Cryogenic Spectroscopy of Anionic Biochromophores
by
Wyatt Zagorec-Marks
B.S. University of Utah

A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillmentof the requirements for the degree ofDoctor of Philosophy
Department of Chemistry2021
Committee Members:
Carl Lineberger
Heather Lewandowski
J. Mathias Weber
Sandeep Sharma
Veronica Vaida

Zagorec-Marks, Wyatt (Ph.D., Chemistry, Department of Chemistry)
Cryogenic Spectroscopy of Anionic Biochromophores
Thesis directed by Professor J. Mathias Weber

Abstract
Biochromophores are ubiquitous and have the potential to be modified to suit our needs inmany applications involving the absorption or emission of visible light. Many chromophores havealready been adapted for this purpose and leading to radical changes in their field, such as theimplementation of the chromophore of the Green Fluorescent Protein in bioimaging. Not only arethese molecules incredibly useful and versatile, they are also interesting from a more fundamental
standpoint. They all involve the excitation of electrons residing in conjugated π systems, and boththe excited states and the resulting de-excitation dynamics are influenced by the structure of thechromophore and the functional groups present. A thorough understanding of these properties willaid in their modification.
This thesis presents my contribution to the understanding of several biochromophores. The
first chapter presents a more in-depth introduction to the field and my place in it. This is followed
by a chapter detailing the methodology. The next two chapters focus on two examples of a common
functional group in chromophores, the phenolate moiety. This group consists of a deprotonatedphenol ring with another functional group branching off the ring, typically in the para- position.
In the first of these chapters, the functional group is an NO2 entity, and we explore substitution
effects by comparing electronic spectra from the ortho-, meta-, and para- isomers. In the second
chapter we look at the chromophore of the Green Fluorescent Protein including experiments on
the temperature dependence as well as the effects of microsolvation on the electronic spectra. Iniithe last two chapters we look at a second common structural motif, tetrapyrrole. This structure
consists of four pyrrole rings linked by methine bridges, with the conjugated system spanning all
four rings. The first of these chapters details the properties of protoporphyrin, in which the four
pyrrole rings form a conjugated ring. Here we investigate the effect of a strong electric field on thephotophysical properties. The final chapter details ongoing experiments on a linear tetrapyrrole
known as biliverdin.

iii

For Livi

iv

Acknowledgements
While the molecules in this thesis were in vacuo, I certainly was not. I received help,
guidance, and support from a wide variety of people who deserve mention. First, I have to thankMathias for everything he has done for me as an advisor. When I originally visited CU Boulder, it
wasn’t on one of the prescribed graduate student visiting weekends (due to a scheduling conflict).
I arrived in town and soon learned that my plans to visit had been misplaced in all the excitement
surrounding the hectic event that is graduate recruiting, and no arrangements for faculty meetings,
or housing, had been made. I reached out to several of the faculty I had been hoping to meet withto see if they had been contacted at all, and if they had any free time in their schedule to meet withme. Mathias responded quickly, was available to meet, and was immediately contacting therelevant administrators to get things sorted out. This sort of behavior is characteristic of his
managing style. He addresses any problems efficiently, always provides useful feedback, andmaintains a positive group environment. This is an incredibly important aspect of advisors that is
typically not emphasized enough.
While Mathias is an important aspect of the Weber group, he is not the only important part.
I have had the privilege to overlap with many fantastic people during my time in the group, all ofwhich I think of as friends first and coworkers second. When I first joined the group, two graduate
students, James E. T. Smith and “Xavier” Shuang Xu, were leaving the group and I had minimal
interaction with them at that time. I would later collaborate with James on the protoporphyrin
project where his expertise and positive attitude were very helpful. There were two other graduate
students, Michael Thompson and Curtis Beimborn, who were not on my project and so provided
a way to escape the day to day struggles. We frequently had hilarious conversations over lunches
or coffee breaks the made every day much more enjoyable. Curtis and I would eventually coauthorv

a paper which required frequent ski trips to discuss the nature of the reflection of sun light off acrystalline surface, such as snow, and the implications for nanosecond laser experiments on
crystalline rubrene. The final member of the Weber group when I joined was a post-doc named
Leah Dodson. Typically, Mathias will recruit a new student for a project when the current student
on the project is in their 3rd/4th year and knows what they are doing, creating a good amount ofoverlap with the new student to transfer knowledge. That schedule did not work out when I joined.
Instead I got to work with Leah, who had been on the project for a few months and was stilllearning how to operate what was, at the time, a very temperamental instrument. The skills one
arrives with as a first year graduate student are typically limited, to say the least. Mine were not
only limited, but they were also only applicable to biochemistry. I knew nothing about vacuum
technology, oscilloscopes, wrenches, screwdrivers, or really anything that would be helpful on this
instrument. Leah was able to not only teach me these basic things, but she did so in a way that hadme operating and troubleshooting the instrument by myself in a relatively short amount of time.
My graduate experience would have had a significantly rockier start had it not been for her
patience, good sense of humor, and willingness to stand back let me try to fix things. When it cametime for me to train my replacement, Madison Foreman and Rebecca Hirsch joined the group.
Madison would be working with me and she brought some much needed enthusiasm to a catalysis
project which had been on ice for some time. I had a wonderful time training her and am sure her
graduate career will be filled with excitement. I overlapped less with Rebecca, as she was on theother low pressure experiment, but because of the culture of the Weber group we still enjoyedmany lunches, hiking trips, coffee breaks, etc. together and I wish her luck with her graduate career
as well. None of this work would be possible without our funding sources. I was fortunate enoughto be funded by the Department of Education through a GAANN fellowship, and the Nationalvi

Science Foundation through the JILA AMO Physics Frontier Center (award no. PHY-1734006)
and through an NSF Chemistry grant (award no. CHE-1764191). I also had access to resourcesfrom the University of Colorado Boulder Research Computing Group, which is supported by the
National Science Foundation (awards ACI-1532235 and ACI-1532236), the University ofColorado Boulder, and Colorado State University
There are many people outside the Weber group that deserve acknowledgement as well.
Every member of the machine shop staff has fielded countless questions from me and have always
been incredibly nice and exceedingly helpful. I also had a significant amount of help from thosein the electronics shop, as much of our instrument is run by devices they have made. They arealways very willing to look at a device that may be misbehaving, help you troubleshoot, provide
potentially life-saving tips for working with high voltage after you have broken a high voltage
switch. I need to acknowledge James Fung-A-Fat specifically for help with that last item. With his
help, the switch was replaced without incident. Like many experiments, much of this experimentrelies on old (ancient?) computers talking to each other, running specific programs, and surviving
long enough to be useful. None of these things would happen were it not for the work of thecomputing staff here at JILA. Last but not least, there are a plethora of people in the JILA
administration who work tirelessly to make sure graduate students can focus on research. These
people do their various jobs so well that most students never have to interact with them at all.
I would also like to thank a few professors outside of JILA. First, my undergraduate
research professor Matthew Kieber-Emmons who guided me through my first experiences inscientific research and who is undoubtedly the reason I ended up in a graduate program at all. I
also need to thank Jan R. R. Verlet who worked in our group as a visiting professor for a few
months and brought with him the molecule which would lead to nearly half of my publications.

vii

He also taught me that while choosing interesting projects is important, it is also good to
occasionally pick the low lying fruit (in this case, a very juicy pineapple).
I also need to thank my family for their constant support and interest in what I was doing,even if they didn’t really understand the specifics. They certainly made it easier to get through thisprogram, and remain interested in science even when it wasn’t cooperating.
Lastly, I need to thank my wife, Livi. Rather than being worried about me moving out ofstate for a graduate program, she encouraged it and, without hesitation, packed up her things andbrought her dog. Throughout my life and graduate career, she has been an excellent sounding
board, source of stress relief, and genuinely curious about how things went in the lab each day.
She been an incredible motivator by reminding me that no matter how well or terribly things go in
the lab, I am still a wonderful trophy husband. For these things, I am eternally grateful.

viii

CONTENTS

Chapter 1 Introduction .................................................................................................................... 1
1.1

Why study biochromophores? .......................................................................................... 1

1.2

Why in vacuo? .................................................................................................................. 3

1.3

Scope of this thesis ........................................................................................................... 5

Chapter 2 Methods .......................................................................................................................... 6
2.1

Overview .......................................................................................................................... 6

2.2

Ion preparation ................................................................................................................. 7

2.2.1

Electrospray ionization ............................................................................................. 7

2.2.2

Pre-trap ion guides .................................................................................................... 8

2.2.3

Cryogenic ion trap................................................................................................... 10

2.3

Time-of-flight mass spectrometry .................................................................................. 13

2.4

Photodissociation spectroscopy...................................................................................... 16

2.5

Laser system ................................................................................................................... 17

2.5.1

IR light source ......................................................................................................... 17

2.5.2

UV-Vis light source ................................................................................................ 18
ix

2.6

Data collection................................................................................................................ 19

2.7

Computational ................................................................................................................ 20

2.8

References ...................................................................................................................... 22

Chapter 3 Nitrophenolates, a ‘simple’ model system ................................................................... 25
3.1

Introduction .................................................................................................................... 25

3.2

Computational ................................................................................................................ 27

3.3

Results and discussion.................................................................................................... 28

3.3.1

Overview ................................................................................................................. 28

3.3.2

para-Nitrophenolate ................................................................................................ 31

3.3.3

ortho-Nitrophenolate............................................................................................... 34

3.3.4

meta-Nitrophenolate ............................................................................................... 36

3.3.5

Ion temperature estimation ..................................................................................... 44

3.3.5

Charge transfer character of NP excitations ........................................................... 47

3.4

References ...................................................................................................................... 51

Chapter 4 The Green Fluorescent Protein Chromophore ............................................................. 54
4.1

Introduction .................................................................................................................... 54

4.2

Computational ................................................................................................................ 57

4.3

The isolated chromophore .............................................................................................. 57

4.4

Effects of microsolvation ............................................................................................... 68

4.5

Hot band assisted infrared spectroscopy ........................................................................ 85x4.6

References ...................................................................................................................... 96

Chapter 5 Protoporphyrin ........................................................................................................... 104
5.1

Introduction .................................................................................................................. 104

5.2

Computational .............................................................................................................. 107

5.3

Discussion .................................................................................................................... 108

5.4

References .................................................................................................................... 123

Chapter 6 Biliverdin .................................................................................................................... 128
6.1

Overview ...................................................................................................................... 128

6.2

Computational .............................................................................................................. 129

6.3

Discussion .................................................................................................................... 130

6.4

References .................................................................................................................... 145

Chapter 7 Bibliography ............................................................................................................... 148

xiFigures

Figure 1.1. Image of a ficus lyrate (fiddle-leaf fig) from my personal collection. The green
coloration is due to various chlorophylls which perform the initial absorption of sunlight inphotosynthesis. ................................................................................................................................ 2
Figure 1.2. Image of organ pipe coral of the genus Tubipora from my personal collection. The redcoloration is due to a combination of biochromophores, including carotenoids which produce thebright orange color of carrots. ......................................................................................................... 3
Figure 2.1. Cartoon of the electrospray source. .............................................................................. 8
Figure 2.2. Instrument drawing of the source region from the desolvation capillary to the 3D
quadrupole trap. ............................................................................................................................ 10
Figure 2.3. Zoom in on 3D quadrupole trap and nearby ion optics. ............................................. 11
Figure 2.4. Diagram of the mass spectrometer. ............................................................................ 16
Figure 3.1. Structures and resonances of meta, para, and ortho isomers of NP, adapted from Wanko
et al.10 ............................................................................................................................................ 26
Figure 3.2. Electronic spectra showing the S1 transition for m-, p-, and o-NP with a trap temperatureof 30 K. In the traces of m- (top) and p-NP (middle), full lines connect the data points. In the caseof o-NP, the data points are shown as open circles with a full line representing a 15-point gliding
average to guide the eye. ............................................................................................................... 29
Figure 3.3. Electronic spectrum of p-NP in the band origin region. ............................................. 32
Figure 3.4. Potential energy surface of the S0 and S1 states in the FC region of p-NP. The blackarrow indicates a vertical excitation. Calculations for the surface were performed at the CAM-

xii

B3LYP/aug-ccpVDZ level of theory using a relaxed scan over the nitro torsion (tors.) andpyramidalization (pyr.) coordinates. ............................................................................................. 33
Figure 3.5. Potential energy surface of the S0, S1, and S2 states in the FC region of o-NP. The redarrow indicates a vertical excitation. Calculations were performed at the same level as for p-NP.
....................................................................................................................................................... 35
Figure 3.6. The top panel contains the experimental electronic spectrum in the band origin regionof m-NP (a), compared to FC simulations at 80 (b) and 0 K (c) which have been shifted match theexperimental band origin. The lower panel contains only the first 2400 cm-1 above the band origin,along with assignments of selected peaks (d); a more extensive assignment is given in Table 3.2.
Hot bands are marked in red, with red asterisks labeling peaks whose primary contribution comesfrom a hot band with one quantum in the nitro torsion vibration. ................................................ 37
Figure 3.7. Potential energy surface of the S0 and S1 states in the FC region of m-NP. The red arrow
indicates a vertical excitation. Calculations were performed at the same level as for p-NP. ....... 38
Figure 3.8. Potential energy scans of m-NP along the torsion (tors.) coordinate of the S0 and S1
states using CAM-B3LYP/aug-cc-pVDZ and a NO2 pyramidalization angle constrained to zero(corresponding to a planar nitro group). Energies are given relative to the minimum energy
structure for each surface, in cm−1. ............................................................................................... 40
Figure 3.9. Temperature dependent spectra of m-NP in the band origin region. Spectra have beennormalized to the peak absorption of the band origin at 16003 cm-1............................................ 45
Figure 3.10. Frontier orbitals for m-, p-, and o-NP, calculated using CAM-B3LYP. Both meta andpara isomers exhibit pure HOMO  LUMO transitions for S0  S1. The S0  S1 transition for
o-NP is HOMO  LUMO+2, while the S0  S2 transition for o-NP is a mixture of HOMO−1 
LUMO+2, HOMO−1  LUMO+6, and HOMO−3  LUMO+2. .............................................. 48

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Figure 3.11. Electron density difference plots for m- (top), p- (center), and o-NP (bottom) in the
FC region. Purple regions indicate an increase of electron density upon electronic transition, while
yellow regions indicate a decrease of electron density upon electronic transition. The density
difference for o-nitrophenolate was calculated for the S1 state. Listed are the charges on the NO2
and O groups for each isomer in the S0 and S1 states. Charges calculated using a Natural Population
Analysis, given in e. ...................................................................................................................... 50
Figure 4.1. Deprotonated p-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI-) as the Z- andE- isomer. The two isomers are related by rotation about the carbon double bond of the methine
bridge. ........................................................................................................................................... 55
Figure 4.2. Electronic spectrum of [HBDI·N2]-. The open circles are raw data points, the purple
full line is a 5-point adjacent average to guide the eye. The black full lines are Franck-CondonHerzberg-Teller (FCHT) simulations of the vibrational substructure in the S1  S0 electronic band,with the vibrational states indicated as vertical sticks. The insert shows the band origin region, andthe data in this trace were taken with a step size of 2.6 cm-1, while the step size in the overview
spectrum was 13 cm-1 in the origin region. The FCHT simulation has been shifted by ca. -4000
cm-1 for the best match to the experimental spectrum (see section 4.2). ...................................... 59
Figure 4.3. Infrared spectrum of [HBDI·N2]- (top trace) compared to simulated spectra of the Z
isomer (center trace) and E isomer (bottom trace) of HBDI-. The calculations have been scaled by
0.98. A detailed assignment of the vibrational features is listed in Table 4.1. ............................. 60
Figure 4.4. Comparison of the experimental spectrum of [HBDI·N2]- with calculated spectra fromthe literature. The calculated curves have all been shifted to optimally match the experimental data.
The literature data have been adapted from the references.24, 38-39 ............................................... 63xiv

Figure 4.5. Electronic spectra of HBDI- at various trap temperatures. The open circles are raw data
points, the full lines are adjacent averages to guide the eye, with 3, 5 and 20 points for [HBDI·N2], 30 K and the other experimental conditions, respectively. The vertical dashed and dotted lines
show the peak positions for bare HBDI- (30 K) of the (v=0)  (v=0) band at (20800 ± 10) cm-1,
and of the high energy feature around 22100 cm-1, respectively. ................................................. 64
Figure 4.6. Electronic spectrum of [HBDI·(N2)2]-. Open circles represent the raw data, the purple
line is a 5-point gliding average to guide the eye. ........................................................................ 66
Figure 4.7. Comparison of the experimental photofragment action spectrum of HBDI- at 30 K trap
temperature (blue curve) with the parent ion depletion spectrum taken at the same temperature(black curve). ................................................................................................................................ 68
Figure 4.8. Infrared spectra of [HBDI·N2]- and [HBDI·(H2O)n]- (n = 1, 2) from 1000 to 2000 cm1. The photofragment signals were due to the loss of N2 for [HBDI·N2]- and of a single H2O

molecule for [HBDI·(H2O)n]-, respectively. ................................................................................. 69
Figure 4.9. Calculated structures for all solvated species. Monohydrated species (left) are shownwith energy relative to (Ia), dihydrate species (right) are presented with energy relative to (IIa).
All energies are calculated at 0 K. ................................................................................................ 71
Figure 4.10. IR spectra of of [HBDI-·N2] (top, black) and [HBDI-·(H2O)n] (n = 1, 2 magenta andblue) from 2700 to 3800 cm-1. The photofragment loss channels are the same as in the low energy
region. ........................................................................................................................................... 74
Figure 4.11. Calculated infrared spectra of selected conformers of monohydrated HBDI- (lower
traces, see Figure 4.9 for other conformers), compared to the experimental spectrum (upper trace).
The spectrum contains signatures for CH stretching vibrations (CH), H-bonded OH stretching on
the phenolate (red-shaded region) and imidazole (blue-shaded region) groups, and the free OH

xv

stretching mode (F). All calculations have been scaled by 0.957 to match the experimentallyobserved free OH stretching vibrations. The calculated energies (at 0 K) of the conformers relative
to the lowest energy conformer (Ia) are given in each simulated trace. ....................................... 76
Figure 4.12. Calculated hydrogen bonded OH stretching frequency as a function of the in-plane
pivot angle for both a phenolate bound and imidazole bound water molecule. ........................... 77
Figure 4.13. Calculated hydrogen bonded OH stretching frequency as a function of the out-ofplane pivot angle for both a phenolate bound water molecule. .................................................... 78
Figure 4.14. Calculated IR spectra of several conformers of [HBDI·(H2O)2]- (lower traces),compared to the experimental spectrum (upper trace). The points in the upper trace are raw data,
the full black line is an 11-point gliding average to guide the eye. The spectrum contains signatures
for CH stretching vibrations (CH), OH stretching H-bonded to the phenolate (red-shaded region)and imidazole (blue-shaded region) groups, and the free OH stretching mode (F). All calculations
have been scaled by 0.957 to match the experimentally observed free OH stretching vibrations.
The calculated energies (at 0 K) of the conformers relative to the lowest energy conformer (IIa)are given in each simulated trace. Arrows in the lowest trace show how the infrared features ofconformer IIa change as the H-bond between the water molecules is lost (see text). Arrows in the
trace for conformer IId show the expected analogous behavior upon breaking the water-water Hbond for a conformer with both water molecules on the imidazole. The full and dashed lines in the
trace for conformers IIc and IIc’ correspond to the water molecule positions indicated by full anddashed circles, respectively. .......................................................................................................... 80
Figure 4.15. Electronic spectra of HBDI- at 30K and [HBDI·(H2O)n]- (n = 1, 2). The photofragment
signals were the loss of a methyl group for bare HBDI-, and of all H2O molecules forxvi

[HBDI·(H2O)n]-, respectively. The vertical dashed line marks the band origin of bare HBDI- at
20930 cm-1. ................................................................................................................................... 83
Figure 4.16. From top: Absorption spectrum of wild-type GFP at 77 K (green); photodissociation
spectra of HBDI- at 30K (black) and [HBDI·(H2O)n]- (n = 1, 2, magenta and blue) in the visible
spectral region; UV/vis absorption spectrum of aqueous HBDI- at pH 11. All traces werenormalized to the same peak height. Photofragment signals were the loss of a methyl group forbare HBDI-, and of all H2O molecules for [HBDI·(H2O)n]-, respectively. The vertical dashed line
marks the band origin of bare HBDI- at 20930 cm-1. The absorption spectrum of wild-type GFP
was digitized from ref.48 ............................................................................................................... 85
Figure 4.17. Excitation schemes for resonant IR-UV 2-photon photodissociation spectroscopy.
Fragmentation occurs upon excitation to state |𝑓. ........................................................................ 87
Figure 4.18. UV-Vis spectra of HBDI- at 300K (red trace) and 30K (blue trace) in the band origin
region. The dots are data points, the lines are a 5-point adjacent average (300 K) and an Akima
spline (30 K) to guide the eye. The dashed vertical line shows the band origin at 20930 cm-1. Notethat no IR irradiation was done to acquire these spectra. ............................................................. 88
Figure 4.19. Electronic spectra in the S1 band origin region of HBDI- at 30, 180, and 300 K trap
temperatures. Full lines are spectra without (black) and with (red) resonant IR irradiation at 1536
cm-1; open triangles show the difference between the two signals, where the difference values for
300 K have been amplified by a factor 3 for clarity. .................................................................... 90
Figure 4.20. Infrared spectra of HBDI- under different conditions. The lowest traces are simulated
spectra for the dominant Z isomer (full black line) and the E isomer (dotted black line). The othertraces show experimental infrared spectra using N2 tagging and HAIR data for different trap
temperatures as indicated on the traces. Open circles are raw data points, the full lines are 5-pointxvii

adjacent averages. The vertical lines show the calculated band positions of the dominant band for
the Z and E isomers. The HAIR spectra were acquired at 20580 cm-1, 20160 cm-1, and 19800 cm1

for 30 K, 180 K, and 300 K trap temperature, respectively. ...................................................... 92

Figure 4.21. Constant volume heat capacity (black trace) and average vibrational internal energy(red trace) of HBDI- as a function of temperature. The circles and curved arrows show the change
in molecular temperature upon absorption of a 1000 cm-1 infrared photon, starting from 30 K (blue)and from 300 K (orange). The horizontal arrows indicate which curve belongs to which verticalscale............................................................................................................................................... 94
Figure 5.1. D4h and D2h macrocycle structures and their schematic electronic spectra for a general
metalloporphyrin (left) and protoporphyrin IX (right). .............................................................. 106
Figure 5.2. Electronic spectrum of [(PP–H)·N2]-, showing the Qx and Qy absorption bands. The Qx
band origin is at 15868 ±10 cm-1, and the Qy band origin is at 18760 ± 30 cm-1. Both band origins
exhibit a qualitatively similar vibronic progression which extends ca. 2000 cm-1 above each origin.
..................................................................................................................................................... 109
Figure 5.3. The experimental Qx absorption band of [(PP–H)·N2]- (blue) compared with the FCHT
simulation of neutral porphine (black) and FCHT simulation (using the vertical gradientapproximation) of (PP-H)- (green). All calculations (ground and excited state) were done at the
CAM-B3LYP/cc-pVDZ level of theory using a scaling factor of 0.97. The open circles in theexperimental trace are data points and the full line is a B-spline to guide the eye. The assignments
are based on the porphine simulation. The simulations have been shifted to align with theexperimental band origin. ........................................................................................................... 111

xviii

Figure 5.4. IR spectra of [(PP-H)·N2]- (blue) and [(PP-2H)·N2]2- (purple) in the fingerprint region
from 1000 cm-1 to 2200 cm-1. Each trace is accompanied by a simulated spectrum (scaling factor
0.95) for the structure shown. ..................................................................................................... 114
Figure 5.5. IR spectra of [(PP-H)·N2]- (top panel) and of [(PP-2H)·N2]2- (bottom) in the CH andOH stretching region. .................................................................................................................. 117
Figure 5.6. Electronic spectrum of [(PP-H)·N2]- (blue) and [(PP-2H)·N2]2- (purple) in the Q band
region. Dashed vertical lines mark the positions of the Qx and Qy band origins of the singly
deprotonated species to emphasize the differences. ................................................................... 119
Figure 6.1. Examples of conformer families of singly deprotonated biliverdin, [BV-H]-, with theremaining proton shared between the carboxylate groups of the propionic acid tails. Numbering
the carbon atoms in the conjugated system as shown in structure H, the abbreviated Z/E labels referto the bonds between carbon atoms 4/5, 9/10, and 14/15 in sequence. The abbreviated syn/antilabels encode the conformations with respect to the bonds between the carbon atoms 5/6, 10/11,
and 15/16. .................................................................................................................................... 129
Figure 6.2. Top: IR spectrum of [(BV-H)·N2]-, monitoring the loss of N2. Center: IR spectrum of[(PP-H)·N2]-, monitoring the loss of N2. Bottom: predicted IR spectrum of conformer J (see Figure1J), with frequencies scaled by 0.9375. ...................................................................................... 131
Figure 6.3. IR spectrum of [(BV-H)·N2]- in the 2800 – 3600 cm-1 spectral region, monitoring theloss of N2, compared to computational IR spectra of selected conformers from conformer families
as shown in Figure 6.1, with frequencies scaled by 0.9392. The insert shows a zoom of the NH
stretching region with assignments. ............................................................................................ 135

xix

Figure 6.4. Electronic spectrum of [(BV-H)·N2]-, monitoring the loss of N2 (black trace) and theUV-vis spectra of BV dissolved in H2O at pH 13 (blue trace). The black, open circles are raw data
points, the full black line is a 20-point gliding average to guide the eye. .................................. 137
Figure 6.5. Comparison of IR spectra of [(BV-2H)·N2]-2 with simulated spectra for three selectconformers from 1000-1800 cm-1. The vertical dashed lines indicate the location of prominent
features in the experimental spectrum. Simulated spectra have been scaled by 0.9325. ............ 139
Figure 6.6. Comparison of IR spectra of [(BV-2H)·N2]-2 with simulated spectra for three selectisomers from 2800-3600 cm-1. The vertical dashed lines indicate the location of prominent featuresin the experimental spectrum. Simulated spectra have been scaled by 0.96. ............................. 140
Figure 6.7. Electronic spectrum of [(BV-2H)·N2]-2. As was the case for [(BV-H)·N2]-, the vibronic
progression is unresolved despite being taken at a trap temperature of 20 K. The main absorption
feature occurs at 10800 cm-1 with a shoulder at +1650 cm-1 and a third feature at 14200 cm-1. 142
Figure 6.8. Electronic spectrum of the band origin region of [(BV-2H)·N2]-2 (top trace) and [(BV2H)·(H2O)n]-2 for n=1-4 (lower traces). The black circles are data points, the red lines are 10 point
adjacent averages to guide the eye. ............................................................................................. 144

xx

Tables

Table 3.1. Calculated S1 and S2 properties (vertical excitation energies, band origins, and oscillator
strengths) compared with experimental data. Calculations shown here were performed with the
CAM-B3LYP functional and aug-cc-pVDZ basis set. Transitions are given in cm-1. ................. 30
Table 3.2. List of calculated harmonic vibrational frequencies (cm−1) and symmetries for o-, m-,and p-NP. Note that the point group is Cs for o- and m-NP in both the X (ground) and A (excited)state. For p-NP, the X state belongs to C2v, while the A state belongs to Cs. ............................... 43
Table 4.1. Vibrational mode assignments for [HBDI·N2]- based on the calculated spectrum of theZ isomer. ....................................................................................................................................... 61
Table 4.2. Vibrational mode assignments for [HBDI-·N2] and [HBDI-·(H2O)n] (n = 1, 2). ......... 70
Table 4.3. Calculated selected charges (in e) and bond lengths (in Angstroms) in HBDI- anddifferent position isomers for [HBDI·(H2O)n]- (n = 1, 2); isomer labels refer to Figure 4.9. ....... 72
Table 5.1. Calculated and experimental vertical transition energies for PP and porphine in various
charge states expressed in cm-1. .................................................................................................. 120
Table 5.2. Solvatochromic data for PP (transition energies in cm-1) .......................................... 122

xxi

Chapter 1 Introduction
1.1

Why study biochromophores?

A molecule is considered a chromophore if it absorbs visible light, including the moleculesthat make bruises blue-green, oranges orange, and ladybugs red, to name a few examples.
Biochromophores are those which have biological origins. These molecules typically have a highdegree of conjugation and either act alone, or as a cog in a larger machine (such as those present
in the chlorophylls of plants) and are often housed in a protein pocket. These protein pockets willoften hold a chromophore in a specific configuration, which may not be stable in solution, andprovide a specific chemical environment to tune the photophysical properties (absorption
spectrum, fluorescence quantum yield, etc.) of the chromophore to suit the needs of the process
they are involved in.

1

Figure 1.1. Image of a ficus lyrate (fiddle-leaf fig) from my personal collection. The green
coloration is due to various chlorophylls which perform the initial absorption of sunlight inphotosynthesis.

Nature has developed a wide variety of chromophores and variants thereof to accomplisha plethora of tasks. Researchers have been able to coerce some of these chromophores to perform
other tasks, and one of the best examples of this is the use of the Green Fluorescent Protein (GFP).
This protein was originally isolated from the jellyfish Aequorea victoria and has since become awork-horse in fluorescence labeling in biology and biochemistry (which lead to the Nobel Prize in
Chemistry in 2008). The fluorescence of GFP originates from a relatively small chromophore
consisting of a phenol ring linked to an imidazole ring. This chromophore has been modified, bothby nature and by researchers, to emit at a variety of wavelengths.

2

Figure 1.2. Image of organ pipe coral of the genus Tubipora from my personal collection. The redcoloration is due to a combination of biochromophores, including carotenoids which produce thebright orange color of carrots.
In order to expand the capabilities of chromophores, a great deal of research has gone into
understanding how they work. Most of these experiments have been performed on chromophores
in the condensed phase (e.g., in solution, crystals, or a protein environment), which makes sense
as that is where they are found and will be used. The present work involves the application of laser
spectroscopy to cryogenically prepared ionic biochromophores in vacuo.

1.2

Why in vacuo?
When studying chromophores in the condensed phase, one must be cognizant ofinteractions between the molecule of interest and the chemical environment. Electronic transitionenergies of the same molecule dissolved in different solvents, or housed in a protein pocket, canvary significantly (solvatochromism). These shifts can range from barely noticeable to thousands3

of cm-1, and can result in higher or lower transition energies. This makes it important to specify
the solvent, as well as characteristics such as pH, when reporting an electronic transition insolution. Speciation in solution can be an issue if there are breakdown products, mixtures ofisomers, varying degrees of protonation, or aggregation effects to name a few situations. Whentaking spectra of such a solution, one acquires the response from every species present, which canbe challenging, if not impossible, to disentangle. Together, these effects, among others, can makeit difficult to compare experimental data to theoretical results, or even to other experimental data.
Having experimental data on the intrinsic photophysical properties of a chromophore (i.e.,
independent of its chemical environment) is highly desirable when one is looking to modify its
properties, but this is impossible to achieve in the condensed phase. Studying molecules in vacuo
allows investigation of their properties as an isolated system, free from any interaction with achemical environment, and provides clean access to their intrinsic photophysics. By looking ationic chromophores, we are able to employ mass spectrometric techniques and select ions by theirmass-to-charge ratio, eliminating many problems that can arise in the condensed phase due tospeciation.
Spectra taken in solution are often plagued by other effects that limit the detail that can beextracted from them. A typical solution UV/vis spectrum is taken at room temperature and consistsof broad and featureless absorption bands, which makes a detailed understanding of the underlying
photophysics inaccessible. To gain valuable information from a spectrum, one must be able toresolve individual transitions which typically requires substantially cooling the molecule ofinterest to suppress the broadening induced by thermal excitation (hot bands). This can be achievedin frozen solutions, but here one encounters matrix effects, which additionally complicate
interpretation. We are able to cool molecules in vacuo to cryogenic temperatures using buffer gas

4cooling techniques, which, in cases where temperature was a significant cause of the spectral
broadening, can greatly improve spectral resolution.

1.3

Scope of this thesis
This thesis involves infrared (IR) and electronic photodissociation spectroscopy ofcryogenically prepared, mass-selected ions in vacuo used to investigate the intrinsic electronic andmolecular structure of anionic biochromophores. The experimental methods are described in detailin Chapter 2. Chapters 3 and 4 will explore molecules containing a phenolate moiety, a functionalgroup observed in many chromophores. Nitrophenolates will be the subject of Chapter 3, where
we will observe the power of the experimental technique on a nearly ideal molecular system.
Chapter 4 describes work on the GFP chromophore. It also introduces the idea of a microsolvation
environment (where the ion is solvated by only a few solvent molecules), which can be thought ofas bridge between experiments done in vacuo and those done in solution. Chapters 5 and 6 discuss
tetrapyrroles, which consist of four pyrrole rings linked by methine bridges, and occur in a wide
range of biological systems. Protoporphyrin, where the pyrrole rings form a macrocycle, is the
subject of Chapter 5. In addition to the spectroscopy of this molecule, we explore the effect of theelectric field of a point charge located a few Å away from the conjugated system. Ongoing workon biliverdin is discussed in Chapter 6. Biliverdin is structurally similar to protoporphyrin, as it
has similar building blocks, but the conjugated macrocycle found in porphines is broken, forming
a linear tetrapyrrole instead. In this chapter, we again explore the effect of a negative charge near
the conjugated system of a very promising near-IR emitter which may not be being used to its fullpotential.

5Chapter 2 Methods
2.1

Overview
The experiments described here were done using a home built photodissociation mass

spectrometer.1 In a typical experiment, the ions are initially generated through electrosprayionization using either water, methanol, or a mixture of the two as the solvent. The ions are thenguided through a series of differential pumping stages by octopole ion guides and lenses until theyreach a 3D quadrupole ion trap (Paul-trap). This trap is mounted on a closed cycle helium cryostat
and is equipped with a resistive heater to allow control of the cold head temperature from 4-300
K. Ions are held in this trap with an inert buffer gas for an amount of time dependent on therepetition rate of the experiment (45 or 95 ms for 20 Hz or 10 Hz, respectively). During this time,the ions are colliding with the buffer gas molecules, which are in turn colliding with the walls of
the trap, cooling the ions. Three body collisions in the cold buffer gas can lead to formation of
weakly bound clusters between the ions of interest and the buffer gas. These clusters are often
useful in our experiments, as will be discussed in greater detail below. Once the ions are cooled,they are ejected as a packet into a time-of-flight mass spectrometer. After a few s flight time, thepacket separates by mass-to-charge ratio (m/z) and encounters a pulsed mass gate, which allows
transmission of a particular m/z, while discarding the rest. The selected ion species is thenirradiated by the output of a tunable light source (600-4000 cm-1 for IR spectroscopy, 220-2500
nm for UV-Vis spectroscopy). Absorption of a photon (or in some cases a few photons) places theion in an excited state with few relaxation options. If the ions absorb enough energy, the ions candissociate into fragments. A reflectron is used to separate the fragments from any parent ions thathave not dissociated, and the formation of fragments is monitored as a function of the laser

6

wavenumber. Results are compared to calculations performed on the RMACC Summit
supercomputing cluster at CU Boulder.2

2.2

Ion preparation

2.2.1 Electrospray ionization
Many biochromophores exist as charged species which enables the use of mass
spectrometric techniques to study them. A widely used method of getting biologically relevantmolecules into the gas phase is to use electrospray ionization (ESI).3 This method is often chosenbecause it can get large, fragile molecules into the gas phase without fragmenting them. The
experimental setup involves taking a molecule of interest dissolved in a volatile, polar solvent suchas water or methanol, and spraying the solution out of a small highly charged needle (ca. -3-4 kV)
aimed at a desolvation capillary that serves as a counter electrode (ca. -300 V) and provides theentry into the vacuum system of the apparatus. The potentials given here are typical for anionicspecies, but we note the apparatus can also produce cationic species in which case the polarity isswapped to produce positive values. In our experiment (see Figure 2.1), the spray assembly is acommercial product (Analytica of Branford), and the solution is pumped through a silica capillary(75 m ID, 193 m OD, Polymicro Technologies) inside the needle at a rate of ca. 50 L/h by asyringe pump (New Era, model NE-1000). At the tip of the silica capillary, a cone forms as a resultof surface tension of the solution, the Coulomb repulsion of the dissolved ions, and the electricfield of the needle attempting to expel the ions (Taylor cone). The tip of this cone emits a fine mistof small, highly charged droplets. As solvent evaporates from these droplets, the density of charged
species increases, leading to the eventual breakdown of the droplets. This process repeats itself
until all that remains are bare ions in the gas phase. This spray is entrained in a nebulizing gas of7

dry N2. The desolvation capillary is heated to aid in evaporation of any remaining solvent
molecules as ions pass through it into the first differential pumping stage of the experiment.

Figure 2.1. Cartoon of the electrospray source.

2.2.2 Pre-trap ion guides
The electrospray process occurs at ambient pressure and time-of-flight mass spectrometry(TOF-MS) requires high vacuum conditions (<10-6 mbar). A series of four differential pumping
chambers are used to step the pressure down to this level. For the ions, the first chamber comprises
a ca. 1 cm flight distance from the exit of the capillary, through a focusing lens, and into a skimmer
that separates the first chamber from the second. The first chamber is pumped by a 65 m3/h rotary
vane pump (Leybold, Trivac D65 B) and operates at ~1 mbar. The second chamber contains anoctopole ion guide that is ~14 cm long, at the end of which is another skimmer. The chamber ispumped by a second 65 m3/h rotary vane pump (Leybold, Trivac D65 B) and reaches pressures of~0.1 mbar. The second skimmer is pulsed from a high potential (ca. -100 V) to a lower potential(ca. -50 V). While the skimmer is held at the high potential, this region of the experiment8

accumulates ions until the skimmer is pulsed to the low potential. We refer to this region as the“pre-trap” and it allows us to take the continuous source of ions that ESI generates and create apulsed source. A pulsed source is advantageous as it provides a more uniform cooling time for theions when they reach the cryogenic trap. After passing through the second skimmer, the ions passinto a third chamber which contains a ~30 cm long octopole ion guide. The third chamber ispumped by a 685 L/s turbomolecular pump (Pfeiffer HiPace 700) and reaches operating pressuresof 10-4 mbar. An electrostatic lens bridges the gap between the third and fourth differential
pumping chamber where the ion packets are guided by another ~30 cm long octopole into a homebuilt quadrupole bender. The bender turns the ion path by 90 degrees to separate out any remaining
neutrals before the ions pass through a set of electrostatic lenses and a final octopole before they
enter the 3D quadrupole trap. This chamber is pumped by a 1100 L/s turbomolecular pump
(Leybold Turbovac 1000 C) and has a typical operating pressure of 10-6 mbar.

9Figure 2.2. Instrument drawing of the source region from the desolvation capillary to the 3D
quadrupole trap.

2.2.3 Cryogenic ion trap
Our 3D quadrupole trap was modeled after a stainless-steel trap purchased from Jordan
TOF products but was instead machined from copper for better thermal properties. The trap ismounted on the cold head of a closed cycle helium cryostat (Sumitomo F-70L, 1.5 W at 4.2 K)
and is equipped with a resistive heater to allow temperature control from 4 K to 300 K. The trap
is enclosed in an OFHC copper heatshield which is mounted to the outer stage of the cold head,keeping the heatshield at ca. 40 K. Helium buffer gas is admitted into this trap at the same time as
the ions by a pulsed valve (Parker General Valve Series 99). Depending on the repetition rate of aparticular experiment, 10 Hz or 20 Hz, the ions are held in the trap for 45 or 95 ms. During this10

time, the ions collide with the buffer gas, which is in turn colliding with the cryogenic walls of thetrap, cooling the ions.

Figure 2.3. Zoom in on 3D quadrupole trap and nearby ion optics.

As the ions cool, complexes with other molecules in the trap can be formed in three-bodycollisions. In the experiments described in this thesis, complexes with N2, D2, and H2O werestudied. N2 and water molecules are part of the residual gas from the ESI source. Complexes with
D2 are formed by seeding the helium buffer gas with D2 (10%). These complexes form over uniquetemperature ranges, which are determined by the balance of their temperature dependent vapor
pressure, and the binding energy of the complexes (temperatures leading to internal energies higher
than the binding energy of the complex lead to a dissociation event). This complexation isdemonstrated by dodecanedioic acid dianion (DDDA2-) in Figure 2.4, which we often employ for11diagnostic purposes. All of these complexes can be useful in photodissociation spectroscopy,
which will be discussed in section 2.4. The choice of running an experiment at 10 Hz or 20 Hz
depends on several factors. 10 Hz allows more cooling time, and thus the stored ions get colder
resulting in larger complexes with weakly bound molecules. For some molecules which are
reluctant to form complexes at all, using a lower repetition rate can be critical to forming them.
The advantage of operating at 20 Hz is a reduction in the time required to collect data. This can bedone when the ion signal is intense enough, and any desired complexes still form despite theshorter cooling period.

12

DDDA2- m/z 214

ion signal [arb. units]

20 Hz

D2

120

N2

140

10 Hz

160

180

220

200

240

260

280

300

m/z

Figure 2.4. Mass spectrum of dodecanedioic acid dianion (DDDA2-) taken at 33 K trap
temperature and a repetition rate of 20 Hz (top) and 10 Hz (bottom). All other parameters are
kept constant. This molecule complexes with D2 and N2 at this temperature at both repetition
rates, but significantly more complexes are observed with a 10 Hz repetition rate, indicative of alower ion temperature.

2.3

Time-of-flight mass spectrometry
After the ion packets have cooled, and formed any desired complexes, they are ejectedfrom the trap by pulsed potentials on the endcaps of the cryogenic trap, creating a ~50 V/cm
potential gradient across the trap pushing the ions out through an exit lens. The contents of thecryogenic trap drift into series of three electrodes, one grounded and two with pulsed potentials
which are initially held at ground. The two later electrodes are pulsed to ~300 V and ~4000 V in a13

Wiley-McLaren arrangement.4 When the ion packet is between the first and second electrode. The
resulting electric field accelerates the ion packets into the final set of chambers of our experiment,
the first of which is a differential pumping stage pumped by a 230 L/s turbomolecular pump(Pfeiffer TMU261) and operating at 10-7 mbar. In this chamber, we have a set of x and y deflectors
which consist of 2 sets of metal plates each, as well as an Einzel lens to adjust the ion trajectories.
The second chamber is the flight tube of the TOF-MS, which is pumped by a 520 L/sturbomolecular pump (Pfeiffer TMU521) and operates at 10-8 mbar.
If one assumes that the drifting packet of ions all have identical starting positions, thenevery ion in the packet receives the same amount of initial kinetic energy (KE) from theacceleration pulse. This can be expressed as:
𝐾𝐸 =

𝑚 2
𝑣 =𝑈
2𝑧

Where m/z is the mass-to-charge ratio of the ion in question, v is the velocity, and U is the potential
difference between the plates causing the acceleration. Assuming a constant flight length L for allions (about 2 m in this experiment) a relationship between the m/z of a pair of ions and their flighttimes assuming the same initial KE is given by:
𝑚1 𝑡1
=
√
𝑚2 𝑡2
This gives a simple way to assign a mass spectrum assuming one knows the m/z of at least one
feature in the spectrum. Of course the underlying assumption that all the ions have identical starting
positions is not true in any experimental situation, and in particular ours where the ions have drifted
ca. 2 cm to arrive at the acceleration region. The packet that is actually accelerated is moreaccurately represented as a cloud of ions spread between the two electrodes.

14In a Wiley-McLaren setup, ions closer to the first electrode receive a higher initial KE thanthose closer to the second electrode when the voltages are pulsed on. All of the ions receive thesame, and substantially larger, KE from the potential drop across electrodes 2 and 3. The difference
in initial KE incurred in the first region results in a slightly higher velocity for ions that were closer
to the first electrode and a point in space where eventually these ions overtake those that started
closer to the second electrode. The location where this occurs can be tuned by adjusting thepotentials of the first two electrodes and is called the space focus of the mass spectrometer. In our
experiment, the space focus occurs at the laser interaction region, where we have a microchannel
plate (MCP) detector that can be moved into the ion beam for diagnostic purposes. Just before thelaser interaction region, we have a home built mass gate which is a collection of parallel shimelectrodes that can be biased with alternating polarity. The mass gate is pulsed such that when an
ion of interest impinges upon it the potentials of all shims are held at ground, and the rest of thetime they are held at +/-100 V to cause ions to be deflected, preventing their detection. This allows
us to select a particular ion by its mass-to-charge ratio to continue to ion detection, while removing
all others. All of this is contained in our flight tube which is the final chamber of the experiment.
Ions that have been selected to pass through the mass gate are irradiated by the output of
our laser system (see section 2.5), and the photodissociation products continue to drift until theyencounter a two-stage reflectron (Jordan TOF), which reflects the ions towards another MCP
detector, giving the ions flight path a “U” shape. The reflectron has three purposes in thisexperiment. It nearly doubles the flight distance, which leads to an increase in the resolving powerof the mass spectrometer. It also allows us to separate fragment ions formed before entering thereflectron from any parent ions who did not dissociate. Finally, after the ions pass through the laserinteraction region where they were tightly focused they will become unfocused again. The

15

reflectron introduces a second focal point to the trajectory of the ions that is tuned to occur at thedetection MCP.

Figure 2.4. Diagram of the mass spectrometer.

2.4

Photodissociation spectroscopy
Photodissociation spectroscopy involves a molecule absorbing a photon and subsequently

dissociating. In our experiment, the laser light is introduced at the first space focus of our WileyMcLaren setup. If the ions absorb an incident photon (or several depending on the molecule, laser
intensity, and experiment) the energy is redistributed throughout the modes of the molecule
producing a vibrationally “hot” ion with no solvent bath to dissipate this excess energy. One way
the ions can dissipate the energy is to dissociate. In the case of UV-Vis experiments, with photon
energies on the order of a couple eV, a single photon (or multiple) can cause dissociation of some16

chemical bonds. In the case of IR experiments, photon energies are substantially lower, in therange of 1000-4000 cm-1. If one has access to a more intense light source, such as a free electron
laser5-7 it is possible to get absorption of many photons and break chemical bonds with IR photons.
We rely instead on a technique referred to as messenger tagging. This entails forming clusters of
the ion of interest with weakly bound messenger tags, such as N2 or D2 whose binding energy tothe ion is on the order of a few hundred cm-1.8-9 Using messenger tagging provides a low energy
pathway to dissociation while introducing minimal perturbation to the molecular system. The
degree of this perturbation is investigated in Chapter 4 where another path to dissociation is
explored. As discussed earlier, we also form clusters with water molecules which, while binding
energies are typically weaker than chemical bonds (ca. 0.5 eV), can have significant perturbations
on the ion of interest which is also visited in Chapter 4. If dissociation occurs before the ions reachthe reflectron (ca. 10 μs after irradiation) the reflectron is used to separate the fragments from theundissociated parent ions, which would otherwise have the same velocity and thus the same arrival
time at a detector in a linear detection setup.

2.5

Laser system

2.5.1 IR light source
The IR light source used in these experiments is an IR optical parametric converter (OPC)
system (Laservision) which produces 900 to 4000 cm-1 light with linewidth of ~2 cm-1 and a pulse
duration of 5 ns. The system is pumped by a Nd:YAG laser (Innolas Spitlight 600, 5 ns pulse, 500
mJ/pulse, 1064 nm) which is run at half the repetition rate of the mass spectrometer for reasons
discussed in Section 2.6. The output of the 1064 nm pump beam is split into two beams, one ofwhich is frequency doubled to produce 532 nm light, which then pumps an optical parametric17

oscillator (OPO) consisting of two potassium titanyl phosphate (KTP) crystals. This generates asignal and idler wave (νsignal 710–880 nm, and νidler 1345–2122 nm). The photon energy andmomentum of these waves adds up to the 532 nm pump energy and momentum. The signal
wavelength and any residual 532 nm light is removed using a Si filter which is transparent to theidler beam. The idler beam propagates through a ʎ/2 wave plate, rotating its polarization by 90º,and then mixes with the second 1064 nm pump beam in an optical parametric amplifier (OPA),
which consists of a set of four potassium titanyl arsenate (KTA) crystals. The idler beam from the
OPO and the 1064 nm pump beam are difference-frequency mixed to create mid-IR light (νmid)from 2100 to 4000 cm-1. Any residual 1064 nm pump is removed from the beam using a dichroic
beam splitter. Depending on the experiment, the idler beam is either removed by a polarizer at thispoint, or the mid IR and idler beam are routed together into a silver gallium selenide (AgGaSe 2)
crystal, where difference frequency mixing of the beams produces our far-IR (νfar) beam withwavenumbers of 900 to 2150 cm-1. Residual mid-IR and idler light is removed using a germanium
filter. Because ambient water and CO2 are strong IR absorbers in the spectral region of this system,
the entire beamline is enclosed and continuously purged with dry nitrogen.

2.5.2 UV-Vis light source
Like the IR light source, the UV-Vis source in these experiments is an OPC system (GWU)
which produces irradiation from 220 – 2500 nm with a linewidth of 5 cm-1 and a pulse duration of
5 ns. The system is pumped by the third harmonic (355 nm) of another Nd:YAG laser (Innolas
Spitlight 600, 5 ns pulse, 100 mJ/pulse, 355 nm) which is run at half the experimental rep. rate forreasons discussed in section 2.6. The 355 nm pump beam is introduced to one of two Type II beta
barium borate (BBO) crystals that convert the pump beam into a signal and idler beam under18

photon energy and momentum conservation. One of the BBO crystals, referred to as the “red”
crystal, generates a signal beam from 505-710 nm and an idler from 710-1190 nm. The othercrystal, referred to as the “blue” crystal, generates a signal beam from 415-515 nm and an idler of1140-2450 nm. The output of the OPO passes through a UV filter to remove any residual pump
beam. In order to generate UV irradiation, an additional set of BBO crystals can be installed after
the OPO. With the output of only the OPO, second harmonic generation can be performed togenerate 220-257 nm light from the “blue” OPO setup or 257-354 nm from the “red” OPO setup.
One can also use sum frequency mixing (SFM) with 1064 nm pulses from the pump laser togenerate 299-350 nm or 350-404 nm irradiation from the “blue” and “red” OPO crystals,
respectively. Any light generated in the OPC system passes through a Pellin-Broca prism to allow
selection of a specific wavelength to route to the molecular beam apparatus. All other beams are
dumped before exiting the OPC system.

2.6

Data collection
All ion detection is done using an MCP detector after the reflectron (see section 2.3). The

signal generated is visualized on two oscilloscopes (Tektronix TDS 2022). These oscilloscopes
and the laser system in use are triggered at half the experimental repetition rate of the mass
spectrometer (10 Hz for a 20 Hz experiment, 5 Hz for a 10 Hz experiment). One of theoscilloscopes is triggered in phase with the laser system, while the other is triggered out of phase,providing us a display of the ion signal with and without laser irradiation. The intensity of the ion
signal is averaged over 16 shots per data point to reduce noise. When collecting data, we set theoscilloscopes to display only the fragment peak and monitor the intensity of this peak as a functionof laser wavelength, using the oscilloscopes as boxcar averagers for the ion signals with and19without laser irradiation. The IR laser pulse energy is monitored by reflecting a small portion of
the beam into a pyroelectric joule meter using a KBr window before the beam enters the vacuum
apparatus. The UV-Vis laser pulse energy is also monitored by a pyroelectric joule meter, but thisis placed at the exit window of the vacuum apparatus and is thus monitored after interacting with
the ions. Absorption of laser light by the ions of interest is too low to have a detectable effect (dueto the low density of ions, 100-104 ions per ion packet) on the measured laser pulse energy, someasurement before and after interaction of the ions yields the same result. The laser pulse energy
is monitored by the second channel of the oscilloscope that measures the “laser on” signal. Whilethe ion signal and laser pulse energy are being monitored, the wavelength of the OPC system in
use is scanned through the desired tuning range by a LabVIEW program, which is simultaneously
collecting output from the oscilloscopes. We subtract the “laser off” signal from the “laser on”
signal for the fragment peak and then correct for photon fluence by dividing the signal intensity
by the photon number per pulse as a function of photon energy. Individual scans are collected forat least 2 days to verify reproducibility (often more time is often required to achieve an adequate
signal-to-noise ratio).

2.7

Computational
Calculations are used in all our experiments in order aid in the interpretation of spectra. Allof our calculations were performed using Gaussian 16.10 Specifics of calculations vary byexperiment, but in general we use density functional theory (DFT, typically B3LYP11 or the range
corrected CAM-B3LYP12, but occasionally ω-B97XD13) to calculate ground state geometries andharmonic frequencies which we then compare to experimental IR spectra. In many cases we
employ time-dependent DFT to calculate excited state structures in efforts to interpret UV-Vis

20

spectra. Initial structural guesses are often based on chemical intuition or previous experiments
but these frequently had to be modified to reach a structure that best reproduced the experimental
results. The basis sets employed are reported in the relevant sections, but they include def2TZVPP14 and cc-pVDZ15.

21

2.8

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Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrosprayionization for mass spectrometry of large biomolecules. Science 1989, 246 (4926), 64.

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Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J. Gaussian 16 Rev. C.01,
Wallingford, CT, 2016.

11.

Becke, A. D., Density-Functional Exchange-Energy Approximation with Correct
Asymptotic-Behavior. Phys. Rev. A 1988, 38 (6), 3098-3100.

23

12.

Yanai, T.; Tew, D. P.; Handy, N. C., A new hybrid exchange–correlation functional using
the Coulomb-attenuating method (CAM-B3LYP). Chemical Physics Letters 2004, 393 (13), 51-57.

13.

Chai, J.-D.; Head-Gordon, M., Long-range corrected hybrid density functionals withdamped atom–atom dispersion corrections. Physical Chemistry Chemical Physics 2008, 10(44), 6615-6620.

14.

Weigend, F.; Ahlrichs, R., Balanced Basis Sets of Split Valence, Triple Zeta Valence andQuadruple Zeta Valence Quality for H to Rn: Design and Assessment of Accuracy. Phys.
Chem. Chem. Phys. 2005, 7 (18), 3297-3305.

15.

Scolaro., L. M.; Castriciano., M.; Romeo., A.; Patane., S.; Cefali., E.; Allegrini., M.,
Aggregation Behavior of Protoporphyrin IX in Aqueous Solutions: Clear Evidence of
Vesicle Formation. J. Phys. Chem. B 2002, (106), 2453-2459.

24Chapter 3 Nitrophenolates, a ‘simple’ model system

This chapter has been adapted from Ref.1 with permission from the PCCP Owner Societies.

3.1

Introduction
The phenolate moiety is a chemical structure observed in many biochromophores such as

the chromophore of the Photoactive Yellow Protein,2 Luciferase,3 Rhodopsin,4 and Green
Fluorescent Protein,5 to name a few. Nitrophenolates (NPs) serve as a small model system forphenolate containing chromophores, as well as charge-transfer chromophores,6 and can exist as
one of three isomers shown in Figure 3.1. Exciting these chromophores with visible irradiation
causes a transfer of electron density from the negatively charged phenolate group to the nitro
group.7 These molecules are small enough that quantum chemical calculations are expected to bestraightforward and NPs provide an interesting opportunity to investigate substitution effects on
the electronic behavior. The combination of these two features, along with others, has garnered
NPs a lot of attention in the literature.8-17

25

Figure 3.1. Structures and resonances of meta, para, and ortho isomers of NP, adapted from Wanko
et al.10

Much of what is known about NPs comes from solution phase experiments. One key
observation is that the substitution position has a drastic effect on the excited state dynamics. Thiswas shown in a recent study by Michenfelder et al.17 where they observed that o- and p-NP havemuch shorter excited state lifetimes than m-NP (300 fs compared to 5 ps). The nature of the solvent
also had a pronounced effect on these lifetimes, and they were generally shorter in aqueous solution
than in organic solvents.
NPs have been investigated in vacuo as well, specifically by Brøndsted Nielsen andcoworkers.8-11, 13-14, 16 They observed excitation energies of the S1 state of p- and o-NP to besignificantly to the blue of the excitation energy of m-NP. This difference can be explained throughresonance stabilization arguments.10 Their experiments were carried out at room temperature, andso the spectra are largely unresolved, leaving key details about the excited state electronic structureand dynamics unanswered. In this chapter, we present the electronic photodissociation spectra in
the S1 region for o-, p-, and m-NP at cryogenic temperatures, showing that the spectra of o- and pNP remain broad, but m-NP exhibits an extended and assignable vibronic progression.
26

3.2

Computational
Several theoretical approaches have been taken in the literature to describe the electronic

spectra of NPs. These include MP2 geometry optimizations of the ground state, with verticalexcited state energies calculated from the optimized ground state geometry.8-10 CC2 and TD-DFT
calculations have been used to calculate vertical excitation energies and achieved reasonableagreement with experiments.13 However, diagnostics in these CC2 calculations revealed that NP
anions have multi-reference character. This makes the use of any single-reference method
problematic. Despite this, the CC2 calculations compare favorably with SORCI vertical excitationenergies.10
In this experiment, we sought to compare a Franck-Condon (FC) simulation with theexperimental UV-Vis spectrum to assign observed vibronic transitions and assess broadening
mechanisms. Performing a FC calculation requires optimized geometries for both the ground andexcited state (S1 in this case) as well as harmonic vibrational frequency calculations. We also
required vertical excitation energy calculations. These calculations would be computationallydemanding using multi-reference methods, and so we employed DFT methods instead. This ofcourse means the accuracy of our computational results may suffer. We used the range-separated
hybrid functional CAM-B3LYP as it has recently been used by Panja, et al. to evaluate the UV/vis
absorption spectra of substituted NPs, where they found good agreement between the experimental
spectra and the values obtained using CAM-B3LYP.15

27

3.3

Results and discussion

3.3.1 Overview
Figure 3.2 shows the electronic spectra in the S1 region for all 3 isomers of NP, acquired
at a trap temperature of 30 K. These spectra represent the two-photon induced loss of a neutral NOgroup as observed previously by others.8-11, 13-14 Despite being taken at cryogenic temperatures,the spectra of o- and p-NP remain broad with no resolved features and onsets at 20500 and 22450
cm-1, respectively. At first glance, the spectrum of m-NP appears to be noisy, but this is vibronic
structure, with an onset beginning at ca. 16000 cm-1, which will be discussed in detail in section
3.3.3. Table 3.1 compares the calculated and experimental values of electronic properties for all
three isomers.

28

wavelength [nm]
556

500

455

417

385

357

normalized intensity [arb. units]625

16000 18000 20000 22000 24000 26000 28000

photon energy [cm-1]

Figure 3.2. Electronic spectra showing the S1 transition for m-, p-, and o-NP with a trap temperatureof 30 K. In the traces of m- (top) and p-NP (middle), full lines connect the data points. In the caseof o-NP, the data points are shown as open circles with a full line representing a 15-point gliding
average to guide the eye.

29

Table 3.1. Calculated S1 and S2 properties (vertical excitation energies, band origins, and oscillator
strengths) compared with experimental data. Calculations shown here were performed with the
CAM-B3LYP functional and aug-cc-pVDZ basis set. Transitions are given in cm-1.
m-NP
p-NP
o-NP
a
Experimental onset
16003
22450
20500
Experimental vertical
17300
22960
23000
transition
Calculated S1 properties
Adiabatic transition energyb
15849
18497
17604
Vertical transition energy
18761
28772
27130
Oscillator strength
0.02680.50480.2033
Orbitals (contribution)
HOMO → LUMO HOMO → LUMO HOMO → LUMO+2
Calculated S2 properties
Adiabatic transition energyb
N/A c
N/A c
21700
Vertical S2 (calc.)
27540
30980
26963
S2 oscillator strength (calc.)
0.00000.00000.0001a

For m-NP, this entry is the energy of the 000 band; for p- and o-NP, the onset of the spectrum islistedb

Values have been corrected for zero-point energies.

c

Adiabatic excitation energies for m- and p-NP were not calculated in the present work.

The difference in absorption energies has been explained previously by Brøndsted Nielsen
and coworkers,10 using resonance arguments. The ground state structures of the o- and p- isomers
can both be described with a low-lying charge transfer resonance, where the charge transfer state
places a formal negative charge on the nitro group. The analogous charge transfer resonance isenergetically unfavorable for m-NP isomer, as seen in Figure 3.1. This leads to ground statestabilization in o- and p-NP which causes an increase in the S0  S1 gap, pushing their absorptions
to higher energy. The individual spectra will be discussed in the following sections.

30

3.3.2 para-Nitrophenolate
The spectrum of p-NP shown in Figure 3.3 shows an onset at 22450 which peaks at 22960
cm-1 and is followed by a second feature at ca. 23690 cm-1. There is some additional structure,most clearly seen on the first peak, consisting of shoulders spaced by 100-130 cm-1, but otherwise
the absorption features are broad and unresolved. Excited state calculations place the verticalexcitation energy at 28772 cm-1, about 6000 cm-1 higher than experiment, and give an oscillator
strength of 0.5048 which is the highest of the three isomers.

31

normalized intensity [arb. units]
0
22000

23000

24000

25000
-1

wavenumber [cm ]Figure 3.3. Electronic spectrum of p-NP in the band origin region.

Calculations on the ground and excited states of this isomer revealed large changes in theminimum energy geometry between the two states, which are predominantly localized on the nitro
group of the molecule. The molecule has a planar ground state, but the minimum energy geometry
in the excited state is realized by twisting the nitro group around the CN bond by 90°, andpyramidalizing the nitrogen to more closely resemble an sp3 hybridization, rather than sp2. Figure
3.4 shows the potential energy surfaces of S0 and S1 in the Franck-Condon (FC) region. The groundstate minimum energy geometry is a saddle point on the excited state surface, ca. 9300 cm-1 above
the minimum of the S1 state. This means that a vertical excitation of the molecule will be exciting
into highly excited vibrational motion in both the nitro torsion and pyramidalization coordinates,
and thus there will be a large density of states in these vibrational modes, which can both be32

expected to be FC active. This large density of states high above the band origin results insignificant spectral congestion, this effect is clearly demonstrated in the spectrum of m-NP which
is discussed in section 3.3.4.

Figure 3.4. Potential energy surface of the S0 and S1 states in the FC region of p-NP. The blackarrow indicates a vertical excitation. Calculations for the surface were performed at the CAMB3LYP/aug-ccpVDZ level of theory using a relaxed scan over the nitro torsion (tors.) andpyramidalization (pyr.) coordinates.

The potential energy surface sheds some light on why the spectrum remains unresolved at
low temperatures. Exciting into a high density of vibrational states in both the torsion andpyramidalization modes (as well as any state coupling to these modes) will lead to significant
spectral congestion. The torsion mode is calculated to be 96 and 257 cm-1 in the S0 and S1
respectively. The difference of these frequencies is comparable with the observed experimental
33

spacing of 100-130 cm-1 for the shoulders on the main absorption feature (see Figure 3.3). The
difference for the pyramidalization modes is ca. 250 cm-1 which does not match the experimental
spacing. As mentioned before, the excited state lifetime of p-NP has been measured byMichenfelder et al. to be 300 fs,17 which leads to lifetime broadening of ca. 18 cm-1. This
broadening combined with a high density of vibrational states on the S1 surface provides aplausible explanation for the lack of resolved features in the electronic spectrum. Michenfelder et
al. also invoked a conical intersection coupling S1 and S0 along the nitro torsion mode as themechanism of relaxation.17 Neither our calculations nor theirs were able to corroborate theexistence of this conical intersection, but both should only be taken as qualitative.

3.3.3 ortho-Nitrophenolate
Like p-NP, o-NP has a broad electronic spectrum in the S1 region, despite being taken at
cryogenic temperatures. The spectrum has an onset at ca. 20500 cm-1 and a set of peaks at 21930,
23000, 23600, and 24200 cm-1. We observe no regular features that could be assigned to specificvibrational transitions. The vertical excitation is calculated to be 27130 cm-1, well above theexperimental features as was also observed in p-NP. Excited state calculations reveal that there are
two close-lying electronic states in the FC region with a calculated separation of 167 cm-1 (seeFigure 3.5).

34

Figure 3.5. Potential energy surface of the S0, S1, and S2 states in the FC region of o-NP. The redarrow indicates a vertical excitation. Calculations were performed at the same level as for p-NP.

Geometry optimizations of the excited states shows that the S1 state has a similar minimum
energy geometry to that of p-NP with a rotated and pyramidalized nitro group. The S2 state has aminimum energy geometry that is planar like the ground state and the calculations place its
potential energy surface energetically below the S1 state in the FC region. The two electronic states
being in such proximity in the FC region suggests they may mix, and there may be a conicalintersection in this region between the two. We note that these calculations should be taken as
qualitative at best due to the low level of theory and the multireference character of the system. In
the absence of the S1 state, vertical excitation into the S2 state may have had well-structured
vibronic features. However, excitation into the S1 state should have a similarly congested spectrum
to that of p-NP due to the high density of vibrational excitation in the S1 state. Also, like p-NP, oNP has an excited state lifetime of 300 fs. The combination of these two sources of congestion
with the additional congestion due to S1/S2 mixing makes deeper analysis of the experimental
spectrum unfeasible.

35

3.3.4 meta-Nitrophenolate
Unlike p- and o-NP, the band origin region of m-NP contains sharp, assignable vibronic
features. The band origin (excitation between the vibrational ground states of the ground and firstexcited electronic states) occurs at 16003 cm-1 (000 ). In the remainder of this work, we denote′𝜈transitions by 𝑄𝜈", where Q is the label of the vibrational mode numbered in order of increasing

vibrational frequency in the S1 state, and ν” and ν’ are the numbers of quanta in mode Q of thelower and upper electronic states, respectively.

36

anormalized intensity [arb. units]

exp.

b
80 K

c
0K
16000

17000

18000

0
0

d0

1010

122

3
120 11 4
12

710

*

*

20000

12114171012101220 0 0110101210 260
7101010

1210

910

111

19000

*

1410

*

71091016000 16200 16400 16600 16800 17000 17200 17400

wavenumber [cm-1]

Figure 3.6. The top panel contains the experimental electronic spectrum in the band origin regionof m-NP (a), compared to FC simulations at 80 (b) and 0 K (c) which have been shifted match theexperimental band origin. The lower panel contains only the first 2400 cm-1 above the band origin,along with assignments of selected peaks (d); a more extensive assignment is given in Table 3.2.
Hot bands are marked in red, with red asterisks labeling peaks whose primary contribution comesfrom a hot band with one quantum in the nitro torsion vibration.

The vibronic bands in the experimental spectrum shown in Figure 3.6 (a) exhibit a regularspacing of ca. 45 cm-1 near the band origin and throughout the band envelope. There are several
regions of high signal intensity, spaced by 620-640 cm-1, and extending to the end of the measured
region (ca. 3500 cm-1). Although the vibronic features are sharp, the photodissociation signal does
not return to baseline in between peaks, indicating increasing spectral congestion.
37

Figure 3.7. Potential energy surface of the S0 and S1 states in the FC region of m-NP. The red arrow
indicates a vertical excitation. Calculations were performed at the same level as for p-NP.

The minimum energy geometry of m-NP is calculated to be planar in both the ground andexcited state (see Figure 3.7). The vertical transition energy is calculated at 18761 cm -1 which is
too high, but still within the experimental band. The calculated band origin is at 15849 cm-1 which
is close to the experimental value, but this is likely fortuitous. The largest geometry changes that
occur upon excitation are a 3% decrease in the CN and a 4% increase in the NO bond length.
Typical values for stretching frequencies of modes involving these bonds are greater than 1000
cm-1, inconsistent with the spacing of the vibrational features. The mode assignments in Figure 3.6
are from FC simulations based on harmonic wave functions. A more extensive summary is given38

in Table 3.2. Overall, the simulation at 80 K qualitatively recovers the experimental spectrum.
This temperature is significantly higher than the experimental trap temperature of 30 K, an
observation which will be discussed following the assignment of the spectrum.
The FC simulation at 80 K produces hot bands in the nitro torsion mode, ⍵1, which is thelowest frequency mode in both the ground and excited electronic states, with harmonic frequenciesof 41 and 77 cm-1, respectively. This mode, however, is significantly anharmonic (see Figure 3.8).
It can be described as a hindered internal rotor around the CN bond axis, with an 1890 cm-1 barrierat 90º in the S0 state, while in the S1 state there is a local minimum at this rotation angle, 1400
cm-1 above the planar geometry. The excited state torsion frequency was set to 87 cm -1 in the FC
simulations to optimize the agreement with experiment. Every transition belonging to a principal
FC active mode is accompanied hot hands on the high energy side, originating from thermally
populated levels in S0 with one or two quanta in ⍵1.

39

2000

S0
S1

energy [cm-1]

1500

1000

500

0
0

30

60

90

120

150

180

tors. []Figure 3.8. Potential energy scans of m-NP along the torsion (tors.) coordinate of the S0 and S1
states using CAM-B3LYP/aug-cc-pVDZ and a NO2 pyramidalization angle constrained to zero(corresponding to a planar nitro group). Energies are given relative to the minimum energy
structure for each surface, in cm−1.

The simulation at 0 K provides a straightforward assignment of the principal FC activemodes. The first feature we assign is due to the overtone of the nitro torsion mode, 120(transitions with odd Δv are forbidden by symmetry), which coincides with the onset of featuresat 16175 cm-1. The next set of FC active modes are a set of intense peaks between 16390 and
16910 cm-1. The first peak, at 16434 cm-1, we assign to 710 where ⍵7 is characterized as an inplane phenolate bending motion coupled with an in-plane nitro wagging motion, as well as a ring
deformation. Next, we assign the 910 (in-plane phenolate bending and ring deformation), 1010
(ring deformation), 1210 and 1410 (both of which are ring deformation and nitro scissoring modes)
40

modes. Modes belonging to the group of transitions in the spectral region close to the band origin
will be referred to as group 1 modes.
Group 2 transitions will refer to modes with fundamental transitions in the region of 1690017590 cm-1 which also contains second order combination bands of ⍵7, ⍵9, ⍵10, ⍵12, and ⍵14, as
well as overtones of ⍵10 and ⍵12 (Δv = 2). Other overtones are assignable but are weaker.
Additionally, transitions involving CN/NO2 stretching motions (⍵26, ⍵28, and ⍵31) appear in thisregion of the spectrum. The third region of high intensity (ca. 18000 cm-1) is comprised of second
order combination bands of group 1 modes with group 2 modes, as well as third order combination
bands of group 1 modes (see Table 3.2). Above this region, the spectrum becomes too congested
to assign. This is not an unexpected result, as excitation in this region will result in vibrational
levels that are above the calculated barrier in the torsional potential energy curve (see Figure 3.8).
Thus, the corresponding density of vibrational states, along with coupling effects between
vibrational modes, will result in a high degree of spectral congestion, corroborating theinterpretation of the spectral congestion in the spectra of p- and o-NP.
The features in the vibronic progression of m-NP have a linewidth we can cleanly
determine (average FWHM for the first ten well-resolved lines of 10.4 ± 1.2 cm-1), which allows
a rough estimate of a lower limit for the excited state lifetime. We performed a simulation using
the PGOPHER program18 and rotational constants obtained through our electronic structure
calculations to obtain an expected width of the rotational envelope of 8 cm-1 (individual rotationallines are not resolved). Convoluting the bandwidth of our light source (5 cm-1) with the result fromPGOPHER leads to an expected linewidth of 9.4 cm-1. Assuming the width is due to lifetime
broadening, a lifetime of ca. 1200 fs is compatible with the observed 10.4 cm-1 linewidth and thisvalue is in the range observed for m-NP in recent ultrafast experiments.17 Our result is, of course,

41

crude, but we note that the longer lifetimes reported by Michenfelder et al. from ultrafast
spectroscopy experiments were measured in nonpolar solvents (5000 fs). We would expect theabsence of solvent molecules to be more comparable with the nonpolar environment and therefore
our estimate of 1200 fs as a lower limit for the S1 lifetime is in rough agreement with the ultrafast
experiments.

42

Table 3.2. List of calculated harmonic vibrational frequencies (cm−1) and symmetries for o-, m-,and p-NP. Note that the point group is Cs for o- and m-NP in both the X (ground) and A (excited)state. For p-NP, the X state belongs to C2v, while the A state belongs to Cs.
Mode

o-NP
X

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35

18.30
107.85232.88253.02381.73429.30438.88546.34565.75572.62662.60723.45750.86810.20825.06870.95893.67989.44998.411037.801088.28
1146.37
1169.231241.751306.43
1379.711407.01
1495.561540.071581.74
1639.01
1691.943152.443194.28
3200.83

m-NP
X

A
A''
A''
A''
A'
A'
A'
A''
A''
A'
A'
A'
A''
A''
A''
A'
A''
A'
A''
A''
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'

41.89
133.33152.85243.41337.54385.02456.38533.96551.90636.22642.78673.86761.27783.74804.02857.18873.30959.42988.951075.131098.151163.38
1169.50
1260.46
1287.421344.05
1360.851389.56
1440.25
1559.89
1651.33
1693.123180.483201.213219.28

A''
A''
A'
A''
A'
A''
A'
A''
A'
A'
A''
A'
A''
A''
A'
A'
A''
A''
A''
A'
A'
A'
A'
A'
A'
A''
A'
A'
A'
A'
A'
A'
A'
A'
A'

41.45
169.14
191.49228.67387.09439.62452.86526.84563.49571.67683.07693.17730.67804.41
843.22876.43902.22941.83982.56999.611086.55
1094.06
1159.89
1256.621314.931400.131425.16
1498.421572.311578.25
1616.85
1669.723158.843189.66
3248.6

p-NP
X

A
A''
A''
A''
A'
A'
A'
A''
A'
A'
A''
A'
A''
A''
A''
A'
A''
A''
A'
A''
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'

43

76.95
150.68213.41
219.36
246.72384.46439.41443.86524.99563.57578.51654.18690.02760.99792.14880.08920.26944.17992.64996.771067.83
1077.58
1149.67
1240.35
1307.92
1374.151424.24
1454.57
1489.51524.57
1613.77
1647.133179.163217.543261.33

A''
A''
A''
A'
A''
A'
A'
A''
A'
A'
A''
A'
A''
A'
A''
A''
A'
A''
A''
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'
A'

95.84
103.56235.67258.73369.92439.32451.21487.26551.72635.38648.04731.44801.08801.17822.78859.05883.99994.961002.071002.50
1104.901138.071204.68
1271.77
1333.941371.931429.24
1501.01
1544.21
1591.06
1619.211674.01
3191.61
3192.30
3228.38

A
A2
B1
B2
B1
A1
A2
B2
B1
B2
B2
A1
B1
B1
A2
A1
A1
B1
A1
B1
A2
B2
A1
A1
B2
B2
A1
A1
B2
B2
B2
A1
A1
B2
A1
B2

88.66
178.59195.32257.46342.62373.87449.46526.02546.87586.49638.26684.33772.77785.47830.39891.18983.00994.741005.411106.33
1121.61
1174.081248.25
1276.04
1318.99
1349.641387.721490.38
1500.701548.00
1628.51
1680.803204.363204.913224.74

A''
A''
A'
A''
A'
A''
A'
A''
A'
A'
A'
A''
A'
A''
A'
A''
A''
A'
A''
A'
A'
A'
A''
A'
A'
A''
A'
A'
A'
A'
A'
A'
A'
A'
A'

3.3.5 Ion temperature estimation
As mentioned before, the trap temperature in the experiment was 30 K, but the best
agreement between the simulation and experiment is with a simulation temperature of 80 K. To
explore this discrepancy in more detail, we performed a series of experiments to measure thetemperature-dependence of the spectrum of m-NP close to the S1 band origin over the experimental
temperature range of 30-124 K. Figure 3.9 shows the band origin region and group 1 modes of theresulting spectra normalized to the peak absorption of the band origin. As the temperature of theions increases, the intensity of the hot bands relative to the band origin should also increase. Thiscan be used to extract an estimate of the ion temperature, assuming the FC factors and vibrationalenergies of the relevant bands are known.

44Normalized Intensity (a.u.)

30 K

40 K

80 K

100 K

120 K

16000

16200

16400

16600

16800

wavenumber [cm-1]

Figure 3.9. Temperature dependent spectra of m-NP in the band origin region. Spectra have beennormalized to the peak absorption of the band origin at 16003 cm-1.

The ratio of intensities of a hot band, I1 to the origin band, I0–0 is equal to:
𝐼1
𝐼0−0

=

|⟨𝜙1′′ |𝜙1′ ⟩|2 𝑒 −𝐸1/𝑘𝑇′′ |𝜙 ′
2|⟨𝜙0−00−0 ⟩|

where ⟨𝜙1′′ |𝜙1′ ⟩ is the FC overlap, E1 is the vibrational energy of the vibrational level in theelectronic ground state which gives rise to the hot band, and T is the temperature of the ion. Thiscan be rearranged to yield:

45

𝑇=

−𝐸1 /𝑘|⟨𝜙 ′′ |𝜙 ′ ⟩|2𝐼
𝑙𝑛 (𝐼 1 ) − 𝑙𝑛 ( ′′ 1 ′1)|⟨𝜙0−0 |𝜙0−0 ⟩|2
0−0

where the right-hand side can be plotted against the experimental trap temperature and should
result in a straight line. The trap temperature will not be the same as the ion temperature, 𝑇 =𝑇𝑚𝑒𝑎𝑠 + 𝑇𝑐𝑜𝑟𝑟 , where Tmeas is the measured temperature of the ion trap, and Tcorr is an additive
correction term. Plotting the last equation as a function of Tmeas produces a y-intercept of Tcorr. The
values for E and 𝜙 are taken from the FC simulations.
We evaluated the temperature correction term Tcorr for the lowest two energy hot bands, 111
and 122 , at 16051 and 16097 cm−1, respectively. The excited state torsion frequency was set to 87
cm-1 as mentioned above. Each data set was fitted with a line with a fixed slope of one. For the 111
peak, Tcorr = (35 ± 3) K, and for 122 , Tcorr = (37 ± 5) K. Therefore, data collected at a trap
temperature of Tmeas = 30 K are expected to represent the spectrum of ions with a temperature T =30 + 36 K = (66 ± 5) K.
Radio frequency traps are known to cause heating by driving collisions between the buffer
gas and trapped ions.19-21 Additionally, the presence of residual buffer gas in the trap during
extraction can lead to heating. Finally, it is possible that collisional cooling of the ions is
incomplete after the storage time in the trap (95 ms in this experiment). None of these effects iseasily quantified in our experiment due to the high sensitivity of the ion intensity on the relevant
experimental parameters, and it is likely that they all contribute to the difference between ion
temperature and trap temperature. The deviation between the ion temperature and the temperatureof the optimal FC simulation is also influenced by computational parameters, such as choice offunctional (using B3LYP instead of CAM-B3LYP yields a best-fit temperature of 150 K) and basis
set, and the anharmonicity of the nitro torsion mode (⍵1). With these facts in mind, we

46conservatively assume that the ion temperature is in the range of 60-80 K. This estimate is in linewith previous experiments at similar trap temperatures, where complexes of ions with N2
molecules were formed and the temperature estimation was made based on the calculated binding
energy of these adducts.18

3.3.5 Charge transfer character of NP excitations
All three of the NP isomers exhibit some degree of charge transfer character. Looking at
the orbitals involved in the electronic transitions (see Figure 3.10), the charge transfer character ismost apparent in m-NP. The HOMO has significant density on the phenolate oxygen whereas the
LUMO has significant density on the nitro group. The extent charge transfer is less in the othertwo isomers, and their transitions are better described as primarily ππ* transitions. Charge transfer
transitions generally have lower oscillator strengths, due to low overlap between the involved
orbitals. The oscillator strength is lowest for the m-NP isomer, followed by that of o-NP, and pNP which has the largest calculated S1 oscillator strength of the three isomers.

47

m-NP

p-NP

o-NP

LUMO

LUMO

LUMO+6HOMO

HOMO

LUMO+2HOMO

HOMO−1

HOMO−3Figure 3.10. Frontier orbitals for m-, p-, and o-NP, calculated using CAM-B3LYP. Both meta andpara isomers exhibit pure HOMO  LUMO transitions for S0  S1. The S0  S1 transition for
o-NP is HOMO  LUMO+2, while the S0  S2 transition for o-NP is a mixture of HOMO−1 
LUMO+2, HOMO−1  LUMO+6, and HOMO−3  LUMO+2.

To make a more quantitative argument, we calculated the change in dipole moment upon
excitation for each of the isomers as well as plotting electron density difference plots (Figure 3.11).
The change in dipole moment was 7.5 D, 2.4 D, and 1.6 D for m-, o-, and p-NP respectively. The

48

large change for the meta isomer is consistent with it having the most charge transfer character.
The other two isomers exhibit changes which are significantly lower, consistent with theirtransitions having less charge transfer character.

49

O:
S0 −0.80
S1 −0.58

NO2:
S0 −0.35
S1 −0.85

O:
S0 −0.74
S1 −0.70

NO2:
S0 −0.52
S1 −0.70

O:
S0 −0.71
S1 −0.59

NO2:
S0 −0.45
S1 −0.64

Figure 3.11. Electron density difference plots for m- (top), p- (center), and o-NP (bottom) in the
FC region. Purple regions indicate an increase of electron density upon electronic transition, while
yellow regions indicate a decrease of electron density upon electronic transition. The density
difference for o-nitrophenolate was calculated for the S1 state. Listed are the charges on the NO2
and O groups for each isomer in the S0 and S1 states. Charges calculated using a Natural Population
Analysis, given in e.

50

3.4

References1.

Dodson, L. G.; Zagorec-Marks, W.; Xu, S.; Smith, J. E. T.; Weber, J. M., Intrinsic
photophysics of nitrophenolate ions studied by cryogenic ion spectroscopy. Physical
Chemistry Chemical Physics 2018, 20 (45), 28535-28543.

2.

Borgstahl, G. E. O.; Williams, D. R.; Getzoff, E. D., 1.4 Å Structure of Photoactive Yellow
Protein, a Cytosolic Photoreceptor: Unusual Fold, Active Site, and Chromophore.
Biochemistry 1995, 34 (19), 6278-6287.

3.

Shimomura, O.; Johnson, F. H.; Masugi, T., Cypridina Bioluminescence: Light-Emitting
Oxyluciferin-Luciferase Complex. Science 1969, 164 (3885), 1299.

4.

G. S. Harbinson, S. O. S., J. A. Pardoen, J. M. L. Courtin, J. Lugtenburg, J. Herzfeld, R.
A. Mathies, R. G. Griffin, Solid-State C13 NMR Detection of a Perturbed 6-s-trans
Chromophore in Bacteriorhodopsin. Biochemistry 1985, 24, 6955-6962.

5.

Shimomura, O., Discovery of Green Fluorescent Protein (GFP) (Nobel Lecture).
Angewandte Chemie International Edition 2009, 48 (31), 5590-5602.

6.

Grabowski, Z. R.; Rotkiewicz, K.; Rettig, W., Structural Changes Accompanying
Intramolecular Electron Transfer: Focus on Twisted Intramolecular Charge-Transfer
States and Structures. Chemical Reviews 2003, 103 (10), 3899-4032.

7.

Bureš, F., Fundamental Aspects of Property Tuning in Push–Pull Molecules. RSC
Advances 2014, 4 (102), 58826-58851.

8.

Kirketerp, M.-B. S.; Petersen, M. Å.; Wanko, M.; Leal, L. A. E.; Zettergren, H.; Raymo,
F. M.; Rubio, A.; Brøndsted Nielsen, M.; Brøndsted Nielsen, S., Absorption Spectra of 4‐
Nitrophenolate Ions Measured in Vacuo and in Solution. ChemPhysChem 2009, 10 (8),1207-1209.

51

9.

Kirketerp, M. B. S.; Petersen, M. Å.; Wanko, M.; Zettergren, H.; Rubio, A.; Brøndsted
Nielsen, M.; Brøndsted Nielsen, S., Double‐Bond versus Triple‐Bond Bridges: Does it
Matter for the Charge‐Transfer Absorption by Donor–Acceptor Chromophores?
ChemPhysChem 2010, 11 (12), 2495-2498.

10.

Wanko, M.; Houmoller, J.; Stochkel, K.; Kirketerp, M.-B. S.; Petersen, M. Å.; Brøndsted
Nielsen, M.; Brøndsted Nielsen, S.; Rubio, A., Substitution Effects on the Absorption
Spectra of Nitrophenolate Isomers. Physical Chemistry Chemical Physics 2012, 14 (37),12905-12911.

11.

Houmøller, J.; Wanko, M.; Støchkel, K.; Rubio, A.; Brøndsted Nielsen, S., On the Effectof a Single Solvent Molecule on the Charge-Transfer Band of a Donor–Acceptor Anion.
Journal of the American Chemical Society 2013, 135 (18), 6818-6821.

12.

Kozłowska, J.; Wielgus, M.; Bartkowiak, W., TD-DFT Study on the Charge-Transfer
Excitations of Anions Possessing Double or Triple Bonds. Computational and Theoretical
Chemistry 2013, 1014, 49-55.

13.

Brøndsted Nielsen, S.; Brøndsted Nielsen, M.; Rubio, A., Spectroscopy of Nitrophenolates
in Vacuo: Effect of Spacer, Configuration, and Microsolvation on the Charge-Transfer
Excitation Energy. Accounts of Chemical Research 2014, 47 (4), 1417-1425.

14.

Houmøller, J.; Wanko, M.; Rubio, A.; Brøndsted Nielsen, S., Effect of a Single Water
Molecule on the Electronic Absorption by o- and p-Nitrophenolate: A Shift to the Red or
to the Blue? The Journal of Physical Chemistry A 2015, 119 (47), 11498-11503.

15.

Panja, S. K.; Dwivedi, N.; Saha, S., Tuning the Intramolecular Charge Transfer (ICT)
Process in Push-Pull Systems: Effect of Nitro Groups. RSC Advances 2016, 6 (107),105786-105794.

52

16.

Stockett, M. H.; Boesen, M.; Houmøller, J.; Brøndsted Nielsen, S., Accessing the Intrinsic
Nature of Electronic Transitions from Gas‐Phase Spectroscopy of Molecular
Ion/Zwitterion Complexes. Angewandte Chemie 2017, 129 (13), 3544-3549.

17.

Michenfelder, N. C.; Ernst, H. A.; Schweigert, C.; Olzmann, M.; Unterreiner, A. N.,
Ultrafast Stimulated Emission of Nitrophenolates in Organic and Aqueous Solutions.
Physical Chemistry Chemical Physics 2018, 20 (16), 10713-10720.

18.

Western, C. M., PGOPHER: A Program for Simulating Rotational, Vibrational andElectronic Spectra. Journal of Quantitative Spectroscopy and Radiative Transfer 2017,
186, 221-242.

19.

Xu, S.; Gozem, S.; Krylov, A. I.; Christopher, C. R.; Weber, J. M., Ligand Influence on
the Electronic Spectra of Monocationic Copper–Bipyridine Complexes. Physical
Chemistry Chemical Physics 2015, 17 (47), 31938-31946.

20.

Asvany, O.; Schlemmer, S., Numerical Simulations of Kinetic Ion Temperature in a
Cryogenic Linear Multipole Trap. International Journal of Mass Spectrometry 2009, 279(2), 147-155.

21.

Wester, R., Radiofrequency Multipole Traps: Tools for Spectroscopy and Dynamics ofCold Molecular Ions. Journal of Physics B: Atomic, Molecular and Optical Physics 2009,42 (15), 154001.

53

Chapter 4 The Green Fluorescent Protein Chromophore

This chapter has been adapted with permission from:(a)W. Zagorec-Marks, M. M. Foreman, J. R. R. Verlet, J. M. Weber, "Cryogenic Ion

Spectroscopy of the Green Fluorescent Protein Chromophore in Vacuo", J. Phys. Chem.
Lett. 10 (2019) 7817 - 7822.
(b)

W. Zagorec-Marks, M. M. Foreman, J. R. R. Verlet, J. M. Weber, "Probing theMicrosolvation Environment of the Green Fluorescent Protein Chomophore in Vacuo", J. Phys.
Chem. Lett. 11 (2020) 1940-1946.
(c)

W. Zagorec-Marks, M. M. Foreman, J. M. Weber "Tag-Free, Temperature Dependent

Infrared Spectra of the GFP Chromophore – Revisiting the Question of Isomerism", J. Phys. Chem.
A 124 (2020) 7827−7831.
Copyright (2019/2020) American Chemical Society.

4.1

Introduction
The Green Fluorescent Protein (GFP) and its variants are widely used for fluorescencelabeling in biology.1-5 The chromophore (and fluorophore) is a small molecular unit, which
consists of a phenol ring linked to an imidazole ring by a methine bridge in the para-position,
residing inside the beta barrel of the protein. The chromophore is accompanied by a single watermolecule, which binds to the anionic phenolate group of the chromophore.6 A commonly used
model

of

the

GFP

chromophoreisdeprotonatedp-hydroxybenzylidene-2,3-

dimethylimidazolinone (HBDI-, see Figure 4.1). In this model, the linkers to the protein have been
replaced with methyl groups on the imidazole ring. The deprotonated form is used because only

54

the anion fluoresces in the protein, while the neutral form undergoes excited state proton transfer
upon photon absorption, and relaxes without photon emission.3 As can be seen in Figure 4.1, there
are two isomeric forms referred to as Z- and E-HBDI- which are related by a twisting around the
double bond of the methine bridge. This twisting motion is reminiscent of the torsional motion of
p-nitrophenolate (p-NP), discussed in Chapter 3. As was the case in p-NP, excitation with visible
irradiation initiates a twisting motion during relaxation on the S1 excited state. This occurs both inlow viscosity solvents7-8 and in vacuo9 and results in loss of fluorescence due to low FranckCondon factors. In GFP, this motion is blocked, giving rise to the fluorescence.

Figure 4.1. Deprotonated p-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI-) as the Z- andE- isomer. The two isomers are related by rotation about the carbon double bond of the methine
bridge.
The S0 → S1 band envelope informs on the initial photophysics of the S 1 transition of
HBDI- and its interactions with the chemical environment (the chromophore pocket of the protein,solvent, etc.). Consequently, there have been many experiments on HBDI- in vacuo aimed atunderstanding its intrinsic photophysics. Interestingly, the spectrum of isolated HBDI- is very
similar to that of the chromophore in the protein, and in stark contrast to the absorption spectrum
in aqueous solution. This suggests that the protein environment results in initial photophysics more55

like those in vacuum than aqueous solution. All previous photodissociation experiments on HBDIhave been done at room temperature. The broadening resulting from hot bands obscures details of
the S0 → S1 band envelope. In addition, the effect of a microsolvation environment mirroring thewater molecule present in the chromophore pocket of the protein has only been exploredcomputationally so far.10
The relaxation pathway from the S1 excited state in vacuo results in dissociation or loss ofan electron, since the energy deposited in the molecule cannot be dissipated into the chemical
environment. As a consequence, the intrinsic photophysics of HBDI- has been primarily
investigated using photodissociation and photoelectron spectroscopies.7-9, 11-32 Photodissociationof bare HBDI- occurs primarily through the loss of a CH3 group. In a one-photon absorption eventwith sufficient energy, this dissociation typically takes milliseconds.33 This time scale is notaccessible in our experiment, and thus we rely on absorption of two photons when studying bare
HBDI-, which causes dissociation on a microsecond timescale.32 The threshold for electron
detachment is only slightly higher than the energy of the absorption band maximum, but still within
the FC envelope (22017 for electron detachment vs 20833 cm-1 band origin).34 This means thatelectron detachment and unimolecular dissociation are competing relaxation pathways upon
photoexcitation with two photons. Moreover, electron detachment can occur either directly, or
through vibrational autodetachment, the latter of which is thought to dominate.9, 16, 18-21, 25-28, 35 A
recent study by Bieske and coworkers13 revealed a further complication, when they showed that
under typical preparation conditions at room temperature, a significant fraction of the produced
HBDI- ions may exist as the E isomer, while previous interpretations have been largely focused
the Z isomer. In this chapter, we discuss the spectra of bare, messenger-tagged, and microsolvated
HBDI- in the visible and infrared spectral regions. The spectra are interpreted using DFT

56

calculations. In the final section of the chapter, we introduce an IR-UV double resonancetechnique, which we will refer to as hot band assisted infrared (HAIR) spectroscopy, which can beused to circumvent the use of a messenger tag, and allows the acquisition of temperature dependent
IR spectra, which is not possible with messenger tagging.

4.2

Computational
Ground state geometry optimizations and harmonic frequency calculations were performedat the DFT level of theory using the B3LYP functional and def2-TZVPP basis sets. As previously
noted by Oomens and coworkers, there is a strong basis set dependence of the IR features for
HBDI.36 Exploratory calculations show that this problem only occurs for basis sets smaller thanthe one used here. Excited state geometry optimizations and harmonic frequency calculations were
performed using the range-corrected CAM-B3LYP functional and def2-TZVPP basis set. Excitedstate frequency calculations were necessary to carry out Franck-Condon (FC) simulations. In thesesimulations, the temperature is 55 K and the full width at half-maximum (FWHM) of thevibrational features is 80 cm-1 fit the observed vibrational structure. Vertical excitation calculations
using both CAM-B3LYP and -B97XD functionals yield similar results that overestimate theelectronic band origin by about 0.5 eV for the Z isomer and were thus shifted by ca. -4000 cm-1 tomatch the experimental spectrum of [HBDI-·N2]. All calculations were carried out in Gaussian 16.

4.3

The isolated chromophore
The electronic spectrum of the complex [HBDI·N2]- is shown in Figure 4.2. The loss

channel for this spectrum was the loss of neutral N2, which has a calculated binding energy of 630
cm-1 and 720 cm-1 when bound to the imidazole and phenolate groups, respectively. Due to the57low binding energy of the messenger tag, dissociation of this complex is likely to occur with near
unit probability upon absorption of a photon, even down to the far infrared (ca. 1000 cm-1). The
low binding energy also guarantees that the complexes are cold, and the temperature can be
estimated to be < 75 K by describing cluster formation in the ion trap as an evaporative ensemble.37
Afforded by the low temperature, the spectrum of [HBDI·N2]- shows a sharp onset at 20530 ± 25
cm-1 followed by peaks at 20780 ± 10 cm-1 and 20835 ± 10 cm-1. Additionally, we partially resolve
vibrational substructure with a spacing of ca. 70 cm-1. Another feature appears with an onset at
21830 cm-1 and a peak at 22130 cm-1.

58

Figure 4.2. Electronic spectrum of [HBDI·N2]-. The open circles are raw data points, the purple
full line is a 5-point adjacent average to guide the eye. The black full lines are Franck-CondonHerzberg-Teller (FCHT) simulations of the vibrational substructure in the S1  S0 electronic band,with the vibrational states indicated as vertical sticks. The insert shows the band origin region, andthe data in this trace were taken with a step size of 2.6 cm-1, while the step size in the overview
spectrum was 13 cm-1 in the origin region. The FCHT simulation has been shifted by ca. -4000
cm-1 for the best match to the experimental spectrum (see section 4.2).

As mentioned above, there is the possibility of two isomers arising from the electrospraysource. The infrared photodissociation spectrum of [HBDI·N2]- is presented in Figure 4.3 alongwith calculated harmonic spectra of the Z and E isomers. The two isomers have similar calculated
spectra, but a distinction can be made using the spacing between the three most intense features in
the 1400 – 1700 cm-1 region. The spacing between the upper two features is 80 cm-1 and 60 cm-1
59

for the Z and E isomers respectively, compared to 80 cm-1 in the experimental. For the lower two
features, the spacing is 50 cm-1 and 80 cm-1 for the Z and E isomers respectively, compared to 55
cm-1 in the experimental. We therefore conclude that the Z isomer is present, and the narrow widthof the main features indicates that only one isomer is significantly populated. This assignment is
consistent with the fact that calculations place the Z isomer lower in energy by 800 cm-1, and withion mobility experiments that showed the presence of the Z isomer exclusively when HBDI- iscomplexed with solvent molecules.13 All vibrational features are assigned and described in Table
4.1.

Figure 4.3. Infrared spectrum of [HBDI·N2]- (top trace) compared to simulated spectra of the Z
isomer (center trace) and E isomer (bottom trace) of HBDI-. The calculations have been scaled by
0.98. A detailed assignment of the vibrational features is listed in Table 4.1.

60

Table 4.1. Vibrational mode assignments for [HBDI·N2]- based on the calculated spectrum of theZ isomer.
modeobserved [cm-1] calculated a [cm-1]
in-plane CH bending / imidazole ring deformation

1151

1137

phenolate CH in-plane bending

1170

1159

methyl umbrella / imidazole deformation

1466

1430

methyl umbrella / phenolate deformation

1477

1451

phenolate in-plane CH bending / phenolate deformation 1512

1500

in-plane CH bending / imidazole ring deformation

1544

1544

phenolate CO stretching / ring deformations

1578

1571

in-plane CH bending / imidazole ring deformation

1600

1593

phenolate CO stretching / bridge deformation

1632

1627

imidazole CO stretching

1680

1667

a

B3LYP/def2-TZVPP scaled by 0.98.

Figure 4.2 includes an FCHT simulation of the S1  S0 absorption band for the Z isomer,
which after shifting the band origin by ca 4000 cm-1 qualitatively matches the experimental
spectrum. Using this simulation, we assign the low-frequency mode, corresponding to the
70 cm-1 spacing prominently appearing in the experimental electronic spectrum, as a methine
bridge bending/ ring scissoring mode (B, calculated at ca. 80 cm-1 on the S1 surface but set to 65
cm-1 to make the agreement better). We assign the peak at 20780 ± 10 cm-1 as the electronic band
origin, based on the hot band structure on its low-energy side. The broad feature which peaks at
22130 ± 40 cm-1 is due to combination bands of B building on an imidazole ring breathing/inplane CH bending mode on the S1 surface (I, calculated at ca. 1350 cm-1), consistent with previous

61assignments.24, 38-39 The observed spectral congestion is largely due to the B mode which is
responsible for the two hot bands below the band origin and the high-energy shoulder of the Ifeature. The width of the experimental features is not limited by the experimental resolution (seeChapter 2). We estimate the measured linewidth has a full width at half maximum of ca. 80 cm-1,
based on a comparison with the calculated spectrum, and note that in the previous chapter we wereable to resolve narrow lines of the vibronic progression of meta-NP. The width is instead attributed
to congestion of the spectrum along with intrinsic dynamics of the system, where the initial excitedstate dynamics are expected to occur on a sub-100 fs timescale.
The unshifted value of the calculated band origin is 26200 cm-1 which is substantiallyhigher than the experimental value. Our FC profile is substantially different from calculations done
at much higher levels of theory,24, 38-39 which yielded more accurate values for the band origin but
vibronic progressions that deviate from the experimental progression (see Figure 4.4 for a directcomparison). We note that the experimental data presented in Figure 4.2 provide the best
benchmark to date to gauge the quality of computational approaches to this molecular system.

62

experiment HBDI-·N2
calculated HBI- (Olivucci 2004)

intensity [arb. units]

calculated HBDI- (Krylov 2011)
calculated HBDI- (Bochenkova 2013)
calculated, this work

20000

22000

24000

wavenumber [cm-1]

Figure 4.4. Comparison of the experimental spectrum of [HBDI·N2]- with calculated spectra fromthe literature. The calculated curves have all been shifted to optimally match the experimental data.
The literature data have been adapted from the references.24, 38-39

Prior electronic spectroscopy experiments using N2 as a messenger tag suggest that a small
shift of spectral features is expected relative to the bare species, so it is of interest to contrast the
spectrum of [HBDI·N2]- presented in Figure 4.2 with that of bare HBDI-.40 Figure 4.5 shows thiscomparison at various ion trap temperatures.

63Figure 4.5. Electronic spectra of HBDI- at various trap temperatures. The open circles are raw data
points, the full lines are adjacent averages to guide the eye, with 3, 5 and 20 points for [HBDI·N2], 30 K and the other experimental conditions, respectively. The vertical dashed and dotted lines
show the peak positions for bare HBDI- (30 K) of the (v=0)  (v=0) band at (20800 ± 10) cm-1,
and of the high energy feature around 22100 cm-1, respectively.
The electronic spectrum of bare HBDI- has an onset at 20250 ± 50 cm-1, which is at much
lower wavenumbers than that of [HBDI·N2]-, and the peak of the spectrum is at 20930 ± 10
cm-1, with partially resolved shoulders on the lower energy side. The high-energy side of this peak64

has the same spacing of vibrational features in both the bare and [HBDI·N2]- spectrum. We assign
the peak at 20930 cm-1 as the band origin of HBDI-. The presence of the first N2 messenger tag
results in a shift of the spectrum by 150 ± 27 cm-1 to the red. Addition of a second N2 tag, shownin Figure 4.6, leads to an additional red shift of 80 ± 27 cm-1 (measured from the highest point of
the band envelope). The spectrum of [HBDI·2(N2)]- has a similar band envelope, but is in generalless well resolved than the spectrum of [HBDI·N2]-. Because there are now two N2 molecules
which can each bind to HBDI- at either oxygen site, there are possibly many distinct positionisomers present, each with a potentially different band origin. The spectral congestion caused by
the presence of these isomers, in combination with low overall signal-to-noise ratio, is likely thereason we do not resolve the 70 cm-1 spaced features on the main absorption peak.

65

photofragment action [arb. units]

20000

21000

22000

23000

24000

wavenumber [cm-1]

Figure 4.6. Electronic spectrum of [HBDI·(N2)2]-. Open circles represent the raw data, the purple
line is a 5-point gliding average to guide the eye.

With increasing temperature, a prominent shoulder below the band origin develops, until
we observe a partially resolved peak at 20700 ± 25 cm-1 at 180 K, which eventually dominates the
spectrum at 300 K. This feature could be due to hot bands. However, our FC simulations do notrecover this behavior regardless of the simulation temperature, consistent with FC simulations
performed by others.24, 38-39 Alternatively, this feature could be evidence for the presence of the E
isomer, which would be consistent with recent results by Bieske and coworkers. They observed
the presence of the E isomer in ion mobility experiments on HBDI- ions prepared at room
temperature, and found that the E isomer absorbs at slightly longer wavelengths than the Z66isomer.13 The barrier to isomerization has been calculated to be 1.24 eV, which would prevent
thermal isomerization.13 We will revisit this problem in Section 4.5, where we will argue that only
the Z isomer is present in our experiment.
There are subtle differences between the band envelopes of bare and N2-tagged HBDI-.
The relative intensities of the feature around 22000 cm-1 and the band origin are different for thetwo species. This could be due to a change in the FC factors, but this seems unlikely given thesmall perturbation the N2 tag induces. Alternatively, it could be due to a change in the branchingratio between internal conversion and electron detachment, as the adiabatic electron affinity of
HBDI is 22017 cm-1.34 To test this hypothesis, we present a comparison of the parent ion depletion(which would include both fragmentation and electron loss) and fragment ion formation signal
channels in Figure 4.7. As can be seen in the figure, the two traces show a near perfect overlap
indicating that electron detachment does not play a role in our experiment. A third possibility issimply that there is a difference in ion temperatures, despite similar trap temperatures between[HBDI·N2]- and bare HBDI-. As discussed in Section 4.3, the presence of the N2 messenger tag
limits the internal energy of the complex to ca. 720 cm-1, while the bare ion has no such limit, andcould be affected by radiofrequency heating (see Chapter 3).

67photodestruction yield [arb. units]

depletionaction

20000

21000

22000

23000

24000

wavenumber [cm-1]

Figure 4.7. Comparison of the experimental photofragment action spectrum of HBDI- at 30 K trap
temperature (blue curve) with the parent ion depletion spectrum taken at the same temperature(black curve).

4.4

Effects of microsolvation
In the chromophore pocket of GFP, a single water molecule binds to the phenolate moietyof the chromophore.41 This interaction has only been investigated computationally for HBDI-,10and one of the questions in this context is whether the water molecule has a strong influence on
the electronic spectrum of the chromophore. By raising the trap temperature to 180 K, we are able
to form complexes between HBDI- and water molecules ([HBDI·(H2O)n]-, where n = 1, 2). Figure
4.8 shows the experimental IR spectrum of the water complexes compared with [HBDI·N2]-.

68

Figure 4.8. Infrared spectra of [HBDI·N2]- and [HBDI·(H2O)n]- (n = 1, 2) from 1000 to 2000 cm1. The photofragment signals were due to the loss of N2 for [HBDI·N2]- and of a single H2O
molecule for [HBDI·(H2O)n]-, respectively.

Based on the similarities between the three traces in Figure 4.8, we conclude that thechromophore in hydrated clusters is in the Z isomer. We also note the positions of the intense
vibrational features of HBDI- are only weakly influenced by the microsolvation environment. The
mode assignments for all three complexes in Figure 4.8 are presented in Table 4.2. The smallchanges to vibrational frequencies are in line with computational predictions by Krylov andcoworkers.10 There are several ways in which each water molecule can bind to the two oxygen
molecules, and the binding motifs considered here are displayed in Figure 4.9.

69

Table 4.2. Vibrational mode assignments for [HBDI-·N2] and [HBDI-·(H2O)n] (n = 1, 2).
mode
HBDI-·N2n=1
n=2in-plane CH bending / imidazole ring deformation

1151

1148

N/A

phenolate in-plane CH bending / CO stretching

N/A

N/A

1396

methyl umbrella / imidazole deformation

1466

1461

N/A

methyl umbrella / phenolate deformation

1477

1478

N/A

phenolate in-plane CH bending / phenolate deformation 1512

N/A

1506

in-plane CH bending / imidazole ring deformation

1544

1535

1526

phenolate CO stretching / ring deformations

1578

N/A

1554

in-plane CH bending / imidazole ring deformation

1600

1595

1593

imidazole CO stretching

1680

1687

1695

70

Ia (0 meV)

IIa (0 meV)

Ia’ (+3 meV)

IIb (+5 meV)Ib (+100 meV)IIc (+70 meV)Ic (+110 meV)IIc’ (+92 meV)IId (+180 meV)IId’ (+196 meV)

Figure 4.9. Calculated structures for all solvated species. Monohydrated species (left) are shownwith energy relative to (Ia), dihydrate species (right) are presented with energy relative to (IIa).
All energies are calculated at 0 K.

Natural bonding orbital calculations, done at the B3LYP/def2-TZVPP level of theory,
indicate that in bare HBDI- both oxygen atoms have roughly equal charge (see Table 4.3). Whena single water molecule is added, the negative change on the oxygen it binds to is increased slightly(ca. 0.04 e), which is followed by an increase in the corresponding CO bond length by about 1%.
As noted in Table 4.2, the peak at 1680 cm-1 corresponds to the CO stretching vibration of the71imidazole group. This peak shifts by +7 cm-1 in the monohydrate spectrum, and another +8 cm-1
in the dihydrate spectrum. These shifts are consistent with a loss of electron density from theimidazole oxygen and a strengthening of the CO bond of the imidazole group. This suggests thatthe water molecules are attached to the phenolate oxygen, but the evaluation of other features in
the fingerprint region and in the OH stretching region shows that this is too simple an interpretation(see below).

Table 4.3. Calculated selected charges (in e) and bond lengths (in Angstroms) in HBDI- anddifferent position isomers for [HBDI·(H2O)n]- (n = 1, 2); isomer labels refer to Figure 4.9.
Absolute values
charge on charge on phenolate
imidazole
phenolateimidazoleCObond CO

bond

O

O

lengthlengthBare-0.662-0.6681.248821.23223n = 1 on phenolate (Ia)-0.699-0.6571.26178

1.22972n = 1 on imidazole (Ib)-0.653-0.713

1.247011.24317n = 2, both on phenolate (IIa)-0.748-0.6481.26932

1.22787n = 2, both on imidazole (IIc)-0.643-0.762

1.24525

1.25365-0.691-0.703

1.25972

1.24032n = 1 on phenolate (Ia)-0.0370.011

0.01296-0.00251n = 1 on imidazole (Ib)0.009

-0.045-0.00181

0.01094n = 2, both on phenolate (IIa)-0.0860.02

0.0205-0.00436n = 2, both on imidazole (IIc)

0.019-0.094-0.00357

0.02142-0.029-0.0350.01090.00809n = 2, one each on phenolate andimidazole (IIb)
Differences for hydration isomers
w.r. to bare HBDI-

n = 2, one each on phenolate andimidazole (IIb)

72

The next two significant features of the HBDI- IR spectrum are the peaks at 1600 cm-1 and
1544 cm-1, and both can be described as in-plane CH bending/imidazole ring deformation modes.
The upper feature shifts by -6 cm-1 upon addition of the first water molecule, and shifts -2 cm-1
further with addition of the second water molecule. The peak at 1544 cm-1 shifts steadily by ca. 10
cm-1 per water molecule. The CO stretching motion of the phenolate ring is present as a shoulder
on the high energy side of the dominant feature at 1578 cm-1. This feature becomes clearly
identifiable in the dihydrate spectrum when it has shifted to 1554 cm-1 and is now a resolved peak.
The red-shift of this mode is consistent with hydration at the phenolate. However, an additional
weak feature around 1650 cm-1 is characteristic of an isomer with a water molecule bound to theimidazole oxygen. Finally, a feature at 1396 cm-1, which corresponds to an in-plane HCH
bending/CO stretching mode on the phenolate moiety, gains intensity in the dihydrate spectrum.
This behavior is only reproduced in calculations with both water molecules bound to the phenolate
moiety.
In general, the spectral region from 1000 cm-1 to 2000 cm-1 shows weak effects of
microhydration on the vibrational spectrum of the chromophore and suggests that we have a
mixture of conformers, with some hydrated at the phenolate and some at the imidazole for boththe mono- and dihydrated species. To investigate this question from the point of view of thesolvent, we move to the OH stretching region of the IR, where the OH stretching frequencies of
the water molecules will serve as sensitive IR probes of their hydrogen bonding environment.
Figure 4.10 shows the IR spectra of [HBDI·N2]- and [HBDI·(H2O)n]- (n = 1, 2) over therange of 2700 cm-1 to 3800 cm-1. The spectrum of [HBDI·N2]- contains only CH stretching modesin this region (as no OH groups are present) in the range of 2800 cm-1 to 3100 cm-1. This region
can contain Fermi interactions between HCH bending mode overtones and CH stretching73

fundamentals, making assignments of vibrational features difficult. Despite this problem, we
assign the peaks at 2848 cm-1 and 2878 cm-1 to the totally symmetric stretching modes of the CH3
groups on the imidazole moiety and the unresolved group of peaks around 2930 cm-1 to theantisymmetric version of these modes. The two peaks at 3033 cm-1 and 3059 cm-1 are tentatively
assigned as CH stretching modes of the phenolate ring. All of the CH stretching features exhibit
minimal shifts upon hydration.

Figure 4.10. IR spectra of of [HBDI-·N2] (top, black) and [HBDI-·(H2O)n] (n = 1, 2 magenta andblue) from 2700 to 3800 cm-1. The photofragment loss channels are the same as in the low energy
region.

The remainder of this spectral region contains the OH stretching vibrations of the watermolecules, which dominate the spectra of the hydrated ion. All structures calculated here have at74

least one free OH oscillator that does not participate in a hydrogen bonding interaction. The
signature for the free OH appears at 3703 cm-1 and is present in both the singly and doubly hydrated
species. The large features in the 3100 cm-1 to 3500 cm-1 range belong to hydrogen bonded OH
stretching vibrations.
Figure 4.11 shows a comparison of the experimental and calculated IR spectra for several
conformers of the monohydrated species. We assign the broad feature at 3180 cm-1 to the signatureof a water molecule solvating the phenolate oxygen. The corresponding vibrational mode can becharacterized as the symmetric OH stretching mode, and is primarily localized on the OH group
participating in the hydrogen bond. A conformer (Ia’, see Figure 4.9) with the water moleculebinding to the phenolate, but anti to the methine bridge CH group is isoenergetic with conformer
Ia and has an identical infrared spectrum. The feature at 3340 cm-1 is representative of thesymmetric OH stretching mode of a water molecule solvating the imidazole oxygen. The presenceof both features confirms that we indeed have a mixture of conformers present in the experiment.

75

Figure 4.11. Calculated infrared spectra of selected conformers of monohydrated HBDI- (lower
traces, see Figure 4.9 for other conformers), compared to the experimental spectrum (upper trace).
The spectrum contains signatures for CH stretching vibrations (CH), H-bonded OH stretching on
the phenolate (red-shaded region) and imidazole (blue-shaded region) groups, and the free OH
stretching mode (F). All calculations have been scaled by 0.957 to match the experimentallyobserved free OH stretching vibrations. The calculated energies (at 0 K) of the conformers relative
to the lowest energy conformer (Ia) are given in each simulated trace.

The broad widths of the hydrogen bonded OH stretching features reflect the fact that thecomplexes are formed through an evaporative ensemble,37 with internal energies on the order ofthe binding energy of a water molecule (about 4000 cm-1, discussion to follow). Because of this,the water molecule is likely to explore a significant range of OH···O- angles through large
amplitude motions both in the plane of the HBDI- and perpendicular to it, resulting in the observed
broadening. Figures 4.13 and 4.14 show the calculated frequency of the hydrogen bonded OH76

stretching mode as a function of the in-plane and out-of-plane pivot angle respectively. As can beseen in the figures, motion in either of these coordinate can lead to a change in OH stretchingfrequency of ca. 200 cm-1.

in-plane pivot angle
3550

Phenolate Bound

OH - - O Stretching Frequency (cm-1)

3500

3450

3400

q

3350

3300

Imidazole Bound

3600

3550

q

3500

3450

3400

3350
-60

-40

-20

0

20

40

60

q [degrees]

Figure 4.12. Calculated hydrogen bonded OH stretching frequency as a function of the in-plane
pivot angle for both a phenolate bound and imidazole bound water molecule.

77

OH - - O Stretching Frequency (cm-1)

3600
Phenolate Bound

3550
3500
3450

q

3400
3350
15

20

25

30

35

40

45

50

q [degrees]

Figure 4.13. Calculated hydrogen bonded OH stretching frequency as a function of the out-ofplane pivot angle for both a phenolate bound water molecule.

Overall, we obtain similar structures and energetic ordering as found by Krylov andcoworkers.10 The lowest energy structure of the monohydrate complex corresponds to hydration
at the phenolate oxygen (Ia). Hydration at the imidazole oxygen is ca. 100 meV higher in energy,
with the oxygen lone pair being able to weakly bind to either the nearest methyl group (Ib) or to
the CH group of the methine bridge (Ic).
A coarse estimate of the relative populations of the two binding sites was achieved byfitting the two H-bonded OH features with Gaussian profiles and comparing the areas, scaled by
the calculated IR intensities for conformers Ia and Ib. The result suggests that ca. 60% of thecomplexes exist with the water bound to the phenolate group and ca. 40% have the water boundto the imidazole group. We note that this ratio is not consistent with a thermal population. One
possible explanation for this behavior is through the differing binding energies of water molecules
to each site. In principle, the larger binding energy at the phenolate site (calculated at 4550 cm-1)
could suppress the fragment ion signal generated from this complex compared to that of the78

complex with the water bound to the imidazole (binding energy calculated at 3740 cm-1). However,as noted previously, there is considerable internal energy in the complexes, because they areformed from an evaporative ensemble.37 The internal energy in a complex formed this way is on
the order of the binding energy of a water molecule in the dihydrate, which is calculated between
3300 cm-1 and 4500 cm-1. As a result, the internal energy of the complex assists in dissociation
after photon absorption. This is also reflected in the fact that we are able to record IR spectra inand below the carbonyl stretching region (see Figure 4.8). A more plausible explanation for thedeviation of the population ratio from that of a thermal distribution is that migration of watermolecules between the two binding sites involves breaking of the hydrogen bond to the ion.
Interconversion between the two binding sites is therefore likely to be hindered by a large barrier,leading to kinetically trapped species. This means that the population ratio is better described by across section for the capture of a water molecule by the two different binding cites. Future
experiments could employ a separation of water adduct formation and messenger tagging, 42
together with hole-burning spectroscopy43-44 to allow observation and assignment of individual
hydration conformers.
The spectrum of the dihydrate species in the OH stretching region is presented in Figure
4.14 and is remarkably similar to that of the monohydrate. It is again dominated by two broadfeatures at 3190 cm-1 and 3340 cm-1 corresponding to hydrogen bound OH stretches and again has
the signature of the free OH stretching vibration at ca. 3700 cm-1. The two water molecules can beeither bonded to the same oxygen, or one to each binding site, resulting in three possible bindingsite occupation combinations. For each binding site occupation there are again several possible
conformers due to the conformational flexibility within each site, and these conformers are all79

close in energy. Figure 4.15 shows a comparison of the experimental spectrum with several
calculated conformers.

Figure 4.14. Calculated IR spectra of several conformers of [HBDI·(H2O)2]- (lower traces),compared to the experimental spectrum (upper trace). The points in the upper trace are raw data,
the full black line is an 11-point gliding average to guide the eye. The spectrum contains signatures
for CH stretching vibrations (CH), OH stretching H-bonded to the phenolate (red-shaded region)and imidazole (blue-shaded region) groups, and the free OH stretching mode (F). All calculations
have been scaled by 0.957 to match the experimentally observed free OH stretching vibrations.
The calculated energies (at 0 K) of the conformers relative to the lowest energy conformer (IIa)are given in each simulated trace. Arrows in the lowest trace show how the infrared features ofconformer IIa change as the H-bond between the water molecules is lost (see text). Arrows in the
trace for conformer IId show the expected analogous behavior upon breaking the water-water Hbond for a conformer with both water molecules on the imidazole. The full and dashed lines in the
trace for conformers IIc and IIc’ correspond to the water molecule positions indicated by full anddashed circles, respectively.

80Conformer IIa is the lowest energy structure with both water molecules binding to thephenolate and forming a water-water hydrogen bond (see Figure 4.9). We note, however, that theexperimental spectrum does not show any feature that would indicate a water-water hydrogenbond, which is predicted at ca. 3600 cm-1. Experiments by Johnson and coworkers on I-·(H2O)2
showed that the water-water hydrogen bond in that cluster does not survive in trap temperatures in
the present experiment, easily explaining the lack of this feature in our experiment.45 Based on
their work, we expect the two ionic hydrogen bond features to shift closer together (see arrows inlowest trace of Figure 4.14), behaving like independent monomers rather than dimers. The
corresponding conformer (IIb) in the present case is a stable minimum energy structure, which ispractically isoenergetic with IIa. At the internal energies present in these complexes, the higher
entropy of conformer IIb will likely drive the breaking of the water-water hydrogen bond, and we
expect that conformer IIa is not populated at this temperature. As a result, the lower frequency
feature in the experimental trace is still due to hydration of the phenolate oxygen, while the higher
energy feature is indicative of hydration of the imidazole moiety. Attempts at generating a stablestructure with two independent water molecules bound to the imidazole moiety failed. We do,
however, expect the same entropy driven loss of a water-water hydrogen bond to occur for thisconfiguration, which will lead to the behavior indicated by the arrows shown in the trace ofconformer IId in Figure 4.14. The experimental spectrum is again consistent with a mixture of
hydration conformers, but we refrain from estimating the ratio of the different conformers due tothe large number of possible structures. Hydration of the phenolate by both water molecules islowest in energy, followed by hydration by one water at each binding site (+70 meV), with twowater molecules on the imidazole being highest in energy (+180 meV), which is consistent withcalculations by Krylov and coworkers.10

81While HBDI- is hydrated at both the phenolate and imidazole moiety in our experiment, it
is only hydrated at the phenolate in the protein. While this hinders the immediate comparison of
hydration effects in clusters with hydration in the protein, it is still interesting to explore the effectof hydration on the electronic spectrum of HBDI-. Figure 4.15 presents the electronic spectra for
HBDI- at 30K and [HBDI·(H2O)n]- (n = 1, 2) at 160 – 180 K. The band envelope for bare HBDIand the singly hydrated species are strikingly similar. The vibronic features of the hydrated species
are less well resolved than for bare HBDI-, but we assign the band origin to a partially resolved
peak at 20985  30 cm-1, which is only 55 cm-1 blue-shifted from bare HBDI-. Additionally, we
have a partially resolved peak at ca. 21120 cm-1 and a shoulder at 21195 cm-1. These features arelikely due to low-frequency bending motions of the methine bridge (see section 4.3). We also
assign the set of peaks around 22200 cm-1 as an imidazole ring breathing/in-plane CH bending
mode and its combination bands with the methine bridge bending mode. While the addition of asingle water molecule has a minimal effect on the electronic spectrum of HBDI-, the spectrum of
the dihydrate exhibits a significantly altered envelope. Here we observe a broad feature around21400 cm-1, followed by another around 22130 cm-1. It is impossible to extract a clean band origin
from the spectrum, but we find that the half-maximum point on the rising edge of the first featureis blue shifted by ca. 180 cm-1 from the analogous point of the monohydrate envelope.

82

photodissociation action [arb. units]

HBDI-

HBDI-·H2O

HBDI-·(H2O)2

19500

21000

22500

24000

-1

wavenumber [cm ]Figure 4.15. Electronic spectra of HBDI- at 30K and [HBDI·(H2O)n]- (n = 1, 2). The photofragment
signals were the loss of a methyl group for bare HBDI-, and of all H2O molecules for[HBDI·(H2O)n]-, respectively. The vertical dashed line marks the band origin of bare HBDI- at
20930 cm-1.

We assume the vertical excitation energies are equal to the band origin energies in the FC
region based on the discussion of the bare HBDI- spectrum, where the calculations predict ashallow minimum energy geometry on the S1 potential energy surface. Our calculations, done at
the CAM-B3LYP/def2-TZVPP level of theory, predict a shift of -9 cm-1 and +54 cm-1 for hydration
at the phenolate and imidazole oxygen, respectively. This is in contrast to calculations done by
Krylov and coworkers10 at the SOS-CIS(D)/cc-pVTZ level of theory which predicted a shift of+240 cm-1 and -160 cm-1 upon hydration at the phenolate and imidazole oxygen, respectively. The
first feature in the monohydrate spectrum (at ca. 20985 cm-1), which is broader than that of bare
HBDI-, likely contains the band origin of both hydration conformers. The splitting between the83calculated band origins in our calculations are consistent with the experimental width of thisfeature, although this agreement is likely fortuitous, while the splitting calculated by the Krylov
group is too large to fit the experimentally observed width of the FC profile.
Calculations of vertical excitation energies for the three hydration families of the dihydrate
span ca. 300 cm-1 (see Table 4.4 for a summary), and some are red shifted from that of the bareion. These calculations do not explain the observed blue shift in the experimental spectrum or thedeviation from the characteristic envelope of HBDI-. It is likely that the change in envelope of thedihydrate spectrum is at least in part due to a mixture of hydration conformers. However, becauseof the many complications arising from the mixture of conformers and the lack of a clear
computational description, we refrain from a more definitive assignment. We note that thedramatic shift induced by the second water molecule is unexpected, as most often the first one or
two molecules induce a significant change, while additional water molecules have less pronounced
effects.46-47 This is clearly not the case here, where the second water molecule brings the electronic
spectrum significantly closer to bulk solution than the first.
Figure 4.16 is a summary of all the electronic spectra we have discussed thus far, alongwith a comparison to the spectrum of GFP taken at 77 K48 and that of HBDI- in aqueous solution.
The figure shows the striking similarity of the absorption band envelopes of HBDI- in the protein
and in vacuo, even when solvated by a single water molecule. As discussed previously, additionof a second water molecule shifts the band dramatically towards the solution spectrum. Thishighlights the value of experiments done on isolated chromophores. Not only can they provide aclean access to intrinsic photophysics, but also solution phase data can be representative of entirely
different behavior than that of a protein environment.

84

Figure 4.16. From top: Absorption spectrum of wild-type GFP at 77 K (green); photodissociation
spectra of HBDI- at 30K (black) and [HBDI·(H2O)n]- (n = 1, 2, magenta and blue) in the visible
spectral region; UV/vis absorption spectrum of aqueous HBDI- at pH 11. All traces werenormalized to the same peak height. Photofragment signals were the loss of a methyl group forbare HBDI-, and of all H2O molecules for [HBDI·(H2O)n]-, respectively. The vertical dashed line
marks the band origin of bare HBDI- at 20930 cm-1. The absorption spectrum of wild-type GFP
was digitized from ref.48

4.5

Hot band assisted infrared spectroscopy
In the visible and UV regions of the electromagnetic spectrum, absorption of one or two

photons is often sufficient to lead to photodissociation of a given molecule, but this is not typicallythe case for IR spectroscopy, since the bond dissociation energy of most molecules is significantly
larger than the energy deposited by one or two IR photons. Therefore, to take IR photodissociation85

spectra of molecules, one must form weakly bound complexes with molecules like N2, withbinding energies on the order of a few hundred cm-1. Combining the absorption of an IR photonwith absorption of a UV photon, or photons, leads to a set of IR-UV double reesonance methods
which can be used to investigate the structures of molecules. Though these methods require theimplementation of two light sources, they are free from ion temperature limitations, difficulties in
the preparation of messenger tagged species, and the technical requirements of infraredmultiphoton dissociation (IRMPD) experiments, which typically require the use of expensive freeelectron lasers. There are several ways to carry out IR-UV double resonance spectroscopy,
summarized in Figure 4.17. One such technique is referred to as ion dip spectroscopy (Figure
4.17.a) which involves tuning the UV/vis light source to a specific, narrow transition |0⟩ → |𝑓⟩starting from the electronic and vibrational ground state, |0⟩, where fragmentation occurs uponelectronic excitation into the fragmenting final state |𝑓⟩. One then monitors the fragment ion signal
while tuning the infrared light source. When the infrared light source becomes resonant with atransition, the fragment ion signal dips as the population of |0⟩ is depleted. Another technique isto tune the UV/vis light source to be on resonance with a specific vibrationally excited state |𝑣𝑖𝑏⟩
to a specific vibronic resonance in the fragmenting state |𝑓⟩ ((|𝑣𝑖𝑏⟩ → |𝑓⟩), true IR-UV double
resonance (Figure 4.17.b).
In this chapter, we discuss another double resonance approach which works well for largeions due to their ability to undergo rapid intramolecular vibrational relaxation (IVR).49 In thisapproach, the target molecule (HBDI- in our experiment) absorbs an infrared photon and undergoes
IVR, resulting in a hot ion with nowhere for the excess energy to go. The distribution of energy
depends on the time after absorption and is not necessarily a thermal distribution. We then tune
the UV/vis photon to an energy where hot bands are found in the UV/vis spectrum of HBDI-,

86

which results in excitation into the fragmenting state |𝑓⟩ (Figure 4.17.c), here the S1 state. We referto this technique as hot band assisted IR (HAIR) spectroscopy, and note it is sometimes referred
to as IR-UV gain spectroscopy in the literature.

Figure 4.17. Excitation schemes for resonant IR-UV 2-photon photodissociation spectroscopy.
Fragmentation occurs upon excitation to state |𝑓⟩.

In Section 4.3 we posed the possibility of the increase of intensity on the low energy sideof the band origin region of the electronic spectrum observed for HBDI- as a function of increasing
temperature being due to the development of a population of the E isomer, which has been
observed in room temperature ion mobility experiments.13 To address this question, we will use
HAIR spectroscopy to investigate the temperature dependence of the IR spectra of HBDI-.
This type of spectroscopy requires a slight modification to our experimental operation, as
both infrared and visible light pulses will interact with the target ions. The two beam paths are
spatially separated by 8 cm, and the infrared pulse irradiates the target molecules ca.1.5 s before
the visible pulse (parent ion kinetic energies are ca. 3.2 keV, the mass of the ion is 215 u). The
mass spectrometer and visible OPO are operated at a repetition rate of 20 Hz, while the IR light87source is fired every other experimental cycle (10 Hz). This provides photodissociation signals
with and without IR irradiation, and the difference between these signals can be traced to theabsorption of IR light.
Figure 4.18 shows a comparison between the electronic spectrum of HBDI- at 30 K and
300 K, focusing on the S0  S1 band origin at 20930 cm-1. These spectra, discussed in Section 4.3,
were taken without prior exposure to infrared irradiation. Comparing the two reveals that the
spectral region below ca. 20750 cm-1 contains only hot bands and shows no absorption features at
low temperatures. We use this region of the spectrum as a probe for infrared spectroscopy in the
HAIR approach.

Figure 4.18. UV-Vis spectra of HBDI- at 300K (red trace) and 30K (blue trace) in the band origin
region. The dots are data points, the lines are a 5-point adjacent average (300 K) and an Akima
spline (30 K) to guide the eye. The dashed vertical line shows the band origin at 20930 cm-1. Notethat no IR irradiation was done to acquire these spectra.
Absorption of an infrared photon prior to electronic excitation adds vibrational energy into
the molecule, which leads to an increase of intensity in hot bands, compared to irradiation by88

visible photons only. This effect is demonstrated in Figure 4.19. The increase in hot band intensity
due to infrared irradiation is observable for all trap temperatures studied here, from 30 K to 300
K, which allows us to record infrared spectra of HBDI- at any temperature in this range. This would
not be feasible relying on messenger tagging, given the narrow range of temperatures for attachingmessenger species (e.g., 25 K – 40 K for N2 in our experiment). As can be seen in Figure 4.19, theinfrared-generated hot bands are more pronounced at low trap temperature than at hightemperature, and the signal-to-noise ratio is correspondingly higher at low temperature.

89

Figure 4.19. Electronic spectra in the S1 band origin region of HBDI- at 30, 180, and 300 K trap
temperatures. Full lines are spectra without (black) and with (red) resonant IR irradiation at 1536
cm-1; open triangles show the difference between the two signals, where the difference values for
300 K have been amplified by a factor 3 for clarity.
Figure 4.20 shows the HAIR spectra of HBDI- in the carbonyl stretching region at various
temperatures, compared to that of the N2 messenger tagged spectrum. Calculated spectra for the E
and Z isomers of HBDI are also presented. The spectra show a weak temperature dependence of
the IR signatures. The peaks at 1544 cm-1 and 1600 cm-1 shift by -5 cm-1 and -7 cm-1 respectively

90

over the range of 30 K to 300 K trap temperature. Recall from Section 4.3, the electronic spectrumof HBDI- had developed a partially resolved peak at 20700 cm-1 at 180 K. The shifts of both the1544 cm-1and 1600 cm-1 feature are nearly indiscernible at 180 K. The peak at 1680 cm-1 broadenswith increasing temperature, until at 300 K it is no longer clearly observable.

91

Figure 4.20. Infrared spectra of HBDI- under different conditions. The lowest traces are simulated
spectra for the dominant Z isomer (full black line) and the E isomer (dotted black line). The othertraces show experimental infrared spectra using N2 tagging and HAIR data for different trap
temperatures as indicated on the traces. Open circles are raw data points, the full lines are 5-point
adjacent averages. The vertical lines show the calculated band positions of the dominant band for
the Z and E isomers. The HAIR spectra were acquired at 20580 cm-1, 20160 cm-1, and 19800 cm1
for 30 K, 180 K, and 300 K trap temperature, respectively.

To understand the decrease in HAIR signal with increasing temperature, we consider thetemperature dependence of the vibrational molecular heat capacity and assume that IVR results ina thermal distribution of energy throughout the vibrational modes of the molecule by time thevisible irradiation arrives (ca. 1.5 s after irradiation by the IR source). Figure 4.21 shows thetemperature dependence of the vibrational contributions to the internal energy (Uvib), which was92

calculated from equation (1), and constant volume heat capacity (CV,vib), which is simply thetemperature derivative of (1), of HBDI-.
ℏ𝜔

𝑈𝑣𝑖𝑏 = ∑𝑗 ℏ𝜔𝑗/𝑘𝐵𝑗𝑇
𝑒

−1

(1)

Here j are the frequencies of the vibrational modes (obtained from harmonic frequency
calculations), ħ is the reduced Planck constant, kB is the Boltzmann constant, and T is thetemperature. If we consider an ion of initial temperature of 30 K absorbing a single IR photon of
frequency 1000 cm-1, after IVR, this increase in internal energy represents a substantial increasein the molecular temperature (demonstrated by the blue dots in Figure 4.21). If we instead consider
an ion with an initial temperature of 300 K absorbing that same 1000 cm-1 photon, the change inion temperature is less pronounced (demonstrated by the orange dots in Figure 4.21) as the heat
capacity has increased. Therefore, the HAIR intensity will in general be more difficult to measurewith higher starting temperatures due to the relatively small change in ion temperature resulting
from absorption of the IR photon. This problem is exacerbated when dealing with larger molecules,
which have correspondingly larger heat capacities to begin with, and when dealing with relatively
low energy photons.

93

Figure 4.21. Constant volume heat capacity (black trace) and average vibrational internal energy(red trace) of HBDI- as a function of temperature. The circles and curved arrows show the change
in molecular temperature upon absorption of a 1000 cm-1 infrared photon, starting from 30 K (blue)and from 300 K (orange). The horizontal arrows indicate which curve belongs to which verticalscale.

We present the carbonyl stretching region in Figure 4.20 because this region was used in
the initial Z isomer assignment in Section 4.3, based on the spacing of the three dominate features.
We observe neither a significant change in the spacing between these features of the spectrum at
180 K, nor the emergence of any new features at that temperature, and therefore conclude the
HAIR spectra do not suggest the development of a population of the E isomer.
This leaves the questions about temperature dependence of the electronic spectrum of
HBDI- unanswered, specifically the observation that behavior of the low energy side of theabsorption band that was not recovered by FC simulations. This behavior is reminiscent of thoseobserved by Roithova and coworkers50 who found a pronounced temperature dependence of thespectra of rhodamine 123, which was also not recoverable by FC simulations. They concluded thatthe cause of this dependence lies in the fact that photodissociation required 3-4 photons. An
94efficient return to the ground state after successive absorption events resulted in an increase ofvibrational temperature of the ions for the absorption of all photons after the first, drasticallyskewing the spectrum towards lower photon energies. These effects would not be accounted forby FC simulations. We assume similar processes are affecting the spectrum of HBDI-, since two
photons are required for photodissociation in the S1 band origin region. HBDI- is known to undergo
radiationless relaxation with ps lifetime7-8, 19, 23-24, 27, 51 after absorption of the first photon, which
results in the population of vibrationally hot ions in the electronic ground state, which then absorb
the second photon. A simple FC simulation is unlikely to predict the spectral envelope in thesecircumstances.

95

4.6

References

1.

Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C., Green FluorescentProtein as a Marker for Gene-Expression. Science 1994, 263 (5148), 802-805.

2.

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103

Chapter 5 ProtoporphyrinThis chapter has been adapted from Ref.1 with permission from the PCCP Owner Societies.

5.1

Introduction
Porphyrins are a class of macrocycles with a characteristically intense visible absorption

and are a vital component of many biological processes such as participating in electronic/chemicaltransport,2 and photosynthesis,3 among others. Their abundance in biology has prompted manyefforts to apply them in areas centered around their visible absorption properties such as
photosensitizers in solar cells,4-5 photocatalysts for CO2 reduction,6-7 and as candidates for nonlinear optical materials8 to name a few. Optimization of these applications requires a thoroughunderstanding of the intrinsic electronic structure of the porphyrin system being used, as well as
how interaction with the chemical environment can affect their properties.
Porphyrins are tetrapyrroles (four pyrrole rings connected by methine bridges forming an
extended conjugated system) which form a conjugated ring. Varieties exist with or without a metal
atom in the center (see Figure 5.1). The incredible biological versatility is introduced by modifying
the side chains of the pyrrole rings or changing the metal center if one is present. The simplest way
to model the electronic properties of a porphyrin ring is by treating the 18 π electrons as particles
on a ring. This was used by Gouterman9-10 to develop the four-orbital model which predicts two
separate pairs of degenerate transitions, where the lower energy pair is weaker and is referred toas the Q band, and the higher energy pair is more intense and is referred to as the B or Soret band.
While the Soret band typically presents as a single, wide feature, the Q band typically consists of
two features spaced by ca. 1500 cm-1, which are part of the vibronic progression. These two104

features are resolved even in low-resolution spectra. The Q band appears either in of two patterns,
which are named after the symmetries of the macrocycles as the D4h and the D2h pattern (see Figure5.1). The D4h pattern consists of a single set of two features and is observed primarily in the spectraof metalloporphyrins, where the macrocycle has approximate D4h symmetry. This spectral pattern
can also be achieved by protonating all 4 of the pyrrole nitrogen atoms. The D2h pattern appears if
the metal center is removed and only two of the pyrrole nitrogen atoms are protonated, thus
lowering the symmetry and breaking degeneracy of the electronic state giving rise to the Q band.
This leads to the observation of two separate pairs of features, which are referred to as the Qx andQy bands. The B band undergoes an analogous splitting and gives rise to a Bx and By band, thoughtypically the splitting in these bands is too small to be observed, and is outside the spectral range
considered in the present experiment. The D2h pattern is also referred to as the free-base pattern
and is found in the case of protoporphyrin IX (PP), which is the focus of this chapter.

105

Figure 5.1. D4h and D2h macrocycle structures and their schematic electronic spectra for a general
metalloporphyrin (left) and protoporphyrin IX (right).
The electronic structure of porphyrin systems has been studied for decades9, 11-14, includingthat of PP.15 Many of these experiments were performed at room temperature in solutions, causing
vibronic structure, beyond the previously mentioned characteristic two-peak structure of the Q
band, to largely be obscured by hot bands and solvent effects. PP has an added complication inthat it is amphiphilic and forms polydisperse aggregates in aqueous solution, with a pH dependent
number of monomer units.16 Porphyrins have also been studied extensively in cryogenic matrices9,15which were able to provide vibronic resolution, but in these experiments each absorption featureappears as a multiplet, due to matrix effects, complicating interpretation. As a result, condensedphase results are difficult to interpret and do not reflect the intrinsic photophysics of thischromophore.

106

Experiments have been done on PP in gaseous environments as well. Brøndsted-Nielsen
and coworkers17 investigated the PP Q bands in an experiment similar to ours but at room
temperature and they were unable to observe the lowest energy peak of the Qx signature. The
features of the vibronic progression they did observe were unresolved due to the elevatedtemperature. Experiments have also been done on neutral PP using supersonic jet techniques whichyielded congested spectra,18 likely due to incomplete vibrational cooling. In general, an electronic
spectrum in the Q band region of PP with vibronic resolution has remained elusive, leaving key
details about the intrinsic photophysics of this important species unknown.
Here we present the electronic spectra of cryogenically prepared PP in a singly and doubly
deprotonated form, where we achieve vibronic resolution of the Q bands. Additionally, we are able
to investigate the electronic Stark effect, a shift of the electronic bands induced by the electric fieldof a charge, or set of charges, located only a few Å away from the conjugated macrocycle, which
is on the order of 109 V/m. We also present infrared spectra of both deprotonated species to assess
structural differences.

5.2

Computational
Ground and S1 excited state geometry optimizations and harmonic frequency calculationsof PP and porphine (a simplified model of PP) were performed using DFT at the CAM-B3LYP/ccpVDZ level of theory. Vertical transition energies of the S1 and S2 states were calculated using
TDDFT with both the ⍵B97XD and CAM-B3LYP functionals and the cc-pVDZ basis sets afterground state optimization with the same functionals/basis sets.
Results from these calculations were then used to perform Franck-Condon-Herzberg-Teller(FCHT) simulations. We interpret our experimental spectrum using the FCHT simulation for107porphine. We employed the vertical gradient approximation19 when performing FCHT simulationsof PP in the S1 state, as calculations on PP not done under this approximation lead to a failure toconstruct a Duschinsky rotation matrix. All FCHT simulations were done at 0 K, and all DFT,TDDFT, and FCHT simulations were performed using Gaussian 16.
We also used multireference methods to calculate the vertical excitation energies asprevious work has shown that CASSCF/NEVPT2 calculations predict the excitation energies of
the Q bands well,20-21 and Angeli et al. note that using an active space of 4 electrons in 4 orbitals
provides nearly quantitative splitting of the Q bands.22 The active space for all reference wave
functions was chosen to include the two highest (and lowest) lying doubly occupied (andunoccupied) π-orbitals from the conjugated system of PP and the reference wave functions weregenerated using state averaged CASSCF (denoted SA-N-CASSCF, where N is the number of states
averaged). The energies of these wave functions were corrected using NEVPT2.23-25 Exploratory
calculations were performed averaging 3 (X, Qx, Qy) and 5 states (X, Qx, Qy, Bx, By). Both results
are reported here as both produced qualitatively similar results with the N = 3 case providing thebest agreement with experiment. All correlated calculations used the cc-pVDZ basis set, were
performed using the PySCF26-28 package, and were checked with the ORCA29-30 package. All
multireference calculations were performed by James E. T. Smith in the group of Prof. Sandeep
Sharma.

5.3

Discussion
Figure 5.2 shows the D2h patterned electronic spectrum of [(PP–H)·N2]- in the Q band

region. The experimental spectrum fails to return to baseline on the high energy end of thespectrum which we attribute to the onset of the Soret band. The Qx band origin is at 15868 ±10108cm-1 and is followed by a vibronic progression that extends about 2000 cm-1. The Qy band origin
is at 18760 ± 30 cm-1 and has a similar, though less resolved, vibronic progression. This reductionof resolution is likely due to a shorter lifetime of the S2 state. Exploratory measurements on thecomplex with two N2 molecules attached indicate a ca. 10 cm-1 induced shift of the band origin
per N2 adduct.

Figure 5.2. Electronic spectrum of [(PP–H)·N2]-, showing the Qx and Qy absorption bands. The Qx
band origin is at 15868 ±10 cm-1, and the Qy band origin is at 18760 ± 30 cm-1. Both band origins
exhibit a qualitatively similar vibronic progression which extends ca. 2000 cm-1 above each origin.

Figure 5.3 shows the Qx band of the experimental electronic spectrum (blue trace) alongwith the FCHT simulation for porphine (black trace) and (PP-H)- (green trace). Recall, the FCHT
simulation for (PP-H)- was done under the vertical gradient approximation. Both simulations
acceptably recover the experimental pattern, however the porphine simulation has a slightly better
match. This suggests that the observed vibronic progression is primarily due to excitations in thevibrational modes of the macrocycle, rather than the propionic side chains. This is consistent with109the observation of the same general Q band structure (and spacing) for most porphyrins, as was
discussed in the introduction. Others have shown previously that while the Herzberg-Teller
treatment underestimates the relative intensity of the band origin, it does produce a better matchwith the vibronic progression in general when compared to FC simulations.12 FC simulations do
however capture the experimental band origin behavior well, and establish the vertical transitionenergies at the band origin.

110

shift from origin [cm-1]
0

750
710

000

1500

1310

7010

7510

2250

9110

4110

intensity [arb. units]

3310

5310

15750

91101310

6810

16500

6110

17250

18000

wavenumber [cm-1]

Figure 5.3. The experimental Qx absorption band of [(PP–H)·N2]- (blue) compared with the FCHT
simulation of neutral porphine (black) and FCHT simulation (using the vertical gradientapproximation) of (PP-H)- (green). All calculations (ground and excited state) were done at the
CAM-B3LYP/cc-pVDZ level of theory using a scaling factor of 0.97. The open circles in theexperimental trace are data points and the full line is a B-spline to guide the eye. The assignments
are based on the porphine simulation. The simulations have been shifted to align with theexperimental band origin.
As stated above, the Qx band origin of [(PP–H)·N2]- is at 15868 ± 10 cm-1 and is followed
by a vibronic progression that extends until ca. 18000 cm-1. In the remainder of this chapter, we′𝜈
denote transitions, as in Chapter 3, by 𝑄𝜈", where Q is the label of the vibrational mode numberedin order of increasing vibrational frequency in the S1 state, and ν” and ν’ are the numbers of quanta111

in mode Q of the ground and S1 electronic states, respectively. For clarity, experimental
frequencies are reported in terms of their offset from the band origin, not as absolute values. Theband origin is accompanied by a shoulder at 77 cm-1 and a partially resolved peak at 227 cm-1. The
shoulder is due to the 710 band, which can be described as a macrocycle deformation mode whereelongation along the x-axis is out of phase with elongation along the y-axis. The peak at 227 cm-1
is due to the 1310 mode and is the in-phase combinations of these motions.
The next set of peaks we assign are the two partially resolved peaks at 642 cm -1 and 722
cm-1, which are the 3310 and 4110 modes, respectively. These modes include deformations of thepyrrole rings coupled with distortions of the macrocycle. This low energy portion of the absorption
band exhibits minor spectral congestion, but beyond ca. 1000 cm-1 from the band origin, significant
spectral congestion sets in because of multiple FCHT active states being close in energy resulting
in the spectrum failing to return to baseline between features, as was similarly observed in m-NP
in Chapter 3. The remaining assignments are based on states with the largest FCHT factors, but
the weaker transitions are likely present as unresolved shoulders and certainly contribute to theoverall band pattern of the experimental spectrum. The 5310 and 6110 modes contribute to twopartially resolved features centered on 972 cm-1. Mode 53 is a combination of pyrrole andmacrocycle deformations, and mode 61 is an in-plane pyrrole CH wagging mode. The 6810 band,
which appears as a shoulder at 1102 cm-1, is a methine CH wagging mode with all four methine
bridges contributing. The set of three intense peaks at 1202 cm-1, 1332 cm-1, and 1532 cm-1
primarily correspond to the 7010 , 7510 , and 9110 modes respectively. Mode 70 is predominantly an
in-plane NH bending motion with minor contributions from methine CH wagging motions. Mode
75 is a CN stretching motion also coupled with methine CH wagging motions. Mode 91 is a CC
stretch involving the bridging carbons combined with NH wagging.

112

In the case of (PP-H)-, the excess negative charge is placed ca. 4 Å from the rim of the
conjugated macrocycle. This produces an electric field strength on the order of 109 V/m at the rimof the conjugated system. The Stark effect from an electric field of this magnitude will have aneffect on the electronic energy levels of the molecule. Unfortunately, our experiment is limited toions, so we cannot measure the spectrum of neutral PP to say anything about the shift induced by
the first deprotonation event. We can, however, investigate the effect of the second deprotonation
event by comparing the results of the singly deprotonated (monoanion) to a doubly deprotonated(dianion) PP. In addition to the change in the overall charge of the molecule, the mono- and dianion
must have different structures. In the dianion, the two carboxylate groups will both carry a negative
charge and therefore repel each other, while in the monoanion no such repulsion exists, as only
one group is deprotonated.

113

Figure 5.4. IR spectra of [(PP-H)·N2]- (blue) and [(PP-2H)·N2]2- (purple) in the fingerprint region
from 1000 cm-1 to 2200 cm-1. Each trace is accompanied by a simulated spectrum (scaling factor
0.95) for the structure shown.
Figure 5.4 shows the IR spectra of [(PP-H)·N2]- and [(PP-2H)·N2]2- in the region from 1000
cm-1 to 2200 cm-1 and simulated harmonic spectra of the conformers shown. These conformers
were the lowest energy structures found in a structural search. The lowest energy monoanion
structure has the two carboxylic groups sharing the remaining proton, while in the case of the
dianionic structure this proton has been removed and the groups repel each other. Overall, thesimulations reproduce the experimental spectra well. For the monoanion, the two peaks at 1726
cm-1 and 1617 cm-1 represent the antisymmetric OCO stretching vibrations of the two carboxylategroups which are slightly coupled. The peak at 1617 cm-1 is accompanied by an unresolved
114

shoulder at ca. 1592 cm-1 which is due to the two CC stretching vibrations of the ethylene groups.
The broad feature at 1430 cm-1 is due to the bending motion of the shared carboxylic acid proton.
The final two features we assign are at 1280 cm-1 and 1245 cm-1 which are HCH bending modes
localized on the carboxylic acid side chains coupled with the symmetric OCO stretchingvibrations.
The purple trace in Figure 5.4 shows the IR spectrum of [(PP-2H)·N2]2-. The twoantisymmetric OCO stretching vibrations observed in the monoanion spectrum have merged intoa single feature at 1609 cm-1 which, based on the calculation, contains the two unresolved
antisymmetric stretching signatures which are of similar intensity and have now been decoupled.
The CC stretching vibrations of the ethylene groups are again present as an unresolved shoulder
on the low energy side of this peak. The lower two features of the monoanion spectrum, whichcorrespond to HCH bending modes localized on the carboxylic acid side chains coupled with thesymmetric OCO stretching vibrations, have similarly collapsed into a single feature at 1324 cm-1
in the spectrum of the dianion. The fact that both sets of features, which were both two separate
peaks in the monoanion spectrum, now exist as single features, reflects the fact that the two
carboxylic acid chains are now nearly completely decoupled. A similar result was reported in work
by Johnson and coworkers on singly and doubly deprotonated dodecanedioic acid,31 where thevibrational signatures of the monoanion species are significantly more complex than those of the
dianion, as the carboxylate groups in the monoanion share a proton, and the two groups become
fully decoupled in the dianion. The OHO bending signature has vanished from the spectrum of(PP-2H)-2, and this is to be expected as the final carboxylic acid proton is no longer present. The
structure shown for the dianion is the lowest energy isomer of four which produce identical IR
spectra. These conformers consist of different combinations of orientations of the carboxylate tails

115being either above or below the plane defined by the macrocycle. All of the conformers in this set
are calculated to lie within 30 meV of the structure shown, which has one carboxylate group (onthe left in the figure) below the plane defined by the macrocycle, and the other (on the right on the
figure) above that plane. We do not attempt to identify which isomer is present in this experiment
as none of our measurements can distinguish between them.
Figure 5.5 shows the IR spectrum in the CH and OH stretching region, 2800 cm-1 to 3800
cm-1, of the two protonation states of PP. Both spectra show the anti-symmetric NH stretchingvibration as intense features at 3324 cm-1 and 3327 cm-1 for the mono- and dianion, respectively.
The symmetric combination of these motions is present as a subtle feature in the monoanion
spectrum at 3368 cm-1, but is too weak to be observed in the dianion spectrum. The features justbelow 3200 cm-1 are CH stretching vibrations as well as Fermi-resonances of these modes withovertones and combination bands of lower frequency modes. There is no evidence of a free OH
stretching vibration in the spectrum of the monoanion (or the dianion), conforming the priorassertion that the hydrogen is shared between the two carboxylate groups (or in the case of the
dianion, no longer present).

116

photodissociation action [arb. units]

0

0

2800

3000

3200

3400

3600

3800

wavenumber [cm-1]

Figure 5.5. IR spectra of [(PP-H)·N2]- (top panel) and of [(PP-2H)·N2]2- (bottom) in the CH andOH stretching region.

The IR spectra inform on the molecular structure of PP when moving from a singly to
doubly deprotonated species. To investigate the effect on the electronic structure we look at theelectronic spectrum. Figure 5.6 shows the Q band region for the two species. The local electricfield created by the charged side chains on the macrocycle most likely plays the main role in theobserved band shifts through the Stark effect. The band structures of the two spectra are
qualitatively very similar, with only slight changes in the splitting of the Qx and Qy bands and in117the relative intensities of a few features in their vibronic progressions. The similarity of thevibronic progressions is not surprising, because the structure of the macrocycle has not changed
substantially and, as we saw in the discussion of the FCHT simulation of the monoanion, themacrocycle largely determines the vibronic structure in PP. There is literature on neutral PP in thegas phase18 but their data have relatively large error bars compared to our results. Comparing thisdata on the neutral to our data on the monoanion suggest that the Qx band shifts very little to thered upon loss of a single proton, while the Qy band shifts by ca. -300 cm-1, reducing the Qx – Qy
splitting (see Table 5.1). In our experiment, the Qx band in the dianion is shifted by -110 cm-1
relative to the monoanion, while the Qy band shifts by -490 cm-1. This again reduces the splitting
between the Qx and Qy bands. Also of note, the onset of the Soret band at 22000 cm-1 is now moreclearly visible in the spectrum of the dianion, suggesting that it too has shifted to lower
wavenumbers.

118

Figure 5.6. Electronic spectrum of [(PP-H)·N2]- (blue) and [(PP-2H)·N2]2- (purple) in the Q band
region. Dashed vertical lines mark the positions of the Qx and Qy band origins of the singly
deprotonated species to emphasize the differences.

Table 5.1 shows a comparison of experimental and calculated vertical transitions energies.
As can be seen in the table, the TDDFT calculations generally overestimate the Qx band origin
energy while underestimating the Qy band origin energy. This results in a Qx – Qy splitting which
is roughly half the experimentally observed value. The results of the TDDFT calculations arequestionable, however, because for the dianion they contain charge transfer contributions from thecarboxylate groups to the macrocycle even though both functionals used here are range-corrected.
The NEVPT2 calculations on the other hand generally underestimate both Qx and Qy band origins
but produce a better Qx – Qy splitting for all charge states. The active space for these calculations
consists of orbitals localized on the macrocycle and transitions involving the carboxylate groups
are therefore excluded by design.
119Table 5.1. Calculated and experimental vertical transition energies for PP and porphine in various
charge states expressed in cm-1.
Species
ChargeMethod(a)E(Qx)(b)E(Qy)(b)
ΔE(c)
State
porphine
0
CAM-B3LYP
17500
19321
1821
0
ωB97XD
17029
18926
1897
(d)
PP
0
experiment
15900
19050
3150
±100
±100
± 141
0
CAM-B3LYP
17312
18738
1426
0
NEVPT2
15810
18606
2796
(e)
-1
experiment
15870
18760
± 2890
± 10
30
± 32
-1
CAM-B3LYP
16923
18426
1503
-1
ωB97XD
16518
18095
1577
-1
NEVPT2
15377
18217
2840
(e)
-2
experiment15760 ±10 18270 ±30 2510(-110)(-490)±32-2

CAM-B3LYP-2

ωB97XD-2

NEVPT216970(+452)
16660(+142)
15472
(+95)

18240
(-186)
18048
(-47)
18123
94)

1270
1388
(- 2651(a)

See Methods section for basis sets.

(b)

Numbers in parentheses represent the shift of bands in the dianion from the position of eachband in the monoanion.
(c)

E refers to the Qx-Qy splitting: E = E(Qy) – E(Qx).

(d)

Supersonic jet experiment, from ref 18.

(e)

FC

simulations

establishthevertical120

transitionenergies

at

the

band

origin.

While the NEVPT2 calculations recover the band positions and splittings quite well, they
are unable to reproduce the more subtle changes that occur upon further deprotonation. They
predict a red shift for the Qx band going from the neutral to the singly deprotonated monoanion
and yield a blue shift as PP is deprotonated a second time rather than another red shift. The
calculated Qx – Qy splitting first becomes wider (from neutral to -1 overall charge), then narrows
again (from -1 to -2 overall charge) while the experimental data suggests it narrows both times.
The comparison of these calculations with our experimental results highlight the difficulties thatmolecules such as PP present even when treated at a high computational level, and shows how
cryogenic ion spectroscopy serves as a benchmark for increasingly sophisticated theory.
In many situations, such as the nitrophenolates of Chapter 3 or HBDI in Chapter 4,cryogenic ion spectroscopy in vacuo provides the opportunity to determine absolutesolvatochromic shifts. In aqueous solution, PP forms pH dependent aggregates due to its
amphiphilic character as noted in the introduction, and monomers of PP are only present at low
pH (< 3). Examining pKa values of similar molecules32 suggests that PP should be neutral in thisrange, not ionic, and so a direct comparison to our results should not be made. As a result, shiftsof electronic absorptions observed upon bringing PP into a solvent can be the result of manyeffects: (i) solvatochromic effects due to differential solvation of charge distributions; (ii)aggregation effects; (iii) charge screening effects that affect the impact of the intramolecularelectric field coming from the deprotonated carboxylic acid groups. These effects are not easily
disentangled, and we therefore do not provide a more detailed discussion in this context, but
provide a comparison of the current data with literation data on solutions of PP in Table 5.2.

121

Table 5.2. Solvatochromic data for PP (transition energies in cm-1)
Solvent/ pH
Qx(0)(a)Qx(1) (a)Qy(0) (a)Temperatur (charge)
e

20410
±100

17

15900
±100

in vacuo / (-1)
60

15870 ± 10 17400 ±1018760 ± 30 20090 ±20This workin vacuo / (-1)
RT

N/A

17210
±100

18690
±100

20040
±100

16

in vacuo / (-2)
6015760 ±1017280 ±1018270 ±3019630 ±30This work

water / RT

1

15870

16670

18050

19380

15

water / RT

5

15530

16840

17670

18730

15

water / RT

12

15970

17300

18350

19610

15

15870

17330

18450

19760

15

(a)

19050
±100

ref.

in vacuo / (0)jet

DMSO / RT N/A

17420
±100

Qy(1) (a)Qx(0) and Qy(0) refer to the maximum of each envelope in the band origin region, while Qx(1)and Qy(1) represent the maxima of the envelope in the region of Franck-Condon active modes, ca.
1000 cm-1 above each band origin.

122

5.4

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Carlsen, C. U.; Møller, J. K. S.; Skibsted, L. H., Heme-iron in lipid oxidation. Coordination
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Mamedov, V. A.; Kalinin, A. A., Chapter Two - Quinoxaline Macrocycles. In Advances in
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Tessore, F.; Biroli, A. O.; Di Carlo, G.; Pizzotti, M., Porphyrins for Second Order
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Gouterman, M., 1 - Optical Spectra and Electronic Structure of Porphyrins and Related
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Gouterman, M., Spectra of Porphyrins. Journal of Molecular Spectroscopy 1961, 6, 138163.

11.

Lopes, J. M. S.; Sharma, K.; Sampaio, R. N.; Batista, A. A.; Ito, A. S.; Machado, A. E. H.;
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12.

Santoro, F.; Lami, A.; Improta, R.; Bloino, J.; Barone, V., Effective method for thecomputation of optical spectra of large molecules at finite temperature including the
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13.

Radziszewski, J. G.; Waluk, J.; Nepras, M.; Michl, J., Fourier transform fluorescence andphosphorescence of porphine in rare gas matrixes. The Journal of Physical Chemistry
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Rimington., C.; Mason., S. F.; Kennard, O., Porphin. Spectrochimiea Acta 1957, 12, 6577.

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O. N. Korotaev, R. I. P., Reversible Transformations of Luminescent Impurity Centers in
an N-Paraffin Matrix Under Laser Irradiation. Optics and Spectroscopy 1971, 32 (5), 479480.

16.

Scolaro., L. M.; Castriciano., M.; Romeo., A.; Patane., S.; Cefali., E.; Allegrini., M.,
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17.

Wyer, J. A.; Jensen, C. S.; Brøndsted Nielsen, S., Gas-phase action spectroscopy of
protoporphyrin IX (PP) and zinc-PP anions from 210nm to 720nm. International Journalof Mass Spectrometry 2011, 308 (1), 126-132.

18.

Beames, J. M.; Vaden, T. D.; Hudson, A. J., The spectroscopy of jet-cooled porphyrins: aninsight into the vibronic structure of the Q band. Journal of Porphyrins andPhthalocyanines 2012, 14 (04), 314-323.

19.

Baiardi, A.; Bloino, J.; Barone, V., General Time Dependent Approach to Vibronic
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Sayfutyarova, E. R.; Hammes-Schiffer, S., Constructing Molecular pi-Orbital Active
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Falahati, K.; Hamerla, C.; Huix-Rotllant, M.; Burghardt, I., Ultrafast photochemistry of
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Angeli, C.; Pastore, M.; Cimiraglia, R., New perspectives in multireference perturbation
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Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H., A consistent and accurate ab initio
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Angeli, C.; Cimiraglia, R.; Malrieu, J.-P., n-electron valence state perturbation theory: A
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127Chapter 6 Biliverdin
6.1

Overview
Fluorescent proteins (FPs) have become widely used for fluorescence labeling in biology

and biochemistry where they have been engineered to emit at a variety of wavelengths spanning
the visible range. FPs typically incorporate a relatively small molecular fluorophore, which is
responsible for the fluorescence. While much of the initial work on FPs has been based on Green
Fluorescent Protein and its variants, the strong visible and UV absorptions of biological tissue, its
yellow autofluorescence, and the resulting shallow penetration depth for fluorescent probes in thevisible spectral range are problematic for many applications in tissue imaging. Efforts havetherefore been directed at developing fluorophores that emit in the 650-950 nm (15384 - 10526
cm-1) range, the so-called “first near infrared (NIR) window” of biological tissue, where absorption
by common biomolecules is minimal.1-2 One promising fluorophore for these applications isbiliverdin (BV) (see Figure 6.1) whose photophysical properties have been studied for decades.3-6
Its typical emission wavelengths (700-750 nm)3, 6-10 are in the first NIR window, but they cover a
relatively small portion of the desired wavelength range.
Biliverdin is a breakdown product of heme11 and is structurally similar to protoporphyrin,
which was the subject of Chapter 5. While the tetrapyrrole structure of BV is inherited from its
porphine-type precursor, it does not form a macrocycle, resulting in a molecule with large
conformational freedom. In aqueous solution, BV exists primarily in a helical conformer,12 eventhough other conformers have been found as well,13-15 and it is likely that the absorption andfluorescence properties of BV will depend on its chemical environment and its conformation. It
may be possible to extend the absorption and emission wavelengths of BV further to the red, if its
response to chemical environment and conformation is known and can be modeled. To achieve

128this goal, we need a detailed understanding of the photophysics governing the low-lying electronic
excited states of the molecule, in particular its intrinsic properties, without the influence of achemical environment. Here we present the electronic and infrared (IR) spectra of BV in two
different deprotonation states.

Figure 6.1. Examples of conformer families of singly deprotonated biliverdin, [BV-H]-, with theremaining proton shared between the carboxylate groups of the propionic acid tails. Numbering
the carbon atoms in the conjugated system as shown in structure H, the abbreviated Z/E labels referto the bonds between carbon atoms 4/5, 9/10, and 14/15 in sequence. The abbreviated syn/antilabels encode the conformations with respect to the bonds between the carbon atoms 5/6, 10/11,
and 15/16.

6.2

Computational
Biliverdin has a high degree of conformational flexibility. However, using characteristic

signatures in the IR we were able to narrow down the conformational space considered. We
generated ca. 15 conformers with the restriction that rings B and C were in a Zs configuration and129either sharing the remaining proton on the propionic acid tails (in the case of the singly
deprotonated monoanion) or allowing these tails to repel each other (in the case of the double
deprotonated dianion). A few representative conformers generated in this way are shown in Figure
6.1, differing in the orientation of rings A and D, where the NH groups of the rings can be oriented
towards the other rings, or away from them. All conformers were subject to ground state geometry
optimizations, harmonic frequency, and vertical excitation calculations at the CAM-B3LYP/ccpVDZ level of theory.

6.3

Discussion
Rotation around the bonds of the methine bridges between the pyrrole rings allows theexistence of many conformers. They can be characterized in terms of the relative orientation of the
N atoms on neighboring rings, and the orientation of the apex of the methine bridges between eachring. To describe the structures of different conformers, we will use the notation explained in thecaption of Figure 6.1. In porphine and in the helical conformer of BV (Figure 6.1H ), which is thedominant structure in solution, all methine bridges have the CH groups on their apex point to the
opposite side of the N atoms (or NH groups) of all rings,13 so both structures are ZZZsss in thisnotation. In aqueous solutions, BV also exists in a small part of the population as a slightly
unfolded conformer, where the bridge between rings C and D is inverted, corresponding to ZZEssa.
For a molecule with the conformational freedom of BV, a key question in the context ofthe current work is whether the structure in solution is retained in vacuo. We use IR spectroscopy
in comparison with predicted IR spectra of various conformers to address this question. Different
regions of the IR spectrum encode different structural characteristics. Figure 6.2 shows acomparison of the spectra of [(BV-H)·N2]- and the analogous singly deprotonated protoporphyrin130

IX complex, [(PP-H)·N2]- (which was discussed in Chapter 5) and a simulated spectrum ofconformer J from Figure 6.1 over the range of 1000 cm-1 to 2100 cm-1. PP does not have the same
conformational freedom as BV because the porphine macrocycle is intact in this molecule, whereas
it is open in BV. This region of the IR spectrum of deprotonated PP carries mainly features of the
propionic acid tails. The striking similarity between the two IR spectra indicates that these
propionic acid tails in [BV-H]- and in [PP-H]- adopt similar structures, with the intact and the
deprotonated carboxylic groups sharing the remaining proton. This is a remarkable structuralfeature, since the deprotonated carboxylic group could in principle undergo H-bonding to any of
the NH groups of the open tetrapyrrole system, if the latter is sufficiently unfolded.

[(BV-H)·N2]-

nas
OCO

ndHCH

nas
OCO

nNH(B)nCH

initensity [arb. units]

ndOHO[(PP-H)·N2]-

Conformer J

1000

1200

1400

1600

1800

-1

wavenumber [cm ]Figure 6.2. Top: IR spectrum of [(BV-H)·N2]-, monitoring the loss of N2. Center: IR spectrum of[(PP-H)·N2]-, monitoring the loss of N2. Bottom: predicted IR spectrum of conformer J (see Figure1J), with frequencies scaled by 0.9375.

131

The comparison between BV, PP, and the predicted IR spectra of conformers whose local
structure of the B and C rings and the bridge connecting them resembles that of PP, allows us toassign the most prominent peaks in this spectral range. The following spectral assignment is basedon the comparison with structure J from Figure 6.1, but we note that this conformer is not the only
one which reasonably reproduces the experimental spectrum in this spectral region. We assign the𝑠
two peaks around 1710 cm-1 to CO stretching vibrations. The peak at 1704 cm-1 (𝜈𝑂𝐶𝑂) contains

two overlapping features, one of which is the symmetric combination of the antisymmetric CO
stretching vibrations of the carboxylate groups. The other feature is the CO stretch of the carbonylgroup on ring D. The higher energy peak at 1719 cm-1 (𝜈𝐶𝑂 ) contains the carbonyl stretchingvibration of the carbonyl group on ring A. The symmetric combination of the carboxylate CO
stretching vibrations of the carboxylate group on ring B contributes to the low energy side of the𝑠𝑠𝜔broad feature at 1590 cm-1 (𝜈𝑂𝐶𝑂). The high energy side of this feature (𝜈𝐶𝐻
) is due to a CC stretch

combined with a CH wagging motion of the methine bridge between rings B and C. This feature
is only calculated to be intense when it is sterically hindered, which reinforces our assumption of
𝑒𝑡the Zs relationship between the BC rings. The very weak feature at ca. 1530 cm-1 (𝜈𝐶𝐶
) is thesignature of the CC stretching vibrations of the ethylene groups on rings A and D. Centered around𝛿ca. 1450 cm-1 (𝜈𝑂𝐻𝑂) is a broad feature that is due to primarily OHO bending motions of the shared

proton. We note that these harmonic frequency calculations predict the OH stretching mode
involving this shared proton to be at ca. 2100 cm-1. However, as was the case in the spectrum of
the monoanion of PP, this feature is not present in the experimental spectrum because the potential
energy landscape for proton motion between the two carboxylate groups is strongly anharmonic
and has been found to be significantly red shifted and broadened in similar molecules.16 The feature𝛿
at 1337 cm-1 (𝜈𝐻𝐶𝐻) is caused by a delocalized combination of HCH bending motions coupled with132

symmetric CO stretching vibrations of the carboxylate group on ring B. The feature at ca. 1275
𝜔
cm-1 (𝜈𝑁𝐻(𝐵)) is due to the combination of HCH and NH bending motions of the sp3 carbons of the

propionic acid tails and the NH group of ring B coupled with symmetric CO stretching vibrationsof the carboxylate group on ring C.
While the fingerprint region of the IR spectrum, discussed above, informs on the propionicacid tails and the conformation of rings B and C, the orientations of rings A and D have only weakeffects on the main features in this region. However, information on the conformation of rings A
and D is encoded in the mid-IR region though the NH stretching modes of the pyrrole rings, as theinteraction between the NH groups and the lone N atom on ring C changes the IR spectra incharacteristic ways. The NH groups on rings A, B, and D can either undergo H-bonding with the
N atom on ring C or behave like free (or nearly free) NH oscillators. The NH group of ring B(NHB) forms a H-bond with the N atom on ring C (NC) for all structures found in our restricted
search. Nearly every structure generated in this restricted structural search reproduce the 1000 –2000 cm-1 region acceptably. Their differences lie predominantly in the 2800 – 3600 cm-1 spectral
region, which has other complications.
Figure 6.3 shows the 2800 – 3600 cm-1 region of the experimental IR spectrum of [(BVH)·N2]- compared with the computational spectra of several conformers. Four potential NH
𝑓𝑟𝑒𝑒stretching signatures are observed. A sharp, intense peak at 3444 cm-1 (𝜈𝑁𝐻 ) which is thesignature of a free NH group. A weaker feature, which appears to be a doublet, at 3405 cm-1 and𝐵/𝐷

3391cm-1 (𝜈𝑁𝐻 ), is characteristic of a NH group participating in a weak hydrogen bond. Finally,a very broad feature that barely rises above the baseline and is centered at ca. 3250 cm-1 could be
representative of a more strongly hydrogen bonded NH group. The fact that there are four potential
NH signatures presents several possible explanations: (i) we are looking at a mixture of at least
133

two conformers; (ii) the feature which appears to be a doublet is actually a singlet with anunfortunate dip in the center due purely to the noise level of the spectrum; or (iii) the broad feature
at 3250 cm-1 is a fluctuation in the baseline that escaped our baseline tracking, not a real signal.
Since many scans were averaged on several different days, the latter explanation is very unlikely.
A comparison of the experimental spectrum of [(BV-H)·N2]- with [(PP-H)·N2]- reveals that allfeatures below 3150 cm-1 are not due to NH stretching vibrations as they appear with remarkablesimilarity in both the PP and BV spectrum, and all NH stretching vibrations are accounted for in
the PP spectrum. As can be seen in Figure 6.3, this spectral region is unable to eliminate any ofthe presented conformers due to the weak signal intensity of the hydrogen bonded NH stretchingvibrations. It is also possible that the red shifts of the hydrogen bonded NH stretching modes areincorrectly predicted in the DFT calculations.

134

nB/D
NH

-

[(BV-H)·N2]

nfree
NH

intensity [arb. units]

0

Conformer J

0

Conformer H

0

Conformer D

0

2800

3000
3200
3400
wavenumber [cm-1]

3600

Figure 6.3. IR spectrum of [(BV-H)·N2]- in the 2800 – 3600 cm-1 spectral region, monitoring theloss of N2, compared to computational IR spectra of selected conformers from conformer families
as shown in Figure 6.1, with frequencies scaled by 0.9392. The insert shows a zoom of the NH
stretching region with assignments.

In the lowest energy conformer (structure J from Figure 6.1), the NH group on ring A(NHA) is pointing away from the others, while the NH group on ring D (NHD) is pointing toward
the N atom on ring C. The distances of NHB and NHD to NC are comparable (2.085 Å and 2.037
Å, respectively), and their NH stretching modes are significantly coupled in symmetric andantisymmetric combinations. Using a scaling factor of 0.9392 for the NH stretching frequencies toalign the experimental and calculated free NH features, the antisymmetric and symmetricstretching modes of NHB and NHD are calculated at 3313 cm-1 and 3348 cm-1, respectively. In theother two conformers presented, this coupling between NH stretching modes is removed. In
135conformer H, steric repulsion pushes rings A and D away from each other and forces the molecule
to adopt a helical structure. This puts the NH groups in separate planes, and we observe two nearly
free NH stretching vibrations due to the NH groups on rings A and D, and a hydrogen bonded NH
stretching vibration due to the NH group on ring B. The latter can still interact with the nitrogenof ring C. Conformer K is similar to conformer H but instead of the NH group on ring A being
turned away from the rest of the molecule, now the NH group on ring D is pointing out instead. In
this situation, the NH groups on rings A and B do not couple as strongly as in conformer H, but
the NH group of ring A is able to weakly hydrogen bond with the nitrogen on ring C. Out of all
calculated IR spectra, the pattern for these conformer families recovers the experimentallyobserved features best.
We note that we observe an intense peak at 2930 cm-1 in the CH stretching region, whichis recaptured in all calculated spectra shown in Figure 6.3. This is the signature of the CH stretchingmode of the CH group on the methine bridge between rings B and C, which is particularly intense
for the Zs geometry governing these two rings and their connecting bridge, corroborating thestructural motif we identified for this part of the molecule.
Figure 6.4 shows the electronic spectrum of [(BV-H)·N2]- and the UV-vis spectrum of BV
dissolved in water brought to pH 13 with addition of KOH. In the spectrum of [(BV-H)·N2]-, weobserve a peak at 15000 ± 100 cm-1 (ca. 667 nm) with an onset beginning at ca. 14200 cm-1, and ashoulder at 14500 ± 40 cm-1. Additional groups of features appear at higher photon energies, butthey are rather broad, despite trap temperatures of 20-35 K. Based on the data shown in Figure 6.4,
BV in aqueous solution shows a solvatochromic shift of -220 cm-1 compared to singly
deprotonated BV in vacuo. This solvatochromic shift is not large enough to indicate a significant
charge transfer component in the transition. Vertical transition energies to the S1 states, calculated

136

using TDDFT, for the lowest energy conformers found in our structural search have an average
vertical excitation of 18390 cm-1, with all of them falling within ca. 1100 cm-1 of each other,overestimating the experimental band maximum by 3390 cm-1. The calculated excitation energies
for the S2 states are on average 6900 cm-1 higher than the S1 states indicating that the observed
band structure is due to the FC envelope of the S1 band and could potentially be due to more than
one conformer. The narrow range of observed S1 transition energies shows the weak relation
between the conformation of the macrocycle and the excitation energy of the monoanion, but does
not provide insight into the conformer(s) responsible.
704

wavelength [nm]
699
694
690

685

intensity [arb. units]

709

14000

16000
18000
wavenumber [cm-1]

20000

Figure 6.4. Electronic spectrum of [(BV-H)·N2]-, monitoring the loss of N2 (black trace) and theUV-vis spectra of BV dissolved in H2O at pH 13 (blue trace). The black, open circles are raw data
points, the full black line is a 20-point gliding average to guide the eye.
Figure 6.5 contains the experimental IR spectrum of [(BV-2H)·N2]-2 along withsimulated spectra for the same three conformer families presented for the singly deprotonatedspecies from 1000 cm-1 to 1800 cm-1. Simply removing the shared hydrogen from the conformers
137considered previously, and allowing the molecule to relax its geometry from these starting points,
results in the conformers used to produce the traces in Figure 6.5. Deprotonating in this location
causes the propionic side chains to electrostatically repel each other, and breaks the coupling
between their vibrational modes. As discussed for the monoanion spectrum, the carbonyl region
informs on the propionic side chains. The following mode assignment is based on conformer K
𝐷which best matches the experimental spectrum. The peak at 1704 cm-1 (𝜈𝐶𝑂) is the carbonyl

stretching motion of ring D which couples to the NH bend of the same ring. The same motion, but𝐴for ring A (𝜈𝐶𝑂) shows up as an unresolved shoulder at 1690 cm-1.The mode corresponding to theasymmetric combination of CO stretches on the individual carboxylate groups are now nearly𝑎𝑠degenerate and appear as the peak at 1650 cm-1 (𝜈𝑂𝐶𝑂). The third intense feature in the carbonyl
𝜔𝐴𝐵region, at 1596 cm-1, is due to the CH wag of the bridge between rings A and B (𝜈𝐶𝐻
) . There are𝜔𝐵𝐶several unresolved features centered around 1518 cm-1 (𝜈𝐶𝐻
) which are due to the CH wag of

the bridge between rings B and C coupling with CC stretches of the pyrrole rings. The next featureof the low energy region we assign corresponds to a diffuse mode involving coupled CH wagging
𝜔motions of all three bridges, and ring deformations of all 4 rings, which is at 1350 cm-1 (𝜈𝐶𝐻). Thesymmetric combination of the CO stretching vibrations of the carboxylate groups are responsible𝑠for the feature at 1300 cm-1 (𝜈𝑂𝐶𝑂). The final feature we assign in this spectral region is the peakat 1250 cm-1 which is due to the NH and CH wagging motions from the NH groups on rings A and𝜔
B and the CH of the BC methine bridge (𝜈𝑁𝐻).

138

[(BV-2H)·N2]-2

intensity [arb. units]

Conformer K

Conformer J

Conformer H

1000

1200

1400

1600

1800

wavenumber [cm-1]

Figure 6.5. Comparison of IR spectra of [(BV-2H)·N2]-2 with simulated spectra for three selectconformers from 1000-1800 cm-1. The vertical dashed lines indicate the location of prominent
features in the experimental spectrum. Simulated spectra have been scaled by 0.9325.
Figure 6.6 contains a comparison of the IR spectra of [(BV-2H)·N2]-2 with the same
three conformers used in Figure 6.5 in the CH and NH stretching region. In this range, we observea similar pattern of CH stretches as in the monoanion, with the important note that the CH
stretching peak of the methine bridge between the BC rings, at ca. 2920 cm-1 is now of lower
relative intensity. The three highest energy features are likely due to NH stretching vibrations. The
𝑓𝑟𝑒𝑒peak at 3507 cm-1 corresponding to a free NH stretching motion (𝜈𝑁𝐻 ) which is present in all
three conformers in the figure. The remaining two features, at 3345 cm-1 and 3155 cm-1 correspond139

to hydrogen bonded NH stretching vibrations. As can be seen in the figure, none of thesesimulations reproduce the pattern of the NH stretching modes. This is not entirely surprising as
these are harmonic frequency calculations, and hydrogen bonded modes can be significantly
anharmonic.17[(BV-2H)·N2]-2

intensity [arb. units]

Conformer K

Conformer J

Conformer A

2800

3000

3200

3400

3600

-1

wavenumber [cm ]Figure 6.6. Comparison of IR spectra of [(BV-2H)·N2]-2 with simulated spectra for three selectisomers from 2800-3600 cm-1. The vertical dashed lines indicate the location of prominent featuresin the experimental spectrum. Simulated spectra have been scaled by 0.96.
The electronic spectrum of [(BV-2H)·N2]-2 is presented in Figure 6.7. The main absorption
feature is red shifted by about 5000 cm-1 relative to that of the singly deprotonated species, and theabsorption profile has changed qualitatively. The absorption maximum is at 10800 ± 100 cm-1 (926nm), well into the NIR window of biological tissue, indicating that this chromophore can access a140

larger portion of the range than is currently achieved in proteins. We observe a broad peak at +1650
cm-1 above the absorption maximum and a second feature of similar width at +3400 cm-1. For[(BV-2H)·N2]-2, the calculated (TDDFT) S1 vertical excitations place the first absorption
maximum on average at ca. 16300 cm-1, and they are spread further apart (ca. 3400 cm-1) than for
singly deprotonated BV. Conformers K and J are on the low energy end of this spread withcalculated S1 transition energies of 15084 cm-1 and 14561 cm-1 respectively. Recall from thediscussion of the singly deprotonated species, calculations overestimated the experimental
spectrum by ca. 3400 cm-1. If we assume a similar offset is present here, the calculated S1transitions for both conformer J and K come close to the experimental value. While we can offer
no unique structural assignment based on our IR and electronic spectra, we do note that the large
observed red-shift between the mono- and dianion is qualitatively recovered by the calculations
for the conformational families discussed here. This suggests that subjecting the conjugated systemof the chromophore to an electric field is playing a substantial role in the position of the S1 band
in BV. However, because we are unable to provide a unique structural assignment to confirm only
small structural changes are occurring between the mono- and dianion, we cannot completely ruleout other effects (such as a large conformational change) .

141

Figure 6.7. Electronic spectrum of [(BV-2H)·N2]-2. As was the case for [(BV-H)·N2]-, the vibronic
progression is unresolved despite being taken at a trap temperature of 20 K. The main absorption
feature occurs at 10800 cm-1 with a shoulder at +1650 cm-1 and a third feature at 14200 cm-1.

Despite being taken at pH 13, the UV-vis spectrum of BV in aqueous solution in Figure
6.4 contains no features red of the peak absorption at 14780 cm-1. This indicates that either the
dianion species is not present in solution, which is unlikely at this pH,18 or that it undergoes asubstantial solvatochromic shift. Figure 6.8 compares the electronic spectra of [(BV-2H)·N2]-2
and [(BV-2H)·(H2O)n]-2 for n=1-4. Addition of each water molecule shifts the absorption feature
blue by ca. 290 cm-1. If this trend were to continue in a linear fashion, one expects ca. 10-12
molecules are required to shift the absorption to where the solution phase absorption occurs. Of
course, this trend is not likely to be linear, and this estimate represents a lower bound for the actual
number of water molecules required to fully reach the value for aqueous solution. While we did
not perform calculations on any hydrated clusters, one would expect the water molecules to solvate
the negatively charged carboxylate groups. As the number of water molecules increases, they142change the charge distribution of the molecule and therefore the electric field experienced by the
conjugated system. As we have already seen, the S1 excitation energy is sensitive to the electricfield and so the shift is not surprising.
At the time of the writing of this thesis, investigations of this molecule are ongoing, aimed
at addressing the question of which conformer, or conformers, is (or are) responsible for theobserved electronic absorptions. The conformer (and charge distribution) responsible for theabsorption spectrum of the dianion is of particular interest as an absorption wavelength of 926 nm
would represent a significant increase in the spectral range accessible by this chromophore
compared to what has been achieved previously. The hydration experiments suggest that one
would be able to tune the absorption band throughout the first NIR window of biological materials.
Future experiments will involve using ion mobility19 to determine how many conformers arepresent (in both charge states), and hole burning20 techniques to then determine which spectralfeatures are due to which conformer.

143

Figure 6.8. Electronic spectrum of the band origin region of [(BV-2H)·N2]-2 (top trace) and [(BV2H)·(H2O)n]-2 for n=1-4 (lower traces). The black circles are data points, the red lines are 10 point
adjacent averages to guide the eye.

144

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