Dynamic Nuclear Polarization of Amorphous and Crystalline Small Molecules
by
Ta-Chung Ong
B.A., Colby College (2007)
Submitted to the Department of Chemistry
in Partial Fulfillment of the Requirement for the Degree of
MASSACHUSETS NTfTE
OF TECHNOLOGY
Doctor of Philosophy
JUN 3 0 2014
at the
LIBRARIES
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June 2014
C 2014 Massachusetts Institute of Technology. All rights reserved.
Signature redacted
............................
Department of Chemistry
June 1, 2014
Signature of Author.............
Signature redacted
Certified by .......................
V
Robert G. Griffin
Professor of Chemistry
Thesis Supervisor
Signature redacted
Accepted by .......................
Robert W. Field
Professor of Chemistry
Chairman, Departmental Committee on Graduate Students
2
This doctoral thesis has been examined by a Committee of the Department of Chemistry as
follows:
Professor Sylvia T. Ceyer..........
Chair
Signature redacted
I
I
Professor Robert G. Griffin.............
Thesis Supervisor
Signature redacted
Signature redacted
Professor Robert W. Field............
3
4
Dynamic Nuclear Polarization of Amorphous and Crystalline Small Molecules
by
Ta-Chung Ong
Submitted to the Department of Chemistry
on June 1, 2014 in Partial Fulfillment of the Requirement for the
Degree of Doctor of Philosophy in Chemistry
ABSTRACT
Solid-state NMR has emerged to become an important technique in the studies of pharmaceutical
formulations consisting of active pharmaceutical ingredients (API) and excipients. Dynamic
nuclear polarization (DNP), which improves NMR sensitivity by 2-3 orders of magnitude, can
potentially reduce the necessary experimental time for formulations that have low API contents.
However, conventionally DNP samples are prepared in cryoprotecting glassing agents such as
glycerol/water or DMSO/water, which may not be suitable for studies of pharmaceuticals. In this
thesis, we examined the performance of solvent-free DNP in amorphous and crystalline orthoterphenyl (OTP) in order to gauge the feasibility of applying DNP to pharmaceutical solid-state
NMR experiments and to study the effect of inter-molecular structure, or lack thereof, on the
DNP enhancement. We found that while DNP of amorphous OTP benefits from greater signal
enhancement due to a more homogeneous distribution of radical polarization agent, DNP of
crystalline OTP features better spectral resolution but requires heavy deuteration to attenuate
proton relaxation. Further application of DNP to nanocrystalline acetaminophen embedded in
cellulose membrane as an undissolved suspension in organic solvent was less successful due to
the fast methyl group motion within the acetaminophen molecule. Deuterium NMR study of
crystalline d3 -acetaminophen showed the methyl group relaxation time is significantly reduced at
low temperature (109 K), which negatively impacts DNP performance. A topical review of
recent developments on high-field (16.4 T) DNP, as well as updates on the temperature-jump
DNP experiment and descriptions of several 2H NMR studies of molecular dynamics, are also
presented as part of this thesis.
Thesis Supervisor: Robert G. Griffin
Title: Professor of Chemistry
Director of the Francis Bitter Magnet Laboratory
5
6
ACKNOWLEDGMENTS
I would like to thank my thesis advisor Prof. Robert G. Griffin for the unique and
invaluable research experience at FBML. Under his guidance, I was introduced to the field and
learned the fundamental principles and practices of solid-state NMR and DNP. None of the
research presented herein would have been possible without Bob's continuous support and
encouragement.
Many thanks go to Christopher Turner and Vladimir Michaelis, whose friendship and
mentorship were indispensible to my training as a NMR spectroscopist and a collaborative
scientist. I would also like to thank my fellow Griffin group members, visiting scientists, and
UROP with whom I had the pleasure of working alongside over the years, Matthew Eddy,
Alexander Barnes, Galia Debelouchina, Marvin Bayro, Marc Caporini, Evgeny Markhasin,
Andrew Casey, Susanne Penzel, Eric Keeler, Thorsten Maly, Patrick van der Wel, Bj$rn
Corzilius, Eugenio Daviso, Michael Colvin, Jennifer Mathies, Marcel Reese, Yongchao Su, Yoh
Matsuki, Anne-Francis Miller, Amanda Shin, Elijah Mena, and Christopher Blake Wilson.
Special thanks go to my cohorts within the group, Loren Andreas, Albert Andrew Smith, and
Rebecca Mayrhofer for being great friends over the years. And very importantly, I would like to
acknowledge the talented research and technical staff who keep the place running, Jeffrey Bryant,
Ajay Thakkar, Ron DeRocher, Mike Mullins, Dave Ruben, and Tony Bielecki.
One of the greatest joys of working at MIT has been the freedom to conduct collaborative
research across multiple disciplines. For our DNP effort, I would like to thank our collaborators
from Prof. Timothy Swager's group, who work hard to design improved biradical polarization
agent for DNP, Matthew Kiesewetter, Joseph Walish, Derik Frantz, and Olesya Haze. And our
collaborators from Dr. Richard Temkin's group at PSFC, who help us keep the gyrotrons healthy,
Emilio Nanni, Sudheer Jawla, Ivan Mastovsky, and William Guss. For the solid-state NMR
collaborations, I would like to thank Prof. Mircea Dinca's group from Inorganic Chemistry,
Natalia Shustova, Guillaume Bertrand, Anthony Cozzolino, and Carl Brozek. And Prof. Allan
Myerson's group from Chemical Engineering, Xiaochuan Yang, Jennifer Huang, and Sydney
Hodges. Most importantly, I would like to acknowledge Melody Mak-Jurkauskas (also a past
Griffin group member), Andrew Clausen, and Janet Cheetham of Amgen Inc. for supporting the
ortho-terphenyl DNP project.
Last and most certainly not least, I would like to thank my family for their unwavering
support of my academic pursuit. I would not have made it to MIT without the encouragement
from Mom and Dad, and also my brother Ta-Hsuan (who is currently conducting his own
graduate research in Prof. Jonathan Sweedler's group at UIUC). Their love and understanding
provide the necessary foundation from whence this thesis is written.
7
8
Table of Contents
Dynamic Nuclear Polarization of Amorphous and Crystalline Small Molecules ........................... 1
Abstract ............................................................................................................................................
5
A cknow ledgem ents........................................................................................................................-7
Chapter 1: Introduction to Solid-State NMR and Dynamic Nuclear Polarization .......
17
Interactions ...............................................................................................
The Zeem an Interaction ........................................................................
The Chem ical Shift Interaction.............................................................
The Dipolar Interaction........................................................................
The Scalar Interaction...........................................................................
17
18
21
22
25
1.1.5. The Quadrupolar Interaction..................................................................
26
1.2. Com m on Solid-State N MR Experim ents................................................................
1.2.1. The Single Pulse Experim ent...............................................................
1.2.2. The Cross Polarization Experim ent ......................................................
1.2.3. The Hahn Echo Experim ent..................................................................
1.3. Introduction to Dynam ic Nuclear Polarization......................................................
27
27
30
32
34
1.1. The Spin
1.1.1.
1.1.2.
1.1.3.
1.1.4.
1.4. Thesis Outline.............................................................................................................40
1.5. References...................................................................................................................41
Chapter 2: Solvent-Free Dynamic Nuclear Polarization of Amorphous and Crystalline
Ortho-Terphenyl...........................................................................................................................45
2.1.
2.2.
2.3.
2.4.
2.5.
2.6.
2.7.
2.8.
Introduction.................................................................................................................46
Experim ental...............................................................................................................48
Results.........................................................................................................................50
D iscussion...................................................................................................................57
Conclusion ..................................................................................................................
A cknowledgem ents.................................................................................................
Supporting Inform ation...........................................................................................
References...................................................................................................................74
61
62
63
Chapter 3: Solid-State NMR and Dynamic Nuclear Polarization of Pharmaceutical
77
Form ulations ................................................................................................................................
3.1.
3.2.
3.3.
3.4.
3.5.
3.6.
Introduction.................................................................................................................77
Experim ental...............................................................................................................81
Results and Discussion ..........................................................................................
Conclusion ..................................................................................................................
Acknow ledgem ents.................................................................................................
References...................................................................................................................96
9
83
95
96
Chapter 4: Progress on Temperature-Jump Dynamic Nuclear Polarization (TJDNP)......101
4.1. Introduction - Challenge to Liquid State DN P.........................................................101
4.2. Optim izing Rotor M aterial and Laser W avelength...................................................107
4.2.1. Experim ental............................................................................................107
4.2.2. Results and Discussion ............................................................................
107
4.3. LCST TOTAPOL Polymer .......................................................................................
113
4.3.1. Experim ental............................................................................................115
4.3.2. Results and Discussion ............................................................................
119
4.4. Conclusion ................................................................................................................
125
4.5. Acknowledgem ents...................................................................................................126
4.6. References.................................................................................................................126
Chapter 5: Investigation of Molecular Dynamic Processes by
2H
NMR ..............................
129
5.1. Introduction to 2H N MR ...........................................................................................
129
5.2.o
e .................................................................................
135
5.3. Lipid Phase Transition in d54 -DMPC/VDAC 2D Crystals ....................................... 140
5.3.1. Experim ental............................................................................................143
5.3.2. Results......................................................................................................144
5.3.3. Discussion................................................................................................151
52 H N M R of Chain Deuterated DPhPC......................................................................157
5.4.
5.4.1. Experim ental............................................................................................159
5.4.2. Results and Discussion ............................................................................
159
5.5. Phenyl Group Dynamics of Zn 2 (TCPE) Metal Organic Framework........................165
5.5.1. Experim ental............................................................................................168
5.5.2. Results......................................................................................................172
5.5.3. Discussion................................................................................................184
5.6. Conclusion ................................................................................................................
191
5.7. Acknowledgem ents...................................................................................................192
5.8. Supporting Inform ation.............................................................................................193
5.9. References.................................................................................................................207
Chapter 6: Topical Developments in High-Field Dynamic Nuclear Polarization................213
6.1.
6.2.
6.3.
6.4.
6.5.
6.6.
6.7.
6.8.
Introduction...............................................................................................................213
Developm ent of CE Biradicals .................................................................................
Direct Polarization of Low-Gam m a Nuclei U sing Trityl .........................................
Sample Preparation Techniques.............................................................................227
Improving DN P Instrum entation at High Fields (; 16 T) ........................................
Conclusion ................................................................................................................
Acknowledgem ents...................................................................................................237
References.................................................................................................................237
Curriculum Vitae.......................................................................................................................243
10
217
223
231
236
List of Figures
Figure 1.1. Zeeman energy diagram for a nuclear spin with I = %................................................20
Figure 1.2. The single pulse experim ent...................................................................................
27
Figure 1.3. The cross polarization experiment...........................................................................
30
32
Figure 1.4. The H ahn echo sequence ........................................................................................
Figure 1.5. Transverse magnetization refocuses during the Hahn echo sequence.....................33
Figure 1.6. A electron-nucleus coupled two-spin system under an external magnetic field ......... 37
37
Figure 1.7. T he solid effect ............................................................................................................
Figure 1.8. A three-spin coupled system involving two electrons and one nucleus..........39
Figure 1.9. The cross effect............................................................................................................39
Figure 2.1. Phase transition scheme of ortho-terphenyl (OTP)...............................................
Figure 2.2. 13C CPMAS DNP enhancement (F) of OTP containing 1 mol% TEMPOL as a
function of levels of deuteration .................................................................................................
48
Figure 2.3. The structure of bis-TEMPO terephthalate (bTtereph) ..........................................
52
51
C CPMAS DNP enhanced spectra of 95% deuterated OTP................53
Figure 2.5. 1H polarization buildup curves of a) amorphous and b) crystalline 95% deuterated
...... ------- 5 5
....
O T P ...........................................................................................................................
Figure 2.4.
13
Figure 2.6. CW EPR field profiles of bTtereph at 9 GHz..............................................................56
Figure 2.7.
13C
CPMAS DNP enhanced spectra of indomethacin glass....................................60
Figure 2.S1. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)
64
plots of bT tereph ............................................................................................................................
Figure 2.S2. NMR field dependent 1H enhancement (c) profile of bTtereph...........................65
Figure 2.S3. DNP enhancement (c) as a function of gyrotron microwave power ..................... 66
Figure 2.S4. DNP enhancement (s) as a function of bTtereph concentration ...........................
67
Figure 2.S5. Experimental and simulated EPR spectra of bTtereph.........................................70
Figure 2.S6. Probablity distribution of Lorentzian linewidth used to simulate the 9 GHz EPR
spectrum of bTtereph in crystalline OTP.......................................................................................71
Figure 2.S7. Pulsed 140 GHz EPR spectra of bTtereph in fully deuterated amorphous OTP.......72
Figure 2.S8. Room-temperature 13C CPMAS NMR spectra of amorphous and crystalline (a and y
73
crystals) indom ethacin ...................................................................................................................
11
Figure 3.1.
13C
CPMAS spectra of form I ibuprofen and cellulose-ibuprofen.............83
Figure 3.2.
13C
CPMAS spectra of form I acetaminophen and cellulose-acetaminophen......85
Figure 3.3. Expanded
Figure 3.4.
13
Figure 3.5.
13C
13
C CPMAS spectra of acetaminophen .................................................
C CPMAS DNP of cellulose membrane in water and EtCl4 ................
86
............ .. . .
CPMAS DNP of cellulose-acetaminophen ......................................................
Figure 3.6. Static
2H
88
90
NMR spectra of d 3-acetaminophen at various temperatures....................91
Figure 3.7.
13C
CPMAS spectra of form I ibuprofen and silica-ibuprofen...............................93
Figure 3.8.
13C
CPMAS spectra of silica-griseofulvin ..............................................................
94
Figure 4.1. Energy level diagram for an electron-nuclear coupled spin system..........................102
Figure 4.2. Experim ental schem e of TJDNP ...............................................................................
106
Figure 4.3. Conceptual diagram of indirect versus direct melting in the TJDNP experiment.....108
Figure 4.4. IR and NIR absorbance profile of zirconia, sapphire, and SiC ................................. 109
Figure 4.5. NIR absorbance of DMSO/H 2 0 and d6-DMSO/D 20................................................110
Figure 4.6. The growth of liquid state proton NMR signal upon laser irradiation ...................... 111
Figure 4.7. The refreezing of TJDNP sample after laser irradiation ........................................... 111
Figure 4.8. TJDNP 13 C NMR spectrum of 800 mM glucose in DMSO/H 2 0.............113
Figure 4.9. Synthesis of the thermoresponsive poly(norbomenyl) polymer bearing TOTAPOL
moieties .............................................
. . .....................................................................................
116
Figure 4.10. 2pESEEM of TOTAPOL moieties in the LCST polymer..................120
Figure 4.11. Solution
13
C NMR spectrum of 800 mM U-' 3 C glucose..................121
13C
NMR spectrum of 13C-urea in d6-DMSO/D 20 ........................ 124
Figure 4.13. 13 C polarization built-up curve of urea sample........................................................125
Figure 4.12. DNP enhanced
Figure 5.1. Pake doublet pattern for 2H in solid powder sample .................................................
Figure 5.2. The quadrupolar echo sequence ................................................................................
131
132
Figure 5.3. Deuterium line shapes at various motional rates for D20 two-fold hop and aromatic
ring flip ........................................-------.............
. . .....................................................................
134
Figure 5.4. A simple N M R circuit diagram .................................................................................
136
Figure 5.5. Basic transmission line circuit for a single resonance NMR probe...........................137
Figure 5.6. The transmission line single channel 2 H probe .........................................................
139
Figure 5.7. Acyl chain deuterated DMPC (d54-DMPC) ...............................................................
143
Figure 5.8. DSC thermograms of pure d54 -DMPC and VDAC1/d 54-DMPC 2D crystal ............. 145
12
Figure 5.9. Static
2H
NMR spectra of d54-DMPC and VDAC1/d 54-DMPC 2D crystals.............147
Figure 5.10. Perpendicular quadrupolar splitting, AvQI, as a function of temperature ............... 148
Figure 5.11. Expansion of 2 H NMR spectra of d54-DMPC and VDAC 1 /d 54-DMPC 2D crystals at
14 9
29 C .............................................................................................................................................
Figure 5.12. Static 2H NMR spectra of VDAC1/d 5 4-DMPC ~1:25 protein-to-lipid ratio and ~1:50
as a function of tem perature.........................................................................................................151
Figure 5.13. Schematic illustrations of a projection of the VDAC1 monomer and dimer .......... 154
Figure 5.14. De-Paked 2 H NMR spectra of d54 -DMPC and VDAC 1 /d54-DMPC 2D crystals . . 156
Figure 5.15. Protonated DPhPC and DPPC .................................................................................
Figure 5.16. Temperature dependent
2H
spectra of d78-DPhPC and d78-DPhPC:M2..................160
Figure 5.17. Static 2H NMR spectra of d78-DPhPC at 173 K and 208 K ....................................
Figure 5.18. Temperature dependent
2H
158
spectra of d62 -DPPC ....................................................
Figure 5.19. Choline headgroup of DMPC and DPhPC ..............................................................
161
162
164
Figure 5.20. The Static 2H NMR spectrum of chain deuterated DPhPC at 290 K ...................... 164
Figure 5.21. X-ray crystal structure of Zn 2(TCPE)......................................................................166
Scheme 5.1. Synthesis of H4 TCPE-d16 . .
. . . . . . ..
.
. . . . . .
. . . . . . . . . . . .. . . . . . . .
169
Figure 5.22. Temperature-dependent X-ray diffraction studies of t...........................................174
Figure 5.23. Static 2H NMR spectra of TPE-d 2 0 . . . .
Figure 5.24. Static
2H
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
176
NMR spectra of 1c taken between 298 and 423 K...................................177
Figure 5.25. Experimental and simulated quadrupolar spin-echo solid-state 2H NMR spectra of
la during heating and transformation into lb and of lb during cooling.....................................178
Figure 5.26. Arrhenius plot of the two-fold phenyl exchange rate in lb during cooling ............ 179
Figure 5.27. 13C CPMAS NMR spectra of fully desolvated lH and ic ...................................... 180
Figure 5.28. DFT-calculated structures of truncated formate-capped models of lb...................181
Figure 5.29. PES for the flipping of one phenyl ring in a truncated model of lb and sum of the
electron density at the C-C single bond critical points................................................................182
Figure 5.30. PES for the flipping of one phenyl ring in a model of TPE ....................................
183
Figure 5.S1. Thermogravimetric analysis plots for 1 and 1H......................................................194
Figure 5.S2. Simulated (red) and experimental (black) PXRD patterns of lc ............................ 195
Figure 5.S3. Variable temperature 'H NMR spectra of TPE and H4TCPE ...............................
196
Figure 5.S4. DFT-calculated PES for phenyl ring flipping in styrene.........................................198
Figure 5.S5. DFT-calculated PES for phenyl ring flipping in benzoic acid................................199
Figure 5.S6. DFT-calculated PES for phenyl ring flipping in vinylbenzoic acid with orthogonal
ethylene and carboxylic acid groups and coplanar ethylene and carboxylic acid groups ........... 200
13
Figure 5.S7. PXRD patterns of ic and fully desolvated lH ........................................................
201
Figure 5.S8. Geometry-optimized conformations of TPE ..........................................................
202
Figure 5.S9. DFT-calculated molecular conformations of truncated lb model .......................... 203
Figure 5.S 10. DFT-calculated PES for phenyl ring flipping in terephthalic acid with coplanar
ethylene and carboxylic acid groups............................................................................................204
Scheme 5.S1. Depiction of the proposed desolvation process that occurs during the conversion of
ib to i c ........................................................................................................................................
206
Figure 6.1.
13
C DNP enhanced CPMAS spectra of 13 C-urea in glycerol/D 2 0/H20 .................... 219
Figure 6.2. 1H DNP field profiles of various bT-thio based radicals...........................................221
Figure 6.3. bTtereph synthetic process ........................................................................................
222
Figure 6.4. Chemical structures and 140 GHz EPR spectra of three narrow-line radicals: Trityl,
TM T , and SA -B DPA ...................................................................................................................
Figure 6.5. Direct polarization of
13 C, 2
H,
223
and 170 field profiles acquired at 5 T ....................... 225
Figure 6.6. Direct polarization of low-gamma nuclei..................................................................227
Figure 6.7. MAS DNP sample preparation protocols for biophysical systems ........................... 230
Figure 6.8. Artistic rendering of the new waveguide designed for the 460 GHz / 700 MHz DNP
N MR spectrom eter.......................................................................................................................233
Figure 6.9. 13C-13C DARR spectrum of U- 3 C-Proline in d 8-glycerol/D 2 0/H2 0 ........................ 234
Figure 6.10. '3 C-' 3 C correlation spectrum of U-13 C-apoferritin at 5 T and 16.4 T ..................... 236
14
List of Tables
Table 2.1. Biphasic DNP ' H Polarization buildup time constants (rB, and rB2) and fraction (/) of
54
crystallized OT P .............................................................................................................................
Table 3.1. T1 ('H) of form I ibuprofen and cellulose-ibuprofen...............................................84
Table 3.2. T, ('H) of form I acetaminophen and cellulose-acetaminophen...............................87
Table 3.3.
13
95
C chemical shifts of silica-griseofulvin polymorphs ............................................
Table 4.1. GPC characterization of TOTAPOL-containing polynorbornenes polymers.............118
Table 4.2. 'H and 13C spin-lattice relaxation time of 800 mM 13C6 glucose in D20 containing
TOTAPOL and TOTAPOL polymer at various concentrations..................................................122
Table 4.3. 'H and 13C spin-lattice relaxation time of 800 mM glucose in D2 0 containing
TOTAPOL or TEMPO with or without blank PEG polymer ......................................................
123
Table 5.1. DFT-calculated low-energy vibrational modes for TPE.............................................184
Table 5 .S 1. X-ray crystal structure refinement data for TPE-d 20 , 1 a and lb at various
temp eratures .................................................................................................................................
193
Table 5.S2. The shortest Ph...Ph contacts, the dihedral angles and the ethylene twist angles in the
determined crystal structures at 93, 298, and 373 K using the indices specified in the sample TPE
19 7
stru cture ........................................................................................................................................
Table 5.S3. Activation energies, C=C bond lengths, and selected angles for geometry-optimized
conformations of TPE at fixed CA-CAr-C=C dihedral angles .................................................... 202
Table 5.S4. Dihedral angles for geometry-optimized conformations of TPE at fixed CAr-CArC = C dihedral angles.....................................................................................................................202
Table 5.S5. Activation energies, C=C bond lengths, and selected angles for geometry-optimized
molecular conformations of a truncated model of lb at fixed CAM-CAM-C=C (1250, 50, and 950)
2 03
dihedral an g les .............................................................................................................................
Table 5.S6. Dihedral angles for geometry-optimized molecular conformations of a truncated
model of lb at fixed CA,-CA--C=C (125', 5', and 950) dihedral angles.....................................203
Table 5.S7. Electron density (e- A-3) at bond critical points for selected bonds along PES for
ring flipping in truncated model of lb.........................................................................................205
Table 6.1. Physical properties for selected biologically relevant NMR nuclei ........................... 224
Table 6.2. Direct polarization of various biologically relevant nuclei using trityl at 5 T............226
15
16
Chapter 1: Introduction to Solid-State NMR and
Dynamic Nuclear Polarization
Since its discovery by Purcell et al.,' nuclear magnetic resonance (NMR) has grown to
become an indispensible analytical method in a variety of scientific disciplines, notably in
synthetic chemistry, biochemistry, structural biology, and materials science. In this chapter, an
introductory background on NMR is provided to give the gentle readers a good basis to
understand the subsequent chapters of this thesis. We will examine the relevant nuclear spin
interactions that take place in NMR, describe common pulse experiments, and give a brief
introduction on dynamic nuclear polarization (DNP), which is an exciting method that can
improve NMR signal-to-noise by 2 to 3 orders of magnitude. For a more detailed treatment on
NMR, the books by Charles P. Slichter,2 Melinda J. Duer, 3 and Malcolm H. Levitt4 are useful
texts for references.
1.1. The Spin Interactions
The full Hamiltonian on a nuclear spin residing inside an NMR magnet, no radio
frequency pulse yet, can be written as follow:
A
A
A
A
H=HZ+ HCH+
H
A
A
(1)
HJ+ Q+
A
Hz is called the Zeeman interaction, experienced by the nuclear spin because it is under the
external magnetic field exerted by the NMR magnet. The rest of the terms are called internal
A
Hamiltonians, which are intrinsic to the spin system under study. Hcs is the chemical shift
17
A
interaction,
HD
A
A
is the dipolar interaction, Hi is the scalar interaction, and HQ is the quadrupolar
interactions. This section examines each interaction and briefly describes their importance.
1.1.1. The Zeeman Interaction
The Zeeman interaction experienced by a nuclear spin is given by
A
A
A
H = -yh Iz Bo = -cooh Iz
(2)
where Bo is the strength of the applied external magnetic field, y is the nuclear gyromagnetic
A
ratio, and Iz is the spin angular momentum operator. By convention, the applied magnetic field
is along the z axis, hence the z designation. It is common to see Eq. (2) written in terms of the
Larmor frequency, oo. Physically, the Larmor frequency describes the nuclear spin precession
about the applied magnetic field. NMR spectroscopists typically use the 'H Larmor frequency to
describe NMR field strengths as opposed to actually using Bo. Therefore, a "211 MHz NMR
spectrometer" has a magnet with a field strength of approximately 5 T.
The eigenvalues of the Zeeman Hamiltonian can be written as
H II, m) = E,,,, II, m)
where Ei,m is the energy of eigenstate
|I,m).
(3)
Substituting Eq. (2) into Eq. (3), we get
A
A
H II, m)= -yhBo I|I, m)
(4)
A
This, and also consider that II, m) is an eigenfunction of Iz with eigenvalue m, we get
A
LZ I,m) = mI I, m)
18
(5)
H II, m) = -yhBom I, m)
(6)
And therefore the energies of the eigenstates are
(7)
EI, = -yhBom
I is the quantum number describing the total angular momentum, and m is referred to as the
azimuthal quantum number that can take any value from -I, -1+1...+1 for a total of 21+1 possible
numbers. For many of the common NMR active nuclei (e.g., 'H,
C,
1N 31P
etc.), I
=
2
, so
therefore m = ±2. For these nuclei, Eq. (7) is thus simplified into
E,
(8)
1=
- hBO
2
And the difference between the two energy levels is
AE = yhBO =wooh
(9)
Eq. (9) gives us the energy level diagram shown in Figure 1.1. As the magnetic field strength
increases, the energy gap between the levels widens, leading to a greater Boltzmann population
difference. At temperature equilibrium, the Boltzmann distribution for each eigenstate is given
by
exp
exp=V
By using a Taylor series approximation
19
-
5
'
(10)
Ev
exp ''(11) ''~1
-Ex
We can obtain the Boltzmann polarization as
P
yhBO
2kT
(12)
From this equation, we can see that nuclear polarization is proportional to BO, and inversely
proportional to temperature. This finding motivates the development of higher field magnets and
cryogenic temperature systems for NMR experiments. Assuming a 10 T magnet at room
temperature (298 K), we find that P is only 5.5x10- 6 for 'H (y
3C
=
42.6 MHz/T) and 1.4x10- 6 for
(y = 10.7 MHz/T). This calculation, coupled with the fact that many NMR active nuclei are
low in natural abundance (e.g.,
13 C
1.1%.
15 N,
0.4%), means that NMR is inherently an
insensitive analytical method. For biological solids, isotopic labeling is commonly employed to
increase NMR signal-to-noise and save acquisition time.
E
0
I(X)
B0
Figure 1.1. Zeeman energy diagram for a nuclear spin with I =
V.
The a and
p
denote the two energy states and will be convention for the remainder of this thesis.
20
designations
1.1.2. The Chemical Shift Interaction
If Zeeman interaction is the only term experienced by the nuclear spins, then each NMR
active nuclear isotope would have only one single NMR peak in a homogeneous magnetic field,
corresponding to its Larmor frequency as shown in Eq. (9). Evidently, this is not true. The
nuclear spins are surrounded by electrons, which are also magnetic and therefore induces a
secondary magnetic field that perturbs the external magnetic field felt by the nuclear spins. The
introduction of this inhomogeneity is called the chemical shift interaction, with the Hamiltonian
A
A
Hcs =
*I-c--BO
(13)
where c is the chemical shielding tensor. In NMR experiments, the absolute Larmor frequencies
(Zeeman plus chemical shift) are not reported directly because the chemical shift contribution is
only on the order of ppm compared to the Zeeman interaction. Instead, the chemical shift is
reported as an offset from a reference frequency. Common reference samples include
tetramethylsilane for solution NMR and adamantane for solids. Assuming the chemical shift
tensor is symmetric, the definition of chemical shift is
9 = 9i + IAs(3 cos2 0 1)
2
=
Vref
(14)
(15)
Vref
where 6iso is called the isotropic chemical shift and Acs called the chemical shift anisotropy. The
isotropic chemical shift is the most useful information provided by NMR, allowing one to obtain
localized molecular information. Any undergraduate organic chemistry textbook is likely to
contain a table of NMR chemical shifts and their corresponding functional groups.
21
From Eq. (14), we can see that the anisotropic part of the chemical shift interaction is
angular dependent. In other words, it depends on the orientation of the chemical shift tensor with
respect to the external magnetic field. In solution NMR, fast molecular tumbling eliminates any
orientation dependence, and therefore averages the anisotropic portion of the chemical shift to
zero, leaving only the 6iso for observation. In solid-state NMR of a static sample, both
components are present and produce a "powder pattern" that includes contributions from all
possible molecular orientations. The powder pattern can be hundreds of ppm wide, and for most
circumstances it is not useful. Magic angle spinning (MAS) is applied to address this concern. If
we spin a solid sample at an angle OR away from the external magnetic field, then the angle
describing tensor orientation becomes time dependent, and the average is
(3cos2 0-1 = 1(3cos2 OR _)(3cos2 8J-)
(16)
where f is the angle between the spinning axis and the tensor principal z-axis. By setting OR to
54.740, we find that 3cos 2OR-
0. This angle is therefore called the "magic angle". By spinning
at this angle at sufficient frequency (a factor of 3 to 4 greater than Acs), we can adequately
minimize chemical shift anisotropy so only the 6isO is observed for solid samples. Spinning at a
lower frequency only partially averages the powder pattern and produces "spinning sidebands"
that collectively trace the shape of chemical shift anisotropy and are each separated by the MAS
frequency.
1.1.3. The Dipolar Interaction
Like electrons, the nuclear spins themselves are also magnetic and each generates its own
magnetic field that interacts with other nuclear spins. This effect is called the dipolar interaction,
and the Hamiltonian is
22
(Ir)(S-r)
AI
Hi
-(2= L7,2sh
4zr
S -3
r3
r5
(17)
for two spins with designation I and S. The above equation is commonly expressed in spherical
polar coordinates, so it takes the form
r17s[A+B+C+D+E+F]
47r
HD=
(4r )
(18)
r
where
A=JzSz(3cos2_1
I.S-+S
_+](3cos2
B=
C=
D =I
z+
++
3
A
[A
zS_
2
z
]
A
A
j sin 0 cos
oi)
e
s
+1I_ Sz sin 0 cos O
3 [=
E =-41+ 8+ ]sin22 Oe -22 '
3A
F=-
4L
-
U- ] sin
2
Oe +2+
The dipolar interaction between nuclei of the same isotope is called homonuclear dipolar
coupling (e.g., 'H-H, 13C- C, etc.), and between nuclei of different isotopes is called
heteronuclear dipolar coupling (e.g., 'H- C, etc.).
23
As we can see from Eq. (18), the dipolar interaction is orientation dependent. In solution
NMR where there is fast molecular tumbling, the dipolar interaction averages to zero. In solidstate NMR, dipolar coupling can be on the order of tens of kHz. In a homonuclear system, the
raising and lowering operators of the dipolar Hamiltonian (the "flip-flop" terms present in the B
term) interfere with MAS averaging and causes homogeneous broadening of resonance lines.
This is the reason why MAS experiments of 1H still result in lines that are tens of kHz wide
unless the sample is heavily deuterated to minimize 1H-1H coupling or the frequency of spinning
is very fast (up to 100 kHz). The same effect is observed for MAS experiments of 13C, since for
most samples the 13C is dipolar coupled to a network of coupled 'H, and consequently MAS only
leads to an incomplete averaging of the 1H- 13 C dipolar coupling.
In order to address the issue of dipolar line broadening, decoupling experiments are
commonly employed to improve NMR spectral resolution. Decoupling works by applying a
high-power multiple pulse sequence to irradiate target nuclei. Application of RF irradiation
perturbs the spin Hamiltonian, and the goal is to obtain an averaged Hamiltonian where the
dipolar coupling is zero. Conceptually, the effect of decoupling RF rapidly causes the nuclear
spins to undergo repeated transitions (a
<-.
f) at a rate larger than the strength of dipolar coupling,
and therefore the time-averaged dipolar coupling becomes zero. Homonuclear decoupling
experiments, in which the decoupling targeted nuclei is the same isotope as the observed nuclei,
include WAHUHA 5 (allegedly6 named after its inventors Waugh, Huber, and Haeberlen) and
MREV-8. 7 Heteronuclear decoupling experiments, in which the decoupled nuclei (typically 1H)
is a different isotope from the observed nuclei (typically
XiX.9
24
13 C, 15N, 3 1P,
etc.), include TPPM8 and
Although in many circumstances the presence of dipolar coupling is problematic, there
are situations where it is experimentally useful. Dipolar coupling is distance dependent (i.e., 1/r 3 ).
Therefore, we can utilize it to determine the distance between two nuclei and use the information
to determine molecular structures. Much like the J-coupling based correlation spectroscopy
(COSY) of solution NMR, the dipolar coupling can be utilized in similar ways by solid-state
NMR. However, MAS reduces dipolar coupling, so the interaction needs to be re-introduced.
This is accomplished by the recoupling experiments. The recoupling experiments work by
applying RF pulses that are synchronized with the MAS rotor period ("rotor synchronization") in
order to interfere with the MAS effect. Notable recoupling experiments include the rotationalecho double-resonance (REDOR),' 0 the transferred-echo double-resonance (TEDOR),"-" and
the radio-frequency driven recoupling (RFDR).13
1.1.4. The Scalar Interaction
The scalar interaction, commonly referred to as the J-coupling, is similar to the dipolar
interaction, but requires mediation through the electrons in chemical bonds. Therefore, it is also
called the indirect dipolar coupling. The effect of J-coupling is not large, only on the order of
tens of Hz, but useful as it permits mapping of chemical bonds within molecules. In solution
NMR, due to the absence of the direct dipolar coupling, J-coupling is apparent as peak splitting
and forms the basis for COSY. However, in solid-state NMR, the various line broadening effects
(e.g. residual dipolar coupling, higher order interaction terms, solid disorders, etc.) generally
overshadow the effect of J-coupling. Therefore, J-coupling only receives little attention in solidstate NMR. Nevertheless, with the advent of very fast MAS (> 65 kHz) that further improves
resolution, it may soon become viable to utilize J-coupling in the solid-state experiments.1
25
1.1.5. The Quadrupolar Interaction
Nuclear spins with I > 1 (e.g.,
2
H, '4N, 170, etc.) have nuclear electric quadrupole
moments that interact with the electric field gradient at the nuclei. This is called the quadrupolar
interaction, which has the following Hamiltonian
eQ
A
HQ =
A
A
21(21 - l)h
(19)
I.lvel
where e is the proton charge, V is the electric field gradient tensor, and
Q
is the nuclear
quadrupole moment. The dot product in Eq. (19) can be fully expanded into
A
A
A
A
A
A
A
A
I.v.I=IxvxxIx+x vx I,+Ix V, IZ+...
As we can see Eq. (19) becomes quite messy. Therefore, secular approximation is commonly
used to remove the excess terms when the quadrupolar interaction is considerably smaller than
the Zeeman interaction. To do this, we break the full Hamiltonian into ordered terms,
A
Full
HQ
A
A (1)
A (2)
=Ho+ H
+...
(20)
A
(1)
(2)
where HQ is the first-order quadrupolar Hamiltonian, HQ is the second-order Hamiltonian,
and so on. For nuclei with relatively small quadrupolar interactions, notably 2 H, on the order up
to hundreds of kHz, only the first-order term needs to be considered. Conversely, for half-integer
nuclei with larger quadrupolar interactions such as 170 and "Cl, which can have quadrupolar
couplings up to the realm of MHz, then the higher-order terms must be considered. For more on
quadrupoles, Chapter 5 of this thesis gives more details on static
to study molecular dynamics.
26
2H
NMR, which is a useful tool
1.2. Common Solid-State NMR Experiments
In this section, we give an introduction to the most common pulsed solid-state NMR
experiments. Starting with the single pulse sequence, or Bloch decay, which is the simplest
experiment.
1.2.1. The Single Pulse Experiment
900
Figure 1.2. The single pulse experiment followed by detection of FID.
The single pulse experiment is the simplest NMR pulse sequence, consists of a single
radio frequency (RF) pulse followed by detection of free induction decay (FID), as shown in
Figure 1.2. The RF pulse introduces an oscillating magnetic field that is time dependent and
perpendicular to the BO field. Combined with Zeeman interaction, the laboratory frame
Hamiltonian in the presence of an RF pulse becomes
A
(A
H =-7h BI z+B, cos(oft)I)
(21)
for an RF field that is oscillating along the x axis. B1 is the magnitude of the RF field and of is
the frequency of the radio pulse. The oscillating B1 field can be considered as two counterrotating fields; the resonant component that rotates in the same direction as the Larmor frequency
27
and the off-resonant component that goes against the Larmor frequency. Only the resonant
component has a major effect on the spin system, so Eq. (21) can be rewritten as
H =-Yh Bo0Iz+Be 'rft Ixerz
(22)
To simplify, we can utilize a rotating frame that is rotating about Bo at frequency Orf, so B
appears static. The rotating Hamiltonian then becomes
H' = hi r(Bo -Wff)I+Z±
7 B,
(23)
Ix
Applying this Hamiltonian into the time-dependent Schr6dinger equation, we get
-.
h aT'A=-h (yBO - ,o )z+
A
YB Ix )'
(24)
which has eigenfunctions
V'=
Ca
(t)|Ia)+ c (t)Lp)
(25)
From Eq. (25), we can see that the eigenstates of the RF perturbed Hamiltonian is a linear
combination of the Zeeman eigenstates. In other words, the RF field mixes the Zeeman states.
This process is called excitation.
We can utilize the rotating frame to better visualize the effect of the RF pulse. In the
absence of RF, magnetization is parallel to B0 along the z axis. This is called longitudinal
magnetization. The addition of the RF field B 1, which is perpendicular to Bo, tips the
magnetization away from the z axis into the xy plane. This magnetization perpendicular to B0 is
28
called transverse magnetization, which induces an oscillating current in the NMR coil that is then
recorded as the FID. The flip angle of magnetization away from the z axis is defined as
(26)
0, = YBrf
In other words, it is the angle turned by B1 in time Trf, and the term yB 1 is commonly called the
nutation frequency. When Orf is 900, as denoted in Figure 1.2, the magnetization is perfectly
flipped to the xy plane, thereby maximizing transverse magnetization and the NMR signal. The
RF pulse can be applied along the x axis or the y axis, this is called the phase of the pulse. Four
phases (x, y, -x, -y) are possible for a single RF pulse.
After the RF pulse, the transverse relaxation slowly decays following the equations
MX = M,, sin (cot)exp
Tj
M, = Meq cos (aOt)exp
T
(27)
2
where T2 is called the transverse relaxation time constant. Meanwhile, the longitudinal
magnetization is slowly rebuilt along the z axis back to Boltzmann equilibrium following the
equation
M, = Meq 1 - exp
(28)
where T, is commonly called the spin-lattice relaxation time constant. Measurements of T, and
T2 can be very informative as both parameters are dependent on molecular motion and they may
uncover subtle spin interactions not easily observable in ID NMR spectra.15 T, can be measured
29
by the inversion-recovery sequence' or, in cases of long TI, the saturation recovery sequence.17
T2 can be measured by the spin echo sequence. 18
1.2.2. The Cross Polarization Experiment
90X
(CP)-ydecoupling
130
r"C
(P)..yAA
Figure 1.3. The cross polarization experiment from 1H to 13C.
While the single pulse experiment described in the previous section can be performed for
all NMR active nuclei, the cross polarization experiment (CP)' 9 is commonly used to detect low
natural abundance nuclei (e.g., 13 C or
15 N)
when they are coupled to 'H. Low natural abundance
nuclei very often have long T, due to the absence of strong homonuclear dipolar coupling,
meaning that the magnetization recovery after each experiment is slow and thus it can take a long
time to properly signal average. Therefore, instead of detecting directly on the low natural
abundance nuclei via single pulse, the CP experiment aims to transfer polarization from the
abundant nuclei (usually 1H) to the surrounding low abundant nuclei. Doing so leads to an
effective improvement of spectral signal to noise, and greatly reduces the necessary experimental
time for signal averaging.
30
As shown in Figure 1.3, the CP experiment is initialized with an excitation pulse on 'H to
generate 'H transverse magnetization. In the figure, a 900 x pulse is used, so the transverse
magnetization is along the -y direction. Following the excitation pulse, a contact pulse is applied
along the -y axis on both nuclei, 'H and 13C. This generates a spin-lock field that can be
designated B1('H) and B1( 3 C). Cross polarization from 'H to 13C occurs when the HartmannHahn matching condition,2 0
C)
YHB, ( H)= (BI ( 13
is satisfied. At the Hartmann-Hahn condition, the energies of the 'H spin states and the
(29)
13 C
spin
states are the same, therefore the larger 'H polarization is transferred to 1C through the
heteronuclear dipolar interaction without net energy change of the whole system. Following CP,
we can then detect the cross polarized
13 C
signal while applying decoupling pulses on 'H. The
experiment can be repeated again after 'H longitudinal magnetization rebuilds, which is
dependent on the shorter 'H T, as opposed to the longer 13C T1. Typically, for 13C that are
strongly coupled to many 'H, such as methyl and methylene, the CP contact time required is
short. Likewise, for
1C
that are relatively farther from other 1H, such as carbonyl, a longer CP
time is often needed. For organic solids, a contact time of 1-3 ms is usually sufficient. Precise
optimization of CP time is system dependent.
In MAS experiment, dipolar coupling is attenuated. Therefore, at higher MAS frequency
CP loses transfer efficiency and longer contact time is sometimes needed. MAS also introduces
time dependence to the dipolar interaction Hamiltonian, which can be compensated by varying,
or ramping,
13 C.
the RF amplitude of the contact pulse. Figure 1.3 shows a ramped contact pulse on
This is the most common CP MAS sequence used in the study of biological solids.
31
1.2.3. The Hahn Echo Experiment
900x
180*y
Figure 1.4. The Hahn echo sequence.
Spectra with broad linewidths have rapidly decaying FIDs. The full-width-half-height
(FWHF) of a given NMR peak is 1/T
2*,
where T2 * is the effective transverse relaxation time
constant accounting for both homogeneous and inhomogeneous broadenings. Shorter T *
2
therefore means broader NMR resonances. In these situations, immediately recording the FID
after the last pulse, such as in the single pulse or the cross-polarization experiment, might not be
suitable due to the required dead time after the last pulse. The dead time is the delay between the
last NMR pulse on the observed channel and the actual beginning recording time of the FID, and
it is usually between 10-20 ps. This delay is needed because immediately after the application of
a pulse, the NMR coil experiences "ringing" that are large oscillatory signals that often
overshadow the FID and therefore introduce significant spectral distortion. They might also harm
the spectrometer receiver by oversaturating it. For most experiments, the 10 ps dead time does
not pose a significant problem because the FID is sufficiently long and thus not much signal
intensity is lost. However, for spectra with broad linewidth and short FID, most notably
quadrupolar spectra that span well over 100 kHz, the FID decays too substantially during the
dead time for proper recording after. One common method that can be employed is an echo
sequence, such as the Hahn echo18 shown in Figure 1.4.
32
The echo sequence works by refocusing the FID away from the last NMR pulse, and
therefore recording the FID is no longer constrained by the spectrometer dead time. For spectra
broadened by heterogeneous interaction such as the chemical shift anisotropy or the
heteronuclear dipolar coupling, the Hahn echo can be used. For spectra broadened by the
quadrupolar interaction or the homonuclear dipolar coupling, the solid echo,
commonly refers
to as the quadrupolar echo (90 x*-t-90y*-r) is used instead. Using the Hahn echo as an example
since it is easily visualized, the vector diagram for echo refocusing is shown in Figure 1.5.
Choosing the appropriate T for maximum echo intensity is dependent on T2 (not the same as T2*,
T2 only depends on the homogeneous broadening), and a
t
array can be set up to measure T2
precisely. In practice, it is typically advisable to make the second r slightly shorter than the first to allow recording to begin before the top of the echo, and then left shift the time domain prior to
Fourier transforming the spectrum. Doing so accounts for the effect of finite pulse width and any
spectrometer hidden delays.
b)
a)
,
y
d)
C)
_.
-
_y
.. y
y
Figure 1.5. Transverse magnetization refocuses during the Hahn echo sequence, a) the transverse
magnetization is along the -y axis after the first 90x pulse, b) the magnetization dephases from
inhomogeneous interactions after a period
t,
c) the 180y pulse flips the magnetization as shown
by the green arrow and allows refocusing to begin, and d) the magnetization is refocused along
the -y axis after a second period -.
33
1.3. Introduction to Dynamic Nuclear Polarization
In our discussion of the Zeeman interaction, it was noted that NMR is an intrinsically
insensitive technique due to the small Boltzmann polarization generated in Eq. (12). In order to
obtain satisfactory signal to noise, an NMR experiment may need to signal average for hours or
even days. Improving the NMR signal to noise and thereby reducing the necessary acquisition
time is therefore a central part of NMR hardware development. To improve the signal, one can
optimize the obtainable Boltzmann polarization from Eq. (12). This strategy has led to the
development of high field NMR magnets over the past several decades. Currently, the highest
field commercial NMR spectrometer available from Bruker is the Avance 1000, boasting a 23.5
T magnet that is a factor of 2 greater than the more conventional 500 MHz spectrometers (11.7
T). In addition to better sensitivity, high field NMR also provides improved resolution if
chemical shift anisotropy can be sufficiently reduced by MAS. Given that the chemical shift
interaction increases with the magnetic field as shown in Eq. (13), the development of high field
NMR magnets has been concurrent with improving MAS probes to allow ever faster MAS
frequencies. Lastly, high field NMR is advantageous for studies involving quadrupolar nuclei, as
the second-order quadrupolar coupling is inversely proportional to the magnetic field.
Other than increasing the magnetic field, better Boltzmann polarization can be achieved
by acquiring the NMR data at cryogenic temperatures. If the sample can be cooled to nearly
liquid nitrogen temperature (80 K), the signal enhancement from the reduced temperature is a
factor of 3.7 compares to the ambient temperature (298 K). Cooling the NMR probe to low
temperature also reduces the Johnson-Nyquist noise of the probe electronics and further
improves signal to noise. Irrespective to low temperature experiments, general improvements
34
made to the spectrometer electronic components and designs have led to a reduction of noise for
the NMR experiment.
While the hardware improvements described above have significantly improved NMR
signal to noise, dynamic nuclear polarization (DNP) has been shown to provide even greater
NMR signal enhancement.
At its very essence, DNP aims to cross-polarize nuclear polarization
using paramagnetic electron polarization that is the basis of electron paramagnetic resonance
(EPR). Since the Zeeman interaction of electrons is much greater than that of nuclei (~660 times
larger compared to 1H, and more for lower y nuclei), the potential non-Boltzmann polarization
that can be generated is significant. DNP was first proposed by Overhauser in 1953,
and
followed by the experimental verification by Carver and Slichter.25 However, as NMR pursued
higher fields beyond 5 T, there was no microwave source with the appropriate frequency (> 140
GHz) and power (> 10 W) that could adequately saturate the EPR transitions necessary to carry
out DNP efficiently. Consequently, despite its early discovery, DNP was thought to only have
limited applications and was not widely adopted. In the 1990s, Griffin and co-workers revived
DNP as a solid-state NMR technique with the introduction of high-frequency (> 140 GHz), highpower (> 10 W) gyrotrons. 26 The introduction of gyrotrons, coupled with the development of
suitable paramagnetic polarization agents, 27 has allowed DNP to achieve NMR signal
enhancements upward of two to three orders of magnitude that translates to significant reduction
of experimental acquisition time.
To understand how DNP functions, we examine briefly here the three common solid-state
DNP mechanisms, the solid effect (SE), 28 -30 the cross effect (CE), 31-35 and the thermal mixing
(TM).36-39 The SE is a two-spin process that is the simplest of DNP mechanisms. It is the
dominant DNP mechanism when the condition oo, > 6, A is matched, where
35
CoOl
is the nuclear
Larmor frequency, 6 is the homogeneous EPR linewidth, and A is the inhomogeneous EPR
linewidth. To visualize the SE, we first consider a two-spin system where a nucleus is coupled to
an electron under an external magnetic field, as shown in Figure 1.6. The Hamiltonian for this
two-spin system in the rotating frame can be written as4 0
I + ASJ + BSJ,,
H =Os s S -co 1o
(30)
where oos is the electron Larmor frequency of the EPR transition, oo, is the nuclear Larmor
frequency of the NMR transition, and A and B are the secular and nonsecular hyperfine
interaction. If the hyperfine interaction were absent in this two-spin system, the cross transitions
would be forbidden. However, the nonsecular hyperfine interaction allows the mixing of states to
occur between the
|alas)
and 1p8 as) states, and also the
|a8 ) and
1,ps) states. This mixing
of states makes the cross transitions /3pcas) to apIs) and Ja~as) to /3pps) allowable. When
microwave is applied at these cross transition frequencies, O)MW =
s ±coo±
, , with sufficient
power to saturate the transitions, the electron and the nucleus spin states undergo a "flip-flop"
and effectively transfer the electron polarization to the nuclear polarization, as shown in Figure
1.7. Saturating the
|p8as)
to japis) transition generates net negative non-Boltzmann
polarization, while saturating the laas) to 1,s)
transition has the opposite effect. The non-
Boltzmann polarization from SE (or the other DNP mechanisms) can be quite substantial.
Theoretically, the maximum achievable enhancement is the ratio 7s/71, which is 660 for 'H and
even greater for lower gamma nuclei.
36
P)s
0a
ccxc
I S)
l
OS
l OS
W0I
I S)
S)
Figure 1.6. A electron-nucleus coupled two-spin system under an external magnetic field. The
red dots conceptually show the relative spin population at each spin state but are not to scale. The
two oos show the electron Larmor frequency of the EPR transitions, and the coo show the nuclear
Larmor frequency of the NMR transitions. Due to the difference in gyromagnetic ratio, the
electron polarization (shown conceptually as the difference in sphere sizes) is far larger than the
nuclear polarization.
a)
b)
0W01
lalas)
WSE
WOS
S
SE
OSS
S
Ia @s)
as
PlI~as)W0
laas)
laPs)
W
Figure 1.7. The solid effect after application of microwave at two matching frequencies, a) the
Iacas) to j1,8ps)
and b) the
transition is saturated and generates net positive non-Boltzmann polarization,
/1
,as)
to
japs)
transition is saturated instead and generates net negative
polarization.
37
When the condition 8 < ooj < A is matched, the CE becomes the dominant DNP
mechanism. While the SE only involves one electron and one nucleus, the CE is a three-spin
process involving two electrons and one nucleus, as shown in Figure 1.8. In this system, the state
jalas1s2) and
/,p1/SIaS2)
are close in energy, and through manipulation of electron-electron
and electron-nuclear dipolar couplings by careful polarization agent design, the two states can be
optimized to be nearly degenerate, matching the condition
0
si -wOs
2 1*
(31)
If this degenerate matching condition is satisfied, when microwaves are applied along the first or
the second electron transition, an energy conserving "flip-flip-flop" process can saturate both the
lalaS1Ps2 )
and the
/,p
1 /sias
2
) state, leading to substantial non-Boltzmann polarization along the
NMR transitions as shown in Figure 1.9.
In practice, the CE generally produces larger polarization enhancement compared to the
SE,41 and therefore it is often the favored DNP mechanism. In recent years, it was found that
utilizing organic biradicals as polarization agents for CE is considerably more efficient at
satisfying the match condition specified in Eq. (31) compared to using monoradicals at higher
concentration. 42 Optimizing the biradicals used for CE is therefore a central part of DNP research
and development. Some notable biradicals that have been successful CE polarization agents
include TOTAPOL,2 ' bTbK,43 bTbtk-py, 44 TEKPol, 45 and AMUPol. 46 The efficacy of biradicals
as polarization agents depend on several factors such as molecular orientation, solubility in
solvent, and electron relaxation rates. A study of how biradical molecular orientation impacts
DNP enhancement is presented in Chapter 6 using variations of bT-thiourea as an example.
38
P2 XSIS2)
W
lI aI
cc 1aS
2)
W
Ln*O
1 2
OOS2
WOS1
WOS2
WOS1
PIASIPLS
W01
I
0
SPS2
2)
SOS2
P
r;
Ia S1aS2)AI*
WOS2
WOS1
WOS2
WOS1
F1
,W01lo
IPS IPS2)
J(XISIPS2
Figure 1.8. A three-spin coupled system involving two electrons and one nucleus, marking all the
allowable nuclear and electron transitions.
a)
b)
PISI(S2
P/aS[(S2)
W0
alas
1 aS
i
2
A
)
SlOS2
OOS2
0OS1
WOS2
WCE
IrX~aSjS2)
WCE
P)aSI3S2)
.....E o,3
(X (SIOS2
5 2)
S0
Ia fP1 S S2)2
PCSI PS2)
.....
Of PSI
A
S2)
W
IFSSSS2)
WOS1
WI0S2
WOS1
CEI SI
S2
I
IP
SI S2)
I ISIPS2
SIPS2)
+
Figure 1.9. The cross effect utilized a "flip-flip-flop" transition between two degenerate states,
leading to either a) positive, or b) negative nuclear polarization enhancement depending on
which electron transition (coosi or OOS2) is saturated.
39
When the paramagnetic electron concentration is large and the condition col < 6, A is
reached, the TM mechanism dominates. The TM is mechanistically similar to the CE. The large
electron concentration creates a strongly coupled electron system, leading to a manifold of
energy states that can undergo energy conserving population transfer between many degenerate
states. In that regard, the TM only differs from the CE by the number of electrons involved in the
process. The high number of electrons required for TM may lead to significant paramagnetic
broadening of the NMR spectra, resulting in a loss of spectral resolution.
1.4. Thesis Outline
Chapter 2 compares the effectiveness of solvent-free DNP of ortho-terphenyl in its
amorphous versus its crystalline state. Currently, most DNP experiments prepare the sample in a
glass-forming solvent matrix such as glycerol/water or DMSO/water that may not be appropriate
for all applications, notably the study of pharmaceutical solids where polymorphic properties
must be preserved. A DNP approach that does not rely on a solvent matrix would make the
technique more adoptable for pharmaceutical solid-state NMR.
Chapter 3 presents the solid-state NMR study of nanocrystalline pharmaceutical
embedded in porous biocompatible excipients, and includes preliminary DNP results that outline
the challenges of pharmaceutical DNP.
Chapter 4 presents recent progress made on temperature-jump DNP (TJDNP), which is a
liquid solution DNP technique. In TJDNP, the sample is polarized while it is frozen at cryogenic
temperature, followed by fast laser irradiation and NMR detection in the liquid state. The chapter
examines improvements that can better preserve non-Boltzmann polarization, including selection
40
of NMR rotor material, laser wavelength, and synthesis of temperature sensitive polymer
polarization agent.
Chapter 5 presents deuterium NMR results on a variety of systems including
phospholipids and metal-organic frameworks. Deuterium NMR is a sensitive probe for studying
molecular dynamics. A new single-channel probe was constructed to study temperature
dependent
2
H NMR from 150 to -173 'C. This chapter examines the phase transition of
phospholipids in the presence of membrane protein, and in a separate section examines the local
dynamic within a metal-organic framework.
Chapter 6 highlights exciting high field DNP development currently ongoing in the
Griffin group, including the new 700 MHz/460 GHz DNP spectrometer, radical polarization
agent development, alternative sample preparation methods, and direct polarization of low-y
nuclei using narrow line radicals.
1.5. References
Purcell, E. M.; Torrey, H. C.; Pound, R. V., Phys. Rev. 1946, 69 (1-2), 37-3 8.
1.
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Sodickson, D. K.; Levitt, M. H.; Vega, S.; Griffin, R. G., J.Chem. Phys. 1993, 98 (9),
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Massiot, D.; Fayon, F.; Deschamps, M.; Cadars, S.; Florian, P.; Montouillout, V.; Pellerin,
N.; Hiet, J.; Rakhmatullin, A.; Bessada, C., Cr Chim 2010, 13 (1-2), 117-129.
15.
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Freeman, R.; Hill, H. D. W., J.Chem. Phys. 1971, 54 (8), 3367-&.
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Hahn, E. L., Phys. Rev. 1950, 80 (4), 580-594.
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20.
Hartmann, S. R.; Hahn, E. L., Phys. Rev. 1962, 128 (5), 2042-&.
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Metz, G.; Wu, X. L.; Smith, S. 0., J Magn. Reson. Ser. A 1994, 110 (2), 219-227.
22.
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23.
Maly, T.; Debelouchina, G. T.; Bajaj, V. S.; Hu, K. N.; Joo, C. G.; Mak-Jurkauskas, M.
L.; Sirigiri, J. R.; van der Wel, P. C. A.; Herzfeld, J.; Temkin, R. J.; et al., J.Chem. Phys. 2008,
128 (5).
24.
Overhauser, A. W., Phys. Rev. 1953, 92 (2), 411-415.
25.
Carver, T. R.; Slichter, C. P., Phys. Rev. 1956, 102 (4), 975-980.
26.
Becerra, L. R.; Gerfen, G. J.; Temkin, R. J.; Singel, D. J.; Griffin, R. G., Phys. Rev. Lett.
1993, 71 (21), 3561-3564.
27.
Song, C. S.; Hu, K. N.; Joo, C. G.; Swager, T. M.; Griffin, R. G., J Am. Chem. Soc. 2006,
128 (35), 11385-11390.
28.
Abragam, A.; Proctor, W. G., Cr HebdAcadSci 1958, 246 (15), 2253-2256.
29.
Jeffries, C. D., Phys. Rev. 1957, 106 (1), 164-165.
30.
Jeffries, C. D., Phys. Rev. 1960, 117 (4), 1056-1069.
31.
Kessenikh, A. V.; Lushchikov, V. I.; Manenkov, A. A.; Taran, Y. V., Sov Phys-Sol State
1963, 5 (2), 321-329.
32.
Kessenikh, A. V.; Manenkov, A. A.; Pyatnitskii, G. I., Sov Phys-Sol State 1964, 6 (3),
641-643.
33.
Hwang, C. F.; Hill, D. A., Phys. Rev. Lett. 1967, 18 (4), 110-&.
34.
Hwang, C. F.; Hill, D. A., Phys. Rev. Lett. 1967, 19 (18), 1011-&.
35.
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36.
Wind, R. A.; Duijvestijn, M. J.; Vanderlugt, C.; Manenschijn, A.; Vriend, J., ProgNucl
Mag Res Sp 1985, 17, 33-67.
37.
Goldman, M., Spin temperatureand nuclear magnetic resonance in solids. Clarendon
Press: Oxford,, 1970; p ix, 246 p.
38.
Duijvestijn, M. J.; Wind, R. A.; Smidt, J., Physica B & C 1986, 138 (1-2), 147-170.
39.
Wenckebach, W. T.; Swanenburg, T. J. B.; Poulis, N. J., Physics Reports 1974, 14 (5),
181-255.
40.
Hu, K. N. Polarizing agents for high-frequency dynamic nuclear polarization development and applications. Massachusetts Institute of Technology, Cambridge, MA, 2006.
41.
Hu, K. N.; Bajaj, V. S.; Rosay, M.; Griffin, R. G., J.Chem. Phys. 2007, 126 (4).
42.
Hu, K. N.; Yu, H. H.; Swager, T. M.; Griffin, R. G., J.Am. Chem. Soc. 2004, 126 (35),
10844-10845.
43.
Matsuki, Y.; Maly, T.; Ouari, 0.; Karoui, H.; Le Moigne, F.; Rizzato, E.; Lyubenova, S.;
Herzfeld, J.; Prisner, T.; Tordo, P.; et al., Angew. Chem. Int. Edit. 2009, 48 (27), 4996-5000.
42
Kiesewetter, M. K.; Corzilius, B.; Smith, A. A.; Griffin, R. G.; Swager, T. M., J Am.
44.
Chem. Soc. 2012, 134 (10), 4537-4540.
45.
Zagdoun, A.; Casano, G.; Ouari, 0.; Schwarzwalder, M.; Rossini, A. J.; Aussenac, F.;
Yulikov, M.; Jeschke, G.; Copdret, C.; Lesage, A.; et al., J. Am. Chem. Soc. 2013, 135 (34),
12790-12797.
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Sauvee, C.; Rosay, M.; Casano, G.; Aussenac, F.; Weber, R. T.; Ouari, 0.; Tordo, P.,
Angew. Chem. Int. Edit. 2013, 52 (41), 10858-10861.
43
44
Chapter 2: Solvent-Free Dynamic Nuclear
Polarization of Amorphous and Crystalline OrthoTerphenyl
Adapted from Ong, T. C.; Mak-Jurkauskas, M. L.; Walish, J. J.; Michaelis, V. K.;
Corzilius, B.; Smith, A. A.; Clausen, A. M.; Cheetham, J. C.; Swager, T. M.; Griffin,
R. G., J. Phys. Chem. B 2013, 117, 3040-3046.
Abstract
Dynamic nuclear polarization (DNP) of amorphous and crystalline ortho-terphenyl (OTP)
in the absence of glass forming agents is presented in order to gauge the feasibility of applying
DNP to pharmaceutical solid-state NMR experiments and to study the effect of inter-molecular
structure, or lack thereof, on the DNP enhancement. By way of 'H-13C cross polarization, we
obtained DNP enhancement (s) of 58 for 95% deuterated OTP in the amorphous state using the
biradical bis-TEMPO terephthalate (bTtereph), and F of 36 in the crystalline state. Measurements
of the 1H T1 and EPR experiments showed the crystallization process led to phase separation of
the polarization agent, creating an inhomogeneous distribution of radicals within the sample.
Consequently, the effective radical concentration was decreased in the bulk OTP phase, and longrange 'H-1H spin diffusion was the main polarization propagation mechanism. Preliminary DNP
experiments with the glass-forming anti-inflammation drug, indomethacin, showed promising
results, and further studies are underway to prepare DNP samples using pharmaceutical
techniques.
45
2.1. Introduction
Solid-state properties of active pharmaceutical ingredients (APIs) and drug formulations
directly impact the safety and efficacy of a drug.1- 3 For example, a drug that is poorly soluble as a
crystal oftentimes is more soluble if it can be prepared in an amorphous form, increasing its
bioavailability. Solid-state NMR is a uniquely informative and versatile analytical technique in
pharmaceutical research that includes analysis of the final solid form of the drug, quantification
of amorphous content, excipient interactions, and salt and polymorph screening. In practice,
solid-state NMR is performed alongside other analytical techniques such as electron microscopy,
x-ray diffraction, IR and Raman spectroscopies, differential scanning calorimetry (DSC), and
thermogravimetric analysis (TGA) to produce a complete characterization of the API and its
formulation. Compared to other techniques, solid-state NMR has the advantage of being an
inherently quantitative method that non-destructively interrogates the whole sample. Depending
on the system of interest, it can be used, for example, to identify and quantify polymorphs, detect
the number of molecules in a unit cell, and elucidate the presence of a hydrate and/or solvate,
investigate structural and dynamic properties over a wide range of time scales, monitor stability
against degradation over time, and analyze crystalline and amorphous environments. 4 5
Although informative, NMR is intrinsically an insensitive analytical technique as a result
of the inherently low nuclear spin polarization. This problem can be further compounded by low
natural abundance NMR active nuclei (e.g.,
3
C,
15N,
170,
etc.). These challenges cause drug
formulation screening by solid-state NMR to be a time-consuming process. Dynamic nuclear
polarization
(DNP) at cryogenic temperatures has been shown to provide significant
enhancements for NMR signals. The gains in sensitivity afforded by DNP are typically one to
two orders-of-magnitude and reduce the need for lengthy signal-averaging thereby dramatically
reducing the data acquisition time.6-14 As a result DNP has found utility in NMR applied to
46
inorganic complexes,' 8 silicon surface functional
membrane proteins, 1-1
amyloid fibrils,"
groups, 1 metabolomics,
and medical MRI. 2- Extension of microwave-driven DNP to analyze
APIs and pharmaceutical formulations will potentially lead to significant savings in cost and time.
Currently most DNP samples are prepared in a glass-forming medium such as
glycerol/water or DMSO/water, which functions as a cryoprotectant to protect biological samples
(e.g., proteins) against freezing damage. 23-24 In addition, the glassy matrix serves to uniformly
disperse the mono- or biradical polarization agents which optimize the DNP enhancement.25-27
However, dissolving APIs or their solid formulations in glassing media eliminates the solid-state
structure under investigation and is unsuitable for studying pharmaceuticals. Previously,
Vitzthum et al. conducted DNP experiments on an amorphous powder mixture of a decapeptide
(DP) containing a spin-labeled decapeptide (DP*) without using solvent. The study obtained a
DNP enhancement (s) of up to 4, and an overall enhancement
(&global)
of up to 10 by taking into
account factors such as Boltzmann population difference at cryogenic temperatures, faster
nuclear relaxation, and nuclear spin bleaching caused by close proximity to paramagnets. 28 In a
separate study, Lilly Thankamony et al. examined solvent-free DNP using mesoporous silica
functionalized with TEMPO moieties, and obtained an enhancement of 3 from direct
29Si
polarization.29
In contrast to previous experiments, the research direction described here ultimately aims
to prepare solvent-free samples for DNP using common pharmaceutical sample-preparation
techniques that create dispersed radical polarization agents (e.g., TEMPO based radicals, BDPA,
trityl, etc.) within the sample. To pursue this goal, and to more thoroughly understand the DNP
process, a solvent-free matrix that displays both amorphous and crystalline states is required.
Importantly our approach results in a rare direct comparison between identical amorphous and
crystalline systems using DNP-NMR.
47
We present herein a comparison of signal enhancements employing DNP for the glassformer ortho-terphenyl (OTP) in its amorphous (i.e., glassy) and crystalline states. OTP is a
well-studied organic glass forming solid 30 consisting of a central benzene ring with two pendant
phenyl rings attached in positions ortho to one another. Steric effects produce an out-of-plane
twisting of the pendant phenyls and allow the molecule to exist as a viscous supercooled-liquid
for an extended period of time upon cooling from the melt. 3 1 Rapid freeze-quenching of the
molten OTP creates a glass with a glass transition temperature (Tg) of -30 'C. At room
temperature supercooled liquids crystallize in a matter of minutes, as shown in Figure 2.1. The
phase behavior of OTP allow for the manipulation of its physical state in situ during the DNPNMR experiment (i.e., permitting us to observe both the crystalline- and amorphous-state DNP
enhancements for a given sample without unpacking the NMR rotor). In turn this allows for
direct comparison of DNP enhancements using the identical OTP sample and packing conditions.
OTP
~ 5-10 minutes
at 25 0C
Crystalline
-30 *C
Supercooled
Glass
Liquid
57.5 'C
Liquid
Liquid N2
Figure 2.1. Phase transition scheme of ortho-terphenyl (OTP)
2.2. Experimental
Materials. Ortho-terphenyl (OTP, >99%) and 4-hydroxy-TEMPO
(TEMPOL) were
purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. d14-OTP
48
(98%) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). The
hydrophobic biradical polarization agent bis-TEMPO terephthalate (bTtereph) was synthesized
from TEMPOL and terephthaloyl chloride as described in the SI.
Sample Preparation. A radical polarization agent (TEMPOL or bTtereph) was
incorporated into OTP at concentrations between 0.25 and 1 mol% by melting and mixing at
elevated temperatures. The solution was then inserted into an NMR probe pre-cooled to 80 K for
rapid quenching by-passing the onset of crystallization and enabling the amorphous state
measurements. The crystalline sample was obtained by removing the amorphous sample from the
pre-cooled NMR probe and waiting until in situ crystallization occurred under ambient conditions
and was checked visually through the transparent sapphire NMR rotor.
DNP NMR. All DNP NMR experiments were conducted on a custom-built 212 MHz (5 T,
H) spectrometer (courtesy of Dr. David Ruben, Francis Bitter Magnet Lab). The magnet is
equipped with a superconducting sweep coil with a range of ±0.05 T, and a field mapping unit for
accurate field measurements. Continuous wave high power (> 8 W) microwaves were generated
by a custom-built 140 GHz gyrotron 32 . All experiments used a custom-designed cryogenic threechannel ('H- 13C- 5N) MAS probe with a commercial 4 mm spinning module (Revolution NMR,
Fort Collins, CO). The probe is equipped with a cryogenic sample eject system3 3 to allow rapid
exchange of samples which is crucial for our in situ studies. All 13C spectra were acquired with
MAS frequency of 4.5 kHz and with two pulse phase modulation (TPPM)34 proton decoupling.
For the cross polarization35 experiments, the CP contact time, -cp, was 2.0 ms at Vrf of 83 kHz.
For the DNP enhanced spectra, the number of scans was 32. For the unenhanced spectra, the
number of scans was 2,000. For the amorphous samples, the recycling time between scans was
60 s. For the crystalline sample, the recycling time between scans was 240 s.
49
EPR. Continuous-wave 9.7 GHz (X-Band) EPR spectra were recorded on a Bruker
Elexsys E580 spectrometer using a dielectric ring resonator ER 4118X-MD5 operating in the
TEO, mode. The measurement temperature of 80 K was reached inside an ER 4118CF-O flow
cryostat using liquid nitrogen as a cryogen. The amorphous sample was prepared by flash
freezing OTP supercooled liquid inside a 4 mm o.d. EPR tube in liquid nitrogen before insertion
into the EPR probe. The crystalline sample was prepared by removing the amorphous sample
from the EPR probe after respective measurements and letting it warm at ambient conditions until
complete crystallization was confirmed by visual means after which it was reinserted into the
probe.
2.3. Results
In consideration of the fact that the 1H concentration affects the polarization transfer and
spin diffusion efficiency responsible for DNP enhancements,36-39 a series of samples were
prepared by incorporating 1 mol% TEMPOL monoradical into OTP with the deuteration level
ranging from 0% to 95%. The
13 C
cross polarization MAS DNP enhancement (F) measurements
from these samples are shown in Figure 2.2 for both amorphous and crystalline OTP. Amorphous
OTP was shown to have , between 10 (100% 'H) and 25 (5% 'H) while crystalline OTP
enhancements were between 1.7 and 7.7, respectively. For both amorphous and crystalline OTP,
deuteration provided significant gains in enhancement consistent with past DNP studies.
50
30-
"
25
*Amorphous
20 -
NCrystalline
15.
10
5
0U
0
20
40
60
80
100
Level of Deuteration (%)
Figure 2.2. 13 C CPMAS DNP enhancement (,) of OTP containing 1 mol% TEMPOL as a
function of levels of deuteration.
Hu et al. has shown that P is substantially larger using nitroxide-based biradicals as
compared to TEMPO monoradicals when cross-effect is the dominant DNP mechanism. 4 0 The
cross-effect mechanism involves a three-spin flip-flip-flop process between two electrons and a
nucleus. 4 1-4 5 Biradicals are more efficient and produce larger enhancements than the equivalent
monoradical electron concentration as a result of their larger e~ - e~ dipolar couplings.
TOTAPOL 46 is an established water-soluble biradical that is used in many DNP experiments,
however we found that this agent was not miscible with OTP, and a new biradical, bis-TEMPO
terephalate (bTtereph, as shown in Figure 2.3), was synthesized for this experiment. Heating is
required to melt OTP, and we conducted thermogravimetric analysis (TGA) and differential
scanning calorimetry (DSC) of bTtereph to establish its thermal stability. The results (Figure 2.S1)
indicated radical stability up to 160 'C, well above the melting point of OTP (57.5 'C); this
enabled bTtereph to be mixed in warm OTP liquid (- 60 C) without fear of thermal degradation.
The DNP analysis of these mixtures revealed that bTtereph provided a larger , in OTP than the
equivalent TEMPOL radical concentration. As shown in Figure 2.4, for 95% deuterated OTP
with 0.5 mol% bTtereph, the 13 C CPMAS &for the amorphous sample increased to 58, and in the
51
crystalline sample the c increased to 36. DNP enhancements by direct
13 C
polarization were also
measured at the same external magnetic fields as the CPMAS z measurements were conducted
and in these measurements the f in the amorphous state was 67, and in the crystalline state was
50. We also determined the
13 C
CPMAS , for 100% protonated OTP with 0.5 mol% bTtereph to
compare with the heavily deuterated sample and found enhancements of 35 and 3.3 in the
amorphous and crystalline states, respectively.
A field-dependent enhancement profile was measured for bTtereph (Figure 2.S2) in 95%
deuterated OTP, and the results were consistent with previously reported TOTAPOL 46 or bTbk2 7
nitroxide-based radicals. We found that f does not vary significantly with either increasing
microwave power from 7 to 11 W, or with bTtereph concentration ranging from 0.125 to 0.5
mol%, for both amorphous and crystalline states, as shown in Figures 2.S3 and 2.S4, respectively.
N
0
0
O
N
0ge 2M
Figure 2.3. The structure of bis-TEMPO terephthalate (bTtereph).
52
NO
a)
Amorphous
E = 58
w/ DNP
w/o DNP x5
b)
Crystalline
E = 36
w/ DNP
w/o DNP
300
13
Figure 2.4.
13C
100
200
0
C Chemical Shift (ppm)
CPMAS DNP enhanced spectra of 95% deuterated OTP with 0.5 mol% bTtereph
for the a) amorphous and b) crystalline states. The spectra are plotted with the DNP off spectra
(no microwaves) to demonstrate the increase in signal-to-noise.
1H
polarization buildup curves of the 95% deuterated amorphous and crystalline OTP
with 0.25 mol% bTtereph were measured to determine the time required to reach signal saturation.
The 1H NMR signal for amorphous OTP reached its maximum very quickly with a buildup time
constant (rB) of 8.3 s, as shown in Figure 2.5a. This result is consistent with past DNP samples
prepared in glass forming solvents such as glycerol/water or DMSO/water.46 For crystalline OTP
a dramatic increase in irradiation time was required to saturate the NMR signal, as shown in
Figure 2.5b. Moreover, the 'H buildup curve for the crystalline state exhibited a biphasic
behavior, which can be described by equation 14 7 :
M(t) = M (1 - fe-'BI -(1- f)_
53
~B2
where buildup of bulk magnetization M(t) is treated as the sum of two first order processes with
time constants,
,rBand
rB2,
and f denoting the fraction of the population polarized by the first
process. Empirically fitting the data with Eq. (1) showed that the initial fast process had rB1 of 22
s, and the slower polarization buildup that followed had rB2 of 202 s, with 35% of the OTP
polarized by the initial fast process. It was found that rB, is inversely related to radical
concentration (i.e., a decrease in the 1H build-up time constant occurred with increasing radical
concentration). Conversely,
TB2
did not show significant dependence on the radical until high
concentrations were reached. This suggests that the bTtereph radical phase separates into distinct
domains during the crystallization of the OTP. Therefore, both a fast radical induced polarization
buildup ('H near the radical clusters) and a slow long-range 'H-1H dipolar coupling of spinpolarization contributed to the overall longer polarization buildup time observed for OTP crystals.
Values of TBI,
TB2,
andfwith respect to bTtereph concentration are reported in Table 2.1.
Table 2.1. Biphasic DNP 'H polarization buildup time constants (TBI and
TB2)
and fraction (/) of
crystallized OTP polarized by the first, fast process at various bTtereph concentrations. The
errors are calculated based on bi-exponential fitting.
ZbTtereph (mOl%)
0.125
TB1
39
(S)
TB2
(s)
f (%)
3.9
201 ±20.3
43
0.25
22 ±1.7
202 ±10.9
35
0.5
16 ±0.5
119 ±6.8
57
54
1.2
a)
C
0.8
8.3 s
S0.6 -TB
0.4
0.2
0'
0
b)
20
40
Microwave Irradiation Time (s)
60
1.2
10.8
9.
10.6
TB1 = 22 s
T = 202 s
f = 35 %
JN
0.4
0.2
0
200
400
600
800
Microwave Irradiation Time (s)
Figure 2.5. 'H polarization buildup curves of a) amorphous and b) crystalline 95% deuterated
OTP with 0.25 mol% bTtereph. The red line shows the exponential curve fitting of the data, with
the amorphous data fitted with a mono-exponential buildup equation: M (t) = M, (1 - e-'IB), and
the
crystalline
MW) = A
(i
-
feIrBl
data
-
(I
fitted
-
f ) etIB2.
with
a
bi-exponential
buildup
equation:
The star in b) marks the amorphous saturation point
obtained in a) to demonstrate the substantial increase in signal saturation time observed for the
crystalline sample.
Continuous-wave, 9-GHz EPR spectra of bTtereph in fully deuterated OTP were
measured for both amorphous and crystalline states, as shown in Figure 2.6. Although the
amorphous sample features a typical well-resolved EPR spectrum of a bis-nitroxide biradical, the
spectrum of the crystalline sample is dominated by a featureless single line with a g-value similar
to the average (isotropic) g-value of the nitroxide. Spectral simulations using the Easyspin
package4 8 (see SI for more details) show that the isotropic line has a Lorentzian shape and
55
underlies a linewidth distribution with mean and variance of both -84 MHz (Figure 2.S5). We
assume that this resonance arises from the bTtereph that phase-separate during crystallization of
the OTP with varying domain size. Strong electron-electron exchange couplings in these clusters
lead to exchange-narrowed, homogeneous EPR lines. Additionally, a small contribution (~7 % of
the overall signal intensity) can be attributed to isolated bTtereph molecules with spectral features
equal to those obtained for the amorphous sample. This points either to a small amount of cocrystallization of bTtereph in OTP or to an incomplete crystallization process. These observations
are further supported by pulsed EPR experiments at 9 GHz and 140 GHz. While bTtereph in
fully-deuterated amorphous OTP allowed for echo detection of the EPR spectrum (see Figure
2.S6 for 140 GHz spectrum) we were unable to obtain echoes in the crystalline state, most
probably due to ultra-fast relaxation of phase-separated bTtereph.
--
Amorphous
Crystalline
338 340 342 344 346 348 350 352 354 356
Magnetic Field (mT)
Figure 2.6. CW EPR field profile of bTtereph at 9 GHz in either amorphous or crystalline fullydeuterated OTP. EPR amplitudes were corrected in order to achieve equal double integral values.
56
2.4. Discussion
DNP of amorphous and crystalline solids. As observed from measurements of DNP
enhancements, 'H polarization buildup time, and EPR spectra, our results are largely in
agreement with current literature that DNP is optimally performed in a glass or amorphous solid
in which radical polarization agents are homogeneously dispersed. Figure 2.6 is a comparison of
the EPR spectra of bTtereph in amorphous and crystalline fully-deuterated OTP. The strong
electron-electron coupling observed for the crystalline sample suggests that bTtereph appears to
cluster in crystalline OTP, meaning that radical-rich and radical-poor regions are created during
crystallization. The inhomogeneous radical distribution leads to smaller DNP enhancements and
longer, biphasic 'H polarization buildup times. This effect has been reported in other systems as
well; Dementyev et al. observed a similar biphasic polarization buildup for DNP of partially
crystalline silicon microparticles at 1.4 K. 49 The amorphous region of these silicon particles
contains high concentration of paramagnetic impurities in the form of "dangling bonds" while the
crystalline region contains few such impurities. This creates an inhomogeneous distribution of
radical polarization agents and as a result, the nuclear relaxation time, T1, becomes longer in the
crystalline region compared to the amorphous region, leading to the biphasic polarization buildup
that we also observed for OTP crystals.
Despite the disadvantage of radical clustering, we nevertheless observed that by using
biradicals as polarization agents and by deuteration, a significant DNP enhancement could still be
obtained for the crystalline system. Moreover, as shown in Figure 2.4, the linewidths were not
significantly affected by the presence of biradicals, meaning that DNP can provide large
enhancements in dry, solvent-free crystalline systems, while maintaining excellent spectral
resolution. In light of the lowered enhancement due to radical clustering, we note that a low 1H
57
concentration, in this case by ~90-95% deuteration, is absolutely crucial for DNP to work by way
of 1H-13 C cross polarization. We observe that 95% deuterated crystalline OTP with 0.5 mol%
bTtereph yields c of 36, while the , decreases to 3.3 in a sample that is 100% protonated. This
finding underscores the role of 1H- 1H spin-diffusion efficiency in propagating polarization in
crystalline samples. The considerably lower enhancement (C = 3.3) observed for the fully
protonated OTP crystal suggests that the radical-poor regions of the sample were largely
unenhanced by DNP, meaning that low 1H concentration is required for polarization to penetrate
into the crystalline core. Importantly, we observed that DNP enhancement remains appreciable (c
= 35) for amorphous OTP even in samples that are 100% protonated. This can be attributed to a
more homogeneous distribution of radicals as observed by the EPR spectrum, which means less
reliance on long-range 'H- 1H spin diffusion to spread polarization.
Implications for DNP of pharmaceutical systems. In terms of preparing solvent-free,
amorphous pharmaceutical samples, we note that our preparation of OTP amorphous samples
emulates hot-melt extrusion50 . During hot-melt extrusion, API, excipients, and polymer carriers
are melted and mixed at elevated temperatures and pressures to achieve a homogeneously
dispersed solid-solution. Based on the results of this work, one can prepare drug samples for
DNP via hot-melt extrusion and in theory should obtain reasonable C, provided the radical
polarization agent survives the process. Experiments are pending to investigate this hypothesis.
Beyond hot-melt extrusion, a number of techniques exist to prepare amorphous, homogeneously
dispersed pharmaceutical samples, including spray-dry dispersions, electrospinning, and freezedrying. All of which can potentially be used to effectively prepare DNP samples.
We have begun DNP studies involving pharmaceutical glass-forming materials, most
notably the anti-inflammation drug indomethacin. As a proof of concept, we prepared an
indomethacin glass from lyophilized indomethacin powder (> 99%, Sigma-Aldrich) doped with
58
0.5 mol% bTtereph. The DNP enhanced
3
1
C CPMAS spectrum showed a 6 of 14, as shown in
Figure 2.7. Taking into account Boltzmann population difference from acquiring the data at 80 K
relative to room temperature, we obtain overall enhancement, S' = 49, which corresponds to a
savings of > 2,000 fold in acquisition time. The room temperature spectra of crystalline and
amorphous indomethacin obtained at 11.74 T (Figure 2.S7) reveal that the resolution of multiple
sites in the DNP enhanced spectrum is hampered as a result of acquisition at a relatively low field
(5 T) and the fact that the majority of the drug is comprised of aromatic resonances confined in a
narrow chemical-shift region. Higher field DNP spectrometers at 400 MHz (Bruker)," 600
MHz,
or at 700 MHz (FBML, MIT)53 are expected to provide improved resolution. The
combination of good DNP enhancement and good resolution will allow for more 2D solid-state
NMR experiments in pharmaceutical research. Currently, 2D solid-state NMR experiments are
not widely applied in pharmaceutical research because they are considered prohibitively timeconsuming. However, recent works have shown that they can be valuable methods to analyze
solid dispersion, particularly H-1 3 C CP-HETCOR.54-56 Successful application of DNP will make
these 2D experiments more practical.
59
12
/
7/ 9
N
2
20
6
CI
'o11
8
20
13
4
5
3
18
1
OH
19
0
11-16
Amorphous
Indomethac in
E = 14
10 6
3,7,8
w/ DNP
w/o DNP
200
Figure 2.7.
13C
160
12
80
4*0
13C Chemical Shift (ppm)
CPM kS DNP enhanced spectra of indomethacin glas s doped with 0.5 mol%
bTtereph.
For DNP of crystalline pharmaceutical samples, we face two challenges:
1)
the
inhomogeneous distribution (clustering) of radical polarization agents during the crystallization
process and 2) bulk isotope labeling of samples (i.e., synthesizing samples that are 95%
deuterated) is not a common pharmaceutical practice. Moreover, deuteration of samples impacts
CPMAS efficiency, so absolute signal sensitivity would be attenuated. To address the challenge
of radical clustering, one can prepare a homogeneous mixture of samples and radicals as a
suspension of protonated nanocrystals or microcrystals in a radical-containing solvent matrix. As
observed by van der Wel et al., the small domain size of the nanocrystals (100-200 nm) results in
a s that is only slightly reduced relative to the glassy matrix.57 Nanocrystals and nanoparticles
with domain sizes less than 100 nm are found in pharmaceutical formulations. 58 To preserve the
60
drug's solid-state structural and physical characteristics, the dispersant of choice should not
dissolve the compound of interest. Recently, Rossini et al. demonstrated the idea in microcrystals
1
by suspending glucose and sulfathiazole with domain sizes up to 500 microns in low H density
organic solvents such as 1,1,2,2-tetrabromoethane and 1,3-dibromobutane. 59 Takahashi et al.
made the interesting observation that simply moisturizing the crystals appear to help DNP
enhancement. 60 In their experiment using TOTAPOL-coated crystalline cellulose with domain
size of 20 microns, F_ of 2.4 was obtained in completely dried cellulose. However, when the
cellulose was moisturized with a small amount of D2 0, the s increased to 20. Since TOTAPOL is
soluble in water, the finding suggests that even a small amount of solvent seems to alleviate the
problem of radical clustering, and thereby improves DNP performance.
2.5. Conclusion
DNP experiments of amorphous and crystalline OTP were compared to evaluate
feasibility of solvent-free DNP for pharmaceutical samples. We found that due to superior
distribution of radical polarization agents, OTP in the amorphous state consistently produced
higher DNP enhancements than in the crystalline state. Considering that a variety of techniques
exists to prepare amorphous, homogeneous pharmaceutical samples, such as hot-melt extrusion,
electrospinning, and spray-dry dispersion, we propose that DNP can be used in combination with
these methods to improve signal-to-noise of solid-state NMR experiments.
The effectiveness of DNP of crystalline systems is hindered by clustering of radical
polarization agents leading to creation of either radical rich or poor regions within the sample.
This leads to longer polarization buildup times and smaller enhancements. However, crystalline
samples maintain resolution due to their long-range order within the sample, whereas the
distribution of sites (both angle and bond length variance) in amorphous solids cause
61
inhomogeneous broadening which will require higher fields and multiple dimension experiments
for further structural information. It is important to note that this is an issue intrinsic to NMR
regardless of DNP. NMR can probe amorphous, locally disordered structure that is not possible
using traditional solid-state techniques such as diffraction methods. Coupling DNP with solidstate NMR now provides a faster method for obtaining structural information about disordered
solids.
2.6. Acknowledgements
The authors thank Jeff Bryant, Blair Brettmann, Matthew Kiesewetter, and Eugene
Cheung for useful discussions. We acknowledge the National Institute of Health for funding
support of DNP projects at the Francis Bitter Magnet Laboratory (EB002804 and EB002026) and
a grant to T.M.S., GM095843. V.K.M. is grateful to the Natural Sciences and Engineering
Research Council of Canada for a postdoctoral fellowship. B.C. was partially supported by the
Deutsche Forschungsgemeinschaft (Research Fellowship C0802/1-1).
62
2.7. Supporting Information
Synthesis of bis-TEMPO terephthalate (bTtereph)
bis-TEMPO terephthalate (bTtereph) was synthesized by the dropwise addition of a
solution of terephthaloyl chloride (2 mM in dichloromethane (DCM)) to a solution of 4-hydroxy2,2,6,6-tetramethylpiperidine-l-oxyl (5.8 mM) and pyridine (6 mM) in DCM at 0 'C. The
solution was then stirred at that temperature for one hour before being warmed to room
temperature where it was stirred for an additional twelve hours. After washing with acidic water
(pH 4) the organic phase was concentrated and purified via column chromatography
(cyclohexane/ethyl acetate). The orange-red, crystalline powder was obtained with an overall
yield of 40%.
63
120-
4
1.845%
(0.07798mg)
100-
1.5-~
- 2
80
2.0
0)0
40-
40
0
50
100
150
200
250
300
Temperature (*C)
Figure 2.S1. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)
plots of bTtereph. Measurements of bTtereph weight (green) and change in weight (maroon)
show decomposition starting at ~160 *C. DSC thermogram (blue) shows two endothermic events
at 160 and 210 'C, followed by two exothermic events at higher temperatures.
64
1.2
0.8
'-
.4
Cl)
0
C
~-0.4
-0.8
-1.2'
4950
4960
4970
4980
4990
5000
Magnetic Field (mT)
Figure 2.S2. NMR field dependent 'H enhancement (c) profile of bTtereph in 95% deuterated
amorphous OTP, taken at 5 T using 8 W of microwave power and MAS frequency of 4.5 kHz. A
line is drawn connecting the data points as a guide.
65
80
70
60
50
W40
30
20
10
0.
U
7
U
U
8
9
10
Microwave Power (W)
11
Figure 2.S3. DNP enhancement (s) as a function of gyrotron microwave power from 7 to 11 W.
A small increase (- 10%) in enhancement was observed for the amorphous (*) and the crystalline
(a) 95% deuterated OTP with 0.5 mol% bTtereph.
66
70
60
++
50
40
U
30
20
10
0
0
0.2
0.4
0.6
0.8
1
XbTter.ph (mol %)
Figure 2.S4. DNP enhancement (s) as a function of bTtereph concentration up to 1 mol% for the
amorphous (*) and the crystalline (m) 95% deuterated OTP using 8 W microwave power and
MAS frequency of 4.5 kHz.
67
Simulations of EPR spectra
EPR spectra have been simulated (Figure 2.S5) using the EasySpin package. 48 In the case
of the amorphous samples the spectra recorded at 9 GHz (X-Band) and 140 GHz have been
simulated with a single set of parameters. The first derivative of the 140 GHz spectrum has been
obtained using EasySpin's "fieldmod" function with 0.3 mT pseudo-modulation amplitude. Two
S = 1/2 electron spins each hyperfine coupled to a 14N nucleus were assumed with the following
set
AY
of interaction
=
18.0 MHz; Az
tensors:
=
g,
=
19.0 MHz; D,
2.00221;
=
gy
=
2.00636;
-7.5 MHz; Dy
=
gzz =2.01016;
A
=
97.5 MHz;
-7.5 MHz; D,, = 15.0 MHz. The g and
hyperfine coupling (A) tensors were assumed to be identical and collinear for both electrons. The
electron-electron interaction (described by D) was assumed to be purely dipolar in nature; no
exchange interaction was considered. Line broadening has been applied independently for each
frequency. For 9 GHz, orientation dependent broadening ("HStrain") has been applied with 21,
28, and 17 MHz in the x, y, and z orientation, respectively; for 140 GHz, 16, 28, and 34 MHz
have been used. General Gaussian line broadening (via the "lw" parameter) of 0.1 mT (full width
at half maximum, FWHM) has been applied for both frequencies while for 9 GHz an additional
Lorentzian broadening of 0.16 mT (FWHM) was used (also via the "lw" parameter).
The spectra of bTtereph in crystalline OTP can be simulated by weighted summation of
the spectrum of bTtereph in amorphous OTP and a featureless line with g = 2.0064. In order to
mimic the shape of this homogeneous line, a distribution of Lorentzian lines with widths between
0.1 and 10 mT (FWHM) had to be assumed. For the simulation, a probability distribution with
(
p(3)
c 64 e
68
07
(1)
reproduced the line shape adequately. The distribution probability is shown in Figure 2.S6. Note
that this function was chosen solely to reproduce the line shape and is not based on physical
models regarding cluster size distribution, for example. The relative intensity (double integral) of
the resolved bTtereph signal is ~8 %.
69
Simulation
Experiment
Amorphous 140 GHz
4975
4980
4985
4990
4995
5000
Magnetic Field (mT)
Simulation
Experiment
Amorphous 9 GHz
340
342
344
346
348
350
352
Magnetic Field (mT)
Simulation
Experiment
Crvstalline 9 GHz
340
342
34
346
348
350
352
Magnetic Field (mT)
Figure 2.S5. Experimental and simulated EPR spectra of bTtereph in amorphous or crystalline
OTP at 9 or 140 GHz.
70
3025-
x
20-
x
x
x
xx
~15-
x
XX
o 10-
.
X
500
50
150
100
200
250
Homogeneous FWHM (MHz)
Figure 2.S6. Probablity distribution of Lorentzian linewidth used to simulate the 9 GHz EPR
spectrum of bTtereph in crystalline OTP. The linewidth distribution peaks at 79 MHz.
71
I
4975
I
4980
I
I
4985
4990
Field [mT]
1
4995
5000
Figure 2.S7. Pulsed 140 GHz EPR spectra of bTtereph in fully deuterated amorphous OTP at 80
K.
The 140 GHz EPR spectrum of 0.0125 mol% bTtereph in OTP were acquired at 80 K,
using a high-field pulsed EPR system described elsewhere.61 A Hahn echo (;r/2-r-w) was used
with a timing of 68 ns - 200 ns - 136 ns, and the echo was integrated at each field point. 4800
shots were acquired at each of 321 field points from 4972 mT to 5004 mT, using a two-step
phase cycle. The derivative spectrum was calculated from the absorption spectrum using the
"fieldmod" function from EasySpin 8 , with a 0.3 mT modulation amplitude.
72
Amorphous
Alpha
zL
GammBiJak
200
80
120
160
13C Chemical Shift (ppm)
Figure 2.S8. Room-temperature
LL
40
C13
CPMAS NMR spectra of amorphous and crystalline (a and y
crystals) indomethacin. Data were acquired on a 11.7 T (500.7 MHz, 'H) home-built
spectrometer (courtesy of Dr. Dave Ruben) using a triple channel MAS probe with spinning
frequency of 8 kHz. All data were acquired for 24 hours using TPPM decoupling.3 4
73
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76
Chapter 3: Solid-State NMR and Dynamic Nuclear
Polarization of Pharmaceutical Formulations
3.1. Introduction
Over the past two decades, solid-state NMR has grown to become an indispensible
analytical method for studying pharmaceuticals, including active pharmaceutical ingredients
(API) as well as formulations that are mixtures of API and excipients.'
2
As mentioned in
Chapter 2, the solid-state properties of API and formulations directly impact the safety and
efficacy of the drug,3-4 and considerable efforts have been invested to improve drug solubility
and bioavailability by researching preparation methods to produce drugs, including forming lessstable amorphous or higher energy polymorphs 5 7 by decreasing particle size8 -9 or by creating
salts.' 0 While methods such as IR, Raman, and powder x-ray diffraction (PXRD) are wellestablished, solid-state NMR offers higher resolution and does not require crystalline samples,
making the technique useful as a fingerprinting tool and also as a probe for localized structural
and dynamic information. In the supporting information Figure 2.S8 of the previous chapter, we
examined the 13C CPMAS spectra of amorphous, a, and y crystals of the anti-inflammation drug
indomethacin at a 500 MHz spectrometer. We can see that the crystalline spectra show narrowlinewidths and thereby good resolution, and also that the chemical shifts are sensitive to
polymorphism, thus allowing us to easily distinguish one crystalline form from another.
Compared to the crystalline spectra, the amorphous spectrum is less resolved due to line
broadening stemming from the distribution of sites, but nevertheless the resolution is good
enough for some site assignment.
77
Although 'H is the most sensitive NMR nuclei, it experiences large dipolar interaction in
the solid state and therefore the resolution suffers. In most circumstances 'H solid-state NMR is
not a suitable method, unless the drug formulation under study exhibits unique dynamic
properties making it "liquid-like"," or heavy deuteration is performed to attenuate the effect of
dipolar interaction, or techniques such as combined rotation and multiple-pulse spectroscopy
(CRAMPS)12-14 or
5
ultra-fast MAS (> 50 kHz) '1
7
are applied to remove the homonuclear
dipolar interaction. Since deuteration or other isotopic labeling is not typically prepared in
pharmaceutical research, the most common solid-state NMR technique for pharmaceutical is the
natural abundance "C CPMAS, as the spectra we showed in Figure 2.S8. Given that the natural
abundance of '3C is only 1.1%, narrow linewidths can be achieved with high-power 'H
decoupling. However, the low natural abundance also means that '3 C is very NMR insensitive.
Although CPMAS provides a sensitivity boost over direct
13 C
13
C detection, it is common to perform
CPMAS of pharmaceutical formulations in large 5 mm rotors with only moderate spinning
frequency (5-7 kHz) and use total sideband suppression (TOSS)' 8 to suppress the spinning
sidebands.
In addition to ' 3 C,
15 N
CPMAS is also commonly performed since '5N chemical shift is
20
sensitive to hydrogen bonding and protonation that lead to shielding or deshielding.19-
Although less sensitive than 13C due to smaller gyromagnetic ratio and lower natural abundance,
the number of nitrogen sites in a molecule is also fewer compared to carbon sites, meaning that
15N
spectra are easier to assign than 13 C spectra. Unlike
13 C
and 15N, molecules containing
fluorine can be studied by 19F NMR that is very sensitive since 19F is 100% natural abundant and
has a high gyromagnetic ratio. As many new API are fluorinated for improved potency,21-22 the
development of fast MAS (> 20 kHz) '1F NMR combined with high power 'H decoupling has
78
had a major impact for new drug design.23 In recent years, pharmaceutical NMR studies
involving quadrupolar nuclei have begun to receive notice as higher field magnets became
commercially available to improve NMR sensitivity. As many API and excipients are designed
as salts to improve solubility, they can be investigated by methods such as 2 3Na and 3 Cl NMR.2 4 26
API that are hydrates can be studied by 2 H and "70 NMR, 27-28 but these methods require
labeling as both nuclei are low natural abundant, and in the case of 170 the isotope is very
expensive.
A popular method to study pharmaceutical polymorphism is NMR relaxometry. Spinlattice relaxation time (T1), in the rotating frame (Tip), and the transverse relaxation time (T2 ) are
all parameters that are structurally and dynamically dependent, meaning they are sensitive to
perturbation in the crystal structures. Measurement of T, and Tip is also important for
standardless quantitative experiments in order to determine the necessary recycling time and CP
contact time for different components of pharmaceutical samples. 29-30 Amorphous materials
generally have shorter T1 than the corresponding crystalline materials due to faster molecular
dynamics caused by the inherent disorder. A good example of NMR relaxometry study is
presented in the work of Lubach et al. where the 'H T, of lactose was compared after undergoing
different
pharmaceutical
manufacturing
processes
such
as
compaction,
cryogrinding,
lyophilization, and spray drying. 3 1 Lubach et al. found that although compacting crystalline
lactose into tablets did not produce differences in the 13C CPMAS spectra, meaning no new
polymorph was generated, the measured 'H Tiwas significantly reduced by a factor of 3.
Cryogrinding, during which particle size of crystalline lactose was reduced by pulverization at 77
K, produced similar effect. The reason was that these manufacturing processes induce small but
increasing amount of crystal defects and amorphous forms, which serve as relaxation sinks and
79
thereby reduce the overall T1. As amorphous materials are typically energetically unfavorable
and recrystallization occurs over time, T, and TIp can also be used in conjunction with
differential scanning calorimetry (DSC) to determine their stability. 32
While 2D solid-state NMR is commonly used for structural analysis of biological solids,
the method is not widely adopted to analyze pharmaceuticals. The reason is because isotopic
labeling is not common in pharmaceutical research due to either synthetic difficulties or costs.
Pharmaceutical process chemistry often produces formulated samples on the order of kilograms,
which can make labeling expensive. The lack of labeling for important nuclei such as 13C or "N
means that the NMR sensitivity is too low for 2D solid-state NMR methods that utilize dipolar
couplings between NMR active nuclei, and consequently the time required for signal averaging
is prohibitively long. However, the advent of high field NMR and fast MAS has allowed 'H
detected 2D solid-state NMR to become feasible. Griffin et al. showed good 2D
'H
resolution
can be obtained by 'H DQ CRAMPS (double quantum combined rotation and multiple-pulse
spectroscopy),' 3 and Zhou and Rienstra showed 13C-1H HETCOR (heteronuclear correlation)
spectra of ibuprofen and acetaminophen formulations under 40-kHz MAS condition.
Aside
from 1H detection based experiments, the CP-HETCOR experiments between various spin pairs
such as IH- 13C and 'H- 19 F have found usefulness in the structural and interaction analysis of
cocrystals, complexes, and amorphous dispersion utilizing the effect of spin diffusion, 34-36 and
the technique has been very recently extended to 'H- 0 as well.37 To address the sensitivity
problem, microcrystalline dynamic nuclear polarization (DNP) works by Rossini et al. and
Takahashi et al. showed promise that DNP may be able to provide the necessary sensitivity boost
38 39
without compromising spectral resolution. -
80
In this chapter, we examine the nano-crystallization of APIs including ibuprofen and
acetaminophen on porous biocompatible excipients such as cellulose membrane and silica
powder by solid-state NMR and DNP. Ibuprofen and acetaminophen are APIs that are poorly
water soluble, and effort to prepare them as more soluble formulations is still an ongoing area of
research. Crystallization of API inside a porous excipient, which is 0.2 to 1 pm for cellulose
membrane and 40 nm for silica powder, constrains the particle size and thereby improve API
solubility. 4 0-42 However, by embedding the API inside the pores, the nano-crystals cannot be
observed by conventional PXRD. This makes solid-state NMR the ideal choice to determine and
quantify polymorph formation inside the excipients.
3.2. Experimental
Crystalline acetaminophen and ibuprofen were acquired from Sigma-Aldrich (St. Louis,
MO). Cellulose membrane was acquired from Whatman, part of GE Healthcare Life Sciences
(Piscataway, NJ). AEROPERL* mesoporous silica powders were manufactured by Evonik
Industries (Hanau-Wolfgang, Germany), and were generously supplied to us by Novartis (Basel,
Switzerland). API-excipient mixtures were prepared by Xiaochuan Yang from Prof. Allan
Myerson's group in MIT Chemical Engineering. Partially deuterated acetaminophen (d3acetaminophen) was synthesized by Joseph Walish from Prof. Timothy Swager's group in MIT
Chemistry. DNP samples were prepared following the protocol published by Rossini et al., 38 as
microcrystalline suspension in 1,1,2,2-tetrachloroethane or 1,1,2,2-tetrabromoethane (SigmaAldrich, St. Louis, MO).
Solid-state NMR experiments were conducted on home-built 500 MHz spectrometers
(courtesy of Dr. Dave Ruben) using either a 3.2 mm or a 4 mm Varian triple resonance ('H- 1C81
'N) probe. For the CPMAS experiments,43 the CP contact time was 2.0 ms at vf of 83 kHz and
the MAS frequency was between 10 to 13.5 kHz. 1H T1 was measured either by the inversionrecovery 44 or the saturation recovery 45 sequence. All experiments utilized the two pulse phase
modulation (TPPM)46 proton decoupling sequence. The recycling time for ibuprofen samples
was 5 s, and for the acetaminophen samples was 120 s. The number of scans was up to 4096
depending on the signal to noise. The spectrometer was referenced by adamantane.
DNP NMR experiments were conducted on a home-built 211
MHz/140 GHz
spectrometer (courtesy of Dr. Dave Ruben). Continuous wave high power (> 8 W) microwaves
were generated by a 140 GHz gyrotron. 47 All experiments used a custom-designed cryogenic
triple resonance ('H- 3 C-15N) MAS probe with a commercial 4 mm spinning module (Revolution
NMR, Fort Collins, CO). As with the solid-state NMR experiment at 500 MHz, CPMAS was
used to acquire all the 13C spectra. The CP contact time was 2.0 ms at vf of 83 kHz and the MAS
frequency was 4.0 kHz. The recycling time between scans was 30 s. The number of scans was
512 for the DNP enhanced spectra and 1,600 for the unenhanced spectra. The polarization agents
used for all experiments were TOTAPOL 8 and bTbK.4 9
Static 2H NMR experiments were conducted on a home-built 400 MHz spectrometer
(courtesy of Dr. Dave Ruben) using a custom-designed single-channel probe (described in detail
in Chapter 5). Spectra were obtained with an 8-step phase cycling5 0-51 quadrupolar echo
sequence52 with a 7r/2 pulse of 2.0 ps and a delay of 30 ps between the two pulses. Static 2H T1
was measured by saturation recovery. For cryogenic temperature experiments, N 2 gas was cooled
by a custom designed heat exchanger53 immersed in liquid N2 with temperature modulated by a
Lakeshore (Westerville, OH) temperature controller before transferring to the probe via a
vacuum jacketed transfer line. The magnet bore was protected from the cryogen by a custom
82
designed vacuum jacketed dewar.54 Sample temperature inside the probe was monitored by a
Neoptix (Quebec, Canada) fiber optic temperature sensor. The number of co-added transients
was between 1,000 and 4,000.
3.3. Results and Discussion
246
7
0
5
7
6,54
10
2
OH1
1
Form I lbuprofen 10
(Sigma Aldrich)
98 7
Cellulose
Cellulose/lbuprofen
250
200
150
100
50
0
"C Chemical Shift (ppm)
Figure 3.1.
13
C CPMAS spectra of form I ibuprofen (top) and cellulose-ibuprofen (bottom) taken
at a 500 MHz spectrometer (11.7 T). The cellulose resonance is located between the ibuprofen
aliphatic and aromatic carbon resonances, and no overlap occurred.
Firstly, we compared the assigned CPMAS spectra of form I crystalline ibuprofen, the
stable polymorph as obtained from Sigma Aldrich, and of cellulose-ibuprofen as shown in Figure
3.1. The form I ibuprofen exhibits sharp resonances with linewidth of approximately 54 Hz, or
0.4 ppm, which is consistent with literature. 55~56 Upon incorporation of ibuprofen into the
83
cellulose membrane (pore size of 0.2 .tm), we found that the resonances of cellulose-ibuprofen
share the same chemical shifts and the same linewidths as the form I ibuprofen. The finding
therefore suggests that ibuprofen exists entirely as form I within the pores of the cellulose
membrane, and no additional polymorph was formed. A comparison of 1H T of form I and
cellulose-ibuprofen only revealed slight differences (Table 3.1), which is further evidence that
the cellulose excipient seemingly does not perturb the structure and the dynamics of ibuprofen.
Table 3.1. Ti (1H) of form I ibuprofen and cellulose-ibuprofen.
Chemical Shift (i, ppm)
185.0
144.1
139.3
47.9
46.1
34.4
27.0
24.0
17.3
Ti (H) (s)
Form I ibuprofen
1.18 ±0.08
1.21 0.05
1.19 ±0.06
1.22 ± 0.02
1.21 ± 0.03
1.17 ±0.03
1.13 ±0.05
1.10 ± 0.06
1.11 ± 0.06
T1 ('H) (s)
Cellulose-Ibuprofen
1.14 0.11
1.15 0.09
1.08 0.10
1.25 0.05
1.27 0.04
1.19 0.06
0.87 0.06
0.81 0.05
0.83 0.06
In contrast with ibuprofen, acetaminophen polymorphism was readily observed within
the cellulose membrane. Compared to the
13C
NMR spectrum of the stable monoclinic form I
acetaminophen, the spectrum for the cellulose-acetaminophen shows resonance peak splitting
that suggests a mixture of polymorphs was formed inside the membrane pores, as shown in
Figure 3.2. The difference in chemical shifts between the polymorphs is not large, but distinct
peaks are clearly resolvable for some resonances as shown in Figure 3.3. The chemical shifts of
these additional peaks are consistent with the data published by Moynihan and O'Hare 57 for the
orthorhombic form II acetaminophen. However, as diffraction method is not possible with the
crystals embedded within the membrane, it is challenging to conclude unambiguously that form
84
II is the additional polymorph within the membrane. The ratio of form I and the possibly form II
acetaminophen within the cellulose membrane is 65:35, calculated by integration of peak areas
assuming Tip is similar for identical sites of different polymorphs.
3
1,-
Form 1
H8'"
42
Acetaminophen
8
7
6
542,3
Cellulose
Acetaminophen
180
140
13C
Figure 3.2.
13C
100
60
20
Chemical Shift (ppm)
CPMAS spectra of form I acetaminophen (top) and cellulose-acetaminophen
(bottom).
85
3
N5
H
8H 64
8
7
6
Form I
Acetaminophen
Cellulose
Acetaminophen
175
165
155
145
13C
135
38
30
22
14
Chemical Shift (ppm)
Figure 3.3. Expanded 13 C CPMAS spectra of acetaminophen focusing on resonances sensitive to
polymorphism to clearly illustrate the onset of form II observed in the cellulose-acetaminophen
formulation.
In addition to the onset of polymorphs, a notable difference between the form I
acetaminophen and the cellulose-acetaminophen is the measured 1H T1. The crystalline form I
acetaminophen has 'H T, that is longer than 100 s, but once inside the membrane acetaminophen
T, reduces to less than 20 s, as summarized in Table 3.2. The finding is evidence that no bulk
microcrystalline acetaminophen was formed on the membrane surface, because the reduction of
H T, can be attributed to spin diffusion through the cellulose membrane, which has 'H T, of
approximately 6 s. Other possible factors such as material disorder and crystal defects can also
serve as relaxation sinks that reduce acetaminophen T, within the membrane.
86
Table 3.2. T, ('H) of form I acetaminophen and cellulose-acetaminophen.
Chemical Shift (8, ppm)
171.8
154.3
135.0
125.3
122.6
118.3
117.7
25.7
T, ('H) (s)
Form I acetaminophen
127.1 ±15.1
122.4± 13.2
128.8 ± 14.9
116.0± 12.6
109.4± 10.6
110.5 ± 13.6
104.5 9.4
126.2 15.9
T, ('H) (s)
Cellulose-Acetaminophen
18.5 ±1.3
19.0± 1.0
17.3 ± 1.7
16.6± 1.2
18.7± 1.9
17.3 ± 1.0
19.9± 1.0
17.8 ± 0.7
DNP experiments were performed on cellulose and cellulose-acetaminophen to gauge the
potential of applying DNP widely to these API-excipient formulations. Since it is important to
preserve the crystal structure of API, and also to obtain the best spectral resolution, we opted for
the crystalline DNP method first pioneered by van der Wel et al.58 59 for nanocrystalline peptides
and Rossini et al.38 for microcrystalline small molecules. In this method, crystals are prepared as
insoluble suspension in solvent containing the polarization agent. Non-Boltzmann electronnuclear cross polarization therefore occurs at the crystal surface, and 'H-'H spin diffusion then
propagates the polarization deeper into the crystal core. Minimizing the crystal size increases the
surface area where electron-nuclear cross polarization can take place, and overall leads to a more
homogeneous distribution of radicals and thereby better DNP performance.
87
TOTAPOL in D2 0/H2 0
s = 42
bTbK in d2 -EtCI 4 /EtCI 4
E
= 10
14
Figure 3.4.
13 C
10
6
2
Frequency (kHz)
-2
-6
CPMAS DNP of cellulose membrane in water and EtCl 4. While the obtained
enhancement was higher using 10 mM TOTAPOL in water, the spectrum was significantly
broadened. Narrower DNP enhanced spectrum was obtained using 10 mM bTbK in EtCl 4 at the
cost of lower enhancement. TOTAPOL performed poorly in EtCl 4 and therefore was not used in
EtCl 4 DNP experiments.
Choosing the appropriate solvent significantly impacts the obtainable DNP enhancement
and spectral resolution. Zagdoun et al.60 found that DNP is optimized in heavily bromonated and
chlorinated organic solvents, which is likely due to the lower IH concentration. Figure 3.4 shows
the DNP of cellulose membrane in water (9:1 D20/H2 0) versus in 1,1,2,2-tetrachloroethane (9:1
d2 -EtCl 4 /EtCl 4 ). While the DNP enhancement was higher in water (, = 42), the spectrum was
significantly broadened in part due to the hydrophilic cellulose forming extensive hydrogen bond
networks. On the contrary, DNP enhancement was lower in EtC4
88
(S =
10), but the observed
linewidth was narrower as well. The choice of paramagnetic polarization agent could contribute
to line broadening too. Recently, it has been proposed that TOTAPOL has a binding affinity to
polymers containing glucose chains such as peptidoglycan and cellulose through hydrogen
bonding, leading to significant paramagnetic broadening and signal bleaching.61 We therefore
chose EtCl 4 as the solvent and bTbK as the polarization agent for subsequent DNP experiments
on the cellulose-acetaminophen.
The DNP experiment on a sample of cellulose-acetaminophen showed an uneven result.
While the cellulose membrane received an enhancement of 10 as was before, the embedded
acetaminophen only received an enhancement of less than 2, as shown in Figure 3.5. The poorer
enhancement on acetaminophen could be explained by the presence of one methyl group within
the molecule, as methyl group dynamics is known to act as a relaxation sink that attenuates DNP
polarization buildup. 38 , 62 The 2 H NMR study on crystalline d3-acetaminophen confirmed that the
acetaminophen methyl group undergoes 3-site hops at the fast limit (k > 107 s )63 from room
temperature to 107 K, as shown in Figure 3.6. Importantly, the 2 H T, of the methyl group
reduced from 8.49 s at 293 K to only 1.13 s at 107 K, near the DNP temperature (90 K). The fast
relaxation introduced by the methyl group presents a challenge for DNP of acetaminophen, but
the problem can be remedied by deuteration as it has been shown in the literature.62 Deuteration
of the methyl group should increase 1H T1 at low temperature, and thereby improves DNP
performance. Plans for DNP experiments of crystalline d3-acetaminophen and cellulose-d 3acetaminophen are underway, and we expect improved enhancement from our current result.
High-field DNP experiments at 16.4 T (700 MHz 'H frequency) 64 are also planned, which would
provide better spectral resolution than at 5 T. In combination with DNP, isotopic labeling using
13 C,
15N,
and possibly
170
are also planned for 2D solid-state NMR experiments.
89
Cellulose with -20% Acetaminophen
9:1 d2 -EtCI 4/EtCI 4 , 10mM bTbK
F
~ 10 (cellulose)
E<
2 (acetaminophen)
MW ON
MW OFF
(Normalized)
14
Figure 3.5.
13 C
10
6
2
Frequency (kHz)
-2
-6
CPMAS DNP of cellulose-acetaminophen. While we clearly observed the
acetaminophen signal in the microwave off spectrum, DNP only effectively enhanced the
cellulose signal. The poor DNP enhancement on acetaminophen can be attributed to its methyl
group, whose fast dynamics acted as a relaxation sink and attenuated non-Boltzmann
polarization.
90
T1 = 8.49 ± 0.12 s
290 K
T1 = 4.20 ± 0.10 s
197 K
T1 = 3.03 ± 0.03 s
167 K
T1 = 1.89 ± 0.01 s
136 K
T1 = 1.13 ±0.02 s
107K
80
40
-40
0
Frequency (kHz)
-80
Figure 3.6. Static 2 H NMR spectra of d3 -acetaminophen at various temperatures. The spectra
show the acetaminophen methyl group undergoes 3-site hop in the fast limit (> 10
7
s-1) at the
temperature range shown. As the temperature lowers, T1 decreases and approaches a minimum.
91
We have started
13 C
CPMAS studies of API-excipient mixtures prepared with
mesoporous silica powders (AEROPERL*), which are granules that are ~30 pm in diameter and
have considerably smaller pore sizes (- 40 nm) compared to that of cellulose membrane (~0.2
pm). The smaller pore sizes in silica further restrict the size of the nanocrystals, and thereby
should further improve aqueous solubility. We have observed onset of additional peaks in the
aliphatic region of silica-ibuprofen, as shown in Figure 3.7, which suggests ibuprofen
polymorphism. By peak integration, we calculated that the ratio of form I ibuprofen and the
previously unknown form observed in silica-ibuprofen to be 79:21.
The same preparation method was used for the anti-fungal griseofulvin to make silicagriseofulvin. The CPMAS
13 C
spectrum of silica-griseofulvin shows that the sample is a
combination of amorphous content and two distinct crystalline polymorphs. However, after
rinsing the silica-griseofulvin with dichloromethane, only one set of polymorph peaks remains
along with the amorphous material, as shown in Figure 3.8. This finding indicates that one
polymorph was formed on the silica surface, while the other was formed inside the silica pores
along with the amorphous content. Complete assignment of peaks is presented in Table 3.3 using
previously published solution NMR spectra.65-66 However, we plan to perform the same
13c
CPMAS experiment on a higher field spectrometer (e.g., 750 MHz), which would provide
improved resolution, especially for the aromatic carbons, compared to the current spectra that
were acquired on the 500 MHz spectrometer.
92
a)
Form I lbuprofen
(Sigma-Aldrich)
Silica/lbuprofen
200
2$0
13C
50
100
1$0
Chemical Shift (ppm)
0
1
4
b)
3
2
2
246
5
10
OH
Form I lbuprofen
(Sigma-Aldrich)
6
3
4
2
5
4'
Silica/Ibuprofen
60
50
20
40
30
C Chemical Shift (ppm)
10
13
Figure 3.7.
13 C
CPMAS spectra of form I ibuprofen and silica-ibuprofen, a) the full spectra and b)
the spectra expanded on the aliphatic region, which clearly shows the onset of additional peaks
for silica-ibuprofen (peak 1' and 4'). Peak integration utilizing peak 1 and 1', and also 4 and 4',
indicates that the ratio of form I ibuprofen and unknown polymorph in silica-ibuprofen to be
79:21.
93
OMe
11
Me 121 13
-
OMe
10
5
8e
14 Cf
2 34
0
11
Silica/Griseofulvin
10-OMe 6-OMe
12-OMe
7,11
Silica/Griseofulvin
(rinsed)
4
200
3 2
9 5 13
6 12 10
40
120
80
Chemical Shift (ppm)
160
0
13 C
Figure 3.8.
13C
CPMAS spectra of silica-griseofulvin before and after rinsing with
dichloromethane. The sharp peaks indicate the presence of crystalline polymorphs and the line
broadening at the base suggests amorphous content. The rinsing removed the surface polymorph,
so only the pore polymorph and the amorphous content remains.
94
Table 3.3.
13 C
chemical shifts of silica-griseofulvin polymorphs
Surface Polymorph
(6, ppm)
197.08
192.86
172.81
171.40
167.04
160.34
106.46
105.15
94.03
61.99
60.97
57.67
41.95
40.00
15.59
Pore Polymorph
(6, ppm)
198.34
194.75
172.81
170.72
168.73
161.65
108.79
106.99
95.29
92.86
58.79
58.30
43.01
40.00
17.77
Assignment
4
8
6
12
14
10
9
5
13
7,11
10-OMe
6-OMe
12-OMe
3
2
1
3.4. Conclusion
Nanocrystallization of poorly soluble API in porous excipient is an effective method to
restrict particle size and produce metastable polymorphs that are more soluble in water. We have
shown here solid-state NMR spectra of several API-excipient formulations and demonstrate that
many of them (cellulose-acetaminophen,
silica-ibuprofen, and silica-griseofulvin) form
crystalline polymorphs or even amorphous content. Successful DNP application on these
samples can potentially improves solid-state NMR signal to noise without compromising spectral
resolution. However, as demonstrated by DNP of cellulose-acetaminophen and 2 H NMR of d3 acetaminophen, the presence of deleterious methyl group dynamics at cryogenic temperatures
can inhibit DNP effectiveness. We plan to conduct selective 2 H labeling on these methyl groups
to neutralize their effect on 1H T1, and consequently should improve DNP polarization of the
whole molecule.
95
3.5. Acknowledgements
We thank Vladimir Michaelis, Ajay Thakkar, and Michael Mullins from MIT-FBML for
many helpful discussions. We thank Xiaochuan Yang, Jennifer Huang, Sydney Hodges, and Prof.
Allan Myerson of MIT Chemical Engineering and Novartis-MIT Center for Continuous
Manufacturing for supplying the API-excipient formulations. We thank Joseph Walish and the
Timothy Swager group for the isotopic labeling experiments. We acknowledge the National
Institute of Health for funding support of DNP projects at the FBML (EB002804 and EB002026).
V.K.M. is grateful to the Natural Sciences and Engineering Research Council of Canada for a
postdoctoral fellowship.
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99
100
Chapter 4: Progress on Temperature-Jump Dynamic
Nuclear Polarization (TJDNP)
This chapter details my contribution to the TJDNP project, pioneered by Chan-Gyu
Joo, Andrew Casey, Christopher J. Turner, Kan-Nian Hu, Jeffrey A. Bryant, and
Robert G. Griffin.
4.1. Introduction - Challenges to Liquid State DNP
DNP in the liquid, solution state was first performed by Carver and Slichter in 1956 in a
demonstrationI that the Overhauser effect 2 could be applied generally beyond the realm of metals.
In the Overhauser effect experiment, a radical's electron paramagnetic relaxation (EPR)
transitions are saturated by microwave, followed by transfer of non-Boltzmann polarization to
the nuclear spins through dipolar cross relaxation. The polarization generated can be described
by Solomon's equation: 3
d (Iz)
_P ((I-
)-40 ((Sz
dt
SO)
(1)
where < 1, > and <Sz> are expectation values for nuclear and electron polarizations, respectively,
with 1o and So denoting the thermal equilibrium polarizations. The total nuclear relaxation rate, p,
and the electron-nuclear cross relaxation rate, u-, can be defined in terms of the transition
probabilities between various energy states shown in Figure 4.1. This gives us the definitions:
p =WO + 2W, +W 2
(2)
U- = W, - W
(3)
101
Ir
P
s)
W,
WS
W2
wo
Wi
(XI PS)
laias)
Figure 4.1. Energy level diagram for an electron-nuclear coupled spin system with the transition
probabilities shown, with I denotes nuclear spins and S denotes electron spins, respectively. The
two W are the NMR transitions, and the two Ws are the EPR transitions. W2 is the double
quantum, and Wo the zero quantum transition.
From these definitions and using the steady-state solution of eq 1, Hausser and Stehlik
derived the Overhauser DNP enhancement that can be obtained under continuous microwave
irradiation of EPR transitions: 4
E _ IZ -O
I0
-
-Sf e
(4)
7n
where ye and y, are the electron and nuclear gyromagnetic ratios. The three parameters
introduced by Hausser and Stehlik are the coupling factor, 4, which describes the efficiency of
cross-relaxation mechanism:
-
p
W -W
W+2W,+W2
(5)
The leakage factor, f takes into account the nuclear spin relaxation caused by the presence of
free electrons. Lastly, the saturation factor, s, describes the efficiency of EPR saturation by
microwave.
102
As NMR development pursued higher external magnetic field in search of better
resolving power, the coupling factor suffered as the condition W >> Wo+ W2 was reached at high
magnetic field. Consequently, the DNP enhancement that could be obtained by the Overhauser
effect approached zero. Moreover, microwave strongly absorbs in certain solvents, most notably
water. This effect introduces severe sample heating that hampers EPR saturation, and can
degrade samples (e.g., biological). Facing these challenges, in situ liquid state DNP by way of
electron-nuclear Overhauser effect was not widely applied in NMR despite its early discovery.
The conventional wisdom that such experiment cannot be done at high field persisted until
recently. Loening et al. showed that for special cases involving the scalar interaction it is still
possible to obtain enhancement of 180 on 1P at room temperature and at magnetic field of 5 T.5
Prisner and coworkers showed that enhancement of -30 can be achieved on water at 9.2 T using
new EPR resonant structures, a gyrotron microwave source, and
15
N-Fremy's salt as the
polarization agent. 6 -7
Aside from in situ methods, ex situ methods based on sample transfers have been
proposed to circumvent the condition imposed by the Overhauser effect. One of the earliest ideas
is the continuous-flow DNP, in which a flowing sample is first polarized at a low field magnet
(0.33 T), followed by transfer to a higher field magnet (4.7 T) via tubing. 89 In this scheme, the
sample is pumped constantly through the two magnets, and can be recycled by closing the tubing
loop. The shuttle DNP experiment allows faster transfer of sample (-300 ms or less) from the
low field to the high field magnet by either mechanically moving the probe' 0 or by pneumatic
equipment.
-
The dissolution DNP experiment, first proposed by Ardenkjaer-Larsen et al.,
included the concept of temperature-jump to further increase enhancement.13 In this experiment,
the sample is cooled to ~1.2 K and polarized at 3.4 T via the solid state DNP mechanisms (solid
103
effect, cross effect, and thermal mixing), followed by rapid dissolution with hot solvent and
transfer of the sample to a higher field magnet for detection. Since Boltzmann polarization
increases at lower temperature, the overall enhancement that could be obtained is
eC
=r
bs
(6)
pwave)
where , is the enhancement obtained through the DNP mechanism,
Tobs
is the temperature at
which NMR detection takes place (- 298 K), and Tgwave is the temperature at which DNP occurs
(~ 1.2 K). The combination of both DNP and low temperature has allowed the dissolution
method to achieve incredibly large signal enhancement factor (> 10,000), especially for small
molecules with long relaxation time, TI.
While the potential of dissolution DNP has been successfully demonstrated, the method
has several drawbacks. The first of which is that at 1.2 K the T, of the targeted nuclei becomes
extremely long as most molecular dynamics slow, and consequently the DNP buildup time for
non-Boltzmann polarization becomes long, usually several hours. Secondly,
since the
temperature-jump is accomplished by injecting hot solvent, the sample cannot be recycled and
used again, therefore
prohibiting
signal averaging and conventional
multidimensional
experiments. And lastly, the transfer time of the solution sample from magnet to magnet is
typically a few seconds, which is similar to T, of many protonated carbons such as methylene or
methyl groups. This leads to a significant polarization loss and an uneven distribution of
achievable enhancements at various sites. The works by Day et al. 14 and Emwas et al.15 showed
that in many molecules studied by dissolution DNP, the heavily substituted carbons (e.g.,
carbonyl, carboxylic acid, etc.), which usually have longer T, due to lack of C-H dipolar
104
interaction, can oftentimes obtain 10 times or more enhancement than the protonated carbons.
The polarization loss is further aggravated by the sample transfer process itself, which passes
through space experiencing only the earth's magnetic field (- 35 pT).16 Recent efforts have
sought to address this problem. Leggett et al. proposed a uniquely isocentered magnet set where
the polarization takes place at an upper magnet at lower field, followed by transfer to the lower
magnet for detection.17 This setup reduces the distance between the two magnets and minimizes
sample exposure to only the earth's magnetic field, thereby retaining more non-Boltzmann
polarization.
The in situ temperature-jump DNP (TJDNP) experiment pioneered by Joo et al.' 8
addresses the aforementioned issues faced by dissolution DNP. By using the high power (> 10
W), high frequency (140 GHz) gyrotron developed by Griffin and coworkers, 19-20 Joo et al.
proposed that both the solid state DNP polarization and the subsequent liquid state detection can
take place at the same field (5 T) in the same magnet. This eliminates the need to transfer sample
which causes polarization loss. As shown in Figure 4.2 (adapted from Ref. 18), the sample is first
cooled to 90 K and polarized by DNP, and after that a IOW 10.6 prn laser quickly melts2-
(~
0.8 s) the sample followed by liquid state NMR detection. After detection the sample is then
frozen again and the process can start over. Joo et al. reported an overall enhancement, , , of 400
on 13C urea. Although the enhancement is smaller compared to dissolution DNP, the difference
can be attributed to the fact that in dissolution DNP the gain from low temperature polarization is
250, but for TJDNP the gain is only 3. However, the TJDNP experiment can be recycled for
detection and signal averaging every 60-90 s, and for dissolution DNP this is all but impossible.
The capability to repolarize and redetect also means TJDNP is compatible with conventional
105
multidimensional experiments. Joo et al. reported an enhanced, well-resolved "C-"C TOCSY
NMR spectrum of a fully deuterated,
Cooling
13C
labeled glucose solution.23
DNP
Melting
90K
Spectrum
300 K
Figure 4.2. Experimental scheme of TJDNP. The sample is cooled to 90 K by cold nitrogen gas,
followed by DNP polarization via cross effect using a 140 GHz gyrotron. The polarized sample
is then melted by a CO 2 laser emitting at 10.6 tm and the solution spectrum is taken soon after.
After detection, the sample is cooled again and the process can restart every 60-90 s. (Figure
adapted from Ref. 18).
While initial experiments have been successful, TJDNP faces challenges in its
experimental design associated with the incorporation of laser heating to the NMR experiment.
Since it is a liquid state NMR experiment using solid state NMR/DNP apparatus, TJDNP uses
solid state NMR rotors to contain the sample and provide low frequency spinning to improve
magnetic field homogeneity. Conventionally, zirconia and sapphire rotors are preferred because
these materials are mechanically robust and provide more reliable, consistent spinning. However,
they are not compatible with high power 10.6 pm laser. 2' Consequently, quartz rotors were
chosen because they could withstand the thermal shock induced by the melting process, but they
are far less reliable than other materials due to their mechanical fragility. Moreover, quartz, like
zirconia and sapphire, absorbs 10.6 ptm infrared. This means much of the laser energy is
106
inefficiently devoted to heating the quartz rotor as opposed to heating the sample itself. Joo et al.
proposed that if a rotor material transparent to 10.6 ptm infrared can be used, it could lead to
faster melting time and less polarization loss. The next section describes in detail efforts to
determine the optimal rotor material and laser wavelength to optimize TJDNP melting time.
4.2. Optimizing Rotor Material and Laser Wavelength
4.2.1. Experimental
All near IR (NIR) measurements were made on a Cary 5000 spectrometer (Agilent
Technologies, Santa Clara, CA) with wavelength ranging from 175 to 3300 nm. Sample cuvettes
were made from IR grade quartz with 2 mm path length. Sapphire, zirconia, and silicon carbide
(SiC) rotor material samples were provide by Insaco, Inc. (Quakertown, PA).
All TJDNP NMR experiments were conducted on a custom-built 212 MHz spectrometer
(courtesy of Dr. David Ruben, Francis Bitter Magnet Lab). Continuous wave high power (> 8 W)
microwaves were generated by a custom-built 140 GHz gyrotron.19 All experiments used a
custom-designed cryogenic three-channel ('H- 1C-2 H) MAS probe with a commercial 2.5 mm
spinning module (Revolution NMR, Fort Collins, CO). The NIR laser used was an ELR 10 W
single mode erbium fiber laser (IPG Photonics, Oxford, MA) with built in Coming SMF-28e+*
(Coming, NY) output fiber optic delivery. For the spectra with TJDNP, the number of
acquisitions was 32. For the off spectra, the number of acquisitions was 1,024.
4.2.2. Results and Discussion
In order to improve the melting process from the indirect melting mechanism to a direct
one, as shown in Figure 4.3, we need to know the optical profiles of conventional solid-state
107
NMR rotor materials (sapphire, zirconia, SiC) and that of typical TJDNP samples mostly consist
of DMSO/water. Ideally, we would like the laser wavelength to have full transmittance through
the rotor material, and only be weakly absorbing in the DMSO/water so the sample can be
uniformly heated. In the most efficient situation, most of the energy would be devoted to heating
the sample, with only small amount of contact heating toward the rotor.
a)
10.6 pm
Quartz Rotor
DMSO/Water
b)
II
Figure 4.3. Conceptual diagram of indirect versus direct melting in the TJDNP experiment. a)
The indirect mechanism, during which the 10.6
sm
laser is fully absorbed in the quartz rotor,
which in turn warms the sample by diffusive heating. b) The ideal, direct mechanism, during
which the laser is fully penetrated through the rotor material and heats the sample, leaving the
rotor relatively cold.
We found that all three rotor materials absorb strongly in the infrared range (A > 2), with
the exception of sapphire which is transparent in the near IR range up to X = 6500 nm as shown
in Figure 4.4. While this finding eliminates zirconia and SiC, two mechanically robust materials,
as suitable rotors for TJDNP, sapphire is preferred for DNP experiments in general due to
somewhat better microwave transmission.
108
I
3.5
.3
C
2.5
2
1.5
0.5
0
2500
4500
6500
1
8500
10500
12500
14500
Wavelength (nm)
Figure 4.4. IR and NIR absorbance profile of zirconia (red), sapphire (blue), and SiC (black)
showing that only sapphire is transparent at the near IR range.
We then obtained NIR absorbance profile of 50/50 vol% DMSO/water (H20 and D2 0)
mixtures, which represents a typical sample for the TJDNP experiment. As shown in Figure 4.5,
the protonated mixture (DMSO/H 20) absorbs weakly at -1450 nm, which arises from a
combination of symmetric and asymmetric stretching mode of the water molecule.25 For the
deuterated mixture (d6-DMSO/D 2 0), the same absorbance peak is redshifted to a lower
wavelength at -2000 nm caused by the kinetic isotope effect. 2 6 The presence of DMSO or d 6DMSO only has a small impact on the water absorbance peak shape at this concentration (50
vol%), so its effect can be safely disregarded.2 7 The NIR spectrometer we used did not permit
temperature dependent measurements at cryogenic temperatures, so we were unable to collect
absorbance profile at 90 K, the temperature at which the DNP polarization takes place and the
laser melting initializes. The effect of temperature is significant in two ways, 1) the phase
transition from liquid water to ice redshifts the absorbance wavelength by -50 nm,5 and 2)
temperature cooling from 270 K to 90 K increases the ice absorption coefficient by a factor of
~10.28 Moreover, it is not clear what is the effect of glassing (from DMSO/water) versus freezing
(from water only) has on the absorbance profile. Considering these factors, temperature
109
dependent and sample specific NIR measurements would clearly be preferred, but nevertheless
the room temperature measurements provided an initial approximation necessary to select a
suitable laser for TJDNP.
3.5
3
2.5
e
0 1.5
0.5
0
1000
1500
2000
2500
Wavelength (nm)
Figure 4.5. NIR absorbance of DMSO/H 2 0 (black) and d6-DMSO/D 2 0 (red). The absorbance
peak observed for the protonated sample at ~1460 nm redshifted to -2000 nm in the
perdeuterated sample.
We were able to borrow a commercial 10 W 1.5 gm erbium fiber laser (courtesy of IPG
Photonics) to perform TJDNP experiment using a sapphire rotor and d6-DMSO/H 2 0 (50/50 vol%)
solvent matrix. Firstly, we evaluated the melting efficiency of the updated setup by measuring
the growth of liquid proton signal as a function of laser irradiation time, as shown in Figure 4.6.
We found that liquid signal could be generated with as little as 0.2 s of laser irradiation. However,
after 3 s of irradiation the growth of liquid signal reached a steady-state and the maximum signal
intensity was only 40% of the intensity obtained at room temperature. This means only a fraction
of the sample was melted by the laser. One possible explanation can be attributed to the uniquely
high thermal conductivity of the sapphire rotor. At 100 K, the thermal conductivity of sapphire is
~ 400 W/mK, 29 -3 0 a value comparable to that of copper. Consequently, laser input energy was
110
efficiently dissipated by the cold rotor, leading to only partial melting. The efficiency of sapphire
energy dissipation was further demonstrated in the sample refreezing experiment, whereas the
loss of liquid signal was measured after some time had passed since laser irradiation as shown in
Figure 4.7. The experiment showed that the sample was completely refrozen after merely 1 s.
Co
.
0
Z
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
T
0
0
I
I
0.5
1
I
I
1.5
2
2.5
3
Laser Irradiation Time (s)
Figure 4.6. The growth of liquid state proton NMR signal upon laser irradiation. Liquid signal
was observed after 0.2 s of irradiation. The growth of signal began to taper after 1 s of irradiation,
ultimately reaching a steady-state after 3 s. Intensity scale is normalized against room
temperature signal intensity.
1
C 0.8
C
0.6
E 0.4
0.2
0
Z
0
i
0
W
1
2
3
4
,on (ms)
Figure 4.7. The refreezing of TJDNP sample after laser irradiation. Sapphire's high thermal
conductivity at cryogenic temperature results in rapid refreezing after 1 s.
11
Lastly, we performed TJDNP of protonated glucose solution in d6-DMSO/H 2 0 (50/50
vol%). We found that with 0.5 s heating we obtained an st of 15, as shown in Figure 4.8.
Comparably, Joo et al. obtained an et of 120 on d 7 -glucose despite a longer melting time at 0.8
S.23
The result highlights the difficulty of performing TJDNP on protonated molecules due to
their inherently shorter T1 , which is also further reduced by paramagnetic relaxation caused by
the presence of TOTAPOL biradical. We might be able to improve DNP enhancement by
decreasing the solvent proton concentration using d6 -DMSO/D 2 0/H2 0 (50/42/8 vol%), and using
a 2.0 pm thulium fiber laser instead of the 1.5 pm erbium fiber laser to accommodate the
redshifted absorbance profile, because according to the PhD dissertation of Rosay, the decrease
of proton concentration would multiply the DNP enhancement by a factor of 2.3' Additionally, it
might be possible to find methods to prolong nuclear T1 of the samples in the solution state. The
next section describes such an effort by incorporating TOTAPOL in temperature sensitive
polymer.
112
Et=15
w/DNP
w/o DNP
11
9
10
8
7
Frequency (kHz)
Figure 4.8. TJDNP
13
C NMR spectrum of 800 mM glucose in DMSO/H 2 0 (50/50 vol%). The
DNP spectrum was taken at 95 K with 32 acquisitions. The spectrum without DNP was taken at
298 K with 1,024 acquisitions. The spectra are plotted on an absolute scale showing the increase
of signal intensity with DNP.
4.3. LCST TOTAPOL Polymer
This section is based on the manuscript prepared by Ta-Chung Ong, Matthew K. Kiesewetter,
and Christopher J. Turner.
While the work of Leggett et al.
7
and the work described in the previous section offer
hardware improvements to the dissolution or the melting process in order to preserve nonBoltzmann nuclear polarization, recent research has shown that it is possible to achieve the same
goal by effectively slow down nuclear relaxation. For dissolution DNP, Midville et al. proposed
injecting the sample with large quantities of sodium ascorbate, which quenches paramagnetic
nitroxide radicals typically used for DNP. 32 Since it is well known that the presence of
113
paramagnetic species reduces nuclear T1,,
quenching them from the sample preserves more
polarization for the liquid state detection. Moreover, organic free radicals are often toxic,
therefore quenching them has the added benefit of making the sample suitable for in vivo
34 5
applications. In addition to simply quenching the radicals, Bodenhausen and co-workers -3
showed recently that non-Boltzmann polarization in liquid can be converted and stored in spin
configurations called long-lived states (LLS) 3 6 -37 that are not NMR observable but can have up to
36 times 38 longer relaxation time constant compares to nuclear T1 . LLS can be sustained by
radio-frequency irradiation, and can be converted back to observable magnetization in small
fractions. However, they require the spins to be inequivalent and scalar-coupled to provide the
necessary delocalization that frees them from the effect of dipolar relaxation.
For Overhauser DNP, Dollmann et al. proposed using a spin-labeled thermoresponsive
hydrogel that leaves the solution when the sample is heated. 39 The hydrogel, spin-labeled with
TEMPO moieties, has a lower critical solution temperature (LCST), Tc, of 63 'C. Below TC, the
hydrogel is in an expanded state and fully soluble in solution. However, as the DNP is underway
and the temperature increases past Tc from microwave irradiation, the hydrogel collapses and
precipitates from the solution, leaving the polarized sample free of paramagnetic relaxation.
Conceptually, a polarizing agent exhibiting LCST behavior might be useful to the TJDNP
experiment as well. Ideally, the radical would be homogeneously distributed while the TJDNP
sample is frozen at cryogenic temperature, and then precipitates from the sample upon laser
melting to room temperature. Recently, ring-opening metathesis polymerization (ROMP) based
poly(norborene)s bearing oligo(ethylene glycol) chains have been shown to exhibit LCST
behavior due to attenuation of hydrogen bonding between the aqueous solvent and the
oligo(ethylene glycol) chains above Tc. 40 Given that poly(norborene)s are compatible with
114
nitroxide radicals,41 we propose a ROMP-based thermoresponsive polymer bearing covalently
attached TOTAPOL radicals. In this section, we evaluate the TOTAPOL polymer's impact on
nuclear T, and present initial DNP results.
4.3.1. Experimental
Material. All labeled chemicals were obtained from Cambridge Isotope Laboratory
(Andover, MA). All materials for norbomenyl monomers and polymer synthesis were purchased
from Aldrich and used as received except for TOTAPOL which was purchased from DyNuPol
Inc. (Newton, MA). Solvents were dried on a solvent purification system and stored in solvent
bombs under inert atmosphere. Standard Schlenk techniques were used for the polymerization
reactions.
Synthesis of 1. To a solution of trans-3,6-endomethylene-1,2,3,6-tetrahydrophthaloyl
chloride (5g, 0.023 mol) in dichloromethane (DCM, 100 mL total), cooled in ice, was added a
DCM solution of the appropriate ethyl capped ethylene glycol (0.050 mol) and triethylamine
(5.07 g, 0.050 mol) dropwise over 30 min. The reaction was allowed to warm to room
temperature overnight under an active pressure of Ar. Reaction mixture was filtered, stripped of
solvent and the resulting material taken into ethyl acetate, filtered of precipitate again. Material
was purified by silica gel chromatography in 100% ethyl acetate mobile phase. Characterization
matched the literature.4 2
115
0
0
CI
Et 3 N
n
0n
0
20DCM
0 CI
n
1 -n
0
OH
0
HN
TOTAPOL
oxalyl chloride
3 drops DMF
DCM
0
0
Et 3N
DCM
CI
0
0
0
0.
2
O
0
0
0
N'/
0
0
HN
/0
0
0
Y
n
0
n
+
metathesis c at.
X
0
DCM
In
0
N
-
N
N
,N,
random co-polymer
3-x-y(n)
Figure 4.9. Synthesis of the thermoresponsive poly(norbomenyl) polymer bearing TOTAPOL
moieties.
Synthesis of 2. A round bottom flask containing 5-norbornene-2-carboxylic acid (mixture
of endo- and exo-) (1.0g, 7.2 mmol), 25 mL dry DCM and a stir bar was equipped with an
addition funnel which was charged with oxalyl chloride (0.94 mL, 8.5 mmol), 3 drops DMF and
25 mL DCM. Under argon, the oxalyl chloride solution was added dropwise over 20 min,
reaction cooled in ice. Reaction was stirred for lh cooled in ice and 30 min at room temperature
after which the reaction was removed of volatiles under vacuum. Conversion to the acid chloride
was confirmed with NMR. The addition funnel was re-attached and charged with TOTAPOL
116
(2.2922 g, 5.7 mmol), pyridine (0.64 mL, 7.9 mmol) and 30 mL DCM. The contents of the
funnel were added dropwise at 0 0C. Reaction mixture allowed to warm to room temperature
overnight and then filtered, removed of volatiles and purified by silica gel chromatography (ethyl
acetate/methanol 95/5) producing an orange oil (1.1431 g, 38% yield). ESI: C29H 4 9 N 30 5+H+
Theory: 520.38 g/mol; found: 520.36. 'H-NMR (500 MHz, 6, CDCl 3 ): 5.91 (in, 2H); 4.51 (in,
1H); 4.35 (m, 2H); 4.24 (m, 1H); 4.07 (m, 2H); 3.79 (m, 2H); 3.16 (s, 2H); 2.98 (m, 2H); 2.89 (s,
3H); 2.21 (m, 1H); 1.90 (in, 4H); 1.39 (bm, 29H).
1C-NMR
(125 MHz, 6, CDCl 3): 188.7; 147.0;
126.2; 71.5; 68.7; 66.8; 62.6; 62.3; 52.0; 48.1; 47.3; 46.8; 46.6; 44.7; 44.2; 41.8; 41.5; 41.2; 40.9;
37.95; 37.7; 31.0; 31.8; 21.88, 20.7.
Polymerization reactions to 3. In a general polymerization reaction, a dichloromethane
(DCM) solution of Grubbs catalyst (as determined by the [M]o/[I]o) is added to a solution of
monomer (0.08 M) in dry DCM (6mL total), reaction progress monitored by TLC. After full
conversion in approximately 90 min, the reaction is quenched with excess ethyl vinyl ether and
stirred for 5 min prior to precipitation of the polymer with hexanes. The polymer is removed of
volatiles under high vacuum. GPC characterization is given in Table 4.1. 1H-NMR (CDCl 3 , 500
MHz, 6): 5.38 (m, 2H); 4.2 (bs, 4H); 3.61 (m, 20H); 3.52 (q, 4H); 3.20 (bs, 2H); 2.95 (s, 1H);
2.01 (bs, 1H); 1.46 (bs, 1H); 1.20 (t, 6H). TOTAPOL content determined via EPR versus known
standards.
117
Table 4.1. GPC characterization of TOTAPOL-containing polynorbornenes polymers
Samplea
Catalyst
M. (GPC, g/mol)
Mw/Mn (GPC)
3-0-100(3)
3-10-90(3)
3-5-45(3)
3-5-45(6)
3-5-20(3)
G3
G3
G3
G2
G2
148,000
144,000
88,000
43,000
49,100
1.06
1.91
1.66
1.58
3.11
a) All samples are labeled as 3-x-y(n). x and y refer to the ratio of TOTAPOL moieties
versus oligo(ethylene glycol) moieties, both were determined from the feed ratio.
Full conversion was observed by TLC for all polymerizations. n refers to the length
of the polyethylene glycol (PEG) chain.
NMR Spectroscopy. 1H and
13
C T, measurements were performed on a custom-built
spectrometer (courtesy of Dr. David Ruben of Francis Bitter Magnet Lab) operating at 591 MHz
for IH using the inversion recovery sequence with Waltz decoupling where appropriate. Samples
consisted of 800 mM uniformly 13 C (U-' 3 C6 ) labeled glucose containing millimolar radicals in
D2 0, with the electron concentration of 3 determined by EPR spin counting. DNP experiments
were performed on a custom-built DNP/NMR spectrometer operating at 5 T (211 MHz for IH,
140 GHz for e~). Microwave radiation was generated with a gyrotron operating at 139.6 GHz.19
The sample was irradiated for 60 seconds at 95 K, followed by 'H-1 3 C cross polarization and
detection with TPPM decoupling. Samples consisted of 2 M
13 C
labeled urea in d6 -
DMSO/D 2 0/H20 with 10-20 mM TOTAPOL equivalent of 3.
EPR Spectroscopy. Low temperature (80 K) continuous-wave and pulsed EPR spectra
were acquired at 9.7 GHz (X-Band) on a Bruker Elexsys E580 spectrometer. For the polymer
expanded state measurement, the sample was first pre-cooled at 270 K to allow the polymer to
118
fully expand and dissolve in solution before flash freezing the sample with liquid nitrogen
followed by insertion to the EPR probe pre-cooled to 80 K. For the contracted state measurement,
the sample was allowed to come to thermal equilibrium at 298 K before the experiment. The
electron T1 was measured by the saturation recovery experiment, and the phase memory time, Tm,
was measured by 2pESEEM. Samples for EPR experiments were dissolved in 50/50 (vol. %)
DMSO/D 2 0 at 2 mM radical concentration.
4.3.2. Results and Discussion
To investigate the impact of polymer LCST behavior on the TOTAPOL moieties, we
measured the electron T1 and TM of the TOTAPOL moieties when the polymer is either in the
expanded or in the contracted state. We observed that electron T1 of the TOTAPOL moieties was
344 ps when the polymer was expanded, and 261 ps when the polymer was contracted. The
effect of LCST behavior was even more pronounced on TM. As observed by 2pESEEM (Figure
4.10), electron TM was 4.98 pts when expanded, and 1.63 pts when contracted, showing a threefold decrease. The T, and TM results are consistent with the fact that when the polymer is
expanded and soluble in solution, the distance between each TOTAPOL moiety is increased.
However, when the polymer contracts and exits the solution, the distance between each
TOTAPOL moiety is decreased, leading to contraction of T1 and TM due to greater electronelectron coupling. The EPR results show that the polymer exhibits LCST behavior as we
intended. The TOTAPOL moieties are homogeneously distributed throughout the sample when
the polymer expands, and they are clustered when the polymer contracts.
119
a)
140000
TM
120000100000*
0
8000060000 40000200000
b)
4.98 ps
2000
4000
6000
8000
(ns)
3Time
300000250000-
TM =1.63 ps
200000Z 150000C
* 100000E50000 -
0
-50000
0
2000
4000
6000
Time (ns)
8000
Figure 4.10. 2pESEEM of TOTAPOL moieties in the LCST polymer showing a) when the
polymer is expanded and b) when the polymer is contracted. The polymer contraction leads to
increased radical clustering, causing the electron TM to decrease due to greater electron-electron
coupling.
Solution NMR T, values for glucose anomeric 'H, carbon C1, and C6 (see Figure 4.11 for
assignments) at 5 'C in the presence of TOTAPOL or 3 are listed in Table 4.2. 5 'C was chosen
because it is below the LCST critical temperature for both polymer samples, which are 20 'C
(for n = 3) and 50 'C (for n = 6). From the result, it is clear that TOTAPOL has a measurable
impact on proton TI, but little effect on 13 C T1. This is in agreement with Solomon's theoretical
work that predicts the effect of paramagnetic relaxation on T1 should be inversely proportional to
the square of gyromagnetic ratio. 3 Remarkably, and in contrast to TOTAPOL, the samples
containing 3-5-45(3) show no decrease of the 'H or 13C T, of glucose despite the polymers being
120
in the expanded, soluble state. Given that EPR experiments had confirmed the polymers are
paramagnetic, this finding suggests that 3-5-45(3) appears to shield the glucose from the free
electrons. We further note that increasing the length of the PEG chains, as in 3-5-45(6), results in
a decreased Ti. These observations suggest that the attenuated effect on T is most likely a
product of steric hindrance caused by hydrophobic and hydrophilic interactions. In other words,
the arrangement of PEG groups in aqueous solution forms a "cage" around the TOTAPOL
moieties in such a way that the hydrophilic PEG groups are outside, while the relatively
hydrophobic TOTAPOL are inside. Such an arrangement means that the glucose molecules,
which are hydrophilic, cannot approach the radicals effectively, thus leading to longer nuclear T,
for the glucose since electron-nuclear dipolar interaction is inversely proportional to the distance
cubed between the two spins.43 It can be theorized that 3-5-45(3) forms a smaller, tighter cage
around the TOTAPOL moieties, and thereby inhibits contact between the radical and the glucose.
Comparatively, the 3-5-45(6) forms a larger, better solvated cage around the TOTAPOL moieties,
allowing glucose to approach closer to the radical and leading to shorter nuclear T1.
OH
6
q
HO
2
HO
OH
OH
6 (a +
1-a
100
90
80
70
60
Frequency (ppm)
Figure 4.11. Solution
13 C
NMR spectrum of 800 mM U- 13 C glucose with assignment showing C1
and C6 of both a and
3rotomers.
121
Table 4.2. 'H and
3
C spin-lattice relaxation time of 800 mM 13C 6 glucose in D 2 0 containing
TOTAPOL and TOTAPOL polymer at various concentrations. n = length of PEG chain
Radical
(none)
TOTAPOL
TOTAPOL
3-5-45(3)
3-5-45(6)
Conc.
(MM)
0
10
20
18.8
18.1
T1 at 5 'C (ms)
'H
C1
C6
(a + p
(a + p)
(a + p)
430
360
180
180
320
170
120
270
160
430
360
180
290
320
180
To further investigate this phenomenon, we examined whether T1 would increase simply
by addition of 3-0-100(3), which contains no TOTAPOL moiety, to a solution of glucose and
radical. The results are listed in Table 4.3. A sample consisting of TOTAPOL (10 mM), U-13C 6
glucose (800 mM) and 3-0-100(3) shows that the mere presence of 3-0-100(3) restores the T of
glucose to longer values compared with the samples containing the radical alone. This could be
due to a partitioning of the TOTAPOL into the solvated polymer because TOTAPOL, while
water soluble, is highly organic soluble.44 Interestingly, when the same experiment was done
with 4-hydroxy-TEMPO (TEMPOL), it was found that the blank polymer only had a minimal
impact on T1 measured. This could be attributed to TEMPOL's better affinity to water compared
to TOTAPOL.
122
Table 4.3. 'H and "C spin-lattice relaxation time of 800 mM glucose in D2 0 containing
TOTAPOL or TEMPO with or without blank PEG polymer. Conc. = radical concentration
T1 at 5 'C (ms)
Radical
TOTAPOL
TOTAPOL + 3-0-100(3)
TEMPOL
TEMPOL + 3-0-100(3)
Conc.
(MM)
20
18
36
36
Hb
C1
C6
(a + p)
120
220
90
100
(a + p)
270
320
250
260
(a + p
160
180
150
160
Finally, we examined the DNP enhancements of the TOTAPOL-pendent polymers, 3, in
DMSO/water glasses at low temperature. The DNP enhancement of a 3-5-45(3) polarized a
sample of 2 M urea in d6 -DMSO/D 2 0/H2 0 (50%/42%/8% by volume) was 21.5. This result is
modest compared with free TOTAPOL, which typically yields enhancement greater than 100 in
similar experiments.
The lower enhancement may be explained by the large number of
additional protons introduced to the matrix by 3. It is known in the literature that DNP
experiments perform optimally with lower concentration of 'H. When we repeated the same
experiment in 50%/50% d6-DMSO/D 20, the enhancement increased to 29, as shown in Figure
4.12. We believe DNP enhancement can be further optimized by deuteration of polymers in
order to lower 'H concentration.
123
DNP Enhanced Spectrum
E = 29
Off Spectrum
16
12
8
4
Frequency (kHz)
Figure 4.12. DNP enhanced 13C NMR spectrum of
13 C-urea
0
in 50% d6 -DMSO and 50% D2 0
containing 25 mM 3-5-45(3) polarizing agent. An enhancement of 29 was obtained compared
with the unenhanced spectrum. Spectra are plotted on an absolute intensity scale.
The microwave irradiation time required to fully saturate polarization is much longer for
3-5-45(3) compared to TOTAPOL, as shown in Figure 4.13. With 3-5-45(3) concentration being
equal (25 mM), the polarization growth time constant for the sample in 50%/42%/8% d6 DMSO/D 20/H20 is 33.3 s, and for the sample in 50%/50% d6-DMSO/D 2 0 the growth time
constant is longer, at 79.6 s. In contrast, the growth time constant for TOTAPOL is 3 s in a
similar solvent matrix and radical concentration. The result is consistent with the finding that
proton T1 is lengthened by the presence of PEG groups. To confirm this, a DNP experiment was
performed using 3-5-45(6) containing 6 PEG groups on each monomer. The growth time
constant reduced to 9 s, much closer to the time constant obtained for TOTAPOL at the same
radical concentration.
124
0.8 -a
cO.6u
00.4
0.2 -
0
50
150
100
Irradiation Time (s)
200
Figure 4.13. 13C polarization built-up curve of urea sample containing 3 (n=3, where n is the
number of PEG groups on each monomer) (*), and 3 (n=6) (m). The dashed line is the built up
curve containing 25 mM TOTAPOL. As explained in the text, the number of PEG groups affects
proton T1 , thereby affects polarization growth time constant. Specifically, smaller number of
PEG groups appears to increase the microwave irradiation time necessary for polarization to
saturate.
4.4. Conclusion
In this chapter, we investigated hardware and radical polarization agent improvements for
the TJDNP experiment to further progress DNP in the liquid state. We found that by optimizing
the NMR rotor material to sapphire and the laser wavelength to NIR, we could obtain a modest
DNP enhancement for protonated small molecule with short T 1. However, we are still unable to
melt the sample uniformly and completely mostly due to sapphire's large thermal conductivity at
cryogenic temperature. Further work addressing this issue will increase TJDNP performance.
We also demonstrated that, by way of polymerization, it is possible to design a radical
polarization agent for DNP without greatly impacting the nuclear T1 of target solutes and still
125
yielding reasonable enhancement. We believe that reducing the 1H concentration by deuterating
the polymer will improve DNP enhancement. Potentially, the new TOTAPOL polymer reported
herein could be an asset to TJDNP and dissolution DNP experiments.
4.5. Acknowledgements
We thank Chan-Gyu Joo, Andrew Casey, Christopher J. Turner, Kan-Nian Hu, and
Jeffrey A. Bryant for many useful and helpful discussions. Darcy Wanger and the Bawendi
group, and also Ziad Ganim and the Tokmakoff group, are gratefully acknowledged for the use
of their IR and NIR spectrometers. We thank IPG Photonics for allowing us to use their
demonstration 1.5 pm erbium fiber laser, and Insaco Inc. for the many discussion on rotor
materials. We thank Matthew Kiesewetter and the Swager group for the synthesis of TOTAPOL
polymer. We acknowledge the National Institute of Health for funding support of DNP projects
at the FBML (EB002804 and EB002026).
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128
Chapter 5: Investigation of Molecular Dynamic
Processes by 2 H NMR
5.1. Introduction to 2 H NMR
Deuterium (2H) is a spin-one (I = 1), low-gamma (y = 6.536 MHz/T), and low natural
abundance (0.0156 %) nucleus. While it is not very NMR sensitive, it is nevertheless an
important nucleus to various 1H and 13C NMR experiments. In liquid state NMR, deuterated
solvents are commonly used to reduce unwanted 'H signal. In solid state NMR, deuteration of
samples can attenuate C-H dipolar coupling that causes line broadening when effective
decoupling experiments are not possible.1 Deuteration is also important in dynamic nuclear
polarization experiments, where excessive 'H concentration reduces the achievable nonBoltzmann polarization.2-4 The advent of labeling chemistry has allowed the synthesis of either
fully or partially deuterated molecules, giving NMR spectrocopists wide options to utilize
2H
to
design experiments.
While less commonly performed than 'H and '3 C NMR experiments,
rich in information as a probe for molecular dynamic processes.
2H
NMR can be
As a spin-one nucleus, 2 H has
a non-zero electric quadrupole moment, adding a quadrupolar interaction term to the internal
spin Hamiltonian. The first-order quadrupolar interaction is given by 8
= co0 (3I
coC(Q)
1(21-1)
129
x
2 - I(I+ 1))
(1)
(3cos2 O -1)
(2)
2
assuming axial symmetry (ij = 0). The constant CQ is the quadrupolar coupling constant and
takes the form
(3)
CQ =e2Q
h
For
2H,
the magnitude of the quadrupolar interaction (140-220 kHz) is larger than other spin
interactions such as the chemical shift anisotropy (~ 10 ppm, or 5 kHz on a 500 MHz
spectrometer) and the dipolar couplings (~10 kHz). Therefore, it dominates the line shape, and
the differences in chemical shift anisotropy and dipolar coupling can be neglected. At the same
time, the 2 H quadrupolar interaction is much smaller than the Larmor frequency. This means the
first-order term alone provides a good approximation and higher order interaction terms can be
ignored. The internal spin Hamiltonian can therefore be simplified as a sum of the Zeeman
interaction and the first-order quadrupolar interaction.
H = H, + H'Q
(4)
HO = wjz
(5)
where HO is the Zeeman Hamiltonian
and o1o is the nuclear Larmor frequency.
Given that deuterium is a spin-i nuclei, there are three nuclear states (-1, 0, 1) separated
by oo. The addition of the quadrupolar interaction perturbs the allowed single quantum
transitions, and therefore the 2 H line shape is a Pake doublet powder pattern consists of two
components, one from each transition. The Pake doublet, shown in Figure 5.1 for axially
symmetric tensors (i=0, where il is the asymmetry parameter), is a characteristic line shape in
130
static solid state NMR that shows the distribution of (1) based on the orientation of the electric
field gradient tensor with respect to the external magnetic field Bo. The inner peaks denote tensor
orientation where Bo is perpendicular to the electric field gradient principal axis, and the weak
shoulders denote tensor orientation where BO is parallel to the principal axis.
A
B0
t
Y
Z
Z
Z
XL
X
3Cj/4
1(
V0
2
0
3C/2
Figure 5.1. Pake doublet pattern for 2 H in solid powder sample. The light blue ovals illustrate the
electric field gradient tensor orientations in the external magnetic field that give rise to each
feature of the pattern.
The width of the Pake pattern is equal to 3C/2, and in solid state deuterium NMR this
can have a wide width of over 200 kHz, corresponding to an FID that decays substantially within
5 p~s, much faster than the spectrometer dead time of NMR experiments. In order to circumvent
this problem, the quadrupolar echo sequence of 90*x- T - 90'y - r - echo (r
131
-
30-60 ps) is
commonly used to acquire deuterium spectra.9 The first pulse generates the FID, and the second
pulse refocuses the magnetization so that the echo appears at t = 2t, as shown in Figure 5.2.
94*)
9%*y
2H
Figure 5.2. The quadrupolar echo sequence. Magnetization is refocused away from the hard
pulses.
As previously mentioned, the magnitude of 2 H quadrupolar interaction is in the range of
140-220 kHz. Coincidentally, this corresponds well to the range of molecular motional rates of
organic and inorganic compounds. Hence, the 2 H NMR powder pattern line shapes are very
sensitive to changes in molecular motion, making 2 H NMR an useful tool to study molecular
dynamics simply by line shape analysis. For many organic compounds, changes in temperature
from 298 K to 50 K take the motional rates from the fast limit (where k ~ 10 7 ~10
intermediate exchange (k
-
8
s-1) to the
104~106 s-') and then to the slow limit (k ~ 102~103 s-1). Each
motional regime yields distinct 2 H powder pattern, and computer programs simulating the line
shape have been developed taking into account tensor orientations, asymmetry parameter, jump
angle, flip angle, site population, and hopping rate. 10-1
A study of methyl three-fold hops at the three motional regimes was conducted by
Beshah et al. using d3-alanine. 12 In the slow exchange limit, each C-D tensor orientation is
magnetically equivalent, and therefore the line shape shows the Pake pattern for axially
132
symmetric tensors, the same pattern as shown in Figure 5.1. In the fast limit, the three C-D
tensors are averaged into a single tensor, producing a reduced Pake pattern that is one-third the
width of the rigid C-D pattern due to motional averaging. In the intermediate exchange regime,
since the motional rates are nearly the size of the quadrupolar interaction, the transverse
relaxation time, T 2, becomes short for many orientations. Therefore, the quadrupolar echo
sequence no longer refocuses magnetization uniformly for all orientations due to loss of
coherence by dephasing, and a reduction of signal intensity is observed. However, for
orientations where the three-fold jump does not affect the transition frequency of the site, T2
remains long and no dephasing occurs in this case. For -CD 3, two such orientations have long
T2's
and therefore the line shape for the intermediate exchange is the sum of each. One
orientation is where B0 is parallel to the C-C axis, making each C-D tensor having a 70.5' angle
with B0 , and this orientation produces a doublet pattern. The other orientation is where B0 is at
the magic angle, 54.7' with the three C-D tensors, assuming a perfect tetrahedral symmetry.
Since the quadrupolar interaction is dependent on the term 3cos 2 0-1 (as shown in eq. 2), the
quadrupolar coupling is therefore averaged by the magic angle (3cos 20-1=0 at 0=54.74)
and
only a singlet is produced. The line shape at the intermediate exchange is therefore a triplet as a
sum of signals from the two orientations.
For two-fold aromatic ring flips 13-14 and also two-fold hop of water' 5 , the tensors are
axially asymmetric in the fast limit (11 # 0). The C-D tensors in an aromatic ring make a 60'
angle with the flip axis, while the O-H tensor in water makes a 53' angle with the flip axis
(experimental value for D-O-D angle is 106
16).
The deuterium line shapes for aromatic ring
flips and water hops at the fast limit are shown in Figure 5.3. An interesting feature to note for
water is that since the tensors are oriented
-
54.74' with respect to the flip axis, the quadrupolar
133
coupling yields a broadened singlet pattern at the fast limit. This is because the splitting is so
small due to the angle being close to the magic angle.
Hop Rate
(s-1)
Aromatic Ring
D20
3.5 x 103
3.5 x 104
3.5 x 105
3.5 x 106
3.5 x 107
I
200
I
Frequency (kHz)
It
-200 200
Frequency (kHz)
-200
Figure 5.3. Deuterium line shapes at various motional rates for D 2 0 two-fold hop and aromatic
ring flip.
Beyond the fast limit (k > 108 s-1), it is difficult to obtain rate information as the line
shape no longer varies dramatically with changes in rate. Therefore, in the fast limit regime,
spin-lattice relaxation anisotropy is used to obtain information on dynamic rate. The spin-lattice
relaxation time constant (TI) of the quadrupolar interaction is related to dynamic rates by' 7
1
3
1=
6 C [J
(wo)+ 4
134
x
J 2 (2wo)]
(6)
where Ji(oo) and J2 (20 o) are the spectral density functions of single and double quantum spin
flips, respectively. Spectral density functions are dependent on the orientation between tensors
and the external magnetic field, Bo. At the fast limit where k >> o (2 H Larmor frequency, ~107
Hz),
JI(weo )+4J
1- 3sin20(1-cos2()
0
2 (2COO)=3
8k L 2
(7)
where 0 and p are polar angles defining the orientation of BO in the molecular frame.' 8
5.2. Transmission Line 2 H Probe
At its very essence, the NMR probe can be simplified to the inductor-capacitor (LC)
circuit diagram shown in Figure 5.4. The sample coil allows transfer of radiofrequency (RF)
power to excite the NMR sample, and also serves as an antenna to detect the signal. The variable
tuning capacitor (CT) changes the resonant frequency of the circuit to match the Larmor
frequency of the nucleus to maximize detection. The resonant frequency (oo) can be calculated
by the equation
Co=
1
(8)
LCT
where L is the inductance of the sample coil. Lastly, the variable matching capacitor (Cm) adjusts
the impedance of the probe circuit to match the impedance of the external electronics, most
notably the RF amplifier (50 Q for NMR). Doing so maximizes the transfer of RF power from
the amplifier to the probe, therefore improving overall efficiency.
135
CM
CT
Sample Coil
Figure 5.4. A simple NMR circuit diagram showing the matching capacitor (Cm), the tuning
capacitor (CT), and the sample coil for a single resonance probe.
Conventionally, the tuning and the matching capacitors are placed closely to the sample
coil to minimize loss. This is called a "locally tuned" circuit, however, for the variable
temperature
2H
NMR experiment, this configuration may not always be ideal. Capacitance is
sensitive to changes in temperature, and commercially available variable capacitors are not
suitable for low temperature (90-100 K) experiments. In other words, it would be valuable to
place the tuning and the matching capacitors away from the sample coil to provide stable tuning
and matching while the sample temperature is varied. One solution is to separate the coil and the
capacitors with a transmission line,' 9 in this case a set of coaxial copper conductors, so that only
the sample coil is placed inside the temperature controlled dewar. The transmission line probe
circuit diagram is shown in Figure 5.5.
136
Transmission Line
CM
Sample Coil
CT
Figure 5.5. Basic transmission line circuit for a single resonance NMR probe. The capacitors are
now separated from the sample coil by a length of transmission line.
A new single-channel 2 H transmission line probe was designed and built using the circuit
diagram shown in Figure 5.5. This probe was used to conduct all the variable temperature 2H
NMR experiments described in this chapter. The capacitors and the coil are separated by 60 cm
of copper transmission line, which has outer diameter of 25 mm and inner diameter of 6 mm.
The coil is enclosed in a copper chamber that protects the sample from the surrounding and also
ensures uniform temperature distribution across the sample. The probe is suitable for both low
and elevated temperature experiments, functional from 90 K to 423 K. The efficiency of a probe
is conventionally described by the quality factor of a resonator, defined as
Q=
(9)
where oo is the resonance frequency and Ao is the half power (-3 dB) bandwidth. An efficient
probe with high
Q
has a narrow Ao such that power is not dissipated over a wide range of
frequency not useful to the experiment, therefore improving the probe nutation frequency (yB 1 )
and the sensitivity. However, it is worth noting that for a 2 H NMR probe
Q
cannot be too high
because 2H spectra are broad up to 250 kHz. If Ao is too narrow, the entire 2H spectrum may not
137
be excited uniformly. Measured with a crystalline sodium acetate sample, we found that the
Q
for the new 2H probe is 196 and the 7 Bi is 167 kHz using a 3.2 mm coil. The current design
allows the coil to be exchanged easily depending on the experiment. A 4 mm coil will have
better sensitivity due to the larger sample volume, while a 2.5 mm or a 3.2 mm coil will have
better RF efficiency. Photographs of the probe are shown in Figure 5.6.
138
a)
b)
sml
o
C)
N2Inlet
CM
CT
E xhaust
Temp. Sensor
Figure 5.6. The transmission line single channel 2 H probe for variable temperature experiments.
a) overview of the probe, b) inside the copper sample chamber showing a 3.2 mm silver plated
sample coil, and c) inside the probe box showing the gas transfer lines and the tuning elements.
139
5.3. Lipid Phase Transition in d54-DMPC/VDAC 2D Crystals
This section is adapted from Eddy, M. T.; Ong, T. C.; Clark, L.; Teijido, 0.; van der Wel, P. C. A.;
Garces, R.; Wagner, G.; Rostovtseva, T. K.; Griffin, R. G., J. Am. Chem. Soc., 2012, 134 (14),
6375-6387, but specifically focusing on the lipid dynamics and transitions of d54-DMPC as
observed by 2H NMR. For a more detailed description of VDAC in 2D crystals, please see the
thesis of Matthew T. Eddy.
Integral membrane proteins are proteins permanently associated with cell membranes
consist of phospholipid bilayers. One of their most important functions is to govern cellular
interaction with the external environment. 20 The membrane receptor proteins serve as a
communicator between the cell and its external environment, and the transport proteins allow
molecules and ions to enter or exit the cell. Because of their functions, understanding membrane
protein is important in the field of drug design, which sought to elegantly neutralize unwanted
cells (such as viruses) by targeting and deactivating key membrane proteins.
However, most membrane proteins are tightly embedded in lipid bilayers that do not
crystallize readily, and the way to extract them is typically by using detergents or denaturing
agents. Consequently, membrane proteins are notoriously difficult to study by conventional
means such as X-ray crystallography, and because the native lipid bilayers are removed, the data
acquired are seldom representative of the proteins' native states. Solid state NMR, which does
not require the proteins to be removed from the lipids, is therefore a powerful technique uniquely
suited to study structural and functional questions of membrane proteins in native, or similar but
non-native, lipid environments.
140
It is well known that the interaction between integral membrane proteins and their lipid
environment impacts the topology and function of membrane proteins and influences the
properties of the lipid bilayers.2 1 23 Lipid phase, composition, and membrane thickness are
known to affect the structure and activity of membrane proteins. A key example has been
observed in cells where most bilayers are reported to be in a liquid-crystalline (La) phase in
which membrane proteins are known to be functional;24 changes from La to the gel phase (Lp)
have been correlated with a loss of activity. 25-27 At the same time, the presence of membrane
proteins in the bilayer affects the properties of the surrounding lipids, which can be directly
observed through changes in phase transition temperatures and enthalpies. The effects of proteinlipid and protein-protein
interactions can be particularly prominent when the protein
concentration is high, such as in the case of ordered unilamellar or multilamellar sheet-like
arrays, known as 2-dimensional (2D) crystals.
The 2D crystals are of great interest within the structural biology community as the
inherent order facilitates
studies with electron microscopy (EM) 2 8 and atomic
force
microscopy. 2 9 Another approach to examine 2D crystals of membrane proteins is magic angle
spinning NMR (MAS NMR). In particular, the microscopic order in 2D crystals results in high
resolution NMR spectra that yield atomic level details of membrane protein structure and
mechanistic information as demonstrated by several investigations. 30-4 1 Thus, 2D crystal
preparations are a promising alternative to 3D crystallization since: (1) membrane proteins can
be reconstituted into a more native-like lipid bilayer, 2 9 (2) membrane proteins in 2D crystals can
retain full functionality, 4 2 and (3) 2D crystals are possibly easier to obtain. Furthermore, unlike
3D crystals, which contain fewer lipid molecules per protein, membrane proteins in 2D crystals
are surrounded by a continuous lipid bilayer. 43
141
Despite the extensive use of 2D crystals in structural studies, little is known about the
lipid dynamics and phase behavior of the lipid bilayers in these systems. Specifically, what is the
nature of the lipid environment in 2D crystals and how does it compares to lipid environments
established for well-studied pure lipids4 4-4 6 or with lower protein concentration? 4 7 Lipid-protein
interactions in a 2D crystal were previously studied by EM for the water channel AQP0
43 ' 48-49
where Gonen et al. found that most annular lipids are tightly packed between adjacent tetramers
of AQPO, mediating lattice interactions. 48 This suggested that channel mobility and
conformational flexibility within the bilayer were very restricted. However, while EM can
provide a picture of lipid arrangement and adaptation to the membrane protein, it does not
directly provide information about the dynamics of the lipid bilayers, including changes in
transition temperatures and lipid order parameters.
In conjunction with differential scanning calorimetry (DSC), solid state
2H
NMR is well
suited to study lipid dynamics and phase transition. The technique has been used extensively to
study lipid dynamics and changes in lipid phase behavior due to heterogenous lipid composition,
lipid-cholesterol interaction,50-4 and hydrophobic mismatch.ss-s Despite its extensive use,
2H
NMR has not been utilized to examine changes in lipid dynamics for 2D crystals and, in
particular, it has not been used to study the effect of p-barrel insertion on lipid dynamics. By
using 2H NMR, we can probe the influence of membrane proteins on surrounding lipids.
Here we examine the changes in lipid phase transition in the 2D crystals of chain
deuterated 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(d54 -DMPC) and voltage-dependent
anion channel isoform 1 (VDAC 1). DMPC is a common saturated lipid with 14 carbons on each
acyl chain, as shown in Figure 5.7. VDAC1 is a 32 kDa integral membrane protein that controls
transport of metabolites between the outer mitochondrial space and the cytosol.57- 60 It is a typical
142
P-barrel ion channel with structures known from detergent based solution NMR and
58
crystallographic studies.61 63 Its function has been extensively studied, '60, 64-66 and 2D crystals
of VDAC1 have been previously characterized by EM.67-70 Analogous to various other
membrane proteins, several studies suggest a significant impact of membrane lipid composition
VDAC gating may be
and protein lipid-interactions on VDAC activity. For example, (1)
regulated by characteristic mitochondrial lipids, 71 (2) VDAC channels isolated from the seeds of
72
Phaseoluscoccineus are sensitive to cholesterol and phytosterols, (3) VDAC has been reported
73
to associate with detergent resistant microdomains isolated from mitochondria, and (4)
interaction of VDAC with proteins such as Bcl-xL are suggested to depend on membrane
composition. 74 Considering these observations, examining the lipid environment surrounding
VDAC is important to understanding the structure and function of the protein.
D2
D2
D
D2
D2
D2
D2
0
D2
D2
D2
D2
D2
02
02
D2
02
02
D2
02
D2
D2
D2
D2
D2
D2
0
/
H
sg
0
Figure 5.7. Acyl chain deuterated DMPC (d54-DMPC)
5.3.1. Experimental
Materials. 1,2-dimyristoyl(d 5 4)-sn-glycero-3-phosphocholine (d54-DMPC) was obtained
from Avanti Polar Lipids (Alabaster, AL). The d54-DMPC/VDAC1 2D crystals were synthesized
by Matthew T. Eddy (MIT-FBML, Cambridge MA) and Lindsay Clark (MIT-FBML, Cambridge,
MA).75 Deuterium depleted H20 was obtained from Cambridge Isotope Laboratory (Andover,
MA). All other reagents were obtained from Fisher.
143
Differentialscanning calorimetry. DSC measurements were performed using a MicroCal
VP-DSC (Piscataway, NJ). Pure d54 -DMPC and VDAC1/d
54-DMPC
2D crystals were each
mixed with excess 25mM phosphate buffer at pH 7.0 at room temperature. All buffers and
samples were degassed for 10 minutes under vacuum prior to the experiments. The scan rate was
1 'C/min from 1-40 'C with 30 minutes between each scan to allow temperature re-equilibration.
The buffer itself was scanned before the samples to obtain a reproducible baseline. Each
experiment was allowed to run overnight to ensure reproducibility. Data analysis was performed
using the Origin DSC software included with the calorimeter.
NMR Spectroscopy. Solid state
2H
NMR experiments were performed on a custom-built
spectrometer (Courtesy of Dr. D. Ruben) operating at 60.8 MHz for 2 H using a single-channel
probe with 4.0 mm coil. Spectra were obtained with a quadrupolar echo sequence with a /2
pulse of 2.5 ps and a delay of 30 gs between the two pulses. Oriented 2 H NMR spectra (0 = 0')
were calculated by de-Pake-ing method described by Sternin et al. 76
5.3.2. Results
Differential scanning calorimetry. Figure 5.8 shows the endotherm of both pure d54 DMPC and VDAC1/d
54 -DMPC
2D crystals (VDAC:d 54-DMPC wt ratio of 2:1, molar ratio
~1:25). Pure d54-DMPC exhibits a sharp transition at 19 'C (rippled gel phase Pp' to lamellar
liquid crystal phase La), as expected, that has large transition enthalpy and is highly cooperative,
with a pretransition at 8 'C (Lamellar gel phase Lp' to rippled gel phase Pp') that is broader with
smaller transition enthalpy.
The effect of VDAC 1 on the lipid phase transition is immediately apparent in the DSC of
the 2D crystals. VDAC1/d 54 -DMPC 2D crystals show a much broader phase transition spanning
144
20 degrees with a maximum at the transition temperature, TM, near 27 'C. The magnitude of the
maximum observed transition enthalpy, AH, of VDAC1/d 54 -DMPC is only 6% of pure d54 DMPC, indicating the transition is almost completely eliminated or severely broadened. These
observations are reminiscent of lipid samples containing a large amount of protein or cholesterol
showing a large disruption in lipid acyl chain packing.54 ' 77 The reduction of transition enthalpy
and broadening of transition temperatures show the transition is less cooperative in the 2D
crystal. The transition profile is asymmetric and can be decomposed into two narrow components
and a broader component as shown in Figure 5.8. The broad component is centered at 16 *C with
a half-width of 8 degrees. The two narrower transitions are centered at 24 and 28 *C, with half
width of 2.5 and 4.5 degrees, respectively.
250
2000
200
, 1501
E
16001
~100.
1200
50.
E
04
0
d54-DMPC
800
10
20
30
40
Temperature (*C)
0
400
-
-
VDAC1/d54-DMPC
oo
o
0
10
20
30
Temperature (*C)
40
Figure 5.8. DSC thermograms of pure d54 -DMPC and VDACl/d
54-DMPC
2D crystal. The
largest peak at 19 *C for pure DMPC is cutoff in this figure so that the 2D crystals can be
visualized on the same scale. Inset: Expansion of the DSC thermogram of VDACl/d
54 -DMPC
2D crystals and simulations using two narrow and one broad component. Experimental data are
graphed by the solid red line, and the simulated values are shown by the black dashed lines.
145
2H NMR
Spectroscopy. The
2H
NMR spectra of pure d54-DMPC (mixed with 2 H depleted
buffer at a ratio of 1:1 (w/w)) and that of VDAC 1/d
54-DMPC
2D crystals (protein-to-lipid molar
ratio of ~1:25) are presented in Figure 5.9. Lipid liquid crystal and gel phases have distinct
2H
NMR lineshapes, therefore the phase boundary is apparent by examining 2 H NMR spectra as a
function of temperature. In the liquid crystal phase, perpendicular edge quadrupolar splittings,
A v., of various 2H's in the hydrocarbon chain are between 5 to 30 kHz depending on their
location in the lipid bilayer and the degree of acyl chain flexibility. The terminal methyl group of
the acyl chain, located in the middle of the bilayer, has the smallest A v., that is also the easiest
to quantify. The width of the plateau, A vPIat, is determined by splittings of the least mobile
methylenes that are near the middle of the acyl chain and closer to the phosphate head group.
Due to extensive overlap, the splittings of individual methylene groups are not easily determined.
The line shapes of d54 -DMPC/VDAC 2D crystals share some features with the pure gel phase
lipid 78 where the gauche-trans isomerization and axial diffusion slows and the spectrum broadens.
146
VDAC/d -DMPC
d 5-DMPC
34 0C
34 *C
29 *C
29 "C
24 0C
27 -C
21 0C
25 C
20 *C
23 ;C
19 *C
21 -C
18 *C
18 "C
16 *C
16 C
14 0C
14 : C
60
20
-20
60
-60
20
-20
-60
Frequency (kHz)
Figure 5.9. Static 2 H NMR spectra of d54 -DMPC (left) and VDAC1/d 54-DMPC 2D crystals (right)
as a function of temperature. For pure DMPC, a sharp transition between the liquid crystalline to
gel phase is observed between 18-19 'C. For the 2D crystals, the transition is more gradual over a
larger temperature range. All spectra are plotted on a normalized intensity scale.
As shown in Figure 5.9, d54-DMPC undergoes a sharp phase transition at 19 'C, with
little evidence of coexistence between liquid crystal and gel phase, as expected. However, for
VDAC 1 /d54 -DMPC 2D crystals, the transition is more gradual. Compared to the spectra of pure
lipid, the spectra of 2D crystals appear to be a superposition of liquid crystal and gel phase
spectra, suggesting coexistence of both liquid and gel phase over a range of at least 10 *C. The
sharp peak in the middle of the spectra is most likely due to residual deuterated water. The phase
transition can be visualized by examining A vPat as a function of temperature. For pure d5 4 -
147
DMPC, the phase transition is apparent by a break in linearity of A
Pat
versus temperature
below 19 'C, the phase transition temperature, as shown in Figure 5.10. For the 2D crystals,
A v"' is larger at temperatures above and smaller below 20 'C compared to the pure lipid (see
also Figure 5.11). Although there is no obvious break in linearity, the fact that the 2 H NMR
lineshape at 39 'C resembles that of liquid crystal phase, but at 14 'C that of gel phase, indicates
that a phase transition must exist, albeit very gradual and hard to quantify. Interestingly, the
terminal methyl group appears to show a decreased splitting in the 2D crystals (Figure 5.11)
suggesting that the rigidification of the acyl chains is not necessarily homogeneous across the
lipid bilayer.
50
45
N4035302520
I
10
15
20
T
25
30
35
Temperature (*C)
40
45
Figure 5.10. Perpendicular quadrupolar splitting, AvQI, as a function of temperature. (+) d 54
DMPC, (m) VDAC1/d
54-DMPC
2D crystal. AvQI were difficult to determine with precision at
lower temperatures due to poorer signal-to-noise ratio caused by spectral broadening in the gel
phase. AvQI were reproducible to within 5% (d54-DMPC) and 10% (VDAC1/d 5 4-DMPC) for
these spectra.
148
AvPlat
29*OC
Pure
DMPC
DMPC-VDAC
2D Crystals
60
-20
20
Frequency (kHz)
-60
Figure 5.11. Expansion of 2 H NMR spectra of d54 -DMPC (top) and VDAC1/d 54 -DMPC 2D
crystals (bottom) at 29 'C. AvQa' is shown as the difference between the two outermost dashed
lines and A vQ, for the terminal methyl group of the acyl chain is shown as the two innermost
dashed lines. For the 2D crystals, A
I
Pa'
is larger at temperatures above and smaller below 20
'C compared to only DMPC while the terminal methyl group appears to show a decreased
splitting for the 2D crystals compared to pure DMPC.
149
Thus, consistent with the DSC experiments, the 2 H NMR experiments show a significant
effect of the protein on the lipid phase behavior, causing a broadening of transition temperatures.
This suggests that the transition is less cooperative in the 2D crystal. In addition, both NMR and
DSC indicate the co-existence of different domains of lipids in distinct phases, with at least part
of the lipids eventually transitioning to a liquid crystalline state that is reminiscent of the bulk
phase of fluid DMPC bilayers.
In addition to the temperature dependent data presented above, we also acquired 2 H NMR
temperature-dependent data using a higher protein-to-lipid molar ratio of 1:50, which is the same
amount used in a previous MAS NMR study. 79 This protein-to-lipid ratio is outside the range
reported by Dolder et al. for forming 2D crystals 67 and under these conditions samples were
likely liposomes. As shown in Figure 5.12, even with higher lipid content we see significant
effects of the protein on the lipids. There appears to be no indication of significant amounts of
bulk lipids, nor does there appear to be separate phases (2D VDAC1 crystals surrounded by
macroscopically separated lipids) since we see no indications of a second component with the
same phase behavior as bulk DMPC. This is consistent with the idea that this protein-to-lipid
ratio forms homogenous samples where VDAC is distributed evenly in the membrane and not
forming 2D crystals.
150
VDAC/d 54-DMPC
VDAC/d 54 -DMPC
2/1 wt ratio
1/1 wt ratio
39 0C
39 0C
34 0C
34 *C
29 *C
29 C
27 *C
27 0C
25 *C
25 0C
23 *C
23 0C
21 0C
21 0C
18 "C
18 C
16 "C
16 0C
14 0C
14 0C
60
20
-20
60
-60
20
-20
-60
Frequency (kHz)
Figure 5.12. Static
2H
NMR spectra of VDAC1/d 54-DMPC ~1:25 protein-to-lipid ratio (left) and
~1:50 (right) as a function of temperature. The protein-to-lipid ratio of 1:25 corresponds with 2D
crystals while 1:50 likely corresponds with the formation of liposomes.
5.3.3. Discussion
DSC thermogram of VDAC 2D crystals and estimation of amounts of bulk and annular
lipids. DSC and 2 H NMR measurements show a broadened, multicomponent phase transition
with a maximum at 27' C for lipids in the VDAC1/DMPC 2D crystals. This observation suggests
the existence of bulk and bound lipids in the 2D crystals that contribute to the phase transition.
However, considerations of the area of the VDAC surface and protein-to-lipid ratio do not
appear consistent with this simple interpretation. The formation of 2D crystals occurs over a
specific molar ratio of protein to lipids and permits us to measure of the minimum number of
151
lipids required to prevent protein aggregation and properly "solvate" the protein molecules.
These solvating lipid molecules could occupy an annulus, and we can estimate the number of
annular versus bulk lipids by considering the size of the pore as determined from diffraction and
NMR structures for VDAC1. These dimensions permit us to estimate the number of lipid
molecules required to cover the surface area.
We first consider the case where the 2D crystals could be composed entirely of VDAC 1
monomers. The pore of VDAC1 is elliptical with dimensions of 27 A and 24 A for the longer
and shorter axes.6 2 With an effective lipid diameter of 8.7 A,80 approximately 24 lipid molecules
are required to form the first shell of the bilayer around each channel. This is illustrated in Figure
5.13, which shows a projection of VDAC 1 surrounded by a single shell of 12 DMPC molecules
per monolayer. The optimal molar ratio used to form homogenous samples of 2D crystals was
approximately 1:25 (protein:lipid) as determined by optimization of the quality of NMR spectra.
For the case of VDAC 1 monomers, this ratio would be approximately the minimal amount
required for each protein molecule to have one annulus, and there would be no bulk lipid present
in the 2D crystals. The lack of bulk lipid contrasts the conventional wisdom that at least some
bulk lipids contribute to the phase transition, and contrasts the observation of regular occurring
depressions observed on the surface of the 2D crystals which are attributed to areas of lipid
density.67 Since samples with DMPC were prepared using the same dialysis conditions as Dolder
et al. it is more likely that VDAC 1 forms dimers in our 2D crystal samples as was previously
reported.67 For the case of dimers, if two protein molecules share annular lipids along one face,
the number of lipids required to occupy a shell around both protein molecules is 20 to 21; if
protein-protein interactions occlude the presence of lipids entirely around both molecules, the
number of required lipids reduces to 18. This case is illustrated in Figure 5.13. For the case of
152
dimers, approximately 20% to 28% of lipid molecules could be available as bulk. It is interesting
to note that Dolder et al. reported the formation of 2D crystals between relative weight ratios of
2:1 (protein:lipid) and 5:1 (protein:lipid), corresponding to a molar range between 1:25 and
1:12.5 (protein:lipid).67 We observed the formation of 2D crystals over this range as well, but a
protein-to-lipid molar ratio of less than 1:25 produced samples that appeared to be heterogeneous
mixtures of 2D crystals and amorphous precipitates. Consequently, the quality of resulting NMR
spectra was compromised, especially for the case where a protein-to-lipid molar ratio of 1:12.5
was used. Given our estimation of the number of annular and bulk lipids present for VDAC
dimers, this suggests that at protein:lipid ratios lower than 1:20 to 1:18 there are insufficient
number of lipids for the formation of a complete annulus, leading to possible VDAC 1
aggregation in order to minimize exposed hydrophobic regions for some fraction of the
protein:lipid mixture. It also suggests that some bulk lipids are required to form homogenous
populations of purely 2D crystals. However, a detailed molecular interpretation of the DSC curve
and its connection to the 2 H spectral lineshapes requires additional experiments.
A second interesting feature is the increase in TM from 19 to 27 'C. This is also a
different result from consideration of two-component systems. The increase in TM was predicted
by Marcelja in the context of lipid-mediated protein-protein interactions present in bilayers with
high membrane protein concentration. 81 According to Marceja's model, attractive forces
between protein molecules promote clustering, which decreases the total free energy of the
membrane by decreasing the total amount of boundary lipid. Furthermore, stronger protein-lipid
interactions shift the bulk phase transition temperature higher than for pure lipid bilayers. These
general theoretical results agree with our experimental data. The idea of strong protein-lipid
153
interactions was also supported in the observation of very small variations in lattice parameters
for VDAC 2D crystals. 6 7
8.7A
24A
VDAC
27A
VDAC
Monomer
Dimer
Figure 5.13. Schematic illustrations of a projection of the VDAC1 monomer and dimer and the
surrounding ~12 and ~18 DMPC molecules in the one half of the bilayer. The VDAC1 pore
forms an ellipse with approximate dimensions of 24 and 27 Angstroms, while the diameter of a
DMPC molecule was reported to be 8.7 Angstroms. Left: 12 DMPC molecules in the two halves
of the bilayer are required to form a shell around a monomer of the protein. Right: VDAC1
dimers would require 18-20 DMPC molecules to form a shell if some lipids are shared between
molecules or overlapping protein-protein interactions preclude the need for a complete annulus
between molecules.
154
2H NMR
spectra and acyl chainpacking in the lipid hydrophobic core. The DSC and
2H
NMR spectra observed are similar to previous studies of lipid reconstituted with large amount of
cholesterol50 or peptides77 relative to phospholipid, consistent with the fact that formation of the
2D crystal can be induced by decreasing lipid content, 69 thereby increasing the protein to lipid
ratio. In this lattice, the liquid crystal phase of the bilayer becomes more ordered and takes on gel
phase characteristics with slower dynamics, while the gel phase becomes disordered and more
fluid, leading to a smoother, gradual phase transition as observed by the width of A vPa' in
Figure 5.10, determined by the section of acyl chain closer to the lipid headgroup. This finding is
actually in contrast with previous studies of lipid-protein systems, which showed disordering of
the gel phase, but not ordering of the liquid crystal phase above Tc. 82 -84 At first glance, the 2D
crystals studied here appeared to be more cholesterol-lipid like, rather than protein-lipid.
Nevertheless, this ordering effect is predicted for a system with strong protein-lipid interaction
according to the theoretical model by Mar'elja. 8 1 Therefore, this observation might be a unique
feature of 2D crystals compared to other model and biological membranes.
In contrast with the ordering effect observed at the top of the acyl chain, the behavior
observed at the hydrophobic core of the bilayer, formed by the lower part of the acyl chain, is not
quite the same. Although the dynamics is slowed, as shown by line broadening, the terminal
methyl group of the lipid is more disordered as indicated by the decrease of quadrupolar splitting,
as seen in the 2 H spectra above 20 *C and also the de-Paked spectra (see Figures 5.11 and 5.14).
This suggests that the presence of VDAC1 perturbs the hydrophobic core of the bilayer and
disrupts acyl chain packing. Similar disordering at the terminal methyl group was previously
observed in the study of DMPC with cytochrome oxidase, showing that boundary lipid is
disordered.
83
This behavior would also explain the large decrease in transition enthalpy and
155
increase in transition temperature range measured by DSC, as the creation of a protein perturbed
region in the bilayer would lead to loss of cooperativity as lipid-lipid contacts are disrupted.
a)
b)
I
C)
I
40
I
I
20
0
-20
Frequency (kHz)
Figure 5.14. De-Paked 2 H NMR spectra of a) d54-DMPC b) VDAC1/d
-40
54 -DMPC
2D crystals at
27 'C, and c) at 34"C. De-Pake-ing is a commonly applied mathematical transform that allows
one to enhance resolution in spectra of overlapped powder patterns. 76' 85 The de-Paked lines of
VDACl/d
54-DMPC
show pronounced line broadening, and thus lines from various methylenes in
the acyl chain cannot be resolved A
P'.at
QI1
156
Since the de-Paked spectrum shows only one lipid environment exists in the liquid crystal
phase, perturbation of the lipid bilayer by VDAC 1 is universal. This shows that there exists very
little conformational flexibility for lipids in the space between VDAC1, similar to a previous
study8 6 involving cholesteryl-p-cyclodextrin
(PCC) derivatives as well as the electron
crystallography study 48 with AQPO. The tight packing of proteins in the lattice is expected since
2D crystals are known to be rigid and stable, thus is possible that no lipid is left unexposed to
VDAC1. Tight lipid packing can also explain the increase of the main phase transition
temperature, TM, observed in DSC as electrostatic attraction between VDAC1 and the lipid
headgroups increases. However, the presence of three distinct transitions in the deconvoluted
DSC thermogram eludes a complete explanation. The asymmetric DSC profile suggests there
may very well be a second lipid environment not seen in the de-Paked NMR spectrum (which,
taken at 27 'C, is between the two transition maxima). Potentially the two phases could be
similar to each other, and line broadening in the 2D crystal
2
H spectrum makes them
indistinguishable. Alternatively, the transition might be more complicated than the simple twostate transition model assumed by DSC deconvolution, as discussed by Huang et al.54
2f
5.4. H NMR of Chain Deuterated DPhPC
Diphytanoylphosphatidylcholine (DPhPC), shown in Figure 5.15, is a popular lipid used
for membrane protein studies. Unlike other saturated straight-chain lipids like DMPC and DPPC,
DPhPC has four additional methyl groups on each acyl chain. The lipid is found naturally in the
membranes of halobacteria, 87 and is known to form very stable bilayers. The bilayers also
experience low ion leakage, making them suitable for electrophysiological ion-conducting
studies.88 An unique and important property of DPhPC bilayers is that they are phase stable at
157
liquid crystalline phase for an extended temperature range from -120 IC to 120 'C according to
DSC studies.8 9 Given that most membrane proteins are functional in the liquid crystalline phase,
the absence of gel phase makes DPhPC an ideal candidate for temperature dependent membrane
protein studies. To date, DPhPC has been used to study gramicidin, 90-9' alamethicin,92
rhodopsin,93 94 VDAC 195 and M2.96
CH3
CH3
CH3
CH3 0
0
0
SH
CH3
CH3
0~
CH3 0
CH3
0
0
Y
H
0~
0
Figure 5.15. Protonated DPhPC (top), compares to DPPC (bottom).
While fully protonated DPhPC is commercially available, the chain deuterated version is
not. Recent advancements in solid-state NMR have created a new need to synthesize more
deuterated lipids. Dynamic nuclear polarization (DNP), 97-98 which improves NMR signal-tonoise by one to two orders of magnitude, is optimal at low proton concentration. Deuterating the
proton-rich acyl chains (39 protons per chain for DPhPC) effectively decreases overall sample
proton concentration and is expected to improve DNP efficiency by a factor of two.2 In general,
deuteration has the advantage of reducing heteronuclear dipolar coupling between
13
C and 1H,
leading to sharper line width and better resolution. It also eliminates unwanted lipid signals in
H-3 C cross-polarization experiments when one only wishes to observe the protein. In this
158
section, the temperature dependent
2H
spectra of a newly synthesized chain deuterated DPhPC
are examined to better understand the lipid's dynamic properties at cryogenic temperatures.
5.4.1. Experimental
Materials. 1,2-dipalmitoyl(d 62)-sn-glycero-3-phosphocholine (d62-DPPC) was obtained
from Avanti Polar Lipids (Alabaster, AL). The d78-DPhPC was synthesized by Loren Andreas
(MIT-FBML, Cambridge, MA) and Vlado Gelev (FBReagents, Cambridge, MA).
NMR Spectroscopy. Solid state
2H
NMR experiments were performed on a custom-built
spectrometer (Courtesy of Dr. D. Ruben) operating at 60.8 MHz for 2 H (10 T) using a singlechannel probe with 4.0 mm coil. Spectra were obtained with a quadrupolar echo sequence with a
7E/2 pulse of 2.0 pis and a delay of 30 ps between the two pulses. For cryogenic temperature
experiments, cold N2 gas was cooled by a custom designed heat exchanger9 9 with temperature
modulated by a Lakeshore (Westerville, OH) temperature controller before transfer to the probe.
The magnet bore was protected from the cold probe by a custom designed vacuum jacketed
dewar. 100 Temperature inside the probe was monitored by Neoptix (Quebec, Canada) fiber optics
temperature sensors.
5.4.2. Results and Discussion
The temperature dependent
2H
spectra for pure DPhPC and DPhPC incorporated with M2
at 1:1 wt ratio are shown in Figure 5.16. Three distinct temperature regimes can be identified.
For the spectral line shapes from 180 K and below, the inner two peaks are separated by 36 kHz
that is indicative of methyl groups undergoing fast 3-site hop (> 106 s1). The outer two peaks are
separated by 120 kHz, which is the expected splitting for rigid C-D (< 104 s-). The overlap of
159
DPhPC with M2
1:1 wt, pH 7.8
DPhPC Only
pH 7.8
243 K
240 K
232 K
231 K
221 K
218 K
212 K
208 K
201 K
197 K
19 K
191 K
190 K
183 K
180 K
170 K
173 K
163 K
160 K
120 80 40 0 -40 -80-120
120 80 40 0 -40 -80 -120
Frequency (kHz)
Figure 5.16. Temperature dependent
2H
spectra of d78-DPhPC and d78-DPhPC:M2.
160
these two sets of powder patterns suggest that at these temperatures the acyl chains only have
slow motions at these temperatures, while the methyl groups retain fast local motions. As
temperature increases from 180 K to 220 K, an isotropic component begins to emerge and
coincide with the diminishing of the powder pattern, as shown in Figure 5.17. From 220 K and
above, only the isotropic component is observed with some broadening at the base. The
incorporation of M2 into DPhPC increased the transition temperature slightly by 7 degrees,
which suggests that pure lipids are more mobile.
173 K
208 K
80'
1 0
40
0
-40
-80
-120
Frequency (kHz)
Figure 5.17. Static
2H
NMR spectra of d78-DPhPC at 173 K (black) overlapped with that of 208
K (red) on an absolute scale. The isotropic peak emerges while the powder pattern is reduced by
50%.
The observation of a sharp isotropic peak at higher temperature was unexpected. As
shown in Figure 5.18, DPPC H spectra are gel phase powder patterns for the same temperature
range. The comparison shows that the additional four methyl groups in DPhPC appeared to have
led to major motional difference respective to DPPC. Nonetheless, isotropic peaks are typically
161
associated with fast, randomized motions, which would suggest the lipid is somehow liquid-like.
MD simulation work by Shinoda et al. comparing DPhPC and DPPC shows the contrary. 101 In
DPPC
pH 7.8
245 K
235 K
223 K
213K
201 K
193: K
183K
171 K
161 K
120 80 40 0 -40 -80 -120
Frequency (kHz)
Figure 5.18. Temperature dependent 2 H spectra of d62-DPPC.
162
the simulation, the methyl groups of DPhPC inhibit gauche-trans isomerization in the acyl chains,
so the rate of acyl chain motions in DPhPC are actually slower compared to DPPC. The
hypothesis for fast DPhPC acyl chain motions would also suggest that DPhPC has a phase
transition that is previously unobserved by DSC, 89 a sensitive method. However, this phase
transition, if it exists, is gradual and spans well over 30 degrees, which can be difficult to detect
by DSC.
Since DPhPC is not likely to exhibit fast, liquid-like motion that would yield the
observed isotropic peak, the most likely explanation would be a slow motion that provides
additional motional averaging that also removes orientation dependence. A 2 H NMR study by
Hsieh and Wu showed that well-hydrated deuterated DMPC headgroup exhibits similar
2H
spectra as the ones we observed for DPhPC acyl chain.' 0 2 At first glance, this finding may seem
counterintuitive. While DMPC has the same headgroup as DPhPC, the headgroup, shown in
Figure 5.19, is the hydrophilic portion of the lipid while the acyl chains are hydrophobic. They
are at opposite end of the bilayer and there is little reason to expect the two regions to exhibit
similar dynamics. However, Hsieh and Wu argued that motions in the headgroup constantly
reorient the headgroup methyl groups along the C-N bond, thereby eliminates orientation
dependence and the powder pattern is averaged into an isotropic peak. In that regard, it is not
farfetched to argue that motions in the acyl chain must be having a similar effect to the DPhPC
methyl groups. We observed that the methyl groups have localized fast motions at lower
temperature (< 160 K) at which the acyl chain motions are frozen. As the temperature increases
and acyl chain motion returns, the acyl chains averages away the angular dependence of the fast
methyl groups, and therefore collapse the powder pattern into the observed isotropic component.
Any residual angular dependence then merely appears as broadening at the base of the isotropic
163
peak, as shown in Figure 5.20. Partially deuterated DPhPC, if synthetically possible, may
provide further elucidation into our observation by examining the motional difference between
the chain methyl groups and the acyl chain deuterons.
0
II
0
Figure 5.19. Choline headgroup of DMPC and DPhPC. Motion along the C-N bond (red)
reorients the three methyl groups and averages NMR angular dependence into only the isotropic
peak.
20
10
-10
-20
requency (kHz)
Figure 5.20. The Static
2H
NMR spectrum of chain deuterated DPhPC at 290 K. At this
temperature, acyl chain motion constantly reorients the methyl groups so an isotropic peak
dominates the spectrum. Residual angular dependence therefore only appears as broadening at
the base.
164
5.5. Phenyl Group Dynamics of Zn 2(TCPE) Metal Organic
Framework
This section is adapted from Shustova, N. B.; Ong, T. C.; Cozzolino, A. F.; Michaelis, V. K.;
Griffin, R. G.; Dinc5, M., J. Am. Chem. Soc., 2012, 134 (16), 15061-15070.
The relaxation of singlet excited states in light-absorbing molecules occurs either by
emission of a photon, giving rise to fluorescence, or nonadiabatically, through nonradiative
decay pathways. 103 In most cases, chromophores that show high fluorescence quantum yields in
dilute solutions become nonfluorescent in colloids and in the solid state, where intermolecular
interactions, such as 7-stacking, often cause self-quenching.' 04 This effect, sometimes referred to
as aggregation-caused quenching, poses significant difficulties for the development of solid-state
fluorescence devices, such as organic light-emitting diodes and luminescence-based sensors. 104106 However, the opposite effect also exists for a select class of chromophores that exhibit weak
or almost no fluorescence in dilute solutions, but show high-fluorescence quantum yields in
colloidal aggregates and in the solid state.1 07~108 This phenomenon, known as aggregationinduced emission (AIE), is typically observed in molecules that contain groups executing fast
discrete diffusion, such as two- or three-fold hops by phenyl or trimethylsilyl rotors. These
moieties are bonded to relatively inflexible backbones, such as ethylenic C=C bonds, or rigid
rings, such as silole.10'-"' In situations where the moieties can undergo uninhibited rotation or
discrete motions, such as in a dilute solution, fluorescence is quenched. 12-114 But once the
moieties are inhibited by short intermolecular interactions in solid aggregates, fluorescence
becomes activated. The discovery of the AIE effect and its wide potential for applicability in
biological
and environmental
sensors, 115~119 solid-state lighting devices,10 6 , 108,
120
luminescent polymers 2 1 - 2 2 have sparked a rapid expansion of the field in the past decade.
165
and
Despite these advancements, the exact mechanism of AIE continues to be a subject of
interest for theoreticians and experimentalists alike; deciphering it unequivocally would clearly
be beneficial for the ab initio development of new classes of AlE molecules."i 4 , 123 Generally,
AIE arises because rotor-containing molecules exhibit low-frequency vibrational modes in the
gas phase or in dilute solutions. These modes are responsible for very fast nonradiative decay of
the singlet excited state but are eliminated in the solid state due to intermolecular steric
interactions. For instance, tetraphenylethylene (TPE), one of the most accessible and simplest
AIE-type chromophores, exhibits low-frequency phenyl torsion modes and C=C twist modes
(Figure 5.21) that are deactivated in the solid state by close intermolecular arene- -H and Ph- -Ph
interactions.10 7,
114, 124
Understanding the relative contribution and effect of these vibrational
modes and conformational changes is one of the keys to making more efficient and more
sensitive fluorescence turn-on sensors from rotor-containing chromophores.
Ph
Ph
Ph
Ph
Figure 5.21. The planes used to define the twist in the ethylene core (left) and a portion of the Xray crystal structure of Zn 2(TCPE) that is representative of both 1H and 1 (right). Orange, red,
blue, and gray spheres represent Zn, 0, N, and C atoms, respectively. H/D atoms were removed
for clarity.
166
To this end, we sought to understand the mechanism that induces fluorescence in a TPEbased metal-organic framework (MOF) reported recently by us.
An MOF is a crystalline
structure formed by repeating network of metal ions and organic linkers. Although the formation
of close intermolecular contacts had previously been presumed necessary for turning on AIE in
rotor-containing chromophores,107 we showed that coordination of phenyl groups to metal atoms
within MOFs also turns on the fluorescence of the TPE cores. One such material,
Zn 2(TCPE)(solvent)2 (1H, TCPE = tetrakis(4-carboxyphenyl)ethylene), as shown in Figure 5.21,
exhibits arene - H and Ph
Ph interactions on neighboring TPE cores that are 1.5 A longer than
in molecular TPE aggregates.12 5 Although these distances are sufficiently large to allow
unimpeded rotation/flipping of the phenyl rings,12 6 1H is fluorescent. We surmised that because
the carboxylate groups in H4TCPE are installed in the para position, phenyl ring flipping and/or
libration in 1H is not completely eliminated, and that understanding the mechanism of
fluorescence turn-on in 1H would therefore aid in the design of efficient emitters and more
sensitive, guest-induced turn-on fluorescence sensors. Our interest in studying the dynamics of
phenyl ring motion in TPE-based MOFs was therefore motivated by the possibility of providing
general principles toward the formation of high-surface area turn-on fluorescent sensors from
AIE-type chromophores. In doing so, we were also hoping to shed more light on the mechanism
of aggregation-induced emission and thereby provide guidance for the development of new
chromophores in this rapidly expanding area.
With these goals in mind, we synthesized a deuterated TPE-based MOF that is
structurally analogous to 1H and employed 2 H NMR spectroscopy and 13 C cross-polarized magic
angle spinning solid-state (CP MAS) NMR spectroscopy to determine the activation barrier for
phenyl ring flipping in this material. In conjunction with temperature-dependent single-crystal
167
and powder X-ray diffraction analysis, and density functional theoretical calculations, these
results reveal that fluorescence is turned-on in TPE-based MOFs by drawing of the TPE core
rather than the presence of close intermolecular Ph - Ph interactions, as is typical for molecular
constructs of rotor-containing chromophores. Accordingly, we propose that both the C=C bond
twist and the torsion of the phenyl rings are important for quenching emission in TPE but that the
former may gate the latter. We use these findings to propose a set of design criteria for the
development of tunable turn-on porous sensors constructed from AIE-type molecules.
5.5.1. Experimental
Materials. Zn(NO 3)2 -6H 2 0 (98%, Strem Chemicals), Br 2 (>99.5%, Sigma-Aldrich),
CuCN (99%, Strem Chemicals), Zn (dust, 98.6%, Mallinckrodt), oxalyl chloride (98%, Alfa
Aesar), TiCl 4 (>99%, Sigma-Aldrich), MgSO 4 (98%, VWR), AlCl 3 (>99%, Sigma-Aldrich),
N,N'-dimethylethylenediamine
(99%,
Sigma-Aldrich),
dichloromethane
(HPLC
grade,
Honeywell), methanol (99.9%, VWR), DEF (>95%, TCI America), ethanol (ACS grade,
Mallinckrodt), ethylene glycol (AR grade, Mallinckrodt), ethyl acetate (VWR), tetrahydrofuran
(ACS grade, Mallinckrodt), toluene (Sigma-Aldrich, ACS), C6 D6 (Cambridge Isotopes), CDCl
3
(Cambridge Isotopes), CD 30D (Cambridge Isotopes), and DMSO-d 6 (Cambridge Isotopes) were
used as received.
Tetraphenylethylene-d2o (C 2 6D 20 , TPE-d 20). The synthetic sequence for the preparation of
this material is shown in Scheme 5.1. Benzophenone-dio was synthesized from benzene-d6 based
on a known procedure 2 7 and was then heated (5.47 g, 0.03 mmol) to reflux in the presence of
TiCl 4 (8.60 g, 0.05 mmol) and Zn dust (5.90 g, 0.09 mol) under McMurry conditions
4.60 g (0.01 mol) of perdeutero-tetraphenylethylene (87% yield).
ppm;
13C
NMR (CDCl 3 , 500 MHz): 6
=
2H
to give
NMR (CHCl 3): 6 = 7.05 (br)
126.20 (t), 127.24 (t), 131.00 (t), 140.93 (s), 143.68 (s)
168
ppm. IR (neat, cm-1): 2281 (s), 2269 (s), 1617 (w), 1563 (m), 1385 (w), 1322 (s), 1279 (w), 1202
(w), 959 (w), 878 (w), 855 (s), 841 (m), 822 (vs), 788 (w), 763 (w). Elemental analysis
calculated for C2 6D2 0 : C, 88.57; H(D), 6.07. Found: C, 88.67; H(D), 5.87.
C6136
C20 2C12
NC
D Ds
O
1-Br 2
TiCI 4, Zn
HF2THF
D
D sD
D5D D5
'
D
NC
5
TPE-d
O
D4 D4
OH
ethylene glycol
2. CuCN, DMF
s
O
CN
>KHO
KOH
D4 D4
D4 D 4
CN
)(
HO
4 DO
H
O
20
O
H4TCPE-d
16
Scheme 5.1. Synthesis of H4TCPE-d1 6.
Tetrakis(4-cyanophenyl)ethylene-d6 (C3 oD 6N 4, H4 TCNPE-d]6).
H4TCNPE-d1 6 was
prepared from TPE-d20 following a recently published synthetic route for the protonated
analogue.1
"C NMR (CD 2Cl 2, 500 MHz) 6
=
111.78 (s), 118.56 (s), 131.56 (t), 132.14 (t),
141.75 (s), 145.82 (s) ppm. IR (neat, cm-1): 2294 (w), 2225 (vs), 1573 (s), 1414 (w), 1321 (m),
1291 (w), 1109 (m), 869 (w), 827 (m), 759 (w), 743 (w), 718 (w), 677 (w). Elemental analysis
for calculated for C30 D 16N4 : C, 80.36; H(D), 3.72; N, 12.49. Found: C, 80.10; H(D) 3.75; N,
12.30.
Tetrakis(4-carboxyphenyl)ethylene-d 6
(C3 oH4D 16 0 8 , H 4 TCPE-d 6).
H4 TCPE-d1 6 was
synthesized by hydrolysis of the corresponding nitrile following the published procedure for the
protonated analogue.1
2H
NMR (CH 30H, 500 MHz): 6 = 7.19 (br), 7.86 (br) ppm;
13C
NMR
(CDCl 3, 500 MHz): 6 = 128.8 (m), 129.29 (s), 130.49 (m), 141.10 (s), 146.32 (s), 166.96 (s) ppm.
IR (neat, cm-1): 2972 (w, b), 2225 (w), 1687 (vs), 1578 (s), 1542 (w), 1439 (m), 1376 (w), 1327
(w), 1259 (b, s), 1206 (s), 1078 (w), 871 (w), 841 (w), 816 (w), 786 (w), 746 (w), 691 (w).
Elemental analysis calculated for C3 0H4 D160 8 -H2 0: C, 66.4; H(D), 4.21. Found: C, 66.09; H(D),
3.93.
169
Synthesis of Zn2(TCPE-d16)(DEF)2-2DEF (]a). This compound was synthesized in an
identical procedure as 1H. IR (neat, cm'): 2979 (w), 2939 (w), 2878 (w), 2272 (w), 1634 (vs),
1578 (m), 1559 (m), 1442 (s), 1382 (vs), 1309 (w), 1265 (w), 1215 (w), 1106 (w), 881 (w), 832
(w), 820 (w), 703 (w), 677 (w). Elemental analysis calculated for la-H2 0: C, 55.92; H(D), 5.91;
N, 5.22. Found C 55.74, H 5.73, N 5.10.
X-ray Crystal Structure Determination.Diffraction-quality single crystals of la, 1b, and
TPE-d 20 were mounted using mineral oil and epoxy on Kapton loops. Diffraction data ((p- and oscans) at 100, 298, and 373 K were collected on a Bruker-AXS X8 Kappa Duo diffractometer
coupled to a Smart APEX II CCD detector with MoKa radiation (k = 0.71073 A) from an IpSmicro source. Absorption and polarization corrections were applied using SADABS.
129
The
structure was solved by direct methods using SHELXS and refined against F2 on all data by fullmatrix least-squares with SHELXL-97.130 All nonhydrogen atoms were refined anisotropically
and were included in the model at geometrically calculated positions. The crystallographic data
for TPE-d 20 and 1 are shown in Table 5.S1.
2H
NMR Spectroscopy. Experiments were conducted on a home-built spectrometer
(courtesy of Dr. Dave Ruben) operating at 61 MHz for 2 H using a single-channel transmission
line probe with 3.2 mm coil. Spectra were obtained using a quadrupolar echo sequence with an
8-step phase cycling 5 using a 2/2 pulse of 2.0 gs and a delay of 30 ps between the two pulses.
Phenyl ring motional dynamics were determined by simulations of the experimental 2 H powder
lineshapes using TURBOPOWDER. 10
'3 C MAS NMR Spectroscopy. Experiments were performed at 16.4 T (697.8 MHz, 1H)
using a home-built spectrometer (courtesy of Dr. Dave Ruben) and a 3.2 mm Chemagnetics
170
triple-channel magic-angle spinning probe. Samples were ground using a mortar and pestle and
packed in 3.2 mm ZrO2 rotors (-28 pL sample volume). Spectra were acquired at spinning
frequencies of 10 kHz, with 512-4096 coadded transients and recycle delays between 3 and 120
s, using either a Bloch decay or cross-polarization' 3 ' (Urf of 83 kHz for 'H and 13C,
TCp=
2.0 ms)
and two pulse phase modulation (TPPM) proton decoupling132 for naturally abundant
3c
deuterated and protonated samples. 13 C experiments were referenced to adamantane at 38.48
ppm relative to TMS.133
Computational Details. Calculations were performed using the ORCA 2.8 quantum
chemistry program package from the development team at the University of Bonn.'3 4 In all cases
the LDA and GGA functionals employed were those of Perdew and Wang (PW-LDA, PW9 1).35
Calculations were performed using the TZV basis set for hydrogen, the TZV(p) basis set for
main group atoms, and TZV(2pf) for zinc. 13 6 Spin-restricted Kohn-Sham determinants have
been chosen to describe the closed-shell wave functions, employing the RI approximation and
the tight SCF convergence criteria provided by ORCA. Numerical frequency calculations were
performed on the optimized structures when size would permit. The atoms-in-molecules analysis
was performed using Xaim.137
Other Physical Measurements. TGA was performed on a TA Instruments Q500
thermogravimetric analyzer at a heating rate of 0.5 'C/min under a nitrogen gas flow of 90
mL/min. Infrared spectra were obtained on a PerkinElmer Spectrum 400 FT-IR/FT-FIR
spectrometer equipped with a Pike Technologies GladiATR attenuated total reflectance
accessory. Solution NMR spectra were collected on a Varian 300 or a Varian Inova-500 NMR
spectrometer. 2H spectra were referenced to the natural abundance 2H peak in protonated
solvents; '3 C and 1H spectra were referenced to natural abundance
171
1C
peaks and residual 'H
peaks of deuterated solvents, respectively. PXRD patterns for la and lb were recorded on a
Bruker Advance D8 diffractometer using nickel-filtered Cu-Ka radiation (A = 1.5418 A), with
accelerating voltage and current of 40 kV and 40 mA, respectively. A PXRD pattern for le was
collected at station 11-B at the Argonne National Laboratory using synchrotron radiation (X =
0.413073 A). Samples for PXRD were prepared by placing a thin layer of the appropriate
material on a silicon (510) crystal plate for la and 1b and by sealing Ic in a Kapton capillary.
5.5.2. Results
Synthesis and Temperature-DependentStructuralStudies. Synthesis of a deuterated TPEbased MOF started from deutero-tetra(4-carboxy)phenylethylene, H4 TCPE-dl 6 , which was
accessed from perdeuterated benzene in four steps, shown in Scheme 5.1. Treatment of C D
6 6
with oxalyl chloride in carbon disulfide produced benzophenone-dio, which was subsequently
homocoupled under McMurry condensation conditions128 to yield TPE-d 20. Bromination of TPEd20 with neat Br 2 followed by copper-catalyzed halide-for-cyanide exchange and basic hydrolysis
of the nitrile groups gave the desired tetracarboxylate ligand, H4 TCPE-d16 in 31% overall yield.
Heating a solution of H4TCPE-d16 and Zn(NO 3)2 -6H 2 0 in a mixture of ethanol and NNdiethylformamide (DEF) at 75 'C for 3 days produced yellow block-shaped crystals of
Zn 2 (TCPE-dl 6)(DEF) 2 -2DEF (la).
An X-ray diffraction study of a single crystal of la revealed a structure in which
Zn 2 (0 2 C ) 4 paddlewheel units are bridged by TCPE4--d16 ligands in infinite two-dimensional
(2D) sheets whose connectivity is identical to that found in H.12 5 The sheets adopt a staggered
conformation to give similar but not identical lattice parameters to those of H, as shown in
Table 5.S1. Despite the slight shift in the stacking arrangement of the 2D sheets in la relative to
1H, the two related structures exhibit almost identical fluorescence spectra and thermal behavior,
172
evidenced in the TGA traces shown in Figure 5.S1. As in 1H, thermal treatment of la produces
several significant structural transformations. Since these are crucial for the interpretation of the
NMR data, we undertook variable-temperature X-ray diffraction studies of both TPE-d 20 and 1.
Thus, the X-ray crystal structure of TPE-d 20 was determined at 93, 298, and 373 K. TPE-d 20
maintains the monoclinic P2 1 space group at all three temperatures, with no significant changes
in lattice parameters, molecular packing, or Ph -Ph ring intermolecular distance. As shown in
Table 5.S2, the shortest interchromophore contacts (Ph- -Ph ring contacts) are 3.583(3)-3.635(5)
A, while the twist angle of the C=C bond (Figure 5.21) is 8.84-10.16'. Over the entire
temperature range, the shortest intermolecular TPE contacts change by no more than 0.052(6) A,
and the change in the C=C twist angle is less than 1.320. Single crystal X-ray structures of 1
were also determined at 100 and 373 K. As shown in Figure 5.22, 1 adopts a monoclinic
structure at 100 K (la) but undergoes a symmetry-increasing transformation to an orthorhombic
phase while heating to 373 K, which we designate as 1b. Importantly, powder X-ray diffraction
(PXRD) analysis revealed that the 2D sheets do not change their relative positioning upon
transformation from la to 1b. Determination of the unit cell parameters of la at room
temperature confirmed only very small deviations from the orthorhombic cell determined at 373
K for 1b. Apart from the small deviation in overall symmetry, important structural differences
between the structures of la and lb include the lack of guest DEF molecules in the latter, an
extension of the shortest Ph - Ph contacts from 4.744(9) to 5.10(1) A and a reduction of the
ethylene twist angle from 5.35' to 3.83'. The lack of guest DEF molecules in 1b, formulated as
Zn 2 (TCPE-di6 )(DEF) 2 , is in agreement with the thermogravimetric analysis (TGA) and
elemental analysis data (vide infra).
173
900
13.734(1) A
17.530(2) A 126.487(2)*
a
90*
1a26.446(2) A
22.143(4) A 90
c
1a
13.531(3) A 900
18.291(4) A 900
22.172(6) A 900
lb
."...
A
191A
d
1b
1I
200*C
e
12.66 A 90*
8.40 A 90*
21.62 A 900
1c
10
4.2 A
I
A
20
30
40
20, deg
1C
Figure 5.22. Temperature-dependent X-ray diffraction studies of 1. Left column: PXRD patterns
of (a) la calculated from the X-ray crystal structure determined at 100 K, (b) la collected at
room temperature, (c) lb calculated from X-ray structure at 373 K, (d) the sample used in the 2 H
NMR study, and (e) 1c. Right column: X-ray crystal structures of la collected at 100 K, lb
collected at 373 K, and the simulated structure of 1c based on the PXRD data. Golden, red, blue,
and gray spheres represent Zn, 0, N, and C atoms, respectively. Guest DEF molecules are shown
in pink. H/D atoms have been removed for clarity.
174
Continued heating at 200 IC caused a complete loss of Zn-coordinated DEF molecules.
PXRD analysis revealed that this is also accompanied by a drastic structural rearrangement to a
desolvated form of 1, Zn 2(TCPE-di 6 ) (1c). Because single crystals of lb do not survive their
transformation into 1c, we sought to match the observed PXRD pattern of 1c with a structural
model. This was accomplished by implementing an original computational routine in Matlab,
which simulates PXRD patterns of possible phases by changing the interlayer distance and
relative displacement of 2D layers. In this case, the structure of lb was used as an initial model,
and we considered the possibility that 1c is related to the former by simple translations of the 2D
sheets in the ab plane and/or by changes in the intersheet separation. Modulation of these
parameters using our routine provided a structural model for 1c that exhibited a good match with
the observed pattern (Figure 5.S2). Although the relatively poor crystallinity of ic prevented a
full Rietveld refinement even from synchrotron-collected data, our computational routine
revealed that 1c is a new orthorhombic phase with parameters of 12.66, 8.40, and 21.62 A.
The one notable difference between lb and ic is the much reduced interlayer distance,
which decreases from 8.7 A in the former to 4.2 A in the latter (Figure 5.22). The contraction of
the interlayer distance brings the Zn 2 (0 2 C~) 4 paddlewheel units in neighboring 2D sheets in close
proximity and prompts the formation of covalent linkages between Zn atoms in one sheet and
carboxylate oxygen atoms in adjacent sheets. The absence of all DEF molecules from 1c was
confirmed by TGA, which showed a mass loss of 36.2% below 200 'C, in agreement with the
35.4% expected for the elimination of four DEF molecules from la (Figure 5.S1).
IH,
13 C,
and 2H NMR Spectroscopic Studies. Variable-temperature 'H NMR spectra of
TPE and H4TCPE were recorded in CD 2 Cl 2 and CD 30D, respectively, between 183 and 293 K.
The phenyl ring protons appear as a pair of doublets with chemical shifts of 7.14 and 7.81 ppm
175
( 3.JHH =
8 Hz) for H4TCPE and two multiplets with chemical shifts at 7.03 and 7.10 ppm for TPE
itself. As shown in Figure 5.S3, cooling to 183 K broadens the 'H resonances in both TPE and
H4 TCPE, but the two different proton signals do not coalesce, suggesting that the phenyl rings in
both molecules are in the fast exchange regime in solution even at 183 K. To confirm the
expected slow exchange regime of phenyl rings in TPE-d 20 in the solid state,
2H
NMR spectra of
crystalline samples of this molecule were recorded between 298 and 423 K. As shown in Figure
5.23, the
by
Q=
2H
NMR spectra of solid TPE-d 20 in this temperature range exhibit two peaks separated
128 kHz. This yields a Pake pattern characteristic of C- H vectors in the slow exchange
regime (x- 10-3--10-4 s).
p1
1'
423K
393K
328K
/
298K
150
Figure 5.23. Static
2H
-150
-50
50
Frequency, kHz
NMR spectra of TPE-d 20 taken between 298 and 423 K.
176
2H
NMR was also used to investigate the phenyl ring dynamics in la and 1c. As shown in
Figure 5.24, ic showed almost identical Pake patterns up to 423 K, the highest temperature
achievable with our NMR probe.
298K
333K
I
349K
369K
it
423K
150
Figure 5.24. Static
2H NMR
-150
-50
50
Frequency (kHz)
spectra of 1c taken between 298 and 423 K.
In contrast, freshly synthesized la showed Pake patterns only between room temperature
and approximately 323 K. Heating la above 323 K caused the line shape to evolve into a pattern,
wherein a second set of symmetric peaks with a smaller splitting of Q/4 = 32 kHz emerged along
with a third wider splitting being -5Q/-4
=
160 kHz. As shown in Figure 5.25, the intensity of
this central set gradually increased at the expense of the original outer signal up to 423 K. An
177
isotropic signal also became apparent above 323 K, likely indicative of the increased mobility of
the guest DEF molecules. Indeed, this isotropic signal disappeared after prolonged heating at 423
K, indicating the loss of the guest molecules and conversion to 1b. Upon cooling of 1b, the
reverse evolution of the quadrupolar signal was observed; the two-fold flip pattern at 423 K
gradually evolved into a typical slow-exchange Pake pattern at 321 K.
Experimental
323
Simulated
421K
1.2 x 10 6 Hz
369 K
2.0 x 105 Hz
345 K
3.2 x 1Hz
321K
1.8 x 1 4 Hz
300K
1.0 x 1 4Hz
1.0 X104H
2.7 x 10
5
Hz
396K
1.6 x lHz
423K
3.0 x le Hz
150
Experimental
Simulated
50
-50
-150
150
50
-50
-150
Frequency (kHz)
150
50
-50
-150
150
50
-50
-150
Frequency (kHz)
Figure 5.25. Experimental and simulated quadrupolar spin-echo solid-state
2H
NMR spectra of
la during heating and transformation into lb (left) and of lb during cooling (right).
To simulate the spectra, we assumed a model consisting of a single population of phenyl
rings undergoing discrete two-fold flips. The model was used for simulations of the 2H
quadrupolar line shapes for five temperatures during the cooling cycle of lb between 421 and
321 K. 14'
' The simulations yielded flipping rates of 1.2 x 106, 2.0 x 105 , 3.2 x 104, 1.8 x 104,
and 1.0 x 104 Hz at 421, 369, 345, 321, and 300 K, respectively. To obtain an activation energy
and pre-exponential factor for phenyl ring flipping in 1b, the natural logarithm of the rates was
plotted against the inverse of the respective temperatures to give an Arrhenius plot. A line fit to
178
this graph, shown in Figure 5.26, gave activation energy and pre-exponential factor values of
43(6) kJ/mol and 2.2 x 1011 Hz, respectively.
14-
U
1312
U
11
1091
2.2
2.4
2.6
2.8
3.0
Iooo/T, K1
3.2
3.4
Figure 5.26. Arrhenius plot of the two-fold phenyl exchange rate in lb during cooling.
Although
2H
NMR revealed a wealth of information about the phenyl ring dynamics in
1b, it was not suitable to interrogate the same in ic, where the phenyl ring motion remains in the
slow regime (<104 Hz) regardless of the temperature (vide supra). Because
spectroscopy can be used to probe motions down to frequencies of
_ 102
13 C
CP MAS NMR
Hz,'3 9
13C
CP MAS
NMR spectra were acquired for 1c and its protonated relative (fully desolvated 1H) at room
temperature. As shown in Figure 5.27, both deuterated and protonated versions of the MOFs
exhibit isotropic peaks at 135, 137, and 153 ppm for the phenyl ring carbon atoms and 147 and
181 ppm for the ethylene and carboxylate carbon atoms, respectively.
179
d
e
/
b
a
a
C
/
d
b
ce
190
170
150
130
110
13C Chemical Shift (ppm)
Figure 5.27.
1C
CPMAS NMR spectra of fully desolvated lH (top) and ic (bottom).
Theoretical Studies. Density functional theory (DFT) was employed to calculate the
activation barrier for ring flipping in 1b. The barrier was estimated by modeling the potential
energy surface (PES) of TCPE4 - bound by four Zn 2 (0 2 C~) 4 paddlewheels. The metal
coordination sphere was completed with three bridging formate ligands and two terminal water
ligands (Figure 5.28).
The PES was constructed by varying one CAr-CAr-C=C dihedral angle from 0 to 180'
and is depicted in Figure 5.29. The Zn and oxygen atom coordinates were fixed in order to
mimic the rigidity imposed by the framework. Notably, a very similar PES could be obtained
using H4 TCPE with the oxygen atom coordinates fixed to those found in the 1 (Figure 5.S5) with
significant savings in computational resources. The lowest energy structure from the PES, which
was deemed closest to the absolute minimum energy conformation, was used as a starting point
180
for a geometry optimization to find the absolute minimum. Because of the size of the system
under investigation, a transition state was not modeled. Under these parameters, the activation
energy for a ring flip in lb was estimated at 49 kJ/mol.
Figure 5.28. DFT-calculated structures of truncated formate-capped models of lb with a fixed
orientation of one phenyl ring at 1250 (left) and 5* (middle) and of TPE with a fixed orientation
of the phenyl ring at 0' (right). The scheme illustrates the distortion in the TPE core that occurs
to minimize the steric repulsion, namely in-plane bends of the CA-C=C angles and the C=C
twist. The models are depicted without hydrogen atoms for clarity. Yellow, red, and gray spheres
represent Zn, 0, and C atoms, respectively. The carbon atoms that define the dihedral angles
used to model the PESs are shown in purple.
181
-1.252
40 -
1.256 e
0
30-(
1.260 G
E2o C
0
10
\1.264
tS
0 -
0
100 120 140 160 180
CArCAr-C=C Dihedral Angle (0)
20
40
60
80
Figure 5.29. PES for the flipping of one phenyl ring in a truncated model of lb (0) and sum of
the electron density at the C-C single bond critical points (grey e). The electron density axis has
been reversed and scaled for clarity. Lines have been added as a visual guide.
The DFT-estimated activation barrier for phenyl ring flipping in TPE in the gas phase is
24 kJ/mol. This value was determined by first modeling the PES by varying the CA,-CA1-C=C
dihedral angle from 0 to 1800 and 180 to 0' with no additional constraints (Figure 5.30). To
correct for false maxima that could arise due to the high number of degrees of freedom, a
minimum energy PES was constructed by convoluting PESs calculated in the forward and
reverse directions of phenyl ring rotation.
182
3530
-
/
25
4
E
20 -
/!
E2 15 10
50- 100
50
0
CAr
150
Dihedral Angle (0)
Ar-CC
Figure 5.30. PES for the flipping of one phenyl ring in a model of TPE. The solid line with black
circles (e) indicates the lowest energy surface constructed from the forward (solid gray line and
open circles, "1)and the reverse (hashed gray line) direction ring-flip PESs.
As before, the lowest energy structure on the convoluted PES was used as a starting point
for a geometry optimization and was confirmed by a frequency calculation that provided no
negative values. Notably, we calculated a barrier of 49 kJ/mol for the truncated model of lb in
the vicinity of 00, a value that is approximately 25 kJ/mol higher in energy than that calculated
for TPE at the same angle. The structure of TPE at the maxima reveals an ethylene core that has
undergone significant structural deviation from the minimum energy structure involving the CArC=C-CAr and CAr-CA-C=C dihedral angles as well as the CAr-C(ethylene)-CAr and CA-CC
bond angles (Figure 5.28), whereas the constraints imposed by the rigid framework in lb prevent
the ethylene core from undergoing similar distortions (Table 5.S3-5.S6). The structural
183
distortions in TPE correspond to the lowest energy vibrational modes (Table 5.1) that occur well
below kT (206 cm- at 298 K).
Table 5.1. DFT-calculated low-energy vibrational modes for TPE.
Energy (cm-1 )
Vibrational Mode
6
v1
C-C=C-C torsion
29
V2
CAr-CAr-C=C torsion
39
V3
CArCAr-C=C
54
V4
Aryl rocking
58
V5
CArCAr-CC torsion
65
V6
Aryl rocking
69
V7
CAr-CAr-CC
torsion
72
V8
CArCArCC
torsion
78
V9
CAr-CArCC
torsion
torsion
In order to deconvolute the steric from the electronic effects in the barriers in the PESs
for the truncated model of lb and for TPE, PESs for the ring flipping of the phenyl ring in
styrene and benzoic acid were constructed under the assumption that the PESs for these two
systems provide a rotational barrier that is free of steric effects. In both cases, the minimum in
energy occurs when the CArCArC=C dihedral angle is 00, which corresponds to the geometry
that maximizes the conjugation between the phenyl ring and the pendant group (Figure 5.S4).
The associated calculated activation energies for phenyl ring flipping in styrene and benzoic acid
are 18 and 27 kJ/mol, respectively.
5.5.3. Discussion
The structure of la consists of a 2D framework composed of paddlewheel Zn 2 (0 2 C )4
secondary building units that are bridged by TCPE 4 --d16 ligands (Figure 5.21 and 5.22). The
184
structure contains both bound DEF molecules, which occupy the axial sites on Zn atoms in the
Zn 2 paddlewheels, and guest DEF molecules, which occupy the pores. The latter likely prevents
fast flipping of the TPE phenyl rings, and
2H
NMR spectra of la accordingly reveal Pake
patterns characteristic of slow exchange (<104 Hz). The Pake patterns persist up to 373 K, but
heating la above this temperature starts liberating the guest DEF molecules, thereby activating
the phenyl ring flips. Indeed, 2H NMR spectra at 373, 396, and 423 K reveal an isotropic signal
that can be attributed to solvent motion and dynamic quadrupole patterns that can be fit to
discrete 1800 phenyl ring flips with respective frequencies shown in Figure 5.25. Because both
guest solvent loss and activation of phenyl ring dynamics take place during heating of la, the
two processes are convoluted and prevent an Arrhenius analysis. Instead, the sample was kept at
423 K for 24 h to eliminate all of the guest solvent molecules and complete conversion of la into
2
1b, as identified by the disappearance of the isotropic signal attributed to mobile DEF. H NMR
data were again collected for lb on cooling back to room temperature, with data points at 421,
369, 345, 321, and 300 K. Because no guest solvent molecules are present in 1b, the data could
be plotted in Arrhenius fashion, as shown in Figure 5.26. The experimentally determined
activation energy for the phenyl ring flip in 1b, 43(6) kJ/mol, is larger than that expected for free
TPE by approximately 20 kJ/mol. This suggests that, indeed, the torsion of the phenyl ring in lb
is impeded relative to solution-phase TPE and is likely the cause of fluorescence turn-on in the
TPE-based MOF. The pre-exponential factor, which can be interpreted as the barrier-less
flipping rate of the pure phenylene bridge,140-141 is 2.2 x 1011 Hz and is somewhat smaller than
those of phenylene bridges in related porous materials, such as MOF-5,
142-143
and periodically
ordered mesoporous organosilica. 2 6 It is conceivable, however, that intramolecular steric effects
converge to decrease the pre-exponential factor in TPE derivatives relative to phenylene itself.
185
One essential aspect of the NMR data interpretation relates to the stability and identity of
the sample during the heating cycle. As for 1H, heating of la above 150 'C causes loss of both
bound and unbound DEF molecules and is accompanied by significant structural changes and
formation of a new phase, lc. In 1c, fused 2D sheets bring phenyl rings on adjacent TPE cores in
close proximity, giving rise to short Ph -Ph contacts of -5 A (measured between the centroids
of the phenyl rings), in line with those observed in molecular crystals of TPE derivatives and
solid TPE itself.144
Expectedly, just like TPE, ic exhibits Pake patterns at both low and high temperature,
reinforcing the observation that close-packed TPE cores prohibit torsional motion of their
phenyl(ene) components. Importantly, however, if lb is heated below 150 'C (i.e., the
temperature range of our NMR experiments), its structure and the large Ph
.
Ph separation
conducive to fast phenyl ring flipping are maintained. This important fact was verified by both
single crystal and powder X-ray analysis. Thus, single crystal X-ray diffraction of lb at 100 'C
showed that no significant structural changes occur relative to la. Although single crystals of lb
do not survive heating at 150 'C, powder X-ray analysis of the sample used for the NMR
experiments showed a pattern that matched that of 1b, with only small peaks corresponding to
the completely desolvated phase, 1c (see Figure 5.22 and below). Because phenyl ring motion in
1c is in the slow-exchange regime in this temperature range, its presence does not affect the
dynamic line shapes used for the Arrhenius plot for lb and is a minor contributor only to the
Pake singularities with large quadrupolar splitting. In addition, 13 C CP MAS NMR spectra of lc
and of fully desolvated 1H illustrate that both of these compounds exhibit similar resolution and
line shape, which is consistent with a rigid lattice with motion that is slower than what is
186
detectable with this technique (<102 Hz) (Figure 5.27). This finding agrees with the
2H
NMR
results, which show that the ring motions are in the slow exchange regime.
To understand the origin of the activation barrier in 1b, especially in comparison to TPE
itself, the ring flipping process in both lb and TPE was probed by DFT calculations. The
calculated values of the activation barriers for ring flipping in a truncated model of lb and gasphase TPE are 49 and 24 kJ/mol, respectively. Clearly, despite the axial symmetry of phenylene
rings in H4TCPE, which should allow fast flipping in a sterically unhindered environment such
as the pores of 1b, phenyl ring flipping in lb is much more sluggish than in TPE itself. To
understand the origin of the increased barrier in lb and the differences between the PESs of lb
and TPE, a more detailed look at the steric and electronic contributions to these was performed.
The electronic contribution was probed by considering ring flipping in styrene and benzoic acid,
as well as vinylbenzoic acid (Figure 5.S4 and 5.S6). These molecules have similar electronic
structures to the benzoate units in the truncated model of 1b, but their phenyl groups lack vicinal
phenyl rings that could sterically hinder rotation. The barrier to phenyl ring flipping in these can
therefore be assumed to be completely electronic in origin. The electronic component of the PES
for ring flipping in TPE could therefore be reconstructed from the PES of styrene, even though
the energy contributions were not necessarily expected to be additive. This implied that the
barrier for ring flipping in gas-phase TPE is almost completely electronic in origin, and the steric
interactions expected to occur at a CAr-CAr-C=C dihedral angle of 0' are avoided due to a
number of small geometrical distortions that correspond to low-energy vibrational modes (Table
5.1). Rationalizing the shape of the PES of lb is more complicated because it cannot be
reconstructed by simply summing the contributions from the PESs of styrene and benzoic acid.
Because in lb itself the ethylene core is perpendicular to the carboxylate groups, the effect of the
187
electronic contribution to the overall barrier for ring flipping is expected to be rather insignificant
(Figure 5.S6). To attest this, an atoms-in-molecules analysis of the C-C single bonds at select
points on the PES was performed (Table 5.37).145
Since the density at the critical point is indicative of the bond order, the sum of the
electron densities at each of the C-C single bond critical points should be indicative of the
amount of electron delocalization throughout the molecule and, by extension, the stability of the
conformation at each point. Figure 5.29 illustrates how the sum of the densities at the C-C
critical points mirrors the shape of PES. A key point is that the lowest total density, which should
correspond to the least stable conformation, is found at a local maximum. This indicates that the
global maximum found at 5' (49 kJ/mol) is not entirely electronic in origin and must have a
considerable steric contribution. Investigation of the geometry at the maximum in the PES of 1
(Figure 5.28) shows that the ortho-hydrogen atom on one phenyl ring is directed into the 7-cloud
of the vicinal cis-phenyl ring. Unlike in gas-phase TPE, where low-energy geometric distortions
to the TPE core allow the steric maximum to be avoided (Figure 5.28), the TPE core in lb is
drawn tight, thereby forcing the phenyl rings to remain in close proximity during the ring
flipping process.
This computational analysis highlights the following points: (1) Low-energy vibrational
modes in the TPE core minimize inter-ring steric interactions and allow ring flipping to occur
with a low barrier (25 kJ/mol); and (2) The drawing of the TPE core by the framework forces
these steric interactions to occur, leading to a significantly higher barrier for ring flipping (49
kJ/mol) that is in good agreement with the experimentally derived barrier.
188
The relative importance of the C=C bond twist and phenyl ring torsion in quenching the
fluorescence in molecular TPE derivatives has been addressed before, and it was concluded that
the latter has a higher contribution to the nonradiative decay of the excited state. 14 6 Our results
are in line with this observation and allow us to establish a connection between the two:
diminution of the C=C twist angle by drawing of the TPE core in lb causes a larger steric barrier
for phenyl torsion/flipping, suggesting that a relatively large C=C twist angle or a flexible core is
required for a low-barrier phenyl ring torsion. The activation barrier for ring flipping in lb is
comparable to the activation energies for phenylene-linked porous materials.1 2 6, 141-142,
147
For
instance, activation energies for 1,4-aromatic dicarboxylate-based MOFs range from 21-53
kJ/mol. 12 6,
142, 147-159
Although some of these are higher than the activation energy for ring
flipping in 1b, the differences can be entirely attributed to conjugation-stabilized conformations
in which the carboxylate groups and the phenylene ring are coplanar. We confirmed
computationally that the PES constructed for ring-flipping in terephthalic acid gives an activation
energy of 50 kJ/mol when both carboxylate groups are held coplanar, in good agreement with
experimentally observed activation energies for MOFs constructed from this ligand. In the
absence of conjugating groups, exemplified by the pyrazine-bridged structures, a much lower
activation energy is found. In these, because pyrazine is primarily a a-donating ligand, there is
little energetic cost for ring flipping, which must only overcome a weak 2-type interaction with
the d10 metal ion.
Importantly, because the origin of the activation barrier in lb is enforced partly by
coordination in a rigid lattice and is therefore not inherently borne in the ligand, strategies can be
envisioned for reducing the activation barrier for ring-flipping. These strategies include: (1)
Designing MOFs where AIE-type chromophores are well separated spatially. This is necessary
189
to avoid aggregation in the empty material and to ensure porosity for analyte adsorption; (2)
maintaining the flexibility in the TPE core to ensure that low-energy vibrational modes are not
eliminated in the empty material. This could be implemented, for instance, by leaving two
dangling/unsubstituted phenyl rings which should maintain dynamics in the fast flipping regime;
and (3) minimizing ligand conjugation to reduce the contribution of an electronic component to
the ring-flipping barrier. This could be achieved, for instance, by enforcing a perpendicular
orientation between the ethylene core and the metal-binding functional groups, as in 1, by using
acetylene spacers to 'insulate' the phenyl ring from orientation-inducing conjugation, or by using
nonconjugating ligating groups.
The criteria outlined above would allow the design of true turn-on MOF sensors. In such
sensors, AIE-type chromophores with low-barrier ring flipping would completely quench the
fluorescence in the empty porous materials. Fluorescence would then only be turned-on in the
presence of analyte guests that can hinder the rotation of the phenylene ring, thereby eliminating
the low-energy nonradiative excited-state quenching pathways. MOFs are ideal candidates for
55
incorporating such strategies because they lend themselves to modular synthetic design.1 ' 160-168
We envision that these strategies are not limited to TPE-based ligands and should be more
broadly applicable to the construction of switchable luminescent MOFs from a wide variety of
AIE-type ligands.
190
5.6. Conclusion
A new transmission line
2H
NMR probe was built to study temperature dependent
dynamic processes and phase behaviors of the following novel systems:
1. We studied the effect of protein-lipid interaction on DMPC phase transition in
DMPC/VDAC1 2D crystals. We found that the phase transition temperature (TM) of DMPC
increased by 8 degrees and the lipid transition was less cooperative in the presence of VDAC1
compared to pure lipid. The finding qualitatively agrees with theoretical molecular field model
proposed by Marceija, whereby lipid-mediated protein-protein interactions and protein-lipid
interactions are necessary to explain the broadened phase transition and the elevated TM found in
2D crystals. A minimum number of lipids is required to form homogeneous samples of 2D
crystals. This can be understood by examining the number of lipids required to occupy the
VDAC1 annulus, which appears to be roughly 2/3 of the total amount of lipids required to form
2D crystals.
2. We acquired
2H
NMR spectra of chain deuterated DPhPC down to cryogenic
temperatures. We unexpectedly discovered that above 220 K the 2 H spectra yielded isotropic
signals with only residual broadening at the base, despite MD simulation predicting slow chain
motion at these temperatures. We hypothesized that the chain motion effectively reorients the
various fast hopping methyl groups and averages to zero the angular dependence of these spectra.
3. We studied the motion of phenyl group linkers in a zinc based MOF that exhibits
aggregation-induced emission (AIE). We found that the elimination of solvent guest molecules
from the MOF reduces the core size, leading to the loss of phenyl group motions as observed by
2H
NMR. Arrhenius analysis of phenyl group motions at various temperatures agreed well with
191
theoretical DFT calculation, which showed the increased activation barrier for MOF phenyl ring
is predominantly steric in origin and is a result of the ligand being drawn tightly in the rigid
framework.
5.7. Acknowledgments
The authors would like to thank Alexander Barnes, Jeffrey Bryant, Ajay Thakkar, and the
staff of MIT Central Machine Shop for their work in constructing the
2H
NMR probe and the
supporting cryogenic temperature system. We wish to thank Dr. David Ruben for help with dePake-ing. The Biophysical Instrumentation Facility for the Study of Complex Macromolecular
Systems (NSF-0070319 and NIH GM68762) is gratefully acknowledged for help acquiring the
DSC data. R.G.G. is supported through the National Institute of Health (NIH grants EB001960
and EB002026). V.K.M. thanks the Natural Sciences and Engineering Research Council of
Canada for a postdoctoral fellowship.
The MOF work is supported as part of the Center for Excitonics, an Energy Frontier
Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic
Energy Sciences under award number DE-SCOOO 1088 (MIT). We would like to thank the 11 Bsector team at the Advanced Photon Source, Argonne National Laboratory, which was supported
by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under
contract no. DE-AC02-06CH1 1357. Grants from the NSF also provided instrument support to
the DCIF and single crystal X-ray diffraction facility at MIT (CHE-9808061, DBI-9729592,
CHE-0946721). We thank Tarun Narayan for writing the Matlab routine for modeling the
structure of lc.
192
5.8. Supporting Information
Table 5.S1. X-ray crystal structure refinement dataa for TPE-d 20 , la and lb at various
temperatures.
formula
FW
T, K
cryst. syst.,
space group
z
a, A
b, A
c, A
,0
V, A3
daic, g/cm 3
PM-1
pu, mm
F(000)
si ze,
crystal
mm
theta range
index ranges
352.54
298(1)
Monoclinic
P2 1
2
9.837(8)
9.489(8)
10.720(9)
107.12(1)
956(1)
1.224
0.065
352.0
0.08x0.lxO.1
352.54
373(1)
Monoclinic
P2 1
2
9.822(1)
9.600(1)
10.672(1)
106.837(2)
963.2(2)
1.216
0.065
352.0
0.08x. 1 xO. 1
1055.94
100(1)
Monoclinic
C2/c
8
26.446(2)
13.734(1)
17.530(2)
126.487(2)
5119.0(7)
1.320
0.999
2088.0
0.1x.2x0.3
lb
(373 K)
Zn 2C30 D160 8 (
DEF) 2
853.64
373(1)
Orthorhombic
Ibam
4
13.531(3)
18.291(4)
22.172(6)
90
5488(2)
1.006
0.915
1640
0.1x.2x0.3
1.99 to 26.35
-12 < h < 12
-11 <k< 11
-13 1 <13
15417
3902/1 /236
1.99 to 25.68
-11 <h< 11
-10 <k< 11
-12 <k< 13
15580
3442/1 /236
1.77 to 27.16
-33 < h < 33
-9 < k < 17
-22 < k < 22
47494
5650/ 310 / 325
1.84 to 24.72
-15 < h < 6
-19 < k < 21
-25 < k:< 18
14787
2373/40 /142
TPE-d2 '
(298 K)
TPE-d
(373 K)
C 26D 20
C 26D 20
la
(100 K)
Zn 2C30D160(DEF) 2 -2DEF
refl. collected
data/restr./para
m.
1.073
1.089
1.072
GOF on F 2
1.550/-0.722
0.104/-0.102
0.135/-0.135
large
peak/hole,
e/A 3
6.97 (22.07)
3.90 (11.59)
4.40 (12.24)
R1 (wR 2), %
[I>2sigma(I)]b
a Mo-Ka (= 0. 71073 A) radiation
2
b R, = YEljFoj - I Fel/ y IFO1, wR 2 = { y [w(F 0 2-FC
)2]/ E [W(F02)2 1
193
1.090
1.658/-1.157
7.07 (27.64)
100-
IH
-2 DEF molecules
802 DEF molecules
370 0C
.c60So-
40-
-~-
200100
200
300
400
temperature, *C
,
Soo
Figure 5.S1. Thermogravimetric analysis plots for 1 (blue) and 1H (red).
194
600
itL -A
o£
0
10
20
30
2e, deg
40
Figure 5.S2. Simulated (red) and experimental (black) PXRD patterns of 1c.
195
50
TPE
CD2Ck2
20 OC
H4TPEC
CD3OD
20 OC
I
oc
AA
7A
00 C
-20 OC
-20 PC
-50 C
-50 OC
-70 0 C
-70 QC
IA -90
0
7.0
ppm
.A
C
8.0
.
-90
pC
-. p
Figure 5.S3. Variable temperature IH NMR spectra of TPE (left) and H4TCPE (right)in CD 2CL2
and CD 3 0D, respectively.
196
3
4
12
5
6
Table 5.S2. The shortest Ph...Ph contacts, the dihedral angles and the ethylene twist angles
in the determined crystal structures at 93, 298, and 373 K using the indices specified in the
sample TPE structure shown above.
T, K
Ph.. Ph contacts, A
C=C, A
Z3126,
93
298
373
3.583(3)
3.592(6)
3.635(5)
1.356(2)
1.354(3)
1.348(3)
10.2(1)
10.6(2)
11.1(2)
197
0
Z4215,
6.3(1)
8.4(2)
8.9(2)
Ethylene
twist,
8.84
9.69
10.16
20
18
16
14
- 12
E
10
>8
6
4
2
0
0
50
CAr
A-C=C
100
150
Dihedral Angle (deg)
Figure 5.S4. DFT-calculated PES for phenyl ring flipping in styrene (o). A line has been added
as a visual guide.
198
30
25
20
E
15
10
5
0
0
100
50
CAr-CAr-C=O
150
Dihedral Angle (deg)
Figure 5.S5. DFT-calculated PES for phenyl ring flipping in benzoic acid (o). A line has been
added as a visual guide.
199
50
45
40
%0
35
%
I
5 30
E
25
1
20
8 15
i10%%
5
0
50
100
150
CAr-C,-C=O Dihedral Angle (deg)
Figure 5.S6. DFT-calculated PES for phenyl ring flipping in vinylbenzoic acid with orthogonal
ethylene and carboxylic acid groups (o) and coplanar ethylene and carboxylic acid groups (o).
Lines have been added as a visual guide.
200
0
10
20
20, deg
30
40
Figure 5.S7. PXRD patterns of 1c (top) and fully desolvated 1H (bottom) after desolvation at
200 0C.
201
4-
3
1
2
Figure 5.S8. Geometry-optimized conformations of TPE at 47.8', 00, and 900 dihedral
angles (depicted by pink) as obtained by DFT calculations.
Table 5.S3. Activation energies, C=C bond lengths, and selected angles for geometry-optimized
conformations of TPE at fixed CAr-CAr-C=C (47.80, 0', and 900) dihedral angles.
Fixed
E,
C=C,
Z315,
Z312,
Z421,
kJ/mol
A
Z426,
Z512,
Z621,
dihedral
0
0
0
0
0
0
angles,
47.8 (min)
0 (max)
90
(local max)
0
31.6
22.2
1.372
1.375
1.365
115.0
115.5
114.1
114.9
112.8
114.4
122.5
126.5
121.9
122.5
124.5
122.4
122.5
117.5
123.8
122.5
122.5
123.1
Table 5.S4. Dihedral angles for geometry-optimized conformations of TPE at fixed CAr-CAC=C (47.80, 00, and 900) dihedral angles.
Fixed dihedral
/3124,
/5126,
Z3126,
angles, 0
0
0
0
47.8 (min)
0 (max)
90
(local max)
12.8
23.4
1.87
12.7
17.5
12.8
12.8
8.6
7.3
202
Z5124,
Ethylene
twist, 0
12.8
14.5
7.4
12.75
15.28
7.15
1'
2
5 6
Figure 5.S9. DFT-calculated molecular conformations of truncated lb model at fixed CACpr-C=C (1250, 50, and 950) dihedral angles (depicted in pink).
Table 5.S5. Activation energies, C=C bond lengths, and selected angles for geometry-optimized
molecular conformations of a truncated model of lb at fixed CAr-CA--C=C (1250, 50, and 95 0)
dihedral angles.
Fixed dihedral
angles, 0
E,
kJ/mol
C=C,
125 (min
5 (max)
95 (local max)
0
59.4
20.1
1.367
1.366
1.355
A
Z315,
Z426,
Z312,
Z512,
0
0
0
0
Z621,
0
116.4
117.2
116.4
116.1
113.6
117.5
121.7
125.3
121.9
121.9
125.9
121.2
122.0
117.6
121.3
122.0
120.5
121.2
/421,
0
Table 5.S6. Dihedral angles for geometry-optimized molecular conformations of a truncated
model of lb at fixed Co--CM-C=C (125', 50, and 950) dihedral angles.
Fixed dihedral
/3124, 0
Z5126,0
/3126,0
/5124,0
125 (min)
5 (max)
95 (local max)
Ethylene
twist, 0
angles, 0
4.7
2.3
5.0
4.6
2.6
5.5
4.7
1.3
2.1
203
4.7
1.0
2.6
4.65
1.73
3.78
60
50
40
E
30
20
10
0
0
50
100
CArCAr-C=O
150
Dihedral Angle (deg)
Figure 5.S 10. DFT-calculated PES for phenyl ring flipping in terephthalic acid with coplanar
ethylene and carboxylic acid groups (o). A line has been added as a visual guide.
204
Table 5.S7. Electron density (e A7-3) at bond critical points for selected bonds along PES for
ring flipping in truncated model of 1b.
CAr-CAr-
p(C1=
C2)
p(C1C3)
p(C1C5)
p(C2C4)
p(C2C6)
p(C11
-C15)
p(C12
-C16)
p(C13
-C17)
p(C14
-C18)
Zc-c
0
0.1535
0.1227
0.117
0.1204
0.1233
0.122
0.1277
0.127
0.1196
0.9797
10
0.1544
0.1229
0.1178
0.1204
0.1226
0.122
0.1268
0.1265
0.1202
0.9792
20
0.1539
0.1231
0.1189
0.1211
0.1226
0.1226
0.1259
0.1258
0.1211
0.9811
30
0.1539
0.1231
0.1198
0.1213
0.1224
0.123
0.1255
0.1252
0.1219
0.9822
40
0.1543
0.1228
0.1207
0.1216
0.1224
0.1236
0.1246
0.1245
0.1226
0.9828
50
0.1549
0.1224
0.1213
0.1217
0.1222
0.1238
0.124
0.1241
0.1232
0.9827
60
0.1557
0.1215
0.1219
0.1217
0.122
0.1239
0.1235
0.1238
0.1235
0.9818
70
0.1565
0.1214
0.1216
0.1216
0.1219
0.1239
0.1231
0.1236
0.1237
0.9808
80
0.1572
0.121
0.1214
0.1214
0.1216
0.1238
0.1229
0.1235
0.1238
0.9794
90
0.1581
0.1208
0.1211
0.121
0.1212
0.1235
0.1229
0.1234
0.1238
0.9777
100
0.1594
0.121
0.1208
0.121
0.1206
0.1233
0.1233
0.1231
0.1235
0.9766
110
0.1555
0.1215
0.1223
0.1221
0.1219
0.1237
0.1234
0.1234
0.124
0.9823
120
0.1548
0.1219
0.1223
0.1221
0.1221
0.1237
0.1237
0.1235
0.1239
0.9832
130
0.154
0.1225
0.122
0.1221
0.1226
0.1237
0.1241
0.1242
0.1234
0.9846
140
0.1533
0.1229
0.1214
0.122
0.1229
0.1236
0.1247
0.1247
0.1229
0.9851
150
0.1529
0.1231
0.1203
0.1217
0.1232
0.1234
0.1255
0.1253
0.122
0.9845
160
0.1525
0.1231
0.1192
0.1214
0.1234
0.123
0.1263
0.126
0.1212
0.9836
170
0.1526
0.1228
0.1176
0.1208
0.1235
0.1225
0.1273
0.1267
0.1201
0.9813
180
0.1535
0.1227
0.117
0.1204
0.1233
0.1221
0.1277
0.127
0.1197
0.9799
C=C
dihedral
angle (0)
205
0-Zn-O
N
-2 DEF
0
,-Zn-O 0'
-Zn-0
Scheme 5.S1. Depiction of the proposed desolvation process that occurs during the conversion of
lb to 1c.
206
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212
Chapter 6: Topical Developments in High-Field
Dynamic Nuclear Polarization
Adapted from Michaelis, V. K.; Ong, T. C.; Kiesewetter, M. K.; Frantz, D. K.; Walish,
J. J.; Ravera, E.; Luchinat, C.; Swager, T. M.; Griffin, R. G., Isr. J. Chem. 2014, 54,
207-221
Abstract
We report our recent efforts directed at improving high-field DNP experiments. We
investigated a series of thiourea nitroxide radicals and the associated DNP enhancements ranging
from s = 25 to 82 that demonstrate the impact of molecular structure on performance. We directly
polarized low-gamma nuclei including "C,
2H,
and 170 using trityl via the cross effect. We
discuss a variety of sample preparation techniques for DNP with emphasis on the benefit of
methods that do not use a glass-forming cryoprotecting matrix. Lastly, we describe a corrugated
waveguide for use in a 700 MHz / 460 GHz DNP system that improves microwave delivery and
increases enhancement up to 50%.
6.1. Introduction
During the past two decades, magic-angle spinning (MAS) NMR spectroscopy has
emerged as an excellent analytical method to determine atomic-resolution structures in various
chemical systems including pharmaceuticals,1- 3 membrane proteins, 4 -8 amyloid fibrils,9-12 and
oligomers.13-14 Unfortunately, NMR sensitivity is inherently low and consequently many
experiments require long acquisition times to achieve adequate signal to noise. A promising route
213
to increase NMR sensitivity is via dynamic nuclear polarization (DNP), which seeks to polarize
nuclear spins using electron polarization transferred via microwave irradiation of electron-nuclear
transitions. In particular, the method has been shown to provide increases in polarization upwards
of 2 to 3 orders of magnitude.1 5 -2 1
Dynamic nuclear polarization was initially demonstrated in the 1950s at low magnetic
fields. Following the groundbreaking work of Overhauser,
Carver, and Slichter,
various
polarization-transfer mechanisms in solids were studied in the 1960s and 1970s and termed the
solid effect (SE),2 4 2 6 the cross effect (CE),27-31 and thermal mixing (TM).' 9'
32-35
However, the
theoretical understanding of the DNP mechanisms suggested limited applicability at magnetic
fields beyond 1 T. This was followed by a brief exploration of applications of DNP to polymers
at low fields (1.4 T) by Wind et al.19 and Schaefer and co-workers.3637 Moreover, DNP
experiments at higher fields (> 5 T) were hindered by the lack of stable, high-power microwave
devices operating at the necessary high frequencies (e.g., 100 to 600 GHz) and also by the
absence of low-temperature, high-resolution MAS NMR probes that offer both effective
microwave coupling as well as the required sample cooling. Together these barriers prevented
DNP from being widely applicable in the decades following its discovery. In the early 1990's, the
Francis Bitter Magnet Laboratory at MIT (MIT-FBML) introduced high frequency gyrotron
(a.k.a. cyclotron resonance maser) sources to magnetic resonance and DNP in particular since
they can reliably provide high-frequency microwaves. 38 They have now made high-field DNP
viable for many applications. Combined with the improved resolution offered with higher-field
MAS experiments, DNP can now be used to investigate many chemically challenging systems
and areas of NMR spectroscopy including biological solids3 9-43 , surface chemistry44, and systems
involving difficult NMR-active nuclei (e.g., low natural abundance, low gamma and/or
quadrupolar nuclei).
214
The DNP mechanism involves microwave irradiation of the EPR transitions of a
paramagnetic polarizing agent that transfers the large spin polarization of electrons to nearby
nuclei. In order to accomplish this at contemporary NMR fields (i.e., 200 to 1000 MHz), three
criteria must be met: i.) a stable high-frequency microwave source (> 102 GHz), ii.) a reliable
cryogenic MAS probe with adequate microwave waveguide delivery, and iii.) a suitable
polarizing agent for the sample under study. The first criterion was met by the aforementioned
gyrotrons, which are fast wave devices that can deliver the appropriate frequency range for
stimulation of the EPR transitions at high fields, and they can be operated stably and
continuously over an extended period of time (i.e., weeks to months), delivering output power up
to 25 W.5 2 Alternative to gyrotrons, DNP can also be performed at helium cooled temperature (<
70 K) using a low-power (~ 30 - 200 mW) diode microwave source that tunes to the appropriate
frequency. 53 Second, to date DNP is optimally performed at cryogenic temperatures to decrease
electron and nuclear relaxation rates in order to increase the obtainable non-Boltzmann
polarization. To achieve the desired temperature (80-100 K) typically requires a specially
designed heat exchanger and dewar system,5 4 vacuum-jacketed gas-transfer lines, and optional
pre-chillers.55 ~56 The complexity of this instrumentation is further compounded by the need for
MAS in order to obtain high resolution spectra, meaning that carefully designed and constructed
57
multichannel (e.g., 'H/13 C/ 15N/e) low-temperature MAS NMR probes are essential. The third
requirement is the availability of paramagnetic species (polarization agents) that are the
polarization source for various chemical systems. The polarizing agent can be exogenous or
endogenous and most often comes in the form of a free radical. It should be compatible with the
chemical system (e.g., non-reactive), able to yield large DNP enhancements, and chemically
robust. Depending on the application, the radicals and experimental conditions can be developed
to optimize a specific DNP mechanism5 8 -59 such as SE or CE.
215
Over the past two decades, development of high-field DNP has focused primarily on
using the CE mechanism, since the typical SE enhancements had been considerably lower
because it relies on irradiation of forbidden transitions. 60 Below we make mention of both the SE
and CE mechanism as recent results have shown that the SE may be useful for polarization using
transition-metal based polarizing agents 61 and recently has been observed to provide significant
enhancements at approximately 100.62-63 Furthermore, with the continued development of
equipment producing increased microwave field strengths, the enhancements and sensitivity may
match those of CE.64 The dominant polarization transfer process (SE or CE) depends on the
NMR-active nuclei being polarized and also the EPR characteristics of the specific polarizing
agent. Particularly, the relative magnitudes of the electron homogeneous (6) and inhomogeneous
(A) linewidths, and the nuclear Larmor frequency (coo,) are the most important factors to
determine the dominant polarization mechanism.
The SE mechanism is a two-spin process which is dominant when coo > 6, A and
25 26
microwave irradiation is applied at the electron-nuclear zero- or double-quantum transition. - ,
62-63
This matching condition is given by:
COM = coos ± COW
(1)
where coos is the electron Larmor frequency and comw is the microwave frequency. For SE, since
the microwave frequency required must match the condition given in Eq. (1), a polarizing agent
with a narrow EPR spectrum is typically used, with an electron Tis that is optimized to allow
efficient polarization of nearby nuclei without introducing large signal quenching.
The CE mechanism may be described as a three-spin flip-flop-flip process between two
electrons and a nucleus, which is dominant when A > col > 6. In order to achieve maximum
216
efficiency, the difference between the two electron Larmor frequencies must be near the nuclear
Larmor frequency.2 7' 2 9' 65-66
)01
= COs
2
-
COs,
(2)
For CE 67, a radical with a broad EPR linewidth, particularly a nitroxide based radical, is often
used to satisfy the condition provided in Eq. (2). CE is often the choice for high-field DNP
experiments due to this mechanism being based on allowable transitions unlike the SE.
The descriptions for the SE and the CE DNP mechanism, vide supra, do not incorporate
sample rotation. That is, the effects of MAS on modulating energy levels that creates level
crossings and impact polarization transfer. Recently, Thurber and Tycko68 and Mentink-Vigier et
al.69 discussed the CE mechanism in MAS, showed experimental MAS DNP NMR data on the
SH3 protein and described theoretical models of the effect MAS has on both the CE and the SE
mechanism.
In this chapter, we provide a brief overview of recent developments in high-field DNP at
the MIT-FBML, including polarizing agents, sample preparation methods, and improvements to
the 700 MHz / 460 GHz DNP spectrometer.
6.2. Development of CE Biradicals
Nitroxide monoradicals (e.g., TEMPOL) were popular in early high-field DNP
experiments. They are suited for CE DNP of 1H because the breadth of the EPR spectrum is on
the order of -600 MHz at 5 T. 70 They are also low-cost, commercially available, highly watersoluble, and offer reasonable DNP enhancements between s = 20 to 50.38,
71
For these
monoradicals, a concentration of up to 40 mM usually provides the best signal enhancements.
However, at these elevated electron concentrations, paramagnetic relaxation strongly competes
217
with DNP enhancement and only provides moderate electron-electron dipolar couplings between
0.2 to 1.2 MHz. Increasing the concentration of radical (i.e., beyond 40 mM electrons) further is
unsuitable for high-resolution NMR work because of line broadening and signal quenching
72 74
effects at these higher radical concentrations. -
To improve the CE efficiency, biradicals were introduced for DNP in order to improve the
electron-electron dipolar coupling critical to CE DNP while lowering the overall radical
concentration to minimize paramagnetic effects (i.e., signal quenching and broadening).
By
tethering two TEMPO monoradicals, one such biradical, TOTAPOL, 75 has an effective electron electron coupling of ~ 26 MHz, is water-soluble, and provides greater 'H enhancements than
TEMPO based monoradicals by nearly four-fold at 5 T as shown in Figure 6.1. The discovery of
TOTAPOL as a polarization agent and the then-unprecedented signal enhancements it produced
belies the extreme sensitivity of the CE efficiency to molecular perturbations. Tethering nitroxide
radicals introduces several parameters that can be optimized, and synthetic organic chemistry is
the primary tool of modulating dipolar coupling (i.e. inter-electron distance), g-tensor orientation,
water solubility, and relaxation behaviors. All of these factors impact the resulting DNP signal
enhancement. The large synthetic opportunity has led us and others to pursue new generations of
biradicals in order to achieve even greater DNP enhancements. 76 79
218
TOTAPOL
mw on
TEMPO
=160
A
mw on
=45
mw off
180
140
13C
Figure 6.1.
13 C{'H}
100
60
20
Chemical Shift (ppm)
DNP enhanced CPMAS spectra of 13C-urea in a 60/30/10 v/v d 8 -
glycerol/D 2 0/H20 with 20 mM TOTAPOL (top, 'H DNP) and 40 mM TEMPO (bottom, 1H
DNP) acquired at the 140 GHz / 212 MHz DNP NMR spectrometer with 8 W of microwave
power, 4.5 kHz MAS, and 16 scans (on-signal) and 256 scans (off-signal).
Here we examine a series of biradicals that are structural variants of bT-thiourea to
illustrate the impact of molecular structure upon DNP enhancement. The bT-thioureas were
synthesized to improve aqueous solubility exhibited by bT-urea 67 , but they have a lower
enhancement as shown in Figure 6.2. The reason for this reduction in obtainable signal
enhancement from bT-urea to bT-thiourea (bT-thio-3) may be due to a compression of the
TEMPO moieties from the increased steric bulk stemming from the sulfur (as opposed to oxygen)
in the thiourea, or alternatively it may be due to an undesirable gain in torsional mobility upon
switching the urea group to a thiourea group. We observed a further loss of DNP enhancement
upon utilizing the bT-thionourethane (bT-thio-2) biradical. The increased conformational
flexibility of the bT-thionourethane may be deleterious in that the only other conformation
available to this molecule (versus bT-thiourea) features the oxygen-bound TEMPO moiety
beneath the thionourethane linker. This would result in a reduced inter-electron distance similar
219
to other highly-coupled biradicals. 67 Nevertheless,
it should be noted that increasing
conformational flexibility is not always deleterious. bT-thionocarbonate (bT-thio-1) is the most
conformationally flexible structural variant studied, and it shows a larger enhancement than bTthionourethane. The slightly preferred s-trans orientation of thionocarbonates is apparently more
than enough to compensate for the modestly diminished inter-electron distance resulting from the
shorter C-O (vs. C-N) bonds, therefore producing a DNP enhancement similar to that of bTthiourea (bT-thio-3).
The study of the bT-thiourea-based radicals highlights the multi-dimensional problem of
developing radicals for DNP. As the study continues, more effective radicals will be discovered
for DNP application to different chemistry problems. For example, many biradicals currently are
optimized for dissolution in cryoprotectants such as glycerol/water or DMSO/water for studying
biological samples at cryogenic temperatures. 75-76 The glassing behavior of cryoprotectants
disperses the radical homogeneously throughout the sample and allows uniform polarization.
Amongst organic solids, some systems have meta-stable amorphous phases such as the antiinflammatory drug indomethacin, 808- 1 but they may not be miscible with existing biradicals such
as TOTAPOL for effective DNP experiments. For this reason, we used the organic biradical bisTEMPO terephthalate (bTtereph) for our DNP study on amorphous ortho-terphenyl and
amorphous indomethacin. 82 We found that the biradical exhibits similar EPR and DNP profiles as
TOTAPOL (Figure 6.3) and can be incorporated uniformly within amorphous ortho-terphenyl
and indomethacin samples without needing other glassing agents.
220
- = 120 (8)
TOTAPOL
51
s120 (8)
05
IIA4
F
-0
A~A
141
A.
~N~8r~N
F
78 (7)445
4
BT-thio-1
BT-thio-
M
A
Ahoue
0I
7'A:
w
2H
p (NP
A
-0a5mAe
A
TOTAPOL
*BT-urea
A
AATti-
(BTbhhiourea
bT-thio-2
s 2()4955
BT-tio-3Magnetic
4965
4975
4985
499-5
Field (mT)
Figure 6.2. 1H DNP field profiles of various bT-thio based radicals extracted from 13 C_-1H
CPMAS spectra ofI1 C-urea in DMSO/D 2 0/H12 0 (60:30:10, v/v) and 10 mM biradical polarizing
agent (20 mM electrons) acquired at 140 GHz / 212 MIflz DNP NMR spectrometer with 8 W of
microwave power. 'H DNP enhancements were scaled with respect to TOTAPOL using three
thiourea variants. From top to bottom five radicals were studied including TOTAPOL (black),
bT-urea (red), bT-thio- 1 (thionocarbonate, grey), bT-thio-2 (bT-thionourethane, blue) and bTthio-3 (BT-thiourea, green). The spectra inset are the on/off 13 C{1H} CPMAS spectra scaled to
the TOTAPOL enhancement in DMSO/water mixture.
221
+aC
DCMlo
N0
3%
OH
b
75
C
50
25
E
C
0
~-25
-50
-75
4950
4965
4980
4995
Field (mT)
Figure 6.3. bTtereph synthetic process (a) and resulting 140 GHz EPR spectrum (b) and 'H DNP
field profile (c) of 10 mM bTtereph incorporated in 95% deuterated amorphous ortho-terphenyl.
More recently, a new truxene-based radical, TMT, was found to be persistent, having a
half-life (tj/2) of 5.8 h in a non-aqueous solution exposed to air. 83 EPR at 140 GHz shows a gvalue very close to that of BDPA8 4 and a linewidth of 40 MHz (Figure 6.4). The radical may be
ideal for supporting the CE, either alone for low-y nuclei such as "N, or as part of a biradical or
radical mixture with Trityl OX063 or TEMPO.6 0' 85 The current work is aimed at increasing the
radical's solubility in aqueous solvent mixtures suitable for DNP of biological samples and
improving its stability under ambient conditions.
222
(SONa)n,
(SO.Na).\
\/H
S
5
MaHcH2
C
HCH2
CHC2O
H
\
/
-
3
(WMua),,
A= 50 MHz
4990
>
4992 4994 4996
Magnetic Field (mT)
a- Trityl (OX063)
4998
A=40MHz
4990
4992 4994 4996
Magnetic Field (mT)
>
4998
=30 MHz
4990
4992 4994 4996
Magnetic Field (mT)
4998
c - SA-BDPA
b - TMT
Figure 6.4. Chemical structures and 140 GHz EPR spectra of three narrow-line radicals: (a) Trityl,
(b) TMT, and (c) SA-BDPA.
6.3. Direct Polarization of Low-Gamma Nuclei using Trityl
Currently, the conventional wisdom is that the most efficient electron-nuclear transfer
mechanism in the solid state is the CE. Consequently, many polarization agents are designed
from nitroxide based radicals due to their broad EPR profile easily satisfying the CE match
condition in Eq. (2) for 'H. For many systems, polarizing 'H (indirect polarization) by CE is an
effective method because 'H typically have shorter relaxation times, which enables rapid signal
averaging as well as offers additional gains by means of cross-polarization to other low-gamma
nuclei that are often less abundant. However, direct polarization of low-gamma nuclei is also of
19 42
interest considering the theoretical maximum DNP enhancement is given by the ratio ye/yI, , ,4950, 85-88
and the technique would offer a significant sensitivity boost for samples containing no or
low 'H content. Focusing on the five most common nuclei used in biomolecular NMR, three of
them have 1=1/2 (i.e., 'H, "C and '5 N) while 2 H has 1=1 and
170
has 1=5/2. With the exception of
'H, these nuclei are low-gamma and low natural abundance (Table 6.1). Moreover, the latter two
223
nuclei are quadrupolar and consequently experience additional line broadening brought about by
the interaction between the intrinsic electric quadrupole moment and the electric field gradient
(EFG) generated by the surrounding environment, thereby giving rise to quadrupolar coupling.
This additional interaction negatively impacts NMR sensitivity because the quadrupolar coupling
constant covers a spectral range from tens of kHz up to a few MHz. With these factors in mind,
DNP experiments that directly polarize low-gamma and/or quadrupolar nuclei can potentially be
useful and open new possibilities for high field DNP.
For the direct polarization experiments, we can utilize radicals with narrower lines than
needed to polarize protons by CE but still able to satisfy the CE match condition of low-gamma
nuclei. The water-soluble narrow-line monoradical trityl8 9-90 with its EPR spectrum is depicted in
Figure 6.4. The EPR spectrum is considerably narrower than that of the common nitroxide based
radicals, with a linewidth of approximately 50 MHz at 5 T.5085' 91 This narrow profile creates the
possibility for both SE and/or CE mechanism to contribute to the DNP enhancement depending
on the targeted nucleus. In order to determine the effectiveness of trityl on three low-gamma
nuclei (i.e., 11C, 2 H, and 170), a series of DNP experiments were attempted, followed by the
characterization of the mechanisms with assistance from the DNP field profiles (Figure 6.5).
Table 6.1. Physical properties for selected biologically relevant NMR nuclei.
NMR Active
Isotope
N.A. (%)
'H
99.99
1.07
0.01
0.037
0.37
13c
2H
17o
1N
Gyromagnetic
Ratio
(MHz / T)
42.57
10.71
6.53
5.77
-4.31
224
Sensitivity
relative to 'H
Theoretical cma,
1
1.7 x 104
1.11 x 10-6
1.11 x 10-5
3.8 x 10 6
658
2616
4291
4857
6502
For direct polarization of
13 C,
we obtained an enhancement of 480 (Figure 6.6a) using
trityl, which is nearly 180% larger than using TOTAPOL.85 '
88
Examining more closely at the
positive and negative maxima of the DNP profile, we can see there is a clear asymmetry (i.e., 380 vs. 480) present. However, unlike the 'H field profile of trityl 62 there is no feature in the
center of the profile between the two maxima. This suggests that CE polarization mechanism is
making some contribution to the DNP mechanism. Nevertheless, the nuclear Larmor frequency
of
13 C
is slightly larger than the breadth of the trityl EPR spectrum at 5 T, and therefore by
definition the SE must be considered. Looking at the positive and negative maxima of the
13
C
DNP field profile, the positions are in remarkably good agreement (Figure 6.5, blue dotted lines)
with those predicted for the SE mechanism, suggesting a significant contribution.
i
"C -ve SE
H
veSE
j
"C +'ve SE
H-veSE
-
TrItyt
Cente
0.9.
0:
-1.2
0+2H
I
A:
ft
of
-0.3
407
7
497
4A24*4
4A&
486
4
Field (mT)
Figure 6.5. Direct polarization of
13C
(circle, blue), 2H (diamond, red) and 170 (triangle, grey)
field profiles acquired at 5 T using 40 mM Trityl radical. 140 GHz EPR spectrum of trityl (black,
top) with the appropriate SE matching conditions illustrated with the corresponding colored
dashed lines.
225
The nuclear Larmor frequencies of 2 H and 170 are separated by only ~ 4 MHz at 5 T and
appear to behave similarly as the field profiles are nearly overlapping. Although the electron
inhomogeneous linewidth of the trityl radical is small, it is still large enough to satisfy the CE
match condition for both nuclei. Both field profiles do not exhibit resolved features at frequencies
corresponding to oos*± ool (Figure 6.5, red and grey lines), which assures that the CE mechanism
is dominant for both 2 H and 170. For static DNP experiments acquired at 85 K, the
2H
and 170
enhancements are 545 and 115, respectively (Figure 6.6b and 6.6c). This makes trityl still one of
the most effective radicals to polarize such nuclei.49 -s'
92
The EPR spectrum is nearly symmetric
which gives rise to the nearly symmetric positive and negative maxima in the DNP field profile.
The smaller enhancement for 170 may be attributed to the comparably short polarization build-up
time constant (TB = 5.0 ± 0.6 s) inhibiting saturation. This suggests a relatively fast nuclear
relaxation rate that inhibits the build-up of non-Boltzmann polarization. In the case of 2 H and 13C,
both nuclei exhibit larger DNP gains and both have longer TB (Table 6.2). The large quadrupolar
coupling of 170 may also be a factor, and studies are currently underway to elucidate this. We
would also like to note that for all of these nuclei studied the trityl EPR line was not saturated by
using 8 W of microwave power, and further enhancement gains should be possible by increasing
the available microwave power.
Table 6.2. Direct polarization of various biologically relevant nuclei using trityl at 5 T.
Nucleus
__________
1H62
1C0
2H
t
&(positive)
~ 1 % ~
90
480
545
115
c (negative)
10 %)
TB (S)
-81
-380
-565
-116
22
225
75
5.5
226
VL (MHz)
V
Mz
212.03
53.3
32.5
28.7
Mechanism
ehns
SE
CE/SE
CE
CE
a
E =480
mw on
MW off/
x
-6
2
2
6
10
80
'ICFrequency (kHz)
b
E=545
mw on
MW off
35
150
2
-50
50
H Frequency (kHz)
-150
C
E =115
M w on
40
-40
0
170
-80
Frequency (kHz)
Figure 6.6. Direct polarization of low-gamma nuclei using 40 mM trityl on (a)
(b) 2 H (vL
=
13C
(vL = 53 MHz),
32 MHz) and (c) '7 O (vL = 28 MHz) in a glycerol/water cryoprotectant. DNP
enhanced signals were acquired using 8 W of CW microwave power with the magnetic field set
to the optimum field position (positive) shown in Figure 6.5.
6.4. Sample Preparation Techniques
The effective DNP polarization of a biological solid requires a few key criteria to be met.
The first is to disperse the polarizing agent, which allows uniform polarization across the whole
sample followed by effective spin-diffusion. For biological samples such as membrane proteins,
227
amyloid fibrils, and peptides, a cryoprotecting matrix such as glycerol/water or DMSO/water,
which forms an amorphous "glassy" state at low temperatures to protect the sample against
freezing damage, can be used to homogeneously disperse the polarizing agent for DNP. Labeling
of the cryoprotecting matrix, in particular D2 0, deuterated glycerol, and deuterated DMSO, can
be used to fine tune IH-1H spin-diffusion to optimize the obtainable DNP enhancement, while
reverse labeling the matrix (e.g.,
12C-glycerol)
can minimize solvent background. In our
experience, a cryoprotecting matrix that is heavily deuterated is optimal for DNP, and typically
we prepare our samples in a 60/30/10 v/v d8 -glycerol/D 20/H20. However, the NMR of a
homogeneous, amorphous chemical system can be limited in resolution due to line-broadening
stemming from a distribution of chemical shift, a commonly observed occurrence for many
organic and inorganic amorphous materials, as well as from slower side-chain dynamics at
cryogenic temperatures.
Despite this limitation, DNP has been successfully applied to
heterogeneous systems like the membrane protein bacteriorhodopsin 15 , 39-40, 52,
93
and M2 94 , and
by combining with methods including specific labeling95-97 and crystal suspension in liquid 41' 44'
98-100
DNP NMR also has been demonstrated on various chemical
systems without adding a
cryoprotectant, due to either thermal stability or self-cryoprotecting ability.82 ' 101-04
Figure 6.7 illustrates the various sample preparation methods both with and without
cryoprotecting matrix. Figure 6.7a and b show DNP of amorphous and crystalline 95%
deuterated ortho-terphenyl. While both samples show large 'H DNP enhancements, the
crystalline sample has somewhat improved resolution of the various 13C resonances. The
resolution as described above is not impacted by temperature, but by the distribution in chemical
shift brought about by the formation of a disordered homogeneous solid. Figure 6.7c and d show
DNP enhanced spectra of apoferritin complex (480 kDa) prepared using either a traditional
glycerol/water cryoprotectant (Figure 6.7d) or the new sedimentation method (SedDNP) (Figure
228
06
6.7c) where the amount of free water is significantly reduced' -1 either by ultracentrifugation
101
(ex situ)'0 2, 107-109 or via fast magic angle spinning (in situ). ' 110-111
Either sedimentation
method results in a "microcrystalline" glass that effectively distributes the polarizing agent within
the sample, allows efficient spin diffusion through the whole sample, and protects against
potential damage from ice crystal formation. Both approaches provide high sensitivity, however
the sedimentation method minimizes the solvent present and so reduces the solvent resonances
(e.g., glycerol at ~60-70 ppm) while improving the overall filling factor when using the ex situ
method. The sedimentation technique has an added advantage where cooling to cryogenic
temperatures and employing DNP can offer additional structural information and constraint not
observed at experiments performed at ambient condition. The low temperature spectra can
provide extensive information on side chain motion and details concerning aromatic regions that
95
,112
are often lost due to decoupling interference at room temperature.
Finally, nanocrystalline preparation of GNNQQNY 98 ' 113 (Figure 6.7e) by suspension in a
cryoprotecting
matrix provides high resolution and DNP enhancement
for structural
understanding in both crystalline and amyloid forms. Wetting of microcrystals has also been
attractive for the study of various surface science questions whereby a nitroxide biradical is
dispersed into an organic solvent and added to the crystalline material of choice prior to
cooling.44 '100'114 Furthermore, a solvent-free dehydration approach whereby the radical is placed
onto the system such as glucose or cellulose, followed by evaporation has also recently shown
promise for natural abundance systems. 103 -104 Although these methods lead to a more
heterogeneous distribution of radicals and hence polarization is not uniform within the samples,
they maintain excellent sensitivity and produce excellent spectral resolution from an overall
smaller effect from paramagnetic broadening.
229
sans
cryoprotectant
Amorphous
E = 58
Crystalline
E=
b
36
Sedimented
E = 42
Dissolved
avec
E
cryoprotectant
= 100
d
Microcrystalline
E
200
160
120
80
40
13
C Chemical Shift (ppm)
=20
0
Figure 6.7. MAS DNP sample preparation protocols for biophysical systems. Without
cryoprotecting solvents (sans) include distributing a polarizing agent within the organic solid:
amorphous (a) or crystalline (b) 95% deuterated ortho-terphenyl with 0.5 mol% bTtereph or
using the SedDNP approach, U-' 3 C,' 5N-Apoferritin (2 mM TOTAPOL) (c). Alternative is
distributing the radical in a cryoprotecting solvent (avec) homogenously, U-'3 C, 15N-Apoferritin
in d8 -glycerol/D 2 0/H20 (v/v 60/35/5) and 15 mM TOTAPOL (d) or heterogeneously using
microcrystals, [U
13C, 15N
GNNQ]QNY in d8 -glycerol/D 20/H20 (w/w 70/23/7) and 35 mM
TOTAPOL (e).
230
6.5. Improving DNP Instrumentation at High Fields (2 16 T)
In recent years, high-field DNP has evolved beyond 9.4 T (400 MHz, 'H). The innovation
in gyrotron technology has led to more adoptions of high-field DNP spectrometers such as the
600 MHz / 395 GHz 5'115 (Osaka University, Japan and University of Warwick, UK), the 700
MHz / 460 GHz5 5 (MIT, Cambridge, MA), and the commercial 600 MHz/ 395 GHz and 800
MHz / 527 GHz from Bruker Biospin. However, DNP theory predicts the experiment to be less
effective at high fields, with an inverse scaling of CE DNP and an inverse-squared scaling of SE
DNP enhancement
with respect to increasing magnetic
field. 5
This is because the
inhomogeneous EPR linewidth of the polarizing agent increases proportionally with respect to
the magnetic field (A a B0), meaning that the CE matching condition becomes harder to satisfy.
The challenge is compounded by the difficult tasks of maintaining effective cooling capabilities
at elevated MAS frequencies (e.g., limiting frictional heating) and also coupling gyrotron
microwaves to the NMR sample. Therefore, considerable effort has been made to improve
instrumentation in order to gain reasonable DNP enhancement at these fields. Given the inherent
better resolution of high field NMR (vide infra), successful DNP can become a valuable approach
to obtain structural information on challenging biological samples.
One particular difficulty in implementing DNP at higher magnetic fields is the
transmission of high-power microwaves from the gyrotron to the sample with minimal loss. This
can be achieved by using corrugated overmoded waveguides, which are more efficient than the
previously used fundamental mode waveguides, to minimize mode conversion and ohmic loss. At
the MIT-FBML, the microwave source of the 700 MHz DNP system is a 460 GHz gyrotron
operating in the second harmonic, in a TE,1, 2 mode."16 The produced microwaves are guided
through a ~ 465 cm long, 19.05 mm inner diameter (i.d.) corrugated waveguide that connects the
231
16.4 T NMR magnet and the 8.2 T gyrotron magnet. The alignment is critical to maintain a clean
microwave mode with minimum energy loss through the long waveguide, and we were able to
achieve less than 1 dB loss from the gyrotron window to the final miter-bend that directs the
microwaves into the probe body. The final -107 cm of the waveguide is located within the NMR
probe, and it was initially constructed by a series of down tapers reducing the i.d. from 19.05 to
4.6 mm. using a combination of smooth-walled macor, aluminum and copper waveguide portions.
However, due to the significant loss of microwave power associated with 4.6 mm waveguide and
macor sections at 460 GHz (X = 0.65 mm), several changes were implemented to improve
microwave transmission to the sample. A newly designed waveguide for our home-built DNP
NMR probes now includes a modified tapered and corrugated aluminum waveguide section from
19.05 to 11.43 mm i.d. at the base of the NMR probe (Figure 6.8), and at which point the
microwaves are directed toward the stator via a 450 miter-bend. The microwaves are then
reflected off a copper mirror into a multi-section corrugated waveguide with an 11.43 mm i.d.
consisting of a stainless steel section at the base which acts as a thermal break followed by two
copper sections. The final 50 mm portion approaches the reverse magic-angle microwave beam
launcher and features an aluminum corrugated part that is tapered from 11.43 to 8 mm i.d. in
order to direct and focus the microwave beam into the 3.2 mm MAS stator housing. A small
Vespel* washer is installed prior to the final taper to act as an electrical break between the
microwaves and the RF. Finally, the waveguide is terminated by a copper microwave launcher at
the reverse magic-angle, and aligned using three brass set screws. With these modifications, the
new probe waveguide design reduces the loss of microwave power being transmitted to the
sample while maintaining the effective Gaussian beam content. The new design has improved the
high-field DNP enhancements by 40-50%, from -38 (4) to -53 (5) on a sample of 1 M
1C-urea
at
80 (2) K with MAS frequency of 5.2 kHz, and from -21 to -33 on a sample of 0.5 M U- 13 C232
proline with MAS frequency of 9.2 kHz. Figure 6.9 shows a DNP enhanced "C-"C DARR
spectrum of U- 3 C-proline that illustrates the good resolution and sensitivity gain that can be
achieved with high field DNP.
Figure 6.8. Artistic rendering of the new waveguide designed for the 460 GHz / 700 MHz DNP
NMR spectrometer (FBML-MIT). The inset is an
13
C{ 'H}
CPMAS spectrum (mw on/off) of 1 M
13
C-Urea in d8-glycerol/D 20/H20 (v/v 60/30/10) with 10 mM TOTAPOL and packed into a 3.2
mm sapphire rotor, acquired at 80 K and a spinning frequency of 5.2 kHz. Abbreviations: copper
(Cu), aluminum (Al) and stainless steel (SS).
233
-0
E = 33
Et = 115
Is
0
9
A
f
e
-50
0
-100
130
13C
OH
13C
a
13C
130.....-
150
NH
CO,
200
200
150
100
50
0
B
.-
C10
20
CP
30
0v
40
50
CO
0
170
160
70
60
50
40
C60
30
20
10
70
13
C Chemical Shift (ppm)
Figure 6.9. (A) 13C-13 C DARR spectrum of U-13C-Proline (0.5 M) in d8-glycerol/D 2 0/H2 0 (v/v
60/30/10) with 10 mM TOTAPOL (1H enhancement of 33 (3)) using a 20 ms DARR mixing
period. (B) An enlarged aliphatic and carbonyl region illustrating the connectivity of U-1 3CProline. Sample was packed into a 3.2 mm sapphire rotor, data was acquired with 8 scans, rd = 20
s, 64 increments, 11 W of microwave power, sample temperature 82 (2) K and a spinning
frequency of 9.2 kHz.
234
We recently used the improved 700 MHz DNP system to study apoferritin, which is an
important protein for maintaining available non-toxic soluble forms of iron in various
organisms. 117-118
Apoferritin, the iron-free form, is a 480 kDa globular protein complex
consisting of 24 subunits, with each unit being 20 kDa in size. The protein is a challenging
system for NMR
19-120
due to its large size comprised of nearly 4,000 residues.1
Nevertheless,
chemical shift separation can be achieved at higher magnetic fields, and structural insight can be
gained through a combination of approaches including solution and solid-state methods (i.e.,
SedNMR)111 , 119-120 as well as combining with DNP (i.e., SedDNP). 10' Figure 6.10 is an overlay
of U-13 C-apoferritin collected at 212 MHz / 140 GHz and 697 MHz / 460 GHz employing a 13C13 C
PDSD dipolar recoupling experiment. Although the DNP enhancement is lower at the higher
field (, = -6, with s_ = -21 accounting for Boltzmann population difference between cryogenic
and room temperature, defined as ef = E(TRT/TDNP)) compares to the lower field enhancement (s =
42), we can see that the aliphatic region is significantly more dispersed in the higher field
spectrum enabling differentiation between the Ca and Cp region. Continuing effort at improving
instrumentation and developing new radicals will potentially increase enhancement further than
what is currently obtainable.
235
0-
r_J. .Q
A
-a---
-
-
B
20
50-
E.
a
e
40
0
C)
100-
700 MHz/460 GHz - 15 pl - E -6 / El
212 MHz/140 GHz - 60 p1 - E.=42 / E
-C
0
21 N
147
60
150170
/
180
150
200
100
40
60
50
20
13
C Chemical Shift (ppm)
Figure 6.10.
13C- 13 C
correlation spectrum of U- 3 C-apoferritin at 5 T (red) and 16.4 T (blue)
using DNP MAS NMR.
6.6. Conclusion
In this topical review, we discussed the recent DNP efforts at MIT-FBML including new
radical polarization-agent development, direct polarization of low-gamma nuclei, various sample
preparation methods, and hardware improvements to the MIT-FBML 700 MHz / 460 GHz DNP
NMR spectrometer.
As developmental
efforts
continue
and
along with
the recent
commercialization of DNP systems, we foresee the method achieving greater sensitivity for
NMR and becoming a more general method to study various biological and chemical systems.
We expect the wider adoption of DNP to be a very fruitful endeavor leading to many new and
exciting scientific discoveries.
236
6.7. Acknowledgements
The authors would like to thank Bjorn Corzilius, Eugenio Daviso, Albert Smith, Loren
Andreas, Galia Debelouchina, Jennifer Mathies, Michael Colvin, Emilio Nanni, Sudheer Jawla,
Ivan Mastrovsky and Richard Temkin for helpful discussions during the course of this research.
Ajay Thakkar, Jeffrey Bryant, Ron DeRocher, Michael Mullins, David Ruben and Chris Turner
are thanked for technical assistance. The National Institutes of Health through grants EB002804,
EB003151, EB002026, EB001960, EB001035, EB001965, and EB004866 supported this
research. This work has been supported by Ente Cassa di Risparmio di Firenze, the European
Commission, contract Bio-NMR no. 261863, and Instruct, part of the ESFRI, MIUR PRIN
(2009FAKHZT_001) and supported by national member subscriptions. Specifically, we thank the
EU ESFRI Instruct Core Centre CERM, Italy. V.K.M. acknowledges the Natural Science and
Engineering Research Council of Canada for a Postdoctoral Fellowship.
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Ta-Chung Ong
EDUCATION
Massachusetts Institute of Technology
Ph.D in Physical Chemistry
June 2014
Cambridge, MA
Colby College
B.A. in Physics and Chemistry. Summa Cum Laude
May 2007
Waterville, ME
Member of Phi Beta Kappa and Sigma Pi Sigma, the Physics Honors Society
American Institute of Chemists Award (2007) - Awarded for outstanding
achievement, ability, leadership, and professional promise in chemistry.
RESEARCH
EXPERIENCE
Cambridge, MA
Massachusetts Institute of Technology
2008-Present
GraduateResearch Assistant
Group of Prof. Robert G. Griffin
Department of Chemistry
Francis Bitter Magnet Laboratory
Thesis title: "Dynamic nuclear polarization of amorphous and crystalline
small molecules"
Colby College
Waterville, ME
UndergraduateResearch Assistant
2005-2007
Group of Prof. D. Whitney King
Department of Chemistry
Thesis title: "Detailed mechanistic and optimization of the photochemical
production method of superoxide"
PUBLICATIONS
Michaelis, V. K., Ong, T. C., Kiesewetter, M. K., Frantz, D. K., Walish, J. J., Ravera, E.,
Luchinat, C., Swager, T. M., Griffin, R. G. Topical developments in high-field dynamic nuclear
polarization. Isr. J. Chem. 2014, 54, 207-221
Li, X., Michaelis, V. K., Ong, T. C., Smith, S. J., McKay, I., Muller, P., Griffin, R. G., Wang, E.
N. One-pot solvothermal synthesis of well-ordered layered sodium aluminoalcoholate complex:
a useful precursor for the preparation of porous A12 0 3 particles. CrystEngComm. In press
Li, X., Michaelis, V. K., Ong, T. C., Smith, S. J., Griffin, R. G., Wang, E. N. Designed singlestep synthesis, structure, and derivative textural properties of well-ordered layered pentacoordinate silicon alcoholate complexes. Chem. Eur. J.In press
243
Bertrand, G. H. V., Michaelis, V. K., Ong, T. C., Griffin, R. G., Dincd, M. Thiophene-based
covalent organic frameworks. Proc. Nat. Acad Sci. USA 2013, 110, 4923-4928
Ong, T. C., Mak-Jurkauskas, M. L., Walish, J. J., Michaelis, V. K., Corzilius, B., Smith, A. A.,
Clausen, A. M., Cheetham, J. C., Swager, T. M., Griffin, R. G. Solvent-free dynamic nuclear
polarization of amorphous and crystalline ortho-terphenyl.J.Phys. Chem. B 2013, 117(10),
3040-3046
Shustova, N. B., Ong, T. C., Cozzolino, A. F., Michaelis, V. K., Griffin, R. G., Dinca, M.
Phenyl ring dynamics in a tetraphenylethylene-bridged metal-organic framework: Implications
for the mechanism of aggregation-induced emission. J.Am. Chem. Soc. 2012, 134(36), 1506115070
Eddy, M. T., Ong, T. C., Clark, L., Teijido, 0., van der Wel, P. C. A., Garces, R., Wagner, R.,
Rostovtseva, T. K., Griffin, R. G. Lipid dynamics and protein-lipid interactions in 2D crystals
formed with P-barrel integral membrane protein VDAC1. J.Am. Chem. Soc. 2012, 134(14),
6375-6387
POSTER PRESENTATIONS
Ong, T. C., Mak-Jurkauskas, M. L., Walish, J. J., Michaelis, V. K., Clausen, A. M., Cheetham, J.
C., Swager, T. M., Griffin, R. G. Solvent-free dynamic nuclear polarization of amorphous and
crystalline ortho-terphenyl. 53 rd Experimental Nuclear Magnetic Resonance Conference, Miami,
Florida. Poster Presentation on April 15, 2012.
Ong, T. C., King, D. W. Photochemical production of micromolar superoxide standards in
aqueous solution. 233rd ACS National Meeting, Chicago, Illinois. Poster Presentation on March
25, 2007.
Ong, T. C., Lin, H., King, D. W. Spatial distribution of Gloeotrichia Echinulada in Great and
Long Pond, Maine. Maine Water Conference, Augusta, Maine. Poster Presentation on March 22,
2006.
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