combined yield. Four additional steps furnished the natural product
(±)-6-epiplakortolide E 8, which bears a flexible phenyl-terminated polymethylene chain. It is not
1 obvious how 6 and 7 form in the same 1.8:1.0 ratio since it is expected that the O2 [2 + 4] cycloaddition is less feasible due to distortion of the 3Z,5E diene to reach the s-cis geometry. For example in 2,4-dimethyl-2,4-hexadienes, [2 + 4] addition is favored for E,E, but not E,Z where the ene reaction and [2 + 2] addition also occur.139
133
Scheme 5. Synthesis of (±)-6-epiplakortolide E
Third, an approach reported in 1999 features a TPP-sensitized photooxidation of 9 and 14 to give hydroperoxides 10 and 15 regioselectively in 62% and 69% yields, respectively (Scheme
140,141 1 6A and 6B). The regioselectively was the result of O2 addition to the Z-allylic alcohol with
H-bonding. The hydroperoxides reacted with DTBN causing a peroxyl radical Schenck rearrangement142 to hydroperoxides 11 and 16. A series of additional steps with hydroperoxide 11 and 16 afforded chondrillin (+)-12 and (-)-17, and plakorin (+)-13 and (-)-18. Chondrillin has shown antitumor activity against P388 leukemia cells, and plakorin is an activator of sarcoplasmic reticulum Ca(II)-ATPase.143 We also note that stereoselective synthesis of (Z)-3-tributylstannyl-2-
1 144-147 alkenyl hydroperoxides from the O2 ‘ene’ reaction of allylstannanes have also yielded synthons for peroxy natural products (Scheme 6C).
134
Scheme 6. Synthesis of (±)-chondrillin, (±)-plakorin and a stannyl alkenyl hydroperoxide
Singlet oxygen chemistry is an efficient way to reach highly oxygenated compounds, such as carbasugars (polyhydroxy cyclohexanes) as we will see next.
135
6.3.3 Carbasugars
Singlet oxygen acts in some way as a liver enzyme (cytochrome P450) by processing hydrophobic compounds to increase their water-solubility by introducing hydroxyl groups. A 1998 report described the TPP-sensitized photooxidation of dioxolane 21 which gave hydroperoxide 22 in 95% yield (Scheme 7).148 Hydroperoxide 22 was reduced with thiourea to form alcohol 23, and subsequent reactions led to DL-vibo-quercitol 24 and DL-talo-quercitol 25. There are literature examples of highly oxygenated compounds, such as pinitol, cyclitols, and polyols synthesized by
148-150 1 other oxidation methods, e.g., epoxidation, osmylation, and ozonolysis. Relatedly, O2 reactions have been previously reported for the synthesis of conduritols,151-157 and proto-quercitol and gala-quercitol as is discussed in the next example.158
Scheme 7. Synthesis of DL-talo-quercitol and DL-vibo-quercitol
136
A synthesis reported in 1997 used the TPP-sensitized photooxidation of 1,4-
1 cyclohexadiene 26 to give hydroperoxide 27, which upon addition of a second O2 molecule led to hydroperoxy endoperoxides 28 and 29 in 72% yield (88:12 ratio) (Scheme 8).158 The reduction of peroxides 28 and 29 with LiAlH4 was followed by protection of the alcohol group with acetic anhydride affording triacetates 30 and 32, respectively. Further reactions led to DL-proto-quercitol
31 and DL-gala-quercitol 33. In a related report in 2010, hydroperoxide 28 was found to afford oxazolidinone 34, leading to DL-gala-aminoquercitol 35.159
Scheme 8. Synthesis of DL-proto- and DL-gala-quercitol and a gala-aminoquercitol derivative
137
From 2014, a report described the TPP-sensitized photooxidation of cyclohexa-1,3-diene
160 1 36 (Scheme 9). The addition of O2 to 36 occurred in the expected [2 + 4] pathway leading to endoperoxide 37, which was reduced and acetylated to afford diacetate 38, and two additional steps led to DL-tetrol 39. In a related report in 2014,161 other carbasugars such as DL-pentaol 40 arise from endoperoxide 28 following a similar strategy.
Scheme 9. Synthesis of a DL-tetrol and a DL-pentaol
A 2013 paper describes the TPP-sensitized photooxidation of 4,5-dimethylenecyclohex-1-
162 1 ene 41 (Scheme 10), where the tandem addition of three O2 molecules was observed. Singlet oxygen first added to 41 by a [2 + 4] cycloaddition to form endoperoxide 42 in 86% yield. A second equivalent of singlet oxygen added by an ene reaction, and a third singlet oxygen equivalent
138
by another [2 + 4] cycloaddition. As can be seen, resulting tris peroxides 43 and 44 contain a high degree of oxygenation. In addition to the evidence for 43 and 44 from NMR spectroscopy, X-ray crystal data were also obtained for 44. The reduction of 43 was carried out with thiourea. The alcohol groups reacted with acetic anhydride in pyridine to form triacetate 45 in 82% yield.
Isomeric heptols DL-46 and DL-47 were then formed in further reactions. Other reports also show
1 that hydroperoxides are efficiently prepared by reacting cyclohexa-1,4-dienes with O2 in an ene- reaction.163-165
Scheme 10. Synthesis of isomeric heptols
In 2011, a report appeared describing the TPP-sensitized photooxidation of 2-(6- acetoxycyclohex-3-en-1-yl)ethyl acetate 48 to form hydroperoxide 49 and enone 50 in 72% and
14% yields, respectively (Scheme 11).166 Reduction and acetylation of hydroperoxide 49 followed by a series of reactions afforded 5a-carba-6-deoxy-α-DL-galacto-heptopyranose (51) and 5a- carba-6-deoxy-α-DL-gulo-heptopyranose (52).
139
Scheme 11. Synthesis of 5a-carba-6-deoxy-α-DL-galacto-heptopyranose and 5a-carba-6-deoxy-
α-DL-gulo-heptopyranose
Lastly, a 2003 report described the TPP-sensitized photooxidation of trans-8-(acetyloxy) bicyclo[4.2.0]octa-2,4-dien-7-yl acetate 53 which gave endoperoxide 54 in 70% yield (Scheme
12).167 Reduction and acetylation of 54 led to tetraacetate 55 in 73% yield, and further reactions led to DL-bis-homoinositol 56. Leaving carbasugar synthesis, epoxides are described next.
140
Scheme 12. Synthesis of DL-bis-homoinositol
6.3.4 Epoxides
1 Even though O2 is a high-energy species, epoxide products are tolerant of it. Four
1 preparative examples of O2 for reaching epoxide-containing natural products are described. First, a 2005 paper describes the MB-sensitized photooxidation of diastereomeric bicyclo[4.2.0]octanes
57 and 60 separately gave endoperoxides 58 and 61, respectively in 69% and 72% yield (Scheme
168 13). Endoperoxides 58 and 61 were then isomerized with RuCl2(PPh3)3 using Noyori’s method to the diepoxides, elysiapyrones A 59 and elysiapyrones B 62 in 68% and 50% yields, respectively.
Additionally, (±)-ocellapyrone B has been used as a precursor to 14-methylelysiapyrone A.169
141
Scheme 13. Synthesis of elysiapyrone A and B
Secondly, a 2015 paper describes the MB-sensitized photooxidation of diene 63 which gave endoperoxide 64 selectively in 92% yield (Scheme 14).170 Endoperoxide 64 was converted to diepoxide 65 by treatment with DIBAL, followed by acetic anhydride and DMAP, and in a subsequent step by Noyori’s ruthenium (II) catalytic method. Seven additional reactions then led to (±)-limonin 66.
142
Scheme 14. Synthesis of (±)-limonin
Third, a 1994 report described the TPP-sensitized photooxidation of a mixture of fusicocca-
2(6)-3-diene 67 and fusicocca-2,5-diene 68 (ratio 2:3) (Scheme 15).171 Attack of singlet oxygen on the cyclopentadiene ring of 67 and 68 occurred forming endoperoxides 69 and 70. Upon warming, rearrangements of endoperoxides 69 and 70 proceed to diepoxide and epoxy ketone products. Namely, 69 led to fusicogigantepoxide 71 and fusicogigantone A 72, and 70 led to
2α,3α:5α,6α-fusicogigantepoxide B 73 and 2α,3α-fusicogigantone B 74. NMR spectroscopy was used to help deduce the structures and X-ray data were collected for 71. Dienes 67 and 68 are plausible but not yet identified as natural precursors to reach 71-74 by singlet oxygenation. Studies of the rearrangement mechanism of 1,4-endoperoxides to 1,2:3,4-diepoxides have been reported.172 We point out that conversion of endoperoxides to cis-epoxides and epoxy ketones has
143
also been successfully carried out with spiro[2,4]heptadiene.173 Furthermore, a photooxidation strategy was successful in the synthesis of the natural product senepoxide.174
Scheme 15. Synthesis of fusicogigantepoxides and fusicogigantones
H
O O H H H O2,h , TPP 69
toluene-d8, -78 °C H
67 68 H
O O 70
144
Fourth, a 2014 paper describes the RB-sensitized photooxidation of furan 75 in the presence of diisopropylethylamine (DIPEA) (Scheme 16).175 Preferential base abstraction of the left-hand bridgehead proton of the endoperoxide causes its decomposition by O–O bond heterolysis to butenolide 76. The early finding on the furan mechanism of forming two C–O bonds and breaking of an O–O bond to reach butenolide is interesting,176 which has been exploited to reach other butenolide and spiroketal natural products (as mentioned in Section 3).43-47
1 Paradoxically, tertiary amines (such as DIPEA) physically quench O2 yet is essential for the base abstraction process. X-ray crystallographic data were also collected for 76. The preparation of four additional triptolide derivatives was accomplished, for an evaluation against ovarian (SKOV-3) and prostate (PC-3) cancer cells showing the activity depends critically on the D ring in these structures.
Scheme 16. Synthesis of a triptolide derivative
145
6.3.5 Tropones and Tropolones
Tropones and tropolones are widely seen in nature and represent highly oxygenated natural products. We have located four studies that use or form tropones and tropolones; first, a 2001 report described the hematoporphyrin-sensitized photooxidation of atropisomers of (-)- isocolchicine (78/79) which led to diastereomeric endoperoxides 80/81 and 82 in 54% and 8%
177 1 yield, respectively (Scheme 17). Preferential anti addition of O2 to (M,7S)-(-)-78 atropisomer is due to molecule twisting negating the steric interactions from the benzenoid ring led to 80/81 as major endoperoxide products. Endoperoxides 80/81 and 82 were separated by column chromatography, in which the latter was favored (ratio of 80/81:82 was 7:1). Further synthetic steps then led to 83.
146
Scheme 17. Photooxidation of isocolchicine atropisomers
1 Second, a 1997 paper described the reaction (-)-colchicine 84 with O2 to reach endoperoxide 85 (Scheme 18).178 This reaction has high regio- and facial selectivity due to the
1 importance of sterics and hydrogen bonding of the acetamide N–H with O2. Endoperoxide 85 was then treated with triphenylphosphine or silica gel to reach N-acetylcolchinol O-methyl ether (87)
147
or androbiphenyline (88) via the 4-membered ring lactone 86. Allo-colchicinoids, 87 and 88, are responsible for cell growth and tubulin polymerization inhibition,179 but until this synthetic work, preparative photooxidations of helimers were largely neglected to reach natural product targets.
Direct photolysis studies in the last few years has also shown the virtues of using helimers in synthetic applications.180
Scheme 18. Photooxidation of (-)-colchicine
OMe MeO OMe O MeO OMe 1 MeO O2 OMe O O2,h , O OMe H H H hematoporphyrin O HN HN CHCl3, 0-20 °C syn O O addition
(M,7S)-(-)-colchicine, 84 85
MeO OMe
MeO OMe H
HN PPh3 R=H O
- O=PPh3 MeO OMe - CO2 (M,7S)-(-)-N-acetyl-colchinol (40%) MeO O OMe O-methyl ether, 87 85 H O R MeO OMe silica gel HN R= OH O MeO OMe (60%) - CO2 H HO 86 HN O
(M,7S)-(-)-androbiphenyline, 88
148
Third, a 2006 report on the TPP-sensitized photooxidation of a dioxin-fused cycloheptatriene (89) was found to produce tricyclic endoperoxide 90 in 94% yield (Scheme
19).181 Treatment of 90 with thiourea led to O–O bond reduction to tropolone 91 or other tautomers
92-94 which bear some similarity to the natural product colchicine 84. Tropone endoperoxide 90
1 releases O2 in a reverse Diels-Alder reaction forming starting material 89, in a similar fashion to
1 182-184 naphthalene and anthracene endoperoxides which are storage pools to get back O2. Previous reports have also shown the photooxidations of benzotropones give endoperoxides that are stable at low temperatures.185-187 Encouragingly, benzotropones possess carbonic anhydrase isoenzymes inhibition properties.188
Scheme 19. Synthesis of tropolone compounds
Fourth, a 1991 report described the MB-sensitized photooxidation of clavukerin A 95 with pyridine in methanol which led to hydroperoxide 96 in 79% yield (Scheme 20).189 Hydroperoxide
96 was treated with acetyl chloride in pyridine giving the acetyl peroxide, which underwent a
Hock-like 1,2-allyl shift to the oxonium ion 97. Addition of TFA leads to a stereoselective
149
protonation that can be explained by 1,2-neighboring group induction from the methyl center with ring opening to the tetrahydrotropones, for (±)-clavularin A 98 and (±)-clavularin B 99 in a 9:1 mixture. Other than tropones and tropolones,190 natural products containing multiple rings common,191 as we will see next.
Scheme 20. Synthesis of (±)-clavularin A and B
6.3.6 Polycyclic Ethers and Polyols
There have been reports in the synthesis of polycyclic ethers and polyols featuring [2 + 4]
1 O2 cycloaddition strategies. A 1999 report describes brevetoxin A 104 in which a key step was a
TPP-sensitized photooxidation of the diene site of 100 (Scheme 21).192 This led to the formation of a diastereomeric endoperoxides 101. Reduction of the O–O bond of 101 with aluminum amalgam in THF/H2O led to the formation of diastereoisomeric diols 102 and 103 in a 1:1 ratio in
58% yield over two steps. Additional steps in the synthesis lead to 104. Such polycyclic ethers and biomimetic pathways to reach them have been of interest due to their therapeutic and toxic properties.193,194
150
Scheme 21. A Singlet oxygen reaction in the synthesis of brevetoxin A
Recently, a 2014 report described the RB-sensitized photooxidation of furan 105 followed by reaction in pyridine with DMAP and acetic anhydride at led to butenolide 106 in 76% yield
(Scheme 22).195 Desilylation of butenolide 106 with tetrabutylammonium fluoride followed by a
Michael addition/cyclization led to (-)-(1S,3R,7R,9R,11S,12R)-furopyranopyranones 107 and 108
151
in 20% and 60% yields, respectively. Compounds 107 and 108 that arose from a furan precusor have potential synthetic utility as building blocks of polycyclic ether natural products.196-199
Schemes 22. Synthesis of polycyclic ethers
In a different approach, one variation of the furan photooxidation topic is its substitution with a silyl group. A 1997 report described the TPP-sensitized photooxidation of trimethylsilylfuran 109 (Scheme 23).200 Here, the reaction involved an intramolecular silyl migration201 leading to butenolide 110 in 95% yield. Further steps were then required to reach milbemycin E 111.
152
Scheme 23. Synthesis of milbemycin E
A paper 2007 reported the synthesis of the C29-C46 subunit I (116) of oasomycin A 117, in which one step included the RB-sensitized photooxidation of oxazole 112 (Scheme 24).202 Here,
1 singlet oxygen was taken up by the oxazole site in 112. The reaction was a O2 [2 + 4] cycloaddition and not a [2 + 2] cycloaddition, as was previously deduced by singlet oxygen 18O-tracer studies with oxazoles.203 The intermediate endoperoxide 113 underwent a Baeyer-Villiger type rearrangement to form imino anhydride intermediate 114 which upon O-acyl to N-acyl migration led to triamide intermediate 115. The triamide 115 released N-benzoylbenzamide in a lactonization process to form 116 in 90% total yield. Such rearrangements of imino anhydrides to triamides are known and are highly useful.204-208
153
Scheme 24. Synthesis of the C29-C46 subunit of oasomycin A
OH OH OH OH
N N Ph Ph O O O OPMB OTBS OTBS O2,h , RB OPMB OTBS OTBS O Ph Ph HO OTBS CH2Cl2,0 °C HO OTBS
112 113
O Ph O Ph OH OH OH OH N N Ph
O O O O OPMB OTBS OTBS OPMB OTBS OTBS Ph HO OTBS HO OTBS
114 115
O
OH O
O O OPMB OTBS OTBS (90%) Ph N Ph H HO OTBS
116
OH OH OH O O
O O 46
HO OH OH OH OH OH OH OH
29 OH OH oasomycin A, 117
1 Next, we describe sterol natural products and mimicks that are reachable by O2 chemistry.
154
6.3.7 Sterols
1 209 Themes on the synthesis of sterols via O2 chemistry have been reported. A 2011 report described the biomimetic synthesis of 5,6-dihydroglaucogenin C 124 (Scheme 25).210 One step involved the TPP-sensitized photooxidation of 118 yielding the hydroperoxide 119 in 99% yield.
The resultant hydroperoxide 119 reacted by a ferrous ion-catalyzed homolysis of the O–OH bond, forming an alkoxy radical and subsequently a 3° carbon radical as transient intermediates. Addition of I2 led to an iodolactone, prior to a regioselective HI elimination affording 123 in 69% yield in
2 steps. Evidence for the existence of a 3° carbon radical was due to trapping with TEMPO, when used in place of I2.
155
Scheme 25. Synthesis of 5,6-dihydro-glaucogenin C
A 2013 report described the TPP-sensitized photooxidation of 126 in pyridine led to an ene reaction where treatment of the resulting hydroperoxide 127 with triphenylphosphine gave allylic alcohol 128 in 61% yield in 2 steps (Scheme 26).211 The synthesis of withanolide A 129 required three additional steps. Additional fifteen unnatural anaolgs of 129 were prepared to probe their neuronal differentiation and neurite outgrowth activity.
156
Scheme 26. Synthesis of withanolide A
A 1996 report featured the synthesis of bis(trifluoromethyl)imidazoline 134 from a MB-
212 1 sensitized photooxidation of pyrrole 130 (Scheme 27). A [2 + 2] cycloaddition on 130 with O2 led to a dioxetane species in situ, which cleaved apart to the dicarbonyl compound, 131. To a lesser extent, the photooxidation of pyrrole 130 proceeded by a Diels-Alder reaction with formation of a methanol adduct 132 and water adduct 133 in 18% and 6% yields, respectively. Further steps led to compound 134, where an inhibition connection was found between 134 and active cholesterol acyltransferase (ACAT).
157
Scheme 27. Synthesis of an imidazoline steroid mimic
6.3.8 Opioids
1 Strategies have also been developed for accessing opioids via O2 chemistry, as the next two examples show. First, in 2000, a paper described the TPP-sensitized photooxidation of thebaine 135 which led to an opioid endoperoxide 136 in the expected [2 + 4] fashion, which on
213 1 loss of an electron led to cyclohexene-1,4-dione 138 (Scheme 28). A second O2 molecule added by a [2 + 2] cycloaddition to form dioxetane 139 in situ. This di-singlet oxygenation process was followed by dioxetane ring-opening to formamide 140. Benzofuran 141 was a minor product and its origin suggested from a double Norrish type I photocleavage of dione 140.
158
Scheme 28. Photooxidation of thebaine
Second, in 2015, a report described the TPP-sensitized photooxidation of diene (R)-142, in which the R stereochemistry is at nitrogen (Scheme 29).214 Here, the singlet oxygenation of the diene quaternary salt led to the corresponding endoperoxide (R)-143 in 93% yield. The formation of (R)-methylnaltrexone 144 occurred after the hydrogenation of (R)-143.
Further examples of natural products bearing ring-fused structures are described next.
159
Scheme 29. Synthesis of (R)-methylnaltrexone
6.3.9 Ring-Fused Examples
1 Researchers have examined reactions of ring-fused compounds with O2, and four such examples are presented here. A 2011 report describes the TPP-sensitized photooxidation of
1 cyclopentadienyl allo-cedrane 145 where the anti-endoperoxide 146 arose from attack of O2 on the less shielded bottom face of the diene (Scheme 30).215 This was followed addition of Zn and acetic acid to furnish cis-1,4-enediol 147 in 77% yield after 2 steps, which contained a paddlane core similar to tashionin 148. Other allo-cedrane derivatives were also examined in this 2011 paper.215
160
Scheme 30. Synthesis of a tashironin-like compound
Another intriguing example is from a 2009 report describing the synthesis of natural product (±)-phomactin A 152 (Scheme 31).216,217 One step in the synthesis involved a RB- sensitized photooxidation of diene 149 that led to endoperoxide 150 in 65% yield.216 Singlet oxygen reacted from the more exposed site above the diene to reach endoperoxide 150, which reacted with KOAc and 18-crown-6 through a deprotonation reaction to give hydroxypyranone
151 in 94% yield. Attempts to reduce the endoperoxide with as Lindlar’s catalyst, thiourea or triphenylphosphine did not occur as would have been expected to reach ene-dione 151.218
161
Scheme 31. Synthesis of (±)-phomactin A
A 2014 report describes the RB-sensitized photooxidation of 153 and 154 which gave
4β,5β-epoxyxanthatin-1α,4α-endoperoxide 155 and 4α,5α-epoxyxanthatin-1β,4β-endoperoxide
156 as a mixture in 70% yield (Scheme 32).219 Pure 155 and 156 were isolated by preparative
HPLC. The formation of endoperoxide 155 was favored (ratio of 155:156 was 5:1) as the result of
1 O2 addition to the diene anti to the methyl substituent.
162
Scheme 32. Synthesis of epoxyxanthatin endoperoxides
A 2009 report describes the MB-sensitized photooxidation of tridachiahydropyrone (157) which gave (±)-158 as colorless oil in 99% yield (Scheme 33).220 In this work, the structure of 157 was revised, and (±)-158 was named as (±)-oxytridachiahydropyrone. HMBC, HMQC, and COSY
1 data were collected, where O2 reacts selectively via attack on the bottom face of 157 for the exclusive formation of the endo product (±)-158.
Scheme 33. Synthesis of (±)-oxytridachiahydropyrone
163
6.3.10 Phenols
1 Strategies have been developed for accessing natural product phenols from O2 reactions, as we will see in this subsection. An approach reported in 2014 uses a the TPP-sensitized photooxidation of phenol 159 which led to its dearomatization (Scheme 34).221 Singlet oxygen added selectively to the opposite side of the shielding trimethylsilyl ether furnishing the hydroperoxy quinol 160. Quinol 161 was formed as a pink solid after the hydroperoxide group of
160 was reduced with triphenylphosphine in 74% yield in 2 steps. X-ray crystal data were obtained for 161. Similar strategies on reduction of O–O bonds in peroxyquinols to form tertiary alcohols have been reported.222 Tertiary alcohol 161 was a substrate for further reactions leading to model compound 162, which has a [5-7-6] tricyclic core similar to the natural product prostratin 163.
164
Scheme 34. Synthesis of tigliane and daphnane-type compounds
A 2015 report described the RB-sensitized photooxidation of (+)-methyllinderatin 164
1 223 which led to dioxetane 165 by a O2 [2 + 2] cycloaddition (Scheme 35). An adjacent phenol oxygen attack of the nearby of the dioxetane 165 carbon accounts for the C–O ring opening to a diastereomeric mixture of hydroperoxides 166 and 167 in a 2:1 ratio with 76% yield. X-ray data were collected for adunctin E 166 to help establish its absolute stereochemistry.
165
Scheme 35. Synthesis of (-)-adunctin E
A 2003 report described the synthesis of a bicyclo[4.3.0] compound (170) to reach a BCE ring system similar to the natural product ryanodine 171 (Scheme 36).224 Introduction of the endoperoxide group in 169 was accomplished regioselectively by the MB-sensitized photooxidation of 168. The hydrogenation of the endoperoxide O–O bond in 169 gave diol 170 in
98% yield, where X-ray crystallography was used to help establish its stereochemistry.
166
Scheme 36. Synthesis of the BCE Ring structure of ryanodine
An approach reported in 2011 featured the MB-sensitized photooxidation of trans-
225 1 resveratrol (172) (Scheme 37). Notably, the main pathways are a [2 + 2] cycloaddition of O2
1 that led to aldehydes 175 and 176, and a [2 + 4] cycloaddition of O2 that led initially to an endoperoxide, which upon heating rearranged to moracin M 177 and 2-hydroxacetaldehyde. This study is a rare example in natural products synthesis where the reaction rate constants were
1 quantitated. The chemical quenching rate constant (kr) of O2 with trans-resveratrol 172 was found
6 -1 -1 to be 1.5 × 10 M s , where the kr accounted for 25% of total rate constant (kT).
167
Scheme 37. Synthesis of moracin M
A 2008 report describes the MB-sensitized photooxidation of isoeugenol (178) (Scheme
226 1 38). Here, a [2 + 2] cycloaddition of O2 led initially to a dioxetane species, which cleaved apart to vanillin 179 and acetaldehyde. The reaction is a mixed photooxidized system since dehydroisoeugenol 180 and other products arise by a type I photosensitized oxidation41 forming oxygen radical intermediates. Compound 181 may form by phenolic hydrogen abstraction in 179 and radical addition to the aldehydic group of a second 179 molecule followed by hydrogen atom transfer. Mechanistically, condensation of 179 to furnish ester 181, likely involved a type I photosensitized oxidation to provide radicals for the ester coupling. Coniferyl alcohol also led to vanillin 179 (reaction not shown), although ferulic acid underwent a trans-cis C=C bond isomerization likely through zwitterionic peroxy intermediate analogous to (E,Z)-2,4-dimethyl-
2,4-hexadiene139,227,228 and trans-propenyl anisole,229 which was also examined.
168
Scheme 38. Synthesis of vanillin, dehydroisoeugenol and derivatives
2+ A 2014 report described the RB or Ru(bpy)3 —sensitized photooxidation of triacetate 182 which was followed by reduction with triphenylphosphine, giving the tertiary alcohol 183 and secondary alcohol 184 in 62-71% yields (Scheme 39).230 The ratio of 183:184 was 2:1 for RB, but
2+ 1 Ru(bpy)3 produced an 8:1 ratio suggesting not only O2 but other oxygen species in the reaction.
Three additional steps, including dehydration and a [2 + 4] cycloaddition led to kuwanon I (185) and kuwanon J (186). Related natural products, brosimone A and B, were also synthesized in a similar manner to kuwanon I 185 and kuwanon J 186.231
169
Scheme 39. Synthesis of kuwanons I and J
A 2004 report used a the TPP-sensitized photooxidation of natural prenylated coumarins mammea A/AA (187), mammea A/BA, and mammea B/AA followed by reduction with triphenylphosphine led to disparinol A 189, isodisparinol A and disprorinol A in 60%, 63% and
65% yields, respectively (Scheme 40).232 In addition to the prenylcoumarins, the importance of natural prenylxanthone photooxidations was also realized. For example, xanthone 190 led to hydroperoxides 191 and 192, and after reduction with triphenylphosphine, to allylic alcohol 193 and a pyranoxanthone natural product 6-deoxyisojacareubine 194. A puzzling effect of regioselective formation of 188 but not in the case of 191 and 192 suggests to us the regioisomer
170
of 188 was formed, but not isolated. The oxidations of prenylated natural products were of interest in terms of their bioactivity.233
Scheme 40. Photooxidation of prenylated coumarins and xanthones
O OH O OH O OH PPh3, O2,h , TPP CH2Cl2, r.t.
HO O O CH2Cl2, 15 °C HO O O silica gel HO O O OOH (60% after OH 2 steps)
mammea A/AA, 187 188 disparinol A, 189 (other regioisomer likely formed)
O OH O OH O OH
O2,h , TPP
O OH CH2Cl2, 15 °C O OH O OH OH OH OOH OH
OOH 190 191 192
O OH O OH PPh3, CH2Cl2, r.t.
silica gel O OH O O OH OH OH
193 194 (8%) (17%)
1 While the vast majority of O2 reactions use an external sensitizer as a strategy to synthesize natural products, this is not always necessary as will be seen in the next section.
171
6.3.11 Self-sensitized Examples There are self-sensitized examples to reach natural products, including a 1994 report described a self-sensitized photooxidation of anthracene 195 was used in the synthesis of (−)-
234 1 balanol 199 (Scheme 41). A O2 [2 + 4] cycloaddition with 195 led to endoperoxide 196.
Protonation of the peroxide oxygen destabilizes the C–OO bond toward heterolysis, where hydration and then methoxide ion attack and ring-opening led to benzophenone derivatives 197 and 198. Additional synthetic steps were carried out leading to (−)-balanol 199, which contains a benzophenone and a chiral hexahydroazepine. (−)-Balanol 199 is a potential inhibitor of protein kinase C.235
Scheme 41. Synthesis of (-)-balanol
172
A paper in 1991 has described a self-sensitized photooxidation of anthracene-1,5-diol 200
(Scheme 42).236 Singlet oxygen was captured by 200 regioselectively at the 9,10-carbons leading to cycloadduct 201, which was reduced with NaBH4 and upon air oxidation led to anthraquinone
202. Additional steps were required to reach vineomycinone B2 methyl ester 203.
Scheme 42. Synthesis of vineomycinone B2 methyl ester
Moving on from self-sensitized reactions, we now describe the synthesis of natural product indoles.
173
6.3.12 Indoles
Strategies have been developed that use indole photooxidation in the synthesis of natural products and analogs. We have located four studies, including a report in 2009 that TPP-sensitized photooxidation of indole 204 which led to hydroperoxide 205 (Scheme 43).237 Under acidic conditions, protonation of the hydroperoxide group in 205 causes water loss and carbonyl formation in oxindole 206 in 88% yield in 2 steps. Hydrogenation of 206 led to epimeric indoles
207 and 208 in a 1:2 ratio in 97% yield. Indoles 207 and 208 contained the [2.2.1] oxobicycloheptane core and thus are similar to the natural product welwistatin 209. Further synthesis on a range of alkylated and acylated indole derivatives of 207 and 208 was conducted where the alkylated derivatives were furnished in higher yields.
Scheme 43. Synthesis of oxindoles
174
A report in 2004 describes a secoporphyrazine-sensitized photooxidation of tryptophan 210
(Scheme 44).238 Afterwards, deoxygenation of the hydroperoxide by dimethylsulfide formed hydroxypyrroloindole diastereoisomers 212 and 213 in 1:1 ratio in 58% combined yield. The anthelmintic natural compound (-)-CJ-12662 214 was prepared in 4 further steps.
Secoporphyrazine is less commonly used in synthesis, but is a good sensitizer239 which gave a high
1 240,241 yield of a triplet state that is capable of efficient energy transfer to oxygen to form O2. The
1 hydroxypyrroloindoles 212 and 213 originated from a O2 [2 + 2] cycloaddition and deoxygenation of dioxetane by dimethylsulfide followed by nucleophilic attack at C2 by the amide nitrogen, and
1 not through a O2 ene reaction. Examples on tryptophan and indole photooxidations have been of interest.242-245
175
Scheme 44. Synthesis of the natural product (-)-CJ-12662
A report from 2001 describes the synthesis of okaramine N 218 (Scheme 45).246 Because
1 the azocane-fused indole reacts readily with O2, it was protected with the enophile N- methyltriazolinedione (MTAD). The N–H allylic bond in diketopiperazine 215 reacted with
MTAD by an ene reaction to reach urazole 216, which enabled a regioselective photooxidation of the tethered indole in intermediate 216. The RB-sensitized photooxidation of 216 was followed by deoxygenation with dimethylsulfide and the intramolecular cyclization led to 217. Additionally,
176
the thermal release of MTAD in a retro-ene reaction led to okaramine N 218 in 70% yield in 4 steps. This reaction is unique because it protected the fused indole site and more substituted C2–
C3 indole bond,247 which capitalized on ene reactions with MTAD that have been characterized previously.248-257
Scheme 45. Synthesis of (+)-okaramine N
N N
N H O O H N H N H O N N O 1. O2,h , MB, N O H O MTAD H O MeOH, -28 °C N O N CH2Cl2, -5 °C HN N 2. Me2S, -10 °C
N N H 215 216
N OH N OH H H 110 °C N H N H O O N O (70% over O O H O H N 4 steps) N HN N
N N H
217 (+)-okaramine N, 218
An approach reported in 2012 described the synthesis of (-)-melohenine B (223) in 99% yield from the MB-sensitized photooxidation of epimers (±)-219 and (±)-220 (Scheme 46).258
177
Notably, the ratio of epimers 219 and 220 was unimportant because the path sequence (dioxetane cleavage then hydroxypiperidine ring-opening or visa versa) ended with a stereospecific ring reclosure to reach (-)-223.259,260 Molecule curvature was of importance where singlet oxygen added regioselectively via the convex side of 219 and 220. The dioxetane intermediates (221 and 222) were formed, where C14 equilibration through an aldehyde intermediate 226, which also equilibrated with dioxetane 221. The position of the hydroxyl group at C14 was assessed from X- ray data of (-)-223.
Scheme 46. Synthesis of (-)-melohenine B
178
6.3.13 Lactams and Related Examples
This subsection provides eight examples where singlet oxygen was used in the synthesis of natural product lactams. The first is a historical example with the synthesis of intermediate 229 reported in 1984 by an enamine—singlet oxygen reaction (Scheme 47).261 A bisacenapthalenethiophene (BANT)-sensitized photooxidation of 227 led to tricarbonyl 228 in
1 42% yield. The enamine site in 227 was cleaved by O2 to form the vicinal tricarbonyl system in
228. The facile cleavage of enamines by singlet oxygen to form carbonyl derivatives has utility not only in the synthesis of β-lactams, but also ketones, lactones and esters.262-264 Upon desilylation and intramolecular cyclization by attack of the lactam nitrogen on the center carbonyl group of
228 followed by reaction with TMSI led to 229. Compound 229 has served as a precursor in the synthesis of antibiotic (±)-PS-5 230.265
Scheme 47. Synthesis of antibiotic (±)-PS-5
179
A 2015 report266 describes the MB-sensitized photooxidation of difuran 231 where MB played a dual role, one as a sensitizer and the other as a redox catalyst (Scheme 48). After the photoreaction, dimethylsulfide was added and subsequently ammonia, which led to the formation of 1,5-dihydro-pyrrol-2-one 232. After concentrating the mixture, it was dissolved in chloroform containing molecular sieves (4 Å) and TFA to give pandamarine 233 in 30% overall yield. This
MB sensitizer and redox catalyst approach266,267 has enabled the access to other units
(diazaspiro[4,5] and 5-ylidenepyrrol-2(5H)-one) common in natural products and drugs.268-272
Scheme 48. Synthesis of pandamarine
A 2014 report describes the RB-sensitized photooxidation of difuran 234 which led to a
1 273 tandem addition of O2 (Scheme 49). After solvent removal, the residue was placed in pyridine with acetic anhydride, followed by the addition of TMSBr leading to methoxybutenolide 235 as a single diastereomer in 75% yield. Addition of H2SO4 to a solution of butenolide 235 led to pandamarilactone-1 236 in 12% yield, and pandamarilactonines A and B 237 in 48% yield with
180
the latter in a 7:3 ratio. While not lactam forming, we thought it is logical to be included in this subsection since a unique spiro-N,O-acetalization and elimination took place to form a spiro- lactone-piperidine structure in 236 and its rearranged lactone-pyrrolidine structure in 237. Another example of the furan topic is a 1995 report on the RB-sensitized photooxidation of trimethylsilylfuran 238 in the presence of DIPEA which led to 239 in 99% yield (Scheme 50).274
The presence of the trimethylsilyl group improved the yield of butenolide formation.201,275
Butenolide 239 underwent further reactions to reach (-)-PI-091 240, a platelet aggregation inhibitor.274
Scheme 49. Synthesis of pandamarilactone-1
1. O2,h , RB MeOH, 0 °C OMe MeO O O N O 2. Ac O, Py O N O O 2 H Boc 3. TMSBr, 234 CH2Cl2, r.t. 235 (75%)
O O H2SO4 N O N O
CH2Cl2, r.t. O O O O pandamarilactone-1, 236 pandamarilactonines A and B, 237 (12%) (48%) A:B = 7:3
181
Scheme 50. Synthesis of (-)-PI-091
An approach reported in 2013 featured the RB-sensitized photooxidation of furan 241 followed by the addition of dimethylsulfide and then of 3,4-dimethoxyphenethylamine led to lactam 243 in 54% yield (Scheme 51).276 The cyclization process is rationalized by intermediate
242, where an additional step led to 1,5-dihydro-pyrrol-2-one 244. Compound 244 contains the bis-spiro structure similar to that seen in the natural product erysotramidine 245.
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Scheme 51. Synthesis of a tetracyclic compound similar to the natural product erysotramidine
1 A 1993 report described the reaction of bipyrrole 246 with O2 by a [2 + 4] cycloaddition reaction (Scheme 52).277 This reaction led to the D,L and meso forms of isochrysohermidin (248 and 249) in 42% combined yield in a 1:1 ratio. The photooxidation of pyrroles have been shown to have utility in the synthesis of 1,5-dihydro-pyrrol-2-ones.278 An example is shown below. The singlet oxygenation of pyrrole 250, in which RB was the photosensitizer gave 1,5-dihydro-pyrrol-
2-one 252 in 92% yield (Scheme 53).279 The reaction produced the endoperoxide 251 before the decarboxylation and formation of 252. Pyrroles and related indolizines also tend to undergo type
I photooxidation reactions.280-284
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Scheme 52. Synthesis DL and meso isochrysohermidin
Scheme 53. Synthesis of a 1,5-dihydro-pyrrol-2-one
1 In conclusion, Section 4 described batch reactions which have been established using O2 in the synthesis of natural products and analogs. There is continuity between batch photooxygenations and reactions conducted in flow settings, as is discussed next.
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6.4 Singlet Oxygen in Flow Synthesis
6.4.1 Background
1 We now turn to O2 flow photoreactors and discuss their success in synthesis to date.
Schemes 54-66 show the extent of compounds synthesized thus far, including juglone, butenolides, rose oxide and artemisinin. Flow experiments to synthesize compounds of high complexity as seen in Section 4 have not yet emerged. Also, reports of tandem addition of singlet oxygen molecules in flow reactions are absent from the literature.
1 In what follows, O2 microflow and macroflow photosystems are described, where Figures
1 and 2 provide illustrations, respectively. There are 20 studies that have used micro- or macroflow
1 1 O2 photoreactors in synthesis. Photoreactors have also been used to produce airborne O2, but will
285-289 1 285 not be described. For example, a Pyrex-tube flowing O2 was developed, where reaction
1 rate data for O2 with alkenes was reported in the gas phase (Figure 3), but this device is not compatible with organic synthesis because of a very rapid oxygen sparge rate, which would cause solvent evaporation.
1 We provide accounts of four O2 photoreactor geometries (i) microflow reactor, (ii) macroflow reactor, (iii) supercritical carbon dioxide reactor, and (iv) bubbling reactor in subsections numbering from 6.4.1-6.4.5 below. We start our discussion by presenting examples of
1 O2 microflow reactors that used dissolved sensitizers (Section 6.4.1).
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Scheme 54. Flow reactor photooxidation of α-terpinene
1 O2 O O
-terpinene, 253 ascaridole, 254 p-cymene, 255 (can form as a byproduct)
Scheme 55. Flow reactor photooxidation of α-pinene
Scheme 56. Flow reactor photooxidation of (-)-β-citronellol
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Scheme 57. Flow reactor photooxidation of cyclopentadiene
Scheme 58. Flow reactor photooxidation of alkenes
A
B
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Scheme 59. Flow reactor photooxidation of 1,5-dihydroxynaphthalene and phenol
A
B
Scheme 60. Flow reactor photooxidation of 2-(3-methoxyphenyl)-3-methyl-1-benzofuran
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Scheme 61. Flow reactor photooxidation of 9,10-dimethylanthracene and 9,10-anthracene dipropionate dianion
A
B
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Scheme 62. Flow reactor photooxidation of cholesterol
Scheme 63. Flow reactor photooxidation of methionine derivatives and an organic sulfide
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Scheme 64. Flow reactor photooxidation of dihydroartemisinic acid
Scheme 65. Flow reactor photooxidation of amines
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Scheme 66. Flow reactor photooxidation of furan derivatives
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1 Figure 1. A microflow system capable of generating O2 in micrometer sized channels. LED or other light sources are often positioned above the plate.
1 Figure 2. A macroflow system capable of generating O2 a in millimeter sized channel. Optical energy is delivered from a light source with transparent tubing coiled around it. Reagents are conveyed through the tube and products drain out the end.
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1 Figure 3. Murray’s gaseous O2 photoreactor device. A Pyrex tube was coated on the inside with 1 rose bengal. Oxygen gas flowing through the illuminated tube exits enriched with O2.
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6.4.2 Singlet Oxygen Microphotoreactors
We have located 11 studies that use microflow photoreactors with solution-phase and immobilized sensitizers (Sections 6.4.2.1 and 6.4.2.2).
6.4.2.1 Microphotoreactors with Solution-Phase Sensitizer
In 2013, a paper reported that ascaridole 254 and byproduct p-cymene 255 were formed in
90% total yield from a RB-sensitized microflow photooxidation of α-terpinene 253 (Scheme
54).290 Byproduct p-cymene 255 arises from a type I photosensitized oxidation process. The device consisted of a microcapillary film (MCF) with 10 parallel channels. Oxygen mass-transport was enhanced by using degassed solvent, where O2 was transported through a fluorinated ethylene propylene (FEP) microcapillary film. Here, space-time yields were found to be 20 times greater compared to a batch reaction. A similar reaction was studied in 2002,291 where ascaridole 254
(85% yield) was also formed from RB-sensitized flow photooxidation of α-terpinene 253 using a glass microchip device with etched channels.292,293
A 2014 report described pinocarvone 257 was synthesized from a TPP-sensitized microflow photooxidation of α-pinene 256 (Scheme 55).294 The system was a microreactor-LED where the reaction led to pinocarvone 257 in pyridine with DMAP and acetic anhydride, as was previously established.295 Space-time yields for the microreactor were found to be 3-7 times higher than other reactors, including an immersed-well and an annular flow system.
2+ A report in 2007 focused on a Ru(bpy)3 —sensitized microflow photooxidation of (S)-(-)-
β-citronellol 259 (Scheme 56).296,297 The device consisted of a blue LED light source and meandering channels where the solution came full circle, i.e. it was pumped in a loop while being
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irradiated for 60-70 h. The yield for 260 was 60%, and for 261 was 40% by HPLC. The
corresponding alcohols were formed after reduction with Na2SO3. Yields were found to be only slightly higher for the microflow reactor compared to the batch reactor, which used a Xe-lamp.
A falling-film microreactor was reported in 2005 to synthesize 2-cyclopenten-1,4-diol 267 in 20% yield from RB-sensitized microflow photooxidation of cyclopentadiene 265 (Scheme
57).298,299 The reactor consisted of a plate with 32 parallel microchannels along with a Xe lamp.
The film flowed downward simply as a consequence of gravity. After exiting the reactor, the endoperoxide 266 is reduced to cis-cyclopent-4-ene-1,3-diol 267 with thiourea. There was no optimization of this flow reaction.
In 2011, a paper described a dual microreactor to synthesize allylic alcohol 262 and 263 in
95% yield (262:263 = ratio 1.0:1.5) from a MB-sensitized microflow photooxidation of citronellol
259 (Schemes 56 and 58A).300 The products were analyzed after treatment of hydroperoxides 260 and 261 with NaBH4 in methanol. The device used a white LED light source, but was unique. It contained a polyvinylsilazane (PVSZ)-line dual-channel where liquid flow and oxygen flow channels were separated by a PDMS layer. The PDMS is gas permeable and permitted rapid saturation of the solution with oxygen. Reaction times ranged from 2-3 min. Other substrates were photooxidized, including α-terpinene 253 that gave ascaridole 254 (91% yield), and allylic alcohols 268 (R = H or Me) to give hydroperoxides 269 (R = H or Me) in 99% yield. The relative efficiency of the dual channel system was found greater than a mono-channel system due to a more efficient oxygen mass transfer process. Furthermore, the efficiency of the dual-channel system was
2.6-fold higher compared to a batch reaction.
A 2014 report describes a microreactor that was fabricated to synthesize ascaridole 254 in
92% yield from RB-sensitized microflow photooxidation of α-terpinene 253 (Scheme 54).301 There
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1 are also data for the O2 microreaction with naphthalene-1,5-diol 272 to give juglone 273 in 92%
1 yield (Scheme 59A) and also the O2 microreaction with citronellol 259 to give allylic hydroperoxides 260 and 261 in 92% yield. Ring opening of an initially formed dioxetane can take place (Scheme 60). For example, the reaction of 2-(3-methoxyphenyl)-3-methyl-1-benzofuran 278
1 with O2 formed 2-acetylphenyl-3-methoxybenzoate 280 in 97% yield. The device consisted of borosilicate glass with channels and a serpentine section, and used an OLED light source. This particular microphotoreactor did not outperforming a batch reaction. However, the thinking was to wrap the flexible OLED white light source around the reactor, which seemed very reasonable.
Lastly, in 2012, a report described the sulfamidic Zn phthalocyanine-sensitized microflow photooxidation with 9,10-dimethylanthracene 281 in DMF (Scheme 61A).302 In this case, a high- pressure mercury lamp was the light source, and samples were followed by HPLC. The data showed that the microreactor could photooxidize the anthracene 281 more rapidly than in a batch reactor.
6.4.2.2 Microphotoreactors with Immobilized Sensitizers
1 Heterogeneous O2 sensitizers have been used in flow, although they are few in number.
We have located 3 studies that use microflow photoreactors with immobilized sensitizers, i.e. the solution is devoid of any sensitizer. It is expected that sensitizers such as porphyrins and fullerenes
1 would remain powerful sensitizers for the production of O2 when immobilized in the photoreactor.
However, restoring of photobleached sensitizer sites has not yet been accomplished.
A 2012 paper reported on immobilized tri(N-methyl-4-pyridyl)porphyrin sensitized microflow photooxidations of α-terpinene 253, citronellol 259, and cholesterol 285 and other
303 compounds were carried out in 30 s in methanol/CH2Cl2 (Schemes 56, 62 and 67). The device
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consisted of a glass microfluidic system with 16 parallel channels that had tetraaryl porphyrin
1 covalently attached through a thiourea bridge to photogenerate O2. Samples collected from the α- terpinene 253 and citronellol 259 experiments were monitored by HPLC and LCMS. The singlet oxygenation of cholesterol 285 led to 5R-hydroperoxycholesterol 286, where the reaction also provides a route to 7R/7β-hydroperoxy and 6R/6β-hydroperoxy cholesterol byproducts by oxygen
1 radicals and not a O2 reaction. The product forming efficiency of the microfluidic system was higher than in a batch reaction. Other reports also describe cholesterol hydroperoxides and epoxides that are carcinogenic or mutagenic,304 and their formation by photosensitized production of singlet oxygen (type II) and oxygen radicals (type I).
Scheme 67. Structure of the glass-attached porphyrin
A 2006 paper describes the generation of p-benzoquinone 276 from a silica bead-supported tetraaryl porphyrin-sensitized microflow photooxidation of phenol 274 after 42 s in buffer solution
(pH 10) (Schemes 59B and 68).305 There was evidence for the existence of the endoperoxide
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intermediate, but the p-benzoquinone oxidatively processed to CO2 and maleic or fumaric acid. p-
Catechol is also a known product of phenol photooxidation.306 The device consisted of PTFE microchannels with silica beads bearing a covalently bound porphyrin through a pyridinium ion
1 bridge to photochemically generate O2. The silica beads were stationary and resided at the bottom of the microchannels. The activity of this microflow system was higher than the silica beads in suspension based on the photooxidative decomposition of phenol.
Scheme 68. Structure of a silica-attached porphyrin
In 2008, a report appeared on a microreactor to synthesize ascaridole 254 in 94-97% yields in 40-50 s from [60]fullerene-sensitized microflow photooxidation of α-terpinene 253 in toluene
307 (Schemes 54 and 69). L-Methoinine methyl ester 288 was also photooxidized in D2O which
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gave the corresponding sulfoxide nearly quantitatively (Scheme 63). The device consisted of thiolene microfluidic channels and a white LED light source. Tentagel-supported or silica gel- supported [60]fullerene beads were packed into the microchannels and held in by a filter. The microreactor led to higher product yields compared to a batch reactor that used a tungsten halogen
1 lamp. In the past, fullerene-containing polymers in O2 jet-type generators pumped by LEDs have also been used.308
Scheme 69. Structures of the tentagel-supported (A) and silica gel-supported (B) [60]Fullerene
1 290- In conclusion, some eleven O2 microflow reactions have been reported in the literature.
303,305,307 As we will see below, relative to microflow about half of this number has been reported
1 for O2 macroflow reactions.
6.4.3 Singlet Oxygen Macrophotoreactors
1 There have been five reports of O2 macroflow reactors that feature dissolved sensitizers in solution (Section 6.4.3.1).
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6.3.4.1 Macrophotoreactors with Solution-Phase Sensitizer
A 2012 report described the synthesis of artemisinin 301 in a 1.36 g quantity (39% yield) from a TPP-sensitized macroflow photooxidation of dihydroartemisinic acid 297 (Scheme 64).309
The device was a macro fluorinated ethylene polymer (FEP) tubing system coiled around an Hg lamp. A flowing dihydroartemisinic acid 297 in an acidic solution of CH2Cl2 led to formation of hydroperoxide 298 in 91% conversion and 75% yield. Through a protonated hydroperoxide, Hock
3 cleavage of 298 led to 299, which added O2 and followed by condensation steps, reaches artemisinin 301.310 Only minor quantities of other hydroperoxides were formed, where byproducts included a 5-membered and 6-membered lactone. Due to therapeutic interest, there are additional reports on synthetic artemisinins as powerful antimalarial APIs.311 Also, it may be noted 2015
1 Nobel Prize in Physiology or Medicine was for the antimalarial drug artemisinin where O2 was used in its total synthesis.312-314
In 2014, a paper appeared where α-aminonitriles were synthesized in 71-99% yields from
TPP-sensitized macroflow photooxidation of primary and secondary amines followed by the
315 addition of TMSCN in CH2Cl2 (Scheme 65). Oxidative cyanation could be accomplished over oxidative coupling. For example, 302 was converted to its corresponding nitrile 304. The device consisted of macro FEP tubing and a 420-nm LED light source, and had variable-temperature control. The oxidative cyanation was favored at -50 °C, whereas at higher temperature (25 °C), the dimer formed from oxidative coupling and cyanide addition. The product percent yield was high and only depended slightly on the amine structure as was studied in detail. This macroflow method
1 may be useful for O2-based syntheses of unprotected amino acids on an industrial scale, which could in turn serve as templates for peptide synthesis.316 Previous synthetic batch reactions have
1 317,318 been carried out on the O2 oxidation of amines to imines.
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1 We have only located one study that provided a macroflow reaction of O2 with sulfides.
In 2011, a paper described the formation of sulfoxide 295 and sulfone 296 (95% yield, 295:296 ratio 3:1) in a TPP-sensitized macroflow photooxidation of 2-(ethylmercapto)-ethanol 294 in a biphasic mixture (Scheme 63).319 The device consisted of a macro FEP tubing was coiled around an Hg lamp. Further evidence for the utility of the flow system was the photooxidation of other substrates, α-pinene 256 (alcohol product 258, 63% yield), α-terpinene 253 (ascaridole 254, 85% yield), 2-methylfuran 306 (to reach ketoacid 309, 68% yield) and citronellol 259 (after reduction with sodium sulfite to reach diols 262 and 263, in a ratio of 1.1:1.0, 22.8 g, 88% yield) (Schemes
55 and 66). In the case of 2-methylfuran 306,319 the approach features the presence of 1.25 eq
DIPEA followed by addition of pyridine in THF and acetone to reach ketoacid 309, 68% yield. As
1 noted in Sections 4.1 and 4.13, O2 reaction efficiencies can be reduced in the presence of amines such as DIPEA. The flow photooxidation of methionine was less productive (4.5 mg/h) compared to the photooxidation of α-terpinene (10 mg/h),307 which may not be unexpected. For some sulfide—singlet oxygen reactions, cleavage occurs through a hydroperoxysulfonium ylide that affords aldehydes or disulfides.320-322
1 A 2008 report found that 1,3-diphenylisobenzofuran 320 reacted with O2 and formed 322 in 34% yield from a porous silicon nanocrystal-sensitized macroflow photooxidation in CH2Cl2
(Scheme 66).323 The device consisted of an annular flow reactor with either an Ar+ laser (488 nm) or a green LED (524 nm) light source, although evidence was not provided on whether the silicon nanocrystal was functioning as a sensitizer.
A 2015 report described a RB-sensitized macroflow photooxidation of 5- hydroxymethylfurfural 310 in aqueous alcohol mixtures formed intermediate 311 which led to products 312-314 (Scheme 66).324 The device consisted of a perfluoroether (PFA) tubing, which
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was coiled around a fluorescent lamp. One optimized condition used a solution of 5-
(hydroxymethyl)furan-2-carbaldehyde 310 in i-PrOH/H2O (1:1), which first led to the corresponding endoperoxide 311, and then to butenolide 312 (93% yield). As desired, trace amounts of 313 and 314 were formed with i-PrOH/H2O, whereas greater amounts were formed with methanol or ethanol. A thermal isomerization of butenolide 312 led to oligomers of 315.
Previously, the syntheses of butenolide 312 from endoperoxide 311 has been reported.325-327
1 Butenolides have been extensively studied in batch reactions of O2. For example, observations that singlet oxygen react with furans to produce butenolides and spiroketal natural products was an important discovery.328-342 Much synthetic utility has been found, one example of a batch reaction is shown in Scheme 70.328 Here, the synthesis of a tetraquinane oxa-cage bislactone structure in parasecolide 325 occurred from the MB-sensitized photooxidation of furan
323 at 0 °C.328 Afterwards, 5-hydroxybutenolide 324 was heated to 110 °C for a [2 + 4] dimerization of 324 to reach paracaseolide A 325 in 59% yield.
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Scheme 70. Synthesis of (±)-paracaseolide A
In conclusion, Section 6.4.3 describes macrophotoreactors that have been established for
1 using O2 in synthesis. Singlet oxygen macrophotoreactors with immobilized sensitizers with conventional solvents have so far not been reported.
1 6.4.4 Supercritical Carbon Dioxide O2 Photoreactor
1 Next, we describe examples of O2 flow reactors that used supercritical CO2 with sensitizer in the liquid phase (Section 6.4.4.1) and immobilized (Section 6.4.4.2). There are advantages in using supercritical CO2 such as safety (it is non-flammable), and enhanced O2 solubility and the
1 reactions give higher product yields, due to longer O2 lifetimes (5.1 ms under the conditions) to overcome mass-transfer limitations of conventional solvents. O2 solubility in CO2 is higher than perfluoronated solvents, which are higher than organic solvents.
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6.4.4.1 Supercritical Carbon Dioxide Photoreactors Using Dissolved Sensitizer
A 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TPFPP)-sensitized macroflow photooxidation in supercritical CO2 was reported in 2009 to generate hydroperoxides 260 and 261 in 52% and 48% yields, respectively, from citronellol 259 after 4 h (Scheme 56).343 The TPFPP sensitizer was dissolved in the liquid. The device was tubular in shape with a sapphire cell mounted in a flow system where the light source was a white LED. The product hydroperoxides 260 and
261 were reduced after exiting the apparatus in an aqueous solution of Na2SO3. Space-time yields were ~9 times higher in this macroreactor compared to a batch reaction.
6.4.4.2 Supercritical Carbon Dioxide Photoreactor Using an Immobilized Sensitizer
A macroflow photooxidation in supercritical CO2 was reported in 2011 to convert citronellol 259 to hydroperoxides 260 and 261 in 88% yield, and α-terpinene 253 to ascaridole 254 in 85% yield (Schemes 56 and 71).344 The device was a packed sapphire flow system, in which 4 immobilized sensitizers were examined (Scheme 71A-D). For example, 5,10,15,20-tetrakis(2,6- dichlorophenyl)porphyrin (TDCPP) was tethered through an amide bridge to polyvinyl chloride
1 (PVC) and loaded in the device with glass wool. Under the conditions, the O2 lifetime was found to be 5.1 ms, which far surpasses typical tens of microsecond lifetimes in organic solvents.
1 345-348 Previously, supercritical CO2 reactions have been used with O2. Other photochemical reactors have been reported for the synthesis of organometallic compounds in supercritical CO2 or other supercritical solvents and is the subject of recent interest.349-351
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Scheme 71. Structures of the immobilized sensitizers in flow devices: (A) rose bengal covalently bound to polystyrene, (B) TDCPP derivative covalently bound to PVC, (C) TDCPP adhered to
PVC, and (D) tetracation porphyrin immobilized on SiO2 gel.
6.4.5 Bubbling Photoreactors
Singlet oxygen bubbling reactors have been scarcely studied. In this section we describe
1 examples of two O2 bubbling reactors. One used a dissolved sensitizer in solution (Section
6.4.5.1), and the other used a heterogeneous sensitizer shielded from solution behind a membrane
(Section 6.4.5.2).
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6.4.5.1 Bubbling Photoreactor Using a Dissolved Sensitizer
A 2012 report describes juglone 273 synthesized in 70% yield in a RB-sensitized bubbling
352 photooxidation of 1,5-dihydroxynaphthalene 272 in t-AmOH/H2O (9:1) (Scheme 59A). The apparatus consisted of a Pyrex glass tube which was situated in between two fluorescent lamps.
This bubbling reactor is unique and is somewhat similar to a batch system, but with a rising airflow.
Bubble diameters were ~50-100 µm, where water-poor media flowed gas via bullet-shaped
bubbles. Various solvents were used, such as i-PrOH, i-PrOH/H2O (9:1), t-AmOH/H2O (9:1),
EtOH/H2O (9:1), MeOH/H2O (9:1). High product yields were observed for the conversion of α- terpinene 253 to ascaridole 254 (71% yield), citronellol 259 to hydroperoxides 261 and 262 (88% yield, ratio 1.1:1.0), and furfural 317 to 5-hydroxyfuran-2(5H)-one (γ-hydroxybutenolide) 318
(>95% yield).
6.4.5.2 Bubbling Photoreactor Using a “Shielded” Heterogeneous Sensitizer
An SMA modified device with dry Si-phthalocyanine particles was reported in 2012 in a bubbling photooxidation of trans-2-methyl-2-pentenoic acid 270, 9,10-anthracene dipropionate dianion 283, N-benzoyl-D,L-methionine 290 and N-acetyl-D,L-methionine 292 in D2O and H2O
(26-46% yields) (Schemes 51 and 63).353,354 The device chamber was loaded with Si- phthalocyanine glass sensitizer particles, which resided behind a microporous membrane with pores that excluded water. An O2 gas feed tube and a red diode laser via a fiber optic were coupled
1 to the SMA device. Bubbles were generated enriched with O2, which left behind no waste or by-
3 products other than O2. The photooxidation reaction rate was shown to increase in O2-poor than
355 O2-saturated solutions where mass transfer was facilitated in an important way.
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1 In conclusion, there are several O2 photoreactor types that have been developed. These include micro and macroreactors. We have seen that (i) high product yields were achieved for flow systems in short periods of time, (ii) sensitizers immobilized in the reactor itself were 2-times less
1 studied than homogeneous sensitizers, and (iii) about half of the studies reported O2 flow relative to batch conditions with an improved efficiency for flow.
6.5 PROSPECTIVES
1 6.5.1 State of O2 Synthetic Science
1 Sections 6.3 and 6.5 present the interplay of basic and applied reactions of O2 in synthesis, respectively. Batch photooxidations in Section 4 have been used with success for decades, but are subject to fundamental limitations. Problems emerge when batch reactions are scaled up, including
(i) long reaction times, (ii) sensitizer photobleaching, (iii) mass-transfer limitations between oxygen and the substrate, and (iv) inner filter effects (large volumes increase the optical pathlength where light can be blocked from reaching the sensitizer).
1 Even though fewer publications exist for O2 flow reactions compared to batch reactions, the former has advantages, including (i) short reaction times when scaling up, (ii) reduced
1 sensitizer photobleaching, (iii) high surface-to-volume ratios for high O2—substrate mixing, (iv) lack of inner filter effect problems, (v) high sensitizer concentrations can be used concurrent with
1 high transmittance and photon flux, (vi) O2-deprived solutions enhance O2 delivery by mass transfer assistance (while paradoxical, less oxygen in solution leads to greater oxygenation).
The utility of singlet oxygen photoreactors in synthetic organic chemistry has emerged.
1 Disappointingly, O2 flow reactions are not as popular as batch reactions, leading us to ask: Why
1 are O2 photoreactors underutilized by synthetic chemists? Some answers may be suggested (i)
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assembly of the photoreactor is required, (ii) there are questions about interchangeability of parts and system configurations, and (iii) flow reactors cannot commonly be purchased “off the shelf”.
6.6 Summary and Outlook
1 Progress has been made regarding O2 in the organic synthesis of complex targets.
1 Synthesis of natural products is often modeled on possible O2 biosynthetic routes. Thus,
1 biomimetic O2 reactions that use alkene and diene precursors are common; however, fewer examples are known with polyenes, di- and polysulfides, and amines due to their facile physical
1 3 356-361 quenching of singlet oxygen ( O2 O2). Singlet oxygen can be used widely, but except for
362-365 366 1 artemisinin and rose oxide, no O2 reactions have yet been used in the pharmaceutical industry. Consequently, one may look forward to estimate what is likely to happen in the field.
1 What are the future prospects of O2 in synthetic chemistry? It seems the combination of fundamental and applied research is beneficial in a reciprocal manner (compare Sections 6.3 and
6.4). The connection between flow technology and synthesis of simple natural products was noticed. It was recognized that the ongoing use of batch reactions for large quantities has drawbacks, therefore new flow options can provide answers.
1 3 In closing, we look back to pioneers of O2 in synthetic and natural products chemistry.
This brings organic chemistry to the front, in reminding us of the first efforts of Foote and Wexler some 50 years ago.1,2 Here, the success of research was not only assured, but also reaffirmed by
1 the notion to look backward and forward as the field of O2 evolves.
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6.7 References
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