PHOTOAFFINITY LABELING VIA NITRENIUM ION CHEMISTRY: THE PHOTOCHEMISTRY OF 4-AMINOPHENYLAZIDES.
Valentyna Voskresenska
A Dissertation
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
May 2011
Committee:
Dr. R. Marshall Wilson, Advisor
Dr. Zhaohui Xu, Graduate Faculty Representative
Dr. Michael Y. Ogawa
Dr. Thomas H. Kinstle
ii
ABSTRACT
Dr. R. Marshall Wilson, Advisor
Phenyl azides with powerful electron-donating substituents are known to deviate from
the usual photochemical behavior of other phenyl azides. They do not undergo ring expansion,
but form basic nitrenes that protonate to form nitrenium ions. The photochemistry of the widely
used photoaffinity labeling system 4-amino-3-nitrophenyl azide, has been studied by transient
absorption spectroscopy from femtosecond to microsecond time domains and from a theoretical
perspective. The nitrene generation from 4-amino-3-nitrophenyl azide occurs on the S2 surface, in violation of Kasha’s rule. The resulting nitrene is a powerful base and abstracts protons extremely rapidly from a variety of sources to form a nitrenium ion. In methanol, this protonation occurs in about 5 ps, which is the fastest intermolecular protonation observed to date. Suitable proton sources include alcohols, amine salts, and even acidic C-H bonds such as acetonitrile. The resulting nitrenium ion is stabilized by the electron-donating 4-amino group to afford a diiminoquinone-like species that collapses relatively slowly to form the ultimate cross- linked product. In some cases in which the anion is a good hydride donor, cross-linking is replaced by reduction of the nitrenium ion to the corresponding amine.
However, the efficiency of 4-amino-3-nitrophenyl azide in generating the reactive nitrenium is impaired by associated photochemistry of the nitro group and a tendency for the initially formed nitrene to undergo reduction to the corresponding amine. We have examined possible alternative molecules that might yield reactive nitrenium ion more efficiently, and thus, provide more effective photoaffinity labeling agents. The 2-(N,N-diethylamino)-5-azidopyridine
has been investigated as this regard and found to offer substantial advantages over 4-amino-3- iii nitrophenyl azide as a photoaffinity agent. Ultrafast transient spectroscopy confirms that 2-(N,N- diethylamino)-5-azidopyridine proceed via a reactive nitrenium ion species, and the same reactive species is formed both photochemically and thermally at relatively low temperatures.
The coupling reactions of 2-(N,N-diethylamino)-5-azidopyridine proceed more rapidly than those of 4-amino-3-nitrophenyl azide in reactions with fewer side products, including reduction to the amino analogs. In addition, 2-(N,N-diethylamino)-5-azidopyridine displays fluorescence that ceases upon conversion to products, a property that might offer distinct advantages in photoaffinity labeling studies in complex biological systems.
iv
ACKNOWLEDGMENTS
There are so very many great people to whom I owe my recognition and gratitude
which has accumulated during my years of graduate study.
I would like to start by thanking my adviser, Professor R. Marshall Wilson. Dr. Wilson,
this work could not have been accomplished without your intellectual support and thoughtful
guidance. I wish to thank you for your enthusiasm, encouragement and concern during my
graduate career.
I want to express my gratitude to our collaborator, Professor Alexander N. Tarnovsky for
sharing his knowledge the course of discussions that proved highly useful. I thank Maxim Panov for
beautiful ultrafast spectroscopic studies and calculation studies. I also thank Mike Ryazancev for
help in calculation studies and useful discussions. Working on those projects together was a pleasure
for me. I also would like to thank our collaborators from the Ohio State University Dr. C. M. Hadad,
S. Vyas and A. H. Winter.
I am very thankful to my MS thesis advisor and current committee member Professor T.
H. Kinstle, who had the great impact on my scientific career. I also wish to thank Professor M.
Ogawa, committee member during my MS and PhD programs, for his compassion and time.
I thank all members of our group, Denis Nilov, Dmitry Komarov and Alexei Shamaev for creating friendly and inspiring atmosphere in the lab.
I would also like to express my appreciation to Professor Castellano for opportunity to use instrumentation in his laboratories; Dr. Jedrzej Romanowicz, and Dr. D. Y.Chen for their help with mass spectrometry and NMR experiments.
My special thank-you goes to my dear friends, especially, to Elena and Vadim Glik,
Anna and Andrey Fedorov, Kate and Alex Mejeritsky, for their constant support, encouragement v
and our friendship. My beloved parents and sister deserve undying gratitude for being very
supportive and unconditionally loving. And finally, I want to thank my husband, Sergey, and my lovely daughter, Anastasia, who fill my life with joy and happiness.
vi
TABLE OF CONTENTS
Page
CHAPTER I. BACKGROUND ...... 1
1.1 Photoaffinity Labeling ...... 1
1.2 Laser Thechniques in the Study of Photoaffinity Labeling ...... 6
1.3 Photochemistry of Aryl Azides ...... 9
Fluorosubstituted Azides ...... 14
1.4 Photochemical Formation and Reactions of Nitrenium Ions ...... 16
1.5 References ...... 23
CHAPTER II. EXPERIMENTAL METHODS ...... 30
2.1 UV-VIS Absorption Spectroscopy ...... 30
2.2 Steady-State Fluorescence Spectroscopy ...... 30
2.3 Nanosecond UV-VIS Time Resolved Absorption Spectroscopy ...... 31
2.4 Femtosecond UV-VIS Time Resolved Absorption Spectroscopy ...... 33
2.5 Computational Details ...... 36
2.6 X-Ray Crystallographic Structure Determination of 31 ...... 36
2.7 References ...... 37
CHAPTER III. THE PHOTOCHEMISTRY OF 4-(N,N-DIETHYLAMINO)-3-NITROPHENYL
AZIDE: PROTONATION OF THE NITRENE TO AFFORD REACTIVE NITRENIUM ION
PAIRS EXPERIMENTAL METHODS ...... 41
3.1 Introduction ...... 41
3.2 Synthesis of 4-(N,N-Diethylamino)-3-nitrophenyl Azide and Study of its
Photolysis Products...... 44
Alcohols as Nucleophiles ...... 49 vii
Phenols as Nucleophiles ...... 50
Dimethylamine Hydrochloride as Nucleophile ...... 50
3.3 Photophysical Studies ...... 55
UV-VIS Steady State ...... 55
Ultrafast transient Absorption and Nanosecond LPF Spectra of
Azide 25 in 2-Propanol ...... 56
Reactivity of Azide 25 in the Presence of Different Solvents ...... 65
3.4 Discussion ...... 76
Theoretical Considerations ...... 76
Nitrene Electronic Configuration ...... 78
3.5 Conclusions ...... 98
3.6 References ...... 99
CHAPTER IV. AN EFFECTIVE NITRENIUM ION PRECURSOR FOR PHOTOAFFINITY
LABELING: 2-(N,N-DIALKYLAMINO)-5-AZIDOPYRIDINE ...... 105
4.1 Introduction ...... 105
4.2 Synthesis of 5-Azido-2-(N,N-diethylamino)pyridine (65) and Studies
of the Products of the Photolysis ...... 109
4.3 Photophysical Studies ...... 114
Ground State Absorption ...... 114
Fluorescence Studies of 5-Azido-2-(N,N-diethylamino)pyridine 65 ...... 116
Ultrafast Transient Absorption Spectra of Azide in Protic Solvents ...... 120
Nanosecond Laser Flash Photolysis Studies of 65 in Protic Solvents ...... 125
Ultrafast and Nanosecond Experiments in Non-Polar Solvents ...... 128
4.4 Discussion ...... 130 viii
Mechanistic Pathways of the Formation of Products
Photolysis of Azide 65 ...... 133
4.5 Conclusions ...... 137
4.6 References ...... 138
CHAPTER V. METHODS AND MATERIALS ...... 140
5.1 Pump-probe Ultrafast Transient Experiments ...... 140
5.2 Calculations of Fluorescence Quantum Yield of Azide 65 ...... 141
5.3 General Information ...... 142
5.4 Synthesis of 4-(N,N-Diethylamino)-3-nitrophenyl Azide (25) ...... 143
5.5 General Procedure For Photolysis Of 4-(N,N-Diethylamino)-3-nitrophenyl
Azide (25) In Various Solvents ...... 144
5.6 Photolysis Products ...... 144
5.7 General Procedure for the Photolysis of 4-(N,N-diethylamino)-3-nitrophenyl
Azide (25) in the Presence of Targeted Functional Groups ...... 148
5.8 Acetylation of Adducts 29 and 30 ...... 150
5.9 Synthesis of 5-Azido-2-(N,N-diethylamino)pyridine (65) ...... 151
5.10 Photolysis Products ...... 153
5.11 References ...... 156
APPENDIX. 1H NMR AND 13C NMR SPECTRAL CHARACTERIZATION ...... 157
ix
LIST OF FIGURES
Figure Page
1.1 Schematic illustration of conventional Photoaffinity Labeling ...... 2
1.2 Three major photoreactive groups and their reactive species generated
photochemically (a) nitrenes from azides; (b) carbenes from diazirines compounds;
(c) biradicals from enones ...... 4
1.3 Cross-linking of the DNA by 3-hydroxyphenyl azide...... 5
1.4 Polar mesomeric structures of organic azides ...... 10
1.5 Three low-lying spin states rise from the near-degeneracy of two 2p orbitals:
open-shell singlet (OSS), closed-shell singlet (CSS) and triplet states (T) ...... 11
1.6 Electronic structure of the lowest energy singlet open-shell state
of phenylnitrene ...... 11
1.7 Nitrenium ions ...... 16
1.8 Geometry of singlet nitrenium ions calculated by DF Theory ...... 17
2.1 Ti:Sapphire regenerative amplifier (Hurricane, Spectra Physics) and two
interchangeable, computer-controlled pump and probe TOPAS-C optical parametrical
amplifiers (Light Conversion Lt.)...... 35
3.1 Model compounds for photochemical studies by Kanaoka ...... 42
3.2 Analysis of structure of 30 by Nuclear Overhauser Effect (NOE) ...... 47
3.3 X-ray structure of acetanilide 31 ...... 48
3.4 Products obtained upon irradiation in acetonitrile and toluene ...... 53
3.5 (4-Azido-2-nitrophenyl)carbamic acid ethyl ester (37) and
4-Ethoxy-3-nitrophenyl azide ...... 54 x
3.6 UV-VIS absorption spectra of azide 25 in cyclohexane (blue solid line), acetonitrile
(pink dashed line), methanol (purple dushdot line) and 2-propanol (green solid line) ...... 55
3.7 Room temperature emission spectra of azide 25 in 2-propanol at three excitation wavelengths: a 350 nm (red), 360 nm (black), 380 nm (blue) ...... 56
3.8 Frames A-C: Transient absorption (ΔA) spectra of a 16 mM solution of azide 25 in
2-propanol shown for various delay times between the 350 nm pump and probe pulses.
These data were provided by Dr. Tarnovsky’s group. Data in frame C represent smoothed spectra by adjacent-averaging (bandwidth, 3.5 nm). The solvent contribution to the ΔA spectra is minor at delay times equal to or longer than 100 fs, except for the 388.9-nm feature that corresponding to stimulated Raman scattering from
2-propanol and yields the instrument response function, 150 fs ...... 57
3.9 Nanosecond transient absorption (ΔA) spectra of azide 25 in 2-propanol, excited at 350 nm, with delay and growth times specified on the graph ...... 59
3.10 The kinetic traces of azide 25 recorded at a) 440 nm and b) 490 nm in
2-propanol along with one-exponential solid line fit superimposed with time constants of a) 556±28 ns and b) 567± 8 ns ...... 59
3.11 Reconstructed decay-associated spectra (εi) extracted from the ΔA transient absorption spectra of azide 25 in 2-propanol irradiated with 350 nm laser pulses...... 60
3.12 A, B, and C: The solution was flowed through a 0.2 mm flow cell and excited with
a 305-nm, 3.8 μJ pulse. The solvent contribution to the ΔA spectra is minor at delay times ≥ 100 fs. For 305 nm excitation, the UV region (280-375 nm) of the ΔA spectra was measured using the probe light generated by TOPAS ([azide 25] = 1.7 mM,
3.1 μJ pump pulse) and subsequently scaled to the visible (360-665 nm) ΔA spectra xi measured using the white-light continuum probe. The inset compares the ΔA kinetic traces recorded at probe wavelengths of 350 and 465 nm ...... 62
3.13 Transient absorption (ΔA) spectra of 25 (1.2 mM) in 2-propanol for various delay
times (in picoseconds, shown in the legends) between the probe and pump pulses...... 64
3.14 (a) Nanosecond transient absorption (ΔA) spectra of azide 25 in ethanol, excited
at 350 nm, with delay and growth times specified on the graph; (b) The kinetic trace
of azide 25 recorded 490 nm in ethanol along with one-exponential solid line fit superimposed with time constants of 305±32 ns ...... 66
3.15 Nanosecond transient absorption (ΔA) spectra of azide 25 in n-butanol, excited
at 350 nm, with delay and growth times specified on the graph ...... 67
3.16 The kinetic traces of azide 25 in n-butanol along with one-exponential solid line fit superimposed with time constants of a) 325±13 ns recorded at 490 nm and b) 567± 8 ns at 440 nm ...... 67
3.17 Nanosecond transient absorption (ΔA) spectra of 25 in 2M solution of phenol in acetonitrile, excited at 350 nm ...... 69
3.18 The kinetic traces of 25 recorded at (a) 490 nm along with one-exponential solid line fit superimposed with time constants of 1.39 ± 0.018 μs and (b) 440 nm in 2M phenol in acetonitrile with time constants of 2 ± 0.1 μs ...... 69
3.19 Nanosecond transient absorption (ΔA) spectra of 25 in saturated solution of
dimethyl amine hydrochloride in acetonitrile, excited at 350 nm, with delay and growth times specified on the graph ...... 70
3.20 The kinetic traces of azide 25 in saturated solution of dimethyl amine
hydrochloride in acetonitrile along with one-exponential solid line fit superimposed xii with time constants of (a) 11.6±0.13 μs recorded at 490 nm and (b) 5.65± 0.8 μs at 440 nm ...... 70
3.21 Nanosecond transient absorption (ΔA) spectra of azide 25 in ethyl malonate ester, excited at 350 nm, with delay and growth times specified on the graph ...... 71
3.22 Nanosecond transient absorption (ΔA) spectra of azide 25 in a 2M solution of hydroquinone in acetonitrile, excited at 350 nm, with delay times specified
on the graph ...... 72
3.23 (a) Nanosecond transient absorption (ΔA) spectra of azide 25 in ethyl glucolate, excited at 350 nm, with delay and growth times specified on the graph; (b) The kinetic traces of azide 25 recorded at 490 nm in ethyl glycolate along with the bi-exponential solid line fit superimposed with time constants of 49 ns and 1191 ns ...... 72
3.24 Transient absorption (ΔA) spectra of azide 25 in acetonitrile upon
350 nm excitation ...... 73
3.25 (a) Transient absorption (ΔA) spectra of azide 25 in toluene upon 350 nm excitation. The solution (16 mM) was circulated through a 0.2-mm pathlength flow cell; (b) Transient absorption (ΔA) spectra of azide 25 in cyclohexane upon 350 nm excitation. The solution (16 mM) was circulated through a
0.2-mm pathlength flow cell ...... 74
3.26 Nanosecond transient absorption (ΔA) spectra of azide 25 in saturated solution of glycine, excited at 350 nm, with delay and growth times specified on the graph ...... 75
3.27 Kinetic traces of azide 25 recorded at a) 490 nm and b) 420 nm in saturated solution of glycine in 2-propanol along with a) one-exponential solid line fit xiii superimposed with time constant of 236+6.9 ns and b) biexponential solid line fit superimposed with time constants of 22.66±1.89 ns, and 1.28±0.2μs ...... 75
3.28 Electron density redistribution in the S1 and S2 states of 25 calculated at the
TD-B3LYP/TZVP level of theory. The green contours depict the accumulation of electron density in the excited state, and the red contours illustrate the loss of electron density from the S0 ground state. The contour values are ±0.005 a.u...... 76
3.29 Excited state energies (kcal/mol) and bond lengths for the optimized geometries
(TD-B3LYP) of the ground state, and the first and second excited states of the N,N-dimethyl analog of azide 25 ...... 77
3.30 (A) Closed-Shell Nitrene optimized at CASSCF(10,10)/pVDZ level of theory using MOLCAS suite of programs (B) Opened-Shell Nitrene optimized at
CASSCF(10,10)/pVDZ level of theory using MOLCAS suite of programs. These results were obtained from Dr. Hadad’s group ...... 81
3.31 (A): conserved angular momentum in intersystem crossing (ISC) of carbonyl group. (B): Relationships between ISC and open- to closed-shell singlet nitrene interconversion (OCSI) ...... 82
3.32 Energy selective photochemistry of 4-(N,N-diethylamino)-3-nitrophenyl azide ...... 83
3.33 Possible reaction pathways connecting excited states of azide 25, nitrene states, and nitrenium ion 33 states. The vertical energy axis is not drawn to scale ...... 85
3.34 Alternative scenarios for closed-shell singlet nitrene reaction ...... 87
3.35 Two regions of space-surrounding the closed-shell singlet nitrene nitrogen and alcohol approach geometries leading to protonation transition states ...... 90 xiv
3.36 Structures of 40, 41, and 42 ...... 95
3.37 Attack profile for protonation of nitrene with approach from the 2- or
6-side of the nitrene nitrogen (-O-H in Å) ...... 97
4.1 Products of photolysis of 65 in t-butanol, the acetonitrile solution of imidazole hydrochloride, 72, and in cyclohexane, 73 ...... 113
4.2 UV-Vis absorption spectra of 65 in acetontrile (solid pink line), 2-propanol
(dot blue line), cyclohexane (dashed dark cyan) and 50 % solution of acetic acid
(red short dash) ...... 114
4.3 Three excited states calculated at the DFT level of theory ...... 116
4.4 Excitation (blue dash) and emission (purple solid line) spectra of 65 in argon-saturated 2-propanol ...... 117
4.5 (a) Emission spectra of 65 in protic and aprotic solvents; (b) Normallized intensity of emission spectra of 65 in protic and aprotic solvents ...... 118
4.6 The kinetic traces of fluorescence of 65 at 420 nm (a) in n-butanol with biexponential solid line fit superimposed with time constants of 36±0.6 ns and 855± 18 ns;
(b) In 2-propanol along with biexponential solid line fit superimposed with time constants of 77±0.14 ns and 623± 6 ns with emission transient spectrum on nanosecond LFP ...... 119
4.7 Hydrogen bonding between 65 and molecule of alcohol ...... 119
4.8 Ultrafast transient absorption (ΔA) spectra of 65 in 2-propanol upon 310 nm excitation A, B, and C: The solution was flowed through a 0.2 mm flow cell and excited with a 305-nm, 3.8 μJ pulse. The solvent contribution to the ΔA spectra is minor at delay times ≥ 100 fs ...... 121
4.9 Transient absorption (ΔA) spectra of 65 in 2-propanol (a) upon 360 nm xv excitation and (b) upon 255 nm excitation ...... 123
4.10 Nanosecond transient absorption (ΔA) spectra of 65 in 2-propanol, excited at 355 nm, with delay and growth times specified on the graph ...... 124
4.11 The kinetic traces of 65 recorded at 510 nm in 2-propanol along with biexponential solid line fit superimposed with time constants of τ1= 354±26 ns
and τ2 = 2.54± 0.12 μs ...... 126
4.12 (a) Nanosecond transient absorption (ΔA) spectra of 65 in n-butanol, excited at 355 nm,with delay times specified on the graph; (b) The kinetic trace of 65 recorded
in n-butanol at 500 nm: along with double-exponential solid line fit superimposed with time constants 43 ± 0.21 ns and 0.67± 0.033 μs ...... 127
4.13 (a) Nanosecond transient absorption (ΔA) spectra of 65 in tert-butanol, excited at 355 nm; (b) nanosecond transient absorption (ΔA) spectra of 65 in acetonitrile solution of imidazole hydrochloride, excited at 355 nm with delay times specified on the graphs ...... 127
4.14 The kinetic traces of azide 65 recorded at 500 nm in 2-propanol along with
biexponential solid line fit superimposed with time constants of τ1= 354±26 ns and
τ2 = 2.54± 0.12 μs ...... 128
4.15 Transient absorption (ΔA) spectra of 65 in cyclohexane upon 310 nm excitation .... 130
4.16 (a) Emission of azide 65 in 2-propanol: one minute of irradiation at 355 nm
(olive solid line); six minutes of irradiation at 355 nm (orange dash line), nine minutes of irradiation at 355 nm (cyan solid line); (b) emission of azide 65 (navy solid line) in 2-propanol; emission of azide 65 after addition of substitution product 69 ...... 132
4.17 Amine 67 in 2-propanol: excitation spectrum observed at 322 nm xvi
(wine solid line); emission spectrum excited at 355 nm (violet solid line) ...... 132
4.18 (a) Fluorescence spectrum of azide 65 (purple solid line), amine 67
(violet dashed line), and adduct 69 (pink dash-dot) in 2-propanol; (b) normalized
intensities of azide 65 (purple solid line) and amine 67 (violet dashed line)
in 2-propanol ...... 133
4.19 Diagram of pathway of the formation of closed-shell nitrene ...... 134
xvii
LIST OF SCHEMES
Scheme Page
1.1 The mechanism of the photolysis of simple aryl azides ...... 12
1.2 Trapping of benzazirine upon the photolysis of 1 in ethanethiol ...... 13
1.3 Products formed upon the photolysis of phenyl azide 1 via triplet pathway...... 13
1.4 The photochemical behavior of fluorinated aryl azides ...... 15
1.5 Transformation of aryl amines into nitrenium ions in biological systems ...... 18
1.6 Addition of alcohols and water to nitrenium ion ...... 18
1.7 Irradiation of p-aminophenyl azide in weakly acidic aqueous solution ...... 19
1.8 Photochemistry of phenyl azide in the presence of acetic acid and ethanol ...... 20
1.9 Photochemical reaction of p-substituted aryl azides in water ...... 21
3.1 Application of NAP as a photoaffinity labeling reagent for binding of antibody ..... 41
3.2 Irradiation of 1-azido-3-nitrobenzene ...... 43
3.3 Synthesis of the 4-(N,N-diethylamino)-3-nitrophenyl azide...... 45
3.4 Photolysis of 4-(N,N-diethylamino)-3-nitrophenyl azide ...... 46
3.5 The conventional mechanism of the formation of the addition product ...... 46
3.6 Photolysis of azide 25 in 2-propanol ...... 48
3.7 Possible mechanism for formation of addition products upon the photolysis
of azide 25 ...... 52
3.8 Products formed upon the photolysis of 25 in cyclohexane ...... 53
3.9 Photolysis of 37 in 2-propanol ...... 54
3.10 The mechanistic pathways of formation of products photolysis of azide 25 ...... 92
3.11 The mechanism of formation of the dimer 35 ...... 98 xviii
4.1 New azide, 5-azido-2-fluoro-pyridine (44), which might be used as more effective
photoaffinity-labeling agent than the nitroanalog NAP...... 105
4.2 The photolysis of methyl-substituted 3-azidopyridines (45) and 3-azido-4-
methoxypyridine (47) in the presence of methoxy anion ...... 106
4.3 Photolysis of the 3-azidoquinoline (49) in bromobenzene ...... 107
4.4 The photolysis of 3-azidoquinoline in the presence of trapping reagents ...... 108
4.5 Ring-opening of 3-pyridylnitrene to nitrile ylide ...... 108
4.6 Synthesis of 5-azido-2-(N,N-diethylamino)pyridine (65) ...... 109
4.7 The alternative route of synthesis of 65 ...... 110
4.8 Irradiation of 65 in the presence of different nucleophiles ...... 111
4.9 Oxidation of the addition product 69 ...... 111
4.10 The mechanism of oxidation of the adduct 69 by air...... 135
4.11 Possible mechanism of the photolysis of azide 65 in 2-propanol and aprotic solvents ...... 136
xix
LIST OF TABLES
Table Page
1.1 Absorption maxima and rate constants for decay of substituted nitrenium ions in water.
While most constants were determined in ionically buffered aqueous media, it should be
noted that some less stable ions are better generated and stabilities enhanced if they are
generated in slightly acidic media ...... 21
3.1 Reduction to addition ratio for photoreaction of 25 with various nucleophiles...... 51
3.2 Transient cascade characteristics observed upon irradiation of azide 25 in various
solvents. Frames A-E refer to the corresponding time frames in Figure 3.11, their
associated λmax’s and time constants for the solvents indicated ...... 61
3.3 Vertical transitions and absorption properties of possible reactive species derived from
the irradiation of azide 25 as calculated at the TD-B3LYP/TZVP level of theory ... 88
3.4 Transition state properties for proton abstraction of closed-shell singlet nitrene from
methanol. The geometries of these transition states are shown in Figure 3.35 ...... 93
4.1 Reduction/Addition Ratios for photoreactions of 65 with various nucleophiles ...... 112
4.2 Electronic steady-state absorption spectra calculated at the TD-B3LYP/TZVP and
CASPT2 levels of theory ...... 115
4.3 Vertical transitions and absorption properties of possible reactive species derived from
the irradiation of ADP as calculated at the TD-B3LYP/TZVP and CASPT2 levels of
theory ...... 122
4.4 Transient cascade characteristics upon irradiation of 65 in protic solvents ...... 124
4.5 Transient cascade characteristics observed upon irradiation of azide 65 in
aprotic solvents ...... 129 xx
4.6 Comparison of the photochemical characteristics of nitrophenyl azide 25 with the new
azidopyridine 65 ...... 131
1
CHAPTER I. BACKGROUND
1.1 Photoaffinity Labeling (PAL)
Discovered more than forty five years ago, photoaffinity labeling has gained by now an important position as one of the most powerful tools for the structural investigation of active binding sites and macromolecular interaction in molecular biochemistry.1-5
Westheimer and co-workers first applied photogenerated reagents to study biological
macromolecules in 1962; they introduced diazoacetylchymotrypsin as the first photoaffinity
labeling reagent.6 Photoaffinity labeling has since been applied for identifying complex
biological receptors and transport proteins, membrane structure and function. protein-nucleic
acid interaction and antibodies in addition to enzymes.7-14 The experimental procedure comprises
the attachment of a photosensitive small molecule to a large biological ligands or inhibitors of
interest and the subsequent exposure to the biological target to form a ligand-receptor complex.
This is followed by irradiation of this mixture, which leads to a highly reactive species derived
from the PAL tag, which then reacts with any suitable functional groups and forms a permanent
covalent bond between ligand and receptor (Figure 1.1). Finally, the covalently labeled
macromolecule can be examinated by a variety of analytical techniques. Thus, photoaffinity
labeling could be applied in two levels. At the macro-level, the method is useful for the screening
of early leads for the evaluation of affinity by cross-linking to determine which ligand
preferentially binds to which macromolecule. In that case, ligand – biomolecule complex is
analyzed by electrophoresis or plate screening.4,7-9 If binding site analysis of a target biomolecule
is important for defining a particular pharmacophore, the photoaffinity labeling gives structural 2
information of the receptor binding domain at the micro-level and can track the
transport/metabolism pathways of substrates, using techniques such as spectroscopic analysis,
MS, HPLC, labeling with radioactive probes, etc.1-5,12,13
reversible binding Electrophoresis
structural h elucidation
High-throughput
target identif ication
MS analysis
-Y-W-L-
Figure 1.1. Schematic illustration of conventional Photoaffinity Labeling.
There are many options in photoaffinity labeling experiments: choice and synthesis of the
photophore, photolysis conditions, and choice of tags for the identification of biocomponents.
Both organic chemistry (the preparation of the photophore and ligand modification) and 3
biochemistry (handling of labeled components), are needed in order to perform photoaffinity labeling experiments. The search for efficient and selective PAL reagent is one of the most important aspects of the technique.
According to the literature,1,2,5,10,12an effective photoaffinity labeling agent:
1) Is a small molecule and does not affect the binding properties of the biological molecule
to which it is attached.
2) Is chemically inert in the dark and becames highly reactive upon irradiation at a
wavelength longer than 280 nm in order to prevent photochemical damage to the
biological sample.
3) Forms a highly reactive intermediate that reacts with nucleophiles and C-H bonds.
4) Has lifetime of the photochemically generated reactive intermediate, which is long
enough to find a target site for covalent binding, but shorter then dissociation of the
ligand –receptor complex.
5) Forms a covalent bond between ligand and macromolecule, which leads to formation of
stable products to enable their isolation, purification and analysis.
Different types of photoreactive species generally suitable for PAL are azides, diazo
compounds, benzophenones, diazonium salts, and diazirines.1,2,5,16-19 Figure 1.2 presents these
commonly used PAL reagents and the conventional wisdom mechanisms: a) generation of the
nitrene from azides; b) carbenes from diazo and diazirines compounds; c) aryl cations from
diazonium salts and d) biradicals from enones.
The most fundamental question in this field of research is: “Which photoreactive groups
should be used as PAL agents?” Several excellent reviews have been provided to answer on this
question by comparing the efficiency of various PAL agents.1,2,5,9,15 4
HN Protein'
N Protein N3 Protein' h
N2 Protein Protein H N Protein' nitrene
Protein (a)
N N CF3 C F C HC Protein' C CF3 3
Protein' h
N2
Protein Protein Protein
(b)
OH O O
h Protein' Ph Ph
protein' protein' Protein protein'Protein Protein +
Protein'
HO Protein'
Ph
protein'Protein
(c)
Figure 1.2. Three major photoreactive groups and their reactive species generated photochemically (a) nitrenes from azides; (b) carbenes from diazirines compounds; (c) biradicals from enones. 5
Ideally, the photoreactive molecules should satisfy all of the aforementioned requirements, but none of the mentioned above groups meets all requirements for perfect photoaffinity labeling agent. In recent time, most of the PAL reagents have been based on nitrene or carbene chemistry. Carbenes and nitrenes, isoelectronic species, display fundamentally almost the same chemistry.19 By far, phenyl azides are the most widely used PAL agents, because they
have almost all properties indicated above.1-5,21,22 Moreover, they are easily synthesized or are
commercially available.
Several hundreds of publications in biological and medicinal journal have been dedicated
to studies of RNA and RNA-protein complexes, DNA-protein, protein-protein, and protein-
ligand interactions using of aryl azide derivatives as PAL agents.
Figure 1.3. Cross-linking of the DNA by 3-hydroxyphenyl azide. XL – Cross Linking, AA – Aryl Azide. 6
The photoafinity labeling can also be carried out in an intramolecular fashion, which also leads to cross-linking.23-27 One current example is the covalent bonding between an RNA duplex strand and photoactivated 3-hydroxyphenyl azide in aqueous solution. Platz, Weeks and co- workers have established the mechanism of this addition process27 (Figure 1.3).
Photoaffinity labeling will continue to evolve as a major technique for studying molecular
interactions in biological systems. Great progress in the development of new PAL agents and
understanding of photoaffinity labeling processes has been made for past 10 years. However, it is
of critical important to determine the photochemical mechanisms of PAL agents in considerable
details to aid in the design of more effective agents and increase their labeling selectivity and
efficiency.
1.2 Laser Thechniques in the Study of Photoaffinity Labeling
In order to elucidate the mechanisms of PAL agents, it is essential to be able to monitor
the formation of transient species. For that purpose researchers use many diiferent spectroscopic
methods, such as UV/VIS spectrophotometry, conductivity, IR, Raman, mass spectrometry, and
chemiluminescence.
Laser Flash Photolysis (LFP) spectroscopy is one of the most effective methods for the
study of mechanisms of Photoaffinity Labeling process by direct observation and
characterization of wide range of very reactive species such as radicals, singlet and triplet excited
states or ions, in chemical and biological systems.28 LFP was developed by Porter, Eigen and
Norrish, who had shared the Nobel Prize for their studies of “extremely fast reactions” in 1967
29,30 This spectroscopic method is based on the very rapid generation of reactive intermediate 7
using a pulsed light source, such as a pulsed laser, and their detection spectroscopically.
Different forms of LPF are presently available in the femtosecond (fs) and nanosecond (ns) regimes.
The technique of Nanosecond LFP has been utilized for the last twenty years, and consists of the tree major parts: a pulsed laser source (pump) that generates the chemical species to be studied, an optical and electronic system capable of sensing optical changes in the sample, and a computer suitably equipped to capture, process and display the data. Nanosecond LFP uses lasers which emit pulses of light with pulse widths around 10 ns. The solid-state Nd: YAG and laser is commonly used and emits a fundamental laser pulse at 1064 nm and using harmonic generation gives the more useful wavelengths of 266, 355, or 532 nm. The intensity of output from lasers is in the 50-200 mJ range. The laser pump source provides a single wavelength excitation with nanosecond resolution and rapidly generates transient species in the sample.
These excited states and reactive intermediates undergo chemical and physical interactions and hence their varying absorption characteristics can be directly observed. The varying absorption characteristics are measured via the detection system. The detection system is a UV-VIS absorption system, which consists of an intense light source (Xenon arc lamp), a monochromator and a photomultiplier tube (PMT) with a fast time response. The use of a xenon lamp permits the acquisition of transient spectra from 250 nm to 850 nm, and its output intensity is enhanced by a factor of 5-100 for a few milliseconds. Modern photomultipliers are able to detect from 200 nm to the near-infrared. The spectra are usually recorded on a point-by-point basis, where the time of capture is set before acquisition, and the wavelength is changed during the experiment. The third component is computer control. Operations, such as shutters which open just before laser 8
irradiation and close right after acquisition is complete, filter wheels, and motor-driven monochromators, are usually under computer control.28,29
After transient signal is measured, the resulting spectrum is called an absorption
difference spectrum (ΔOD), which reflects changes in transmitted light from the probe beam
before (It=0) and after (It=0) laser excitation (Equation 1.1).
The probe beam transmitted intensity before and after laser excitation is shown by
Equations 1.2 and 1.3, respectively (from Lambert Beer’s law).
Where IPB is the probe beam intensity incident in the sample, εgs and εT are molar
absorptivities at the probe wavelength of the ground state and the transient formed after
excitation, respectively; l is the cell optical path; Cgs and CT are concentration before and after
excitation, respectively.
The observed spectrum is however precursor dependant in those wavelengths where the
ground state absorbs: if εgs > εT, the negative signal is obtained; if εgs < εT , the observed
signal is positive, and if both molar absorptivities are equal, ΔOD = 0 and no signal is detected.
At the end of 1980s, Ahmed Zewail had performed series of experiments that were to
lead to the new era in the research of reactive intermediate for which he was awarded with the
Nobel Prize in 1999.31He decreased the observable time from the 10-9 to the 10-12 second range.
Since then, numerous papers devoted to ultrafast transient laser spectroscopy have been 9
published. The basic components of ultrafast laser systems are similar to those used in nanosecond experiments, except the same laser pulse is used for both for excitation of the sample and as a source of probing light.
1.3 Photochemistry of Aryl Azides
Since the discovery of organic azides by Peter Grieb and synthesis of phenylazide by
Greiss more than 140 years ago,32 aryl azides and many of their derivatives had been the subjects
of intensive investigations. Aryl azides are known for their ease of preparation and
transformation into various substituents (amine, triazene, aza-ylide, and isocyanate), reactive
species (nitrene, nitrenium ion), and nitrogen-containing heterocycles (azirine, aziridine, triazole,
triazoline, and azole).1,3,33 In more recent times, completely new approaches have been
developed for their use as photoaffinity labeling agents in peptide chemistry, as well as
combinatorial chemistry. Moreover, there have recently been a number of applications in
medicinal chemistry and industry (propellants, explosives, polymer cross-linkers, rubber
vulcanisations, reactive dyes, blowing agents, and biologically and pharmaceutically active compounds).34,35
The chemical behavior of azides strongly depends on their electronic structure, which has
been determined by Curties and Hantzsch:36 in the ground state there are a three nitrogen atoms
linearly linked by bonding and non-bonding electron pairs (Figure 1.4). Some physicochemical
properties of the organic azides can be explained by a consideration of polar mesomeric
structures. For example, aromatic azides are stabilized by conjugation with aromatic system; 10
moreover, the stable resonance structure 1.4a can explain the decomposition of azide under photochemical or thermal conditions to nitrene.37
R-N3 = RNN N RNN N RNN N
abc
Figure 1.4. Polar mesomeric structures of organic azides.
The photochemistry of simple aryl azides has been well established and reviewed many times.38-41Their irradiation initiates a complex series of chemical reactions that lead to different
products depending on the details of the experiment. The mechanistic details of this complex
decomposition of phenyl azide have been studied extensively through product analysis, matrix-
isolation spectroscopy,42 laser flash photolysis (LFP)43,44 and modern molecular orbital (MO)
theory.45,46
The typical photochemical behavior of aryl azides is to initially undergo elumination of
molecular nitrogen from the singlet excited state of the azide and the formation of singlet phenyl
nitrene, which is considered the first intermediate.
The electronic structure of phenyl nitrene consists of a 2sp orbital, the hybrid orbital of
the Ph-N bond, and two nonbonding electrons in 2p orbitals: a p-π orbital and a p orbital on
nitrogen that lies in the plane of the benzene ring. Three low-lying spin states arise from the
near-degeneracy of these two 2p orbitals: open-shell singlet (OSS), closed-shell singlet (CSS)
and triplet states (T) (Figure 1.5).45-47
11
N N N
Figure 1.5. Three low-lying spin state,s which arise from the near-degeneracy of these two 2p orbitals: open-shell singlet, closed-shell singlet and triplet states.
It is reasonable to suggest that the closed-shell singlet is stabilized by the aromatic ring.
However, according to the high-level calculations, the open-shell configuration is more stable with strong delocalization of one electron between the π orbital of nitrogen and π-system of the aromatic ring shown on Figure 1.6. The other non-bonding electron occupies the p-orbital on nitrogen, which lies in the plane of the benzene ring. So, the corresponding two electrons with opposite spins lie in different region of space which minimizes their Coulombic repulsion energy.
Figure 1.6. Electronic structure of the lowest energy singlet open-shell state of phenylnitrene.
This geometry leads to a short C-N bond (1.276 Ǻ) in comparison with closed-shell
singlet and triplet (1.338 Ǻ). In 1997 MO calculations have established that the singlet nitrene
has an open-shell character and 1,3-biradical nature.45 12
This short-lived intermediate has been directly observed for the first time in 1997 by laser flash pholysis (LFP) spectroscopic techniques as an intense band at 350 nm wavelength.48 Thus,
singlet phenyl nitrene 2 generates the bicyclic benzazirine BA (Scheme 1.1). This process is very
rapid and occurs within a few hundred picoseconds (10-100 ps). BA then rapidly undergoes ring
expansion to dehydroazepine DA with time constant of ca. 0.1-1.0 ns. When irradiation is
conducted in the presence of a nucleophile, such as diethylamine, DA undergoes attack by the nucleophile to form the azerpine AZ. The DA intermediate has been observed by either time resolved UV (λ =320-380 nm, broad) or IR (1890 cm-1) spectroscopy. The rate constant for the reaction of DA with diethylamine was found to be 6.5x106 M-1 s-1 and its life-time is 32 min at
194 K. 41,49
Scheme 1.1. The mechanism of the photolysis of simple aryl azides.
In spite of the benzazirine intermediate has never been observed directly,50-51
experimental evidence of this intermediate has been provided by trapping with ethanethiol,
which affords o-thioethoxyaniline 3 in 39% yield (Scheme 1.2).52 13
H CH CS N 3 2 NH SCH2CH3 NH2 HSCH2CH3
BA 3
Scheme 1.2. Trapping of benzazirine upon the photolysis of 1 in ethanethiol.
Intersystem crossing (ISC) process from singlet phenyl nitrene 2 to triplet nitrene 4
dominates at 77 K. The absolute rate constant of intersystem crossing for phenylnitrene is found
to be 3.2×106 s-1. The triplet nitrene behaves like a diradical, but it has been termed “totally
useless” in photoaffinity labeling, becouse two types of products have been obtained in such
experiments, Scheme 1.3: the azobenzene 5 and aniline 6 were formed via dimerization and the latter via hydrogen abstraction from the solvent.53-55
Scheme 1.3. Products formed upon the photolysis of phenyl azide via triplet pathway.
The decay of triplet arylnitrenes and/or formation of corresponding azocompound were
studied by LFP techniques, and found to obey second-order kinetics with rate constants in the
range of (0.55-2.1)x109 M-1 s-1 in benzene at room temperature. 14
Yields of photolabeling with phenyl azides are generally less than 30 %.1 There are
several explanations for the low efficiency of PAL with phenyl azides: high yield of triplet
nitrene, short excitation wave-length (280 nm), and formation of diazirine DA. Unfortunately, this major trappable intermediate produced upon photolysis of phenyl azide, is not the ideal species for an efficient PAL experiment. However, the ring expansion of an aromatic singlet nitrene to DA is strongly dependent on the substituents on the ring, as well as the reaction temperature. Researchers have evaluated the effect of many substituents on the aryl azide photochemistry and found substituents that have potential for improving the effectiveness of photolabeling. Nitro, imino, carboxy and fluoro groups are substituents that seem to afford better
PAL reagents.
Fluorosubstituted Azides. The photochemical behavior of fluorinated aryl azides is an area of significant importance, since the resulting fluorinated nitrenes possess properties that have made them more desirable PAL agents. Specifically, fluorinated nitrenes undergo ring expansion to diazirines much more slowly (ΔE# = 7-8 kcal/mol) than their nonfluorinated
analogs (ΔE# = 2-3 kcal/mol) that ring expand ca. 170-1700 times more rapidly.56 Due to this
slow ring expansion, nitrenes derived from fluorinated aryl azides are thought to have extended
singlet lifetimes (200-250 ns),57 and to undergo insertion into C-H bonds of cyclohexane or
reaction with pyridine, and what appears to be insertion into N-H bonds of secondary amines as
indicated in Scheme 1.4.57-63 15
Scheme 1.4. The photochemical behavior of fluorinated aryl azides.
This type of indiscriminate reactivity is thought to be ideal for fixing the ephemeral
associations of biomolecules into covalently bonded units that can be readily identified and
studied in detail via careful disassembly of the cross-linked systems. For these reasons, many
PAL applications have been based upon modified fluorinated aryl azides. In all cases studied to
date, the cross-linking processes of fluorinated nitrenes are attributed to direct nitrene insertion or ring expansion followed by nucleophile addition to the resulting ketenimine.
16
1.4 Photochemical Formation and Reactions of Nitrenium Ions
It turns out that some of the more effective photoaffinity cross-linking agents generate aryl nitrenium ions as their pivotal intermediates. Nitrenium ion were first described in 1964 by
Abramovitch.65
Nitrenium ions (NI) are highly reactive intermediates which are structurally characterized
by dicoordinate nitrogen with a formal positive charge (Figure 1.7). They are similar to the
nitrenes in that they possess the nitrogen atom with six valence electrons, to the carbenium ions
in that both are cationic, and to the carbenes in that both have coordinate atoms that have two
nonbonding orabitals and two nonbonding electrons.66 Moreover, nitrenium ions, like carbenes, exist in singlet or triplet ground states depending on the ligands.
R R N NH N R'
R' R'
Figure 1.7. Nitrenium ions.
According to the computational and theoretical studies, phenyl nitrenium ions exist as a
singlet planar ground states67 in contrast to alkylnitrenium ions that are predicted to be in triplet
ground state.68 Aryl substitutions stabilize the singlet due to the significant delocalization of the
positive charge of nitrenium ion into the benzene ring. The triplet phenyl nitrenium ion is 21 kcal/mol less stable than the singlet (Figure 1.8). 17
Figure 1.8. Geometry of singlet nitrenium ions calculated by DF Theory.
Substituents on the aromatic ring of a phenyl nitrenium ion, especially π-donating groups,
greatly stabilize singlet state due to delocalization of the positive nitrenium charge (Figure 1.8).
Consequently, knowledge of the reactivity of nitrenium ions with biological molecules
becomes critical in designing effective PAL strategies. In the early 1970’s, Gassman and co-
workers have studied the decomposition of N-chloro-N-alkylanilines and shown that nitrenium
ions can be involved in many reactions in solution.69 Aryl amines occur in a wide variety of
industrial and consumer products, and many of these have been considered as possible
environmental carcinogens. In addition, nitrenium ions are generated via amino acid pyrolysis in
broiled and fried protein-containing foods and tobacco smoke. As a result, aryl amines have been
studied extensively in efforts to understand how they initiate carcinogenesis and mutagenesis.70-
73 The process by which aryl amines are transformed into nitrenium ions in biological systems is
outlined in Scheme 1.5. Thus, the aryl amines are initially converted to nitrenium ions by
metabolic oxidation to hydroxylamines. Then, esterification of the amine nitrogen leads to
hydroxylamine acetate or hydroamic acid acetate. Finally, the reaction of the ester with a guanine
residue of DNA gives a covalent addition product 7. 18
OAc Ar NH2 Ar NH Ar NH Ar NH O N R dG HN OH OAc NAr N H2N N dRibose OAc Ar NHAc Ar NAc Ar NAc Ar NAc 7
OH OAc
Scheme 1.5. Transformation of aryl amines into nitrenium ions in biological systems.
Nitrenium ion can be generated by two methods: thermal and photochemical. There are
many aromatic amines, N-chloroamines and azides, that upon heterolysis form nitrenium ion
precursors.74-76 The chemistry of NI’s with different nucleophiles has been well-studied and
reviewed.77-80
Scheme 1.6. Addition of alcohols and water to NI.
The generation of NI in aqueous solution or in alcohols usually leads to addition of nucleophiles to para- or ortho-positions (Scheme 1.6). The formation of the addition products requires an imino-cyclohexadiene intermediate. 19
The application of nitrenium ions as PAL intermediates has been shown to be viable through the photolysis of substituted aryl azides that are effective nitrenium ion precursors (vide infra). The capability to observe these short-lived reactive intermediates directly is proving to be a mechanistic tool of great value. The first photochemical studies using LPF methods were performed by Baetzold and Tong in 1971.81 During the experiment with dimethylaminophenyl azide 8, they had detected an intermediate with absorption at 325 nm and life-time more than 100
ms which was unassigned for the next several decades by the photochemical community. In these studies, the photolysis of p-aminophenyl azide leads to the formation of the corresponding
quinodiimines 9 upon irradiation in weakly acidic buffered aqueous solutions, Scheme 1.7.
N3 N NH NH
hν H2O
-N2
NEt 2 NEt2 NEt2 NEt2 Singlet Nitrenium Quinodiiminium Nitrene Ion Ion 89
Scheme 1.7. Irradiation of p-aminophenyl azide in weakly acidic aqueous solution.
The authors had selected an optimal system for switching the aryl nitrene chemistry from its
usual ring-expanding, ketenimine mode to its nitrenium ion mode; however, they did not
unequivocally establish the structure of the unstable nitrenium ions formed in these reactions, or
the products of their reaction. Later, the photochemistry of phenyl azide in the presence of acetic
acid and ethanol was investigated by Takeuchi and Koyama.82,83 In that study, three major 20
products were obtained (10, 11 and 12). Based on these results, these authors had suggested that two branches were in competition with each other: a convential ketenimine branch described in
Part 1.3 and a nitrenium ion branch, collapse of a nitrenium ion pair which in turn arose from protonation of the nitrene by the acidic solvent.
H Ketenimine N O Branch N3 N 10
hν 1.4 NHAc AcOH NHAc 1 OR -N2
R=H,Ac Nitrenium 11 12 Ion Branch OR
Scheme 1.8. Photochemistry of phenyl azide in the presence of acetic acid and ethanol.
McClelland has pioneered the application of LPF in the study of aryl nitrene/nitrenium
ion chemistry.84,85 In 1995, LFP spectroscopy was used to directly observe the nitrenium ions
formed in these reactions. The aryl nitrenium ions in aqueous acetonitrile and water alone were
formed within the laser pump pulse width of 20 ns via the rapid protonation of the nitrene by solvent. Then, the nitrenium ion undergoes a relatively slow reaction with water to form either benzoquinone or 4-substituted 4-hydroxy-2,5-cyclohexadienones86 depending upon the substitution in the 4-position of the phenyl ring as outlined in Scheme 1.9.
21
N3 N NH NH NH O hν H2O R=OH -N2 R'OH H2O -NH3 ps s ms fs μ minutes HO R R R R O O Aryl Nitrene Nitrenium Dienone Iminoquinone Quinone Azide Ion Imine
Scheme 1.9. Photochemical reaction of p-substituted aryl azides in water.
-1) Substituient λmax (nm) kdecay (sec Reference
R NH R= -H ~2x1010 88 -Cl ~2x108 89 -OPh ~2 x 107 90 7 -OC6H4-p-OCH3 1.9 ± 0.2 x 10 90 -Ph460 2.84 ± 0.04 x 106 87
-OCH3 305 2.7±0.2x106 90 -OCH2CH3 300 1.8 ± 0.1 x 106 90 5 -OCH(CH3)2 295 8.0 ± 0.1 x 10 90 4 -OC(CH3)3 290 6.4±0.2x10 90 NH 525-550 -Br 6.5±0.3x106 91 R -H 6.3±0.3x106 91
-CH3 1.5±0.1x106 91
-N(CH3)Ac 5.6 ± 0.3 x 105 91 -OCH3 6.1±0.1x104 91
NH 3.4±0.2x104 87
Table 1.1. Absorption maxima and rate constants for decay of substituted nitrenium ions in water. While most constants were determined in ionically buffered aqueous media, it should be noted that some less stable ions are better generated and stabilities enhanced if they are generated in slightly acidic media.
22
87 From these studies, it became clear that some nitrenes are rather strong bases, pKa > 12.4, capable of being protonated by water and alcohols.
The rate constants for decay of various nitrenium ions in water are listed in Table 1.2.
According to these data, arylnitrenium ions are kinetically stable in water and their stability depends upon substituents on the phenyl ring.
The next generation of ultra fast laser transient spectroscopy has made it possible to visualize the extremely rapid events that were obscured by the long-probe laser pulses in the previous studies. Platz, Burdzinski, and Bally have examined unadorned polycyclic aryl nitrenes and observed them to undergo protonation to form nitrenium ions, but in order for protonation to be competitive with intersystem crossing to the triplet, the nitrenes must be generated in acidic solvents. Thus, the following singlet nitrene lifetimes were observed when the nitrenes were generated in 88% formic acid: 1-naphthylnitrene, τ = 8.4 ps92; p-biphenylnitrene, τ = 11.5 ps92,
phenylnitrene, τ = 12.0 ps (100% formic acid)93, and 2-fluorenylnitrene, τ = 10 ps94, but much
less rapid protonation occurs in alcoholic solvents, for example in methanol where 2-
fluorenylnitrene has a lifetime of τ = 250 ps.94
The valuable information gleaned from these transient studies was that, if properly substituted, aryl nitrenes are surprisingly strong bases that can abstract protons from water without the aid of added acid. In order for the nitrenium ion-forming branch of aryl nitrene chemistry to be favored over the ring-expansion branch in Scheme 1.8, the nitrene must be conjugated to strong electron donating groups or extended π-systems. Detailed theoretical studies of phenyl azides substituted in the para-position with a wide variety of substituents reinforces the observation that para-electron donating groups greatly stabilize the singlet state of nitrenium ions.95 The nitrenium ion pairs that result from protonation of these nitrenes are surprisingly 23
stable and do not collapse quickly, as might be expected, but survive into the microsecond and even millisecond domains. Furthermore, the mode of collapse observed in these early studies was nucleophilic attack of water at the para-position, which results in hydrolysis to a quinone with loss of the original aryl azide nitrogen (Scheme 1.9)
Many of these properties of aryl nitrenium ion are not particularly favorable for development into photoaffinity labeling systems. Thus, an ideal photoaffinity label will form a strong covalent bond with neighboring molecules, and this bond must survive isolation and analysis. As can be seen from the aforementioned examples, the nitrenium ion branch often affords cross-linking bonds that are easily broken by hydrolysis. In addition, while the nitrenium ion pair is formed very rapidly, it collapses to form the pivotal cross-linking bond very slowly in the micro- to millisecond domain. So information about specific binding sites derived from such slowly formed cross-links may be spurious.
1.5 References
(1) Fleming S.A. Tetrahedron, 1995, 51, 12479-12520.
(2) Brunner, Annu.Rev.Biochem. 1993, 62, 483-514.
(3) Gartner, C. A. Curr. Med. Chem. 2003, 10, 671-689.
(4) (a) Chuang, V. T. G., Maruyma, T., Otagiri, M. Drug Metab. Pharmacokinet. 2009,24,
358–364; (b) Grammer,J. N.; Kuwayama,H.; Yount, R.G. Biochemistry, 1993, 32, 5725-
5732; (c) Dormán, G.; Prestwich, G. D. Trends Biotech. 2000, 18, 64-77.
(5) Hatanaka, Y.; Sadakane, Y. Curr. Top. Med. Chem. 2002, 2, 271-288.
(6) Singh, A.; Thomton, E.R.; Westhermer, F. H. J.Biol.Chem. 1962, 237, 3006-3008. 24
(7) Fedan, J. S.; Hogaboom, G. K.; O’Donell, J. P. Biochem. Pharmacol. 1984, 33, 1167-
1180.
(8) Driessen, A. J. M.; Manting, E. H.; van der Does, C. Nature Struct. Biol. 2001, 8, 492-498.
(9) Karwatsky, J. M.; Georges, E. Curr. Med. Chem. - Anti-Cancer Agents, 2004, 4, 19-30 b)
Zanirato, P. ARKIVOC 2009, I, 97-128.
(10) Goeldner, M., Angew.Chem.Int.Ed.Engl. 1995, 34, 1296-1312.
(11) Hashimoto, M.; Hatanaka, Y. Eur.J.Org.Chem. 2008, 2513-2523.
(12) Peer, M.; Csaszar, E.; Vorlaufer, E.; Kopp, S.; Chiba, P. Mini-Reviews in Medicinal
Chemistry, 2005, 5, 165-172.
(13) Schuster, D. I.; Probst, W. C.; Ehrlich, G. K.; Singh, G. Photochem.Photobiol. 1989, 49,
785-804.
(14) Addona, G. H.; Kloczewiak, M. A.; Miller, K. W. Anal. Biochem. 1999, 267, 135-140.
(15) Dormán, G. Topics in Current Chemistry 2000, 211, 169-225.
(16) Weber, P. J. A.; Beck-Sickinger, A. G. J. Peptide Res. 1997, 49, 375-383.
(17) Tate, J. J.; Persinger, J.; Bartholomew, B. Nucleic Acids Res. 1998, 26, 1421-1426.
(18) Scott, A. P.; Platz, M. S.; Radom, L. J. Am. Chem. Soc. 2001, 123, 6069-6076.
(19) (a) Bayley, H. Photogenerated Reagents in Biochemistry and Molecular Biology;
Elsevier: New York, 1983. (b) Bayley, H.; Staros, J. In Azides and Nitrenes; Reactivity and
Utility; Academic Press: New York, 1984; p. 434-465.
(20) Watanabe, Y.; Hirata, M.; Ogasawara, T.; Koga, T.; Ozaki, S. Bioorg. Med. Chem. Lett.
1991, 1, 399-402.
(21) Kemnitz, C. R.; Karney, W. L.; Borden, W. T. J. Am. Chem. Soc. 1998, 120, 3499-3503.
(22) Platz, M. S. Acc. Chem. Res. 1995, 28, 487-492. 25
(23) Bartholomew, B.; Tinker, R. L.; Kassavetis, G. A.; Geiduschek, E. P. Methods Enzymol.
1995, 262, 729-753.
(24) Capson, T. L.; Benkovic, S. J.; Nossal, N. G. 1995, 262, 449-456.
(25) Pal, P. K.; Coleman, P. S. J. Biol.Chem. 1990, 265, 14996-15002.
(26) Schneider, K.; Zimmermann, H. W. Histochemistry 1994, 101, 135-142.
(27) Buchmueller, K. L.; Brian, T. H.; Platz, M. S.; Weeks, K. M. J. Am.Chem.Soc. 2003, 125,
10850-10861.
(28) McClelland, R. A.; Mathivanan, N.; Steenken, S. J. Am. Chem. Soc. 1990, 112, 4857-
4861.
(29) Lackner, M. Lasers in Chemistry, vol. 2, WILEY-VCH, 2008.
(30) Porter, G.; Topp, M. R. Proc. R. Soc. Lond. A 1970, 315, 163-184.
(31) Small Jr., R. D.; Scaiano, J. C. J.Am.Chem.Soc. 1978, 100, 296-298.
(32) Grieb, P. Philos. Trans. R. Soc. London 1864, 13, 377.
(33) Lin, T. S.; Prusoff, W. H. J. Med. Chem. 1978, 21, 109-112.
(34) Meijer, E. W.; Nijhuis, S.; Von Vroonhoven, F. C. B. M. J. Am.Chem. Soc. 1988, 110,
7209-7210.
(35) (a) Cai, S. X.; Glenn, D. R.; Keana, J. F. W. J. Org. Chem. 1992, 57, 1299-1304. (b)
Niino, H.; Koga, Y.; Yabe, A. J. Photochem. Photobiol.A: Chemistry 1997, 106, 9-13.
(36) Hantzsch, A. Ber. Dtsch. Chem. Ges. 1933, 66, 1349-1354.
(37) Pauling, L.; Brockway, L. O. J. Amer. Chem. Soc. 1937, 59, 13-20.
(38) Reiser, A.; Leyshon, L. J. J. Am. Chem. Soc. 1970, 95, 7487.
(39) Platz, M. S. In Reactive Intermediate Chemistry, Moss, R. A., Platz, M. S., Jones, M., Jr.,
Eds.: John Wiley & Sons: Hoboken, N.J., 2004; pp 501-560. 26
(40) Gritsan, N. P.; Platz, M. S. Chem. Rev. 2006, 106, 3844-3867.
(41) Gritsan, N. P.; Pritchina, E. A. Russ. Chem. Rev. 1992, 61, 500-530.
(42) Wang, J.; Burdzinski, G.; Zhu, Z. D.; Platz, M. S.; Carra, C.; Bally, T. J. Am. Chem. Soc.
2007, 129, 8380-8388.
(43) Wang, J.; Kubicki, J.; Burdzinski, G.; Hackett, J. C.; Gustafson, T. L.; Hadad, C. M.;
Platz, M. S. J. Org. Chem. 2007, 72, 7581-7586.
(44) McCulla, R. D.; Burdzinski, G.; Platz, M. S. Org. Lett. 2006, 8, 1637-1640.
(45) Karney, W. L.; Borden, W. T. J. Am. Chem. Soc. 1997, 119, 1378-1387.
(46) Kim, S. J. I.; Hamilton, T. P.; Schaefer, H. F., III J. Am. Chem. Soc. 1992, 114, 5349-
5355.
(47) Borden, W. T., Gritsan, N. P.; Hadad, C. M.; Karney, W. L.; Kemnitz, C. R.; Platz, M. S.
Acc. Chem. Res. 2000, 33, 765-771.
(48) Gritsan, N. P.; Yuzawa, T.; Platz, M. S. J. Am. Chem. Soc. 1997,119, 5059-5060.
(49) Schuster, G. B.; Platz, M. S. Adv. Photochem. 1992, 17, 69-143.
(50) Chapman, O. L.; LeRoux, J. P. J. Am. Chem. Soc. 1978, 100, 282-285.
(51) Donnelly, T.; Dunkin, I. R.; Norwood, D. S. D.; Prentice, A.; Shields, C. J.; Thomson, P.
C. P. J. Chem. Soc. Perkin. Trans. 2 1985, 307-310.
(52) Carroll, S. E.; Nay, B.; Scriven, E. F. V.; Suschitzky, H. and Thomas, D. R. Tetrahedron
Lett. 1977, 18, 3175-3178.
(53) Liang, T.-Y.; Schuster, G. B. J. Am. Chem. Soc. 1986, 108, 546-548.
(54) Reiser, A.; Willets, F. W.; Terry, G. C.; Williams, V.; Marley, R. Trans. Faraday Soc.
1968, 64, 3265-3275.
(55) Schrock, A. K., Schuster, G. B. J. Am. Chem. Soc. 1984, 106, 5234-5243. 27
(56) Poe, R.; Schnapp, K.; Young, M. J. T.; Grayzar, J.; Platz, M. S. J. Am. Chem. Soc. 1992,
114, 5054-5067.
(57) Pandurangi, R. S.; Lusiak, P.; Kuntz, R. R.; Volkert, W. A.; Rogowski, J.; Platz, M. S. J.
Org. Chem. 1998, 63, 9019-9030.
(58) Pandurangi, R. S.; Karra, S. R.; Kuntz, R. R.; Volkert, W. A. Photochem.Photobiol.
1997, 65, 208-221.
(59) Keana, J. F. W.; Cai, S. X. J. Org. Chem. 1990, 55, 3640-3647.
(60) Cai, S. X.; Glenn, D. J.; Keana, J. F. W. J. Org. Chem. 1992, 57, 1299-1304.
(61) Pandurangi, R. S.; Katti, K. V.; Barnes, C. L.; Volkert, W. A.; Kuntz, R. R. J. Chem.
Soc., Chem. Commun., 1994, 1841-1842.
(62) Pandurangi, R. S.; Katti, K. V.; Kuntz, R. R.; Volkert, W. A. J. Org. Chem. 1997, 62,
2798-2807.
(63) Pandurangi, R. S.; Kuntz, R. R.; Volkert, W. A. Appl. Radiat. Isot. 1995, 46, 233-239.
(64) Pandurangi, R. S.; Kuntz, R. R.; Sharma, U.; Volkert, W. A. J. Org. Chem. 2002, 67,
6748-6757.
(65) Abramovitch, R. A.; Davis, B. A. Chem. Rev. 1964, 64, 149.
(66) Boche, G.; Andrews, P.; Harms, K.; Marsch, M.; Rangappa, K. S.; Schimeczek, M.;
Willeke, C. J. Am. Chem. Soc. 1996, 118, 4925-4930.
(67) Cramer, C. J.; Dulles, F. J.; Falvey, D. E. J. Am. Chem. Soc. 1994,116, 9787-9788.
(68) Ford, G. P.; Herman, P. S. J. Am. Chem. Soc. 1989, 111, 3987-3996.
(69) (a) Gassman, P. G.; Cryberg, R. L. J. Am. Chem. Soc. 1969, 91, 5176-5177; (b) Gassman,
P. G.; Hartman, G. D. J. Am. Chem. Soc. 1973, 95, 449-454. (c) Gassman, P. G. Acc. Chem.
Res. 1970, 3, 26-33. 28
(70) Schut, H. A. J.; Snyderwine, E. G. Carcinogenesis, 1999, 20, 353-368.
(71) Benigni, R.; Giuliani, A.; Franke, R.; Gruska, A. Chem. Rev. 2000, 100, 3697-3714.
(72) Hoffmann, G. R.; Fuchs, R. P. P. Chem. Res. Toxic., 1997, 10, 347-359.
(73) Verna, L.; Whysner, J.; Williams, G. M. Pharmacol.Ther. 1996, 71, 83-105.
(74) Schut, H. A. J.; Snyderwine, E. G. Carcinogenesis, 1999, 20, 353-368.
(75) Neumann, H.-G. J. Cancer Res. Clin. Oncol 1986, 112, 100-106.
(76) Sugimura, T.; Sato, S.; Wakabayashi, K. “Mutagens/Carcinogens in Pyrolysates of
Amino Acids and Proteins and in Cooked Foods: Heterocyclic Aromatic Amines” Appendix
III in “Chemical Induction of Cancer”, Vol. IIIc, Y.-T. Woo, D. Y. Lai, J. C. Arcos, M. F.
Argus, Ed., Academic Press, New York, 1988, pp. 681-710.
(77) Arcos, J. C.; Arcus, M. F. “Chemical Induction of Cancer”, Vol. IIB, Academic Press,
New York, 1974.
(78) Burdzinski, G. T.; Gustafson, T. L.; Hackett, J. C.; Hadad, C. M.; Platz, M. S. J. Am.
Chem. Soc. 2005, 127, 13764-13765.
(79) Falvey, D. E. “Nitrenium Ions” in “Reactive Intermediate Chemistry” R. A. Moss, M. S.
Platz, M. Jones, Jr., Eds., Wiley-Interscience, Hoboken, N. J., (2004) 593-650.
(80) Thomas, S. I.; Falvey, D. E. J. Phys.Org. Chem. 2006, 19, 291-294.
(81) Baetzold, R. C.; Tong, L. K. J. J. Am. Chem. Soc. 1971, 93, 1347-1353.
(82) H. Takeuchi, K. Koyama J. Chem. Soc. Chem. Comm. 1981, 202-204.
(83) Takeuchi, H.; Koyama, K. J. Chem. Soc. Perkin Trans. I. 1982, 1269-1273.
(84) Sukhai, P.; McClelland, R. A. J. Chem. Soc., Perkin Trans. 2, 1996, 1529-1530.
(85) Gadosy, T. A.; McClelland, R. A. J. Am. Chem. Soc. 1999, 121, 1459-1465. 29
(86) McClelland, R. A.; Davidse, P. A.; Hadzialic, G. J. Am. Chem. Soc. 1995, 117, 4173-
4174.
(87) McClelland, R. A.; Kahley, M. J.; Davidse, P. A.; Hadzialic, G. J. Am. Chem. Soc. 1996,
118, 4794-4803.
(88) Fishbein, J. C.; McCelland, R. A. Can. J. Chem. 1996, 74, 1321-1328.
(89) Novak, M.; Kahley, M. J.; Lin, J.; Kennedy, S. A.; James, T. G. J. Org. Chem. 1995, 60,
8294-8304.
(90) Ramlall, P.; McClelland, R. A. J. Chem. Soc., Perkin Trans. 2, 1999, 225-232.
(91) Bose, R.; Ahmad, A. R.; Dicks, A. P.; Novak, M.; Kayser, K. J.; McClelland, R. A. J.
Chem. Soc., Perkin Trans, 2, 1999, 1591-1599.
(92) Wang, J.; Burdzinski, G.; Zhu, Z.; Platz, M. S.; Carra, C.; Bally, T. J. Am. Chem. Soc.
2007, 129, 8380-8388.
(93) Wang, J.; Kubicki, J.; Platz, M. S.Organic Letters 2007, 9, 3973-3976.
(94) Wang, J.; Burdzinski, G.; Platz, M. S.Organic Letters 2007, 9, 5211-5214.
(95) Sullivan, M. B.; Brown, K.; Cramer, C. J., Truhlar, D. G. J. Am. Chem. Soc. 1998, 120,
11778-11783.
30
CHAPTER II. EXPERIMENTAL METHODS
2.1 UV-VIS Absorption Spectroscopy
The ground state electronic absorption spectra were measured at room temperature on either with an Agilent 8453 or a Hewlett Packard 8453 diode array spectrometer, using 10 mm or
2 mm path-length quartz cuvettes. In each experiment the sample was referenced to background.
2.2 Steady-State Fluorescence Spectroscopy
Steady state fluorescence spectra were obtained using the instrumentation available in the Dr. Castellano’s group.
Steady state fluorescence spectra were obtained at room temperature using an Edinburgh
Instruments FL/FS 900 spectrofluorimeter. A 450 W Xe lamp optically coupled to a single- grating monochromator with an accuracy of ± 2 nm was used to excite the sample. The emission was gathered at 90o excitation and passed through a second identical monochromator. All photophysical measurements were taken with optically dilute solutions (OD<0.1) prepared with spectroscopic grade solvents in 1 cm2 quartz cells (Starna cells). Prior each measurements
sample solutions were degassed by bubbling of argon through the solution for 20-35 minutes.
The comparative method of Williams1 was used for the determination of fluorescence
quantum yields (Φfl) for diethylaminopyridine azide in Chapter IV. The standard sample was
diphenylanthracene; excitation wavelength was set to 355 nm. The standard sample was prepared
in ethanol; absorbance of all samples at 355 nm was adjusted to ~ 0.1~0.12.
The equation for estimation of fluorescence quantum yields by the Comparative Method is: 31
Where Φx and Φs are quantum yields of sample and standard, correspondingly; Ix and Is are the
integrated area under the corrected fluorescence spectra; Ax and As are absorptions at excitation
wavelength; the n’s are the refractive indices of the solvents used for the two solutions.
All steady-state absorption and fluorescence data were analyzed using Origin 6.1
software (Origin Lab).
2.3. Nanosecond UV-VIS Time Resolved Absorption Spectroscopy
Nanosecond excited state absorption spectra and decay kinetics for azides 25 and 65 were obtained using the instrumentation available in the Ohio Laboratory for Kinetic Spectrometry.2a
Nanosecond (ns) time-resolved laser flash photolysis was performed on a Spectra Physics
Quanta Ray GCR-230 Nanosecond Transient Absorption spectrometer (Ultrafast Systems) equipped with a 150 W Xe-arc lamp (Oriel Corporation), Spex 1681 (0.22 m) monochromator and Hamamatsu R928 photomultiplier tube (5 stage dynode amplifier) operating with a Keithley
247 high voltage supply. Excitation at 350 nm from a computer-controlled Q-switched Nd:YAG laser operating at 10 Hz was directed to the sample with an optical absorbance of 0.64 at the excitation wavelength. Pump pulse energy is about 4-5 mJ at the sample position. The probe was focused through the sample onto the entrance slit of a monochromator, than monochromatic light was detected by aforementioned photomultiplier tube. The resulting signal was routed through a
DC-coupled back-off circuit that stored and displayed digital readouts for I0. The real time signal was fed to a back-off circuit in order to eliminate a DC offset from the PMT. 32
The absorbance of the samples was typically ~0.6-0.7 in a 1 cm path length cuvette at the excitation wavelength. In order to prevent the decomposition of sample and the accumulation of decomposition products on the walls of the cell, 25 mL of the solution was prepared and degassed for at least 20 min before the measurements. Then a flow cell set-up was applied and solution kept under an argon or nitrogen atmosphere for the duration of experiments.
Experimental solutions were pumped through a quartz flow cell (Starna Cells) with three polished windows at a rate of 150 mL per minute. The number of spectra acquisitions was from
16 to 32. Lab VIEW Software (National Instruments) was applied for collection of data.
Transient UV-vis spectra were measured every 10 nm over 360-650 nm spectral windows. The kinetic profiles were recorded using the same software, at each single emission wavelength and averaged over a predetermined number of pulses. The ground state absorption spectra of each sample were measured before and after each experiment to verify the stability of the sample to the experimental conditions.
Nanosecond transient absorption spectra for azide 65 were obtained using the instrumentation available in the Dr. Castellano’s group.
Nanosecond transient absorption spectra were collected on a Proteus spectrometer2b
(Ultrafast Systems) equipped with a 150 W Xe arc lamp (Newport), a Chromex monochromator
(Bruker Optics) equipped with two diffraction gratings blazed for visible and near-IR dispersion, respectively, and Si or InGaAs photodiode detectors (DET 10A and DET 10C, Thorlabs) optically coupled to the exit slit of the monochromator. Excitation at 355 nm with a power of
3.0–5 mJ per pulse from a computer-controlled Nd:YAG laser/OPO system from Opotek
(Vibrant LD 355 II) operating at 10 Hz was directed to the sample with an optical absorbance of 33
0.6-0.8 at the excitation wavelength. The data consisting of a 128-shot average were analyzed by
Origin 8.0 software and difference spectra at select delay times are presented.
2.4 Femtosecond UV-VIS Time Resolved Absorption Spectroscopy
Femtosecond transient absorption spectra and decay kinetics were obtained by Maxim Panov and Dr. Tarnovsky using the instrumentation available in the Dr. Tarnovsky’s group. Spectrometer information also was provided by Dr. Tarnovsky3
The femtosecond time-resolved transient absorption spectrometer used in this work is
based on the combination of a Ti:Sapphire regenerative amplifier (Hurricane, Spectra Physics)
and two interchangeable, computer-controlled pump and probe TOPAS-C optical parametrical
amplifiers (Light Conversion Lt.) (Figure 2.1). The amplified output is a train of 800 nm laser
pulses with pulse widths of ~100 fs and pulse energy of 0.92 mJ with a repetition rate of 1 kHz,
it is divided by a beam splitter into two beams. One beam (50%) pumped a TOPAS-C pump
amplifier to generate 305, 350 and 420 nm light pulses used for sample excitation. The typical
-1 excitation energy was kept between 3 and 4 μJ pulse , which was focused into a 300 μm
diameter spot at the sample position. The second beam was focused onto a 3-mm CaF window 2
-1 to produce a white light continuum. The energy of that beam was ~4 μJ pulse . The white-light
continuum beam was further split into reference and probe beams, the latter of which was
o focused to a 100 μm diameter spot and overlapped with the pump beam at an angle of 8 at the sample position. Alternatively, for the 305 pump, the other half of the 800 nm amplified output was delivered to the TOPAS-C probe amplifier to produce UV-probe pulses tunable from 280 to
390 nm. The reference and probe beams were focused through the sample and sent to a 34
monochromator/ spectrograph (Spectra-Pro 2358, Acton Research) and registered on a 512 pixel dual diode array for simultaneous accumulation of kinetic traces within 274 nm spectral windows (white-light continuum) or two Si-photodiodes (TOPAS-C probe). The excitation beam is chopped (on/off) at a 500 kHz repetition rate. Probe and reference diode array signals (I , I ) pr ref
are read after each laser shot for adjacent pairs of excitation on and off pulses and the transient
absorption for each pair of pulses is obtained as follows: ΔA = log (I /I ) – log (I /I ) . For pr ref on pr ref off a kinetic trace, 300 pairs of excitation on/off ΔA points are collected at ~120 time delayed positions between –10 ps and 1200 ps. Spectral data obtained in the complementary 274 nm ranges are averaged for about 10 successive scans of the delay line (total acquisition time, 45 min), and subsequently linked together to yield the resultant ΔA spectra from 345 to 765 nm.
Time zero at different probe wavelengths is obtained by using the non-resonant or two-photon absorption pump-probe signals from neat solvents.
The resultant group velocity dispersion curve (chirp rate, 2.0 × S42 fs) is used to correct the ΔA spectra. A strong Gaussian-like emission feature (165 ± 15 fs fwhm) due to stimulated Raman scattering (Raman-active CH symmetrical vibrational mode, v = 2853 cm) observed in neat cyclohexane in the Stokes region with respect to the excitation wavelength delivers a cross- correlation signal between pump and probe pulses. The pump light polarization was set using a
Berek compensator to be at 54.70 with respect to the probe light polarization, so all
measurements are performed at magic angle polarization conditions. The samples were
circulated through a Spectrosil quarz flow cell with a 0.2 or 0.5 mm path length (Starna) at a 35
Figure 2.1. Ti:Sapphire regenerative amplifier (Hurricane, Spectra Physics) and two interchangeable, computer-controlled pump and probe TOPAS-C optical parametrical amplifiers (Light Conversion Lt.).The picture was taken from official site of Dr. Tarnovsky’s group.
linear velocity of 0.6-1.6 m/s to avoid secondary excitation. All samples were prepared in
25 or 50 mL of solvent with typical sample absorbance in the 0.4-0.9 range at the excitation wavelength per 0.2 or 0.5 mm thickness of the flow cell used.
36
2.5 Computational Details
All of the calculations are performed by Dr. Hadad’s group, using Turbomole-5.914 at the Ohio Supercomputer Center.5
4-(N,N-diethylamino)-3-nitrophenyl azide (25)was optimized using Becke’s three-
parameter hybrid exchange functional with Lee-Yang-Parr correlation functional (B3LYP)6 methodology and using second-order couple cluster method with resolution-of-identity approximation (RI-CC2)7 with triple-zeta valence polarized (TZVP) basis sets as developed by
Ahlrichs and co-workers.8 Other than the azide 25, all of the other species were optimized at the
B3LYP/TZVP level. Vertical excitations were obtained at these geometries using time- dependent density functional theory (TD-DFT).9 The open-shell and closed-shell nitrenes were
optimized at the CASSCF(4,4)/6-31G(d) level using the Gaussian 0310 program. However,
further CASSCF optimizations and CASPT2 single-point energies of open-shell and closed-shell
nitrenes using larger active space and more active electrons, i.e., CASSCF(10,10), were
performed using MOLCAS 6.2.11 These CASPT2//CASSCF computations were accomplished
using the pVDZ basis set of Pierloot, et al.12
2.6 X-Ray Crystallographic Structure Determination of 31
The X-ray examinations were performed by Dr. Jeanette A. Krause from Department of Chemistry, at University of Cincinnati.
For X-ray examination and data collection, a suitable crystal, approximate dimensions
0.30 x 0.05 x 0.02 mm, was mounted in a loop with paratone-N and transferred immediately to
the goniostat bathed in a cold stream. 37
Intensity data were collected at 150 K on a standard Bruker SMART6000 CCD diffractometer using graphite-monochromated Cu Kα radiation, λ=1.54178Å. The detector was
o set at a distance of 5.165 cm from the crystal. A series of 10-s data frames measured at 0.3
increments of ω were collected to calculate a unit cell. For data collection frames were measured
o o for a duration of 8-s at 0.3 intervals of ω with a maximum θ value of ~135 . The data frames
were processed using the program SAINT. The data were corrected for decay, Lorentz and
polarization effects as well as absorption and beam corrections based on the multi-scan
technique.
The structure was solved by a combination of direct methods SHELXTL v6.14 and the
13 difference Fourier technique and refined by full-matrix least squares on F . Non-hydrogen
atoms were refined with anisotropic displacement parameters. All hydrogen atoms were located
directly in the difference map and their positions refined. The isotropic displacement parameters
for the H-atoms were defined as a*U of the adjacent atom, (a=1.5 for methyl and 1.2 for all eq others). The refinement converged with crystallographic agreement factors of R1=3.99%,
wR2=10.00% for 2451 reflections with I>2σ(I) (R1=5.44%, wR2=10.79% for all data) and 268
variable parameters.
2.7 References
(1) Williams, A. T. R.; Winfield, S. A.; Miller, J. N. Analyst, 1983, 108, 1067.
(2) (a) Rihter, B. D.; Kenney, M. E.; Ford, W. E.; Rodgers, M. A. J. J. Am. Chem. Soc.
1990, 112, 8064-8070; (b) Danilov, E.O.; Rachford, A.A. Goeb, S.; Castellano, F.N. J.
Phys. Chem. A 2009, 113, 5763-5768. 38
(3) Voskresenska, V.; Wilson, R. M.; Panov, M.; Tarnovsky, A. N.; Krause, J. A.; Vyas,
S.; Winter, A. H.; Hadad, C. M. J. Am. Chem. Soc. 2009, 131, 11535–11547
(4) (a) Ahlrichs, R.; Bär, M.; Häser, M.; Horn, H.; Kölmel, C. Chem. Phys. Lett. 1989,
162,165. (b) For the current version of TURBOMOLE, see http://www.turbomole.de. (c)
Treutler, O.; Ahlrichs, R. J. Chem. Phys. 1995, 102, 346.
(5) Spartan ’06, Wavefunction, Inc.,Irvine, CA, Shao, Y.; Molnar, L. F.; Jung, Y.;
Kussmann, J.; Ochsenfeld, C.; Brown, S. T.; Gilbert, A. T. B.; Slipchenko, L. V.;
Levchenko, S. V.; O’Neill, D. P.; DiStasio Jr., R. A.; Lochan, R. C.; Wang, T.; Beran, G.
J. O.; Besley, N. A.; Herbert, J. M.; Lin, C. Y.; Van Voorhis, T.; Chien, S. H.; Sodt, A.;
Steele, R. P.; Rassolov, V. A.; Maslen, P. E.; Korambath, P. P. Adamson, R. D.; Austin,
B.; Baker, J.; Byrd, E. F. C.; Dachsel, H.; Doerksen, R. J.; Dreuw, A.; Dunietz, B. D.;
Dutoi, A. D.; Furlani, T. R.; Gwaltney, S. R.; Heyden, A.; Hirata, S.; Hsu, C.-P.;
Kedziora, G.; Khalliulin, R. Z.; Klunzinger, P.; Lee, A. M.; Lee, M. S.; Liang, W. Z.;
Lotan, I.; Nair, N.; Peters, B.; Proynov, E. I.; Pieniazek, P. A.; Rhee, Y. M.; Ritchie, J.;
Rosta, E.; Sherrill, C. D.; Simmonett, A. C.; Subotnik, J. E.; Woodcock III, H. L.; Zhang,
W.; Bell, A. T.; Chakraborty, A. K.; Chipman, D. M.; Keil, F. J.; Warshel, A.; Hehre, W.
J.; Schaefer, H. F.; Kong, J.; Krylov, A. I.; Gill, P. M. W. and Head-Gordon, M. Phys.
Chem. Chem. Phys., 8, 3172 (2006).
(6) (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648-5652.(b) Lee, C.; Yang, W.; Parr,
R. G. Phys. Rev. B 1988, 37, 785-789.
(7) (a) Hättig, C.; Weigend, F J. Chem. Phys. 2000, 113, 5154–5161. (b) Hättig, C.;
Kőhn, A.; Hald, K. J. Chem. Phys. 2002, 116, 5401–5410. (c) Hättig, C. J. Chem. Phys.
2003, 118, 7751-7761. 39
(8) Weigend, F.; Häser, F.; Patzelt, H.; Ahlrichs, R. Chem. Phys. Lett. 1998, 294, 143-
152.
(9) Olivucci, M. In Computational Photochemistry Elsevier; Amsterdam, 2005, p 92-
128.
(10) Gaussian 03, Revision C.02, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.;
Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, Jr., J. A.; Vreven, T.;
Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.;
Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada,
M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda,
Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; 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.; Ayala, P. Y.; Morokuma, K.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels,
A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D.
J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P.
M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; and Pople, J. A.; Gaussian,
Inc., Wallingford CT, 2004.
(11) Karlstrom, G.; Lindh, R.; Malmqvist, P.-A.; Roos, B. O.; Ryde, U.; Veryazov, V.;
Widmark, P.-O.; Cossi, M.; Schimmelpfennig, B.; Neogrady, P.; Seijo, L. Comp. Mat.
Sci. 2003, 28, 222. 40
(12) Pierloot, K.; Dumez, B.; Widmark, P. O.; Ross, B. O. Theor. Chim. Acta. 1995,
90, 87-114.
(13) SMART v5.631 and SAINT v6.45A data collection and data processing
programs, respectively. Bruker Analytical X-ray Instruments, Inc., Madison, WI;
SADABS v2.10 for the application of semi-empirical absorption and beam corrections.
G.M. Sheldrick, University of Göttingen, Germany; SHELXTL v6.14 for structure
solution, figures and tables, neutral-atom scattering factors as stored in this package.
G.M. Sheldrick, University of Göttingen, Germany and Bruker Analytical X-ray
Instruments, Inc., Madison, WI.
41
CHAPTER III. THE PHOTOCHEMISTRY OF 4-(N,N-DIETHYLAMINO)-3-
NITROPHENYL AZIDE: PROTONATION OF THE NITRENE TO AFFORD
REACTIVE NITRENIUM ION PAIRS
3.1 Introduction
The PAL agent, 4-fluoro-3-nitrophenyl azide (NAP) (13), is a system that has been widely applied in photoaffinity studies with virtually ever class of biological molecules in resent years.1 It was initially used as a photoaffinity labeling reagent for binding of antibody sites by
Fleet and Knowles in 1970.2,3 The nitro group, according to Knowles, was selected not only to facilitate preparation of antigens relative to 2,4-dinitro-1-fluorobenzene, but for two other reasons as well. First, the nitro substituent shifts Δλmax of the hapten into the visible region (350
nm), away from the ultraviolet absorption of the protein. Second, a nitro group increases the
reactivity, decreases the life-time of the nitrene.1-5
Scheme 3.1. Application of NAP as a photoaffinity labeling reagent for binding of antibody. 42
After irradiation of the antibody in the presence of hapten, the (NAP)' haptenic group was covalently linked into more than 65% of antibody combining site. Furthermore, this haptenic group selectively coupled to the ε-amino group of lysine, allowing the synthesis of antigenic molecules with varying valence. Scheme 3.1 displays the step by step photoaffinity labeling of antibody experimental procedure.1-2
After the success of that experiment, NAP became the most widely used reagent in
labeling of different biomolecules. In recent time, NAP has been successfully used in protein,6-8
red cell membranes,9-10 DNA,11-14 RNA,15 vitamin D,16-19 steroids22, and other labeling studies. In
spite of a large number of PAL studies that have been conducted with NAP, there is little
information about the photochemical mechanism of the corresponding phenyl azides have been
reported. Kanaoka and co-workers23 had selected three model compounds for the photochemical
studies: p-nitrophenyl azide 14 and two related nitro amides 15, 16 (Figure 3.1).
Figure 3.1. Model compounds for photochemical studies by Kanaoka.
Upon the irradiation of these azides, the formation of azipine or intermolecular insertion
products were not observed. Just two triplet products were obtained: the azo compound and
primary amine. These authors suggested, that introduction of a nitro group into aromatic ring
promotes the intersystem crossing of excited singlet to triplet azide and increases the fraction of
triplet nitrene. 43
Later, the photochemistry of 1-azido-3-nitrobenzene (18) has been the subject of several studies.24 Photoaffinity labeling of meta- and para-nitrophenyl azides gave different results: the
meta-nitro substituent is a more effective photoaffinity labeling agent than the para-isomer.25
Photochemical studies of meta-nitrophenyl azide in the presents of nucleophile, such as diethyl
amine (DEA), indicate that the major pathway at room temperature is formation of singlet
nitrene, which has an open-shell electronic structure and leads to ring expansion or formation of
interception products as outlined in Scheme 3.2.26
Scheme 3.2. Irradiation of 1-Azido-3-nitrobenzene.
44
Studies have shown a very strong DEA concentration dependence. At the lower concentrations of DEA, the major products are the azobenzene 19 and 3-nitroaniline (20). At higher concentration, the yield of 3-nitroaniline increases, that yield of azobenzene decreases, and three additional products were formed: 2-(N,N-diethylamino)-6-nitro-3H-azepine (21) and its
4-nitro isomer 22 and N,N-diethyl-N’-(3-nitrophenyl)hydrazine (23). Authors had observed a transient intermediate with λmax=400 nm that formed with τ=13 ns; however, they did not assign
this absorption species.
After analysis of numerous examples of the application of NAP in biological studies in which the fluorine was replaced by a powerful electron-donating group such as amino nitrogen or ether oxygen; it is very surprising that no mechanistic photochemical studies have been reported prolong the nature of the photoreactions involved in these biological applications.
Consequently, we have studied the photochemistry of this widely applied 4-amino-3-nitrophenyl azide PAL/PCL system in the presence of nucleophiles using ultrafast pump-probe spectroscopy and theoretical techniques in an effort to better understand the mechanism of these reactions.
3.2. Synthesis of 4-(N,N-Diethylamino)-3-nitrophenyl Azide 25 and Study of its Photolysis
Products.
We have chosen 4-(N,N-diethylamino)-3-nitrophenyl azide (25) for this study of the
mechanism of photoaffinity labeling. Precursor 13 was prepared according to a known method
(Scheme 3.3) from the corresponding commercially available amine 24. Diazotization of 24
followed by treatment of sodium azide afforded 13.27 Then, nucleophilic aromatic substitution of fluorine by diethylamine leads to formation of desired azide 25. 45
Scheme 3.3. Synthesis of the 4-(N,N-diethylamino)-3-nitrophenyl azide.
The photolysis of compound 25 was performed in a Pyrex tube using 350 nm light in a
Rayonet photochemical reactor at ambient temperature (20-25oC).
In a typical procedure for the photolysis of the 4-(N,N-diethylamino)-3-nitrophenyl azide
(25), a solution of 25 in alcohol, which serves as both nucleophile and solvent, was degassed by
bubbling nitrogen trough the solution for 30 min. The resulting mixture was irradiated at room
temperature for 4 hours. The reaction was monitored every hour by TLC and 1H NMR. The
separation of the reaction mixture was performed by preparative silica gel TLC using a hexane-
dichloromethane mixtures (1:0; 1:0.5; 0.5:1).
According to the literature there are two processes active in these reactions: nucleophilic
addition of alcohols acting as nucleophiles attacking the benzene ring, and oxidation/reduction.
After analyzing of reaction mixtures by NMR and GC/MS two products were obtained: an
addition product 26 or 27, and reduction product 28 (4-(N,N-diethylmino)-3-nitroaniline) in a
ratio of 92:8, respectively (Scheme 3.4).
46
Scheme 3.4. Photolysis of 4-(N,N-diethylamino)-3-nitrophenyl azide.
The challenge was to establish the structure of the addition product. NMR spectra indicated the addition of the methoxy nucleophile might have place at either of two positions to form the products: 4-(N,N-diethylamino)-2-methoxy-3-nitroaniline (26) or 3-(N,N- diethylamino)-6-methoxy-2-nitroaniline (27). The formation of the isomer 26 would be the result of the aromatic nucleophilic substitution. While in the case of the second isomer 27, the attachment of the methoxy group would have occurred at the position originally occupied by the azido-function as shown in scheme 3.5. The formation of 27 could be explained by the conventional mechanism28 of nucleophilic attack by alcohol on the cyclic azirine to form an aziridine, which subsequently undergoes re-aromatization.
N N N OMe 3 MeO NH NH2 h MeOH
NO2 NO2 NO2 NO2 NO2 NEt2 NEt2 NEt2 NEt2 NEt2 25 27
Scheme 3.5. The conventional mechanism of the formation of the addition product. 47
Further results were obtained from the photolysis of the azide in the 2-propanol. The main addition product 30 was obtained in high yield (94%) (Scheme 3.6). However, the formation of second addition product 29 was observed in small quantity (4%). Interestingly, addition products with this regiochemistry were only detected when 2-propanol was the solvent.
When solvents other then 2-propanol were used in the irradiation, products of this alternative regiochemistry were not observed. In the 1H NMR spectrum of the major product, a pair of the
doublets at 6.76 and 6.87 ppm was observed with spin coupling constant corresponding to the
ortho-positioning of the hydrogen in the aromatic ring, which would be consistent with either
structures 27 or 30. The difference in these two structures is the relative position of the amino
group with respect to the alkoxy-substituent. Due to the minor structure difference and electronic
properties of these substituents, both have electron-donating character, the chemical shifts in 1H and 13C NMR spectra cannot be used as a definition evidence for either structure.
Therefore in order to confirm the structure of this
H NOE addition product, Nuclear Overhauser Effect (NOE) N H
O H spectroscopy was performed. Irradiation of upfield 6.85 ppm
aromatic proton signal resulted of enhancement of the 7.0ppm H NO2
NH2 signal, not the –O-CHMe2 signal (See Chapter V). NOE N
Irradiation of the -CH2 – signal of the diethylamino group Figure 3.2. Analysis of structure of 30 by Nuclear Overhauser Effect (NOE) leads to enhancement of the signals from CH3- group and the downfield aromatic proton signal. Whereas, the irradiation of the signal for the -CH3 of isopropyloxy group enhanced only the the –O-CHMe2 signal. So, these NOE data seemed to be
consistent with structure of product 30 rather then 27.
48
NH2 NH2 NH2 N3 h O OH O
NO2 NO2 NO2 NO2
NEt2 NEt2 NEt2 NEt2 25 28 29 30 4:4:92
Ac2O DMAP
AcHN NHAc O O
NO NO2 2 NEt NEt2 2 31 32
Scheme 3.6. Photolysis of azide 25 in 2-propanol.
AcHN O
NO2
NEt2 31
Figure 3.3. X-ray structure of acetanilide 31. 49
These spectroscopic data seemed to favor structure 30, it was absolutely necessary to prove the structure of the product unequivocally. The only method for such an unequivocal structure assignment is X-Ray analysis. All products were isolated from the reaction mixture as oils. In order to obtain the crystals for X-Ray analysis, the free amino group in the 2-propanol adduct was converted to an acetamide using acetic anhydride. The X-ray study of the resulting crystalline acetanilide 31 confirmed that the structure of the addition product was 4-(N,N- diethylamino)-2-isopropyloxy-3-nitroaniline 30 (Figure 3.3).
In order to evaluate the solvent dependence and products distribution upon the stereoelectronic environment of nucleophiles, series of photolysis experiments were conducted using different alcohols, phenols and dimethylamine hydrochloride.
1) Alcohols as Nucleophiles
Photolytic decomposition of the azide 25 in several of alcohol solvents gave the corresponding amines listed in Table 3.1 with details of the yield ratios for reduction versus substitution.
In the case of the simple aliphatic alcohols (a – d) the substitution product dominated the.
Thus the photolysis of azide in n-butanol as solvent led only to 5% yield of reduction product; whereas use of tert-butanol, a much larger and weaker nucleophile, leads to almost triple the amount of the reduction product. This is thought to be due to steric hindrance between t-butyl group and the amino and nitro groups of the starting azide.
Conducting this reaction in the presence of the esters of α-hydroxyaliphatic acids such as ethyl glycolate (e) and ethyl lactate (f) resulted in complete production of reduction product. 50
Then compounds bearing electron withdrawing groups attached to the α-position also bearing a hydroxy group makes hydrogen(s) readily abstracted in oxidation/reduction processes.
2) Phenols as Nucleophiles
Two phenols were tested in this serious of experiments, namely phenol and hydroquinone
(h and i). In both cases the irradiation was performed in the acetonitrile using large excess of the phenol (CM = 2 mole). Both experiments were consistent with our previous observation. The
strongly nucleophilic properties of the phenol resulted in ring substitution product in high yield
(98%). However, using a strong reducing agent like hydroquinone produced the reduction
product 28 quantitatively.
3) Dimethylamine Hydrochloride as Nucleophile
The experiment with the dimethylamine hydrochloride as the strongest proton source
used so far, lead predominent to the exclusive formation of the adduct 30 with little reduction.
To summarize the results of the above observations, there is a strong correlation between
the nucleophilic/basic properties of the attacking species and the final product ratio. The yields
of the substitution product are highest for the stronger nucleophiles, which surprisingly attacking
at the more hindered aromatic position. In the case of weaker nucleophiles, the acidity of protons
is determinative which leads to the formation of the reduction product. As indicated in Scheme
3.7, upon excitation, the starting azide losses a molecule of nitrogen, which produces a highly
reactive singlet nitrene, posessing strongly basic character. The rapid fast protonation of the nitrene leads to the much more stable quinodiimine or nitrenium ion. Assuming significant electron deficient nature of the nitrenium ion two possible routes for its transformation seems 51
reasonable. First, Michael-type nucleophilic attack on the one of the positions ortho to nitrenium nitrogen might produce substitution products 29 and 30. Another possibility is reduction, which would result aniline 28.
Experiment # Nucleophile Product(s)1,2
Reduction Substitution
a MeOH 10 (2) 90 (98)
b i-PrOH 5 (4) 95 (96)
c n-BuOH 5 (5) 95 (95)
d t-BuOH 14 86
e HOCH2CO2C2H5 100 0
f HOCH(CH3)CO2C2H5 100 (100) 0 (0)
g (CH3)2NH×HCl 10 (5) 90 (95)
h C6H5OH 4 (0) 96 (98)
i Hydroquinone 100 (100) 0 (0)
Table 3.1. Reduction to addition ratio for photoreaction of 25 with various nucleophiles.
1 Determined by mass spectrometry of photoproducts from laser transient studies.
2 Determined from preparative runs using a Rayonet Photochemical Reactor in parentheses.
52
Scheme 3.7. Possible mechanism for formation of addition products upon the photolysis of azide 25.
Further experiments were conducted in solvents that are not good proton sources. Thus, upon the photolysis in cyclohexane, the azide 25 undergoes dimerization and reduction where the dimerization product predomenates (Scheme 3.8). The corresponding azocompound 34 was obtained in 88 % yield. According to the literature, this product forms from the nitrene triplet state. This experiment is consistent with our previous data and suggestions.
53
Scheme 3.8 Products formed upon the photolysis of 25 in cyclohexane
Irradiation in acetonitrile afforded, in addition to dimer 34, the oxidized dimer 35 was
isolated in 15% (Figure 3.3).
PhH2C NH
NO2
NEt2 36
Figure 3.4. Products obtained upon irradiation in acetonitrile and toluene.
Irradiation in toluene afforded the three major products (reduced amine 28, 34 and the benzyl amine 36 in the ratio 32:20:48, respectively) (Figure 3.4). 54
Since, in general, the azide-derivatized biomolecules used in PAL/PCL studies are aryl azides in which the azide (nitrene) is in electronic communication with primary amino nitrogen or an ether oxygen; we have synthesized the additional two molecules shown in Figure 3.5.
N3 N3
NO O NO2 2 NH OEt 37 EtO
Figure 3.5. (4-Azido-2-nitrophenyl)carbamic acid ethyl ester (37) and 4-Ethoxy-3-nitrophenyl azide.
Even if the electron-donating capacity of the para-amino group is attenuated by amide formation such as in acetamide 37, the nitrenium ion formation and addition products still occur effectively as shown in Scheme 3.9. However, irradiation of 4-ethoxy-3-nitrophenyl azide in alcohols provided only reduced anilines.
N3 NH2 OCH(CH ) hν 3 2 48% HOCH(CH3)2 NO2 NO2
HNCO2Et HNCO2Et 37
Scheme 3.9. Photolysis of 37 in 2-propanol.
The presence of the nitro group is also an important component, since it makes the
resulting nitrenium ion much more susceptible to the Michael addition that ultimately produces
the cross-linking covalent bond, Scheme 3.7. McClelland’s work29 has shown that without this 55
type of Michael-facilitating group nitrenium ions tend to undergo hydrolysis to the quinone, which does not produce a cross-linking covalent bond, see Scheme 1.9.
3.3 Photophysical Studies
UV-VIS Steady State
The absorption spectra of azide 25 recorded in several solvents and normalized to unity at the visible absorption maximum are displayed in Figure 3.6. The UV/VIS absorption spectrum of azide 25 in alcohols consists of two broad structureless bands; the low energy band is centered at
450 nm, the intense higher energy band is located at about 280 nm.
2.0 Cyclohexane CH CN 3 CH OH CH OH 3 3 i-PrOH 1.5 C H 6 12 Absorbance (a.u.) ΔΑ
1.0
280 300 320 340 Wavelength (nm)
0.5
0.0 300 400 500 600 700 wavelength (nm)
Figure 3.6. UV-VIS absorption spectra of azide 25 in cyclohexane (blue solid line), acetonitrile (pink dashed line), methanol (purple dushdot line) and 2-propanol (green solid line).
Based on results of TD-B3LYP calculations, the broad absorption band at lower energy
-1 -1 with extinction coefficients of ~1390 M cm is assigned as S0 → S1 transition. According to 56
calculations this transition should be centered at λmax = 477 nm (f = 0.0042), and the S0 → S2
transition is centered at λmax = 353 nm, but it carries no oscillator strength (f = 0.0000). At the
same time, the RI-CC2 calculations locate the 352 nm transition (f = 0.0062), but do not show
the 477 nm band.
The absorption spectra in polar protic and aprotic solvent are similar. In non-polar
solvents such as cyclohexane, a blue shift (1000-2000 cm-1) of the spectrum is observed. Finally,
it should be noted that azide 25 did not exhibit any noticeable fluorescence in polar or non-polar
solvents (excitation 280-360 nm) (Figure 3.7).
1.0x106 350nm 360nm
8.0x105
6.0x105
5 Emission 4.0x10
2.0x105
0.0 400 450 500 550 600 650 wavelength, nm
Figure 3.7. Room temperature emission spectra of azide 25 in 2-propanol at three excitation wavelengths: at 350 nm (red), at 360 nm (black), at 380 nm (blue).
Ultrafast Transient and Nanosecond LPF Absorption Spectra of Azide 25 in 2-Propanol
Ultrafast photolysis of azide 25 in 2-propanol upon 350 nm excitation at time delays
starting from 100 fs results in the spectral changes presented in Figure 3.8. 57
Figure 3.8. Frames A-C: Transient absorption (ΔA) spectra of a 16 mM solution of azide 25 in 2-propanol shown for various delay times between the 350 nm pump and probe pulses. These data were provided by Dr. Tarnovsky’s group. Data in frame C represent smoothed spectra by adjacent-averaging (bandwidth, 3.5 nm). The solvent contribution to the ΔA spectra is minor at delay times equal to or longer than 100 fs, except for the 388.9-nm feature that corresponding to stimulated Raman scattering from 2-propanol and yields11 the instrument response function, 150 fs.
The corresponding figure consists of three frames with different time scales. Short time, from 0.1 to 1 ps, is presented on frame A. Intermediate times, 1-20 ps, and long times, 20ps-1.1ns, are shown on frames B and C, respectively. Flow cell was used in these experiments. The solution was flowed through a 0.5-mm cuvetts and excited with energy of 4 μJ-pulse-1 .
The transient absorption spectrum, recorded after 100 fs, indicates two bands: extensive broad
UV absorption centered at 350-400 nm and a bell-like visible absorption band centered at 580 58
nm (Figure 3.8 A). This transient absorption signal grows and in the same time undergoes a small blue shift to 560 nm at 200 fs. At 300 fs, transient absorption rapidly decays to the blue- shifted band at 541 nm, which in turn undergoes a further blue shift to a new band at 528 nm (τ =
1.76 ps). As time progresses from 1 to 6 ps (Figure 3.8 B), the shifting of visible band occurs to the blue region with the formation of a broad absorption spectrum centered at about 495 nm, and the entire ΔA spectrum decays. From 6 to 20 ps, the visible absorption continues to rise and sharpens to a peak at 470 nm that dominates the ΔA spectrum. Frame C displayed the changes from 20 ps to 1.1 ns. During this time, absorption slowly decays in the 350-400 nm and 525-700 nm ranges, with formation of a narrowed, long-lived band centered at 486 nm with a broad shoulder around 390 nm.22
Laser flash photolysis of azide 25, performed in 2-propanol upon 350 nm excitation at time delays starting from 12 ns, produces the transient ΔA absorption spectrum shown in Figure
3.9. The solution was flowed through a 1 cm flow cell and excited with 5 mJ pulse. This
spectrum contains an absorption narrow red-shifted band with the maximum around 486 nm transient which is similar to that produced at 50-1000 ps in the ultrafast experiment. The band
486 nm forms faster than the time resolution of the spectrometer and decays relatively slowly to yet another intermediate absorbing at 445 nm, producing a very clear isosbestic point at 460nm.
The 445 nm transient eventually decays to baseline in the μs domain. Kinetic traces for the decay
(490 nm) and the growth (440 nm) follow first-order kinetics and can be fit to an exponential function (Figure 3.10). The time constants of decay of intermediate at 486 nm and growth of intermediate at 440 nm are the same, within experimental error, τ1 = 557±8 ns and τ2 = 556±28
ns, respectively, 59
0.016 54ns 0.014 240ns 508ns 0.012 622ns 802ns 0.010 1002ns 1624ns 0.008 1852ns A
Δ 0.006
0.004
0.002
0.000
400 450 500 550 600 wavelength,nm
Figure 3.9. Nanosecond transient absorption (ΔA) spectra of azide 25 in 2-propanol, excited at 350 nm, with delay and growth times specified on the graph.
0.0150 0.0150
0.0125 0.0125
0.0100 0.0100 A A Δ Δ
0.0075 0.0075
0.0050 0.0050
0.0025 0.0025
0 200 400 600 800 1000 1200 1400 1600 1800 2000 0 200 400 600 800 1000 1200 1400 1600 1800 2000 time, ns time, ns
Figure 3.10. The kinetic traces of azide 25 recorded at a) 440 nm and b) 490 nm in 2-propanol along with one-exponential solid line fit superimposed with time constants of a) 556±28 ns and b) 567± 8 ns.
60
Figure 3.11 illustrates step by step a good-quality global fit using a sum of four
exponential functions and an offset (permanent background spectrum) for azide 25 in 2-propanol
excited at 350 nm. The validity of the fitting procedure is evident by comparison of the
reconstructed decay-associated spectra maxima with the maxima observed in the time-evolving
ΔA spectra (cf. Table 3.2, entry 7 vs. the maxima at 565, 539, 528, 469, and 486 nm in Figure
3.8).
Figure 3.11. Reconstructed decay-associated spectra (εi) extracted from the ΔA transient absorption spectra of azide 25 in 2-propanol irradiated with 350 nm laser pulses. The time constants (τi) obtained from a least-square global fit of the 512 ΔA kinetic traces to a sum of four exponential functions and a permanent offset are shown in the insets. The final frame shows the ns/μs decay of the long-lived 487 nm intermediate to a final 445 nm intermediate. The data were provided by Dr. Tarnovsky’s group. 61
62
An experiment exciting to higher-lying excited electronic state was achieved using 305- nm pump pulses. Transient absorption spectrum is presented on Figure 3.12. At the short-time
(less than 200 fs) ΔA spectra are dominated by the 350 nm absorption band. As in the previous cases (λexc=350 nm and 420 nm), after 100 fs transient absorption at 550 nm was recorded.
Figure 3.12. A, B, and C: The solution was flowed through a 0.2 mm flow cell and excited with a 305-nm, 3.8 μJ pulse. The solvent contribution to the ΔA spectra is minor at delay times ≥ 100 fs. For 305 nm excitation, the UV region (280-375 nm) of the ΔA spectra was measured using the probe light generated by TOPAS ([azide 25] = 1.7 mM, 3.1 μJ pump pulse) and subsequently scaled to the visible (360-665 nm) ΔA spectra measured using the white-light continuum probe. The inset compares the ΔA kinetic traces recorded at probe wavelengths of 350 and 465 nm. 63
The 350 nm band starts to decay at 1 ps and almost disappeared over 50 ps (time constant, 19 ps) with a 900 cm-1 blue shift. The 350 nm band and 550 nm band decay with different rate constants: the 350 nm band has decayed by 50%, when the 550 nm band vanished
completely, which indicates that these bands are not related to each other. The 550nm band decays to the species exhibiting the narrow 487 nm band with the 390 nm shoulder. It is noteworthy that the decay of the 350 nm band, with a time constant of 19 ps, correlates very well
with growth of the ~465-nm band (19 ps), producing an isosbestic point at 375 nm.
The photolysis upon 305 and 350 nm excitation produce similar spectra with exception
that the intensity of ΔA signals is much larger in the 350 nm spectrum. This indicates that 350
nm and 305 nm excitation leads to the formation of several species, which have similar
absorption spectra, but that 305 excitation produces mainly the more weakly absorbing
components of the 350 nm spectrum.
The aforementioned transient behavior was compared with that upon excitation at a
longer wavelength, 420 nm (Figure 3.13A and B). The solution was flowed through a 0.5 mm
flow cell and excited with a 420 nm, 3.5 μJ pulse. From 100 to 200 fs, ΔA spectra display bands
with maximum at 550 nm and 360-380 nm, which are absolutely identical to those observed
upon 350 nm excitation in their shape, signal amplitude, and decay constants. However, upon
350 nm excitation, the absorption spectra are much broader in this time range; that indicates a
larger excess of internal energy content of the contributing species, and possibly the formation of
new species. With 420 nm excitation, no discernible ΔA (positive ΔA) signal is observed beyond
20 ps. The vanishing of both bleach (negative ΔA) and transient absorption signals beyond 20 ps
indicates the quantitative reformation of the relaxed ground state of azide 25. Thus, a 420 nm
photon delivers photoexcitation into the lowest energy band of the absorption spectrum of azide 64
25, most likely the S0→S1 electronic transition, which does not give rise to either the 470 nm or
the long-lived 486-nm intermediates.
0.03 0.1 ps 0.2 ps 0.3 ps 0.02 A 0.5 ps 1 ps
A Δ 0.01
0.00
350 400 450 500 550 600 650 700 7501 ps 3 ps 0.004 B 6 ps 10 ps 20 ps 100 ps 0.002 A Δ
0.000
-0.002
400 500 600 700 wavelength, nm
Figure 3.13. Transient absorption (ΔA) spectra of 25 (1.2 mM) in 2-propanol for various delay times (in picoseconds, shown in the legends) between the probe and pump pulses. The data were provided by Dr. Tarnovsky’s group.
The 305 and 350 nm photoexcitation lead to a complicated spectral evolution, which
exhibits several kinetically and spectrally different features, namely, sub-ps, and picosecond
components, ~350-nm and 465 nm bands, and a ~490 nm product band. Lower quality fits are
obtained with a smaller number of time constants, while the increase of a number of time
constants leads to no further improvement of χ2 values. Only two time constants and a very small 65
permanent background spectrum are required for a satisfactory global fit of the spectrum generated by 420 nm excitation. The resulting time constants are summarized in Table 3.2, together with the maximum absorption wavelengths (λmax) of the resolved component spectra.
A detailed analysis of the formation of nitrenes that undergo aromatic ring expansion,
including ortho- and para-biphenylyl azide and 1-naphthyl azide, has recently been reported.23
The absorption maxima of these singlet nitrenes are observed at 345 nm, 410 nm, and 397 nm, respectively. Comparing the transient spectra observed in this work with these literature data, we note that the initial absorption spectrum, presumably that of the singlet excited state of 25, occurs at significantly longer wavelength. Thus, in the case of 25 in 2-propanol, an initial broad absorption band centered at 562 nm (τ = 333 fs, Table 3.2, entry 7 and Figure 3.12 A) is
observed in contrast with the previously observed singlet excited state of para-biphenylyl azide,
which has an absorption maximum at 480 nm (τ ~ 100 fs).23 In general, the lifetimes of aryl
azide excited singlet states fall is between 100 and 600 fs. Consequently, the 333 fs lifetime of
the 562 nm species observed in this system is consistent with the singlet excited state of the azide
25. However, this absorption is assigned to the lowest excited singlet state of azide 25 (S1), which does not afford the nitrene (vide infra and Table 3.2, entry 10).
Reactivity of Azide 25 in the Presence of Different Solvents
Ultrafast pump-probe and nanosecond LFP were performed for azide 25 in the presence of variuos nucleophiles, such as alcohols, phenol, ethyl glycolate, and dimethylamine
hydrochloride. The measurements of ultrafast transient absorption of azide upon 350 nm
excitation at time delays starting from 100 fs were done. The spectrum all are identical with
different kinetic trace, which is presented in Table 3.2. 66
Nanosecond Laser Flash Photolysis. The transient absorption of azide 25 in other alcohols produces spectra, similar to those in 2-propanol. Surprisingly, no transient absorbtion has been observed for MeOH in nanosecond experiment. This might be explained due to short lifetimes of corresponding intermediates.
The transient spectrum of nanosecond experiment of 25 in ethanol is presented on Figure
3.14a. The transient absorption, recorded immediately after the flash, generates two bands (486 nm and 440 nm) at the same time. The life time of the intermediate at 490 nm is much shorter in ethanol than in 2-propanol and this band decays for 305 ns. The intensity of 440 nm band rises for 1.3 μs, than starts decaying. The dynamics of the process can be modeled by a one- exponential fit, illustrated on Figure 3.14b.
0.005 220ns (a) 368ns 0.004 (b) 0.004 676ns 1350ns 1804ns 0.003 0.003 A
Δ ΔΑ 0.002 0.002
0.001 0.001
0.000 0.000 400 425 450 475 500 525 0 100 200 300 400 500 600 wavelength, nm time, ns
Figure 3.14. (a) Nanosecond transient absorption (ΔA) spectra of azide 25 in ethanol, excited at 350 nm, with delay and growth times specified on the graph;(b) The kinetic trace of azide 25 recorded 490 nm in ethanol along with one-exponential solid line fit superimposed with time constants of 305±32 ns.
67
0.005
94ns 0.004 362ns 528ns 862ns 0.003 1804ns
ΔΑ 0.002
0.001
0.000 400 450 500 550 600 wavelength, nm
Figure 3.15. Nanosecond transient absorption (ΔA) spectra of azide 25 in n-butanol, excited at 350 nm, with delay and growth times specified on the graph.
0.006 0.005
(a) (b) 0.005 0.004
0.004 0.003
0.003 0.002 A
Δ A Δ 0.002 0.001
0.001 0.000
0.000 -0.001 0 500 1000 1500 2000 0 250 500 750 1000 1250 1500 time, ns time, ns
Figure 3.16. The kinetic traces of azide 25 recorded at (a) 490 nm and (b) 440 nm in n-butanol along with one-exponential solid line fit superimposed with time constants of a) 325±13 ns and b) 567± 8 ns.
68
Transient Absorption spectrum of 25 in n-butanol, presented on Figure 3.15, produced
490 nm band over 94 ns, which decays with a rate constant (3.07±0.07)x107 (τ=325±14 ns); the decay of this species is accompanied by growth of 440 nm band over 567 ns, which shows no decay after 1.8 μs.
Figure 3.17 shows the transient absorption of azide 25 in solution of 2M phenol in acetonitrile. Three bands with maximums at 380 nm, 440 nm and 490 nm are detected at 170 ns.
A strong sharp band at 380 nm corresponds to aromatic ring of phenol. The decay of 490 nm band is accomplished with the rises of absorption at 440 nm. This process is much slower than in case of alcohols. The band 440 nm continues to rise at ~4μs, after that very slow decay occurs.
The kinetic trace of 490 nm (Figure 3.18a) was fitted to single exponential with the time constant
1.39 ± 0.018 μs. The kinetic trace for 440 nm (Figure 3.18b) was fitted with a biexponentional with time constants of 323±6.9ns, and 2 ± 0.1 μs.
This general pattern is repeated for saturated acetonitrile solution of dimethylamino hydrochloride (Figure 3.19, 3.20). The clear isosbetic point occurs on the transient absorption spectra of azide in diethyl malonate (Figure 3.21).
69
0.007 170ns 280ns 0.006 460ns 1390ns 0.005 2200ns 3330ns 0.004 4090ns 5870ns A
Δ 0.003
0.002
0.001
0.000 400 450 500 550 600 wavelength,nm
Figure 3.17. Nanosecond transient absorption (ΔA) spectra of 25 in 2M solution of phenol in acetonitrile, excited at 350 nm, with delay and growth times specified on the graph.
0.006 0.004 (a) 0.005 (b)
0.004 0.003 0.003
0.002 A A
Δ Δ
0.001 0.002
0.000
-0.001
-0.002 0.001 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 0 1x103 2x103 3x103 4x103 5x103 6x103 time, ns time, ns
Figure 3.18. The kinetic traces of 25 recorded at (a) 490 nm along with one-exponential solid line fit superimposed with time constants of 1.39 ± 0.018 μs and (b) 440 nm in 2M phenol in acetonitrile with time constants of 2 ± 0.1 μs. 70
0.0100
130ns 500ns 0.0075 780ns 1520ns 2500ns 3500ns 4000ns
A 0.0050
Δ 5740ns
0.0025
0.0000 400 450 500 550 600 wavelength,nm
Figure 3.19. Nanosecond transient absorption (ΔA) spectra of 25 in saturated solution of dimethyl amine hydrochloride in acetonitrile, excited at 350 nm, with delay and growth times specified on the graph.
0.008 0.010 (a) 0.007 0.009 (b) 0.008 0.006 0.007 0.005 0.006
0.004 A 0.005 A Δ
Δ 0.003 0.004 0.003 0.002 0.002 0.001 0.001 0.000 0.000 0.0 2.0x103 4.0x103 6.0x103 8.0x103 1.0x104 0.0 2.0x103 4.0x103 6.0x103 8.0x103 1.0x104 time, ns time, ns
Figure 3.20. The kinetic traces of azide 25 recorded at (a) 490 nm and( b) 440 nm in saturated solution of dimethyl amine hydrochloride in acetonitrile along with one-exponential solid line fit superimposed with time constants of (a) 11.6±0.13 μs and (b) 5.65± 0.8 μs. 71
0.008 140ns 0.007 290ns 980ns 0.006 2090ns 4330ns 0.005 7510ns 9060ns 0.004
ΔΑ 0.003
0.002
0.001
0.000
-0.001 400 450 500 550 600 wavelength, nm
Figure 3.21. Nanosecond transient absorption (ΔA) spectra of azide 25 in malonate ester, excited at 350 nm, with delay and growth times specified on the graph.
Measurements of the transient absorption of the azide 25 in acetonitrile solution of 2 M
hydroquinone (Figure 3.22) and ethyl glycolate (Figure 3.23) displayed just the 490 nm band, no
440 nm band was detected. Figure 3.23 b presents the decay of the 490 nm band in ethyl
glucolate. The intermediate is also very long-live and does not decay to the baseline after 1.8 μs.
The LPF experiments in nonpolar solvents; such is benzene, tetrachloromethane, do not detected
490 nm and 440 nm bands.
72
0.014 40ns 120ns 0.012 382ns 608ns 1050ns 0.010 1550ns 1872ns 0.008 A
Δ 0.006
0.004
0.002
0.000 400 450 500 550 600 wavelength, nm
Figure 3.22. Nanosecond transient absorption (ΔA) spectra of azide 25 in a 2M solution of hydroquinone in acetonitrile, excited at 350 nm, with delay times specified on the graph.
0.016 68 ns 0.020 0.014 (a) 288 ns (b) 522 ns 876 ns 0.012 1858 ns 0.015 0.010 A Δ 0.008
A
Δ 0.010 0.006
0.004
0.005 0.002
0.000 350 400 450 500 550 600 0 200 400 600 800 1000 1200 1400 1600 1800 wavelength, nm time, ns
Figure 3.23. (a) Nanosecond transient absorption (ΔA) spectra of azide 25 in ethyl glucolate, excited at 350 nm, with delay and growth times specified on the graph; (b) The kinetic traces of azide 25 recorded at 490 nm in ethyl glycolate along with the bi-exponential solid line fit superimposed with time constants of 49 ns and 1191 ns. 73
Ultrafast Pump-probe Experiments. The full range of transients is observed when the azide 25 is irradiated at 350 nm in acetonitrile (Table 3.2, entry 13), but irradiation at 420 nm in acetonitrile leads only to the initial transients similar to those observed at this wavelength in 2- propanol (Table 3.2, entry 10).
0.010 t1ps t0.8ps t0.5ps t0.3ps t0.2ps 0.005
t0.1ps A
Δ t0ps tM0.1ps tM0.2ps 0.000
0.006350 400 450 500 550 600 650 t100ps t50ps 0.004 t20ps t10ps t6ps
t3ps A Δ 0.002 t2ps t1ps
0.000 350 400 450 500 550 600 650
t100ps 0.003 t200ps t500ps 0.002 t800ps
A t1ns Δ 0.001
0.000 350 400 450 500 550 600 650 wavelength, nm
Figure 3.24. Transient absorption (ΔA) spectra of azide 25 in acetonitrile upon 350 nm excitation.
Ultrafast Pump-probe Experiments in Aprotic Solvents. In non-acidic solvents, such as
toluene (Table 3.2, entry 18), the sub-ps absorption is very broad extending beyond 700 nm. The
ensuing broad transients occur at 514, 543, and 492 nm. The latter 492 nm transient persists
beyond a time delay of 1 ns (Figure 3.25a), and the longer-lived 465, 486, and 445 nm bands,
usually observed, never develop (Table 2, entry 18, and Figure 4D-F). 74
Figure 3.25. (a) Transient absorption (ΔA) spectra of azide 25 in toluene upon 350 nm excitation. The solution (16 mM) was circulated through a 0.2-mm pathlength flow cell; (b) Transient absorption (ΔA) spectra of azide 25 in cyclohexane upon 350 nm excitation. The solution (16 mM) was circulated through a 0.2-mm pathlength flow cell.
Nanosecond Experiments in the Presence of Aminoacids. Figure 3.26 displays the
nanosecond transient absorption spectrum of azide 25 in solution of glycine in 2-propanol. The
presence of amino acid in 2-propanol increases the rate of decay of 490 nm band. The kinetic
trace of 490 nm was fitted as single exponential with the time constant 236 ± 0.08 ns (Figure
3.27). However, the band, that observed at 425 nm, grows slowly compare to the decay of the
440 nm band in pure 2-propanol.
75
0.006 68ns 0.005 114ns 134ns 280ns 0.004 428ns 728ns 0.003 962ns
A 1416ns
Δ 0.002
0.001
0.000
-0.001 400 450 500 550 wavelength,nm
Figure 3.26. Nanosecond transient absorption (ΔA) spectra of azide 25 in saturated solution of glycine, excited at 350 nm, with delay and growth times specified on the graph.
0.003 0.008 (a) 0.007 (b) 0.006
0.005 0.002 0.004
0.003 A
ΔΑ Δ 0.002 0.001 0.001
0.000
-0.001
0.000 -0.002 0 500 1000 1500 0 500 1000 1500 2000 time, ns time,ns
Figure 3.27. Kinetic traces of azide 25 recorded at a) 420 nm and b) 490 nm in saturated solution of glycine in 2-propanol along with a) biexponential solid line fit superimposed with time constants of 22.66±1.89 ns, and 1.28±0.2μs; b) one-exponential solid line fit superimposed with time constant of 236+6.9 ns.
76
3.4 Discussion
Theoretical Considerations
Active Excited State and Nitrene Formation. Detailed calculations have been conducted on the azide 25 and its associated nitrene. The first question to be addressed was that of the excited state behavior of the azide 25 in an effort to determine which of the several low-lying excited states would be most likely to produce nitrogen extrusion (Figure 3.28). The lowest excited state,
S1, is (π,π*) with a shift of electron density from the aromatic ring and the o-amino group to the
nitro group at the TD-B3LYP level of theory, λmax = 477 nm. On the other hand, the second
excited state, S2, is (π,π*in-plane) is localized on the azide group with a significant loss of electron
density between the first and second nitrogen atoms of the azide moiety (TD-B3LYP), λmax =
353 nm (f = 0.0000), and suggests subsequent elongation of the –N—N2 bond.
Figure 3.28 Electron density redistribution in the S1 and S2 states of 25 calculated at the TD- B3LYP/TZVP level of theory. The green contours depict the accumulation of electron density in the excited state, and the red contours illustrate the loss of electron density from the S0 ground state. The contour values are ±0.005 a.u.
77
Furthermore, TD-B3LYP optimization of the S0, S1, and S2 states of the N,N-dimethyl analog of 25 indicates that the –N—N2 bond lengthens appropriately for the loss of nitrogen and
formation of the nitrene in the S2 excited state, but not in the S1 excited state, Figure 3.29. These
correlations clearly indicate that S1 is not an effective precursor state for loss of nitrogen and
formation of the nitrene. Consequently, S2 or higher excited states, but not S1, seem to be ideal
candidates for the extrusion of nitrogen and formation of the nitrene. These calculated energy relationships correlate very nicely with the observed transient behavior of 25. Thus, irradiation of
25 at 420 nm might selectively populate S1 but not S2 (calculated λ(S0→S1) = 477 nm, calculated
λ(S0→S2) = 353 nm).
S1 optimization S0 optimization S2 optimization
173˚ 171˚ 146˚ 1.126 1.131 1.143
1.241 1.229 1.316 1.386 1.417 1.365
1.438 1.471 1.449
S2 84.4 S2 81.8 S 63.7 1 S2 55.5 S 41.3 1 S1 53.9
S 28.6 0 S0 22.9
S0 0.0 Adiabatic Vertical Excitation Adiabatic
Figure 3.29. Excited state energies (kcal/mol) and bond lengths for the optimized geometries (TD-B3LYP) of the ground state, and the first and second excited states of the N,N-dimethyl analog of azide 25.
78
The transient cascades outlined in Table 3.2 indicate that irradiation at 420 nm (entry 10) leads to a broad absorption ca. 559 nm that forms within 400 fs and returns to baseline a few ps later. In contrast, irradiation with 350 nm light (entry 7), which should populate S2 or higher excited states, leads to the complete cascade of transients out into the μs domain. The simplest interpretation of these observations is that 420 nm excitation populates S1 and that this intensely absorbing species returns to the ground state, S0, without loss of molecular nitrogen or nitrene
formation. In contrast, 350 nm excitation populates both S1 and S2, since similar initial transient
spectra are observed, but the transient cascade extends beyond the decay of S1 with a stream of
intermediates arising from the loss of nitrogen and nitrene formation. In this interpretation, the
source of the spectra in Figure 3.11A and B are S1 and S2 or higher. Both of these excited states
may de-energize to S0 within 2 ps, but S2 also extrudes N2 within this same time frame leading to formation of the nitrene which is the source of the spectra in Figure 3.11C-F. The comparison of the τ1 entries in Table 2 for 420 nm and 350 nm excitation shows that the short-time constants are not affected; therefore, it seems likely that the S2 lifetime is shorter than the S1 lifetime.
Nitrene Electronic Configuration
The electronic configuration of the nitrene derived from azide 25 becomes quite
important in interpreting the chemistry of this species. This nitrene might exist in any of three
electronic configurations: the triplet, the open-shell singlet, or the closed-shell singlet.
Considering the time frame of the chemistry occurring in the aforementioned transient section
(Figure 3.11A-C), the triplet configuration can be excluded, since singlet aryl nitrenes usually
require about 100 ps to 10 ns to reach singlet-triplet equilibrium,28,30 and in the systems studied 79
here, the singlet nitrene (Figure 3.11C) proceeds to the next intermediate (Figure 3.11D) in < 20 ps.
Therefore, the remaining questions are whether the configuration of the ground state singlet nitrene is closed-shell or open-shell, and which of these is the reactive intermediate in the chemistry to follow. Extensive CASSCF calculations using large electron/orbital sets
(CASSCF(10,8)/6-311+G(3df,3pd)//CASSCF(4,4)/6-31G(d)) (Figure 3.30), indicate that the open-shell (OS) singlet is about 6.6 kcal/mol lower in energy than the closed-shell (CS) singlet, and that ΔECS-OS is definitely <10 kcal/mol. Therefore, one can say with a fairly high degree of
certainty that the open-shell singlet is the ground singlet state of the nitrene.
However, both ultrafast femto/picosecond and conventional nanosecond spectroscopy, as
well as the products observed in the photochemistry of azide 25 seem to be consistent with the
involvement of a closed-shell nitrene intermediate. Indeed, open-shell singlet states are typically
thought to engage in single-electron, radical chemistry; while closed-shell singlet states are
thought to engage in two-electron, acid/base or nucleophile/electrophile chemistry. In the
photochemistry of 25, the amine 28 (Scheme 3.4) might conceivably arise via a radical process,
but the substitution products 29 and 30 would be more expeditiously formed via two-electron, closed-shell nitrene chemistry, vide infra.31 Furthermore, the transient species observed in
toluene, conditions under which the triplet nitrene and associated radical intermediates should be
formed, are distinctly different from those formed under the conditions of the reaction in question (compare Figure 3.13 and 3.11 with Figure 3.25a). Finally, the calculated C=N bond length for the S2 excited state of the azide 25 is 1.365 Å (Figure 3.29), which is significantly
closer to the calculated C=N bond length for the closed-shell nitrene, 1.364 Å, than for the open-
shell nitrene, 1.447 Å. 80
Thus, theoretical calculations and experimental evidence can be reconciled if the Franck-
Condon excited state of the nitrene leads to the closed-shell nitrene. Since interconversion between the closed- and open-shell singlet nitrene states is forbidden, and thus, a relatively slow process, relative rates of reaction, not singlet nitrene energies, might well determine the course of subsequent reactions.
In support of this relative rate scenario, there is a straightforward correlation between singlet-triplet intersystem crossing (ISC) and closed-to-open-shell nitrene interconversion
(COSI). An example of which is shown in Figure 3.31A for the carbonyl (1n,π*)−(3π,π*) ISC. In
this well understood system, ISC (effective rate < 109 s-1), only occurs rapidly if the change in
electron spin angular momentum is balanced by a corresponding and opposite change in orbital
angular momentum. Thus, the spin flip of the electron is coupled with the redistribution of
electron density between orthogonal molecular orbitals. If orthogonal orbitals of similar energy
are not available for the conservation of angular momentum, then the higher energy singlet
excited state will not readily undergo ISC to the lower energy triplet state, and the singlet state
can persist for hundreds of nanoseconds. This is the case with the singlet (π,π*) excited states of
aromatic hydrocarbons.32 In Figure 3.31B, the relationships between open- and closed-shell
singlet and triplet nitrene states are shown. Thus, interconversion between open- and closed-
shell singlet states (OCSI) is expected to be highly forbidden, since the change in orbital angular
momentum cannot be balanced by a corresponding change in electron spin angular momentum.
Likewise, the ISC of the open-shell singlet to the corresponding triplet also should be highly forbidden, since the change in electron spin angular momentum cannot be balanced by a change in orbital angular momentum. 81
Figure 3.30. (A) Closed-Shell Nitrene optimized at CASSCF(10,10)/pVDZ level of theory using MOLCAS suite of programs (B) Opened-Shell Nitrene optimized at CASSCF(10,10)/pVDZ level of theory using MOLCAS suite of programs. These results were obtained from Dr. Hadad’s group. 82
However, the closed-shell singlet nitrene should readily undergo ISC to the triplet nitrene, since this is a spin-orbit allowed process.33 Therefore, even though the closed-shell
singlet nitrene may be at higher energy than the open-shell singlet nitrene, it can play a
significant role in the chemistry of 25, if it is populated directly upon loss of molecular nitrogen
from the singlet excited state of the azide 25, and if it reacts more rapidly than it undergoes
interconversion to the open-shell singlet or ISC to a triplet state.
A Spin-orbit Allowed C O C O ISC
n,π* singlet π,π* triplet
B Spin-orbit Forbidden N N OCSI
Opened-shell Singlet Closed-shell Singlet Triplet Spin-orbit ISC Spin-orbit ISC Forbidden Allowed
N
Figure 3.31. (A): conserved angular momentum in intersystem crossing (ISC) of carbonyl group. (B): Relationships between ISC and open- to closed-shell singlet nitrene interconversion (OCSI).
83
Nitrene formation. On the basis of these calculations and the experimental data, nitrene formation arises from the second, S2, or higher excited singlet states of azide 25. The S1 singlet state decays back to the ground state without giving rise to any transients that survives longer than a few picoseconds (Figure 3.32 A, Table 3.2 for excitation at 420 nm in 2-propanol, entry
10, and in cyclohexane, entry 16). The source of the spectra in time frames A and B (Table 3.2 and Figure 3.11) is thought to be largely due to the lowest excited singlet S1 state of azide 25 and
the vibrationally hot states of the S0 state formed upon internal conversion of S1. In contrast,
irradiation at 350 and 305 nm leads to population of S1 and higher energy excited states (S1 + S2
+ …), and a cascade of nitrene-derived intermediates extending into much longer time domains
(Figure 3.32, B).
Figure 3.32. Energy selective photochemistry of 4-(N,N-diethylamino)-3-nitrophenyl azide.
For example, excitation of 25 in 2-propanol at 305 nm (Table 3.2, entry 8 and Figure
3.13) leads to a new absorption at 531 nm and intense absorption <350 nm both of which decay
over about 20 ps to a species absorbing at 460 nm that in turn decays over about 833 ps to a
species absorbing at 484 nm. Nanosecond transient absorption spectroscopy shows that this 484
nm species slowly decays (t = 557 ns) to the final species observed that absorbs at 445 nm. In 84
this experiment and others using an excitation wavelength of 350 nm or shorter, S1 is populated along with higher singlet excited states, and the strongly absorbing S1 and associated
vibrationally hot ground states of azide 25 tend to mask the formation of the hot nitrene from
higher excited states to the extent that the singlet nitrene only becomes visible after S1 and hot
ground states have decayed significantly. This occurs after ca. 2-6 ps, Frame C in Table 3.2, and
Figure 3.13 and 3.11 judging from the spectral shift, narrowing and decay of the visible band.
As soon as these processes are complete, the UV kinetic traces (360-380 nm region), after an
appropriate scaling, are super imposable out to about 25 ps, indicating that the spectral range in
this time domain is dominated by the population decay, and not by cooling. By this time, a new
band in the 460-480 nm region is already well developed (Figure 3.12B). Using this time as our
starting point, it is noted that a species absorbing strongly at <380 nm seem to be the source of
the 460 nm species. This <380 nm species is assigned to the closed-shell nitrene (Figure 3.33 and
Table 3.3). Excitation of 25 at 350 nm in cyclohexane (Table 3.2, entry 15) produces the same
singlet nitrene that decays to the triplet nitrene (t = 80 ps to λmax = 452 nm). When the proton
source phenol is added to cyclohexane (Table 3.2, entry 17, also see Figure 3.18), a very
conspicuous new transient is rapidly formed in the 452–480 nm region over ca. 18 ps, and
survives for several hundred nanoseconds. These two experiments indicate that two processes
compete for the closed-shell nitrene, and that both of these processes yield species absorbing in
the 452-480 nm region. One of these is triplet nitrene formation and the other is a proton-
dependent process, probably nitrenium ion formation (Table 3.3).
The aforementioned fs-ps spectroscopic observations are correlated by the reaction
scheme outlined in Figure 3.33. Clearly the lowest excited singlet state of the azide 25 does not
lead to nitrene formation, but higher (π,π∗) excited states localized mainly on the azide unit, 85
particularly the S2 state, do afford the nitrene. Since similar processes in carbene chemistry have been shown to produce both ground and excited carbene states,30b,34 it is quite possible that the
excited closed-shell nitrene is formed initially upon loss of molecular nitrogen in this system. As
indicated in the previous discussion, the closed- and open-shell singlet nitrenes should not be
readily interconverted without some type of second-order vibronic coupling that is thought to be
Figure 3.33. Possible reaction pathways connecting excited states of azide 25, nitrene states, and nitrenium ion 33 states. The vertical energy axis is not drawn to scale.
86
weak at best.33 Therefore, an initially formed closed-shell nitrene might survive long enough to
initiate chemistry unique to its electronic configuration. In fact, the lifetime of this closed-shell
nitrene may be governed by ISC to the triplet nitrene, which is a spin-orbit allowed process
rather than by decay to the open-shell singlet nitrene, which is a spin-orbit forbidden process.30b,
33 Finally, one plausible reaction pathway of the closed-shell nitrene might be protonation, which
correlates with the ground state of the nitrenium ion 33; while protonation of the open-shell
nitrene correlates with the excited state of 33, that is estimated to be approximately 87 kcal/mol
above its ground state. In this scenario, the nitrenium ion 33 would be the pivotal reactive
intermediate leading to the substitution products 29 and 30, Scheme 3.7. Many alternative
scenarios have been considered for the nitrene reaction cascade. One example is shown in Figure
3.34 in which two scenarios are compared: Scenario I in which the closed-shell singlet nitrene
initially undergoes protonation, and Scenario II in which the closed-shell singlet initially
undergoes conversion to the opened-shell singlet nitrene before it becomes protonated. Even
though the calculated spectra are in nearly perfect agreement with the observed spectra in
scenario II, the protonation step occurs in the range of 20 ps not 560 ns. Therefore, scenario I is
the more viable alternative. 87
t = 833 ps Triplet Amine Experimental 450 nm Scenario I Calculated 426 nm (f = 0.0828) H t = 560 ns Nitrenium Nucleophile = 20.5 ps = 20.5 Aromatized t Ion Adduct Adducts 484 nm 445 nm 441 nm (f = 0.0019) CS Singlet < 350 nm t = 560 ns t = ??? μs & 531 nm OS Singlet Nitrenium Nucleophile Aromatized Ion (340 nm 484 nm Adduct Adducts H 445 nm (f = 0.0065) 483 nm (f = 0.0323) 441 nm (f = 0.0019) Scenario II t = 833 ps = 20.5 ps= 20.5
t Triplet Amine 450 nm 426 nm (f = 0.0828)
Figure 3.34. Alternative scenarios for closed-shell singlet nitrene reaction.
In the model outlined in Figure 3.33, the closed-shell nitrene (Figure 3.11, frame C) undergoes nitrene protonation in competition with ISC to the triplet nitrene. If the triplet nitrene is generated in this cascade of intermediates, then one must take into consideration hydrogen abstraction to form the corresponding nitrogen radical, which is a reasonable precursor for the amine 28. TD-B3LYP calculations indicate that these possible intermediates all absorb in the
420-480 nm region as indicated in Table 3.3. Clearly, a combination of triplet nitrene, nitrogen radical, and/or nitrenium ion 33 might give rise to the transient spectrum observed in Figure
3.11, frame D, which is a broad absorption band centered at 465 nm. This band narrows and shifts to longer wavelength, 486 nm, upon going from frame D to E in Figure 3.11. 88
Species λmax (nm) Oscillator Strength (f)
Closed-shell singlet nitrene 340 0.0065
Open-shell singlet nitrene 483 0.0323
Triplet nitrene 426 0.0828
Nitrogen radical 447 0.0978
Nitrenium ion 10 441 0.0019
Adduct 13 360 0.0443
Adduct 14 409 0.0524
Table 3.3. Vertical transitions and absorption properties of possible reactive species derived from the irradiation of azide 25 as calculated at the TD-B3LYP/TZVP level of theory.
The band in frame E is also shifted to the red by polar solvents (compare λmax = 484-501
nm in polar solvents, Table 3.2, frame E, entries 1-9, 11, 12, and 14 with cyclohexane λmax = 466 nm, entry 17). It is interesting to note that the calculated oscillator strengths of the triplet and radical are about 50 times larger than that calculated for the nitrenium ion 33 in Table 3.3.
Consequently, any mixture of (triplet + radical)/nitrenium ion 33 of ca. 2/98 would give overlapping signals of approximately equal intensities as observed in frame D of Figure 3.11.
While it may be a coincidence, this is about the same ratio that is observed for product formation, and amine 28/nitrenium ion adducts 29 and 30 = 5-2/95-98. Apparently in neat protic
-1 solvents, the rate constant for nitrene singlet-triplet ISC (τISC ) is significantly slower than that
-1 for protonation (τH = kH). This is also indicated in Table 3.2, entry 15 in cyclohexane, where
protonation is not possible, τ3 = τISC = ca. 80 ps, and entry 17 in cyclohexane/phenol where
τ3 = τISC+H = ca. 18 ps. Similar time constants of 60-100 ps. have been observed for singlet-triplet 89
ISC in p-amino-substituted aryl nitrenes.35 Apparently the short wavelength component of the
465 nm band in Figure 3.11, frame D is due to small amounts of the triplet nitrene. In support of this assignment, the amine radical was obtained by irradiation of azide 25 in toluene, Table 3.2, entry 18 and Figure 3.25a. Under these conditions, a broad band at 543 nm persists and slowly shifts to an intense and broad band centered at 492 nm over ca. 315 ps. In addition, the
benzylamine corresponding to benzylation of the nitrene nitrogen was isolated as the main
product of this reaction in toluene. Therefore, the source of this 492 nm band is most reasonably
attributed to the nitrogen radical. These considerations indicate that ISC to the triplet and hydrogen atom abstraction to form the nitrogen radical are relatively slow processes compared to
the process shown in Figure 3.11 frames C to D, and that the nitrene triplet would be expected to absorb on the short wavelength shoulder of the nitrenium ion absorption band (Table 3.3).
Finally, additional confirmation of the presence of the nitrene triplet in these reactions has been obtained via the isolation of the typical triplet azo-dimer 34, 28 as well as the unusual oxidized
alternative dimer 35, Scheme 4, from reactions in non-alcoholic solvents such as acetonitrile.
Therefore, the observed rate constant coupling frames C and D is the sum of the rate constants
for the two branches of singlet nitrene reactions, kISC + kH, shown in Figure 3.33. However, so
long as the singlet-triplet ISC branch develops much more slowly, this composite rate constant
will largely reflect the much faster rate of the protonation branch.
A factor that might influence the observed rates of protonation is the initial formation of a
vibrationally hot singlet nitrene, Figure 3.11, frame C, which subsequently undergoes a slight
blue shift of its spectrum as it cools.36 For example, the nitrene derived from para-biphenylyl
azide in acetonitrile has a cooling time constant of 11 ps,37a which is coincident with the time
constants for the intermediates involved in Figure 3.11 frame C to frame D. Consequently, it 90
seems highly likely that the protonations observed in this work involve vibrationally hot singlet nitrenes to a significant extent, which may help to account for the rapidity of these protonation reactions.
The identity of the intense band at 486 nm in both frames E and F of Figure 3.11 is of central importance in determining the pivotal intermediates in this chemistry. The source of this intermediate is thought to be the nitrenium ion 33 arising from protonation of the nitrene, and calculated to have λmax = 441 nm, Table 3.3. In an effort to evaluate this possibility, the effect of
deuterium on the rates of the nitrene protonation step with methanol-d and 2-propanol-d was
investigated. These nitrene reactions display little, if any, deuterium kinetic isotope effect (KIE)
within experimental error. Thus, kH/kD = 1.4 for methanol (Table 3.2, entries 1 and 2), and kH/kD
= 0.9 for 2-propanol (Table 3.2, entries 7 and 9). Similar small KIE’s ranging from 1.0 to 1.7 are observed for alcohol protonation of arylcarbenes.34d, 38a, b Kirmse and coworkers have discussed
the lack of deuterium isotope effects for the reactions of a variety of carbenes with alcohols, and
note that it can be attributed to an ‘early’ transition state with little proton transfer from ROH to
the carbene, and, correspondingly, little charge development.38c This notion is consistent with the
involvement of vibrationally hot species for which an earlier transition state might be expected.
Figure 3.35. Two regions of space-surrounding the closed-shell singlet nitrene nitrogen and alcohol approach geometries leading to protonation transition states. 91
Variations on these suggestions have been explored from a theoretical perspective for the case of the closed-shell singlet nitrene derived from 25 at the B3LYP/6-31G* level as outlined in
Figure 3.35. While the possibility exists that hydrogen bonding between alcoholic solvents and the azide nitrogen occurs prior to photochemical expulsion of molecular nitrogen, no such stable complex could be observed either experimentally (IR) or theoretically. The question also arises as to the influence of the departing nitrogen molecule on the reactivity of the incipient nitrene.
Since the nitrene is formed in a vibrationally hot state, it is assumed that the expelled molecular nitrogen rapidly leaves the immediate vicinity of the nitrene nitrogen, and thus, has little, if any, influence on the reactivity of the nitrene. Furthermore, since nitrenes are only attached to a single substituent, they extend further into solvent space than do carbenes, which are attached to two shielding substituents. As a result, they are sterically more available to solvent molecules and more flexible to geometric modifications during the course of reactions.
Theoretical analyses of the reactions of this nitrene with alcohol have located two transition states for nitrene protonation, and these are described in Table 3.4. These protonation trajectories have small activation energies, ca. ΔE‡ = 4.81−4.43 kcal/mol, and are quite
exothermic reactions with ΔH = ca. −40 kcal/mol. In addition, they both have transition states
that occur fairly early along the reaction coordinate with the RO−H bond being only ca. 28%
broken. Therefore, we assign the 486 nm transient in frame E and F of Figure 3.11 to the
nitrenium ion 33 that results from simple nitrene protonation. The small or lack of a KIE in this
protonation process would seem to be due to a combination of factors including the availability
of the nitrene nitrogen in solvent space, the early and low activation energy barriers for singlet
nitrene protonation, and, at least in the case of methanol, the very rapid protonation of a
vibrationally hot nitrene shortly after its formation. 92
93
Transition ΔH νι A O-H Bond N-H Bond State for Distance (A) Distance (A) (kcal/mol) Protonation
A 4.81 i 1119.48 56.81 1.22 1.21
B 4.43 i 1107.93 41.78 1.22 1.21
Table 3.4. Transition state properties for proton abstraction of closed-shell singlet nitrene from methanol. The geometries of these transition states are shown in Figure 3.35.
In alcohol solvents, nitrene protonation occurs over a range of τ3 = 5−23.4 ps with the most rapid being protonation by methanol and the slowest being protonation by n-octanol. The same general dependence on alcohol structure as has been observed in previous work with carbenes in which the most rapid protonation occurs in methanol with other primary, secondary, and tertiary alcohols protonating at slower rates that are similar to each other.38a,b The
protonation of the nitrene by methanol observed in this work ranks with the fastest
intermolecular proton transfer processes known, which includes the protonation of singlet
diphenylcarbene by methanol, τ = 9 ps,38a,b and the protonation of water by several excited
photoacids.39a,b
Finally, immediately following protonation, a contact ion pair (CIP) would result. This
CIP will rapidly equilibrate to the solvent separated ion pair (SSIP) via a proton relay mechanism
(Grotthuss mechanism40) that will redistribute the alkoxide anions in an equilibrated array
surrounding the nitrenium ions. Again a parallel exists in the protonation of carbenes. Thus,
protonation of singlet di(p-methoxyphenyl)carbene yields a CIP (λmax = 470 nm) which red-
shifts to a SSIP (ca. 500 nm) over ca. 700 ps.38b The time constant for this type of proton 94
exchange is estimated to be 55 ps for methanol/methoxide,40 and is expected to be about an order of magnitude slower in 2-propanol,38b which is very close to the time constant observed for the
formation of the 486 nm, τ4 = 507 ps, in that solvent, Figure 3.11, frame D to E. Therefore, it seems likely that the red shift observed upon going from frame D to frame E in Figure 3.11, is
not entirely due to decay of the short wavelength triplet component, but may have a significant contribution from the equilibration of the CIP to the SSIP.
The nitrenium ion 33 is very long-lived to the extent that it can be easily observed by
nanosecond transient absorption spectroscopy, Figures 3.9, 3.10. It slowly undergoes
transformation over several hundred nanoseconds to the final intermediate(s) absorbing at 440
nm. In the 2-propanol reaction, the source of this absorption seems to be the nucleophile adducts
38 and 39 (Scheme 3.10) arising from collapse of the SSIP. Based upon the structures of the
observed products, 39 will be the major contributor to this absorption, and its absorption is
estimated by TD-B3LYP to have λmax = 409 nm, while that of the minor contributor 38 is
estimated to have λmax = 360 nm (Table 3.3). It is perhaps surprising that this ion pair collapse
does not occur immediately following the protonation step, but this is clearly not the case, and the SSIP survives into the ns-μs time domains.
The aforementioned relationships are summarized in Scheme 3.10, where the absorption characteristics and lifetimes of the intermediates are provided, and related to frames A-F in
Figure 3.11 where their spectra are shown. Several aspects of this general scheme require further comment. The general spectroscopic pattern shown in frame F of Figure 3.11 is repeated for many different types of proton sources. Some of these are shown in Figures 3.15, 3.17, 3.21.
Carbon acids such as malonate esters, ammonium salts, and phenols all clearly display the characteristic nitrenium ion absorption in the 480-490 nm regions. In addition, all of these proton 95
sources clearly display the characteristic absorption for the nucleophile adduct at about 440 nm.
In the cases of phenol and dimethylamine hydrochloride, adducts 40 and 41, respectively, can be isolated. As indicated above, hydroquinone is a proton source that forms the nitrenium ion 33,
Figure 3.22. However, the lack of a 440 nm band indicates that no adduct is formed, and this observation is supported by the fact that hydroquinone yields only the reduction product amine
28, Table 3.1. Exactly the same reduction pattern is observed for the alcohol ethyl glycolate
(Figure 3.23), which affords only reduction to the amine 28, Table 3.1. Consequently, in addition to radical pathways for reduction of the nitrene to the amine 28, there appear to be ionic reduction pathways as well. Thus, 33 might undergo reduction to amine 28 via an internal oxidation-reduction of the nitrenium ion pair via hydride transfer from the hydroquinone anion, or ethyl glycolate alkoxide anion as shown in structure 42. In protic solvents, this ionic pathway seems to be the main route for reduction of the nitrene to the amine. However, one cannot exclude small contributions to reduction via radical pathways such as that outlined in Scheme
3.10.
H H NH2 Ph NH2 N CHCO2Et O N(CH3)2 O
NO2 NO2 NO2 NEt2 NEt2 NEt2 40 41 42
Figure 3.36. Structures of 40, 41, and 42.
Another surprising aspect of these reactions is that the major addition products were always the more hindered regioisomers related to 30, 40, and 41 rather than the less hindered 96
isomers related to 29. This is the case even for the most hindered t-BuOH adduct. The formation of adducts 38 and 39 have been analyzed theoretically at the DFT B3LYP the reasons for the collapse of the ion pair with preferential bond formation at the 2-position between the nitrene nitrogen and the nitro group rather than at the less hindered 6-position (Figure 3.37).
While these transition state imaginary frequencies are small, both initial attack transition states involve hydrogen bonding between the incoming methoxide ion and the =N-H hydrogen atom.
The possibility of a 1-5 methoxy shift between the 2- and 6-positions has been considered, but was found to have very high activation energies in either direction, and thus, not to play an important role in the final product isomer distribution. On the other hand, the transition state for formation of the 2-methoxy adduct has a slightly lower energy, 2.05 kcal/mol, and higher intensity (probability) than that for the 6-methoxy adduct. Both of these transitions state occur with quite long O-C bond distances, 2.43 Å, and therefore, the attack at the 2-position is not particularly susceptible to steric interference from the nitro group, but is facilitated by the additional electron withdrawal of that group. 97
Figure 3.37. Attack profile for protonation of nitrene with approach from the 2- or 6-side of the nitrene nitrogen (-O-H in Å).
Finally, the picosecond transient absorption behavior of the nitrene derived from 25 in dry acetonitrile, Table 3.2, entry 13, parallels that observed in methanol quite closely, and that in other alcohols reasonably closely. Apparently, the nitrene derived from 25 is sufficiently basic
42 to abstract a proton from acetonitrile (pKa = ca. 25). What makes nitrene generation in dry acetonitrile worthy of note is that the usual adducts related to 29 and 30 are not formed. Instead, good yields of the dimer 34 and the oxidized dimer 35 are obtained, 34:35 = 65:35. Analysis of the transient data by any of several kinetic models (see Table 3.2, entry 13) indicates that the protonation step is very rapid, <10 ps. Thus, the nitrenium ion 33 is formed, and in the nanosecond time domain gives rise to a complex of absorption bands in the 440 nm region, which would normally signal the formation of adducts related to 39. However, in acetonitrile, the 440 nm absorption apparently signals formation of the dimer 43, Scheme 3.11, or some 98
related intermediate that carries the same chromophore as the adduct 39. At this juncture, it is unclear as to why simple collapse of the nitrenium ion-acetonitrile anion ion pair is superseded by a nitrenium ion dimerization.
Scheme 3.11. The mechanism of formation of the dimer 35.
3.5 Conclusions
This work has confirmed the earlier observations43 that aryl nitrenes conjugated to
powerful electron-donating groups such as amines do not undergo the usual collapse to azirines
and ring expansion to ketenimines. Instead these electron-rich nitrenes are strong bases that
form nitrenium ions via very rapid proton abstraction from a wide variety of proton sources. The nitrenium ions that result in these processes have iminoquinone structures that in past studies have been observed to undergo hydrolysis to quinones in aqueous media. However, when an auxiliary electron-withdrawing group, such as a nitro group, is attached to the aromatic ring, the nitrenium ion becomes a powerful electrophile, and usually undergoes nucleophilic aromatic substitution with the conjugate base of the acid that led to its formation. 99
This photochemical cross-linking (PCL) reaction has been used in recent biochemical studies to establish the interactions between many classes of biomolecules. In studies of this type, the two primary assumptions are that the introduction of the photocross-linking agent, an amino azide related to 5, will not perturb the natural interactions of the biomolecules, and that when the photocross-linking agent is activated with light, it will react rapidly with critical sites that participate in binding the biomolecules together. Under this set of assumptions, the activated PCL agent will react rapidly with sites in its immediate vicinity before it has time to migrate to alternative sites. In the present work, it has been established that while the initial nitrene protonation step is very rapid in the picosecond time domain, the collapse of the nitrenium ion pair is surprisingly slow, often requiring microseconds for the formation of the critical cross-linking bond. While these critical site/nitrenium ion pairs might be maintained over microseconds by salt bridges before they collapse to form adducts, due caution should be exercised in the interpretation of any PCL studies of biomolecules using this system. This will be particularly true in cases where the cross-linked sites are analyzed in detail in an effort to ascertain the structural nature of associative interactions.
Finally, the results of this study are being used to assist in the development of more effective photocross-linking agents.
3.6 References
(1) Escher, E. H. F.; Robert, H.; Guillemette, G. Helv. Chim. Acta 1979, 62, 1217-1222. (b)
Addo, J. K.; Swamy, N.; Ray, R. Bioorg. & Med. Chem. Letters 2002, 12, 279-281. (c)
Mauri, L.; Prioni, S.; Loberto, N.; Chigorno, V.; Prinetti, A.; Sonnino, S. Glycoconjugate J. 100
2003, 20, 11-23. (d) Sedlák, E.; Panda, M.; Dale, M. P.; Weintraub, S. T.; Robinson, N. C.
Biochemistry 2006, 45, 746-754.
(2) Knowles, J. R. Acc. Chem. Res. 1972, 5, 155-160
(3) Fleet, G. W. J.; Porter, R. R.; Knowles, J. R. Nature, 1969, 224, 511-512.
(4) Fleet, G. W. J.; Knowles, J. R.; Porter, R. R. Biochem. J. 1972, 128, 499-408.
(5) Fisher, C. E.; Press, E. M. Biochem. J. 1974, 139, 135-149.
(6) Smith, R. A. G.; Knowles, J. R. Biochem. J. 1974, 141, 51-56.
(7) Sonnino, S.; Chigorno, V.; Acquotti, D.; Pitto, M.; Kirschner, G.; Tettamanti, G.
Biochemistry 1989, 28, 77-84.
(8) Mauri, L.; Prioni, S.; Loberto, N.; Chigorno, V.; Prinetti, A.; Sonnino, S. Glycoconjugate
J. 2004, 20, 11-23.
(9) Hegyi, G.; Michel, H.; Shabanowitz, J.; Hunt, D. F.; Chatterjie, N.; Healy-Louie, G.;
Elzinga, M. Prot. Science 1992, 1, 132-144.
(10) Hegyi, G.; Mák, M.; Kim, E.; Elzinga, M.; Muhlrad, A.; Reisler E. Biochem. 1998, 37,
17784-17792.
(11) (a) Kim, E.; Phillips, M.; Hegy, G.; Muhlrad, A.; Reisler, D. Biochem. 1998, 37, 17793-
17800; (b) Kim, E.; Bobkova, E.; Miller, C. J.; Orlova, A.; Hegyi, G.; Egelman, E. H.;
Muhlrad, A.; Reisler, E. Biochem. 1998, 37, 17801-17809.
(12) Fra, A. M.; Masserini, M.; Palestini, P.; Sonnino, S.; Somons, K. FEBS Letters 1995,
375, 11-14.
(13) Prioni, S.; Mauri, L.; Loberto, N.; Casellato, R.; Chigorno, V.; Karagogeos, D.; Prinetti,
A.; Sonnino, S. Glycoconjugate J. 2004, 21, 461-470. 101
(14) Pate, E.; Nakamaye, K. L.; Franks-Skiba, K.; Yount, R. G.; Cooke, R. Biophys. J. 1991,
59, 598-605.
(15) Chen, J.-K.; Franke, L. A.; Hixson, S. S.; Zimmermann, R. A. Biochemistry 1985, 24,
4777-4784.
(16) a) Wells, J. A.; Yount, R. G. Proc. Natl. Acad. Sci. USA 1979, 76, 4966-4970; b)
Okamoto, Y.; Yount, R. G. Proc. Natl. Acad. Sci. USA 1985, 82, 1575-1579.
(17) Nakamaye, K. L.; Wells, J. A.; Bridenbaugh, R. L.; Okamoto, Y.; Yount, R. G. Biochem.
1985, 24, 5226-5235.
(18) Tong, S. W.; Elzinga, M. J. Biol. Chem. 1983, 258, 13100-13110.
(19) Luo, Y.; Wang, D.; Cremo, C. R.; Pate, E.; Cooke, R.; Yount, R. G. Biochem. 1995, 34,
1978-1987.
(20) (a) Kerwin, B. A.; Yount, R. G. Biocon. Chem. 1992, 3, 328-336; (b) Kerwin, B. A.;
Yount, R. G. Proc. Natl. Acad. Sci. USA 1993, 90, 35-39; (c) Sedlák, E.; Panda, M.; Dale, M.
P.; Weintraub, S. T.; Robinson, N. C. Biochemistry 2006, 45, 746-754.
(21) (a) Ray, R.; Holick, S. A.; Holick, M. F. J. Chem. Soc., Chem. Commun. 1985, 702-703;
(b) Ray, R.; Holick, S. A.; Hanafin, N.; Holick, M. F. Biochem. 1986, 25, 4729-4733; (c)
Ray, R.; Bouillon, R.; Baelen, H. V.; Holick, M. F. Biochem. 1991, 30, 4809-4813; (d) Ray,
R.; Bouillon, R.; Baelen, H. V.; Holick, M. F. Biochem. 1991, 30, 7638-7642; (e) Swamy,
N.; Addo, J. K.; Ray, R. Bioorg. & Med.Chem. Lett. 2000, 10, 361—364.
(22) (a) Mappus, E.; Chambon, C.; Fenet, B.; de Ravel, M. R.; Grenot, C.; Cuilleron C. Y.
Steriods 2000, 65, 459-481; (b) Coltrera, M. D.; Potts, J. T., Jr.; Rosenblatt, M. J. Bio. Chem.
1981, 256, 10555-10559.
(23) Nakayama, H.; Nozawa, M.; Kanaoka, Y. Chem. Pharm. Bull. 1979, 27, 2775-2780. 102
(24) Liang, T.; Schuster, G. B. J. Am. Chem. Soc. 1987, 109, 7803-7810.
(25) Fleming, S. Tetrahedron, 1995, 51, 12479-12520.
(26) Suginome, H.; Kurokawa, Y.; Orito, K. J.Org.Chem. 1989, 54, 5940-5948.
(27) (a) Leyva, E.; Munoz, D.; Platz, M.S., J. Org. Chem. 1989, 54, 5938-5945, (b) Hagedorn,
M.; Sauers, R. R.; Eichholz, A. J. Org. Chem. 1978, 43, 2070-2072.
(28) Platz, M. S. In Reactive Intermediate Chemistry, Moss, R. A., Platz, M. S., Jones, M., Jr.,
Eds.: John Wiley & Sons: Hoboken, N.J., 2004; pp 501-560.
(29) McClelland, R. A.; Davidse, P. A.; Hadzialic, G. J. Am. Chem. Soc. 1995, 117, 4173-
4174.
(30) (a) Gritsan, N. P.; Tigelaar, D.; Platz, M. S. J. Phys. Chem. A 1999, 103, 4465-4469.
Carbenes require a similar time in order to establish singlet-triplet equilibration, (b) Wang, J.;
Kubicki, J.; Hilinski, E. F.; Mecklenburg, S. L.; Gustafson, T. L.; Platz, M. S. J. Am. Chem.
Soc. 2007, 129, 13683-13690.
(31) (a) Falvey, D. E. In Reactive Intermediate Chemistry, Moss, R. A., Platz, M. S., Jones,
M., Jr., Eds.: John Wiley & Sons: Hoboken, N.J., 2004; pp 593-650. (b) Winter, A. H.;
Gibson, H. H.; Falvey, D. E. J. Org. Chem. 2007, 72, 8186-8195.
(32) Liu, Y. S.; de Mayo, P.; Ware, W. R. J. Phys. Chem. 1993, 97, 5987-5994.
(33) Borden, W. T.; Gritsan, N. P.; Hadad, C. M.; Karney, W. L.; Kemnitz, C. R.; Platz, M. S.
Acc. Chem. Res. 2000, 33, 765-771.
(34) (a) Chang, K. T.; Shechter, H. J. Am. Chem. Soc. 1979, 101, 5082-5084. (b) Langan, J.
G.; Sitzmann, E. V.; Eisenthal, K. B. Chem. Phys. Lett. 1984, 110, 521-527. (c) Wang. Y.;
Sitzmann, E. V.; Novak, F.; Dupuy, C.; Eisenthal, K. B. J. Am. Chem. Soc. 1982, 104, 3238-
3239. (d) Sitzmann, E. V.; Wang, Y.; Eisenthal, K. B. J. Phys. Chem. 1983, 87, 2283-2285. 103
(35) Irradiation of p-(dimethylamino)phenylnitrene in toluene leads to triplet ground state
formation in 120 ps, Kobayashi, T.; Ohtani, H.; Suzuki, K.; Yamaoka, T. J. Phys. Chem.
1985, 89, 776-779, but time constant may have involved competitive ISC to the triplet and
proton abstraction, Lloyd-Jones, G. C.; Alder, R. W.; Owen-Smith, G. J. Chemistry, 2006, 12
(20), 5361-5375.
(36) (a) Laermer, F.; Elsaesser, T.; Kaiser, W.; Chem. Phys. Lett. 1989, 156, 381-386. (b)
Miyasaka, H.; Hagihara, M.; Okada, T.; Mataga, N. Chem. Phys. Lett. 1992, 188, 259-264.
(c) Schwarzer, D.; Troe, J.; Votsmeier, M.; Zerezke, M. J. Chem. Phys. 1996, 105, 3121-
3131. (d) Elsaesser, T.; Kaiser, W. Annu. Rev. Phys. Chem. 1991, 42, 83-107. (e) Gritsan, N.
P.; Polshakov, D. A.; Tsao, M.-L.; Platz, M. S. Photochem. Photobiol. Sci. 2005, 4, 23-32.
(37) (a) Burdzinski, G.; Hackett, J. C.; Wang, J.; Gustafson, T. L.; Hadad, C. M.; Platz, M. S.
J. Am. Chem. Soc. 2006, 128, 13402-13411. (b) McCulla, R. D.; Burdzinski, G.; Platz, M. S.
Org. Lett. 2006, 8, 1637-1640. (c) Gritsan, N. P.; Polshakov, D. A.; Tsao, M.-L.; Platz, M. S.
Photochem. Photobiol. Sci. 2005, 4, 23-32.
(38) (a) Peon, J.; Polshakov, D.; Kohler, B. J. Am. Chem. Soc. 2002, 124, 6428-6438. (b) Dix,
E. J.; Goodman, J. L. J. Phys. Chem. 1994, 98, 12609-12612. (c) Kirmse, W.; Guth, M.;
Steenken, S. J. Am. Chem. Soc. 1996, 118, 10838-10849. (d) Wang, J.; Burdzinski, G.;
Gustafson, T. L.; Platz, M. S. J. Org. Chem. 2006, 71, 6221-6228. (e) Wang, J.; Burdzinski,
G.; Gustafson, T. L.; Platz, M. S. J. Am. Chem. Soc. 2007, 129, 2597-2606. (f) Wang, J.;
Kubicki, J.; Hilinski, E. F.; Mecklenburg, S. L.; Gustafson, T. L.; Platz, M. S. J. Am. Chem.
Soc. 2007, 129, 13683-13690. 104
(39) (a) Pines, E.; Pines, D.; Barak, T.; Magnes, Ben Zion; Tolbert, L. M.; Haubrich, J. E.
Berichte der Bunsen-Gesellschaft 1998, 102, 511-517. (b) Lima, J. C.; Abreu, I.;
Brouillard, R.; Macanita, A. L. Chem. Phys. Lett. 1998, 298, 189-195.
(40) (a) Cukierman S. Biochim Biophys Acta. 2006, 1757, 876-885. (b) Voth, G. A. Acc.
Chem. Res, 2006, 39, 143–150. (c) Markovitch, O. ; Chen, H.; , Izvekov, S.; Paesani, F.;
Voth G. A.; Agmon N. J. Phys. Chem. B, 2008, 112, 9456–9466. (d)
http://www.lsbu.ac.uk/water/grotthuss.html
(41) Grunwald, E. J. Chem. Phys. 1982, 86, 1302-1305.
(42) Pearson, R. G.; Dillon, R. L. J. Am Chem. Soc. 1953, 75, 2439-2443.
(43) Chehade, K. A. H.; Speilmann, H. P. J. Org. Chem. 2000, 65, 4949-4953.
105
CHAPTER IV. AN EFFECTIVE NITRENIUM ION PRECURSOR FOR
PHOTOAFFINITY LABELING: 2-(N,N-DIALKYLAMINO)-5-AZIDOPYRIDINE
4.1 Introduction
According to our studies described in Chapter II, the presence of the nitro group as a strong electron-withdrawing substituent on the benzene ring has a dramatic effect on the nature and reactivity of the intermediates produced by photolysis. However, it has several disadvantages: the nitro group is bulky, so reactions with some big molecules are sterically hindered and the nitro group is photoreactive itself. With these considerations in mind, we set out to design and synthesize the new azide, 5-azido-2-fluoro-pyridine (44), which might be used as more effective photoaffinity-labeling agent than the nitro analog NAP.
Scheme 4.1. New azide, 5-azido-2-fluoro-pyridine (44), which might be used as more effective photoaffinity-labeling agent than the nitro analog NAP.
Numerous researchers have been interested in the effects of σ−heteroatom substitution upon the
nature of conjugated high spin molecules. Nitrogen-containing heterocyclic compounds with
azido group have attracted considerable attention as a photoaffinity labeling reagents.1,2,3,4 The 106
photochemistry of pyridine azides is very similar to photochemistry of aryl azides. The thermal and photochemical ring expansion of pyridyl azides to seven-membered di-N-heterocyclic ring via the singlet nitrenes has been well described.2,3,5,6 Our interest is in photochemical studies of
3-azidopyridine. Thus, for example, in 1987, Tsuchiya and co-workers5 reported that substituted
3-azidopyridines 45a-c upon the photolysis in the presence of methoxy anion gave 5H-1,3-
diazepines (46), while in the case of 3-azido-4-methoxypyridine (47), under the similar
conditions 2,4,7-trimethoxy-5,6-dihydro-2H-1,3-diazepine (48) was obtained in 60% yield
(Scheme 4.2).
Scheme 4.2. The photolysis of methyl-substituted 3-azidopyridines (45) and 3-azido-4- methoxypyridine (47) in the presence of methoxy anion.
The results of this study have shown the great influence of electron-donating group conjugated with azidogroup on the reaction products. 107
An investigation of the thermal and photochemical reactions of 3-azidoquinoline (49) in bromobenzene6 and 1,4-dioxane7 revealed the formation of a mixture of 3-aminoquinoline (50),
3,3-azoquinoline (51), and pyrazino[2,3-c:5,6-c]diquinoline (52) (Scheme 4.3), which are perfectly consistent with formation of triplet 3-quinolylnitrene which dimerizes or abstracts hydrogens.
Scheme 4.3. Photolysis of the 3-azidoquinoline (49) in bromobenzene.
The solution photolysis of 49 in the presence of sodium methoxide solution (Scheme 4.4)
as a trapping reagent afforded disubstituted benzo[e]-1,4-diazepine derivative 55. However, 3-
(N,N-diethylamino)-4-aminoquinoline (56) was obtained in the presence of diethylamine, and no diazepine product was isolated.7
108
Scheme 4.4. The photolysis of 3-azidoquinoline in the presence of trapping reagents.
Recent studies8 of photochemistry of 3-pyridyl azide (57) (Scheme 4.5) showed that photolysis at
low temperature in an argon matrix leads to ring opening of the corresponding nitrene 58 and/or
the seven-membered cyclic cumulene 59 to the nitrile ylides 60. The ylides 60 were directly
observed by IR and UV spectroscopy (at excitation wavelength λ =222 nm; absorption band at
360 nm). Further photolysis led to the conversion of 60 to ketenimine 61 by a 1,7-H shift.
Scheme 4.5. Ring-opening of 3-pyridylnitrene to nitrile ylide. 109
The 5-azido-2-(N,N-diethylamino)pyridine (65) was chosen for initial study.
Photophysical studies and calculations of 65 had been performed to determine the possible application of these new systems and these are described in this Chapter.
4.2 Synthesis of 5-azido-2-(N,N-diethylamino)pyridine (65) and Studies of the Products of the Photolysis
The route used to synthesize 3-azidopyridine, in which the diethylamine is attached in the para position to the azido group, is shown in Scheme 4.6.
Scheme 4.6. Synthesis of 5-azido-2-(N,N-diethylamino)pyridine (65).
In the first step, commercially available 2-chloro-5-nitropyridine (62) and diethylamine were stirred in EtOH at 40oC to afford compound 63. Reduction of 63 with tin chloride dihydrate
afforded the aniline derivative 64. An alternative method, in which the nitro compound was
reduced by hydrogen and palladium on carbon, was more successful (92%). The diazonium salt
was obtained from this amine after reaction with sodium nitrite in aqueous hydrochloric acid,
and then was converted into the azido derivative 65 by the addition of sodium azide. Compound 110
65 was isolated after extraction DCM/KOH. This route has been applied in large scale synthesis.
The alternative route of synthesis of 65 is shown on Scheme 4.7, using a general procedure described in Chapter III.
Scheme 4.7. The alternative route of synthesis of 65.
1H NMR and 13C NMR, GC/MS spectroscopy were applied for characterization of
compound 65. The presence of azido group was also confirmed by IR spectroscopy that displayed the characteristic sharp band at 2094.8 (m) cm-1.
Direct irradiation of the solutions of 65 under a nitrogen atmosphere was conducted at a Pyrex
tube at 350 nm in a Rayonet Photochemical reactor at room temperature (20-25oC). The products
distribution was determined in each case by NMR analysis of the characteristic and well-
resolved aromatic and alkyl signals. GC/MS analysis was also used. The 65 exhibited a rather
similar photochemistry to that of nitroanalog 25; the photolysis of 65, performed in the presence
of alcohols, leads to the formation of addition products 68 in excellent yields (Scheme 4.8). A
pair of doublets at δ 7.00 and 8.18 is produced by the adjacent aromatic ring protons similar to
addition products in Chapter V. The irradiation of 65 in 2-propanol leads to formation only one 111
addition product 69 (the 2-substituted adduct), the 6-substituted product has not been observed.
Adduct 69 is susceptible to air oxidation to dimet 70 if it is kept more than 1 week even under argon atmosphere at low temperature (Scheme 4.9).
Scheme 4.8. Irradiation of 65 in the presence of different nucleophiles.
Scheme 4.9. Oxidation of the addition product 69.
The results of the product studies in various solvents are summarized in the Table 4.1.
The addition process with 65 proceeds much more effectively, in range from 98% to 100%, in 112
comparison to the nitrobenzene analog 25. Moreover, the conversion of starting material to product was more rapid and proceeds to completion in 2.5 to 4 hours. Experiments in saturated solutions of phenol and dimethylammonium salt in acetonitrile provided addition products in ratio 100% and 98%, respectively. Even, irradiation of 65 in tert-butanol leads to full conversion of starting material to the addition product 71 (Figure 4.1).
Ratio of Products Time of
Irradiation (hours) Nucleophiles Reduction Substitution
i-PrOH 0 100 2.5
n-BuOH 0 100 3
MeOH 20 80 2.5
i-PrOD* 2 98 40 min.
Imidazole x HCl 0 100 4
(CH3)2NH×HCl 2 98 3
C6H5OH 0 100 3
tert-BuOH 0 100 3.5
* photolysis of 65 in deuterium solvents was performed in NMR tube
Table 4.1. Reduction/Addition Ratios for photoreactions of 65 with various nucleophiles.
113
Moreover, photolysis with imidazole hydrochloride in acetonitrile led exclusively to corresponding adduct 72 (Figure 4.1). The molecule shows a characteristic pattern in the aromatic region: a pair of doublets (δ 7.1 and 6.4) is produced by pyridine ring protons with J- coupling of 9 Hz, while imidazole ring protons give rise to three singlets at δ 7.21, at δ 7.5, and at δ 8.1 (See Part 4.4). It is noteworthy, that no substitution reaction occurred in the presence of imidazole in acetonitrile.
Figure 4.1. Products of photolysis of 65 in the acetonitrile solution of imidazole hydrochloride (72) and in cyclohexane (73).
Photolysis of 65 in cyclohexane was completed for 3 hours and afforded just one product: dimer 73, which is perfectly consistent with the formation of triplet nitrene in non-protonating solvents. Reduced aniline 67 and dimer 73 in ratio 3:1 were obtained upon photolysis of 65 in toluene.
114
4.3 Photophysical Studies
Ground State Absorption
The normalized ground-state absorption spectra of the 65 recorded in different solvents are shown in Figure 4.2. The position of the absorption bands does not get significantly perturbed by solvent polarity; absorption spectrum of 65 consists of the two bands: a weak band at 340 nm (ε = 5969 cm-1 M-1), and somewhat stronger band at 281 nm (ε = 18690 cm-1 M-1),
which corresponds to the highest energy transition. Ground-state absorption of azide in solution
of 2:1 glacial acetic acid to water displays some shift of maxima: λmax1= 355 nm, λmax2= 273 nm.
Moreover, the intensity of the short wavelength absorbance is weaker in 50% of acetic acid than
in alcohols; the absorption coefficient ε at 355 nm is 4238 cm-1 M-1.
4.5 acetonitrile 4.0 2-propanol cyclohexane 3.5 50% acetic acid
3.0
2.5
ΔΑ 2.0
1.5
1.0
0.5
0.0 200 300 400 500 600 Wavelength, nm
Figure 4.2. UV-Vis absorption spectra of 65 in acetontrile (solid pink line), 2-propanol (dot blue line), cyclohexane (dashed dark cyan) and 50 % solution of acetic acid (red short dash). 115
Electronic absorption spectra were calculated at the TD-B3LYP/TZVP and CASPT2 levels of theory (Table 4.2) by Maxim Panov. These calculations are very close to the experimental data and indicate that the lowest excited state S1 is optical silent; the band with maximum at 341 nm corresponds to second excited state S2, and the band (λ = 281 nm) due to
the third excited state S3.
Table 4.2. Electronic steady-state absorption spectra calculated at the TD-B3LYP/TZVP and CASPT2 levels of theory.
Theoretical studies of electron distribution in these states, S0, S1, S2, S3 of 65 were
conducted using the Density Functional Theory (DFT) method (Figure 4.3).
According to these calculations, the intense band in the blue region indicate that all the observed
transitions corresponds to allowed π−π* transitions, and π−π* interaction between the pyridine
ring and the azide group attached in meta-position is very strong.
116
Figure 4.3. Three excited states calculated at the DFT level of theory.
Fluorescence Studies of 5-Azido-2-(N,N-diethylamino)pyridine (65)
The photophysical studies indicate an emission of the azide 65 in different solvents.9,10 It
is well known that 2- and 3- aminopyridines are fluorescent compounds.9-14 Therefore, both
isomers have been used as fluorescent reporters of hydrolytic activities of multiple enzymes such
as nucleotide pyrophosphatases, transferases, and hyaluronidase.10-12 The photophysics of pyridine and pyridine derivatives, such as aminopyridines, has been the subject of many experimental and theoretical studies of the elucidation of radiative and non-radiative processes
induced by interacting of n,π* and π,π* states.13 117
Figure 4.4 presents the emission and excitation spectra of 65 in the 2-propanol, which displays intense fluorescence in alcohols with broad emission with λmax = 405 nm-410 nm upon
excitation at 355 nm.
4.0x106
3.5x106 emission excitation 3.0x106
2.5x106
6 2.0x10 Emission 1.5x106
1.0x106
5.0x105
0.0 300 350 400 450 500 wavelength, nm
Figure 4.4. Excitation (blue dash) and emission (purple solid line) spectra of 65 in argon- saturated 2-propanol.
The fluorescence behavior of aromatic molecules containing nonbonding electron pairs
are generally expected to show sensitivity to solvent polarity and hydrogen bonding.10,14,15
Measurements of the fluorescence of 65 were performed in the following solvents in order to increasing of polarity: cyclohexane, toluene, chloroform, n-butanol, 2-propanol, methanol, acetonitrile, aqueous acetic acid and a solution of sodium hydride.
Our studies indicate that the effect of solvent polarity on the fluorescence of aminopyridines is significant. Figure 4.5 showed the changes in emission of 65 upon excitation at 355 nm in presence of different organic solvents. Non-polar solvents, such as cyclohexane and 118
toluene, blue shifted the emission profile of 65. The most polar solvents shifted the emission to longer wavelengths, which is typical for π,π* transition.16,17 This fluorescence shift might be
explained by excitation to an excited state that is more polar than the ground state, thus, the
stabilization of dipolar excited state by an increase in dielectric constant of solvent.10,14
6x105 1.2 Toluene n-Butanol 5 CH3CN 5x10 n-BuOH Chloroform CH3COOH 1.0 CH3CN CH3COOH 5 Toluene 4x10 cyclohexane cyclohexane 0.8 chloroform Methanol i-Propanol 3x105
0.6 Emission
5
2x10 Intensity Emission 0.4
5 1x10 0.2
0 0.0 360 380 400 420 440 460 480 500 520 540 400 450 500 550 wavelength, nm Wavelengh, nm
Figure 4.5. (a) Emission spectra of 65 in protic and aprotic solvents; (b) Normallized intensity of emission spectra of 65 in protic and aprotic solvents.
The kinetic of fluorescence was measured with a Proteus spectrometer during the laser
flash photolysis experiment. Life-times of the excited state of 65 in n-butanol and 2-propanol
were measured under optically delute conditions. In both cases the emission intensity decays
were well modeled by biexponential kinetics and recovered lifetimes were constant along the
entire emission profile. The kinetic spectra obtained in n-butanol and 2-propanol are displayed in
Figures 4.6 (a) and (b) respectively. The room temperature emission lifetime of 65 in n-butanol 119
and in 2-propanol: τ1=36±0.6 ns and τ2=855± 18 ns, and τ1=77±0.14 ns and τ2=623± 6 ns, respectively.
1.4
(a) 1.6 4.0x10-5 (b) nanosecond transient 1.2 -5 1.4 2.0x10 0.0 1.0 Emission 1.2 -2.0x10-5
-5 0.8 1.0 -4.0x10
-6.0x10-5
0.8 380 390 400 410 420 430 440
Emission 0.6 wavelengh, nm Emission 0.6 0.4 0.4 0.2 0.2
0.0 0.0 01234 01234 time, μs time, μs
Figure 4.6. The kinetic traces of fluorescence of 65 at 420 nm (a) in n-butanol with biexponential solid line fit superimposed with time constants of 36±0.6 ns and 855± 18 ns; (b) in 2-propanol along with biexponential solid line fit superimposed with time constants of 77±0.14 ns and 623± 6 ns with emission transient spectrum on nanosecond LFP.
While the intensities of the emissions seem to be
strongly depended from the acidity of the solvents,
which is in good agreement with previous
studies.9,10,14 Generally, strong fluorescence for
aminopyridine is detected in acidic or neutral
solutions. The intensity of fluorescence of 65
increases dramatically with increasing acidity and reached maximum in acetic acid. The fluorescence quantum yield of azide in isopropyl alcohol excited at 355 nm is Φfl = 0.065 and 120
about 4 times greater than that in cyclohexane, Φfl = 0.016. This fact might be attributed to
removal of the nonbonding electron pair on the pyridine ring upon protonation.10-14
It was reported, that the 2-amino (2-AMP) and 3-amino (3-AMP) pyridines are more basic in the excited state than in the ground state. The pKa values of 6.86 in the ground state and 8.95 in the
excited state were found for 2-AMP; and an even greater difference in pKa values for 3-AMP:
5.98 and 11.2 in ground and excited states, respectively.10 Therefore, the strong enhancement of
emission in the presence of alcohols, and acid is consistent with its strong hydrogen bonding and
the charge–charge interaction shown in Figure 4.7.
Ultrafast Transient Absorption Spectra of Azide in Protic Solvents
Figure 4.8 displays the transient ΔA absorption spectra of 65 which was measured in 2-
propanol upon 310 nm excitation at time delays starting from 100 fs.
The solution was flowed through a 0.5 mm flow cell and excitation energy was kept
between 4 and 6 μJ-pulse-1 . The delay times in picoseconds are shown in the frame legends:
Frame A (short times, 0.1-1 ps), Frame B (intermediate times, 1-20 ps), and Frame C (long
times, 20-1100 ps). Data in Frame C represent smoothed spectra by averaging of adjacent point
(bandwidth, 3.5 nm).
The short time scale (Figure 4.8, Frame A) of ultrafast transient consists of the hot nitrene
band. At 500 fs, a broad transient absorption was observed with maximum wavelength at 320 nm
(Figure 4.8B). From 500 fs to 20 ps, this transient absorption signal rises and sharpens to a peak
at 315 nm that dominates the ΔA spectrum. This process is accompanied by growth of transient
absorption centered at 472 nm. At longer times, from 20 to 200 ps (Frame C) the visible band 121
continues to grow. After approximately 200 ps, this maximum decays very slowly without changes in its position.
.
Figure 4.8. Ultrafast transient absorption (ΔA) spectra of 65 in 2-propanol upon 310 nm excitation. A, B, and C: The solution was flowed through a 0.2 mm flow cell and excited with a 305-nm, 3.8 μJ pulse. The solvent contribution to the ΔA spectra is minor at delay times ≥ 100 fs.
Following our previous ultrafast studies on aryl azide 25 (Chapter III), the broad transient absorption at 320-340 nm is assigned to closed-shell singlet nitrene. CASPT2 calculations predict that the corresponding nitrene absorbs at 312 nm with significant oscillator strength (f =
0.58431). The 12 ps time constant represents the population decay time of the closed-shell singlet nitrene by protonation to form the nitrenium ion (Table 4.3). The lifetime of 122
corresponding singlet nitrene is shorter than that of singlet of 4-amino-3-nitrophenyl nitrene (25)
(19 ps).
Table 4.3. Vertical transitions and absorption properties of possible reactive species derived from the irradiation of ADP as calculated at the TD-B3LYP/TZVP and CASPT2 levels of theory.
A strong absorption, centered at 315 nm, and a weak absorption tail in the visible region, centered at 472 nm, are identified as a nitrenium ion. This assignment is consistent with CASPT2 calculations which assigns to that nitrenium ion two transitions, one centered at 290 nm (f =
0.00793) and the other at 483 nm (f = 0.095) (Table 4.3). Moreover, Falvey’s group reported that
N-methyl-N-phenylnitrenium ion, PhNMe+, displayed two bands centered at 325 nm and 470
nm.17
The Figure 4.9(a) presents the transient absorption spectra of azide 65 upon 360 nm
excitation, which according to calculations populates the second state S2. Corresponding 123
photolysis produces the same several species, which have similar absorption spectra, but much more intense that 310 nm and 250 nm. Ultrafast photolysis of 65 in 2-propanol with 255 nm excitation wavelength (excitation to the third singlet excited state S3) which is presented on
Figure 4.9b gave results similar to those obtained at 360 nm excitation wavelength with a few
notable exceptions: the intensity of ΔA signals is less in the 255 nm spectrum and life-time of
nitrene is less ether.
(a) (b)
Figure 4.9. Transient absorption (ΔA) spectra of 65 in 2-propanol (a) upon 360 nm excitation and (b) upon 255 nm excitation.
124
Kinetic studies indicated that the life-time of this nitrene increases as the excitation wavelength increases from 255 nm to 360 nm: 12.2 ps, 16.8 ps and 17.5 ps, respectively (Table 4.4).
The measurements of ultrafast transient absorption of azide 65 in series of alcohols, such as methanol, ethanol, n-butanol, upon 360 nm and 310 nm excitation at time delays starting from
100 fs were determined. The resulting time constants are summarized in Table 4.4, together with the maximum absorption wavelengths (λmax) of the resolved component spectra. Upon 360 nm
excitation nitrene protonation in methanol occurs over 4.9 ps. The slowest protonation process
occurs in t-butanol.
Solvent Pump A B τa, τb, λ, nm λmax,nm ps λmax,nm ps CH OH 3 360 325 1.47 475 4.95 (CH ) CHOH 3 2 360 325 1.98 475 17.5 n-C H OH 4 9 360 325 1.47 475 17.7 CH CH OH 3 2 310 324 1.5 475 9.35 Acetonitrile/H O 2 310 328 0.854 475 4.34 1:1 ImidazolexHCl 310 325 2.18 475 9.00 acetonitrile (CH ) CHOH 3 2 310 325 2.07 475 16.8 n-C H OH 4 9 310 325 2.21 475 18.6 (CH ) COH 3 3 310 325 2.15 475 20.0 (CH ) CHOH 3 2 255 325 2.18 475 12.2
Table 4.4. Transient cascade characteristics upon irradiation of 65 in protic solvents.
125
Nanosecond Laser Flash Photolysis Studies of Azide 65 in Protic Solvents
The solution of azide 65 was flowed through a 1 cm flow cell and excited with 5 mJ pulses. Laser flash photolysis of 65 in 2-propanol with 12 ns pulses of 355 nm excitation light produces the transient ΔA absorption spectrum shown in Figure 4.10. The broad transient absorption between 450 nm and 650 nm is detected at the earliest time observable (154 ns). The negative signal that occurs just after pulse in the 400 nm-430 nm regions is due to the bleaching corresponding to fluorescence. This broad band might be assigned as a nucleophile addition intermediate. The significant difference in absoption spectra of the adduct intermediates of azides 25 and 65 might be due to the steric factor. The nitro-group in sigma-complex 39 is apparently twisted out of conjugation which leads to the blue shift in the absorption of 39.
0.016
0.014
0.012 154ns 374ns 0.010 802ns 0.008 1122ns
A 1844ns Δ 0.006
0.004
0.002
0.000
-0.002
-0.004 450 500 550 600 wavelength, nm
Figure 4.10. Nanosecond transient absorption (ΔA) spectra of azide 65 in 2-propanol, excited at 355 nm, with delay and growth times specified on the graph.
126
Kinetic trace for the decay at 510 nm is best fit with a double exponential (Figure 4.11).
The time constants of decay of intermediate at 510 nm are τ1= 354±26 ns and τ2 = 2.54± 0.12 μs,
within experimental error.
0.008
0.006
ΔΑ 0.004
0.002
0.000 0.0 2.0x103 4.0x103 6.0x103 8.0x103
time, ns Figure 4.11. The kinetic traces of azide 65 recorded at 500 nm in 2-propanol along with
biexponential solid line fit superimposed with time constants of τ1= 354±26 ns and τ2 = 2.54± 0.12 μs.
Nanosecond photolysis of 65 in n-butanol, tert-butanol and in acetonitrile solution of
imidazole hydrochloride gave results similar to those obtained in isopropyl alcohol with different
kinetic traces. The adduct product 68 was isolated as the only one product in all these cases.
Therefore, the sources of the broad band at 460-560 nm is the most reasonably attributed to the adduct complex. The transient absorption spectrum of azide 65 in n-butanol exhibits rapid decay of the broad band at 570 nm of τ =670 ns (Figure 4.12). While, the life-time of adduct complex
in tert-butanol decays very slowly (τ ~2.5 μs) (Figure 4.13a). In the acetonitrile solution of imidazole hydrochloride the growth of adduct intermediate is observed (Figure 4.13b). 127
0.014 0.03 b) B 0.012 a) ExpDec2 fit of A500NM_B 208ns 0.010 488ns 1042ns 0.02 1804ns 0.008
ΔΑ ΔΑ 0.006 0.01 0.004
0.002 0.00 0.000 400 450 500 550 600 01234 wavelength, nm time, μs
Figure 4.12. (a) Nanosecond transient absorption (ΔA) spectra of azide 65 in n-butanol, excited at 355 nm, with delay times specified on the graph; (b) The kinetic trace of azide 65 recorded in n-butanol at 500 nm: along with double-exponential solid line fit superimposed with time constants 43 ± 0.21 ns and 0.67± 0.033 μs.
0.030 0.016 a) b) 0.014 0.025
0.012 194ns 114ns 562ns 0.020 672ns 0.010 950ns 1524ns 1798ns ΔΑ 1832ns ΔΑ
0.015 0.008 0.006 0.010 0.004 0.005 0.002
0.000 0.000 400 450 500 550 600 450 500 550 600 650 wavelength, nm wavelength, nm
Figure 4.13. (a) Nanosecond transient absorption (ΔA) spectra of azide 65 in tert-butanol, excited at 355 nm; (b) nanosecond transient absorption (ΔA) spectra of azide 65 in acetonitrile solution of imidazole hydrochloride, excited at 355 nm with delay times specified on the graphs.
128
Ultrafast and Nanosecond Experiments in Non-Polar Solvents
The photolysis of azide 65 upon 310 nm excitation in cyclohexane is presented in Figure
4.14.The spectrum at short time scale (Frame A) are similar to those in alcohols and acetonitrile.
However, the transient species observed in Frame B, conditions under which the triplet nitrene should be formed, are distinctly different from those formed under the conditions of the reaction with alcohols. After 1 ps, an intense sharp band with maximum 359 nm and weaker broad band at 483 nm begin to appear in the spectra.
Figure 4.14. Transient absorption (ΔA) spectra of azide 65 in cyclohexane upon 310 nm excitation. 129
The 359 nm band begins to decay after 2 ps; its decay is accompanied by the growth of the band centered at 458 nm that produces two isosbestic points at 413 nm and 467 nm. Frame C displays the band at 458 nm which continues to rises from 50 ps to 1 ns. This transient develops two bands, centered at 458 nm and 435 nm. In accordance with the products obtained from the irradiation of 65 in cyclohexane, the intensive band at 458 nm might reasonably be identified as triplet nitrene, which forms as a result of the relaxation of singlet nitrene to the triplet nitrene
(τ = 45.7 ps, Table 4.5). CASPT-2 calculation of vertical transitions and absorption properties of
triplet nitrene indicate absorption of the triplet around 422 nm (Table 4.3).
Pump A τ , B τ , a b λ, nm λ ,nm λ ,nm Solvent max ps max ps
Toluene 310 325 2.76 458 36.5
Cyclohexane 310 325 2.93 458 45.7
Table 4.5. Transient cascade characteristics observed upon irradiation of azide 65 in non-polar solvents.
Figure 4.15 presents the transient spectra of 65 in toluene (a) in the ultrafast transient
absorption time domain and (b) in the nanosecond absorption time domain. These spectra look
very similar to those in cyclohexane and, also, might be identified as arising from the triplet
nitrene. Intersystem crossing (ISC) from singlet nitrene to triplet nitrene has τ = 36.5 ps (Table
4.5). Nanosecond absorption spectra has the same pattern, however, the band is centered at 439 nm continues to grow even after 1864 ns. Thus, the 439 nm band can be referred to the corresponding radical. 130
(a) (b)
Figure 4.15. (a) Ultrafast transient absorption (ΔA) spectra of 65 in toluene upon 360 nm excitation; (b) nanosecond transient absorption (ΔA) spectra of 65 in toluene upon 355 nm excitation.
4.4 Discussion
The 5-azido-2-(N,N-diethylamino)pyridine (65) has been investigated and found to offer
substantial advantages over 25 as a photoaffinity agent. Comparison of the photochemical
characteristics of nitrophenyl azide 25 with the new azidopyridine 65 is shown in Table 4.6. The one of the main disadvantages of many aryl azides as photoaffinity agents is their photoreactivity at wavelengths under/or 280 nm that leads to photochemical damage to biological samples.
Azide 65 is highly photoactive from 250 nm to 360 nm excitation wavelengths. Moreover, the photolysis of 65 at different wavelengths leads to more efficient formation of substitution 131
product. It is noteworthy, that even though azide 25 absorbs in the visible region, 420 nm of excitation do not yield reactive nitrene.
Azide 25 Azide 65 Addition products range 90-96% 98-100% Excitation wavelength, nm 350nm-420nm 260-350nm Irradiation time, hours 4-6 hours 2.5-4 hours Conversion of starting material to products 50-70% 100%
Table 4.6. Comparison of the photochemical characteristics of nitrophenyl azide 25 with the new azidopyridine 65.
First, the coupling reactions of azide 65 proceed more rapidly than those of azide 25 (2-
3.5 hours instead of 4 hours) with full conversion of starting material. Second, the photolysis of azidopyridine 65 with different nucleophiles formed mainly addition products and fewer side products, including reduction to the amino analogs.
Azidopyridine 65 displays intense fluorescence that is greatly attenuated upon conversion
to products, a property that might offer distinct advantages in photoaffinity labeling studies in
complex biological systems. In many systems, quenching of fluorescence occurrs due to charge
transfer interactions between the proton donor and acceptor or to hydrogen transfer
mechanisms.15,16 In this case, quenching presumably occurs due to the formation of adduct
product, which quenchs the emission of azide 65 (Figure 4.16). 132
3.0x106 65 5 1 min 6x10 6 min (b) adding of product 69 (a) 6 9 min 2.5x10 5x105 2.0x106 4x105 1.5x106
3x105 Emission 1.0x106 Emission Intensity Emission 2x105 5.0x105 1x105 0.0 0 400 450 500 550 300 350 400 450 500 550 600 650 700 Wavelength, nm Wavelength, nm
Figure 4.16. (a) Emission of 65 in 2-propanol: one minute of irradiation at 355 nm (olive solid line); six minutes of irradiation at 355 nm (orange dash line), nine minutes of irradiation at 355 nm (cyan solid line); (b) emission of 65 (navy solid line) in 2-propanol; emission of 65 after addition of substitution product 69.
excitation 6 emission 2.0x10 (b)
1.5x106
1.0x106
Emission Intensity 5.0x105
0.0 300 350 400 450 500 550 Wavelength, nm
Figure 4.17. Amine 67 in 2-propanol: excitation spectrum observed at 322 nm (wine solid line); fluorescence excited at 355 nm (violet solid line).
133
Measurement of the fluorescence of adduct 69 displayed a band with very low intensity. The fluorescence of 67 in isopropyl alcohol was measured, and its emission specrum consists of the intense band with λmax= 435 nm (Figure 4.17). Thus, dissappearance of fluoresence would
indicate the formation of a covalent bond to targeted biomolecule. While. red shift of fluoresence
would indicate the reduction to amine 67 (Figure 4.18). So, 65 might be used in sensitive
photoaffinity labeling fluorescent assays.
azide 65 2.0x106 (a) 67 1.0 (b) amine 67 69 65 0.8 1.5x106
0.6 1.0x106
0.4 Normallized Intensity Normallized 5.0x105 Emission Intensity Emission 0.2
0.0 0.0 400 450 500 550 400 450 500 550 Wavelength, nm Wavelength, nm
Figure 4.18. (a) Fluorescence spectrum of azide 65 (purple solid line), amine 67 (violet dashed line), and adduct 69 (pink dash-dot) in 2-propanol; (b) normalized intensities of azide 65 (purple solid line) and amine 67 (violet dashed line) in 2-propanol.
Mechanistic Pathways of the Formation of Products of Photolysis of Azide 65
The mechanism of photolysis of azide 65 (Scheme 4.10) in the presents of nucleophiles
seems to be very similar to that of nitro analog 25. According to CASPT2 calculations, 360 and 134
255 nm excitations should populate the second singlet excited state S2 and the third singlet
excited state S3, respectively. The excited state S3 decays by the internal conversion IC to S2 state. Followed by conversion from S2 to S1 occurs. This process proceeds in competition with
radiative emission (fluorescence quantum yield of 65 in isopropyl alcohol Φ= 0.065), which has
a very long life-time, 623 ns. During IC from S2 to S1 the excess energy is converted into heat;
which probably leads to formation of the closed-shell nitrene in a vibrationally hot state on an
ultrafast time scale, ~2 ps (Figure 4.19). Therefore, the released hot nitrene undergoes
protonation in protic solvents on a 4.5-20 ps time scale. Protonation of hot nitrene explains the
shorter protonation time of nitrene generated at 255 nm excitation (third excited state) in
comparison to excitation at 360 nm (second excited state) in isopropyl alcohol.
Figure 4.19. Diagram of pathway of the formation of closed-shell nitrene.
Protonation of the nitrene 74 by isopropanol proceeds for 16.8 ps with excitation at 310 nm and
12.2 ps at 255 nm. According to results of nanosecond experiments, the life-time of nitrenium
ion 75 is <174 ns that is much shorter than that of the nitro analog 33 (~600 ns). Therefore, 75 is
not observed by nanosecond transient absorption spectroscopy. 135
136
The source of the final intermediate absorption at 460-570 nm on nanosecond time domain seems to be the nucleophile adducts 76. This adduct complex 76 in isopropyl alcohol is very long-lived, > 2 μs. As in the case of nitro analog 25, the life-time of adduct complex in n-butanol is shorter than in 2-propanol.
The photolysis in aprotic solvents, such as toluene and cyclohexane, leads to formation of dimer 73 and reduced amine 67 via triplet and radical pathways. The closed-shell singlet nitrene undergoes ISC to triplet nitrene 77 in 35-45 ps.
The adduct 69 is very easily oxidized by air. A possible mechanism for the oxidation of the adduct 69 shown on Scheme 4.11. It is possible that this process might begin with the oxidation of 69 to nitrenium ion 78. Then, Michael addition of 69 to 78 would lead to formation of intermediate 79, which in turn oxidizes in two steps to product 70. This tendancy of adduct 69 to be oxidazed with formation of dimer 70 might be useful in double cross-linking processes.
NEt2 NEt2 N NH2 NH N O-iPr O-iPr iPr-O NH O 70 iPr-O O NH2 N O-iPr + N + N -H HN -H O-iPr -H+ N NEt2 NEt2 N 69 78 79O NEt2 80 NEt2
NEt NEt2 2
N N O iPr-O iPr-O NH N -2H+ HN N O-iPr O-iPr
N N
70 NEt 81 NEt2 2
Scheme 4.11. The mechanism of oxidation of the adduct 69 by air.
137
4.5 Conclusions
During this research it was shown that if the nitro group is replaced by a ring nitrogen atom to produce a pyridinium aryl azide, cross-linking becomes significantly more effective.
PAL agents of this type have not been investigated in biological environments. However, for many applications the ring nitrogen might be much less intrusive than is the much larger nitro group, which is also likely to undergo photochemical transformation.
Investigations of photophysical properties of 65 were performed and the mechanism of the photolysis of azide 65 was established. The elimination of molecular nitrogen from the both higher singlet excited states S2 and S3 of the azide 65 and the formation of closed-shell singlet
pyridine nitrene occur very rapidly in ca 850 fs in 50% aqueous acetonitrile solution to 2.2 ps in
n-butanol. Ultrafast transient spectroscopy confirms that nitrene 74 proceeds via a nitrenium ion
75. The protonation times of this nitrene by the polar solvents are similar to those of nitro analog
(4.56 - 12 ps). The protonation step in 50% aqueous solution of acetonitrile with 310 nm excitation is remarkable fast, 4.34 ps. Ultrafast LFP in aprotic solvents cleanly gives the transient
spectrum of the triplet nitrene 77. The ISC from singlet nitrene 74 to triplet nitrene 77 proceeds
in ~40 ps.
The 5-azido-2-fluoropyridine 44 was shown to be a good labeling agent for photoaffinity
studies. The remarkable ability of 5-azido-2-(N,N-diethylamino)pyridine 65 to form exclusively
the addition products at longer wavelength, short irradiation time, and the advantage of the
fluorescence make this new system attractive for biochemical applications.
While aryl azides are among the most widely used PAL reagents, these entire PAL
studies were conducted before the nitrenium ion branch of nitrene chemistry was fully 138
appreciated, or understood. Thus, these previous studies tend to concentrate on the biochemical aspects of the work and assume a mechanism for cross-linking that was generally accepted at the time that the studies were conducted. In this research we show that many cross-linking studies, that attribute the cross-linking to ketenimine or nitrene insertion chemistry, probably involve nitrenium ion chemistry.18-22
4.6 References
(1) Dias, M.; Mornet, R.; Laloue, M. Bioorg. Med. Chem. 1995, 3, 361-366.
(2) Dias, M.; Richomme, P.; Mornet, R. J.Heterocyc.Chem. 1996, 33, 1035-1039.
(3) De Waal, A.; Hartog, A.F.; de Jong, L. Biochim.Biophys.Acta 1987, 912, 151-155.
(4) Zhang, N.; Tomizawa, M.; Casida, J. E. J. Med. Chem. 2002, 45, 2832-2840.
(5) Sawanishi, H.; Tajima, K.; Tsuchiya, T. Chem.Pharm.Bull. 1987, 35, 4101-4109.
(6) Hollywood, F.; Nay, B.; Scriven, E. F.V.; Suschitzky, H.; Khan, Z.U.
J.Chem.Soc., Perkin I 1982, 421-429.
(7) Kvaskoff, D.; Mitschke, U.; Addicott, C.; Finnerty, J.; Bednarek, P.; Wentrup, C.
Aust. J. Chem. 2009, 62, 275–286.
(8) Bednarek, P.; Wentrup, C. J. Am. Chem. Soc. 2003, 125, 9083-9089.
(9) Babiak, S.; Testa, A. C. J. Phys. Chem., 1976, 80, 1882–1885.
(10) Testa, A. C. J. Phys. Chem. 1981, 85, 2637-2639.
(11) Huang, H.; Nishi, K.; Tsai, H.-J.; Hammock, B. D. Analyt.Biochem. 2007, 363,
12–21. 139
(12) Huang, H.; Tanaka, H.; Hammock, B. D., Morisseau, C. Analyt.Biochem. 2009,
391, 11–16.
(13) (a) Ghosh, K.; Sarkar, A. R.; Patra, A. Tetrahed. Lett. 2009, 50, 6557–6561; (b)
Araki, Y.; Andoh, A.; Fujiyama, Y.; Hata, K.; Jin Makino, J.; Okuno, T.; Fumiyasu
Nakanura, F.; Bamb, T. J.Chrom.B: Biomed. Sc. Appl. 2001, 753, 209-215.
(14) Mutai, T.; Cheon, J.-D.; Tsuchiya, G.; Araki, K. J. Chem. Soc., Perkin Trans. 2,
2002, 862–865.
(15) Faujmoto, A.; Ando, H.; Inuzuka, K.; Nakamura, J. Bull. Chem. Soc. Jpn., 1993,
66, 414-420.
(16) Kimura, K.; Takaoka, H.; Nagai, R. Bull. Chem. Soc. Jpn., 1977, 50, 1343-1344.
(17) Kung, A. C.; Chiapperino, D.; Falvey, D. E. Photochem. Photobiol. Sci. 2003, 2,
1205-1208.
(18) Sonnino, S.; Chigorno, V.; Acquotti, D.; Pitto, M.; Kirschner, G.; Tettamanti, G.
Biochem. 1989, 28, 77-84.
(19) Fra, A. M.; Masserini, M.; Palestini, P.; Sonnino, S.; Somons, K. FEBS Lett.
1995, 375, 11-14.
(20) Mauri, L.; Prioni, S.; Loberto, N.; Chigorno, V.; Prinetti, A.; Sonnino, S.
Glycoconjugate J. 2004, 20, 11-23.
(21) Prioni, S.; Mauri, L.; Loberto, N.; Casellato, R.; Chigorno, V.; Karagogeos, D.;
Prinetti, A.; Sonnino, S. Glycoconjugate J. 2004, 21, 461-470.
(22) See references 1-26 in Chapter IV
140
CHAPTER V. METHODS AND MATERIALS.
5.1 Pump-probe ultrafast transient experiments
Pump-probe ultrafast transient experiments with azides 25 and 65 have been conducted in various solvents at excitation wavelengths of 420, 350, 305 and 280 nm. All transient absorption
(ΔA) spectra were corrected for the group velocity dispersion of the white-light continuum chirp with an accuracy of ± 30 fs by using the non-resonant or two-photon absorption signals from neat solvent.2 Dissolved oxygen had no noticeable effect on the transient absorption spectra as
verified by degassing the azide solution with argon (solvent, 2-propanol). Linearity of the transient absorption signals were verified by attenuating the excitation light with neutral density filters up to one-fourth of the typically used pulse energy; the extrapolated line passed through the origin. The ΔA spectra (solvent, 2-propanol, 350 nm excitation) were found to be independent of azide concentration (16-32 mM). About 120 ΔA data points were collected at each position of the delay line and this procedure was repeated about 10 times for averaging.
Steady-state absorption spectra of the azide solutions measured before and after the pump-probe experiment indicated that the degree of the sample decomposition was always less than 10%. All measurements are performed at magic angle polarization conditions and 22°C.
The possibility that the solvent contributes to the measured transient absorption is checked by measuring the ΔA spectra from the neat solvent immediately prior or subsequent to the azide experiment under the same excitation conditions. We conclude that the short-time ΔA spectra (from -100 to 100 fs) are dominated (under our conditions: relatively low photon energy,
λ = 420, 350, and 305 nm) by cross-phase modulation and impulsive stimulated Raman exc 141
scattering, and, as the one pump/one probe photon process, vary approximately linearly with the pump intensity. The subsequent ΔA spectra (time delay, 200 fs and longer) for the neat solvent are due to the formation of product(s) via non-linear, typically, two-pump-photon absorption. As the result, when azides 25 and 65 is added to the solution, the excitation intensity is reduced by the solute absorption to the extent that the solvent contribution to the transient absorption measured after time delay of 200 fs becomes negligible.
The temporal evolution observed was deconvoluted by a sum of exponential functions with the time constants τi: ΔA(λt) = ∑ εi exp(- t/τι), where εi are the decay-associated spectra re- i
constructed from the resulting time constants based on the assumption of a consecutive reaction
mechanism. For each excitation wavelength used, a global fit4 to obtain the time constants
involved was performed on 512 kinetic traces within the measured 274-nm interval of probe
wavelengths (λ). The region from -125 fs to 125 fs was not used in the global fit because of the
solvent contribution to the measured ΔA spectra. A decay-associated spectrum defines the
absorption, which contributes to the recorded ΔA spectra, and which is characteristic of a
specific decay component obtained by a global fit. Also, a global fit assumes that the absorption
of products species changes only in their strength, not band shape, which may affect the resulting
time constants (τi) up to several picoseconds.
5.2 Calculations of fluorescence quantum yield of azide 65
In this study, diphenylantracene in spectroscopic grade ethanol at the maximal excitation
wavelength 355 nm was used as the standard (Φstd = 0.88).5 Samples were prepared in the
spectroscopic grade isopropanol and cyclohexane. To minimize inner filter effects, the optical 142
densities of samples at the excitation wavelength (λ = 355 nm) were kept in the range of 0.1–
0.12 (less than 0.150–0.165). The integrated area of fluorescent intensity for the standard and the samples was based on the area falling between 365 and 550 nm. The quantum yields were calculated taking into account the difference of the refractive index of solvents (ηisopropanol=
1.377, ηethanol= 1.36, ηcyclohexane= 1.426).
5.3 General Information
Reagents and anhydrous solvents were purchased from Aldrich, EMD, and were used
without further purification. The 4-fluoro-3-nitroaniline was purchased from Alfa. The 2-chloro-
5-nitropyridine and 5-amino-2-fluoropyridine were purchased from Oakwood Inc. All reactions
were conducted using oven-dried glassware under an atmosphere of nitrogen or argon.
Preparative TLC was performed on glass plates (Merck Kieselgel 60 F254; layer thickness, 0.25
and 0.2 mm). Products were purified via flash chromatography using 60 μm silica gel. 1H-NMR
13 and C NMR spectra were recorded on a 300 MHz Bruker spectrometer using CD3CN, CDCl3
as solvents. The chemical shifts (δ) are reported in parts per million (ppm) relative to the residual
1 13 CHCl3 peak (7.26 ppm for H-NMR and 77.0 ppm for C-NMR) and CD3CN peak (2.03 ppm
for 1H NMR and 118.06, 0.85 ppm for 13C NMR), and coupling constants (J) are reported in
Hertz (Hz). UV-Visible absorption spectra were measured with an Agilent 8453
spectrophotometer; IR spectra were measured with a ThermoNicolet IR 200 spectrometer. EI
mass spectra (70 eV) were measured in-house using a direct insertion probe in a Shimadzu
QP5050A spectrometer. Exact mass and MSn determinations were done in the mass spectrometry 143
facility in the Chemistry Department of the University of Cincinnati using a ThermoFinnigan
LTQ Linear Ion-Trap FTMS pESI instrument.
5.4 Synthesis of 4-(N,N-Diethylamino)-3-nitrophenyl azide (25)
Synthesis of 1-Fluoro-2-nitro-4-azidobenzene (13)6
N3 The diazonium salt was prepared from 4-fluoro-3-nitroaniline (24) (0.95 g, 6
mmol) dissolved in warm (40-50oC) concentrated hydrochloric acid (6 mL).
The amine solution was cooled to 5oC and a solution of sodium nitrite (0.5 NO2 mg, 7.5 mmol) in 4 mL of water added. The solution was stirred for 30 min. F 13 at ice/water bath temperature, and the resulting solution added dropwise to a cold solution (0ºC) of sodium azide (0.51 g, 7.5 mmol) in 10 mL of water. The light orange
crystals were formed immediately. The yield of 4-Fluoro-3-nitrophenylazide (13) was 73% (mp
6 1 = 54°C (lit. 53–55°C). H NMR (300 MHz, CD3CN): δ 7.72 (dd, J = 2.7 Hz, J = 6 Hz, 1H), 7.26
13 (m, 2H); C NMR (75.5 MHz, CD3CN): δ 137.25, 128.5 (d, J = 240 Hz), 125.35 (d, J = 30 Hz),
123.13, 118 (d, J = 90 Hz), 112.97. IR (KBr): 2121 (m) cm-1.
Synthesis of 4-N,N-Diethylamino-3-nitrophenyl azide (25)7
A solution of 4-fluoro-3-nitrophenyl azide (13) (1.82 g, 0.01 moles) and N3 diethylamine (7.3 g, 0.1 moles) was heated in acetonitrile (12 mL) at 40oC for
3 h. The crude product was purified by silica chromatography on a short
NO2 column of silica gel eluting with hexane to afford 4-N,N-diethylamino-3-
1 NEt2 25 nitrophenyl azide (25) as a red oil (1.57 g) in 67 %. H NMR (300 MHz, 144
CDCl3): δ 7.36 (d, J = 2.6 Hz,1H), 7.18 (d, J = 9 Hz, 1H), 7.11 (dd, J = 9 Hz, J = 2.6 Hz, 1H),
13 3.05 (q, J = 7.2 Hz, 4H), 1.05 (t, J = 7.2 Hz, 6H), C NMR (75.5 MHz, CD3CN) δ 145.08,
-1 -1 141.57, 133.24, 124.59, 123.07, 115.54, 47.80, 12.59; UV-Vis (CH3CN) λmax (nm) (ε, M cm ):
445 (1390); IR (KBr): 2117(m) cm-1.
5.5 General Procedure For Photolysis Of 4-(N,N-Diethylamino)-3-Nitrophenyl azide (25) In
Various Solvents
A solution of 20 mg of 25 in alcohol (5 mL) was flushed with nitrogen for 15 min and
irradiated for 4 h using 350 nm light in a Rayonet Photochemical reactor. The crude photolysis
mixture was concentrated to dryness in vacuum, leaving an oily residue, which was separated by
preparative TLC (hexane) to afford the aniline 28 and addition products.
5.6 Photolysis Products
a) Irradiation in MeOH.
Irradiation of 25 in methanol afforded 4-N,N-diethylamino-2-methoxy-3- NH2 1 OCH3 nitroaniline (26) as the major addition product (38%): H NMR (300 MHz,
CDCl3) δ 6.92 (d, J = 9 Hz, 1H), 6.75 (d, J = 9 Hz, 1H), 3.5 (s, NH2), 3.71(s,
NO2 3H), 2.9 (q, J = 7.2 Hz, 4H), 0.9 (t, J = 7.2 Hz, 6H); 13C NMR (75.5 MHz,
NEt2 CDCl3) δ 147.6, 137.9, 137.8, 134.3, 121.3, 116.7, 61.2, 49.6, 12.9; HRMS calcd for C11H18N3O3 (M + H) 240.13425, found 240.134817. 145
4-N,N-diethylamino-3-nitroaniline (28)
NH2 Trace amounts of 4-N,N-diethylamino-3-nitroaniline (28) (2%) were isolated:
1 H NMR (300 MHz, CDCl3): δ 7.34 (d, J = 9 Hz, 1H), 7.3 (d, J = 2.1 Hz, 1H),
NO2 7.22 (dd, J = 9 Hz, J = 2.1 Hz, 1H), 3.0 (q, J = 7.2 Hz, 4H), 0.9 (t, J = 7.2 Hz,
13 NEt2 6H); C NMR (75.5 MHz, CDCl3): δ 145.08, 141.57, 133.24, 124.59, 28 123.07, 115.54; 47.13, 12.59; Mass spectrum: m/z (relative intensity): 209(40),
194(47), 174(15), 162(30), 147(20), 134(80), 119(75), 92(18), 65(100); HRMS calcd for
C10H16N3O2 (M + H) 210.12425, found 210.12374.
b) Irradiation in n-BuOH
NH2 Irradiation of 25 in n-butanol afforded 4-N,N-diethylamino-2-n- O(CH ) CH 2 3 3 butoxy-3-nitroaniline as the major addition product (55%): 1H NMR
(300 MHz, CDCl3): δ 6.96 (d, J = 9 Hz, 1H), 6.84 (d, J = 9 Hz, 1H), NO2 3.98 (t, J = 7.2 Hz, 2H), 2.86 (q, J = 7.2 Hz, 4H), 1.67 (m, 2H), 1.41 NEt2 13 (m, 2H), 0.92 (t, J = 7.2 Hz, 6H), 0.85 (t, J = 7.2 Hz, 3H); C NMR (75.5 MHz, CDCl3): δ
145.73, 137.12, 135.93, 133.26, 120.01, 115.63, 79.46, 48.75, 28.02, 17.91 (2C), 11.59; Mass spectrum: m/z (relative intensity): 281(70), 266(20), 264(18), 190(90), 178(17), 163(40),
150(30), 135(17), 107(10), 79(20) UV-vis 280-300 nm; HRMS calcd for C14H23N3O3 (M + H)
282.18176, found 282.18121.
146
c) Irradiation in 2-Propanol
NH2 Irradiation in 2-propanol afforded 4-N,N-diethylamino-2-iso-
OCH(CH ) 1 3 2 proproxy-3-nitroaniline (30) as the major addition product (58%): H
NMR (300 MHz, CD3CN): δ 6.88 (d, J = 9 Hz, 1H), 6.76 (d, J = 9 Hz, NO2 1H), 4.4 (m, 1H), 4.25 (br.s, NH2), 2.9 (q, J = 7.2 Hz, 4H), 1.2 (d, J = NEt2 13 6 Hz, 6H), 0.9 (t, J = 7.2 Hz, 6H); C NMR (75.5 MHz, CD3CN): δ
142.1, 140.16, 134.54, 132.82, 120.87, 116.63, 75.61, 49.74, 21.70, 12.36; Mass spectrum: m/z
(relative intensity): 267(40), 225(10), 210(23), 190(100), 176(15), 163(30), 150(25), 148(15),
135(23), 121(20), 79(30).
NH2 The minor addition product was 4-N,N-diethylamino-6-iso- (H C) HCO 3 2 proproxy-3-nitroaniline (29) (1%): 1H NMR (300 MHz,
CD3CN): δ 7.16 (s, 1H), 6.58 (s, 1H), 4.5 (m, 1H), 3.2 (q, J = NO2 7.2 Hz, 4H), 1.26 (d, J = 6 Hz, 6H), 1.10 (t, J = 7.2 Hz, 6H); NEt2 Mass spectrum: m/z (relative intensity): 267(40), 264(10), 250(12), 224(5), 210(22), 208(100),
193(5), 191(22), 178(25), 166(15), 164(30), 150(50), 136(23), 121(15), 108(20), 94(10), 80(20).
d) Irradiation in t-BuOH
Irradiation of 25 in t-butanol afforded 4-N,N-diethylamino-2-tert-butoxy-3-nitroaniline as the
1 major addition product (36%): H NMR (300 MHz, CD3CN): δ 6.89 (d, J = 9 Hz, 1H), 6.76 (d, J
= 9 Hz,1H), 2.85 (q, J = 7.2 Hz, 4H), 1.27 (s, 9H), 0.9 (t, J = 7.2 Hz, 6H); 13C NMR (75.5 MHz, 147
NH2 CDCl3): ): δ 145.08, 137.34, 133.23, 132.11, 123.01, 120.28, 79.11,
OC(CH3)3 49.16, 28.56, 17.49; HRMS calcd for C14H23N3O3 (M + H) 282.18176,
found 282.18119. NO2
NEt2
e) Irradiation in Acetonitrile
NO2 NEt2
N NO2 N N NEt2
Et2N Et2N N NO 2 34 NO2 35
Irradiation in acetonitrile afforded two dimeric products 34 and 35.
1 34: 45% yield, H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 2.4 Hz, 1H), 7.95 (dd, J = 9 Hz, J =
2.4 Hz 1H), 7.15 (d, J = 9 Hz, 1H), 3.4 (q, J = 7.2 Hz = 7.2 Hz, 4H), 0.9 (t, J = 7.2 Hz, 6H) 13C
NMR (75.5 MHz, CDCl3) δ 160.73, 143.41, 141.8, 126.59, 121.95, 120.24, 46.12, 11.51 HRMS
1 calcd for C20H27N6O4 (M + H) 415.209379, found 415.20915; 35: 15% yield, H NMR (300
MHz, CDCl3) δ 8.05 (d, J = 9 Hz, 1H), 7.63 (d, J = 9 Hz, 1H), 3.5 (q, J = 7.2 Hz = 7.2 Hz, 4H),
0.9 (t, J = 7.2 Hz, 6H); 13C NMR (75.5 MHz, CDCl3) δ 162.73, 141.61, 138.48, 132.92, 131.26,
126.8, 46.03, 13.51; HRMS calcd for C20H25N6O4 (M + H) 413.193180, found 413.19317.
148
f ) Irradiation in Toluene
CH2Ph A solution of 20 mg of 25 in toluene (8 mL) was flushed with nitrogen for 15 HN min and irradiated for 4 h using 350 nm light in a Rayonet Photochemical
reactor. The crude photolysis mixture was concentrated to dryness in vacuo,
NO2 leaving an oily residue, which was separated by preparative TLC (hexane:
dichloromethane in ratio 3:1) to afford the three major products (28, 35 and NEt2 benzyl amine 36 in the ratio respectively 32:20:48). Benzyl amine 36 (33%): 1H NMR (300
MHz, CDCl3): δ 7.35-7.25 (m, 5H), 7.44 (d, J = 2.1 Hz, 1H), 7.23 (dd, J = 9 Hz, J = 2.1 Hz, 1H),
6.86(d, J = 9 Hz, Hz, 1H), 4.49 (broad NH), 4.38 (s, 2H), 3.0 (q, J = 7.2 Hz, 4H), 0.9 (t, J = 7.2
Hz, 6H); HRMS calcd for C17H21N3O2 ( M+H ) 300.17065; found 300.17064.
5.7 General Procedure for the Photolysis of 4-(N,N-diethylamino)-3-nitrophenyl azide (25) in the presence of Targeted Functional groups
A solution of 20 mg of 25 and 200 mg of the molecule containing the targeted functional
group in acetonitrile (7 mL) was flushed with nitrogen for 15 min and photolyzed for 4 h using
350 nm light in a Rayonet Photochemical reactor. The crude photolysis mixture was
concentrated to dryness in vacuo, extracted with water and CH2Cl2, dried over Na2SO4, leaving
an oily residue, which was purified by preparative TLC (hexane/dichloromethane) to give aniline
6 and addition product as yellow oils.
149
a) Irradiation in presence of Phenol
Irradiation in the presence of phenol afforded the phenol adduct in NH 2 the 2-position 40 in 98% yield as an oil that had: 1H NMR (300 O MHz, CDCl3) δ 7.37-7.29 (m, 2H), 7.18 (d, J = 9 Hz, 1H), 7.09 (t,
NO2 J = 7.2, Hz, 1H), 6.8 (d, J = 9 Hz, 1H), 6.91 (dd, J1 = 7.2, J2 = 0.9
Hz, 2H), 4.3 (s, NH2), 2.9 (q, J = 7.2 Hz, 4H), 0.9 (t, J = 7.2 Hz, NEt2 13 6H); C NMR (75.5 MHz, CDCl3) δ 171.4, 156.44, 139.1, 138.4, 132.6, 129.39, 122.87, 122.63,
114.66, 49.36, 12.93; HRMS calcd for C16H19N3O3 (M + H) 302.14989, found 302.150467.
b) Irradiation in the presence of Dimethylamine Hydrochloride
Irradiation in the presence of dimethylamine hydrochloride afforded the NH2 amine adduct 41 in the 2-position in 98% yield as an oil that had: 1H NMR NMe2
(300 MHz, CDCl3) δ 6.92 (d, J = 9 Hz, 1H), 6.75 (d, J = 9 Hz, 1H), 4.2 (s,
NO2 13 NH2), 2.9 (q, J = 7.2, 4H), 2.71 (s, 6 H), 0.9 (t, J = 7.2 Hz, 6H); C NMR NEt 2 (75.5 MHz, CDCl3) δ 147.6, 137.9, 137.8, 134.3, 121.3, 116.7, 61.2, 49.6,
12.9; HRMS calcd for C12H20N4O2 (M + H) 253.16590, found 253.166451.
150
5.8 Acetylation of Adducts 29 and 30
The mixture of addition products 29 and 30, isolated from the irradiation of 25 in 2- propanol, and acetic anhydride (150 mL) were dissolved in THF (10 mL) and heated at 60oC for
3 h. The crude product mixture was separated and purified by silica gel chromatography on a short column using hexane and dichloromethane as eluants. Two products were obtained: the acetamide of the minor addition product 32, and the acetamide of the major addition product 31.
NHAc Acetamide of 32 was isolated as a yellow solid in 2% yield:
(H3C)2HCO 1 H NMR (300 MHz, CDCl3) δ 9.84 (s, 1H), 6.58 (s, 1H), 4.62
(m, 1H), 3.19 (q, J = 7.2 Hz, 4H), 2.2 (s, 3H), 1.4 (d, J = 7.2, NO2 13 6H), 1.2 (t, J = 7.2 Hz, 6H); C NMR (75.5 MHz, CDCl3) δ NEt2 171.3, 147.9, 143.22, 133.8, 124.3, 121.3, 116.7, 105,11, 71.1, 47.15, 21.98, 12.67; HRMS calcd for C15H24N3O4 (M + H) 310.1767, found 310.1765.
NHAc Acetamide 31 was isolated as a yellow solid in 96%: 1H NMR (300
OCH(CH3)2 MHz, CDCl3) δ 8.28 (d, J = 9 Hz, 1H), 7.58 (s, NH), 7.02 (d, J = 9
Hz, 1H), 4.32 (m, 1H), 2.9 (q, J = 7.2 Hz, 4H), 2.2 (s, 3H), 1.29 (d, J NO2 13 = 7.2, 6H), 0.9 (t, J = 7.2 Hz, 6H); C NMR (75.5 MHz, CDCl3) δ NEt2 168.01, 145.18, 139.81, 138.70, 129.43, 122.05, 120.33, 80.01, 48.99, 24.6, 22.78, 12.72; HRMS calcd for C15H24N3O4 (M + H) 310.176682, found 310.1767.
151
5.9 Synthesis of 5-azido-2-(N,N-diethylamino)pyridine
Synthesis of 5-Azido-2-Fluoropyridine (44)
The diazonium salt was prepared from 5-amino-2-fluoropyridine (1.2 g, 10 mmol) N3 dissolved in warm (40-50oC) concentrated hydrochloric acid (10 mL). The amine
o N solution was cooled to (-15 C) and a solution of sodium nitrite (0.8g, 13 mmol) in 1 mL of water added. The solution was stirred for 10 min. at (-10oC), and added F 44 dropwise to the cold solution 0º C of sodium azide (0.77 g, 13 mmol) in 1 mL of
water. After 10 min. of stirring, solution was neutralized by NaHCO3, and then extracted with
dichloromethane. The yield of 5-Azido-2-Fluoropyridine (brown oil) is 62 %. H NMR (300
MHz, CD3CN): δ 7.87 (d, J = 2.5 Hz, 1H), 7.38 (m, 2H), δ 6.87 (dd, J = 9 Hz, J’ = 2.5 Hz, 1H);
13 C NMR (75.5 MHz, CD3CN): 128.5 (d, J = 240 Hz), 125.35 (d, J = 30 Hz), 123.13, 118 (d, J =
90 Hz), 112.97. IR (KBr): 2110 (m) cm-1
Synthesis of 2-(N,N-diethylamino)-4-nitropyridine (63)
NO2 A solution of 2-chloro-5-nitropyridine (5.6 g, 36 mmoles) and diethylamine (10 g, 1.44 moles) was stirring in ethanol (12 mL) at 30oC for 4 h. The precipitation
N was filtered and washed by cold ethanol. 2-N,N-diethyl-4-nitropyridine was
1 obtained as a bright yellow crystals in 98% yield. H NMR (300 MHz, CD3CN): NEt2 63 δ 9.05 (d, J = 2.7 Hz,1H), 8.19 (dd, J = 9 Hz, J’ = 2.7 Hz, 1H), 6.43 (d, J = 9 Hz,
13 1H), 3.65 (q, J = 7.2 Hz, 4H), 1.25 (t, J = 7.2 Hz, 6H), C NMR (CD3CN, 300 MHz) δ 159.49,
147.08, 134.28, 132.67, 103.99, 43.53, 12.72; UV-Vis (CH3CN) λ max= 400nm. 152
Synthesis of 5-amino-2-(N,N-diethylamino)pyridine (64)
NH2 1) Starting material 63 (200mg, 1mmol) was dissolved in 5 ml of ethanol.
Round-bottom flask with condenser was flushed with argonfollowed by
N hydrogen. The reaction mixture was stirred overnight and it changed color to
dark gray. The solution was filtered, and then the EtOH was removed under NEt2 reduced pressure to afford a purple oil in 92%.
2) The nitropyridine (0.7g, 3.37 mmole) was dissolved in MeOH, and three equvalents of
stannous chloride dihydrate(2.46 g, 0.01 mole) was added. Reaction mixture was stirred for 2
days at 450C. Then, the solution was neutralized with sodium bicarbonate and was extracted with
1 dichloromethane to afford a purple oil in 87%. H NMR (300 MHz, CD3CN): δ 7.76 (d, J = 3.3
Hz,1H), 6.96 (dd, J = 9 Hz, J’ = 3.3 Hz, 1H), 6.39 (d, J = 9 Hz, 1H), 3.45 (q, J = 7.2 Hz, 4H),
13 1.21 (t, J = 7.2 Hz, 6H), C NMR (CD3CN, 300 MHz) δ 152.62, 135.58, 131.79, 127.18,
106.32, 42.67, 13.07; HRMS calcd for C9H16N3 (M + H) 166.13387, found 166.13389
Synthesis of 5-azido-2-(N,N-diethylamino)pyridine (65)
N3 The diazonium salt was prepared from 5-amino-2-(N,N-diethylamino)pyridine
(64) (1.5 g, 9 mmol) dissolved in warm (40-50oC) solution of hydrochloric acid
N o (4 mL) in H2O (10 mL). The amine solution was cooled to 0 C and a solution of
sodium nitrite (0.8 g, 13 mmol) in 4 mL of water added. The solution was NEt2 65 stirred for 10 min. at 0oC, and added dropwise to the cold solution (0ºC) of sodium azide (0.77 g,
13 mmol) in 10 mL of water. After 10 min. of stirring, solution was neutralized by NaHCO3, and then extracted with dichloromethane. The crude purple product was purified by silica 153
chromatography on a short column eluting with hexane to afford 4-azido-2-N,N- diethylaminopyridine as a yellow oil (1.1 g) in 72 %. H NMR (300 MHz, CDCl3): δ 7.88 (d, J =
3.3 Hz, 1H), 7.11 (dd, J = 9 Hz, J’ = 3.3 Hz, 1H), δ 6.47 (d, J = 9 Hz, 1H), 3.5 (q, J = 7.2 Hz,
13 4H), 1.25 (t, J = 7.2 Hz, 6H), C NMR (300 MHz, CD3CN): δ 155.30, 139.09, 128.19, 123.98,
-1 106.03, 42.76, 12.89; UV-Vis (CH3CN) λmax = 280 nm, 340 nm IR (KBr): 2094.8 (m) cm ;
HRMS calcd for C9H14N5 (M + H) 192.12439, found 192.12437.
5.10 Photolysis Products
a) Irradiation in MeOH
Irradiation of azide 65 in methanol afforded 3-amino-6-N,N-diethylamino- NH2 1 2-methoxypyridine as the major addition product (48%): H NMR (CDCl3, OCH3
300 MHz) δ 6.92 (d, J = 9 Hz, 1H), 5.92 (d, J = 9 Hz, 1H), 3.5 (s, NH2), N 3.92(s, 3H), 3.5 (q, J = 7.2 Hz, 4H), 1.22 (t, J = 7.2 Hz, 6H); 13C NMR
NEt2 (CDCl3, 300 MHz) δ 150.6, 148.9, 126.8, 119.1, 94.5, 61.2, 49.6, 12.9
b) Irradiation in n-BuOH.
Irradiation of azide 65 in n-butanol afforded 3-amino-2-n-butoxy-6-(N,N-diethylamino)pyridine
1 as the major addition product (67%): H NMR (300 MHz, CD3CN): δ 6.85 (d, J = 9 Hz, 1H),
5.91 (d, J = 9 Hz, 1H), 4.24 (t, 2H), 3.4 (q, J = 7.2 Hz, 4H), 1.7 (m, 2H), 1.4 (m, 2H), 1.16 (t, J =
13 7.2 Hz, 6H), 0.9 (t, J = 7.2 Hz, 3H); C NMR (300 MHz, CD3CN): δ 150.76, 149.03, 125.05, 154
NH2 119.26, 96.31, 64.37, 42.10, 29.59, 18.81, 12.12; UV-vis 280-300
O(CH2)3CH3 nm; HRMS calcd for C13H23N3O (M + H) 238.19126, found
N 238.19139.
NEt2
c) Irradiation in i-PrOH (69)
Irradiation of azide 65 in 2-propanol afforded 3-amino-6-(N,N- NH2 diethylamino)-2-isopropyloxypyridine 69 as the addition product OCH(CH3)2 1 (59%): H NMR (300 MHz, CD3CN): δ 6.88 (d, J = 9 Hz, 1H), 5.82 N (d, J = 9 Hz, 1H), 5.2 (m, 1H), 3.40 (q, J = 7.2 Hz, 4H), 1.28 (d, J = 6
13 NEt2 69 Hz, 6H), 1.18 (t, J = 7.2 Hz, 6H); C NMR (300 MHz, CD3CN): δ
151.33, 150.06, 139.03, 132.82, 96.22, 67.40, 42.74, 22.39, 12.96; HRMS calcd for C12H21N3O
(M + H) 223.16846, found 223.16807; Mass spectrum: m/z (relative intensity): 223(29), 208(06),
182(07), 181(32), 166(100), 152(25), 138(18), 124(05), 110(11), 93(3), 79(20), 54(29)
d) Irradiation in tert-BuOH
Irradiation of azide 65 in tert-butanol afforded addition product 71
NH2 1 (56%) : H NMR (300 MHz, CDCl3): δ 6.73 (d, J = 9 Hz, 1H), 5.3 (d, OC(CH3)3 J = 9 Hz, 1H), 3.5 (q, J = 7.2 Hz, 4H), 1.25 (s, 9H), 1.1 (t, J = 7.2 Hz,
N 13 6H); C NMR (300 MHz, CDCl3): δ 151.31, 142.03, 121.19, 115.08,
89.15, 61.82, 42.77, 24.68, 12.5; HRMS calcd for C13H23N3O (M + NEt2 H) 238.19126, found 238.19139. 155
e) Irradiation in imidazole x HCl/Acetonitrile
A solution of 20 mg of 65 and 200 mg of the imidazole x HCl in N NH 2 acetonitrile (7 mL) was flushed with nitrogen for 15 min and photolyzed N for 2.5 h using 350 nm light in a Rayonet Photochemical reactor. The N crude photolysis mixture was concentrated to dryness in vacuo, extracted
with water and CH2Cl2, dried over Na2SO4, leaving an oily residue. NEt2 1 Addition product 72 (61%): H NMR (300 MHz, CDCl3): δ 8.1 (s, 1H), 7.51 (s, 1H), 7.18 (s,
1H), 7.11 (d, J = 8.7 Hz, 1H), 6.51 (d, J = 8.7 Hz, 1H), 3.43 (q, J = 7.2 Hz, 4H), 3.20 (br.s, NH2),
13 1.16 (t, J = 7.2 Hz, 6H); C NMR (500 MHz, CDCl3): δ 153.17, 151.42, 130.44, 122.92,
118.35, 106.00, 100.55, 42.74, 12.96. Mass spectrum: m/z (relative intensity): 231(29), 216(35),
204(23), 188(32), 175(22), 160(28), 148(12), 132(18), 107(12), 86(59), 84(126), 66(32), 51(88)
HRMS calcd for C12H17N5 (M + H) 232.15622, found 232.15707.
e) Irradiation in Cyclohexane
A solution of 20 mg of 65 in cyclohexane (8 mL) was N NEt2 flushed with nitrogen for 15 min and irradiated for 3 h N using 350 nm light in a Rayonet Photochemical reactor. N The crude photolysis mixture was concentrated to
1 Et2N N dryness in vacuo, leaving an oily residue. Dimer 73: H
NMR (CDCl3, 300 MHz) δ 8.663 (d, J = 2.4 Hz, 1H), 7.95 (dd, J = 9 Hz, J = 2.4 Hz 1H), 6.49
13 (d, J = 9 Hz, 1H), 3.6 (q, J = 7.2 Hz, 4H), 1.25 (t, J = 7.2 Hz, 6H) C NMR (CDCl3, 300MHz) δ 156
159.73, 149.61, 141.3, 127.5, 116.2, 42.74, 12.96; HRMS calcd for C18H27N6 (M + H)
327.22917, found 327.22901; 82% yield.
5.11 References
(1) El-Khoury, P. Z.; Tarnovsky A. N. Chem. Phys. Lett., 2008, 453, 160-166.
(2) Kovalenko, S. A.; Dobryakov, A. L.; Ruthmann, J. Ernsting, N. P. Phys. Rev. A 1999,
59, 2369-2382. Rasmusson, M.; Tarnovsky, A. N.; Åkesson, E.; Sundström, V. Chem. Phys.
Lett. 2001, 335, 201-208.
(3) Kovalenko, S. A.; Ernsting, N. P.; Ruthmann, J. Chem. Phys. Lett. 1996, 258, 445-454.
(4) Van Stokkum, I. H.; Larsen D. S.; van Grondelle, R. Biochim. Biophys. Acta 2004,
1657, 82-104.
(5) Hamai, S.; Hirayama, F. J. Phys. Chem. 1983, 87, 83-89.
(6) (a) Leyva, E.; Munoz, D.; Platz, M.S., J. Org. Chem. 1989, 54, 5938-5945, (b)
Hagedorn, M.; Sauers, R. R.; Eichholz, A. J. Org. Chem. 1978, 43, 2070-2072.
(7) Lormann, M. E. P.; Walker, C. H.; Es-Sayed, M.; Braese, S. Chem. Com 2002, 12,
1296-1297.
157
APPENDIX. 1H NMR AND 13C NMR SPECTRAL CHARACTERIZATION
158
159
160
161
162
163
164
165
166
167
168
169
170
Proton NMR of crude mixture
171
172