Hyperpolarization with Parahydrogen in NMR

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Sören Lehmkuhl, M.Sc.

aus Göttingen

Berichter: Prof. Dr. rer. nat. Dr. h.c. Bernhard Blümich Prof. Dr. rer. nat. Stephan Appelt Prof. Dr. rer. nat. Thomas Theis

Tag der mündlichen Prüfung: 19.02.2019

Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek verfügbar. 2 Hyperpolarization with Parahydrogen in NMR

Hyperpolarization with Parahydrogen in NMR

Parts of this thesis have already been published. My contributions are written in italic:

Suefke, M., Lehmkuhl, S., Liebisch, A., Blümich, B., Appelt, S., 2017. Para-hydrogen raser delivers sub-millihertz resolution in nuclear magnetic resonance. Nature Physics 13, 568-572. All authors wrote the manuscript. I designed, optimized and prepared all SABRE samples to surpass the RASER threshold and selected appropriate substrates. The experiments were conducted together with Martin Süfke, Alexander Liebisch and Stephan Appelt. I discussed the experimental results and contributed to the developed model for the spin order transfer mechanism.

Lehmkuhl, S., Emondts, M., Schubert, L., Spannring, P., Klankermayer, J., Blümich, B., Schleker, P.P.M., 2017. Hyperpolarizing Water with Parahydrogen. Chemphyschem 18, 2426-2429. I wrote the original draft of the manuscript until Meike Emondts and Phillip Schleker revised it. As all authors, I contributed to the final version of the publication. I initiated the project, prepared the SABRE samples, conducted the experiments and analyzed the experimental results together with Lukas Schubert.

Lehmkuhl, S., Wiese, M., Schubert, L., Held, M., Küppers, M., Wessling, M., Blümich, B., 2018. Continuous hyperpolarization with parahydrogen in a membrane reactor. J Magn Reson 291, 8-13. I wrote the manuscript and all authors contributed to its final version. Martin Wiese and I designed the experimental setup and supervised the experiments. I analyzed the experimental results together with Lukas Schubert.

In Preparation:

Appelt, S., Kentner, A., Lehmkuhl, S., Blümich, B., From LASER physics to the para-hydrogen pumped RASER, invited for Prog Nucl Magn Reson Spectrosc.

For this manuscript, I supported Stephan Appelt to write and proofread the manuscript, designed a system, which enabled giant RASER pulses, prepared all SABRE samples and conducted the experiments together with Arne Kentner and Stephan Appelt.

Hyperpolarization with Parahydrogen in NMR 3

Acknowledgement

A large number of people have supported and contributed to this thesis in various ways. Here, I would like to acknowledge some of them. First of all, I would like to thank my doctorate supervisor Prof. Dr. Dr. h.c Bernhard Blümich, who entrusted me with the chance to work in his group. Despite being burdened by many other issues, he always took the time to discuss my research and allowed me to work on the topics I found most interesting and encouraged me to develop and follow my own ideas. Secondly, I would like to thank my co-supervisor Prof. Dr. Stephan Appelt, for the informal atmosphere and patience to discuss current scientific subjects concerning the RASER project and far beyond it. Thanks to all former and present colleagues at the ITMC of the RWTH Aachen University for the nice working atmosphere, enriching discussions and fruitful cooperation. Special thanks go several people: - to Dr. Markus Küppers, who always found time to listen and his administrative support. - to all my students Stefan Benders, Anna Friedrich, Bin Kang, Larissa Klass, Annette Nordhausen and Monica Spritzky for their great work and especially Lukas Schubert, who also supported the lab for more than a year. - to the chemical apprentice Alexander Eltester for good work in the lab - to Dr. Meike Emondts, Dr. Peter Spannring and Dr. Philipp Schleker for the collaboration on the water project, the latter additionally for his initial support on the Augustine project. - to Martin Süfke, Alexander Liebisch and Arne Kentner for the exciting mutual work on the raser project. - and finally to my colleague Martin Wiese from the AVT for the productive cooperation on the DGF project “Mikro-PHIP NMR”, which lead to “Continuous hyperpolarization with a membrane reactor”. Next, I would like to thank Prof. Dr. Simon Duckett who introduced the SABRE approach to me during my ERASMUS exchange as a master student at the University of York. This includes his working group, especially Dr. Marianna Fekete and Dr. Ryan Mewis who supervised me. Special thanks to my friend and colleague Patrick Offer for inspiring discussions, relaxing holidays for distraction when needed and the proofreading of this work. Further personal thanks are dedicated to all my friends, especially Jean-André Schramm and Andreas Schümmer, to help me sustain a healthy work-life balance. This also includes my friend Kathy Schmitz for hosting the reoccurring Sunday evening events and all those who contributed to them. Finally, I am grateful for the support and encouragement of my parents Carola and Frank throughout my whole duration of study and my brother Till, who was always there for me and proofread this work. 4 Hyperpolarization with Parahydrogen in NMR

Table of contents

Hyperpolarization with Parahydrogen in NMR ...... 2 Acknowledgement...... 3 Table of contents ...... 4 List of figures ...... 7 List of tables ...... 10 Abbreviations ...... 11 Variables ...... 15 Important symbols and constants ...... 17

1 Introduction ...... 18 1.1 Sensitivity in NMR ...... 20 1.2 Hyperpolarization in low field NMR ...... 23

2 Theory: Parahydrogen based hyperpolarization in NMR ...... 25 2.1 What is parahydrogen? ...... 26 2.2 Parahydrogen induced polarization (PHIP) ...... 29 2.2.1 The catalytic cycle ...... 30 2.2.2 Heterogeneous PHIP catalysts ...... 32 2.2.3 Pairwise replacement and other exchange mechanisms ...... 32 2.3 Signal amplification by reversible exchange (SABRE) ...... 34 2.3.1 The catalytic cycle ...... 35 2.3.2 Substrate classes and applications...... 38 2.4 Efficiency in hyperpolarization experiments ...... 39 2.5 Spin order transfer mechanisms in PHIP experiments ...... 41 2.5.1 Level anti crossings (LACs) ...... 42 2.5.2 The role of LACs in SABRE experiments ...... 43

3 Devices, synthesis and experimental procedures ...... 48 3.1 Devices...... 48 3.1.1 Magnets as detection fields ...... 49 3.1.2 Magnets for polarization transfer fields ...... 51 3.1.3 Parahydrogen generators ...... 51 3.2 Synthesis ...... 52 3.2.1 Synthesis of [IrCl(COD)(IMes)] ...... 52

3.2.2 Synthesis of [Rh(COD)-(S)-BINAP]BF4 ...... 53 3.2.3 Activation of homogeneous catalyst precursors ...... 55 3.3 Experimental section ...... 56 Hyperpolarization with Parahydrogen in NMR 5

3.3.1 Shake-and-drop experiments ...... 56 3.3.2 Data acquisition and processing...... 57

4 A modular immobilization system ...... 58 4.1 Introduction ...... 59 4.2 Towards a model system for hyperpolarization experiments ...... 62

4.2.1 The homogeneous [Rh(COD)-(S)-BINAP]BF4 catalyst, butylacrylate and parahydrogen – a model system ...... 62 4.2.2 Support materials ...... 64 4.2.3 Activation of the immobilized catalysts ...... 65 4.3 Methods ...... 66

4.3.1 Synthesis of PTA@Al2O3 ...... 67

4.3.2 Immobilization of homogeneous catalyst precursors on PTA@Al2O3 ...... 68 4.3.3 Sample preparation ...... 69 4.3.4 Detection and data processing ...... 70 4.4 Evaluation for hyperpolarization experiments ...... 70 4.4.1 Particle size of the support material ...... 71 4.4.2 Experimental parameters ...... 72 4.4.3 Other catalytic systems ...... 75 4.4.4 High field experiments / PASADENA conditions ...... 82 4.5 Conclusion and outlook ...... 83

5 Hyperpolarizing water with parahydrogen ...... 86 5.1 Introduction ...... 87 5.2 Methods ...... 88 5.3 Results and discussion ...... 89 5.3.1 Hyperpolarization of water ...... 89 15 5.3.2 Hyperpolarization of N3-histidine: ...... 92 5.3.3 Mechanistic considerations ...... 95 5.4 Conclusion and outlook ...... 97

6 Continuous flow hyperpolarization ...... 99 6.1 Introduction ...... 100 6.2 Methods ...... 101 6.3 Results and discussion ...... 103 6.3.1 Single-scan hyperpolarization spectra ...... 104 6.3.2 The liquid and gas phase pressures ...... 105 6.3.3 The volume flow ...... 107 6.3.4 Comparison with traditional SABRE methods ...... 108 6.4 Conclusion and outlook ...... 109 6 Hyperpolarization with Parahydrogen in NMR

7 The parahydrogen fueled NMR RASER ...... 110 7.1 Introduction ...... 111 7.2 Methods ...... 112 7.3 Results and discussion ...... 114 7.3.1 Single mode RASER ...... 115 7.3.2 Initial nonlinear dynamics ...... 116 7.3.3 Heteronuclei ...... 120 7.3.4 Two-spin ordered RASER ...... 124 7.3.5 Multi-mode RASER ...... 125 7.3.6 Implications of the parahydrogen fueled multi-mode RASER ...... 128 7.4 Conclusion and outlook ...... 129

8 Summary ...... 131

9 Supplement: A modular immobilization system ...... 132 9.1 Other immobilization approaches – an excursus ...... 132 9.1.1 Rh nanoparticles on a glass slide ...... 132 9.1.2 Iridium catalysts with phosphine ligands ...... 134 9.2 Further measurements with the modular immobilization system ...... 140 9.2.1 Methylacrylate and styrene ...... 140

9.2.2 [Ir(COD)(IMes)]Cl@Al2O3 and [Rh(COD)(Duphos)][BF4]@Al2O3 ...... 141 9.2.3 Further hyperpolarization experiments at high field ...... 142

Supplement: Hyperpolarizing water with parahydrogen ...... 145

Supplement: The parahydrogen fueled NMR RASER...... 147

References ...... 149

Hyperpolarization with Parahydrogen in NMR 7

List of figures

Figure 1: Guideline through “Hyperpolarization with Parahydrogen in NMR” ...... 18 Figure 2. Zeemann splitting ...... 20 Figure 3. Guideline through the first three sections of chapter 2 ...... 25 Figure 4. Scope of the final two theory sections ...... 26 Figure 5. The four nuclear spin isomers of hydrogen ...... 27 Figure 6. Temperature dependent fraction of parahydrogen ...... 28

Figure 7. Hydrogenation of an olefin with p-H2 ...... 29 Figure 8. Activation of catalyst precursors with hydrogen gas ...... 30

Figure 9. General catalytic cycle of a hydrogenation with p-H2 ...... 31 Figure 10. Pairwise replacement ...... 32 Figure 11. Mechanism of the pairwise replacement of hydrogen ...... 33 Figure 12. The SABRE approach ...... 34 Figure 13. Activation of the precursor IMes ...... 35 + Figure 14. Schematic drawing of the spin order transfer by SABRE in [Ir(IMes)H2Sub3] ...... 36 Figure 15. Catalytic cycle of the SABRE hyperpolarization...... 37 Figure 16. NMR states and transitions in PHIP experiments ...... 41 Figure 17. Energy diagrams to illustrate a LAC ...... 42 Figure 18. Spin order transfer by SABRE ...... 44 Figure 19. Guideline through chapter 3 ...... 48 Figure 20. Low field spectrometers ...... 50 Figure 21. High field Spectrometers ...... 50 Figure 22. Magnets for polarization transfer fields ...... 51 Figure 23. Parahydrogen generators ...... 52 Figure 24. Synthesis of IMes ...... 53 Figure 25. 1H NMR spectrum of the synthesized IMes ...... 53 Figure 26. Synthesis of BINAP...... 54 Figure 27. 1H NMR spectrum of the synthesized BINAP ...... 54 Figure 28. 31P NMR spectrum of the synthesized BINAP ...... 55 Figure 29. Activation of the homogeneous catalyst precursors a) IMes and b) BINAP56 Figure 30. Scope of the chapter “A modular immobilization system”...... 59 Figure 31. HPA as linker ...... 61 Figure 32. Different hyperpolarization mechanisms of butylacrylate ...... 63 Figure 33. 1H NMR Hyperpolarization experiments with a) the homogeneous BINAP catalyst and b) the immobilized BINAP@Al2O3 ...... 64

Figure 34. Crystal structures of α- and γ-Al2O3 ...... 65 225 Figure 35. Synthesis of PTA @Al2O3 with the impregnation technique...... 67

Figure 36. Immobilization on a α-Al2O3 hollow fiber ...... 68

Figure 37. Synthesis of BINAP@Al2O3 ...... 69 8 Hyperpolarization with Parahydrogen in NMR

Figure 38: 1H NMR Hyperpolarization experiments with different particle sizes of the γ- Al2O3 support material ...... 72 Figure 39. 1H NMR hyperpolarization experiments at different polarization transfer field and amount of heterogeneous catalyst ...... 73 1 Figure 40. H NMR hyperpolarization experiments at different p-H2 pressures and temperatures of a pre-heating bath ...... 74 Figure 41. 1H NMR hyperpolarization experiments at particle sizes of the support material ...... 75 Figure 42. Different ligands bound to a transition metal center (M) that are known in hydrogenation reactions and were used in this work ...... 77 Figure 43. 1H NMR spectra from hyperpolarization experiments with other anchored homogeneous catalysts ...... 78 Figure 44. 1H NMR spectra from hyperpolarization experiments with methylacrylate and dimethylitaconate ...... 80 Figure 45. Shake-and-drop hyperpolarization experiments with styrene ...... 81 Figure 46. 1H NMR hyperpolarization experiment at 300 MHz under PASADENA conditions ...... 83 Figure 47. Scope of the chapter “Hyperpolarizing water with parahydrogen”...... 86 Figure 48. a) The IDEG precursor from Spannring et.al49 and b) the additive L-histidine employed to polarize water with parahydrogen...... 88 Figure 49. 1H NMR spectrum recorded under the standard conditions for the hyperpolarization experiments of water ...... 89

Figure 50. Intensity of the water polarization depending on a) the p-H2 pressure and b) the temperature of the pre-heating bath ...... 90 Figure 51. a) 1H NMR spectra of hyperpolarized water depending on the magnetic evolution field Bevo and b) the intensity of the water peak corresponding to these spectra...... 92 1 15 Figure 52. H NMR spectrum featuring hyperpolarized N3-histidine, HDO, HD at a) Bevo = 70 G and b) Bevo = 220 G ...... 93 Figure 53. 1H NMR spectra at different Bevo ...... 94 15 Figure 54. Polarization of water (blue triangles) and N3-histidine (red circles) from the hyperpolarization experiments depending on the polarization transfer field Bevo ...... 95 Figure 55. Scope of the chapter “Continuous flow hyperpolarization” ...... 99 Figure 56. Sketch of the continuous flow hyperpolarization setup ...... 103 Figure 57. 1H Single scan NMR spectra of the continuous flow hyperpolarization of pyridine and nicotinamide ...... 104 Figure 58. Hydride region of the 1H NMR spectra from Figure 57 ...... 105 Figure 59. Pressure-dependent signal enhancement of nicotinamide and pyridine 106 Figure 60. Average 1H NMR signal enhancement of a) pyridine and b) nicotinamide depending on the volume flow rate ...... 107 Figure 61. Scope of the chapter “The parahydrogen fueled NMR RASER” ...... 110

Figure 62. Activation of the IMes precursor under continuous p-H2 bubbling ...... 112 Hyperpolarization with Parahydrogen in NMR 9

Figure 63. Structural formulas of the SABRE substrates employed in the RASER experiments...... 113 Figure 64. Pictures of a RASER experiment with the ultra-low field setup ...... 114 Figure 65. A starting 1H RASER of a) pyridine and b) acetonitrile ...... 116 Figure 66. Simulation of the transverse spin component α (red) and the polarization PZ (blue) in a 1H RASER experiment for a) pyridine and b) acetonitrile ...... 118 Figure 67. Initial nonlinear dynamics of 1H RASER of continuously SABRE pumped a) pyridine and b) acetonitrile from Figure 65 compared to corresponding simulations of α (red) and PZ (blue) for pyridine (c, e) and acetonitrile (d, f) .. 119 Figure 68. Initial measured nonlinear 1H RASER dynamics of SABRE pumped 3-picoline and 3-fluoropyridine ...... 119 Figure 69. 1H and 13C SABRE of 13C-acetonitrile at 41.7 kHz ...... 120 Figure 70. 1H and 19F SABRE of 3-fluoropyridine at 41.7 kHz ...... 121 Figure 71. 1H and 15N SABRE of 15N-pyridine ...... 122 Figure 72. 1H and 15N SABRE and SABRE SHEATH of 15N-acetonitrile at 41.7 kHz ...... 123 Figure 73. 1H RASER of 15N-acetonitrile at 41.7 kHz based on heteronuclear two-spin order...... 124 Figure 74. Multi-mode 1H RASER of 3-picoline at 166.7 kHz ...... 126 Figure 75. Multi-mode 1H RASER of 15N-pyridine at 41.7 kHz ...... 127 Figure 76. SABRE polarized 1H spectrum of 15N-pyridine compared to the 1H multi-mode RASER spectrum ...... 128 Figure S 1. a) Synthesis of Rh-NP from Bis-nbd on a glass slide and b) microfluidic reactor ...... 133 Figure S 2. Experiment with Rh-nanoparticles in a microfluidic reactor module ...... 134

Figure S 3. activation of the IMes and the IMes@PPh3PB precursors ...... 135 Figure S 4. Synthesis of iridium hydride complexes ...... 136 Figure S 5. Impact of polymeric support on the hyperpolarization ...... 137 Figure S 6. SABRE with a polymer bound catalyst ...... 138 Figure S 7. h-PHIP and pr-PHIP with a polymer bound catalyst ...... 139 Figure S 8. PHIP experiments in toluene and water ...... 139 1 Figure S 9. H NMR hyperpolarization experiments with methylacrylate at different p-H2 pressures and temperatures of a pre-heating bath ...... 140 Figure S 10. 1H NMR hyperpolarization experiments with methylacrylate and styrene at different particle sizes of the support material ...... 141 Figure S 11. 1H NMR spectra from hyperpolarization experiments with the anchored homogeneous catalysts IMes and DuPhos ...... 142 Figure S 12. 1H NMR hyperpolarization experiment at 300 MHz under ALTADENA conditions ...... 143 Figure S 13. 1H NMR hyperpolarization experiment at 300 MHz under PASADENA conditions recorded directly after the ALTADENA experiment ...... 144 Figure S 14. Corresponding chemical shifts of the water peaks (HDO) shown in Figure 50b depending on the temperature the sample was exposed to prior to detection ...... 145 10 Hyperpolarization with Parahydrogen in NMR

Figure S 15. Hydride region of the 1H NMR spectra of the hyperpolarization experiments of water at different Bevo corresponding to Figure 51 ...... 145 Figure S 16. Enlarged 1H NMR spectrum of Figure 52a ...... 146 Figure S 17. Hydride region of the 1H NMR spectra of the hyperpolarization 15 experiments of water and N3-histidine at different Bevo corresponding to Figure 53 ...... 146 Figure S 18. Simulated α (blue) and PZ (red) for a 1H RASER experiment with a) pyridine and b) acetonitrile ...... 147 Figure S 19. Simulated α and PZ for a crusher pulse length of 30 s for a) pyridine and b) acetonitrile ...... 148 Figure S 20. Different crusher pulse length in a 1H acetonitrile RASER experiment .. 148 Figure S 21. Starting 1H RASER of SABRE pumped 13C-acetonitrile and 15N-acetonitrile ...... 148

List of tables

Table 1: Weighed-in chemicals for the size-dependent syntheses of PTA@Al2O3...... 67

Table 2: Weighed-in chemicals for the size-dependent syntheses of BINAP@Al2O3. ..69 Table 3: Signal intensity of polarized water under different conditions than the standard experiment ...... 91

Hyperpolarization with Parahydrogen in NMR 11

Abbreviations

abbreviation description (R,R)Me-DuPHOS 1,2-bis[(2R,5R)-2,5-dimethylphospholano]benzene (S)-BINAP (S)-2,2′-bis(diphenylphosphino)-1,1′-binaphthalene

@Al2O3 immobilized on Al2O3 (analogous for other support materials)

[Ir(OMe)(1,5-cod)]2 bis(1,5-cyclooctadiene)di-μ-methoxydiiridium(I)

[RhCl(PPh3)3] tris(tripheny1-phosphine)rhodium(I)chloride shake-and-drop see subsection 3.3.1 1/f -noise noise with intensity inversely proportional to the frequency of the signal (pink noise) 13C-acetonitrile 99% 13C labeled acetonitrile in the quarternary carbon position 13 (CH3 CN) 15N-acetonitrile 99% 15N labeled acetonitrile 15 15 N3-histidine Triply 95% N labeled L-histidine 15N-pyridine 99% 15N labeled pyridine

BINAP [Rh(COD)-(S)-BINAP][BF4] or (1,5-cyclooctadiene)((S)-2,2′- bis(diphenylphosphino)-1,1′- binaphthalene)rhodium(I)tetrafluoroborate

BINAP(a) [Rh(-(S)-BINAP)H2L2] [BF4]

Bis-nbd [Rh(nbd)2]BF4 or bis(2,5-norbornadiene)- rhodium(I)tetrafluoroborate br. s. broad singlet CIDNP chemically induced dynamic nuclear polarization COD 1,5-cyclooctadiene d doublet 2 D or D deuterium

D2O deuteriumoxide or heavy water DCM dichloromethane DIPAMP bis((2-methoxyphenyl)phenylphosphino)ethane DIPP 1,3-bis(diisopropylphosphino)propane DNP dynamic nuclear polarization dppb 1,4-bis(diphenyphoshino)butane

dppb [Rh(COD)(dppb)][BF4] or (1,4-bis(diphenylphosphino)butane)(1,5- cyclooctadiene)rhodium(I) tetrafluoroborate

Duphos [Rh(R,R)Me-DuPhos)(COD)][BF4] or 1,2-bis[(2R,5R)-2,5- dimethylphospholano]benzene(1,5-cyclooctadiene)rhodium(I) tetrafluoroborate 12 Hyperpolarization with Parahydrogen in NMR abbreviation description EHQE external high-quality-factor-enhanced eq equivalents EtOH ethanol FID free induction decay FT Fourier-transformation FWHM full width at half maximum HD dihydrogen gas consisting of one hydrogen and one deuterium atom HDO one time deuterium labeled isomer of water HPA heteropoly acid h-PHIP hydrogenative PHIP HSAB hard and soft acids and bases Hünig base diisopropylethylamine IDEG 1,3-bis(3,4,5-tris(diethyleneglycol)benzyl)imidazole-2-ylidene IDEG [IrCl(IDEG)(COD)] or (chloro)(1,5-cyclooctadiene)( 1,3-bis(3,4,5- tris(diethyleneglycol)benzyl)imidazole-2-ylidene)iridium(I) IMes 1,3-bis(2,4,6-trimethylphenyl)-imidazol-2-ylidene IMes [IrCl(COD)(IMes)] or (chloro)(1,5-cyclooctadiene)(1,3-bis(2,4,6- trimethylphenyl)-imidazol-2-ylidene)iridium(I)

IMes(a) [Ir(H)2(IMes)(L)3]Cl

IMes(b) [Ir(H)2(IMes)(py)3]Cl or (dihydrido)(1,3-bis(2,4,6-trimethylphenyl)- imidazol-2-ylidene)(tripyridine)iridium(III)chloride IPr 1,3-bis(2,6-diisopropyl)-imidazol-2-ylidene L ligand LAC level anti crossing

Ln n-th ligand in a transition metal complex m multiplet M metal (mostly the transition metal center of a catalytic complex) MeOH methanol methanol-d4 99.9% four times deuterated methanol MRI magnetic resonance imaging nbd 2,5-norbornadiene n-H2 normal hydrogen: hydrogen with spin isomers of similar abundance (ortho : para = 3:1) as in equilibrium at RT NMR nuclear magnetic resonance OMe Methoxy

Hyperpolarization with Parahydrogen in NMR 13 abbreviation description oneH-PHIP PHIP mechanism where one hydrogen stemming from parahydrogen is incorporated into a target substrate OR offresonance frequency from the Larmor frequency, where the signal is acquired orthohydrogen mixture of all three symmetric spin isomers of hydrogen parahydrogen antisymmetric spin isomer of hydrogen PASADENA parahydrogen and synthesis allow dramatically enhanced nuclear alignment

PCy3 tricyclohexylphosphine

PCy3 [Ir(PCy3)(py)][PF6] or (1,5- cyclooctadiene)(pyridine)(tricyclohexylphosphine)- iridium(I)hexafluorophosphate known as Crabtree’s catalyst PDMS polydimethylsiloxane PEO-PBT poly(ethylene oxide)-poly(butylene)terephthalate p-H2 para-enriched hydrogen: Note that this refers to the equilibrium mixture at e.g 20 K. Pure parahydrogen and orthohydrogen are not abbreviated and discussed in section 2.1 PHIP para hydrogen induced polarization POM polyoxometallate

PPh3 triphenylphosphine

PPh3PB PPh3 polymer bound: polystyrene polytriphenylphoshine polymer pr-PHIP pairwise replacement PHIP PTA polyoxotungstic acid ptppds 3-(diphenylphosphino)benzenesulfonate py pyridine pyridine-d5 99.9 % five times deuterium labeled pyridine RASER radio wave amplification by stimulated emission of radiation rds rate determining step rf-pulse radio frequency impulse Rh-NP Rhodium nanoparticles RT room temperature s singlet S the solvent as a ligand in a catalytic complex SABRE signal amplification by reversible exchange SABRE-Relay a way to relay SABRE hyperpolarization from one substrate to another by an additional catalytic complex 14 Hyperpolarization with Parahydrogen in NMR

abbreviation description SABRE-SHEATH SABRE shield enables alignment transfer to heteronuclei

SEOP spin exchange optical pumping SIMes 1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene SNR signal to noise ratio Sub substrate t triplet THF tetrahydrofurane TOF turn over frequency

toluene-d8 99.9% eight times deuterated toluene X anionic ligand XRD X-ray diffraction

*Note that metalorganic catalysts are abbreviated in bold. The abbreviation is often chosen similar to the name of the contained ligand, which itself may be abbreviated in regular font. Example: While IMes refers to a carbene ligand, IMes refers to the metalorganic catalyst precursor containing this ligand.

Hyperpolarization with Parahydrogen in NMR 15

Variables

symbol description unit B magnetic field strength T

B0 strength of constant magnetic detection field T

B1 resonant magnetic field strength of the T rf-impulse

Bevo magnetic evolution field G (1 G = 10−4 T) c concentration mol L−1

dpore pore size (average diameter of the pores) Å

dsupport diameter of the support material µm E energy J ΔE energy difference J rotational quantum number -

Jn,m J-coupling between the spins n and m Hz ℐ m magnetic spin quantum number -

M0 equilibrium polarization at constant SABRE pumping J T−1

MT transversal magnetization J T−1

MZ longitudinal magnetization J T−1 n number of spins in the ground state - nα number of spins in the excited state - α nsβ number of scans - β N noise V

Northo population of the symmetric hydrogen spin states -

Npara population of the antisymmetric hydrogen spin state -

NS number of spins in the sensitive volume - p pressure bar

P or PZ polarization -

Pt intrinsic magnetization in B0 (thermal polarization) - Q quality factor of a resonator -

Qm,n transition probability for the population of two - coupled states R resistivity Ω S signal V

Shyp detected signal in a hyperpolarization experiment V or a.u.

Stherm thermal reference signal in the detection field of Shyp V or a.u. SEF signal enhancement factor (alphabetisch, bitte) - SNR signal to noise ratio - 16 Hyperpolarization with Parahydrogen in NMR symbol description unit

Spore specific surface of the pores in a material m2 g−1 t time s T temperature K or °C

T1 longitudinal relaxation time s−1

T2 transversal relaxation time s−1

TC coil temperature K volume flow ml min−1

Vpore pore volume ml g−1 𝑉𝑉̇ α transverse spin component -

γI gyromagnetic ratio of the nucleus I MHz T−1

νI Larmor frequency of the nucleus I s−1 ν detection bandwidth s−1 ρ spin density cm−3 Δ σ chemical shift ppm

σI chemical shift of the species I ppm

τcrusher length of a crusher gradient s

τp SABRE pumping rate s−1

τrd radiation damping rate s−1

ω0 (angular) Larmor frequency rad s−1

Hyperpolarization with Parahydrogen in NMR 17

Important symbols and constants

symbol description B rotational constant h planck quantum reduced Planck quantum ( ) I moment of inertia of a molecule ħ ħ = h/2π kB Boltzmann constant

I gyromagnetic ratio of the nucleus I filling factor of a sample in the sensitive volume of a coil γ 0 vacuum permeability or magnetic constant η R simplified rotational constant μ |m state m Φ |n state n 〉 S0 or S singlet state of H2 〉 T triplet states of H2 (T+1 is also abbreviated as T+ and T 1 as T ) −1,0,1 ground state of the Zeemann splitting − − excited state of the Zeemann splitting α Ψtot overall wavefunction β Ψtrans translational wavefunction

Ψvib vibrational wavefunction

Ψel electronic wavefunction

Ψnuc nuclear wavefunction

Ψspin spin wavefunction

Ψns nuclear spin wavefunction

Ψrot rotational wavefunction A spin species in a coupling system (usually hydrogen) A’ spin species chemically equivalent but magnetically inequivalent with respect to A M spin species chemically and magnetically inequivalent with respect to A (This could be another hydrogen species but also a heteronucleus such as 15N) M’ spin species chemically equivalent but magnetically inequivalent with respect to M X spin species chemically and magnetically inequivalent with respect to A and M X’ spin species chemically equivalent but magnetically inequivalent with respect to X 18 1 Introduction

1 Introduction

Over the past decades, nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) have consolidated as analytical tools in medical and physical sciences. As routine analytical techniques, their high information content and versatile timescale from picoseconds to days allow for manifold applications from synthetic chemistry to clinical diagnosis. They are used to probe complex structures and dynamics ranging from atomic resolution in molecular compounds over biological macromolecules up to macroscopic structures such as the human body. Despite of all its broad applications, the struggle for higher sensitivity is an omnipresent issue since the discovery of NMR (Figure 1). To meet the increasing demands for higher sensitivity and homogeneity, large superconducting magnets were developed converging to a technical realizable maximum. Hyperpolarization techniques offer a way to create high levels of polarization.1-6 The most prominent techniques are spin exchange optical pumping (SEOP)7-9, dynamic nuclear polarization (DNP)10, 11 and parahydrogen based techniques12-15.

Figure 1: Guideline through “Hyperpolarization with Parahydrogen in NMR”: The scope of NMR (green), hyperpolarization techniques to increase the sensitivity of NMR (blue). This work deals with the latter i.e. parahydrogen based polarization (PHIP) and the repetitive16 signal amplification by reversible exchange (SABRE) mechanisms (Figure 1). They allow for manifold applications in physics, engineering, chemistry and life science.5, 17-21 The 1 Introduction 19 trace analysis of organic molecules22-26 and mechanistic studies27-31 demonstrate the increased NMR sensitivity. While easy to handle and cost efficient, parahydrogen based techniques can rapidly create high polarization levels P on different NMR active nuclei32-38, including long-lived states17, 39-46, independent from the magnetic field. Many homogeneous catalysts26, 47-50 have been developed for a variety of substrates22, 33, 38, 41, 51-55. Unfortunately, these complexes are expensive, usually inseparable from the reaction mixture for reuse and potentially disadvantageous for biological applications. Therefore, immobilizing these well- known homogeneous catalysts seems to be a straight forward approach for the design of new catalytic systems. In this thesis a modular system with high activity and negligible leaching allowing easy immobilization of known homogeneous catalysts is introduced and evaluated. Moving away from organic solvents, biomolecules in aqueous solution and even bulk water itself is hyperpolarized with a suitable catalytic system.56 Furthermore, the repetitive SABRE, hitherto limited to batch experiments, is implemented in a continuous flow hyperpolarization setup featuring a two-phase membrane reactor to introduce parahydrogen into solution and a 1 T NMR spectrometer for detection.57 Finally, a new discovery reveals an unconventional way to boost the sensitivity of an NMR experiment: The created population inversion of continuously SABRE pumped proton spins can induce radiowave amplification by stimulated emission of radiation (RASER) based on the nuclear spin states of the hyperpolarized molecules and thus exhibits unprecedented precision measurements of molecular coupling parameters in the sub mHz regime.58 The ambition of this thesis is to enhance the sensitivity of NMR by extending the scope of parahydrogen based hyperpolarization in NMR. Therefore, this work is clustered in seven chapters. As a motivation into this field, this introduction additionally contains a brief excursion about sensitivity in NMR and a summary of other hyperpolarization techniques. The theory (chapter 2) focuses on parahydrogen itself and the mechanisms that to convert the spin order from parahydrogen into NMR detectable magnetization on target molecules. The latter chapter is followed by an experimental chapter 3 describing the used methods, devices and chemicals. The main chapters 4-7 are the results section subdivided into four parts: (4) A new general immobilization approach for known hyperpolarization catalysts. (5) The first hyperpolarization of water with parahydrogen.56 (6) A continuous flow hyperpolarization setup.57 (7) The discovery of a new phenomenon: The parahydrogen fueled NMR RASER.58 These results chapters are based on three published and one planned publication. They have individual conclusions and outlooks. The final chapter 8 summarizes the thesis in the context of the research field of parahydrogen based hyperpolarization. Further material is provided in the supplementary chapter. The following sections of this chapter cover the 20 1 Introduction crucial sensitivity issue in NMR and introduce the different hyperpolarization techniques in NMR offering a way to create high levels of polarization.

1.1 Sensitivity in NMR

A glance at the fundamental principles of NMR and its key parameters elucidates the critical sensitivity issue of NMR experiments.59-62 When nuclear spins are exposed to a constant magnetic field B0, they precess around B0 with the Larmor frequency ω0 = ν0, which depends on the magnetic field strength and the gyromagnetic ratio I of the NMR 2π active nucleus I (equation(1)). γ

= = (1)

𝜔𝜔0 2π𝜈𝜈0 γI𝐵𝐵0 These nuclear spins undergo a Zeemann splitting in a magnetic field B0, which means that their energy levels are split up and their energy difference E can be calculated as shown in Figure 2. For spin ½ nuclei the spin can occupy two energy levels. These energy Δ levels are characterized by the spin quantum number m. The ground state is typically referred to as and the excited state as . This gives the Zeemann levels m = +½ and m = −½

(Figure 2). For a spin ensemble exposed to B0, a population difference between the Zeeman α β levels is created.

Figure 2. Zeemann splitting into energy levels α and β of a spin ½ nucleus exposed to a constant magnetic field B including the corresponding NMR transition between these levels. This population difference, equal to a macroscopic magnetization60, can now be excited by a radiofrequency impulse (rf-pulse), which matches the precession frequency of the nuclei ω0. Its resonant magnetic field strength is defined as B1. The free induction decay (FID) during the relaxation back into the initial thermal equilibrium state is detected with a suitable coil. Applying sophisticated pulse sequences, not only relaxation measurements of the longitudinal (T1) and transversal (T2) relaxation times, characteristic for many materials, can be obtained: A homogeneous B0 provides resolution for spectroscopy, which is mainly 1 Introduction 21 characterized by the chemical shift σ, the indirect spin-spin interaction (J-coupling) J and the spin density ρ. To sample the k-space63, gradient coils are a common additive as they are necessary for imaging64, 65 or even flow66 and diffusion67-69 experiments. The demand for higher sensitivity is a major challenge for NMR and its manifold applications.3 Sophisticated pulse sequences61, 70, rapid sampling71 and data processing adjusted to the individual applications such as compressed sensing72 do their part to reduce measurement time while increasing the precision and information density of NMR experiments. The precision of a NMR spectrum is limited by the natural linewidth of 1/( T2) as well as the signal to noise ratio (SNR) and the measurement time.58 Mostly the transversal π relaxation time in an NMR experiment is shorter than the “true” T2 relaxation time due to magnetic field inhomogeneities and therefore called T2*. When boosting the sensitivity of a NMR experiment, the SNR is maximized by increasing the signal S or reducing the noise N (equation (2)). For longer measurement time t, accomplished by a higher number of scans ns, the signal rises linearly while the statistical noise increases with a squareroot function.3 This means that the SNR of an NMR experiment is proportional to the squareroot of the measurement time:

= ~ (2) 𝑆𝑆 𝑆𝑆𝑆𝑆𝑆𝑆 √𝑡𝑡 The SNR of a NMR experiment considering𝑁𝑁 the primary parameters has been expressed by Türschmann et al. 73 depending on Hoult and Richards74 as

(3) = 1 2S 2 I4k𝐵𝐵 I 0 𝜂𝜂𝜂𝜂 ∙ γ � 𝐼𝐼 � ∙ ħ γ 𝐵𝐵 ∙ 𝑃𝑃 with the number of spins 𝑆𝑆𝑆𝑆𝑆𝑆Ns, the filling factor η of the coil and the coil sensitivity B1/I. √ � B ∙ 𝑇𝑇c ∙ 𝑅𝑅 ∙ Δ𝜈𝜈 The polarization P can originate from thermal polarization build-up by B0 and from hyperpolarization, which will be elaborated in the following. The denominator i.e. 4k in equation (3) considers the dominating Johnson noise, also called thermal noise from the detection coil with alternating current � B ∙ 𝑇𝑇c ∙ 𝑅𝑅 ∙ Δ𝜈𝜈 resistivity R. The skin and proximity effects75 of the preamplifier, which are voltage and current noise sources i.e. shot noise and 1/f -noise (Pink noise) are mostly negligible. The considered thermal noise is determined by the coil temperature Tc, the total, induced by the sample and the coil, and the detection bandwidth ν, where kB is the Boltzmann constant. Despite efforts to optimize these experimental parameters for example by implementing Δ cryo probes76, a realizable threshold for possible noise reduction has been reached for standard NMR applications. Therefore, increasing the signal is crucial when boosting the sensitivity of NMR experiments. The gyromagnetic ratio I is constant for each of the different NMR active nuclei. Some spins, such as 1H or 19F, have a higher I and therefore a higher sensitivity in γ γ 22 1 Introduction

NMR. The detection of spins with a lower I such as 13C ( C H) or 15N ( N ≈ 1/10 H) is more demanding. γ γ ≈ 1/4 γ γ γ A straight forward approach is to increase the spin density and with it the amount of detected nuclei NS in the sensitive volume. Nevertheless this reaches a practicable maximum due to the size of the sensitive volume and solubility in solutions. This approach also includes elaborate spin labelling of to be detected compounds to enrich spin isomers with a low natural abundance such as 13C (1.1%) and 15N (0.4%). In practice most samples contain low concentrated species within mixtures and consequently demand higher sensitivity.

Therefore, the most popular parameter to be optimized is B0, where increased power and homogeneity have advanced NMR to one of the most powerful analytical tools today. This development resulted in today’s large superconducting magnets, which provide ppb homogeneity when combined with suitable shim coils77 at high magnetic fields up to 30 T. While high homogeneity is easier to achieve at low magnetic fields, the spectroscopic resolution at a high B0 is often required. A higher B0 increases not only the coil sensitivity and builds up a higher polarization, but also features a better chemical shift resolution as the chemical shift differences are proportional to B0.

It is generally assumed that the sensitivity can be increased by three halves power of B0 meaning that an increase of B0 by a factor of three would increase the sensitivity by 5.2.74

The effect of a higher B0 can be divided into the coil sensitivity with the corresponding noise and the polarization due to increasing population difference of the Zeeman levels. The coil sensitivity increases linearly with the number of scans ns, while the statistical noise increases by the squareroot.

The sensitivity of the coil (B1/I) as expressed by the field B1 of the rf-pulse over the current I generated by the coil, also contributes to the SNR. Thus, the quality of the signal detection has been optimized by different approaches such as microcoils78-80, minimized detection bandwidth81 or sophisticated coil preparation such as coil arrays82, 83 or tailor-made coils84. When exposed to a magnetic field, a spin ensemble builds up an intrinsic magnetization, i.e. thermal polarization Pt, based on the Zeemann splitting shown in Figure 2 as the ground state α is energetically preferred and therefore higher populated. The polarization P in an NMR experiment is a population difference between energy levels which can be read out. It can be defined as the difference between the number of spins in the ground state n and the number of spins in the excited state n over the total spin number: α

β = 4 + ( ) 𝑛𝑛α − 𝑛𝑛β 𝑃𝑃 For a common NMR magnet, applied 𝑛𝑛forα standard𝑛𝑛β analytical purposes, the energy difference E between the Zeemann levels is small compared to the thermal energy at room

Δ 1 Introduction 23 temperature (RT). The corresponding population difference of its energy levels and thereby

Pt can be calculated following the Boltzmann statistics.85 For example at 400 MHz (B0 = 10 T) this gives a Pt ≈ 10-5, when thermally polarized in the magnetic field. This means that only one out of 10,000 spins is detected while all other spins remain NMR silent. In clinical MRI setups, the magnetic field is mostly even lower (1.5 – 3 T) to be compatible for life science.86 This small population difference in NMR experiments is the source for the inherently lower sensitivity of NMR compared to other techniques such as mass spectrometry.2

1.2 Hyperpolarization in low field NMR

To overcome the polarization limit, determined by the energy difference of the Zeeman splitting at room temperature as described above, different so-called hyperpolarization techniques have been developed.1-6, 17 These create a spin order in the sample, which can be read out in a suitable detection field i.e. the constant magnetic field B0. Reaching polarization levels of several percent or even more, the polarization build-up is independent from B0, separating polarization and detection in NMR experiments. Therefore, inexpensive and compact low field NMR87-92 benefit eminently from the increased polarization from hyperpolarization techniques.1, 2 A higher spin order or hyperpolarization can also be created by exposing the sample to a high magnetic field and then transferring it to a lower detection field B0. When separating the polarization and detection fields in such a brute-force polarization, the sample already loses a significant amount of polarization by relaxation. More efficient hyperpolarization techniques require an initial spin state whose high order is transferred to a sample. In the case of DNP10, 11, microwave irradiation excites unpaired electrons in e.g. radicals. The electron has a significantly higher gyromagnetic ratio than the proton (103-fold) and the resulting higher polarization of the electron can be transferred to different nuclei. Prominent experimental variations for the well-established Overhauser DNP93, 94 are dissolution95 and magic angle spinning DNP96. Applications range from studies of water dynamics, metabolomics, enzyme kinetics and biologically relevant molecules up to in vivo cancer detection and treatment response monitoring.5, 97-104 Nonetheless also chemically induced DNP (CIDNP)105, 106 exploiting spin correlated radical pairs offers the possibility of a higher polarization. At the temperature of liquid helium rotationally hindered methyl groups are the source of nuclear spin order for quantum rotor induced polarization.107, 108 The circular polarized photons of a LASER are exploited in SEOP7, 8 to create a high nuclear spin order on a noble gas such as xenon. In this case the polarization transfer goes via optical pumping of the electrons of an alkaline metal vapor in a buffer gas environment; for example rubidium in nitrogen. Subsequently the spin order is transferred by the hyperfine interaction through Rb-Xe binding or three body collisions to xenon.9 Applications are mainly 129Xe found in vivo 24 1 Introduction

MRI, chemical shift resolved 129Xe spectroscopy and the relaxation measurements with 83Kr.109-113 This work focuses on parahydrogen based hyperpolarization with its different mechanisms PHIP13-15 and SABRE12. They allow for cost-efficient setups, which are easy to handle and enable a rapid build-up of high polarization levels on target molecules. In this case, in contrast to other hyperpolarization techniques, not a dipole polarization but the singlet spin order of parahydrogen is exploited to create detectable magnetization. As SEOP also parahydrogen based hyperpolarization is superior at lower magnetic fields114 compared to DNP, which is limited by the Boltzmann polarization of the electron spin. This parahydrogen based hyperpolarization in NMR is subject to the following theory chapter.

2 Theory: Parahydrogen based hyperpolarization in NMR 25

2 Theory: Parahydrogen based hyperpolarization in NMR

This theory chapter includes five main sections. The first section covers the hydrogen molecule with a focus on its nuclear spin isomers orthohydrogen and parahydrogen. The second one involves the scientific background and the history of p-H2 in NMR, which has been reviewed several times already115, 116. It contains the effective homogeneous and heterogeneous PHIP catalysts with a focus on homogeneous, metalorganic catalysts and their corresponding catalytic cycle. The third section introduces SABRE, a more recent 12 mechanism for spin order transfer from p-H2. This approach for reversible and repetitive polarization transfer on target molecules relies on a sophisticated concept: The choice of the catalytic system with its corresponding catalytic cycle, adjusted kinetics, optimized experimental parameters and suitable magnetic evolution field during the spin order transfer. The scope of these three first sections is summarized in Figure 3.

Figure 3. Guideline through the first three sections of chapter 2: After an excursus about

parahydrogen, its properties and the preparation of p-H2, the PHIP and SABRE mechanisms are introduced. The chemical systems for PHIP and SABRE experiments and important catalytic cycles of these are displayed. The fourth section focuses on the efficiency of parahydrogen based hyperpolarization experiments. It introduces important parameters and conditions and compares the quantities enhancement and polarization. The final section describes the spin order transfer mechanisms from parahydrogen to target substrates. It begins with the evolution of spin order deriving from p-H2 in different magnetic fields in PHIP mechanisms, then introduces level anti crossings (LACs) and closes with a detailed analysis of their role in SABRE polarization transfer. The extent of these sections is sketched in Figure 4.

26 2 Theory: Parahydrogen based hyperpolarization in NMR

Figure 4. Scope of the final two theory sections about the efficiency in hyperpolarization experiments and the spin order transfer mechanisms from parahydrogen to target substrates.

2.1 What is parahydrogen?

First of all parahydrogen itself is to be discussed as the understanding of its nature is essential for further discussions. Following the Born Oppenheimer approximation117 the overall wavefunction Ψtot of hydrogen can be written as a product of the translational, vibrational, electronic, rotational, nuclear and spin wavefunctions:

= (5)

𝛹𝛹tot 𝛹𝛹trans𝛹𝛹vib𝛹𝛹el𝛹𝛹rot𝛹𝛹nuc𝛹𝛹spin To obtain Ψns nuclear wavefunction Ψnuc and the spin wavefunction Ψspin were combined (Ψns Ψnuc Ψspin):Four normalized wavefunctions for the nuclear spin states of a hydrogen molecule= can be formulated from the nuclear spin wavefunction Ψns.116

= (6)

𝑇𝑇+1 αα 1 = ( + ) (7) 2 𝑇𝑇0 αβ βα √ = (8)

𝑇𝑇−1 ββ 1 = ( ) (9) 2 𝑆𝑆0 αβ − βα √ 2 Theory: Parahydrogen based hyperpolarization in NMR 27

They consist of three symmetric nuclear spin functions ( , + ), called ortho- states and one antisymmetric singlet spin function ( ), the para-state (Figure 5). The αα, ββ αβ βα transitions between the three symmetric triplet states T 1,0,1 spin can be detected in NMR as αβ − βα they split up symmetrically when exposed to an −external magnetic field B0. The antisymmetric isomer of hydrogen, the para-state, is a singlet S0 and therefore NMR silent.

Figure 5. The four nuclear spin isomers of hydrogen: (a) Schematic representations of the symmetric orthohydrogen triplet (green) and the antisymmetric parahydrogen singlet (red).

= (10)

𝛹𝛹tot 𝛹𝛹trans𝛹𝛹vib𝛹𝛹el𝛹𝛹rot𝛹𝛹ns A hydrogen molecule consists of two protons which are spin ½ nuclei. The Pauli exclusion principle dictates, that the sign of the wave function changes for an interchange of these two fermions.118, 119 Therefore, the overall wavefunction Ψtot of a hydrogen molecule needs to be antisymmetric regarding the exchange of two nuclei following the Pauli principle for fermions. This quantum mechanical characteristics has significant consequences for the hydrogen. The translational, vibrational and electronic wavefunctions

(Ψtrans, Ψvib, Ψel) of molecular hydrogen are always symmetric. Therefore the overall wavefunction can only be antisymmetric if either one of the nuclear spin wavefunctions Ψns described above or the rotational wavefunction Ψrot is antisymmetric. This means that if the rotational wavefunction in a hydrogen molecule is symmetric, the nuclear spin wavefunction is antisymmetric and vice verca. In other words the spin wavefunction is coupled to the rotational wavefunction: The antisymmetric nuclear spin wavefunction of the para-state ( ) is coupled to even numbered levels of rotation ( 0, 2, ). And symmetric nuclear spin functions of ortho-hydrogen are connected to αβ − βα the odd numbered levels of rotation ( 1, 3, ). ℐ = 4 … The properties of fermions like hydrogen can be precisely described by the Fermi-Dirac ℐ = 5 … statistics. For an ensemble of hydrogen molecules, i.e. with increasing number of hydrogen molecules, this statistics evolves into the Maxwell-Boltzmann statistics.120 With it, the populations of the symmetric hydrogen spin isomers Northo as well as the antisymmetric spin isomer Npara can be calculated. For the ratio of spin isomers this gives 28 2 Theory: Parahydrogen based hyperpolarization in NMR

( ) ( + 1) e = ℐ ℐ+1 ΦR (11) ( 𝑇𝑇 ) 𝑁𝑁para 3∑ℐ=even 2ℐ( + 1)∙ e ℐ ℐ+1 ΦR 𝑁𝑁ortho 𝑇𝑇 with the simplified rotational constant∙ ∑ ℐ=oddR. This2ℐ constant∙ can be determined by equation (12), where B is the rotational constant, h the Planck quantum and I the moment of inertia Φ of the molecule. 116

B h = = (12) k 8 2Ik ΦR 2 The energy difference between the lowestB rotationalπ B levels 0 and 1 is 6 kJ/mol. At room temperature or at even higher temperatures many higher rotational levels are ℐ = ℐ = populated and also the energy difference is not significant considering the entropic advantage of populating different rotational energy levels. Therefore, at room temperature all spin isomers are nearly similar abundant and the ratio of orthohydrogen over parahydrogen is approximately 3:1. This means, that the mole percent of parahydrogen χpara is 0.25 (see Figure 6). Molecular hydrogen with this ratio of spin isomers is from here on referred to as normal hydrogen (n-H2). Nevertheless when considering the first six terms of the sum in (equation (11)), the small population difference i.e. the excess of parahydrogen over the 3:1 ratio at room temperature can be calculated. This ratio at the thermodynamic equilibrium changes drastically when cooling the hydrogen down.

Figure 6. Temperature dependent fraction of parahydrogen (red) at thermodynamic equilibrium: At room temperature (295 K) mostly normal hydrogen consisting of ¾ orthohydrogen (light green) and ¼ parahydrogen, inaccessible for hyperpolarization experiments (light red). Excess parahydrogen content (dark red) in mixtures of parahydrogen and orthohydrogen

with a thermodynamic equilibrium at or below the boiling point of N2 is called para-

enriched hydrogen (p-H2) in this work, with 36 K as the conversion temperature of the commonly used parahydrogen generator (see 3.1.3).

2 Theory: Parahydrogen based hyperpolarization in NMR 29

At low temperatures, the 0 is energetically preferred (enthalpy), while higher rotational states are less populated. Thus the para-state of the nuclear spin function is also ℐ = preferred at low temperatures. Under these conditions an excess of parahydrogen is formed from normal hydrogen in the presence of a suitable catalyst115, 116, 121, 122 (Figure 6, dark red). At the boiling temperature of liquid nitrogen (T = 77 K) more than half of the spin isomers are in the para-state for hydrogen in thermodynamic equilibrium. This mixture of hydrogen spin isomers is accessible for hyperpolarization experiments and referred to as para-enriched hydrogen (p-H2) in this thesis. Cooling even further down leads to higher fractions of parahydrogen. At 36 K the enrichment of parahydrogen is so high (93 %), that 90 % is accessible for hyperpolarization experiments. In absence of the catalyst and other paramagnetic materials, this metastable p-H2 can be stored at room temperature over weeks before application. It’s extremely long lifetime originates in the forbidden singlet triplet transition. The spin order of p-H2 can be exploited to create high levels of polarization. The symmetry has to be broken to convert the high singlet spin order into detectable magnetization. The following sections will focus on the different mechanisms of such a spin order transfer.

2.2 Parahydrogen induced polarization (PHIP)

In 1986 Bowers and Weitekamp discovered the parahydrogen effect on NMR.13, 14 They discovered that using p-H2 for hydrogenation reactions not only enhances the signal of the hydrogenated substrate significantly, but also its antisymmetric nature resulting from the population created by the parahydrogen. This effect was then called parahydrogen induced polarization (PHIP). The spin order of the parahydrogen singlet has to be broken to achieve detectable magnetization. This happens at the catalyst, where both hydrogen atoms are consecutively incorporated into a target molecule by a hydrogenation reaction (Figure 7).

H [cat] H p-H2 R' R' R = R' R R

Figure 7. Hydrogenation of an olefin with p-H2. Such a reaction is referred to as hydrogenative PHIP (h-PHIP) in the following. It has been largely investigated on different unsaturated substrates in the gas and liquid phases to monitor catalytic reactions, for trace analysis or even for biomedical applications.17, 18, 123 The created polarization can be very high (>50%).124, 125 It is also possible to incorporate only one hydrogen atom stemming from parahydrogen into a target molecule. This mechanism is called oneH-PHIP and has been applied to chemical intermediate identification when forming aldehydes by different catalysts. 126, 127 30 2 Theory: Parahydrogen based hyperpolarization in NMR

2.2.1 The catalytic cycle

A catalytically active dihydrogen complex can be prepared in different ways.128 The activation with hydrogen gas from a robust catalyst precursor is the most common and convenient way. Other methods to prepare a desired dihydrogen complex are for example the protonation of a hydride complex, a reduction in the presence of protons or a modification of an existing dihydrogen complex. A free coordination site or an exchangeable neutral (L) or anionic ligand (X) is required to for the dihydrogen to coordinate to the transition metal center (M). The possible ways to form the active species of a homogeneous transition metal catalyst with dihydrogen gas are shown in Figure 8.

1a) LnM H2 LnM(H2)

1b) [LnM] X H2 [LnM(H2)] X

2) LnML' H2 LnM(H2) L'

3) LnMX H2 [Ln 2 X M(H )] Figure 8. Activation of catalyst precursors with hydrogen gas into the catalytically active species of

homogeneous transition metal catalysts. 1a) addition of H2 at a free coordination site of a

neutral complex. 1b) addition of H2 at a free coordination site of a cationic complex. 2)

exchange of H2 with a neutral ligand. 3) exchange of H2 with an anionic ligand. The hydrogenation of an unsaturated hydrocarbon at a homogeneous catalyst can be divided into several key steps. In a first step, molecular hydrogen from the solution enters the catalytic cycle. As described above, p-H2 is chemically equivalent to n-H2. To illustrate the way of p-H2 in a typical catalytic cycle for hydrogenations, both hydrogen atoms stemming from p-H2 are marked in red (Figure 9).

When p-H2 from the solution reaches the catalyst 1, it first binds as a non-classical hydride to the metal center of the catalyst (a) and forms 2. Prior to this, a dissociation step of the solvent (S) can be necessary. In a second step, the symmetry of p-H2 is broken by an oxidative addition (b) at the transition metal center forming the catalytic complex 3 with two classical hydrides. Apart from the hydrogen also the target substrate needs to coordinate to the catalyst. This unsaturated molecule coordinates to the transition metal center by a ligand association (c) and forms 4. The first hydrogen atom stemming from p-H2 is transferred from the catalyst to the substrate by a migratory insertion (d) into the metal organyl 5. The second one is incorporated during the reductive elimination (e) of the aliphatic product from the catalyst after which 1 is formed again. While the catalyst can pass through another cycle, the yielded aliphatic product contains a detectable polarization due to the hydrogen atoms stemming from p-H2. 2 Theory: Parahydrogen based hyperpolarization in NMR 31

p-H2

(a) S S S L1 S L1 M S H M S L2 H L2 L3 H L3 H 1 2 H R' R (e) (b) 3S S

R' R H S H L L1 M 1 H M H L HH L2 L 2 L3 3 5 3

R' R S H L R' R (d) M 1 (c) H L2 L3 4

Figure 9. General catalytic cycle of a hydrogenation with p-H2: (a) non classical coordination of p-H2 to the catalytic center, (b) oxidative addition of hydrogen, (c) ligand association of the substrate (d), migratory insertion of the coordinated substrate and (e) reductive elimination of the hydrogenated aliphatic product. R,R’: organic rests of the substrate; S:

solvent; M: transition metal center; L1-3: ligands. The homogeneous catalysts in this thesis are metal organic catalysts. They consist of a transition metal core (mainly iridium or rhodium) and organic ligands coordinated to the core. The different ligands can be compared with the Tolman principle distinguishing between electronic and steric parameters. Sterically demanding and electron donating phosphine or carbene ligands are suitable ligands for hydrogenation catalysts: A higher electron density at the metal organic center increases the basicity and decreases the activation energy of the oxidative addition. The oxidative addition is often the rate determining step (rds) in hydrogenations, so its acceleration is beneficial. A bulky ligand suppresses side reactions such as dimerization, agglomeration or even the formation or nanoparticles. Suppressing these catalytically inactive species is one reason for sterically demanding ligands. Another reason is the space at the catalyst itself favoring 32 2 Theory: Parahydrogen based hyperpolarization in NMR

certain functional groups or substrates. The hydrogen atoms stemming from p-H2 need to stay close to each other not to lose their correlation/interaction. Depending on the nature of the solvent S it can coordinate to the center M of the metal organic catalyst. The activation of such catalysts from their precursor to an active species is shown in subsection 3.2.3.

2.2.2 Heterogeneous PHIP catalysts

Different heterogeneous PHIP catalysts are reported in literature. They are advantageous compared to homogeneous catalysts in practical use, when the catalyst is to be recovered for reuse or because of its lacking biocompatibility.129 Their disadvantage is their lower polarization efficiency compared to homogeneous catalysts.18, 129, 130 A way around this is the 131 extraction of the catalyst , but this may be demanding and costly. Heterogeneous PHIP have been applied widely. For example a gas phase hydrogenation 130 of propene with p-H2 was studied in detail ; water soluble Pt nanoparticles allowed for 132 hydrogenation reactions with p-H2 and a hollow fiber membranes enabled continuous 37 hydrogenation with p-H2 in the liquid phase . Another way to obtain a heterogeneous catalyst is immobilizing known homogeneous catalyst. There are various immobilization approaches for hydrogenation catalysts.133-140 This has the advantage to rapidly obtain a variety of catalysts and thoroughly study them as they are well defined. The challenge in this approach is that the properties of an immobilized catalyst may change compared to its homogeneous analogues. This includes their PHIP efficiency, but there are elaborate immobilized PHIP catalysts.129, 141-143 In this thesis an immobilization approach on Al2O3 is developed and optimized for p-H2 based applications. In the supplement (see subsection 9.1.2) also an immobilization on a polymer is shown.

2.2.3 Pairwise replacement and other exchange mechanisms

Hydrogenation is not the only way to hyperpolarize unsaturated hydrocarbons. Another way to introduce hydrogen into target molecules is chemical exchange. For terminal olefins a “pairwise replacement” of two hydrogen atoms allows for non hydrogenative hyperpolarization with parahydrogen (pr-PHIP) (see Figure 10).144

H H H [cat] H n-H p-H2 H H 2 R R Figure 10. Pairwise replacement of the terminal hydrogens of an olefin with parahydrogen (pr-PHIP). Compared to the catalytic cycle of h-PHIP (Figure 11), another route is possible for terminal olefins. After the reductive elimination (a) from 4a to 5a, one of the terminal 2 Theory: Parahydrogen based hyperpolarization in NMR 33 hydrogens can undergo a β-H elimination (b) forming a classical hydride at the catalyst (4b). This will leave the olefin chemically unchanged as just the hydrogens are exchanged. Repeating this process can result in a product 4c, where both terminal hydrogens are exchanged for the protons stemming from p-H2. After this the substrate can dissociate and be detected in solution without an altered chemical structure. When the hydrogen is exchanged for new p-H2, a new substrate can coordinate and be polarized again (e).

H (a) H H R H R H H L L H M 1 H M 1 H L2 HH L2 H L3 L3 H n-H2 R 4a 5a

(e) (b) H H p-H H 2 R H H H R H R H L H L M 1 M 1 H L2 H L2 L3 L3 4c 4b

H H R H (c) (d) L H M 1 HH L2 L3 5b Figure 11. Mechanism of the pairwise replacement of hydrogen: (a) migratory insertion of the first hydrogen atom. (b) β-H elimination of a terminal hydrogen after rotation. (c+d)

repetition of (a) and (b), (e) dissociation of the terminal olefin, exchange of n-H2

with p-H2 and ligand association of a new olefin. This mechanism is only rarely used and developed. Apart from a few applications145-147 it is mainly a side reaction. The number of free coordination sites at the catalyst is important for the mechanism. Different polarized species are created as for example also the exchange of only one hydrogen atom occurs. Nevertheless, as a side reaction it can also be a source for higher polarization on terminal unsaturated molecules undergoing pr-PHIP before another p-H2 molecule is incorporated into the same substrate by hydrogenation (h-PHIP). 34 2 Theory: Parahydrogen based hyperpolarization in NMR

Notably, some of the catalysts that feature a significant pairwise replacement efficiency are heterogeneous. 146, 147 Finally, also a single proton exchange analog to oneH-PHIP, where a single proton is transferred to a substrate, is possible. In this case for example water can be hyperpolarized under ALTADENA conditions.56 The evolution time in the strong coupling regime prior to the proton substitution is necessary to obtain detectable magnetization.148 Details about this mechanism including experimental results will be discussed in subsection 5.3.3.

2.3 Signal amplification by reversible exchange (SABRE)

12 A more recent way to exploit the spin order of p-H2 in NMR was reported in 2009. In contrast to the PHIP mechanisms described above, signal amplification by reversible exchange (SABRE) is non-hydrogenative. It allows for a repetitive16, 22, 43, 45, 57, 58 spin order transfer from p-H2 to target substrates leaving these target molecules unaltered (see Figure

12). The core of this approach is a homogeneous, metalorganic catalyst, where both the p-H2 and a target molecules reversibly bind. This substrate coordinates to the catalyst and spin order is transferred to it before it dissociates into the solution again.

Figure 12. The SABRE approach modified from the original publication of Adams et al. in 200912: a) Schematic representation of the magnetization transfer from parahydrogen to a target substrate by a suitable catalyst. b) Single scan 1H-NMR spectra of 6 nmol pyridine in 149 methanol-d4 at RT hyperpolarized using Crabtree’s catalyst and p-H2 with Bevo = 20 mT (orange) and the thermal reference shown in blue and 128-fold enlarged.

2 Theory: Parahydrogen based hyperpolarization in NMR 35

The polarization efficiency of SABRE is highly dependent on a variety of parameters.

Especially the kinetics, including the contact time of both p-H2 and the substrate to be polarized, is crucial. Therefore different catalysts, substrates and reaction conditions are studied.

2.3.1 The catalytic cycle

When SABRE was first published in 200912 it was demonstrated with the so-called

149 Crabtree catalyst [Ir(PCy3)(py)][PF6] (PCy3), an iridium based catalyst with a tricyclohexylphosphine (PCy3) ligand (py = pyridine). In the following more SABRE active catalysts have been reported.26, 47-50 Especially iridium based catalyst with different ligands have shown to be suitable SABRE catalysts. These vary by their different individual ligands.Among them are for example also a solvent responsive catalyst150 or catalysts with 151, 152 an additional phosphine ligand . Along with the PCy3 ligand in the Crabtree catalyst, many other ligands with a single coordination to iridium have shown high polarization levels.26, 47, 48, 50 The most successful among them so far are N-heterocyclic carbenes153 such as IMes, SIMes and IPr (IMes = 1,3-bis(2,4,6-trimethylphenyl)-imidazol-2-ylidene; SIMes = 1,3- bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene; IPr = 1,3-bis(2,6-diisopropyl)- imidazol-2-ylidene). Also a similar, but water soluble version carbene, the IDEG ligand49, has been developed (IDEG = 1,3-bis(3,4,5-tris(diethyleneglycol)benzyl)imidazole-2-ylidene. Up to now most of the catalysts have some things in common. They feature an iridium as the center of the metal organic catalyst. Apart from that, suitable catalysts include an electron donating, sterically demanding ligand accelerating the oxidative addition of the hydrogen and inhibiting the formation of catalytically inactive dimers.

An example for a suitable catalyst is the very efficient [Ir(H)2(IMes)(L)3]Cl, abbreviated as IMes(a), catalyst. This catalyst is formed from the air stable [IrCl(COD)(IMes)]47 (IMes) catalyst precursor as shown in Figure 13. It can be activated in methanol (MeOH) by an oxidative addition of hydrogen and a ligand association of pyridine into [Ir(H)2(IMes)(py)3]Cl (IMes(b)) while 1,5-cyclooctadiene (COD) dissociates from the catalytic complex. + py, p-H N N 2 N N Mes Mes COD Mes Mes py H Ir MeOH Ir Cl py H py

IMes(a) IMes(b) Figure 13. Activation of the precursor IMes with hydrogen and py in MeOH to the SABRE active

complex IMes(b). In this example p-H2 is directly used for the activation.

36 2 Theory: Parahydrogen based hyperpolarization in NMR

In Figure 15 the general catalytic cycle is shown. For known SABRE catalysts, the metal atom M in the center of the complex is mostly iridium. The coordination site L1 is usually occupied by an electron donating and sterically demanding ligand. The additional coordination sites L2 and L3 can be occupied by additional phosphine ligands, substrates, solvent molecules, or other parts of the solution such as additives. For the exemplary catalyst shown in the activation in Figure 13, M is iridium, L1 the carbene ligand IMes and L2 and L3 are most of the times occupied by the substrate pyridine. In this case there are different species of pyridine bound to the catalyst, but polarization however is built up on the equatorial pyridine only, which then exchanges with the solution. The symmetry of the two hydride protons stemming from p-H2 has to be broken for a successful spin order transfer. This is the case in the equatorial plane, when a substrate is in trans position to one hydride and in cis position to the other. The polarization transfer mechanism, for example to the ortho-protons of pyridine with its two different 4J-couplings to the hydrides, are discussed in detail in subsection 2.5.2.

Therefore, for the polarization transfer of the catalytic intermediate [M(H)2(L)3(Sub)] (6), at least one substrate (Sub) is coordinated in the equatorial plane, but mostly two more coordination sites L are occupied by Sub. In Figure 14 the polarization transfer for IMes(b) is illustrated: When a free substrate (Sub) in solution is polarized, the substrate as well as parahydrogen exchange at the transition metal center. The substrate exchange is a dissociative mechanism while the p-H2 exchange is an associative mechanism. Spin order is transferred via the temporary J-coupling network of this intermediate from the two hydrides stemming from parahydrogen to a target substrate. A simple analogy to this process are coupled pendulums. The transfer step in 6, generally formulated in Figure 15 as the step from 6a to 6b, is described in the second part of this subsection.

+ Figure 14. Schematic drawing of the spin order transfer by SABRE in [Ir(IMes)H2Sub3] : Both, p-H2 and the to-be-polarized substrate (Sub), exchange at the transition metal center. With the typical substrate pyridine, this gives the efficient SABRE active complex IMes(b). Some of its shown coordination sites can also be occupied by different components in the solution such as other substrates, additives or the solvent.

2 Theory: Parahydrogen based hyperpolarization in NMR 37

The catalytic cycle to repetitively obtain 6, containing the exchange mechanisms of both hydrogen and the substrate, is shown in Figure 15. At first the polarized substrate dissociates from 6b and the magnetization of the free substrate in solution can consequently be read out (a). For certain ligands, the obtained free coordination site in the 16 electron complex

7a can stabilized and protected. For example a CH3 group from the IMes ligand can coordinate to the free site of this intermediate or even undergo a CH activation and be polarized in the same way as a substrate. At this free coordination site, the hydride protons in the complex can now be exchanged for protons stemming from parahydrogen.

L1 L1 H Sub H Sub a M = a M Hb L2 Hb L2 L3 L3 (e) 6a 6b (a) Sub Sub

L1 L1 Ha Ha M L2 M L2 Hb Hb L3 L3 7b 7a

H2 p-H2

(d) (b)

L1 L1 H Hb Ha Ha H M M H L2 Hb L2 H L3 L3 8a 8b (c) Figure 15. Catalytic cycle of the SABRE hyperpolarization. The route of the spin order which derives

from p-H2 is depicted in red: (a) Ligand dissociation of Sub, (b) Ligand association of p-H2,

(c) exchange of the hydride protons: oxidative addition of p-H2 and reductive elimination

of the hydrides (which are in thermal equilibrium) forming H2, (d) dissociation of H2, (e) ligand association of Sub. Therefore, parahydrogen binds to the free coordination site at the transition metal center (b) and forms the non-classical hydride 8a. Now the parahydrogen at the catalytic center undergoes an oxidative addition (c), as in the activation step, and forms two classical hydrides. The original hydrides at the catalyst do the inverse step which is a reductive elimination (c). This process can happen simultaneously and involve the vibrational modes of the two pairs of hydrogen atoms in the catalytic complex. Subsequently hydrogen dissociates (d) and the catalytic intermediate 7b is formed leaving a binding site for the 38 2 Theory: Parahydrogen based hyperpolarization in NMR substrate to coordinate. In this way the hydrogen atoms at the catalyst are exchanged for hydrogen atoms stemming from the spin ordered parahydrogen. Finally, new to-be- polarized substrate (Sub) can coordinate to the metalorganic complex (e). The spin order transfer step from 6a to 6b is elaborated in section 2.5.

Apart from the desired substrate also a solvent, or a part of L1 may coordinate to the catalytic center.154 In the case of the most used standard substrate pyridine, this ligand association process happens by the nitrogen atom binding to iridium with its free electron pair. In this way complex 6a is formed again and the hydrogen atoms from parahydrogen are incorporated into the catalytic complex. Thus spin order from parahydrogen can be transferred to the substrate again. In contrast to the described homogeneous catalysts, heterogeneous SABRE catalysts are rare. Immobilized SABRE catalysts with a signal enhancement factor (SEF) of around five at 400 MHz were reported for proton155, 156 and around 100 for 15N at the same field.157 The changed properties, especially of the steric and electronic parameters, alter and often enhance the polarization losses (details see section 2.4) due to relaxation of several intermediates. Nevertheless, also other non hydrogenative approaches that transfer spin order from parahydrogen to target molecules have been published. Most interestingly is the 114 approach on a Pt3Sn@mSiO2 surface to polarize water and several alcohols.

2.3.2 Substrate classes and applications

The SABRE approach, as a relatively new method, can be applied to a constantly growing range of substrates. These include chemical motives such as diazarines54, nitriles33, 52, purines55, pyrazoles51, pyridines12, 53, 55, pyriminides55, schiff bases158 or thiazoles41. Also neat substances or solvents, as typical in DNP97-100, have been successfully polarized with the SABRE approach.56, 159, 160 When combined with proton exchange, SABRE-Relay161 allows for a further expansion of the range of substrates such as “amines, amides, carboxylic acids, alcohols, phosphates, and carbonates”38. The SABRE approach is not limited to 1H NMR. Different studies on e.g. 13C, 15N, 19F, 29Si, 31P, 119Sn have been carried out.12, 32-36, 38, 162 With its high sensitivity, SABRE facilitates trace analysis with NMR22-26, 163 and has also been applied to fast 2D measurements22, 24. Since its discovery, SABRE is developed into the direction of biological or medical applications. The polarization of various drugs33, 55, 164, vitamin B353 and also water56, 114 pave the way towards this domain. For clinical studies so far the extraction of the homogeneous catalyst or the polarized substrate165 is state of the art, as the iridium complexes are toxic and heterogeneous catalyst lack the desired high polarization levels up to now.155-157 2 Theory: Parahydrogen based hyperpolarization in NMR 39

2.4 Efficiency in hyperpolarization experiments

Several different conditions, effects and mechanisms determine the hyperpolarization efficiency in NMR experiments. In this section, most of the general contributors that determine the sensitivity in hyperpolarization experiments with parahydrogen are perused, with a focus on the SABRE approach. In many experimental setups, the polarization losses dominate the efficiency. Therefore they will be discussed here first. There are different mechanisms and ways of polarization loss. In the liquid phase, the prominent polarization loss is relaxation. This relaxation can be the relaxation of the substrate in solution, but a crucial point in this case is also the apparent relaxation time T1 at the catalyst.48 It is an average over the relaxation times of the catalytic intermediates with respect to their corresponding abundance and lifetimes. Substrates with long lifetimes can alleviate polarization loss by relaxation. Therefore long-lived states are studied for PHIP as well as for SABRE.166 Such symmetric states with extended lifetimes still suffer from several relaxation mechanisms. These include symmetric and antisymmetric chemical shift anisotropy, singlet-triplet leakage, a so called forbidden transition, as well as intra and intermolecular dipole interactions from couplings to other nuclei such as deuterium.18 Also the spin internal motion and the spin rotation are contribute to the relaxation.166 Paramagnetic impurities, such as molecular oxygen, also significantly diminish the polarization. Therefore, samples are often degassed and thus freed from the paramagnetic oxygen of the air.167 Eliminating coupling partners that induce high polarization losses, especially in substrate or solvent molecules, can help to reduce these effects. Several studies have shown, that exchanging hydrogen for deuterium not only the solvent but also parts of the substrates (e.g. pyridine heterocycles) or even the catalyst drastically increases the detected polarization.43, 151 Finally, also general polarization losses due to the NMR hardware, such as inhomogeneities in B0 but also in B1, are also a source for polarization loss in hyperpolarization experiments. The polarization efficiency of a hyperpolarization experiment of can be described in different ways. The most common ones are the enhancement as well as the polarization. The signal enhancement factor SEF is defined as the detected signal in a hyperpolarization experiment Shyp over the corresponding thermal reference signal Stherm in the same detection field.

= (13) 𝑆𝑆hyp 𝑆𝑆𝑆𝑆𝑆𝑆 This traditional definition is used in many studies.𝑆𝑆therm In DNP, it makes a lot of sense as it is connected to the polarization transfer efficiency: The DNP enhancement depends on the 40 2 Theory: Parahydrogen based hyperpolarization in NMR gyromagnetic ratio of the electron and is therefore limited to SEF ≈ 660 for protons, if polarization and detection field are identical. For parahydrogen based hyperpolarization experiments, a measure that is field independent is more suitable to compare different studies. Otherwise just reducing the detection field while keeping the level of polarization with the same sample increases the enhancement. Therefore the polarization P, as introduced in the sensitivity section 1.1, is more suitable to compare parahydrogen based hyperpolarization experiments. As already described there, the polarization in standard NMR for a spin ½ nucleus is the population difference of the two Zeemann levels in the constant magnetic field B0 over the number of spins in both energy levels. The occupation difference of these energy levels at thermal equilibrium is very small in most common NMR experiments: For a magnetic field of 400 MHz only a P 10-5 is obtained. Nonetheless, exactly this population difference is used in the standard thermally polarized NMR experiments. In hyperpolarization experiments ≈ with parahydrogen, this polarization can be significantly higher as the creation of spin order is independent from the magnetic field. Also the polarization may be negative meaning that the excited state is higher populated than the ground state α. This occupation of the energy levels is also referred to as negative spin temperature. β The whole catalytic system can be optimized to obtain maximum SABRE polarization. This begins with a suitable temperature considering kinetic parameters such as the 36, 48, 49, 55, 56, 116, 168 exchange at the catalyst. It also includes the p-H2 enrichment from the parahydrogen generator as well as the p-H2 supply (i.e. bubbling, shaking, flow) and p-H2 pressure (p) applied to the sample. 16, 47, 55, 56, 58, 116, 168-170 The solvent plays a key role concerning solubility of the catalyst, hydrogen and substrates as well as its own interaction with the catalyst.49, 55, 56, 160, 171, 172 The concentrations (c) of the individual components play an important role as well.23, 48, 53, 56, 163, 168 Further substances in the solution, mostly referred to as co-substrates or additives, can not only stabilize the catalytic complex but significantly enhance the polarization of the target substrate.22, 25, 45, 56 Especially, different new catalysts, where SABRE experiments were motivation, have been developed.26, 47-50 Therefore mostly the ligand L1 is modified to obtain the desired steric and electronic parameters. In this way important parameters such as the exchange rates of different substrates, solvents, hydrogen as well as the lifetimes of important intermediates and so on are adjusted. This can also increase the apparent T1, often small at the catalytic intermediates, and reduce this source of polarization loss.33, 43, 48 Knowing the catalytic cycle and the corresponding experimental parameters, the polarization built up by the SABRE approach can be described by a kinetic model.173 Finally the polarization transfer field plays a major role optimizing SABRE experiments.33, 36, 47, 48, 51, 52, 55, 56, 116, 168 This topic and its connection to LACs will be the subject to the final subsection 2.5.2. 2 Theory: Parahydrogen based hyperpolarization in NMR 41

2.5 Spin order transfer mechanisms in PHIP experiments

The built-up magnetization and the corresponding NMR spectra stemming from p-H2 depend on the magnetic field where the singlet spin order of parahydrogen is broken. This magnetic evolution field Bevo can be divided in two fundamentally different regimes: The strong coupling regime at low magnetic fields and the weak coupling regime at high magnetic fields. The chemical shift depends on the magnetic field B0, but the J-coupling is independent from it. Thus, at high magnetic fields, the chemical shift dominates the spectrum, whereas at low magnetic fields, the energy difference between the states resulting from J-couplings between the spins is larger than from chemical shift differences.115, 174 When the sample is exposed to a high magnetic field, usually the detection field itself, during a hydrogenation with p-H2, this is referred to as “parahydrogen and synthesis allow dramatically enhanced nuclear alignment” (PASADENA).14 Here, in the simplest model of a two-spin system, the levels and in the adduct are populated. The resulting single quantum coherence has the same transitions as in standard NMR (see Figure 16a) but αβ βα enhanced, antiphase and depends on the magnetic detection field strength, which is characteristic for PHIP spectra (Figure 16b).175, 176

Figure 16. NMR states and transitions in PHIP experiments for two 1H spins with their chemical shifts

σa and σb coupled with Jab: Transitions (upper part) and the corresponding NMR spectra (lower part) under (a) the thermally polarized standard NMR (b) polarized in a high magnetic evolution field (PASADENA) and (c) polarized at a low magnetic evolution field (ALTADENA).

A hydrogenation with p-H2 in the strong J-coupling regime at a low Bevo on the other hand is called “adiabatic longitudinal transport after dissociation engenders nuclear alignment” (ALTADENA).177 In this case, the polarization transfer, e.g. a hydrogenation of the 42 2 Theory: Parahydrogen based hyperpolarization in NMR

sample, often takes place in a separate Bevo prior to detection. Thus, it is usually lower than the detection field B0. In this strong coupling regime, the two parahydrogen protons remain strongly coupled and the dominant polarization transfer mechanism is based on LACs.18 These will be discussed in detail for SABRE polarization transfer in subsection 2.5.2. After the spin order transfer, only one energy level is populated and only two of the four single quantum transitions from the standard NMR spectrum are observed, again in giving an antisymmetric spectrum (see Figure 16c). There are also other polarization transfer mechanisms. For example, rf-pulse sequences in both coupling regimes can mediate the spin order transfer to target nuclei.178 Alternatively, magnetic field cycling into near zero field enables efficient polarization transfer to heteronuclei.179 Apart from the pairwise proton transfer, there can be single proton transfer, so-called oneH-PHIP. Also, there are spin order transfer mechanisms, where one or two hydrogens are chemically replaced as described in subsection 2.2.3. These are pairwise and single proton exchange mechanisms. In the case of single proton transfer exchange, an evolution of the spin order at the catalytic center, similar to ALTADENA, prior to the exchange is necessary.148

2.5.1 Level anti crossings (LACs)

At low magnetic fields, the spontaneous spin order transfer from parahydrogen can be understood when accounting for a polarization transfer based on LACs. A convenient way to picture a LAC is by looking at a system with two states of different energies. In Figure 17a, such a situation with the states |m and |n is shown. When the energies of these levels change with another parameter, such as the magnetic field B, their energy levels would 〉 〉 cross at some point if the energy of the higher state is lowered and the one of the lower state increased (see Figure 17b).

Figure 17. Energy diagrams to illustrate a LAC: a) Two states |m and |n of different energy. b,c)

Energy diagrams for the two states depended on the magnetic field B with a crossing 〉 〉 point in b) the uncoupled case and with a LAC and c) the case with coupled |m and |n .

〉 〉 2 Theory: Parahydrogen based hyperpolarization in NMR 43

Such crossing points can exists for a variety of slopes and only one of the energy levels needs to be dependent on the varied parameter. If now these states are coupled, the energy levels do not actually cross. In fact they will never meet and at the crossing point, one energy level will be higher and the other one lower instead. Such an avoided crossing of two energy levels is called LAC and shown in Figure 17c. The transition probability Qm,n for the population of two coupled states was estimated by Ivanov et.al.180 It depends on the strength of the scalar coupling J between the states and the rate at which the difference of the precession frequencies ν of the spins changes during the passage: (2 ) δ = exp (14) , d( )/2d π𝐽𝐽 𝑄𝑄m n �− � Thus, if the coupling is high and the rate at whichδ𝜈𝜈 δν𝑡𝑡 changes during the passage low, Qm,n goes towards its maximum. At this point, population is efficiently transferred from one high energy state to the other and from one low energy state to the other.

2.5.2 The role of LACs in SABRE experiments

In the strong coupling regime, polarization transfer by SABRE can be understood as a spin order transfer based on LACs. In an ALTADENA experiment,177 as introduced in section 2.5, the sample is exposed to a low magnetic field Bevo during the hydrogenation prior to detection. Similarly, for the SABRE approach, the spin order transfer strongly depends on the polarization transfer field Bevo. At certain polarization transfer fields, significantly higher polarization was reported on different substrates.12, 34-36, 48, 51, 52, 55, 151 This matches to the theory of LACs.121, 180, 181 In the case of the SABRE approach even the polarization level as well as the sign can be easily estimated when accounting for LACs.181 Their position and thus the optimum field depends on many parameters in the catalytic system. The catalytic cycle in a SABRE experiment can repetitively generate 6a. In this complex, the singlet symmetry of parahydrogen is broken and the polarization can be transferred to a target substrate, which then dissociates into the solution and is detected. Typically this is performed with excess p-H2 to maximize the polarization. As an exemplary SABRE complex, the efficient IMes(b) catalyst is shown in Figure 18a. The equatorial plane in the catalytic system plays the major role in the polarization transfer. The main reasons for this are the symmetry break as well as the stronger coupling of the substrate spin with respect to the 182 hydrides stemming from p-H2. As the chemical shifts of the hydride protons are often similar or even identical, the symmetry is broken by the J-couplings and the polarization transfer in a SABRE system can be estimated as a four spin system due to the strong interaction of ligands opposite to each other. Opposite i.e. in trans-position to the two hydrides Ha and Hb stemming from parahydrogen bound to the catalyst there are two 44 2 Theory: Parahydrogen based hyperpolarization in NMR coordination sites in the octahedral complex. The couplings in such a four spin system are shown in Figure 18b: The hydride protons Ha and Hb, shown as A and A’ in the spin system, and the two substrate molecules in the equatorial plane as coupling partners, shown as M and M’, build a coupling network. This AA’MM’ spin system contains four different J- coupling constants: The couplings between the identical species JAA (JA,A’) and JMM (JM,M’) as well as the cis and trans J-couplings. The cis-couplings JA,M = JA’,M’ are referred to as JAM and the trans couplings JA,M’ = JA’,M are referred to as J’AM. In the case of a proton experiment with pyridine, M and M’ would be the ortho-protons of two different pyridine molecules, as they are closest to the catalyst and have the strongest coupling to the hydrides. This also fits to experimental results where the highest 1H hyperpolarization is observed on these. The whole system is coupled through its J-coupling network, the hydrides are strongly coupled to each other (2JA,A’ 7 Hz) whereas the other couplings, which are 4J couplings, are smaller.181 ≈ −

Figure 18. Spin order transfer by SABRE: a) 6a as a general representation of a SABRE active catalyst

as shown in the catalytic cycle in Figure 15 and IMes(b) as an example for a catalyst, L1 from 6a is occupied by the IMes ligand and py, which is the only substrate and added in excess compared to the iridium complex and the IMes ligand, occupies three coordination sites. b) J-coupling network of a representary 4-spin system in a SABRE active complex: A

and A’ represent the protons Ha and Hb stemming from parahydrogen, while M and M’ stand for substrate spins such as the ortho-protons in pyridine. A common representation for the states in such a coupling network are singlet triplet basis functions. For the four spin system, decoupled block matrices for the symmetric as well as for the antisymmetric levels can be derived.121 The non-diagonal elements of these contain the strength of the coupling of the corresponding levels while the diagonal elements describe the energy of the individual states. For an efficient SABRE transfer between two spin states, the energy difference of the two involved levels should be as small as possible. A transfer takes place, when the two involved states have the same symmetry group and are connected (coupled) by a non-diagonal element of the density matrix which is not zero. Starting from these requirements, the different level anti crossings for a spin order transfer from parahydrogen can be derived: The J-coupling is independent from the 2 Theory: Parahydrogen based hyperpolarization in NMR 45 magnetic field. Therefore several crossterms can be discarded when looking for level anti crossings. Others depend on the Zeemann interaction and therefore their energy levels can be matched i.e. brought to the same energy with a suitable magnetic field. This is then called polarization transfer field and the probability for a transition from one state to the other is maximized. The sign of this field dictates how the transferred spin order is aligned with respect to the existing magnetic field or in other word whether a detected polarization would be positive or negative. When calculating the energy differences starting from the singlet states, several crossing points of the energy levels where transitions are likely remain. If one of these resonance conditions is met, an efficient polarization transfer to the respective state is possible. In the case of parahydrogen, the singlet state is higher populated and a spin order transfer to other states with a suitable SABRE catalyst is feasible. If the chemical shifts of the hydride protons as well as the substrate protons are identical

(δA δA’ and δM δM’), the spin system can be described as an AA’MM’ coupling network.181, 183, 184 In this AA’MM’ coupling system the LACs between the and the states as = = well as between the singlet state to the triplet states can be matched with a |ST±〉 |T±S〉 suitable Bevo. This matching condition can be calculated from the Larmor frequencies νA and |SS〉 |T±T∓〉 νM of the involved nuclei and the four different J-coupling constants between these spins, which were introduced in Figure 18b. For

|ST± T±S (15)

the matching condition is � ↔ � �

±( ) = (16)

A M AA MM and for 𝜈𝜈 − 𝜈𝜈 𝐽𝐽 − 𝐽𝐽

|SS T±T (17)

∓ there is a matching condition at: ⟩ ↔ � � 1 ±( ) = + ( + ) (18) 2 𝜈𝜈A − 𝜈𝜈M 𝐽𝐽AA 𝐽𝐽MM − 𝐽𝐽AM 𝐽𝐽′AM The precession frequencies νA and νM of the involved substrate and hydride spins can be calculated from their gyromagnetic ratios and chemical shifts. Thus the needed Bevo to match these states is: ±( ) = (19) (1 ) (1 ) 𝐽𝐽AA − 𝐽𝐽MM 𝐵𝐵evo For and and 𝛾𝛾A − 𝛿𝛿A − 𝛾𝛾M − 𝛿𝛿M |ST±〉 |T±S〉 46 2 Theory: Parahydrogen based hyperpolarization in NMR

1 ±( + ) ( + ) = 2 (20) (1 ) (1 ) 𝐽𝐽AA 𝐽𝐽MM ∓ 𝐽𝐽AM 𝐽𝐽′AM for and |T±T . 𝐵𝐵evo 𝛾𝛾A − 𝛿𝛿A − 𝛾𝛾M − 𝛿𝛿M A typical example for such a case is the polarization of the ortho-protons of the substrate |SS〉 ∓〉 pyridine. Their LACs in the IMes(b) complex introduced above can be calculated with the gyromagnetic ratio of hydrogen (42 MHz/T), the chemical shifts (δA = −22.8 ppm and

δM = 8.1 ppm) and the J-couplings (JAA’ = −7 Hz; JAM = 3 Hz; J’AM = 0.3 Hz and JMM’ = −0.11 Hz) taken from Pravdivtsev et al.182 To match this LACs (19) and (20) give Bevo = ±52 G and ±67 G. This fits well to the polarization transfer field dependence found for 1H hyperpolarization experiments with pyridine.12, 47, 151, 185 Even in this simple model, the position and nature of the level anti crossings varies depending on the substrate, catalyst and experimental conditions. When changing the catalytic system also the involved energy levels change. Even in the discussed case of pyridine in IMes(b), the other pyridine protons, the IMes ligand and the solvent impact the effect of the sample exposed to Bevo. However, in this simple model they are approximated to be small or cancel each other out. Interestingly, the LACs for many other heterocycles containing nitrogen atoms are all very similar.55, 121 This includes the resonance condition for polarization transfer to protons at around 60 G. The reason is their similar chemical and magnetical nature including the chemical shift difference, between the ring protons and the hydrides in the formed catalytic complex, which is around 30 ppm. Further coupling partners can also play a role in the coupling network. For example in the case of a phosphine ligand in the position L1 as in the first published SABRE active PCy3 catalyst, the coupling to the 31P has to be taken into account. This five spin system has also been theoretically investigated in the context of SABRE.181 Nevertheless the position and shape of the LACs still very similar. When polarized level is split up due to another coupling, e.g. to a neighboring CH group, it can be shown that there is a sign change within the individual transitions of the multiplet. For example in the case of a LAC with a negative sign from positive to negative polarization of transitions within the multiplet. Larger changes and more interesting cases arise, when looking at mixtures of different substrates. This is discussed in the context of water hyperpolarization in chapter 5. There the catalytic system contains not only water but also histidine as an additive and additional coupling partner are part of the spin system. Such a system is not an AA’MM’ system as the two substrates are not equivalent anymore and vary in their chemical shifts and J-couplings. This is also the case for other additives or when the solvent coordinates to the catalytic center.22, 151, 152, 160 2 Theory: Parahydrogen based hyperpolarization in NMR 47

Also, for an efficient polarization transfer to more insensitive nuclei,186, 187 the knowledge of LACs can also be applied, for example to directly hyperpolarize 15N.34-36 The same model can be applied while M and M’ now are the 15N nuclei of the substrate. In this case, it is a reasonable approximation to neglect the influence of the pyridine protons: In this case the coupling between the substrates but also their coupling to the hydrides is stronger. Especially the frequency difference between the hydrides and the 15N nuclei now differs mainly due to the gyromagnetic ratio instead of the chemical shift, which is significantly higher. In the case of 15N this is a factor of 10 instead of the chemical shift difference which is around 30 ppm ≈ 10-5 so around one million times higher. This huge difference leads to a changed LAC at significantly lower fields. A very efficient transfer is therefore observed at microtesla fields even shielded from earth’s magnetic field. This is referred to as SABRE- SHEATH (SHield Enables Alignment Transfer to Heteronuclei).35 Additionally, introducing such a strong coupling partner also impacts the polarization of the proton. In the case of 15N labeled pyridine, abbreviated 15N-pyridine, it is necessary to consider an AA’MM’XX’ system, where X denotes 15N. Apart from the hydrides, both the 15N nuclei and polarized protons from 15N-pyridine, are not negligible and a further coupling partner X is introduced. The additional heteronuclear coupling within the 15N-pyridine is also 35 significant (JM,X ≈ 11 Hz) . Such a system is discussed in chapter 7. In this case also a significant amount of higher order spin states has been created by distributing the singlet order in the catalytic intermediate. In this case heteronuclear two-spin order and even higher spin order was observed.58 Higher spin order by distributing the singlet order in the catalytic intermediate has already been theoretically studied in the homonuclear case i.e. between proton spins.188 Experimentally it has also been reported very early and can be read out with suitable pulse sequences.169, 189, 190 Generally, when looking at optimum evolution fields in the strong coupling regime, the higher the difference between the νI of the involved nuclei, the lower the optimum Bevo and vice versa. This does not only include very low evolution fields as described for SABRE- SHEATH. An example of a higher evolution field is the evolution of two hydride protons after the oxidative addition of p-H2 to a catalytic center. This is taken advantage of in chapter 5, where water is polarized in this way by a consecutive proton exchange. Apart from this coherent spin mixing in the strong coupling regime, also spontaneous polarization transfer in a high magnetic field by cross relaxation191 is possible. The main motivation for this is the better chemical shift resolution in high magnetic fields. More conventional approaches in high field use trains of pulses to unselectively transfer polarization from parahydrogen to all SABRE substrates that interact with the Ir-complex.18

Finally, there is an alternative to a prepolarization at a low Bevo. An irradiation at high field can also fulfil LAC conditions and enable an efficient polarization transfer from parahydrogen with the SABRE approach at high magnetic fields.182, 192-194 48 3 Devices, synthesis and experimental procedures

3 Devices, synthesis and experimental procedures

This chapter introduces the involved devices, catalyst synthesis and experimental procedures of this work (see Figure 19). Detailed descriptions or adjustments fitting to the experiments of each results chapter are provided in individual methods sections of those chapters.

Figure 19. Guideline through chapter 3: The used devices and catalysts as well as the experimental details during hyperpolarization experiments are introduced. The impact of different experimental parameters and conditions on the hyperpolarization efficiency is discussed in section 2.4.

3.1 Devices

The most important devices involved in the parahydrogen based hyperpolarization experiments of this work are mentioned below: The different magnets providing the detection field for NMR experiments and the ones used as polarization transfer fields as well as the involved parahydrogen generators are described in this section. 3 Devices, synthesis and experimental procedures 49

3.1.1 Magnets as detection fields

As detection fields a 20 – 512 kHz (0.5 – 12 mT) electromagnet in the ultra-low field, a 43 MHz (1 T) compact NMR spectrometer and two superconducting magnet for high field spectroscopy at 300 and 400 MHz (7.1 and 9.5 T) were chosen. All of them feature different sets of shim coils to achieve the needed spectroscopic homogeneity.

1) Ultra-low field spectrometer In the ultra-low field, a custom-made cylindrical electromagnet was used, which generates a field of 1-16 mT (see Figure 20a). It was constructed by the ZEA 1 and ZEA 2 of the Forschungszentrum Jülich under the supervision of Stephan Appelt. Johannes Collel built the magnet during his PhD thesis at the RWTH Aachen University. Its detection coil and excitation coil are separated. The used detection coils were winded by Alexander Liebisch. The cylindrical detection coil, made of copper wire, is connected to a ferrite to obtain an EHQE-NMR81 (external high-quality-factor-enhanced NMR) circuit before the signal is fed into a low noise preamplifier. The EHQE setup including the hardware for the hyperpolarization experiments was built by Martin Süfke. Typical detection frequencies are 41.7 kHz, 83.3 kHz, 166.7 kHz, 333.3 kHz and 512.0 kHz as very sensitive coils were made for these fields and ferrites matched to these frequencies. The signal is preamplified before it is fed into a lock-in amplifier constructed by Stephan Appelt. The preamplification could be reduced and one amplifier turned off to optimize the signal acquisition i.e. not saturate the receiver. With shim coils in z, z2, x and y, it reaches a homogeneity below 1 ppm in a sensitive volume of about 0.5 ml. A glass setup and connected tubing including flow meters as well as pressure and needle valves allow for continuous polarization in a bubble setup. The signal is acquired at a rate of 16 kHz with a 16 bit resolution at an off resonance frequency (OR) about 60 Hz from the Larmor frequency. A Python software written by Martin Süfke is used to analyze experimental results.58

2) Low field spectrometer - Spinsolve For the low field experiments the compact NMR Spinsolve spectrometer (43 MHz, Magritek) is used (see Figure 20b). The magnet is shimmed to a reference sample consisting of 90% D2O and 10% H2O. A typical shim resulted in a linewidth of 0.3 Hz FWHM (full width at half maximum). The samples were prepared in deuterated solvents for a better comparability, although such a solvent is not necessary in low field measurements. Such solvents were e.g. methanol-d4 (99.9% perdeuterated methanol: δ = 3.4 ppm and 4.8 ppm), toluene-d8 (99.9% perdeuterated toluene: δ = 2.08 ppm and 7.0 ppm (m)) or D2O (99,95% perdeuterated H2O: δ = 4.8 ppm). A thermal reference was taken before applying p-H2, between and also after the experiments to be compared with the hyperpolarized signal. The 50 3 Devices, synthesis and experimental procedures spectra were processed with MNova by mestrelab research (apodization 0.4 Hz, baseline correction: polynomial fit zero-order, first order phase correction).

Figure 20. Low field spectrometers: a) Ultra-low field spectrometer (1-16 mT) and b) low field spectrometer (1 T) in the fume hood.

3) High field spectrometers The reference measurements (1H, 13C, 19F and 31P-NMR-spectra) were measured with a Bruker Avance III 400 MHz spectrometer. The samples were dissolved in a deuterated solvents such as methanol-d4, CDCl3, D2O or toluene-d8. The chemical shift δ is given in ppm 1 relative to the residual H signal from the deuterated solvent (δ = 7.24 ppm for CDCl3) or to TMS. Constant spin multiplicity is denoted as: ”s” for singlet, ”d” for doublet, ”t” for triplet and ”m” for multiplet, while “br.” indicates a broad peak. Hyperpolarization measurements were conducted on a Bruker Avance III 300 MHz (see Figure 21). The experimental procedure for data acquisition is described in subsection 3.3.2. Data were acquired and processed with Topspin 3.0 (Bruker).

Figure 21. High field Spectrometers: a) Bruker Avance III, 300 MHz (7.1 T) and b) Bruker Avance III, 400 MHz (9.4 T).

3 Devices, synthesis and experimental procedures 51

3.1.2 Magnets for polarization transfer fields

To create a suitable Bevo, two different setups (see Figure 22) have been used: a) The stray field of a simple self-constructed horse shoe shaped permanent magnet whose magnetic field ranges from 1 G to 3000 G. b) A box, to shield the sample from external magnetic fields, such as the earth’s magnetic field or rf-fields constructed by Stephan Appelt. The shielding comes from a mu-metal frame, which is a ferromagnetic nickel-iron alloy with high permeability, and a small coil inside the shielding to generate minute magnetic fields from 1 µT to 500 µT.

Figure 22. Magnets for polarization transfer fields: a) Horse shoe typed magnet and b) μ-metal shielded box with a small coil inside.

3.1.3 Parahydrogen generators

Standard hydrogen (5.0) purchased from a commercial source is used without further purification. Two different p-H2 generators, which convert n-H2 into p-H2 were employed in this work:

The p-H2-Generator bphp90 from Bruker generating around 92% enriched p-H2 (see Figure 23a): The thermal hydrogen is cooled down to around 35.6 K with 300 W using a closed cycle cryostat setup in the presence of an iron oxide hydroxide catalyst (FeOOH). This means that the polarization is about 0.90 (as shown in section 2.1, Figure 6). The generator provides a constant p-H2 output of about 10 ml/min flow for continuous measurements with p-H2 pressures of up to 7 bar. The actual enrichment can decrease at high flow rates. 195 Alternatively, a custom-made p-H2 generator was applied (see Figure 23b). Jörg Ackermann built it under the supervision of Martin Süfke and Stephan Appelt 196. The conversion temperature can be chosen freely. It is cooled down in Gifford-McMahon cycles 52 3 Devices, synthesis and experimental procedures

(300 K > T > 9 K, ARS Cryo). The high throughput and even storage of liquid p-H2 allows for very high flow rates up to 400 ml/min.

Figure 23. Parahydrogen generators: a) Bruker parahydrogen generator bphp90 and b) home-made

p-H2 generator.

3.2 Synthesis

Unless otherwise stated all chemical work is conducted water free and under inert gas atmosphere (so-called Schlenk conditions). Solvents were dried, degassed and stored over molecular sieve 3a. All chemicals were purchased from Sigma Aldrich and used as provided unless otherwise noted. The synthesis of the vastly used IMes and [Rh(COD)-(S)-BINAP]BF4 (BINAP) catalyst precursors and a corresponding characterization with NMR are described below ((S)-BINAP = (S)-2,2′-Bis(diphenylphosphino)-1,1′-binaphthalene). Additionally, their activation with hydrogen gas into their catalytically active species is shown. The

[Rh(COD)(dppb)][BF4] (dppb) and [Rh((R,R)-Me-DuPhos)(COD)]BF4 (DuPhos) were synthesized according to literature197, 198 and the recorded NMR data of them agreed with the literature values (dppb = 1,4-bis(diphenylphosphino)butane; (R,R)-Me-DuPhos =

1,2-bis[(2R,5R)-2,5-dimethylphospholano]benzene). The PCy3 catalyst (Crabtree’s catalyst) was bought and used without further purification.

3.2.1 Synthesis of [IrCl(COD)(IMes)]

The synthesis of IMes (see Figure 24) was based on Vazquez-Serrano et al.199 and has been conducted multiple times. In an experiment two equivalents (eq) IMes∙HCl ((2,4,6-trimethylphenyl)imidazolium-chloride) were solved in 3 ml acetone. One eq of

[Ir(OMe)COD)]2 (OMe = methoxy) was solved in 2 ml acetone. The solution containing the

IMes ligand was slowly dropped to the solution of the Ir-dimer [Ir(OMe)COD)]2 over a time period of 15 min. The resulting solution was stirred overnight (15 h) at RT. 3 Devices, synthesis and experimental procedures 53

N N 15 h, RT • 2 IMes HCl [Ir(OMe)(COD)]2 2 acetone Ir Cl

Figure 24. Synthesis of IMes from IMes∙HCl and [Ir(OMe)(COD)]2. After drying a yellowish-orange solid is obtained. For further purification the catalyst was recrystallized in methanol twice. A yellow solid was obtained and characterized with NMR:

1 H-NMR (400 MHz, methanol-d4, Figure 25): δ = 1.19 (m, 4H, H9), 1.57 (m, 4H, H10), 2.08 (br. s,

6H, H3), 2.19 (br. s, 6H, H5), 2.26 (s, 6H; H1), 2.98 (m, 2H; H8), 3.2 (s, CD2HOD), 3.90 (m, 2H; H11),

4.8 (s, CD3OH), 6.93 (br. s, 4H; H2, H4), 7.13 (s, 2H; H6, H7) ppm.

1 Figure 25. H NMR spectrum of the synthesized IMes in methanol-d4 at 400 MHz.

3.2.2 Synthesis of [Rh(COD)-(S)-BINAP]BF4

The catalyst precursor BINAP was synthesized as shown in Figure 26: Rh(COD)BF4 (59.6mg, 0.16 mmol) was dissolved in 5 ml dichloromethane (DCM). Whilst stirring, a solution of (S)-BINAP (100.4 mg, 0.16 mmol) in 3 ml DCM was slowly dripped into it and the red solution was stirred for 3.5 h at RT. Hereafter, the solution was concentrated to about one third with a vacuum pump. It was then precipitated and washed with pentane. The 54 3 Devices, synthesis and experimental procedures orange-red solid was stored under argon atmosphere for further use. The yield was 120 mg, 200 92.3% confirmed by NMR in CDCl3. The obtained NMR data agrees with the literature.

Ph Ph 2.5 h, RT P - [Rh(COD)]BF4 (S)-BINAP Rh BF4 DCM P Ph Ph

Figure 26. Synthesis of BINAP.

1 H-NMR (400 MHz, CDCl3, Figure 27): δ = 1.53 (m, 4H; H11), 4.87 (s, 2H; H10), 6.41 (d, 1H; H1,

J1,2 = 8.5 Hz), 6.64 (t, 2H; H8, J = 7.2 Hz), 6.71 (t, 1H; H9, J8,9 = 7.2 Hz), 6.92 (t, 1H; H2, J = 7.7 Hz),

7.19 (CDCl3), 7.26 (t, 1H; H3, J = 7.6 Hz), 7.36 (m, 4H; H7, H7‘), 7,40 (t, 2H; H8‘, J = 7.3), 7.53 (d, 1H;

H4, J3,4 = 8.2 Hz), 7.60 (t, 1H; H9‘,J = 7.0), 7.63 (m, 2H; H5, H6) ppm. 31 P-NMR (162 MHz, CDCl3, Figure 28): δ = 26.0 (d, JRh,P = 156.3 Hz) ppm.

1 Figure 27. H NMR spectrum of the synthesized BINAP in CDCl3 at 400 MHz. The peak assignments are given above. 3 Devices, synthesis and experimental procedures 55

31 Figure 28. P NMR spectrum of the synthesized BINAP in CDCl3 at 162 MHz. It features a doublet at 31 103 δ = 26.0 ppm from the P J-coupling to Rh of JRh,P = 156.3 Hz.

3.2.3 Activation of homogeneous catalyst precursors

The catalyst precursors used in this work form their catalytically active species after a reaction with hydrogen gas (see subsection 2.2.1). In Figure 29 the activation of the IMes and the BINAP catalyst precursors are shown. The IMes consists of the monodentate carbene ligand IMes, an anionic chlorine ligand and COD as a protective group bound to the iridium center. During the activation with hydrogen and pyridine not only the COD is removed, but also the chlorine ion dissociates from the iridium forming the homogeneous cationic catalytically active species of this transition metal catalyst: [Ir(IMes)H2(py)3]Cl (IMes(b)) As introduced in subsection 2.3.1, the activation of IMes with py is just one example and the general catalytically active complex is [Ir(H)2(IMes)(L)3]Cl (IMes(a)). In contrast to that the BINAP catalyst already consist of the cationic complex containing the bidentate (S)-BINAP ligand and cyclooctadiene as a protective group bound to a – rhodium center and the BF4 as counterion. Upon activation with hydrogen gas, COD is removed and the catalytically active complex [Rh(-(S)-BINAP)H2L2] (BINAP(a)) is formed, where L is a free coordination sites for the solvent or the substrate not shown in Figure 29b to make it clearer. 56 3 Devices, synthesis and experimental procedures

a)

N N py, H2 N N COD py H Ir Ir Cl py H py Cl

b) Ph Ph Ph H2 Ph P COD P H Rh Rh BF4 P P H Ph Ph Ph Ph BF 4 Figure 29. Activation of the homogeneous catalyst precursors a) IMes and b) BINAP into their catalytically active species. The oxidative addition of hydrogen changes the oxidation state of Rh and Ir from +I to +III and the cationic, octahedral dihydride complexes are formed.

3.3 Experimental section

Different parahydrogen based hyperpolarization experiments are described over the four results chapters. They include continuous flow and batch experiments with p-H2. The latter are mostly so-called shake-and-drop experiments. They allow a quick and easy sampling of various experimental conditions in hyperpolarization experiments. Their principle experimental procedure and the corresponding data acquisition and processing are described below. Other experiments, like continuous hyperpolarization, as well as adjusted shake-and-drop experiments to fit the experimental demands are described in the individual methods section of each chapter.

3.3.1 Shake-and-drop experiments

For shake-and-drop experiments, Young-type tubes containing the catalyst and the target substrate in a suitable solvent were prepared. The details on the individual sample compositions are described in the methods section of each chapter as well as in the corresponding results sections. Solvents and substrates were dried and degassed. In the hyperpolarization experiments, p-H2 was pressed on top of the samples filling the headspace with up to 7 bar p-H2. For the mostly conducted ALTADENA experiments the sample was shaken, heated up if necessary and exposed to a magnetic evolution field Bevo before detection. A permanent magnet was used to study the evolution of the 3 Devices, synthesis and experimental procedures 57 magnetization in a polarization transfer field. (see subsection 3.1.2) A water bath kept the samples at a higher temperature, except during shaking or detection, if required.

3.3.2 Data acquisition and processing

The shaken samples are dropped into a spectrometer and detected immediately with a single scan. Thermal reference spectra are taken after a suitable waiting time to match the polarization built up from B0. For the experiments 90° impulses are applied unless otherwise stated. As detection fields 0.5 – 12 mT in the ultra-low field, 1 T at low field and 7.1 or 9.5 T magnets for high field are used. Details on the devices and the corresponding software to analyze the results are described in section 3.1. The recorded FIDs are zerofilled and Fourier- transformed. Spectra are phase and baseline corrected and apodization below the linewidth is applied. 58 4 A modular immobilization system

4 A modular immobilization system

In this chapter an immobilization system introduced by Augustine et.al201 is evaluated upon its capability for parahydrogen based hyperpolarization. With this system, hydrogenation catalysts are anchored on different support materials. The resulting immobilized catalysts are heterogeneous catalysts and can be easily recovered or even employed in biomedical applications considering their low metal contamination. While the literature mainly focuses on the high activity, stability, enantioselectivity and manifold applications of these hydrogenation catalysts134, 201-207, they have not been employed in hyperpolarization experiments up to now. Nevertheless the properties of a homogeneous catalyst can change when it is anchored on a support material. The impact of these changes, optimized reaction conditions, and possible catalytic systems for parahydrogen based hyperpolarization experiments are the content of this chapter. The catalytic systems, operating conditions and evaluated experimental parameters during the hyperpolarization experiments in this chapter are summarized in Figure 30. The chapter consists of five parts. The introduction (4.1) covers heterogeneous PHIP catalysis, the immobilization of homogeneous PHIP catalysts and a description of the Augustine system. The following section (4.2) develops a model system to probe the Augustine approach in parahydrogen based hyperpolarization experiments. Based on these results, the activation of the immobilized species is altered to fit the changed properties and obtain catalysts suitable for hyperpolarization experiments. Consecutively, a methods section (4.3) describes the synthesis of a polyoxotungstic acid (PTA) anchored on several support materials, the immobilization of several homogeneous catalysts on the latter as well as the experimental procedures. The main section (4.4) evaluates the Augustine systems for application in hyperpolarization experiments based on a model system. It covers experimental parameters such as temperature, pressure, magnetic evolution field, amount of catalyst, as well as the particle size of the support material. Additionally the large scope of hyperpolarization systems containing other anchored catalysts, further substrates and possible solvents is demonstrated. Finally an exemplary experiment in a higher magnetic detection and polarization transfer field is discussed. At last a conclusion and an outlook (4.5) are given.

4 A modular immobilization system 59

Figure 30. Scope of the chapter “A modular immobilization system”. The general conditions are highlighted in yellow, while solvents and varied experimental parameters of this chapter are shaded in light yellow. Parahydrogen based chemistry i.e. the substrates and catalysts as well as the polarization transfer regime and mechanism are highlighted in red. PHIP

represents the classical spin order transfer mechanisms from p-H2 by hydrogen substitution or incorporation into the substrate. Points not addressed in this chapter are

written in grey. The solvents toluene-d8 and water as well as the ligand PPh3 and SABRE are part of the excursus for this chapter, which can be found in the supplement section 9.1. The abbreviations are introduced as they occur in the text and are summarized in the beginning of this work.

4.1 Introduction

While there are plenty of homogeneous catalysts for parahydrogen based spin order transfer, heterogeneous catalysts are rarer. The main advantage of a heterogeneous catalysts is their facilitated separability from reaction mixtures. Thus, such a catalyst can be reused for another experiment or employed in those biomedical applications which are inhibited by toxic homogeneous catalyst. Compared to chemical transformation reactions, hyperpolarization catalysts additionally require attention considering the detection of the created spin order. Relaxation, transport phenomena and arising susceptibility issues alter the detected 60 4 A modular immobilization system polarization when employing heterogeneous hyperpolarization catalysts.18, 129, 130, 208 While the hyperpolarization catalyst itself can be heterogeneous like nanoparticles132, 146, 147, 209, the focus of this work lies on immobilization systems exploiting the well-known optimized homogeneous catalysts.28, 124-126, 210 Such a homogeneous catalyst can be attached to a support in different ways: Prominent examples are chemical bonds, physical interactions such as adsorption or trapping or ionic interactions.133-140 Some of them were already used in PHIP experiments.129, 141 The immobilized catalyst can then be implemented in a continuous hydrogenation setup37, 130, 133, 211-218, where it is for example held back by a sieve or a membrane if not part of the setup anyway like in the case of fibers. Although this approach is mostly too expensive for industrial applications219 due to the lower rates, lower stability, possible leaching and the additional synthesis step of immobilization, these catalysts are far developed and their well-defined nature should be ideal for spin order transfer as in hyperpolarization experiments.

Rh nanoparticles, formed from [Rh(nbd)2]BF4, physisorpted on glass plates within a hyperpolarization setup in a membrane reactor showed hydrogenation and hyperpolarization of unsaturated substrates with parahydrogen (details see subsection 9.1.1, nbd = 2,5-norbornadiene). Unfortunately, they were subject to a significant catalyst ablation especially at higher flow rates. Such a loss of an immobilized catalyst e.g. being washed out over time is called leaching. This prevents long time applications and also the application in biological systems as the toxic metal catalyst is released into the solution. Thus stronger binding of the homogeneous catalyst to the support is necessary.

A polystyrene polytriphenylphosphine polymer, also called PPh3 polymer bound

(PPh3PB) as support material allows multiple catalytic centers when transition metal precursors, in this case based on iridium, are formed on its triphenylphoshine (PPh3) groups. 151 In this way heterogeneous analoga to the SABRE active [Ir(IMes)PPh3H2(py)(ac)] are obtained (ac = acetonitrile). These synthesized and activated polymerbound catalysts allow for hyperpolarization with parahydrogen (see supplement 9.1.2), but it is less efficient than the homogeneous counterpart due to the altered chemical structure and needed diffusion of the substrates and parahydrogen to the catalytic center. Furthermore polymeric support, which is difficult to hold in place for continuous flow experiments (size: 100−200 mesh), decreases the detected polarization due to the magnetic susceptibility difference at the polymeric surface and relaxation. An easy and convenient way to immobilize homogeneous catalysts on different support materials has been developed: The so-called Augustine system.201, 203 It uses a heteropolyacid (HPA) to link cationic homogeneous catalysts to a metal oxide surface.205 Augustine et al. presented several support materials such as montmorillonite, alumina and lanthana.204 Different heteropolyacids can be used as a linker. The formation of polyoxometallates(POMs), the anions of the HPAs, is most prominent for group V and VI 4 A modular immobilization system 61 heteroatoms in the center (e.g. vanadium (V), molybdenum (VI) or tungsten (VI)) due to their comparably low 5th (or 6th respectively) ionization energies and the ion radius.220 POMs feature a diversity of applications in many scientific fields such as environmental science, medicine, electrochemistry or catalysis as will be the focus here.221-224 When used as a linker for a homogeneous catalyst, the POM binds to the metal oxide support with its hydroxyl groups.225 The oxygen atoms are also the binding site of the POM to the cationic catalyst precursor202, which is immobilized by a simple acid base reaction of the HPA. Following the concept of hard and soft acids and bases (HSAB), an ion exchange replaces a proton of the HPA with the larger catalyst precursor ion with consecutive washing and drying of the support. Thus, the charged transition metal complex is held in place by an ionic interaction with the POM or a covalent bond.202 Duque et al. assume that the transition metal center of the metalorganic complex is anchored to the HPA while its ligands are not involved in the anchoring of the catalyst.134 This agrees with the picture the complex from of Augustine et al. represented in Figure 31b.205

Figure 31. HPA as linker: a) Structure of an α-Keggin ion with its heteroatom in the center (yellow), the twelve metal centers (blue) and the 40 positions of the oxygen atoms (red). b) “Depiction on an anchored homogeneous catalyst composed of a support, a heteropolyacid anchoring agent and a catalytically active organometallic complex” from Augustine et al.205.

Augustine et al. uses a Keggin type POM, a hollow sphere consisting of 12 MO6 octaeders with the heteroatom in its center form shown in Figure 31a named after their founder in 1933.226 Here, the acidic polyoxotungsticacid (PTA), characterized by Keggin as 227 H3PW12O40 5H2O with XRD , is employed. This Keggin type HPAs is very acidic compared to others228 and therefore more electron withdrawing ligand, which increases its reactivity, but also other HPAs can be employed.205 A Dawson type HPA has been employed by Finke et al., 62 4 A modular immobilization system

8− 202 leading to [(COD)RhP2W15Nb3O62] when using Rh (COD)2 as a precursor. This case showed Nb bonding to the HPA and allows for multiple bonding of the HPA. These catalysts show high activity and negligible leaching rates even for polar solvents such as ethanol.204 Furthermore, they allow for continuous, enantioselective hydrogenation reactions, which means that the immobilized catalysts retain the selectivity and chirality of their homogeneous form.134, 206, 215-217 Nevertheless this approach has not been combined with parahydrogen based hyperpolarization yet.

4.2 Towards a model system for hyperpolarization experiments

This section features the development of a model system suitable to evaluate the Augustine system in the context of parahydrogen based hyperpolarization. Such hyperpolarization experiments do not only increase the NMR sensitivity concerning the substrates, but can also allow further insights into the catalytic cycle for example by determining catalytic intermediates or exchange mechanisms such as deuteration. This described modular immobilization approach anchoring homogeneous catalysts on different support materials linked by a HPA enables various chemical applications such as efficient, continuous asymmetric hydrogenations.134, 206, 215-217 Nevertheless, the properties of these catalysts immobilized on a support can differ from their homogeneous analoga in many ways including the created polarization in hyperpolarization experiments. Therefore, in this section a model system and adapted experimental procedures for such experiments are developed stepwise. First, a suitable homogeneous catalyst that will be anchored and a target substrate for the hyperpolarization are introduced. After that, different support materials are compared towards their use in hyperpolarization experiments. Finally, the experimental methods are adjusted. A suitable catalyst activation procedure to obtain an immobilized species which can be used in hyperpolarization experiments is developed.

4.2.1 The homogeneous [Rh(COD)-(S)-BINAP]BF4 catalyst, butylacrylate and parahydrogen – a model system

To probe the modular immobilization system for parahydrogen based hyperpolarization experiments, standard conditions are defined to be able to evaluate different effects. For facilitated comparability, only one experimental parameter was varied and not many simultaneously. Therefore, standard conditions and a model system were developed and defined in the following. For this model system, the [Rh(COD)-(S)-BINAP]BF4 catalyst precursor (BINAP) as a homogeneous catalyst to be anchored and butylacrylate as the to- be-polarized substrate were used. To have an easy and fast sampling, the experiments were 4 A modular immobilization system 63

performed in young type tubes with an excess of p-H2, shaken at earth’s magnetic field and detected in a compact 1 T spectrometer. A spectrum of a shake-and-drop experiment using the homogeneous catalyst precursor BINAP is shown in Figure 33a. It was recorded without prior catalyst activation with hydrogen and is depicted in grey to be compared with its thermal reference in black. Interestingly two different chemical groups from two different chemical compounds are hyperpolarized under ALTADENA conditions: The unsaturated CHCH2 of the substrate butylacrylate itself is hyperpolarized by chemical exchange with p-H2 (pr-PHIP) at around

6 ppm. Secondly, the CH2CH3 group of the formed butylpropionate through hydrogenation from butylacrylate with p-H2 (h-PHIP) shows enhanced polarization. The antiphase nature of the signals from the two chemical groups (CH3 at 1.1 ppm and CH2 at 2.3 ppm) is typical for

PHIP experiments and elaborated in section 2.5. Figure 32 shows both p-H2 based hyperpolarization mechanisms for the model substrate butylacrylate: These are a) hydrogenation and b) pairwise replacement, whose mechanisms are shown in section 2.2. a) H H H O [Rh(S)BINAP] H O p-H2 H -PHIP H H O h H O (hydrogenation) butylacrylate butylpropionate

b) H H H O [Rh(S)BINAP] H O p-H2 n-H2 H O pr-PHIP H O (exchange) butylacrylate butylacrylate

Figure 32. Different hyperpolarization mechanisms of butylacrylate with parahydrogen using BINAP as a catalyst: a) hydrogenation to butylpropionate (h-PHIP) and b) exchange of the terminal protons (pr-PHIP).

In Figure 33b, an experiment with BINAP catalyst immobilized on γ-Al2O3 is shown. The experimental details of the immobilization including this BINAP@Al2O3 will discussed in the following subsections. Already in these preliminary experiments, significant enhancement to a thermally polarized reference spectrum was achieved and the effect cannot be 64 4 A modular immobilization system explained by leaching into the liquid phase as the filtrated solute shows no catalytic activity.

Figure 33. 1H NMR Hyperpolarization experiments with a) the homogeneous BINAP catalyst and b)

the immobilized BINAP@Al2O3. Experiments with 7 bar p-H2 spectra in grey and thermal reference spectra (single scan) in black. The upper grey spectrum in b) features an experiment with the filtrated solute, where no hyperpolarization was observed.

Experiments were conducted at RT with 20 µl butylacrylate in 200 µl methanol-d4 (CD3OH- peak marked with a star) and detected at 1 T. For further details see text.

4.2.2 Support materials

The properties of a homogeneous catalyst can change, when it is immobilized on a heterogeneous support. This may vary depending on the chosen support material. Therefore, different support materials are validated concerning their suitability for hyperpolarization experiments. Homogeneous catalyst precursors are anchored using a 202 HPA as a linker. Therefore the Keggin type and very acidic H3PW12O40 (PTA) is bound to the support. As such forms of alumina, α-Al2O3, γ-Al2O3, a hollow fiber made of α-Al2O3, and montmorillonite ((Na,Ca0,5)0,3(Al,Mg)2[(OH)2|Si4O10] 4H2O), a three layer clay from the smectite group where two tetrahedral layers sandwich an octahedral alumina sheet, were probed. “Aluminium oxide mainly occurs in these two forms: The stable α-alumina called corundum 229 and the most prominent metastable form: γ-Al2O3 , the so-called activated alumina.” The 2– 3+ structure of α-Al2O3 is an hexagonal close packed array of O with the Al ions occupying two thirds of the octahedral interstitial sites (Figure 34a). This gives a rhomboedric crystal –3 structure and therefore a very hard, dense (4.0 g/cm ) and unreactive material. γ-Al2O3 has a defect spinel structure in a cubic crystal lattice, which can be beneficial as a catalyst support due to the associated increased porosity and lower density (3.5 g/cm–3), but is less mechanical stabile (Figure 34b). 4 A modular immobilization system 65

Figure 34. Crystal structures of α- and γ-Al2O3 with aluminum depicted in blue and oxygen in red: a)

α-Al2O3 with its rhomboedric crystal lattice (R-3c) giving a very hard material and b) γ-Al2O3 with its cubic crystal lattice (Fd-3m) as a very porous material from Jiang 2011230.

The catalysts anchored on the α-Al2O3, including the whole and grinded fiber, did not show hyperpolarization in preliminary experiments and were therefore considered less promising for applications in hyperpolarization experiments. In contrast to that, experiments on montmorillonite as well as on γ-Al2O3 deliver promising results. A spectrum of this hyperpolarization experiment with BINAP@Al2O3 is shown in Figure 33b (grey) and compared to a thermal reference spectrum (black) at 1 T. The catalyst was not activated prior to the experiment and handled a room temperature the whole time. Thus, the observed polarization is yet small, but will be increased by optimizing the system, which is discussed in the following sections. The liquid phase of the experiments with the immobilized catalysts is taken for further studies. Neither hydrogenation nor hyperpolarization could be observed using the residual liquid (Figure 33b upper grey spectrum). Also in a 1H spectra at a 300 MHz high field magnet no trace of the catalyst could be detected (ns = 1000). This suggests that the heterogeneous material and thus the immobilized catalyst was responsible for the observed hydrogenation reaction with p-H2. It also responds to the high stability of such catalytic complexes and their minimal leaching rates especially in organic solvents. 204

4.2.3 Activation of the immobilized catalysts

The homogeneous catalyst precursors were activated by filling the headspace of the

NMR tubes with p-H2 at different pressures. The catalytically active species is obtained in the same way as for the homogeneous analoga where the non-classical coordination of hydrogen is followed by an oxidative addition of hydrogen to the transition metal center (see subsection 2.2.1 or Jessop and Morris128). For the heterogeneous catalysts, this activation process was significantly slower. Thus, an activation of the catalyst prior to the hyperpolarization experiments is beneficial. Experiments have shown that the active complex can be identified by its dark grey color. During deactivation, i.e. due to a lack of hydrogen pressure, the metastable active catalytic complex becomes a lighter grey within minutes until it reaches its initial white and 66 4 A modular immobilization system inactive form. When only one hydride is at the cationic transition metal center while the other is found at the metal cluster, such a complex is less active or even inactive in hyperpolarization experiments. The correlation between the two protons stemming from parahydrogen is crucial for hyperpolarization experiments. There is a relaxation of the spin order depending on the relaxation times of the different intermediates and their corresponding lifetimes in the catalytic cycle. Thus, also the mechanism of the hydrogenation with parahydrogen is crucial for efficient hyperpolarization experiments. Many hydrogenation mechanisms are possible with such complexes ranging from inner sphere hydrogenation to ligand assisted ones.128 This involves different hydrides and intermediate complexes. The classical hydrides formed in this case are crucial for the hydrogenation. Bond et al. and Augustine et al. describe a two-step hydrogenation mechanism and not a simultaneous one indicated by double bond isomerization and deuterium scrambling.231, 232 Therefore a fast incorporation of the hydrogen into the target substrate is desired. Different ways to activate the catalyst have been tested in order to find the most efficient one. First H2 was bubbled through a dispersion of the catalyst in ethanol and stored overnight in a H2 atmosphere. Only parts of the catalyst changed from the yellowish-white powder to grey. Second, the catalyst was stored in an analogous experiment in an autoclave at 5 bar H2 pressure. Although the color changed to grey black, which identifies the active species, it deactivated during the sample preparation. Finally, a water bath was added to the activation procedure after the headspace of the prepared NMR tubes was filled with three bars of hydrogen. It provides additional thermal energy for several hours to alleviate the overcome of the activation barrier of the oxidative addition and therefore accelerates the activation. In this case, the whole catalyst turned to the grey black color and remained this way whilst kept at 60°C. Details on the sample preparation are described in the following methods section.

4.3 Methods

Most of the experimental procedures, synthesis and the involved devices are explained in the overall methods (see chapter 3). This section deals with the additional synthesis, i.e. the immobilization and adaptions to the experimental procedures required when dealing with immobilized catalysts. First, the synthesis of PTA@Al2O3 is described. Following this, the immobilization of homogeneous catalyst precursors on the support materials is outlined. After that, the focus is laid on the sample preparation employing the anchored catalysts, while the final subsection deals with data acquisition and processing. 4 A modular immobilization system 67

4.3.1 Synthesis of PTA@Al2O3

Different forms of neutral alumina were used as support materials. The bonding of the PTA to the alumina support was realized in an analogous way for all used forms of alumina as well as for montmorillonite. Below the immobilization on different particle sizes of γ-Al2O3 as well on an α-Al2O3 hollow fiber are described in detail.

1) Immobilization of PTA on Al2O3 particles

Different sizes of γ-Al2O3 were prepared. γ-Al2O3 from Saint-Gobain (specific surface: Spore 2 = 57m /g, pore volume: Vpore = 0.93 ml/g, pore-size: dpore = 741 Å) was sieved into seven fractions with diameters dsupport of <63 μm, 63 – 80 μm, 80 – 125 μm, 125 – 250 μm, 250 – 500

μm, 500 – 630 μm and 630 – 800 μm. No flea was used for the synthesis as the Al2O3 pulverizes otherwise. First, the γ-Al2O3 (500 mg, 4.9 mmol) was heated and degassed by several Ar–vacuum cycles and dissolved in 3 mL ethanol (EtOH). A solution of degassed PTA

(H3PW12O40 6H2O, 101 mg, 33.8 mmol) in 3 mL EtOH were added to the solution containing the alumina and swiveled in 30 min intervals over five hours at RT until it rested for another ten hours (see Figure 35). 15 h, RT γ PTA -Al2O3 PTA@Al2O3 ethanol 225 Figure 35. Synthesis of PTA @Al2O3 with the impregnation technique. The supernatant excess solution was removed via cannula and the solid was washed with EtOH several times and dried afterwards. Finally, PTA@Al2O3 was obtained. Different batches for each particle size were synthesized. The amounts of the involved components described here are used for all alumina versions except for the size dependent synthesis, where it was altered for some particle sizes (see Table 1).

Table 1: Weighed-in chemicals for the size-dependent syntheses of PTA@Al2O3.

d(γ-Al2O3) m(PTA) c(PTA) m(γ-Al2O3) c(γ-Al2O3) batch [µm] [mg] [µmol] [g] [mmol] I < 63 284.3 95.0 1.40 13.8 II 63 – 80 67.0 22.4 0.333 3.24 III 80 – 125 67.0 22.4 0.333 3.24 IV 125 – 250 67.0 22.4 0.333 3.24 V 250 – 500 101.0 33.8 0.500 4.9 VI 500 – 630 101.0 33.8 0.500 4.9 VII 630 – 800 67.0 22.4 0.333 3.24

68 4 A modular immobilization system

2) Immobilization of PTA on a Al2O3 hollow fiber

To immobilize homogeneous catalysts inside an α-Al2O3 fiber (d = 5 mm), an approach analogous to the one with γ-Al2O3 was chosen. The solutions were injected slowly into the fiber with a syringe pump to assure homogeneous impregnation (see Figure 36c). On the α-

Al2O3 fiber DuPhos and PCy3 were immobilized. For comparability to the experiments above, the immobilization was also applied on a grinded α-Al2O3 fiber.

Figure 36. Immobilization on a α-Al2O3 hollow fiber: a) single hollow fiber (d = 5 mm), b) hollow fiber inside a reactor module, c) syringe pump for the injection of solutions into the fiber under inert gas conditions.

3) Immobilization of PTA on montmorillonite The immobilization of PTA on montmorillonite (75% <63 μm) was conducted as described for Al2O3 including the amounts of the involved components. Two batches were prepared accordingly. One with flea and one without. As for the previous synthesis, the flea pulverized the support and decreased its particle size.

4.3.2 Immobilization of homogeneous catalyst precursors on PTA@Al2O3

Different homogeneous catalyst precursors were anchored on PTA@Al2O3. Their synthesis is described in the methods chapter 3. The most used catalyst in this chapter is the BINAP catalyst as it is part of the model system to evaluate the Augustine approach for hyperpolarization experiments. Therefore, its immobilization is described in the following:

4 A modular immobilization system 69

Ph Ph 15 h, RT P [Rh(COD)-(S)-BINAP]BF4 PTA@Al2O3 Rh ethanol P Ph Ph

PTA@Al O 2 3

Figure 37. Synthesis of BINAP@Al2O3 ([Rh(COD)-(S)-BINAP]BF4@PTA@γ-Al2O3).

BINAP was immobilized on different sizes of γ-Al2O3 as well as on other support materials

(reaction scheme see Figure 37). In the case of batch (I) PTA@Al2O3 was dispersed under stirring in 2 mL EtOH. A solution of BINAP (1 mg, 1.33 μmol) in 1 mL EtOH was added dropwise. After the solution was swiveled occasionally it was left overnight at RT. The yellow solid was washed with EtOH, dried and stored under argon for further use. The concentrations of the solution for the batches of the other particle sizes are kept constant to be able to compare the batches with each other. The used amounts are shown in Table 2.

The resulting immobilized catalyst [Rh(COD)-(S)-BINAP]@PTA@γ-Al2O3 is from here on referred to as BINAP@Al2O3.

Table 2: Weighed-in chemicals for the size-dependent syntheses of BINAP@Al2O3.

d(γ-Al2O3) m(PTA@Al2O3) c(PTA) m(Rh-cat*) c(Rh-cat*) # batch [µm] [mg] [mmol] [mg] [µmol] I < 63 199 0.067 1 1.33 II 63 – 80 400 0.13 2 2.66 III 80 – 125 400 0.13 2 2.66 IV 125 – 250 400 0.13 2 2.66 V 250 – 500 601 0.195 3 3.99 VI 500 – 630 601 0.195 3 3.99 VII 630 – 800 400 0.13 2 2.66

Rh-cat*: homogeneous catalyst precursor [Rh(COD)-(S)-BINAP]BF4 Other homogeneous catalyst precursors were anchored in an analogous way. The dppb catalyst was immobilized on batch III (80 – 125 µm particle size). The IMes, PCy3 and DuPhos catalysts were immobilized on commercial neutral γ-Al2O3 with a broader particle size distribution, where 70 % of the particles had a size of 63 − 0.200 µm.

4.3.3 Sample preparation

Young type NMR tubes were prepared under inert gas conditions. The experiments in this chapter were performed with the amounts and conditions given here unless otherwise stated. First approximately 1 mg of the anchored catalyst was filled into the NMR-tube with 70 4 A modular immobilization system

300 μL of a deuterated solvent (e.g. methanol-d4). For activation purposes, the tube was frozen in liquid nitrogen, pressurized with 6 bar p-H2 or thermal hydrogen and immerged in a 60°C water bath overnight. During that period, the catalyst changes from a white-yellow to a grey-black color and remained like that at the 60°C. The catalyst deactivates within hours when not kept under these conditions anymore. Samples that remained or returned to the white/yellow color showed no activity. Therefore, the sample was prepared in a reasonable time period. Before the to-be-polarized substrate (e.g. 5 μL of butylacrylate) was added, the hydrogen atmosphere was removed by a vacuum pump. Otherwise, the n-H2 could begin to hydrogenate the substrate already before p-H2 is added. During this preparation period catalyst slowly changed the color to a lighter grey. The reason for this is the absence of the temperature and hydrogen pressure for a short time. A water bath kept the samples at 60°C between experiments.

4.3.4 Detection and data processing

The measurements are performed as shake-and-drop experiments as described in the methods section. A water bath keeps the samples at the desired temperature until the shaking and detection. In the case of hyperpolarization experiments with parahydrogen, single scan spectra were recorded in a compact 1 T spectrometer. The FIDs were processed with MNova as described in the methods subsection 3.3.2: After a Fourier-transformation (FT), the spectra were phase and baseline corrected as well as line broadening of 0.4 Hz was applied. The thermal reference spectra were taken at 1 T using 4 scans with phase cycling and 90° pulses for excitation and processed identically. Their intensity was halved to be compared with the single scan hyperpolarized spectra (as the SNR of an NMR experiment increases with the squareroot of the number of scans - see sensitivity in section 1.1). The errors on the measurements cannot always be given precisely as the sample composition changes over time during hydrogenation experiments. Mostly 1-3 measurements per data point could be compared. Considering the time-consuming sample preparation, the focus was laid on the scope and trends of the hyperpolarization experiments with the immobilization system instead of many repetitions.

4.4 Evaluation for hyperpolarization experiments

In the previous sections a suitable model system with the BINAP@Al2O3 catalyst at its core has been elaborated to evaluate the possible application of the Augustine approach in parahydrogen based hyperpolarization experiments. Therefore, standard procedures for the synthesis of catalyst precursors, catalyst activation, sample preparation, experiments themselves and data processing have been defined. 4 A modular immobilization system 71

This section focuses on the adaption of experimental parameters for anchored catalysts to hyperpolarization experiments. The significance concerning the choice of the particle size, temperature, p-H2 pressure, magnetic evolution field, synthesis of catalyst precursors, timing during the experiments, chemical composition of the sample, amount of catalyst but also variations of the system with further catalysts (ligands, metals), solvents and substrates are discussed. When screening such a multivariable parameter system, several measurements are necessary to evaluate and compare their impact on hyperpolarization experiments, especially as some parameters are not completely independent. In the following, the trends of the most important parameters under suitable working conditions are shown and discussed. Further results are provided in the supplement for this chapter (see section 9.2).

4.4.1 Particle size of the support material

Besides the structure, morphology, composition of different elements etc. of the support material, especially its particle size has a significant impact on the detected polarization in hyperpolarization. Therefore, an optimum particle size to be used in the model system will be determined in this subsection.

The BINAP precursor has been immobilized on different particle sizes of γ-Al2O3. With these, the influence of the particle size has been investigated. Very small particles, called silt considering their grain size (<63 µm), or very large particles (>630 µm), which are already coarse sand, showed no hyperpolarization. The batch with a particle size from 63 to 80 µm showed polarization over a long period of time, but a small amplitude. A reason for this may be a small amount of active catalyst in this sample. The catalyst can rapidly deactivate during the sample preparation visible by a change in color. More experiments at different particle sizes of the support with this system are shown in section 4.2.2 (see Figure 41). An optimum particle size for hydrogenation reactions with p-H2 is between 80 and 125 µm and is shown in Figure 38. Smaller alumina particles in the sample volume generate a higher polarization loss due to the increased magnetic susceptibility mismatch as for the same mass of catalyst, there are more particles and therefore more surface. Larger particles suffer from a faster relaxation and from a lower loading capacity to immobilize the homogeneous catalyst as the surface area decreases compared to smaller ones. In addition, the pr-PHIP mechanism could be observed (at δ = 6.3 ppm). It was highest for a particle size of the support between 125 to 250 µm. The altered maximum towards a higher particle size can be attributed to the slower conversion of butylacrylate to butylpropionate due to the lower catalyst loading at the surface of these particles. After the activation of the catalyst, i.e. when the two stable hydrides form such transition metal catalyst bound to metal oxide clusters in form of [IrH2(PPh3)2]3PW12O40, they feature H/D 72 4 A modular immobilization system exchange on olefins as well as on aromatic systems.233 Full deuteration, i.e. the exchange of al hydrogen atoms for deuterium, of PPh3 as well as of cyclooctene could be observed with such a system.234, 235 This affinity to undergo CH activation processes, explains the activity concerning the pr-PHIP mechanism of these immobilized catalysts. One side reaction caused by this is deuterium scrambling in a sample over time with deuterium stemming from the solvent methanol-d4.

1 Figure 38: H NMR Hyperpolarization experiments with different particle sizes of the γ-Al2O3 support

material: For each fraction 1 mg of the immobilized BINAP@Al2O3 was activated overnight under hydrogen atmosphere at 60°C in young type tubes. Experiments were conducted

under 7 bar p-H2 pressure at 60°C with 10 µl butylacrylate in 300 µl methanol-d4 and detected at 1 T.

4.4.2 Experimental parameters

Searching for the optimal working conditions for hyperpolarization experiments the following parameters are considered: The polarization transfer field Bevo, the amount of catalyst, the p-H2 pressure and the temperature. Exposing the sample to different magnetic evolution fields prior to detection shows no significant difference in comparison to exposure to the earth’s magnetic field (0.048 mT) or to the detection field of 1 T on the observed polarization under these ALTADENA conditions (Figure 39a). The slightly higher signal at earth’s magnetic field may be explained by relaxation in the inhomogeneous fields of the polarization transfer magnet during the other experiments. Therefore, these hydrogenations are conducted at the earth’s magnetic field. 4 A modular immobilization system 73

During these experiments, the sample tubes were filled with an excess of p-H2 and substrate. In this case, the amount of hydrogenated substrates is expected to increase linearly with the amount of catalyst when neglecting transport limitations. Nevertheless, this is not the case for the detected polarization shown in Figure 39b, where the observed polarization rises with increasing amount of catalyst but by half of the expected linear increase. An increased amount of catalyst can change the relaxation properties of the sample. Homogeneous analogues are more active and more efficient as the alumina support material has a significant impact on the relaxation of surrounding molecules and with it the hyperpolarization efficiency. This polarization loss increases with increasing amount of alumina and one can assume the dominant reason for this to be relaxation at the surface of the support material, but also the loss due to the susceptibility mismatch increases especially at high magnetic fields. More heterogeneous catalyst means that there is more solid matter in the sample which reduces the detected polarization as the relaxation losses increase.

Figure 39. 1H NMR hyperpolarization experiments at different polarization transfer field and amount

of heterogeneous catalyst: The CH2CH3 group signal of butylpropionate resulting from

hydrogenation of butylacrylate with 1 mg BINAP@Al2O3 (particle size: 80-125 µm) at 7 bar

p-H2 depending on a) the magnetic evolution field and b) the amount of catalyst used, measured at 1 T. In b) the earth’s magnetic field, the stray field of a simple horse shoe shaped permanent magnet and the detection field at 1 T were used as polarization

transfer fields. One sample contained 10 µl butylacrylate and 300 µl methanol-d4 and was preheated at 60°C.

The detected polarization rises with increasing partial pressure of p-H2 (see Figure 40a).

This means that the p-H2 supply is critical for the observed polarization. Either the oxidative addition or the supply of p-H2 to the catalyst is rate determining. Similar trends where a high 130, 146 p-H2 pressure is beneficial have been reported. The experiments here were therefore conducted at 7 bar, which is still convenient to handle when using glass ware. When increasing the temperature from 30°C up to 60°C also the observed polarization rises (Figure 40b). A higher temperature accelerates the hydrogenation reaction. The activation barrier of the oxidative addition of parahydrogen to the transition metal center, 74 4 A modular immobilization system which is the rate determining step in the hydrogenation reaction (see subsection 2.2.1), can easier be overcome by the additional provided thermal energy. An increased reaction speed means a higher turnover of substrates and therefore a higher detected polarization as sufficient substrate for multiple experiments and p-H2 excess were added to the tubes. This reaches a plateau, where a higher temperature does not lead to higher turnover of substrate molecules. In this case, the turnover at the catalyst is not the rds anymore, but the hydrogen supply at the catalyst is the limiting factor either due to transport processes or even hydrogen or substrate depletion.

1 Figure 40. H NMR hyperpolarization experiments at different p-H2 pressures and temperatures of a

pre-heating bath: The CH2CH3 group signal of butylpropionate resulting from

hydrogenation of with p-H2 depending on a) the p-H2 pressure and b) the temperature

detected at 1 T. One sample contained 1 mg BINAP@Al2O3 (particle size: 80-125 µm) in

300 µl methanol-d4 and 10 µl butylacrylate at 60°C in a) and for b) together with 7 bar p-H2. A higher temperature and the associated faster conversion also impact the observed polarization depending on the particle size of the support material. Nonetheless, the silt fraction with its smallest particle size (<63 µm) was unsuitable for the hyperpolarization experiments. For the different sand fractions, a shift to an optimum at a smaller particle size was observed for the higher catalytic activity at a higher temperature. Although the smaller amount of catalytic surface at larger particle sizes should be beneficial concerning the efficiency of the observed polarization when considering the susceptibility mismatch, the increased turnover and lower relaxation at a small particle size dominate the picture with intense observed polarization (see Figure 41). 4 A modular immobilization system 75

Figure 41. 1H NMR hyperpolarization experiments at particle sizes of the support material: The

CH2CH3 group signal of butylpropionate resulting from hydrogenation of butylacrylate

with p-H2 depending on the particle size of the γ-Al2O3 support at (a) 25°C and (b) 55°C

detected at 1 T. One sample contained 1 mg BINAP@Al2O3 in 300 µl methanol-d4 together

with p-H2 (7 bar) in excess and butylacrylate (10 µl).

4.4.3 Other catalytic systems

The presented model system in the previous sections was the homogeneous BINAP catalyst precursor immobilized on Al2O3 using PTA as a linker. With this homogeneous catalyst the p-H2 based hyperpolarization of butylacrylate as a reference substrate was studied in methanol. Although this serves as proof of principle experiments to validate the presented system, expanding the scope of this approach by looking at other homogeneous catalysts to anchor (1), substrates (2) and solvents (3) is desirable. The modularity of the presented hyperpolarization system allows to choose a suitable catalytic system.

1) Homogeneous catalysts (to be anchored)

So far BINAP@Al2O3 as an exemplary heterogeneous hydrogenation catalyst where

BINAP is anchored on γ-Al2O3 by PTA as a linker has been studied. The immobilized homogeneous catalyst precursor BINAP consists of a phosphine ligand and COD − coordinated to the rhodium center as well as the non-coordinating anion BF4 . In the homogeneous case, this cationic catalytic precursor is activated by an oxidative addition of hydrogen to the transition metal center forming the homogeneous octahedral catalytically active species BINAP(a) (see 3.2.3). It features a chiral bidentate phosphine ligand: (S)-BINAP. Phosphine ligands can be classified by the Tolman principle distinguishing between electronic and steric parameters.236 While the electronic parameters describe the electron withdrawing or electron donating effect of the ligand, the steric parameters describes the spatial restrictions when accessing the catalytic center created by the ligand. The former can easily be compared to a complex containing a CO ligand when comparing its shift of the stretch vibration in an IR spectrum while the latter is described by the cone angle. When 76 4 A modular immobilization system using three triphenylphosphine ligands, such a catalyst is known as the Wilkinson catalyst referring to its inventor.237 The intermediates during the catalytic cycle of this homogeneous catalyst have been studied with parahydrogen indicating a pairwise hydrogen transfer mechanism in the asymmetric hydrogenation:30, 238 It also showed binuclear species (Dimers) and even Trimers as well as the “putative olefin dihydride catalytic intermediate”238. Hyperpolarization with similar phosphine catalysts using an iridium instead of a rhodium center, are shown in the supplementary subsection 9.1.2. Triphenylphosphine is a monodentate ligand with an average cone angle and electronic parameters compared to 236 other phosphine ligands. For hyperpolarization experiments with p-H2, regardless whether its hydrogenation or exchange, the hydrogen has to coordinate to the catalytic center for the discussed inner sphere mechanisms. This oxidative addition of hydrogen is the rate determining step in the catalytic cycle (see subsection 2.2.1). Therefore lowering the activation barrier by increasing the electron density at the transition metal center with electron donating ligands is beneficial. Sterically demanding ligands determine the accessibility of the catalytic center for hydrogen, the substrate, the solvent, intermediates. Thus a certain steric demand is beneficial as unwanted side reactions or further coordination is suppressed. Following this, phosphine ligands with a larger ligand sphere can be convenient. Bidentate phosphine ligands offer this increased steric demand and are bound stronger to the metal center due to the entropic advantage (chelat effect). Some examples for different ligands coordinated to a metal center are shown in Figure 42. One of them is the dppb ligand239, where two phosphines are connected by an aliphatic butyl chain. This catalyst allows for high turnovers in hydrogenation reactions. This was extended to chiral diphosphine ligands, allowing for enantioselective reactions of prochiral substrates. Examples are DIPP240, DIPAMP241, BINAP242, DuPhos198, 206, while suitable multidentate ligands for enantioselective hydrogenations not necessarily bind with a phosphine group243-246 (DIPP = 1,3-bis(diisopropylphosphino)propane; DIPAMP = bis((2- methoxyphenyl)phenylphosphino)ethane). The (S)-BINAP ligand employed here is known for its enantioselective hydrogenations with a high enantiomeric excess with Ruthenium but also with rhodium.247, 248 A major catalyst deactivation mechanism of homogeneous transition metal complexes is dimerisation.128, 149 The DuPhos ligand is a bidentate phosphine ligand, which allows for enantioselective hydrogenations and is so bulky that it prevents dimerization of the catalytically active species.198 4 A modular immobilization system 77

Figure 42. Different ligands bound to a transition metal center (M) that are known in hydrogenation reactions and were used in this work: Five phosphine ligands triphenylphosphine (a), 1,4-Bis(diphenylphosphino)butane (b), (S)-2,2′-Bis(diphenylphosphino)-1,1′- binaphthalene (c),which was already used for the model system, 1,2-Bis[(2S,5S)-2,5- dimethylphospholano]benzene ((S,S) Me-DuPhos) tricyclohexylphosphine (d) and a carbene ligand: 1,3-bis(2,4,6-trimethylphenyl)-imidazol-2-ylidene (e).

2) Anchored homogeneous catalyst in hyperpolarization experiments Several known homogeneous catalysts were anchored in the same way as the BINAP and tested in hyperpolarization experiments. The properties of a homogeneous catalyst can change when immobilized on a heterogeneous support. The dppb catalyst can be employed instead of the BINAP catalyst, when enantioselectivity is not demanded and a higher turnover, which results in faster exchange and hydrogenation, is favored. The fast hydrogen exchange at the catalyst as well as the rapid hydrogenation of the reference substrate butylacrylate with p-H2 result in a significant detected polarization: The unsaturated CHCH2 group is hyperpolarized by the exchange of terminal protons with p-H2 (5.5 ppm, pr-PHIP) while hydrogenation with p-H2 results in the hyperpolarized CH2CH3 group (1 ppm, h-PHIP) (see Figure 43a). The other signals are lower than in the thermal reference spectrum, as the polarization build-up by B0 before detection is minimal during shake-and-drop hyperpolarization experiments. 78 4 A modular immobilization system

Figure 43. 1H NMR spectra from hyperpolarization experiments with other anchored homogeneous

catalysts. The homogeneous catalyst precursors that were anchored on PTA@Al2O3 are

shown below the spectra. In a) the sample contained 4.5 mg of the PCy3@Al2O3 anchored

on neutral alumina along with 20 µl butylacrylate in 300 µl methanol-d4 at RT and 7 bar p-

H2 while b) contained 2 mg dppb@Al2O3 and 5 µl butylacrylate in 300 µl methanol-d4 at

60°C and 6 bar p-H2. Further explanations see text. The DuPhos198, as a Rh based hydrogenation catalyst with a more bulky enantioselective phosphine ligand which prevents dimerization, a major deactivation mechanism of homogeneous Rh catalysts, is shown in proof of principle experiments in the supplementary subsection 9.2.2 for this chapter. The detected polarization is lower compared to the other tested anchored homogeneous catalysts (see Figure S 11b). This can be explained by the smaller turn over frequency (TOF) of the catalyst, especially as it is probed at RT, and the more difficult accessibility of the catalytic center for hydrogen and especially the substrate. Due to these changes, the operating conditions that were found optimal for the

BINAP@Al2O3 are not ideal for DuPhos@Al2O3. For example, a higher catalyst loading may be beneficial to increase the amount of hydrogenated substrate and with it the detected polarization. In addition, the reaction speed could be increased by working at a higher pressure or temperature. In any case dimerization has not been observed with these heterogeneous catalysts lowering the necessity for bulky ligands. They are only important concerning the olefin hydrogenation and possible side reactions and the BINAP catalyst is very selective247, 248. Finally, when anchoring a homogeneous catalyst its properties change including the space at its ligand sphere. The catalyst precursors are quadratic planar (d8-configuration), while the active catalyst BINAP(a)249 has an octahedral structure. Therefore, also activating the catalyst precursors with hydrogen prior to immobilization has been investigated. This has shown to be no advantage over the immobilization of catalyst precursors. 4 A modular immobilization system 79

Not only rhodium based catalysts as described above were anchored with this approach. Iridium based catalysts are especially interesting as they could extend the scope of this system to the SABRE approach and thus enable an additional range of possible substrate as they remain unaltered after the polarization transfer.

For example, the iridium based catalyst precursor PCy3 was anchored on Al2O3. This famous catalyst, referred to as “Crabtree’s Catalyst”, is known for its high activity and its ability to hydrogenate three and four times substituted alkenes, but was also the first catalyst that has shown SABRE activity12. It contains a monodentate phosphine ligand with aliphatic cyclohexyl groups, which are electron donating groups increasing the electron density at the transition metal center and therefore lowering the activation barrier of the oxidative addition, a rate determining step in the catalytic cycle (see subsection 2.2.1).

The immobilized version of PCy3 changes its color from orange to a very light orange upon activation. In hyperpolarization experiments with p-H2 it showed even more absolute polarization when hydrogenating butylacrylate in a single scan at RT (see Figure 43b), than the dppb@Al2O3 discussed before. This can mainly be attributed to the higher turnover due to the electron donating cyclohexyl groups of its phosphine ligand. Also experiments with a more recent and very efficient SABRE catalyst47 have been conducted. The IMes250 contains a carbene instead of a phosphine ligand, which can increase both activity and selectivity in hydrogenation reactions.251 Its anchored version shows less hydrogenation of butylacrylate at RT compared to PCy3 and therefore also less detected polarization (see Figure S 11a). Similar to the described DuPhos@Al2O3 catalyst this can be attributed to the smaller turnover of substrates caused by the sterically demanding ligand combined with the altered chemical surroundings due to the immobilization on a support and experimental parameters, which were optimized for another catalyst: the

BINAP@Al2O3. Unfortunately, contrary to their homogeneous analoga, no hyperpolarization of pyridine using the SABRE approach was observed with the anchored iridium based catalyst. Therefore, this heterogeneous hyperpolarization system still has room for optimization as it is not SABRE-active yet limiting the range of possible substrates to unsaturated hydrocarbons whose chemical structures are altered by the addition of p-H2. Note that there are only very few heterogeneous SABRE studies with minimal achieved polarization up to now.155, 156 It may still be possible to find ways of heterogeneous SABRE with this system, when immobilizing catalyst precursors whose sterics and the corresponding exchange kinetics of hydrogen and substrate are less altered by the immobilization.

3) Substrates for hyperpolarization experiments with the anchored catalysts So far butylacrylate has been employed as reference substrate, but also other substrates can be hyperpolarized with the BINAP catalyst in methanol. Figure 44 shows hyperpolarized 80 4 A modular immobilization system spectra and thermally polarized reference spectra of a) methylacrylate, a similar substrate to butylacrylate and b) dimethylitaconate, a secondary alkene. While experiments with methylacrylate had higher polarization levels under the chosen conditions, which can be attributed to a higher conversion rate, it is notable that dimethylitaconate shows the pairwise replacement mechanism exchanging protons bound to the unsaturated bond. Furthermore dimethylitaconate is prochiral and has been used to determine the isomers involved in the hydrogenation reaction (chiral dihydride complexes).29 The two catalytically active isomers discussed in literature for the homogeneous case could not be verified as the line broadening caused by the heterogeneous material eliminates the already quite weak and broad hydride signals. Also the lower TOF in the case of the heterogeneous catalysts can be a reason for the missing hydride signals as the detected polarization is lower. To obtain stabile hydrides already high temperatures (>60°C) and high pressure (6 bar) are needed to form stabile hydrides within minutes or hours (see subsection 4.3.3). To achieve this within seconds using p-H2 in a sufficient quantity to obtain sharp hydride signals might demand more drastic experimental conditions or a continuous setup absent solid material in the sensitive volume.

Figure 44. 1H NMR spectra from hyperpolarization experiments with methylacrylate and dimethylitaconate: Hyperpolarized and corresponding thermal reference spectra of a)

10 ml methylacrylate and b) 14 µl dimethylitaconate using BINAP@Al2O3 (particle size: 80

to 125 µm) catalyst precursor preactivated with hydrogen at 55°C and with 7 bar p-H2 in

300 µl methanol-d4. The noise levels differ as the thermal reference spectra were recorded with 4 scans using phase cycling and scaled appropriate to the single scan hyperpolarization experiments (see subsection 4.3.4). The spectra recorded with dimethylitaconate as a substrate are enlarged by a factor of 4 for visualization. Figure 45a shows a hyperpolarized spectrum of the substrate styrene compared to a thermal reference spectrum. In Figure 45b the detected polarization depending on the particle size of the support is depicted. This is also temperature dependent (see Figure S 10) and it follows similar trends as the model substrate butylacrylate for the particle size 4 A modular immobilization system 81 distribution. It has a maximum polarization at a certain particle size as a compromise between relaxation and accessible surface area and susceptibility artefacts caused by the heterogeneous material. Also in the case of methylacrylate, the particle size as well as other reaction conditions such as pressure or temperature (see Figure S 9 and Figure S 10) show the same trends as for styrene and butylacrylate (see supplement 9.2).

Figure 45. Shake-and-drop hyperpolarization experiments with styrene (10 µl) and BINAP@Al2O3

(particle size: 80-125 µm) with 7 bar p-H2 at 25°C and 1 T in 300 µl methanol-d4. a) Hyperpolarized spectrum (black) compared to its thermal reference spectrum (1H, 4 scans) (grey). The hyperpolarized spectrum is enhanced by a factor of 2 to compensate for the 4

scans in the reference spectrum. The modulus of the antiphase signal from the CH2CH3

group of ethylbenzene (0.8 ppm and 2.0 ppm) by hydrogenation of styrene with p-H2 is

given in b) depending on the particle size of the Al2O3 support material.

4) Solvents for hyperpolarization experiments Up to now the hyperpolarization experiments were demonstrated in methanol. Other solvents can be beneficial depending on the catalytic system and investigated substances. The presented immobilization approach provides a variety of heterogeneous catalyst stable and active in various solvents203, 216, 217, 252 including solvent free hydrogenation134. Even in the polar solvent ethanol minimal leaching rates have been reported.204 For example a non- coordinating organic solvent could be of interest for hyperpolarization experiments, leaving − a free coordination site during the exchange as the used anion BF4 is not coordinating as well and therefore changing the exchange kinetics at the catalyst. Also a solvent with a higher or lower polarity than methanol could be of interest. For iridium phosphine complexes, similar to Wilkinsons catalyst, hydrogenations with p-H2 are shown in the supplement subsection 9.1.2. They show hyperpolarization in toluene as a more apolar and non-coordinating solvent as well as for water as a polar, coordinating solvent. 82 4 A modular immobilization system

4.4.4 High field experiments / PASADENA conditions

Finally, the Augustine system was probed in hyperpolarization experiments under PASADENA conditions in a superconducting high field magnet (300 MHz). As elaborated in section 2.5, such a spin order transfer in the weak coupling regime differs from the magnetization build-up in the ALTADENA experiments before. This was achieved by placing the sample in the sensitive volume after the shake-and-drop and repetitively recording single scans every 45 s. This waiting time lets the heterogeneous material sink to the bottom of the tube, which is outside of the sensitive volume, so that it rests to avoid line broadening by arising susceptibility artefacts, which is already the case after a waiting time of 36 s (see Figure S 13). Furthermore the waiting time allows for spin order transfer under pure

PASADENA conditions at 10 T. The p-H2 was added in excess so that the butylacrylate was fully hydrogenated after an hour. During that time, butylpropionate accumulates in the sample and therefore its thermal signal increases with time. An example spectrum taken after 7.25 min, is shown in Figure 46. This spectrum features butylacrylate with the unsaturated bond peaks at around 6 ppm. The CH2 (b) and CH3 (a) - groups of butylpropionate, are a superposition of the thermally and hyperpolarized species (see inset of Figure 46). The CH2 quartet consists of both the hyperpolarized 1:1:-1:-1 species under PASADENA conditions as explained in theory section 2.5 and the 1:3:3:1 quartet following 61 the pascal triangle as known from traditional NMR experiments . The signal of the CH3 group (a) consists of the hyperpolarized species which contains two peaks. The middle one originated from the thermal triplet (1:2:1) of butylpropionate that was created before. The first scans at this higher magnetic field are note pure PASADENA as the sample was shaken outside of the magnet and also feature bit of line broadening due to the still moving solid catalyst after the shake-and-drop. Compared to the spectra recorded at 1 T, the line broadening is higher due to increased susceptibility mismatch (see Figure S 12). The slow turnover allows to monitor the reaction over minutes or even hours until the substrate is finally depleted. Like at 1 T, experiments with an excess of p-H2 were performed depleting the substrate over time. Here, the created polarization, detected in a single scan, is smaller due to the slow turnover. Additionally, the formed butylpropionate builds up a thermal signal during the waiting time in this higher magnetic field. This leads to the visible superposition of signals shown in Figure 46. Overall applying the Augustine system to parahydrogen based hyperpolarization experiments at high magnetic fields allows for the advantages such a field can offer, such as the high chemical resolution. Care should be taken considering the increased susceptibility mismatch from the solid material as well as to superposition arising from increased thermally polarized signals. 4 A modular immobilization system 83

Figure 46. 1H NMR hyperpolarization experiment at 300 MHz under PASADENA conditions with 6 bar

p-H2 at 60°C. The sample was shaken for 10 s and dropped into the spectrometer, where several single scan spectra (45° pulse) were recorded. First an ALTADENA spectrum was taken (see Figure S 12) and after a waiting time of two min the first PASADENA spectrum was recorded. From thereon single scan spectra were taken every 45 s. Shown here is a spectrum taken after an overall time of (2 min + 5 min 15 s = 7 min 15 s) as an example, where the signal of the thermally polarized butylpropionate (structure shown in the figure) from the hydrogenation is still smaller than the hyperpolarized one. 5 µl

butylacrylate along with 2.3 mg dppb@Al2O3 catalyst (particle size: 80-125 µm) in 500 µl

methanol-d4 were prepared in a Young type tube and activated prior to the experiment.

4.5 Conclusion and outlook

In hyperpolarization experiments with parahydrogen, a catalyst is used to convert the singlet spin order of the parahydrogen into detectable magnetization on target molecules. Such experiments not only offer an immense sensitivity enhancement in NMR, but may even allow further insights on the catalytic system itself such as reaction intermediates or exchange mechanisms. In this chapter the so-called Augustine system201 has been evaluated for applications in parahydrogen based hyperpolarization experiments. This modular immobilization system for homogeneous hydrogenation catalysts is of interest as heterogeneous catalysts are beneficial in many ways such as their possibility for a reuse or biocompatibility. The variety of the many and well-defined homogeneous spin order transfer catalysts can be exploited when they are anchored to a support material resulting in a variety of new catalysts. There are many ways to anchor a homogeneous catalyst. The 84 4 A modular immobilization system immobilization approach described in this chapter features a HPA to link the homogeneous catalyst to the support. This system has shown to be highly efficient for chemical reactions including stereo selectivity in hydrogenations134, 206, 215-217 and features minimal leaching204. This proof of principle study shows, that it is applicable to parahydrogen based hyperpolarization experiments. Experiments with the liquid phase or remnant solutions without the immobilized catalyst material showed no hyperpolarization, which indicates that the leaching was minimal and the heterogeneous catalyst responsible for the hyperpolarization. Nevertheless, the properties of a homogeneous catalyst may change, when immobilized on a support material. The reasons for the changes caused by this immobilization approach are based on the linker as well as the support. They effect the electronic properties of the catalyst and the ligand sphere. With that, they change the accessibility and available space for hydrogen as well as for the to-be hyperpolarized substrates to coordinate to the catalytic center. Furthermore, the support influences the NMR detection, if it is in the sensitive volume. The aim of this chapter was to work out the differences, adapt the system to parahydrogen based hyperpolarization experiments. The choice of the support material itself involved in hyperpolarization experiments and its structure are crucial. A suitable support is γ-Al2O3. The activation of the catalytic complexes has shown to be significantly slower than for the homogeneous analoga. Therefore the activation of the catalyst precursors has been adjusted accordingly. A water bath providing thermal energy to facilitate the oxidative addition of hydrogen combined with hydrogen pressure and an increased amount of time resulted in the formation of catalytically active species. The formed classical hydrides are preferred over a split up of the p-H2 into two hydrides, on the linker (PTA) and one the catalyst precursor. In a dihydride complex, the spin order stemming from p-H2 can evolve into detectable states as described in the theory section 2.5. Also during slow two step hydrogenations, proposed as a the mechanism for this system,231, 232 the correlation between the hydrogen atoms stemming from p-H2 can be lost, leading to a less efficient polarization transfer to target molecules. On the basis of these adjustments to the experimental procedures from homogeneous catalysts, a model system featuring BINAP@Al2O3 as the catalyst and butylacrylate as a substrate has been used to probe the Augustine system for hyperpolarization experiments with parahydrogen. These experiments have shown different trends for various experimental parameters: The size of the support material plays an important role due to the magnetic susceptibility mismatch at small particle sizes versus the lower catalyst loading and increased relaxation losses with larger particles. Therefore, an optimum particle size of 80 – 125 µm for the highest polarization levels with the model system was found. While the effect of a polarization transfer field was negligible in these PHIP experiments, by increasing 4 A modular immobilization system 85

the temperature, the p-H2 partial pressure or the amount of catalyst increased the substrate conversion and with it the detected polarization. The scope of the immobilization ranges over a manifold of hydrogenation catalysts and corresponding substrates in various solvents. In this work the homogeneous catalyst precursors BINAP, dppb, DuPhos, IMes and PCy3 have been immobilized on the alumina support and successfully probed in hyperpolarization experiments. As substrates butylacrylate, dimethylitaconate, methylacrylate and styrene were hyperpolarized. Finally this hyperpolarization system was used at different magnetic detection fields. In conclusion, the Augustine system performed well in these proof of principle hyperpolarization experiments with p-H2, but of the obtained PHIP so far is more than an order of magnitude below known heterogeneous catalytic systems in liquid systems. 132, 209, 253 Future improvement and development could include different aspects. Other support materials such as the CeO instead of alumina could reduce the relaxation losses caused by the support. Different immobilized iridium based catalysts have been shown h- and pr-PHIP on olefins, but no hyperpolarization following the repetitive SABRE approach, with its additional range of substrates, has been observed yet. Continuous hydrogenation setups for enantioselective hydrogenation reactions has been demonstrated with the Augustine system.134, 206, 215-217 A prototype setup for continuous flow hyperpolarization, such as the membrane reactor presented in chapter 6, could be beneficial to probe and optimize the efficiency of such a system with a consecutive analysis of multiple test samples. Arising susceptibility issues during the detection can be circumvented by such a continuous flow hyperpolarization setup or by using relatively low magnetic fields. This would allow for spectroscopy or imaging independent from catalyst influences. When using water as a solvent combined with this catalytic system, organic solvents and a separation of the mostly toxic homogeneous catalysts for biomedical applications can be avoided at the same time.

86 5 Hyperpolarizing water with parahydrogen

5 Hyperpolarizing water with parahydrogen

In this chapter the first hyperpolarization of bulk water with parahydrogen which was published in 201756 is discussed. Additionally, the hyperpolarization of triply 15N labeled L- 15 histidine, abbreviated as N3-histidine, is presented. Polarizing both water and L-histidine is a key step towards biomedical applications of parahydrogen based hyperpolarization. It is of special interest, because water is the global medium for life and a biocompatible solvent and L-histidine one of the 22 proteinogenic α-amino acids.254 The catalytic system, operating conditions and evaluated experimental parameters during the hyperpolarization experiments in this chapter are summarized in Figure 47.

Figure 47. Scope of the chapter “Hyperpolarizing water with parahydrogen”. The general conditions are highlighted in yellow, while solvents and varied experimental parameters of this chapter are shaded in light yellow. Parahydrogen based chemistry i.e. the substrates and catalysts as well as the polarization transfer regime and mechanism are highlighted in red. 15 15 In the case of L-histidine this also includes the triply N labeled version N3-histidine. Points not addressed in this chapter are written in grey. PHIP represents the classical spin

order transfer mechanisms from p-H2 by hydrogen substitution or incorporation into the substrate. The abbreviations are introduced as they occur in the text and are summarized in the beginning of this work.

5 Hyperpolarizing water with parahydrogen 87

This chapter is separated into four parts. The introduction (5.1) motivates the hyperpolarization of water and L-histidine with a parahydrogen based technique. The following individual methods section (5.2) describes the experimental procedures for the hyperpolarization experiments in this chapter. The results and discussion section (5.3) addresses key experimental parameters such as pressure, temperature and the chemical composition of the sample when hyperpolarizing water. After this, the hyperpolarization of 15 N3-histidine is discussed. A special focus is laid on the magnetic evolution field Bevo during 15 the polarization transfer to water and also to N3-histidine. The section closes with mechanistic considerations. Finally, the conclusion and outlook (5.4) conclude this chapter.

5.1 Introduction

Water is highly concentrated in many systems and as a biocompatible solvent especially interesting for biomedical applications. Therefore, polarizing water can be advantageous in different ways as shown in literature.97-100, 255 Especially, studies with water hyperpolarized by DNP techniques are dominant until now. Overhauser DNP was applied to investigate local water dynamics at the surface of biomolecules and to gain structural information.97 Transferring polarization from water to heteronuclei of biomedical relevant substances such as urea or arginine proved to be an interesting alternative way followed by dissolution DNP.98 Nevertheless, the first parahydrogen based system polarizing water has been reported in 201756 and will be the subject of this chapter. The polarization of neat substances or solvents like methanol with parahydrogen have been demonstrated before with the SABRE approach allowing for efficient, repetitive polarization with a low cost setup.12, 16, 22, 51, 160 In contrast to DNP, the creation of the singlet order as the source for parahydrogen based hyperpolarization is independent from the magnetic field. This renders especially parahydrogen based hyperpolarization techniques, but also SEOP where the polarization stems from circular polarized photons of a LASER, superior at low magnetic fields. Most recently, the SABRE approach has been advanced further regarding biomedical applicability. Among the polarization of different biomedical relevant substances, different approaches creating a water based system have been published.49, 152, 170, 256 One of them involved the water soluble iridium based catalyst [IrCl(IDEG)(COD)] (IDEG) containing the ligand IDEG (IDEG = 1,3-bis(3,4,5-tris(diethyleneglycol)benzyl)imidazole-2- ylidene)49 (see Figure 48). This carbene ligand differs from the IMes ligand in the well-known

47 IMes catalyst . The IDEG ligand features two additional CH2 groups which connect the benzene rings to the imidazolium center and which has polyglycol groups instead of methyl groups on those to enable water solubility. 88 5 Hyperpolarizing water with parahydrogen

This catalyst has been applied to hyperpolarize e.g. pyridine or nicotinamide in D2O with 15 the SABRE approach. Here, it is used to hyperpolarize water itself and also N3-histidine. Optimized experimental conditions for the polarization transfer from parahydrogen were determined. The polarization depends on the composition of the solution as well as on experimental parameters such as temperature, parahydrogen pressure and especially the polarization transfer field. Finally, the polarization transfer mechanism of this approach is discussed. It differs from the known SABRE mechanism regarding an involved proton exchange.

5.2 Methods

The devices and experimental procedures that were used for this chapter are discussed in chapter 3. Here, a typical experiment, referred to as standard experiment, is introduced. In such, the sample was prepared with 0.66 mmol/l of the catalyst 13.2 mmol/l (20 eq)

L-histidine in D2O with 1 vol% H2O in high pressure NMR young type tubes under inert gas conditions. The catalyst precursor, shown in Figure 48, was synthesized and supplied by Peter Spannring as reported in literature.49 The sample was measured with the shake-and- drop method described in the experimental subsection 3.3.1: A sample was pressurized with

7 bar p-H2 and heated up to 90°C in a water bath within 5 s. Consecutively, it was shaken for

7 s, exposed to a polarization transfer field of Bevo = 220 G for another 10 s and then dropped within 3 s into a 1 T compact NMR spectrometer (43 MHz Spinsolve, Magritek) and detected with a single scan (90° pulse). The data was processed with MNova, as described in subsection 3.3.2, analogous to the other results chapters. a) O O O O O O O O O O O O O O O O O O N N

Ir Cl b) O N OH NH HN 2 Figure 48. a) The IDEG precursor from Spannring et.al49 and b) the additive L-histidine employed to polarize water with parahydrogen.

5 Hyperpolarizing water with parahydrogen 89

5.3 Results and discussion

In 2017 it was shown that under suitable experimental conditions polarization of water itself with a parahydrogen based technique is feasible.56 The first subsection will be based on these results. It therefore features impact of several experimental parameters, such as the pH, temperature, p-H2 pressure, chemical composition and magnetic evolution field, on the observed polarization. In the second subsection, 15N labeled L-histidine is introduced into the system. Most interestingly, histidine polarization is observed here and the dependence of its polarization on Bevo is studied. Finally, the results are discussed in respect of the underlying polarization transfer mechanisms to the hyperpolarized substances.

5.3.1 Hyperpolarization of water

The first hyperpolarization of water with parahydrogen involved the water soluble IDEG 56 catalyst in a H2O/D2O mixture. Additionally, the presence of a substrate such as pyridine or L-histidine seems to be necessary for polarizing water. The reasons for this will be discussed in the context of the mechanistic considerations (see subsection 5.3.3). Experiments without such an additive showed no water polarization. As such, L-histidine is way more efficient than pyridine to polarize water (see Table 3). As a reference to further measurements and for better comparability, a standard experiment is defined (see section 5.2). The spectrum of such an experiment under ALTADENA conditions is shown in Figure 49 (black) and compared to a reference measurement (grey).

Figure 49. 1H NMR spectrum recorded under the standard conditions for the hyperpolarization experiments of water defined in the methods section 5.2. (black) compared to a thermally polarized reference (grey) detected at 43 MHz (1 T): a) region from 0 to 10 ppm containing hyperpolarized water and HD gas and b) the corresponding hydride region from −35 to −15 ppm enhanced by a factor of 24 for better visualization. The FID of the hyperpolarized spectrum was cutoff after 1.5 s. The negative water signal is more than ten times enhanced compared to the thermal reference signal polarized at 1 T. Apart from the water signal, the spectrum contains a triplet (J = 43 Hz)257 identified as hyperpolarized HD gas and 2 groups of asymmetric hydrides from 90 5 Hyperpolarizing water with parahydrogen

oxidative addition of p-H2 to the iridium center. The different hydrides suggest the coordination of histidine trans to one of them and water opposite to the other. The signal intensity of the hyperpolarized water depends on a variety of parameters. In the following the influence of the temperature, p-H2 pressure, pH value and especially the polarization transfer field are discussed. A control experiment with normal hydrogen showed no hyperpolarization of water proving a parahydrogen based effect (see Table 3). Looking at different experimental conditions, the first investigated parameter is the p-H2 pressure. The detected negative polarization of water rises with increasing p-H2 pressure (see Figure 50a). This trend has already been reported polarizing different substrates with an iridium based SABRE catalysts containing carbene ligands with imidazole substructure.47, 48 Thus the system seems to be limited by the p-H2 supply at the transition metal center. Consecutively, the temperature of the pre-heating bath was varied while all other parameters were kept constant. In this case the chemical shift of the water peak decreases linearly with increasing temperature (see supplement Figure S 14).258 The negative polarization of water increases linearly with increasing temperature (see Figure 50b). The highest possible temperature as an optimum has already been reported for the IDEG catalyst49 differing from most SABRE studies in methanol.48, 55, 168 Nonetheless, for 31P polarization of PPh3 ligands in toluene, a higher required temperature like for the IDEG catalyst was reported.36 Thus, the chosen temperature of 90°C, close to the boiling point of water, is adequate. It accelerates the exchange of both parahydrogen and the substrate showing that the optimum operating conditions of this water soluble catalyst are at higher temperatures due to its kinetics and corresponding exchange rates.

Figure 50. Intensity of the water polarization depending on a) the p-H2 pressure and b) the temperature of the pre-heating bath under the defined standard conditions. Furthermore, the pH was varied to modify the concentration of the mobile protons in solution (see Table 3). While adding 20 equivalents of diisopropylethylamine, a non- coordinating base also called Hünig base, only decreases the polarization from –349 a.u. (arbitrary units) to –200 a.u., acidic conditions have a stronger effect on the 5 Hyperpolarizing water with parahydrogen 91 hyperpolarization of water. Already a small amount of HCl (4.0 10-2 eq) diminishes the polarization severely to –45 a.u. while at even larger amount of HCl (40 eq) the negative polarization vanishes leaving only the residual thermal water signal built up before detection. This measured dependence of the pH on the water polarization is comparable to reports on the polarization of methanol as a solvent.53 Deuterating parts of the sample can drastically increase the polarization of target 43 nuclei. Here, the ratio between H2O and D2O was varied in the presence of 40 equivalents

Hünig base. A higher amount of H2O (10 %) slightly diminished the detected polarization from –200 a.u. to –179 a.u. while a smaller amount of H2O (0.1%) increased the polarization up to –353 a.u. (see Table 3)

Table 3: Signal intensity of polarized water under different conditions than the standard experiment detected at 1 T. Experiment I was conducted under the standard conditions defined in the methods section 6.2 (0.66 mmol IDEG, 13.2 mmol/L L-histidine, 7 bar p-H2, Bevo = 350 G). # experiment deviations from standard experiment intensity [a.u.]

I - –349 II no histidine 9 III pyridine[a] –9

IV normal-H2 30 V 0.04 eq HCl[b] –45 VI 40 eq HCl[b] 15 [c] VII 40 eq Hünig base (1.0%H2O) –200 [c] VIII 40 eq Hünig base (0.1%H2O) –353 [c] IX 40 eq Hünig base (10%H2O) –179 15 X N3-histidine –340

[a] 20 eq pyridine instead of L-histidine [b] HCl added: pH (VI) = 6; pH(VII) = 2 (pH-paper) [c] Hünig base added: pH = 8 (pH-paper) As a next step the polarization transfer field was varied to get a better understanding of the polarization transfer mechanism, which takes place in the strong coupling regime. Starting at earth’s magnetic field the negative polarization of water increases steeply up to a maximum at 350 G (see Figure 51). At even higher evolution fields the polarization decreases slowly. When using the 1 T field of the compact spectrometer as an evolution field, hence in the weak coupling regime, no polarization of water was observed. The corresponding hydride region with the two asymmetric hydrides of these NMR spectra is shown in the supplement (see Figure S 15). The intensity of those hydride signals seems to be independent from the polarization transfer field. 92 5 Hyperpolarizing water with parahydrogen

1 Figure 51. a) H NMR spectra of hyperpolarized water depending on the magnetic evolution field Bevo and b) the intensity of the water peak corresponding to these spectra.

15 5.3.2 Hyperpolarization of N3-histidine:

15 15 Finally, triply N labeled (95%) L-histidine, referred to as N3-histidine, was used instead of unlabeled L-histidine containing mainly 14N (99.6 %). 15N is a nucleus with I = 1/2 and thus has no quadrupole moment. As a strong coupling partner, it increases the magnetic inequivalence of the hydride protons at the transition metal center. During the 15 measurements but polarization of both N3-histidine and water protons was detected (see Figure 52). No splitting of the 15N-histidine peak due to the spin half nitrogen is observed. The reason for this is chemical exchange of the protons, which is elaborated in the following 15 subsection. At a polarization transfer field of 70 G negative polarization of the N3-histidine proton at 7.24 ppm is achieved (Figure 52a, highlighted in red). At the same time positive polarization of HD gas, (highlighted in green) and HDO (highlighted in blue) are observed. HD gas is hydrogen gas and HDO water, where one deuterium is substituted for hydrogen respectively. The coupling of the proton to the spin I = 1 deuterium gives a triplet with the intensity 1:1:1 as in Figure 49, but the middle peak is superimposed by the water signal here. Also, the two groups of asymmetric hydride signals, as in the measurements before, are present. An enlarged picture of this spectrum is shown in the supplement (Figure S 16). The water polarization under the defined standard conditions is comparable to the non-labeled pyridine as shown in Table 3. A corresponding spectrum at the polarization transfer field of 220 G, used for the standard experiments, is shown in Figure 52b. In contrast to Figure 52a, the water polarization is negative, as in the experiments with unlabeled L-histidine. In this 15 case the polarization of the N3-histidine proton is smaller and positive. 5 Hyperpolarizing water with parahydrogen 93

1 15 Figure 52. H NMR spectrum featuring hyperpolarized N3-histidine, HDO, HD at a) Bevo = 70 G and

b) Bevo = 220 G with the IDEG catalyst in 99% D2O/1% H2O mixture. The different chemical 15 species that were hyperpolarized with parahydrogen are marked in red ( N3-histidine), blue (HDO), green (HD) and in dark yellow for the hydrides. In b) two insets show the 15 257 antiphase N3-histidine signal, the two outer peaks of the HD triplet (43 Hz ) and the hydrides 8-fold enhanced for better visualization.

15 The impact of the polarization transfer field Bevo on the N3-histidine hyperpolarization has been studied. At earth’s magnetic field as a polarization transfer field, the polarization 15 of both water and N3-histidine is relatively small. In the weak coupling regime at high magnetic field of 1 T (10000 G), no hyperpolarization but only residuals thermal polarization was detected. Note, that this experiment is a PASADENA experiment in contrast to all other hyperpolarization experiments in this chapter, which are under ALTADENA conditions. An even lower polarization transfer field than earth’s magnetic field, i.e. magnetic shielding down to microtesla fields, could be beneficial when directly hyperpolarizing the 15N heteronucleus (SABRE-SHEATH)35. Nevertheless, for 1H polarization, transfer fields between the earth’s magnetic field and the high field are the relevant region.12, 47, 48, 51, 52 In Figure 53 the experimental results for these two limiting cases as well as several polarization transfer fields between them are shown. In Figure 53a, the spectrum from 6 to 16 ppm is shown 15 enlarged for better visibility of the N3-histidine polarization, while in Figure 53b in the range from 0 to 10 ppm the water polarization as well as the HD triplet are prominent. The corresponding hydride region of the spectrum is shown in the supplement (Figure S 17). No effect of Bevo on the polarization of hydride signals has been found. 94 5 Hyperpolarizing water with parahydrogen

1 Figure 53. H NMR spectra at different Bevo during the hyperpolarization experiments under the standard conditions defined in the methods section but at different polarization transfer fields: a) shows the range of the 1H NMR spectra from 6 to 16 ppm with featuring 15 1 hyperpolarized N3-histidine at 7.1 ppm and b) depicts the H NMR spectra from 0 to 10 ppm with the HDO peak and the HD triplet as most prominent species.

15 The enhancement of the N3-histidine proton reaches a maximum negative polarization at Bevo = 70 G and a maximum positive polarization at Bevo = 550 G. The negative polarization at Bevo = 70 G has an enhancement factor of SEF 1.1 compared to a thermally polarized sample at 1 T. For HDO an opposite trend was observed: The highest positive polarization ≈ was observed at Bevo = 70 G while the highest negative polarization was observed at 15 Bevo = 550 G. Looking at the field dependence when using N3-histidine instead of the unlabeled version, an additional optimal polarization transfer field at 70 G with maximum 15 polarization of both water and N3-histidine is observed (Figure 53). This correlates well to the known optimum of 65 G for SABRE experiments polarizing N-heterocyclic substrates, which has already been reported in several studies.47, 116 The second maximum is comparable to the one already shown in the experiments, where water was polarized using unlabeled L-histidine as an additive. The shape looks similar, but the position is slightly shifted to the measurement point at 550 G instead of the one at 360 G reported above. This could be explained by a measurement error in the single shot experiment for 360 G, which may give a higher polarization when repeated. The signal of water is about 50 times larger compared to L-histidine, but also the abundance of water is more than three orders of magnitude higher. Note that the HDO proportion in the water is initially around 2% while the rest is mostly D2O. Although the 15 achieved polarization of N3-histidine is as high as e.g. pyridine or water and should be optimized for future experiments, already looking at its nature can help to elucidate the polarization transfer mechanism, which will be the subject of the following subsection. 15 Therefore, the water and N3-histidine polarizations are directly compared in Figure 54 dependent on Bevo which is plotted in a logarithmic scale. This illustrates, that the 5 Hyperpolarizing water with parahydrogen 95

15 polarization of water and N3-histidine are connected in these experiments. The relative intensities of the polarizations of both species is anticorrelated: For example, at a polarization transfer field with a high negative water polarization, a high positive L-histidine polarization is detected and vice versa. Yet, in the weak coupling regime at Bevo = 10000 G (1 T) only the residual thermal polarization is detected in the measurements and not a parahydrogen based effect anymore. The thermal polarization after a waiting time of one minute in the spectrometer is depicted in grey.

15 Figure 54. Polarization of water (blue triangles) and N3-histidine (red circles) from the

hyperpolarization experiments depending on the polarization transfer field Bevo. A thermal reference measurement at 1 T is shown in grey (open triangle and circle).

5.3.3 Mechanistic considerations

Looking at the experimental results, either the oxidative addition of hydrogen or the solubility of hydrogen seem to limit the polarization of water and therefore are rate determining. The emission signal of water rises with higher partial parahydrogen pressure. Higher temperatures also increase the observed negative polarization, as classical SABRE experiments to polarize pyridine with this catalyst49, due to the varied exchange at the iridium center. This can be attributed to the accelerated exchange of parahydrogen and the substrates at the iridium center as well as proton exchange between them. The two asymmetric hydride signals of parahydrogen (at around -22 ppm and -29 ppm) bound to the iridium center by oxidative addition suggest at least a bidentate coordination of histidine to the catalytic center at the same time. Parahydrogen as well as an additive are necessary for the built-up polarization of water, as proven by the control experiments with normal hydrogen and absent an additive such as pyridine or L-histidine. This still leaves the question about the polarization transfer mechanism from parahydrogen. Known mechanisms with proton transfer are the hydrogenative PHIP, oneH-PHIP and pr-PHIP, while other mechanisms are based on NOE or LAC effects. For example, LAC’s determine the SABRE polarization, where spin order is transferred via a 96 5 Hyperpolarizing water with parahydrogen temporary J-coupling network in a catalytic complex as elaborated in subsection 2.5.2. Several of these mechanisms may contribute to the experimental findings. The polarization of the water can occur in two different ways: Either the water is directly polarized by coordinating to the transition metal center or the polarization is initially built up on the additive and consecutively transferred to water. The presence of the hyperpolarized HD gas signal indicates a coordination of the D2O to the iridium or at least an outer sphere exchange mechanism for the hydrogen substitution with parahydrogen. PHIP experiments covering di-hydrogen incorporation and substitution show hyperpolarization in the weak coupling regime at high magnetic fields and under PASADENA conditions.5, 18 This was not observed in the experiments here, so that the results cannot be explained by a classical additive PHIP mechanism. Nevertheless, the observed water polarization could be created by a PHIP mechanism substituting only one hydrogen atom, analogous to the oneH-PHIP mechanism, where only one proton is incorporated into the substrate as for example in aldehydes. A CH-activation of the ortho-position of pyridine has been shown with a di-iridium catalyst.259 In this way the protons of pyridine and its derivatives can be exchanged by Ir-catalysts.260-263 Water and L-histidine exchange protons even faster254, which could explain the lower efficiency of pyridine as an additive. The polarization from such an exchange experiment should be independent from the proton concentration in the solution apart from effect of the relaxation time. Thus an exchange mechanism is likely, as the polarization slightly diminishes with an increased amount of protons by adding H2O and increases with increasing proportion of D2O in the solution. Nevertheless, this possible PHIP mechanism, describing polarization transfer from parahydrogen by single proton exchange, is not the sole step of in such a mechanism. The observed maximum, negative polarization of water at a higher Bevo (> 300 G) with both the 15N and 14N-histidine additives is atypical for known PHIP and SABRE experiments compared to literature values. A reasons for this deviation is the altered chemical structure of the active catalyst and the therefore different J-coupling network. As strong coupling partners, the two groups of hydrides undergo an efficient spin order evolution into detectable states, prior to an exchange at a suitable Bevo. Their close chemical shifts (Δδ 7 ppm) should result in a significantly higher Bevo than the ones for polarization transfer (SABRE) as described in ≈ subsection 2.5.2. This fits to the positive polarization of HD gas and that its intensity shows a similar trend with varied Bevo as the negative water polarization. Theoretical considerations have shown that indeed a single proton exchange after evolution in the strong coupling regime fits well to the experimental findings.148 15 When introducing a strong coupling partner in the form of N3-histidine for the hydride protons, another optimal Bevo at 70 G is observed (see Figure 54). This matches with the known dipolar resonance condition (LAC) for SABRE at 65 G for N-heterocycles (see 15 subsection 2.5.2). The observed correlation between water and N3-histidine polarization 5 Hyperpolarizing water with parahydrogen 97 suggests a connected polarization transfer mechanism. A direct polarization transfer 15 through proton exchange between water and N3 histidine is possible and seemingly connected polarization of HDO and 15N-histidine could stem from exchange mechanism. But such a mechanism would not change the sign of the polarization. Thus, a spin flip flop, where the two spins interchange their quantum state, might be the reason for the observed positive water and 15N-histidine polarization. Finally, apart from the known dipolar resonance condition a higher spin order might have been created by the singlet transfer mechanism during the SABRE process. Such spin states have been reported and discussed in several studies.58, 188, 194

5.4 Conclusion and outlook

In this chapter, the hyperpolarization of water56 as well as of L-histidine using parahydrogen have been presented. In these experiments the catalytic system contained the water soluble IDEG along with L-histidine as a necessary additive. The latter has proven to be more suitable than for example pyridine, which results in less polarization of water. The chapter addresses the effect of several experimental parameters when hyperpolarizing water with parahydrogen: A high temperature has shown to be beneficial 49 which fits well to previous studies with this catalyst. Also, a higher p-H2 pressure increases the detected water polarization as already reported in different SABRE studies.47, 48 While basic conditions diminish the polarization, a slightly acidic environment (pH = 6) even annihilates the polarization. When the sample was exposed to a polarization transfer field prior to detection, a polarization maximum of water at Bevo = 360 G has been found. Finally, triply 15N labeled L-histidine was used instead of ordinary L-histidine. It introduces 15N as a strong coupling partner for the hydride protons stemming from parahydrogen in the transition metal complex increasing their magnetic inequivalance. In 15 this case proton polarization of N3-histidine was observed at 7.24 ppm. Interestingly 15 maximum water and N3-histidine polarization, with opposite signs each, was observed at two different polarization transfer fields: At around 70 G the negative polarization of pyridine was highest, while water has its highest negative polarization at a higher polarization transfer field of 550 G comparable to the measurements with unlabeled L-histidine. The chapter concluded with mechanistic considerations for the polarization transfer from parahydrogen. The polarization is limited by the p-H2 supply to the transition metal center, which hints which steps in the catalytic cycle may be rate determining. The additive seems to be necessary for a successful polarization transfer. The water polarization could be created by a transfer of a single proton from parahydrogen similar to a oneH-PHIP mechanism. This could explain the absence of polarization transfer in the weak coupling 98 5 Hyperpolarizing water with parahydrogen

15 regime at high field. In the case of N3-histidine and water polarization, their polarization transfer mechanisms seem to be connected. The most likely explanation for this is proton exchange. Nonetheless, this does not explain the SABRE-like dependence on the polarization transfer field at Bevo ≈ 300 G and therefore is not necessarily the sole mechanism. Here, an evolution of the spin system as known from SABRE experiments, prior to the exchange, is suggested. In summary, after three decades of intensive research114, spin order has been successfully transferred from parahydrogen to water56. Naturally, after this proof of principle study there is still room to optimize the setup and maximize the water polarization. Also, a heterogeneous catalyst, either heterogeneous by itself or an immobilized version of a homogeneous catalyst (see chapter 4), would offer several advantages such as the facilitated catalyst separability from the reaction mixture. Less than one year after this study, hyperpolarization of liquid water with parahydrogen on a Pt3Sn surface has been reported.114 Although the polarization in this study is unfortunately still relatively small, this functioning heterogeneous catalyst is a first exciting step towards heterogeneous systems. In conclusion, the hyperpolarization of water and also the hyperpolarization of 15 N3-histidine open the door to manifold applications especially in the life sciences. DNP as a well-established hyperpolarization technique has already shown the great potential of hyperpolarized water. Applications range from studies of local water dynamics97 and drug discovery101 to biomedical imaging.99, 100 Excitingly, even a polarization transfer from the hyperpolarized water to different nuclei in biomolecules was demonstrated.98 In contrast to DNP, parahydrogen based hyperpolarization not only offers a simple, rapid and cost efficient way to hyperpolarize target substrates, but is especially superior at low magnetic fields. It could allow for miniaturization of the setup and with it increased accessibility of hyperpolarization technology. 6 Continuous flow hyperpolarization 99

6 Continuous flow hyperpolarization

Typically, hyperpolarization studies are limited to batch experiments in NMR tubes. Alternatively, this chapter introduces a continuous flow hyperpolarization setup for parahydrogen based experiments, which has been published in 2018.57 At its core, a 3D-printed membrane reactor is combined with a compact low field NMR spectrometer. The setup ensures repeatable maximum polarization by precise control of experimental conditions i.e. liquid pressure and gas pressure or volume flow . The catalytic system, operating conditions and evaluated experimental parameters during the hyperpolarization 𝑉𝑉̇ experiments in this chapter are summarized in Figure 55.

Figure 55. Scope of the chapter “Continuous flow hyperpolarization”. The general conditions are highlighted in yellow, while solvents and varied experimental parameters of this chapter are shaded in light yellow. Parahydrogen based chemistry i.e. the substrates and catalysts as well as the polarization transfer regime and mechanism are highlighted in red. Points not addressed in this chapter are written in grey. This chapter is divided into four parts. The introduction (6.1) addresses continuous hyperpolarization with parahydrogen and motivates the continuous flow hyperpolarization setup. The methods section (6.2) features the experimental design at ultra-low field and operation of the continuous hyperpolarization setup. In the results section (6.3), the functionality of the setup is evaluated. Therefore, pyridine and nicotinamide as model 100 6 Continuous flow hyperpolarization substrates are hyperpolarized under continuous flow conditions. Their recorded hyperpolarization spectra are compared to thermal polarized reference spectra and the dependences of several experimental parameters, e.g. pressure and volume flow, on the detected polarization is shown and discussed. Finally, this proof of principle study is compared to traditional SABRE methods and the chapter finishes with its conclusion and outlook (6.4).

6.1 Introduction

Many parahydrogen based hyperpolarization experiments have been discussed in the previous chapters including h-PHIP13, 14, pr-PHIP144, 145 and SABRE12. Continuous flow hyperpolarization setups with h-PHIP have been reported. For propane gas, a continuous flow hydrogenation was studied in detail130, 214 and a liquid phase hydrogenation reaction 37 with p-H2 using hollow fiber membranes has been described. Nevertheless, no continuous flow system for SABRE has been discussed yet. Polarizing target molecules with the SABRE approach leaves their chemical structure unaltered.12 Therefore, continuous and repetitive polarization of the same substrate could be accomplished in a NMR tube.16 Such a repetitive polarization transfer and detection combined with continuous p-H2 supply has been subject to studies with discontinuous flow.22, 58, 169, 182, 264-268 Furthermore, trace detection is possible with the SABRE approach with its very high polarization levels (P > 0.1) on different target nuclei, such as 13C44, 15N33, 35, 19F34 and 31P32, 36. In batch experiments single samples, e.g. Young type tubes with a p-H2 gas phase pressed on top of a liquid solution, are shaken prior to the detection. These experiments are easy, fast and cheap to conduct and thus allow rapid testing of new substrates25, 33, 43, 51, 52, 55, 158, 257 or catalytic systems47-49. Nevertheless, these batch type experiments suffer from a consistent p-H2 supply into the solution or other problems such as susceptibility artifacts and line broadening when bubbling hydrogen into the target solution and detecting the polarization at a high magnetic field (M0 > 1 T). Thus, a continuous system is advantageous considering reproducibility, determination of the measurement error or thorough studies of experimental parameters especially due to the reduced time needed per scan.

In this chapter, a system for continuous flow hyperpolarization with p-H2 allowing repetitive polarization and consecutive detection is presented. This system contains two main parts: One for the polarization of target molecules and one for their detection. For the polarization part, a membrane reactor combining the properties of a gas liquid mixer with a hyperpolarization reactor in one unit is applied. In such a reactor, the membrane can consist of different materials such as metallic, ceramic or polymeric material and can have a porous or dense structure. Membranes are permeable selective structures allowing certain 6 Continuous flow hyperpolarization 101

molecules or ions to pass while blocking others. In this case, the small p-H2 molecules can pass the membrane, while the liquid solvent and target molecules are blocked. Membranes are used for selective removal of products, controlled dosing of reactants or to enhance the contact between catalyst and reactants. Furthermore, they can be a support material for catalysts attached either to their surface or within their porous structure limiting bleeding (leaching) and clearly defining the reaction zone.269 Considering all of the properties, membranes find manifold applications in different fields such as catalysis270, biotechnology211, 271 and electrochemistry272.

In this case, the membrane allows controlled dosing of the needed gaseous p-H2, while the target molecules are supplied and detected in the liquid phase. A 1 T NMR Spinsolve spectrometer was chosen to read out the magnetization built up in the reactor. A flow cell made of glass for the to-be-detected liquid was installed in the spectrometer. The flow cell contains an enlarged volume of 5 mm in the middle to slow down the liquid and increase the sensitive volume for detection. This system is validated by comparing single scans of the continuous flow experiments to thermally polarized spectra. In this way, the influence of experimental parameters such as pressure or volume flow on the signal enhancement is determined. Possible applications for such a system are, for example, the monitoring of substrates over a certain amount of time, the consecutive analysis of multiple test samples with the same system via separated injection or reaction monitoring of highly diluted substances.

6.2 Methods

For this continuous hyperpolarization system, a membrane reactor has been designed by Martin Wiese from the AVT to bring the p-H2 in solution. Different versions of the reactor for the online monitoring of substances have been 3D printed. Maximizing the residence time of the substrate in the reactor and the gas liquid surface of the reaction mixture and the p-H2, a meandering, rectangular channel with a PolyActive® membrane on top was used. This composite membrane consisting of a polydimethylsiloxane (PDMS) polymer support and a selective skin made of poly(ethylene oxide)-poly(butylene terephthalate) (PEO-PBT) copolymer, which separates the liquid and the gas phase. Most importantly, the hydrogen permeability has to be taken into account choosing the right membrane material. For each measurement series new reactors have been 3D printed, if an organic solvent such as methanol is utilized, which slowly damaged the reactor over time as is shown in long term studies. The liquid phase has been chosen to be the upper phase in order to observe a possible formation of gas bubbles. A schematic drawing of the setup and the reactor as well as a picture of the setup are shown in Figure 56. 102 6 Continuous flow hyperpolarization

The stock solutions of the samples were prepared in Schlenk tubes under inert gas conditions. The methanol was distilled, dried and degassed. It was not necessary to use deuterated solvents in this system. A typical sample contained 30 ml methanol, 7.5 mg IMes47 and 18.5 mg pyridine or 28.7 mg nicotinamide. The latter are the two target substrates and known to be SABRE active12. The setup has been tested with a homogeneous SABRE catalyst and two substrates, which are to be polarized to demonstrate the possibility of repetitive hyperpolarization. Therefore, the IMes precursor has been used in methanol. Both substrates, pyridine as a standard substances to compare with literature results, and nicotinamide, as a substrate closer to biomedical applications, have been utilized. The membrane reactor is mounted onto a “Modular MicroReactor System” from “Ehrfeld Mikrotechnik” to keep the needed parts and control elements of the reactor in place. A picture and a sketch of this setup are shown in Figure 56. To regulate the temperature, a thermostat is connected to the reactor. The samples were kept at a constant temperature of 30°C. When performing an experiment, the reaction mixture is pumped from the storage Schlenk (stock solution) through the system with a peristaltic pump (Knauer P 4.1S). This reservoir is kept under inert gas atmosphere and nitrogen is bubbled through the solution at RT and atmospheric pressure to lower the hydrogen concentration. The solution could be reused as the SABRE approach leaves the hyperpolarized substrates chemically unaltered. An integrated back pressure valve behind the reactor and a pressure sensor allow monitoring and controlling of the liquid phase pressure before and behind the membrane reactor. The gas pressure, or to be more precise p-H2 pressure, can already be controlled at the p-H2 generator from Bruker. Thus, pressure changes in liquid and gas are studied individually. Also, an exhaust valve for the hydrogen is implemented above the membrane at the reactor, which is needed as the metastable p-H2 is converted to normal hydrogen over time. The p-H2 diffuses through the membrane to be absorbed by the solution. After solving the hydrogen in the reactor by mixing, hyperpolarization can occur. For detection, the reaction mixture is pumped through a flow cell within a 43 MHz NMR Spectrometer at 27.5°C. This flow cell with an outer diameter of 5 mm has with a wider inner diameter in the middle, comparable to the one of standard 5 mm NMR tubes, which is over 8 cm long and placed into the sensitive volume the spectrometer. Afterwards, the solution is transferred into a trash container or back into the storage flask. The liquid is to be reused as repetitive hyperpolarization is possible with this system. Tubing was reduced to a minimum as the created polarization attenuates with time due to relaxation. For heterogeneous catalysts a sieve was implemented and the catalyst is infiltrated into the reactor module. The whole setup is kept under inert gas conditions, because oxygen and water can decrease the activity of certain catalysts and therefore diminish the observable polarization.

6 Continuous flow hyperpolarization 103

Figure 56. Sketch of the continuous flow hyperpolarization setup. a) Schematic drawing comprising an illustration of the SABRE polarization transfer in the reactor with IMes(a) and b) corresponding picture of the continuous hyperpolarization setup including tubing, pump, pressure valves (P) and sensor, temperature regulation, storage valve with the reactor mounted on an Ehrfeld system, where different modules can easily be arranged and tightly kept under pressure. A picture of the parahydrogen generator can be found in 3.1.3 c) Image of the 3D printed reactor with its meandering channel structure featuring the membrane, which separates the liquid and gas phases. For detection, single scans with a 90° pulse were applied in a 1 T Spinsolve spectrometer from Magritek every 30 s. To calculate the error and show reproducibility, more single scans are taken for most of the data points depending on how fast the experimental target conditions were achieved and the detected signal reached a constant value. The standard deviation is shown in error bars in all figures of this chapter. The reference spectra were recorded as an average of 40 scans over 10 min. The recorded FID’s were processed with the MNova software from Mestrelab research. The enhancement values were calculated as the ratio of the sums of the integrals of the ortho-protons from the hyperpolarized and thermally polarized species. Adding the integrals of all protons instead is less precise due to superposition of the intense methanol peak as no deuterated methanol was used.

6.3 Results and discussion

The continuous flow polarization setup including the membrane reactor, a p-H2 generator and 1 T NMR spectrometer for detection was evaluated with two SABRE active 104 6 Continuous flow hyperpolarization reference substances, pyridine and nicotinamide. These were polarized under different conditions in the reactor. In the first part their spectra from the continuous flow hyperpolarization are compared to reference spectra polarized and recorded at 1 T. To achieve the enhancements shown in these spectra, several experimental parameters were optimized. The optimum working conditions for p-H2 and the liquid pressure of the system as well as the volume flow rate are shown in the two latter subsections. Finally, the continuous flow hyperpolarization is compared to traditional SABRE methods. During the measurements also the activation of the catalyst could be monitored over time. Additionally, this catalyst can be used to polarize solvents such as water56 or methanol160 itself. However, no significant solvent polarization was observed here.

6.3.1 Single-scan hyperpolarization spectra

Single-scan spectra of the reference substances pyridine and nicotinamide hyperpolarized with the SABRE approach are shown in in Figure 57 (black). They are recorded by taking 90° pulses every 30 s in the continuous mode. After processing, the single scan spectra were compared to reference spectra polarized and detected at 1 T within 40 scans. The signal enhancement of the hyperpolarized species was more than 1000-fold. Thus, the thermally polarized reference spectra were enlarged by a factor of 128 to be in the same order of magnitude as the spectra of the continuous hyperpolarization experiments (Figure 57, grey).

Figure 57. 1H Single scan NMR spectra of the continuous flow hyperpolarization of pyridine and

nicotinamide with for a) 3 ml/min and for b) 5 ml/min at 3 bar p-H2 pressure, 2.5 bar liquid pressure and 27°C in methanol. The reference spectra detected with 40 scans at 1 T are shown enlarged by a factor of 128 for visualization. The solvent signals of methanol at 3 ppm and 4.5 ppm are strong, as no deuterated solvent was used. Due to the higher SNR for both pyridine and nicotinamide in the hyperpolarized spectra, not only the intensity of their lines is higher but also the lines are more distinct. For pyridine in (a), the ortho-proton at 8.2 ppm has the highest negative 6 Continuous flow hyperpolarization 105 enhancement under these conditions, the para-proton (7.6 ppm) shows only a small enhancement and the meta-proton at 7 ppm is positively enhanced. These values vary, when another polarization transfer field than earth’s magnetic field is used.47, 116 Also in the case of nicotinamide, the meta-proton (7.4 ppm) is positively polarized, while the ortho- protons (8.4 ppm and 8.8 ppm) and the para-proton (7.9 ppm) are negatively polarized. Furthermore, the hydrides of the catalytically SABRE active complex are visible in the continuous flow hyperpolarization experiments (see Figure 58). Their concentration was not high enough to be detected in the thermal reference spectra. The hydrides from the hyperpolarization experiments at –23 ppm, which compares well with literature values12, are shown enlarged by a factor of 16 compared to the hyperpolarization spectrum shown in Figure 57. For the continuous flow hyperpolarization experiment with nicotinamide, the hydride signal is more intense. This can be attributed to the higher volume flow of the experiment as well as to a longer lifetime.

Figure 58. Hydride region of the 1H NMR spectra from Figure 57 detected during continuous flow hyperpolarization of a) pyridine and b) nicotinamide. The spectra are depicted enlarged by a factor of 16 compared to Figure 57.

6.3.2 The liquid and gas phase pressures

In this case of a continuous setup, the liquid and the gas pressures could be varied individually. Therefore, in a first experiment the liquid pressure is kept constant while the gas pressure of p-H2 was increased stepwise. In a second step, both were increased simultaneously. At a higher liquid phase pressure than gas phase pressure, the liquid starts to penetrate the membrane and fills the gas chamber inhibiting the p-H2 supply.

Furthermore, a driving force to supply the solution with fresh p-H2 is needed. Thus, a higher gas phase than liquid phase pressure was chosen as operating conditions.

The pressure of the p-H2 gas phase was increased, while the liquid phase pressure was kept constant at 1.1 bar with a volume flow rate of 3 ml/min at 27°C. In the case of pyridine as the substrate, the polarization rises with increasing p-H2 pressure from an enhancement of 20-fold at 1.6 bar up to 210-fold at 3.5 bar (see Figure 59a, brown). For p-H2 pressures 106 6 Continuous flow hyperpolarization above 4 bar, gas bubbles are formed. The lines in the corresponding spectra are broad and the measurement error increases when bubbles are transferred to the spectrometer. Nicotinamide shows a similar trend as depicted in Figure 59a (blue), but gas bubbles already disturbed the measurement at p-H2 pressures above 2 bar. The reason for this may be a bypass at higher pressures enabled by small damages of the membrane.

Figure 59. Pressure-dependent signal enhancement of nicotinamide and pyridine using the IMes precursor as SABRE catalyst in methanol. The polarization was built up in the earth’s magnetic field at 27°C during a continuous flow of 3 ml/min and was detected at 1 T a) Increasing the pressure of the liquid and the gas phase simultaneously. b) Increasing only the pressure of the gas phase above the membrane. Contrary to the depicted nicotinamide enhancements (blue circles) the pyridine enhancements (brown triangles) were not determined under inert gas conditions.

A higher p-H2 pressure leads to a higher polarization as discussed in literature for single 47, 116 experiments . Such a higher pressure increases the partial pressure of p-H2 in solution and therefore accelerates the accessibility and the exchange of the hydrogen at the catalytic center. This relation is also valid for the flowing system. Nevertheless, there are also differences to batch experiments: At very high pressures, in the gas phase compared to the liquid phase, the hydrogen breaks through the membrane in such high amounts that the hydrogen is not fully dissolved anymore and gas bubbles form in the reactor. Although this allows for a high amount of p-H2 to be close to the solution, mixing and also detection become an issue. The magnetic susceptibility difference at the gas liquid interface is very high. Thus, when bubbles are transferred into the sensitive volume of the magnet, the lines in the spectrum broaden and consequently complicate or even prevent proper detection.

The pressure of the liquid phase had to be adjusted to a level close to the p-H2 pressure for optimal operating conditions. A pressure difference of 0.5 bar was found to produce a suitable driving force of p-H2 molecules from the gas phase into the solution. In a next step both the liquid and the gas pressure were increased simultaneously, while the pressure difference was kept at 0.5 bar. For pyridine the enhancement increases from

180-fold at 1.6 bar p-H2 pressure to up to 670-fold at 4.5 bar p-H2 pressure (Figure 59b, 6 Continuous flow hyperpolarization 107 brown). The polarization of nicotinamide increases in a similar way from 210-fold enhancement at 1.6 bar up to 980-fold at 4.0 bar (Figure 59b, blue). This increase in the detected polarization with rising p-H2 pressure was expected due to the increased p-H2 supply at the transition metal center as described above. In the pressure range of 2.25 to 3.5 bar an unexpected plateau is observed where the polarization only increases slightly with increasing p-H2 pressure. This may be attributed to a lack of p-H2 supply as the membrane is pressed tighter against the open channel reactor with increasing pressure, which may cause a nonlinear hydrogen permeation through the membrane. Therefore, a liquid pressure of 2.5 bar and a hydrogen pressure of 3 bar were chosen as suitable and moderate operating conditions.

6.3.3 The volume flow

In a next step, the volume flow rate was varied to find suitable conditions for the hyperpolarization experiments during continuous flow. It was increased stepwise from 1 ml/min up to 9 ml/min. For both nicotinamide and pyridine, the polarization increases steeply up to a maximum. While the maximum for pyridine with 1330-fold (= 0.14% polarization) was reached at = 3 ml/min (Figure 60a), for nicotinamide a higher optimum = 5 ml/min with 670-fold enhancement (= 0.73‰ polarization) was found under these 𝑉𝑉̇ conditions (Figure 60b). A further increase of the volume flow up to 10 ml/min slowly 𝑉𝑉̇ decreases the detected polarization down to 460-fold for pyridine and to 570-fold for nicotinamide. At lower flow rates, the pump is more pulsatile and therefore the observed enhancement varies stronger between scans.

Figure 60. Average 1H NMR signal enhancement of a) pyridine and b) nicotinamide depending on the volume flow rate during continuous flow hyperpolarization detected at 1 T. The polarization was build up using the IMes precursor as SABRE catalyst in methanol at 3.0

bar p-H2 in the gas phase and 2.5 bar in the liquid phase under inert gas conditions in the earth’s magnetic field. Looking at the process parameters, the volume flow of the system is determinant for the created polarization. The sensitive interplay between the residence time within the reactor 108 6 Continuous flow hyperpolarization and the relaxation time of the polarized substrate determine the detected polarization. At a low volume flow there is a loss of polarization due to relaxation. At higher volume flows there are less relaxation losses, but the residence time during the polarization in the reactor and for the detection in the spectrometer limit the observed polarization. Thus, for each system a local maximum in the enhancement with corresponding optimum volume flow as a compromise between these values is found. The higher optimum volume flow of 5 ml/min for nicotinamide in comparison to the maximum at 3 ml/min for pyridine can be attributed to a faster relaxation than that of pyridine.

6.3.4 Comparison with traditional SABRE methods

The continuous flow setup for hyperpolarization experiments introduced here differs in several aspects from traditional SABRE methods. The latter can comprise continuous rehyperpolarization of the same sample, whereas in continuous flow experiments fresh solution is provided for detection in the sensitive volume. While this enables the analysis of multiple test samples or continuous reaction monitoring, more sample is required due to tubing, reactor and sample reservoir if more than a single scan is demanded. In this study with parahydrogen based hyperpolarization 0.14% polarization of pyridine and 0.73‰ polarization of nicotinamide were achieved under continuous flow conditions. This is well below routinely achieved polarization levels of traditional SABRE methods. Several effects diminish the detected polarization of the continuous flow hyperpolarization: Relaxation during the travel time to the spectrometer lowers the detected polarization. To minimize relaxation losses, long-lived states are of particular interest, especially for imaging and therefore studied in literature40, 41, 45, 46. As known from reaction monitoring273, apparent

T2 relaxation, which broadens the NMR lines and the shorter residence time in the sensitive volume, diminish the peak amplitude with increasing flow rate. Thus, an optimum volume flow rate between the sensitivity and the line broadening can be found when detecting a flowing liquid. During these experiments, the reactor was exposed to the earth’s magnetic field. Adding a suitable polarization transfer field would enable level anti crossings (see section 2.5) and provide higher polarization levels. Looking at batch experiments with pyridine and the IMes catalyst, a more than twofold improvement of the signal enhancement is expected.47, 116 This could be realized by a magnetic field around the reactor or by a magnetic shielding when 32-36, 38, 44 polarizing heteronuclei with p-H2. In contrast to most studies, no deuterated substances, which could reduce spin order losses were involved here. A deuterated solvent, but also deuterated catalyst ligands151 or even a partially deuterated substrate43 enable higher polarization levels. The here employed approach was aimed to be a proof of principle study for realistic and affordable application. 6 Continuous flow hyperpolarization 109

6.4 Conclusion and outlook

In this chapter, a setup for continuous flow hyperpolarization reactions was presented. It included proof of principle hyperpolarization experiments of pyridine and nicotinamide as reference substances during continuous flow with the SABRE approach over several hours. The setup is evaluated by a comparison with thermally polarized reference spectra of the involved compact NMR spectrometer. The enhancement as function of volume flow and pressure is investigated. For this setup, a 3D printed membrane reactor was used. In the reactor the gas and liquid phases are separated by a PolyActive® membrane that enables p-

H2 transfer from the gas to the liquid phase. As no deuterated solvent is needed, common methanol can be used. In those experiments, a signal enhancement of over 1000-fold was achieved. In future, a consecutive analysis of multiple test samples or reaction monitoring of highly diluted substances is possible. Signal averaging and 2D spectroscopy could extend its applications. Such a setup may also allow the implementation of heterogeneous catalysts as discussed in chapter 4, which could be integrated into the reactor and held in place with a sieve. This may be of special interest for biological substances25, 26, 43, 48, 53, 98, 257, 274. Finally, this method can be extended to heteronuclei such as 13C44, 15N33, 35, 19F34 or 31P32, 36, which may benefit notably by extending the setup with a magnetic shielding35 to vary the magnetic evolution field. This field could even include the tubing for the transfer into the magnet.

110 7 The parahydrogen fueled NMR RASER

7 The parahydrogen fueled NMR RASER

This final results chapter covers the parahydrogen fueled NMR RASER. It was discovered in 2015 during mutual experiments by the team, which published it in 2017.58 Here, additional experiments and simulations are included to get an idea of the underlying principles of the NMR RASER. High, negative polarization on target substances is continuously built up with the SABRE approach. Such population inversion can cause a coherent oscillation of magnetization with the NMR frequency, a phenomenon similar to the known MASERs and LASERs. Its oscillation frequencies are based on molecular spin states of organic molecules including higher spin orders. RASER experiments shrink the linewidths drastically and increase the SNR. Both limit the resolution in NMR spectroscopy as discussed in section 1.1. Therefore, spin quantum states, correlating to J-couplings, could be resolved with unprecedented precision by a multi-mode RASER. Figure 61 summarizes the important experimental parameters, involved compounds and measurement conditions.

Figure 61. Scope of the chapter “The parahydrogen fueled NMR RASER”. The general conditions are highlighted in yellow, while solvents and varied experimental parameters of this chapter are shaded in light yellow. Parahydrogen based chemistry i.e. the substrates and catalysts as well as the polarization transfer regime and mechanism are highlighted in red. This also includes 13C and 15N labeled versions of acetonitrile and pyridine. Points not addressed in this chapter are written in grey. 7 The parahydrogen fueled NMR RASER 111

This chapter is divided into four sections. At first, an introduction (7.1) about LASERs and MASERs including their properties and applications is given. It also summarizes such coherent oscillations for NMR active nuclei and introduces the 1H NMR RASER fed by SABRE polarization. The second section (7.2) describes the methods used to obtain and sustain the parahydrogen fueled RASER. It contains the experimental procedures of the involved chemistry as well as the ultra-low field NMR hardware. The third section (7.3) presents and discusses the experimental results. It introduces a parahydrogen fueled single mode NMR RASER in the form of 1H RASERs based on different SABRE pumped organic molecules. Then, the initial nonlinear dynamics of the RASER based on theoretical considerations, simulations and the experimental results, is elaborated. In the following, the heteronuclei 13C, 15N and 19F are discussed convening their impact on 1H RASERs as well as their own potential for RASER activity. In a next subsection, a RASER based on heteronuclear two-spin order is introduced and studied in detail. The most prominent subsection features different examples for multi-mode RASER operation characterized by the spin eigenstates of the SABRE pumped molecules. The last subsection discusses the implications and capability of the parahydrogen fueled RASER compared to other known systems that emit coherent radiation. Finally, a conclusion and an outlook (7.4) are given to summarize the obtained results from this approach, but also to motivate the great potential of this newly developed technique.

7.1 Introduction

A device named MASER,275 which emits coherent radiation in the microwave regime by stimulated emission, opened a whole new field in science and technology. The hydrogen MASER276 was established as a frequency standard and the optical MASER, known as LASER, was realized only shortly after that.277 Thus, this research on stimulated emission was awarded with a Nobel prize for C. Townes, N. Basov and A. Prokhorov.278 LASERs play an important part of everyday life with its implementation in many devices and applications in physics, engineering and also medicine. Both LASERs and MASERs are based on population inversion. They require a feeding process that continuously builds up population inversion on a medium in a resonator. In a LASER, the excited states are obtained by e.g. optical or electrical pumping. In the simplest case, the chosen LASER medium is placed between two mirrors within the optical resonator, where only one mirror is partly translucent. A MASER oscillating in the radiofrequency regime (~10 GHz) is fed by pumped NH3 molecules which enter a microwave resonator.275 Townes introduced the acronym “RASER” already in his Nobel lecture,279 for an analogous phenomenon with radio waves. Such NMR RASERs, also known as Zeeman MASERs, were realized with different nuclei such as 1H, 3He, 29Al, and 129Xe. In this case, the NMR resonant circuit is the resonator and there are different examples for 112 7 The parahydrogen fueled NMR RASER

RASER media and pumping mechanisms: A 1H RASER in the liquid phase280 and a 29Al RASER in the solid phase281 based on DNP polarization and 3He and 129Xe Zeeman MASERs based on SEOP were demonstrated.282, 283 Also a liquid state 1H RASER based on a photochemical excitation (CIDNP) was shown.284 The SABRE approach12 creates high spin order on various substrates. These can be equivalent to a population inversion or negative polarization. When sufficient population inversion is built up on substrate in such a way, spontaneous, coherent radiofrequency is emitted. Such a liquid state RASER of the protons of organic molecules, fueled by parahydrogen, has been discovered58 and will be subject to this chapter.

7.2 Methods

The NMR signal was detected with coils and LC-resonant circuits optimized for 41.7 kHz and 166.7 kHz in an electromagnet. Details on the NMR setup with its EHQE81 based detection and also the catalyst synthesis are described in chapter 3. A typical sample contained the catalyst precursor IMes, a to-be-polarized substrate, methanol-d4 as the solvent and was prepared under inert gas conditions. The deuterated solvents were degassed by three pump-freeze-thaw cycles. Unlabeled substrates were dried under molecular sieve and degassed. The labeled isotopologues were used as supplied from Sigma Aldrich. Therefore a stock solution of IMes in methanol was prepared and 600 or 900 µl of it are filled into glass tubes that fit into the sensitive volume of the solenoid coil, which has a volume of approximately 500 µl. A few microliters of the substrate are added to this solution under inert gas conditions. The catalyst precursor IMes is activated by bubbling p-

H2 through the solution for 15 min at 6 bar (see Figure 62). During that time, the sample changes its color from yellow to a transparent solution. The reason for this change is the oxidation of the iridium center. The activation mechanisms is described in subsection 3.2.3.

Figure 62. Activation of the IMes precursor under continuous p-H2 bubbling: Stock solution with

1.17 mg IMes and 1 µl pyridine in 600 µl methanol-d4 (left) and SABRE active sample (right).

During the measurements, p-H2 was constantly bubbled through the solution in the measurement cell. The p-H2 is transported through PTFE tubing and controlled by pressure 7 The parahydrogen fueled NMR RASER 113

valves and flow regulators. A polypropylene pipette tip was used to bubble the p-H2 through this solution directly in the measurement cell (see Figure 62). At these low detection fields, susceptibility artifacts arising from the pipette tip and the gaseous p-H2 are negligible.

For the conversion of dihydrogen into p-H2, two different parahydrogen generators were used: A commercial Bruker bphp90 generator and a custom made generator built by J. Ackermann and M. Süfke. Both are described in detail in subsection 3.1.3. The latter enabled control of the conversion temperature from room temperature down to 8 K and p-H2 storage. This parahydrogen generator also allowed for very high parahydrogen flow rates if necessary. RASER activity could be observed up to a conversion temperature of 155 K,58 which results in a parahydrogen fraction of 28.5% with a p-H2 polarization of 4.7% as depicted in Figure 6. In this experimental setup, various SABRE active substrates were hyperpolarized: pyridine, acetonitrile, 3-picoline and 3-fluoropyridine (see Figure 63). The latter two are similar to pyridine, but both their SABRE polarization and their RASER signals significantly deviate from pyridine. To the acetonitrile samples, a small amount of perdeuterated pyridine

(pyridine-d5) was added for complex stabilization. Also, the impact of heteronuclei is accounted for in the form of 13C, 15N and 19F. While 19F has a high natural abundance (100%) and is already a part of 3-fluoropyridine, 15N labelled isotopologues (99%) of acetonitrile and pyridine were used. Also for carbon 13C labeled acetonitrile (99%), with the label at the quarternary carbon position, abbreviated 13C-acetonitrile, was employed. In the 1 experiments at the Larmor frequency of other nuclei than H, the magnetic field B0 of the electromagnet was cycled to match the Larmor frequency with the same coil instead of exchanging the coil. Thus, switching between different nuclei by changing the magnetic field retains the same Q-factor and sensitivity and is fast and convenient. a) b) c) d)

H N H H H N H H N H N C C H H H H H CH3 H F H H H Figure 63. Structural formulas of the SABRE substrates employed in the RASER experiments: a) pyridine, b) acetonitrile, c) 3-picoline and d) 3-fluoropyridine. Both, an ordinary SABRE pulse experiment and a RASER experiment can be performed.

To detect the underlying longitudinal magnetization MZ during RASER oscillation, a so- called crusher gradient was applied. This gradient in the z direction dephases the coherence in the transversal plane and therefore interrupts the strong coupling of the resonator with the sample. By applying a pulse experiment directly after such a gradient, the residual MZ polarization can be measured as the transverse magnetization MT is dephased and therefore set to zero. The recorded FIDs were Fourier transformed after zero filling with twice the 114 7 The parahydrogen fueled NMR RASER number of points. As many parameters had to be monitored, adjusted and recorded, the measurements were performed with two or three experimentalists at the same time. Depending on the individual measurements, the experimentalists were Stephan Appelt, Arne Kentner, Sören Lehmkuhl, Alexander Liebisch and Martin Süfke. The insertion of a

RASER active sample into the magnet during constant p-H2 flow is depicted in Figure 64a. The observed continuous RASER oscillations and recorded signal are shown in Figure 64b.

Figure 64. Pictures of a RASER experiment with the ultra-low field setup developed by M. Süfke and S. Appelt (FZ Jülich and RWTH Aachen) operating at 166.7 kHz: a) Insertion of the sample

into the electromagnet during constant p-H2 flow. b) Recorded initial nonlinear dynamics of the RASER active sample at the measurement computer and RASER oscillation at the offresonance (OR) frequency detected with an oscilloscope. The sample contained

1.17 mg IMes and 1 µl pyridine in 600 µl methanol-d4. To increase the understanding of the coupled differential equations introduced in subsection 7.3.2, the evolution of the polarization PZ and the transverse spin component α under different experimental conditions was simulated. For this purpose the software Berkeley Madonna (version 9.1.6) with the classical Runge-Kutta method was applied. This four step iterative, numerical algorithm to incrementally solve differential equations was named after their inventors C. Runge285 and M. Kutta286. The time increment used for the simulation points of α and PZ was 0.01 s. The individual simulation parameters are given in the figure captions of the respective simulations in the main text and in the supplement.

7.3 Results and discussion

In this section, experimental findings of the first parahydrogen fueled RASER are summarized. Some of these results were already published 2017 in Suefke et al.58 The single mode proton RASER is introduced by several examples of SABRE pumped molecules. Their initial nonlinear dynamics is studied exemplarily with pyridine and acetonitrile by comparing experiments with simulations based on the theoretical description58. Also, other nuclei than 1H, called heteronuclei, are addressed concerning RASERs on their Larmor frequency instead of 1H as well as their impact on RASERs. One special example of those, a RASER based on heteronuclear two-spin order, is thoroughly elaborated. In the following, two different multi-mode RASERs are presented. In a RASER based on 3-picoline, the 7 The parahydrogen fueled NMR RASER 115 chemical shift difference at the low frequencies was sufficient, while in 15N-pyridine the strong 1H - 15N coupling enabled multi-mode operation. The implications the multi-mode case of this RASER are elaborated in a final results chapter as it fundamentally differs from the known MASERs and lasers.

7.3.1 Single mode RASER

Population inversion is required for MASERs, lasers and RASERs. Here, it is realized by spin order transfer from parahydrogen with the SABRE approach. As described in the methods section 7.2, p-H2 is bubbled through the solution directly in the measurement cell. Therefore, the detection field during the measurements is also the magnetic evolution field (Bevo) for the spin order transfer. Hence in the strong coupling regime at low magnetic field, it allows for an efficient SABRE polarization transfer following the theory of LACs as elaborated in section 2.5. A further advantage of the low magnetic field are negligible susceptibility artefacts. Therefore, p-H2 can be bubbled through the solution constantly, even at high flow rates, whereas studies at high magnetic fields often require e.g. shuttling techniques. Thus, the SABRE pumping into the different molecular spin states is sustained during the experiments.

When bubbling p-H2 through a SABRE active solution, polarization is built up on molecular spin states with a pumping rate τp. This pumping rate is determined by the SABRE process. Thus, it can be controlled by e.g. changing the partial hydrogen pressure of the system or the flow rate (bubbling) through the solution. It also depends on the catalytic system, which has been vastly studied in literature for SABRE as listed in section 2.4. The build-up of polarization in a continuous SABRE experiment saturates at an equilibrium polarization M0. Interestingly, if a certain threshold of negative polarization is overcome, RASER oscillations start. This threshold is referred to as RASER threshold and discussed in detail in the next subsection 7.3.2. At this point, of the detected precessing transverse magnetization MT built up by the strong coupling to the EHQE resonance circuit, described by the radiation damping rate 1/τrd, is higher than its relaxation 1/T2*. This transverse magnetization, also referred to as RASER amplitude, is fed by the constantly built-up

(negative) magnetization MZ or polarization PZ. A starting 1H RASER for the SABRE substrates (a) pyridine and (b) acetonitrile is shown in Figure 65. It is the simplest case of a parahydrogen fueled RASER: A single mode RASER. For both, pyridine and acetonitrile, the RASER amplitude is constant after a certain amount of time, as the experiment is conducted under invariant conditions. The magnetization precesses around MZ with the proton Larmor frequency 166.7 kHz. The RASER amplitude

(MT) in the case of pyridine is significantly higher, as pyridine is more efficiently polarized by SABRE than acetonitrile. 116 7 The parahydrogen fueled NMR RASER

1 Figure 65. A starting H RASER of a) pyridine and b) acetonitrile at 166.7 kHz. In a) p-H2 was bubbled through the solution with 20 ml/min at 6.0 bar. The sample contained 1 µl pyridine and

1.17 mg IMes in 600 µl methanol-d4. One amplifier was turned off in this experiment to be in an optimal detector range. This reduces the detected signal and therefore also the RASER amplitude by a factor of six compared to the other experiments in this chapter. In

b) p-H2 was bubbled through the solution with 20 ml/min at 6.1 bar. The sample contained

4 µl acetonitrile, 1.17 mg IMes and 0.4 µl pyridine-d5 for complex stabilization in 600 µl

methanol-d4. For comparable starting conditions a short (<1 s) crusher gradient was

applied directly before the measurement for both samples to dephase the coherent MT of an already running RASER in a stationary state.

7.3.2 Initial nonlinear dynamics

At the start of a RASER experiment, RASER “bursts” can be observed while it evolves into a stationary state with a constant RASER amplitude. This initial nonlinear dynamics is known from MASERs and lasers and its shape and nature depends on different (experimental) parameters. It is a consequence of the interaction between MT and MZ. For an NMR RASER, it can be described by the nonlinear Bloch equations shown in the supplement of Suefke et al.58 These give a set of two partial differential equations for MT and MZ describing the nonlinear behavior analogous to a single frequency MASER: d ( ) 1 ( ) ( ) = + { ( )} (21) d 2 𝑀𝑀T 𝑡𝑡 𝑀𝑀Z 𝑡𝑡 𝑀𝑀z 𝑡𝑡 − 𝑀𝑀0 − 𝑀𝑀Z 𝑡𝑡 − and 𝑡𝑡 𝜏𝜏rd𝑀𝑀0 𝜏𝜏P 𝑇𝑇1 d ( ) 1 ( ) = (22) d 𝑀𝑀Z 𝑡𝑡 𝑀𝑀T 𝑡𝑡 𝑀𝑀T � − ∗� The radiation damping rate 1/τrd𝑡𝑡 quantifies the𝜏𝜏 couplingrd𝑀𝑀0 𝑇𝑇 2of the resonator with the nuclear spins. It depends on the vacuum permeability 0 , the filling factor , which is 0.3 for the used setup, the gyromagnetic ratio of the proton γH, the quality factor of the resonator Q, μ η ≈ which is 300 for the employed EHQE resonator, and the equilibrium magnetization M0:58

≈ 1 = | | (23) 2 µ0 − η𝑄𝑄 𝛾𝛾𝐻𝐻𝑀𝑀0 The equilibrium magnetization M0 reached𝜏𝜏rd by MZ pumping with the pumping rate 1/τp can be expressed depending on the equilibrium polarization P0:287 7 The parahydrogen fueled NMR RASER 117

1 = (24) 2 0 𝐻𝐻 𝑆𝑆 0 The relation between the longitudinal𝑀𝑀 magnetizationħ𝛾𝛾 𝑛𝑛 𝑃𝑃 MZ and the polarization PZ as well as between the transversal magnetization MT and the transverse spin component α can be expressed in a similar way: 1 = (25) 2 𝑀𝑀𝑍𝑍 ħ𝛾𝛾𝐻𝐻𝑛𝑛𝑆𝑆𝑃𝑃𝑍𝑍 1 = (26) 2 𝑇𝑇 𝐻𝐻 𝑆𝑆 Following equation (22), a necessary 𝑀𝑀conditionħ𝛾𝛾 for𝑛𝑛 RASER𝛼𝛼 activity, the RASER threshold, is given as 1 1 1 1 > = + , (27)

∗ where the apparent transverse relaxation𝜏𝜏rd 𝑇𝑇rate2 1/𝑇𝑇2T2* 𝜏𝜏isp the sum of the transverse relaxation rate 1/T2 and the pumping rate 1/τp. This nonlinear phenomenon was simulated numerically depending on equations (21) and (22). The evolution of the transverse spin component α and the polarization PZ is shown Figure 66 for (a) pyridine and (b) acetonitrile. Details on the simulation software and implementation are given in the methods section 7.2. The parameters for the simulation are deduced from the experimental results in

Figure 65: They are τp = 20 s, τrd = 1.6 ∙ 10–3 s, T2* = 0.7 s and P0 = –3.8 ∙ 10–3 for pyridine and

τp = 10 s, τrd = 0.18 s, T2* = 0.6 s and P0 = –7.3 ∙ 10–5 for acetonitrile as already published.58

The T1 values were estimated as 2.7 s for pyridine58 and 9.0 s for acetonitrile considering their decrease in the low magnetic detection field compared to high field relaxation times.288 As starting value for PZ, the magnetization during stationary RASER conditions is chosen (PZ =

–8.686 ∙ 10–5 for pyridine and PZ = –2.19 ∙ 10–5 for acetonitrile) to have constant and comparable starting conditions. A simulation beginning from PZ = 0 would have a slightly longer initial build-up time of PZ. Prior to the shown experiments, a short, strong crusher gradient with a 1 s duration in z-direction dephases the MT magnetization. Thus, α is set to 10-6 at the time of 0.3 s, as value close to zero after a short crusher gradient including the experimentally observed noise level. From this point on the commencing RASER is observed as shown in Figure 66: At first, PZ (red) grows negatively following equation (22) until the RASER threshold summarized in (27) is reached. From this point on, α (blue) is built up according to equation (22) and the initial nonlinear dynamics sets in. The created MT now reduces PZ again following equation (21). In the case of pyridine, it even reaches positive values for PZ. Consequently, α shrinks down to nearly zero, hence its source PZ is so drastically diminished. Now, PZ slowly (negatively) increases again, fed by the pumping with 118 7 The parahydrogen fueled NMR RASER

rate τp from the SABRE process. This interplay between PZ and α goes back and forth for several times with decreasing amplitude until constant values for both are reached. Comparing the simulations for a) pyridine and b) acetonitrile, the major difference is the lesser SABRE polarization build-up on the acetonitrile protons. This leads to a lower RASER amplitude in the stationary regime, as well as an altered initial nonlinear dynamics. Or, looking at it the other way, the higher polarization in the case of pyridine gives an equilibrium polarization P0 far above the RASER threshold. This leads to a higher equilibrium RASER amplitude and multiple, intense, initial RASER bursts compared to acetonitrile until α and PZ reach their equilibrium values. When the pumping conditions only allow for a smaller

P0 close to the RASER threshold, the time between the individual bursts, i.e. the late time period TL, becomes considerably larger.

1 Figure 66. Simulation of the transverse spin component α (red) and the polarization PZ (blue) in a H RASER experiment for a) pyridine and b) acetonitrile. The chosen simulation parameters are based on the experiments shown in Figure 65 and are given in the text. In Figure 67, the initiating 1H RASER of acetonitrile and pyridine from Figure 65 is compared to the simulations of PZ and α from Figure 66. The simulations of α show similar trends as the measurements for amplitude and frequency of reoccurring RASER bursts for both pyridine and acetonitrile. Nonetheless, the initial RASER bursts in the experiment last longer than in the simulation or in other words the late time period TL of the overall exponential decay of the relaxation oscillations differs. A reason for this could be, that the estimated T1 of 2.7 s for pyridine and 9.0 s for acetonitrile is too short. In the supplement

(Figure S 18) a simulation when neglecting the term –MZ(t)/T1 is shown. This results in pronounced relaxation oscillations, even stronger than observed in the experiments. For this case, an analytical solution for the set of equations (21) and (22) can be found.58

Regardless, this solution may still indirectly comprise T1, which then reduces M0 into an effective equilibrium magnetization M0,effective. The simulation can be extended to further experimental conditions and systems. One interesting possibility is the application of a longer crusher gradient. In this case, PZ is built up to an even higher value than the threshold condition, resulting in a pronounced initial nonlinear dynamics. This is exemplarily shown for a crusher pulse length of 30 s for both, pyridine and acetonitrile in the supplement (Figure S 19). The dependence of the amplitude of the initial nonlinear dynamics from the crusher pulse length is also demonstrated by 7 The parahydrogen fueled NMR RASER 119 experiments with a 1H acetonitrile RASER, where five different crusher pulse lengths were applied (Figure S 20).

Figure 67. Initial nonlinear dynamics of 1H RASER of continuously SABRE pumped a) pyridine and b)

acetonitrile from Figure 65 compared to corresponding simulations of α (red) and PZ (blue) for pyridine (c, e) and acetonitrile (d, f), which are partly shown in Figure 66. Also other SABRE substrates are RASER active. In Figure 68, the starting 1H RASER of a) 3-picoline and b) 3-fluoropyridine in a field of 41.7 kHz is depicted. These two SABRE substrates are chemically similar to pyridine (see Figure 63). Their initial nonlinear dynamics can be understood in the same way as for pyridine and acetonitrile before. Interestingly, the SABRE polarization of 3-fluoropyridine was just slightly above the RASER threshold.

Therefore, no relaxation oscillations are visible, as the long time period TL becomes extensively large, comparable to a critically damped oscillation.

Figure 68. Initial measured nonlinear 1H RASER dynamics of SABRE pumped 3-picoline and 3- fluoropyridine at 41.7 kHz. The sample contained a) 1.5 µl 3-picoline or b) 3.0 µl 3-

fluoropyridine in 900 µl methanol-d4 and 1.76 mg IMes and was detected during a) 50

ml/min at 4.4 bar p-H2 and b) = 20 ml/min and 4.2 bar p-H2. 𝑉𝑉̇ = 𝑉𝑉̇ 120 7 The parahydrogen fueled NMR RASER

7.3.3 Heteronuclei

Also other nuclei can be polarized with the SABRE approach, as discussed in the theory subsection 2.3.2. Therefore, it should be possible to have a RASER based on one of these heteronuclei instead of a 1H RASER. Furthermore, when a molecule is labeled with an additional or different NMR active nucleus such as 13C or 15N, its properties change with respect to SABRE and therefore also the corresponding 1H RASER of the labeled molecule. Thus, several SABRE substrates containing 13C, 15N and 19F were analyzed with the ultra-low field setup. For a 13C experiment, labeled acetonitrile was polarized in the continuous hyperpolarization setup. Acetonitrile labeled with 13C in the quarternary carbon position was chosen, as the polarization transfer from the protons over 2J is more efficient than over the 1J-coupling.52 To illustrate which carbon of acetonitrile has been labeled, its molecular structure is depicted in Figure 69. The 1H spectrum is an antiphase doublet with 2JCH = 9.9 Hz. The proton polarization was very high and a 1H RASER, shown in Figure S 21a, could be observed. The 13C polarization, shown in Figure 69b, was too weak to overcome the 13C 13 RASER threshold. In fact, the quartet of the C coupling, to the protons of the CH3 group in the corresponding spectrum (d), could only just be resolved.

Figure 69. 1H and 13C SABRE of 13C-acetonitrile at 41.7 kHz with the a) 1H and b) 13C FIDs and the corresponding c) 1H and d) 13C spectra recorded at an off resonance frequency (OR). The sample contained 1.76 mg IMes, 7.5 µl 13C-acetonitrile (structure shown in the figure) and

3 µl pyridine-d5 in 900 µl methanol-d4, was supplied with 80 ml/min p-H2 at 3.0 bar.

A nucleus with a gyromagnetic ratio closer to the one of the𝑉𝑉̇ = proton is 19F. High SABRE polarization on 3-fluoropyridine has been reported already with the discovery of SABRE.12 In Figure 70, 1H and 19F FIDs and corresponding spectra of SABRE pumped 3-fluoropyridine at 41.7 kHz are shown. Hence in the strong coupling regime, the proton spectrum (Figure 70c) 7 The parahydrogen fueled NMR RASER 121 shows many NMR transitions due to the proton-proton interactions of the four chemically inequivalent protons.289, 290 In the fluorine spectrum, 16 lines as the doublets from the couplings to the protons in the weak coupling regime can be identified. The antisymmetric nature of the 19F spectrum suggests that it is based on higher spin order such as two spin order. In the proton spectrum such a contribution is minor and the main polarization stems from the coherent spin mixing at the most used LAC around 65 G introduced in subsection 2.5.2. The 1H RASER of 3-fluoropyridine has already been shown In Figure 68b in the previous subsection. Although the polarization of 19F was more than an order of magnitude higher than the 13C polarization in the previous example, the RASER threshold for none of the transitions could be overcome and therefore no 19F RASER has been observed.

Figure 70. 1H and 19F SABRE of 3-fluoropyridine at 41.7 kHz: a) 1H FID with the corresponding spectrum in (c) and b) 19F FID with its corresponding spectrum in (d) recorded at an off resonance frequency (OR). The sample contained 3.0 µl 3-fluoropyridine and 1.76 mg

IMes in 900 µl methanol-d4 detected during = 20 ml/min at 3.4 bar p-H2.

Next, 15N is introduced as a spin label into the𝑉𝑉 ̇system in the form of 15N-pyridine and 15N-acetonitrile, which couples to the protons in these molecules. The SABRE experiments with 15N-pyridine are shown in Figure 71. The 1H spectrum is very complicated and shows many transitions, as the experiment was performed in the strong coupling regime. The spectrum matches with simulations, when the individual SABRE polarization levels of the ortho-, meta- and para-protons are considered.58 Nonetheless, one striking feature is the shape of the spectrum. When the spectrum is separated in the middle, both halves are a mirror image of the other one but with opposite sign. The symmetry originates from the coupling of the protons to 15N, which split each transition of the proton spectrum into a doublet. The antiphase nature suggests a higher and even spin order, most likely two-spin order. The proton RASER of 15N-pyridine will be discussed in more detail in subsection 7.3.5. 122 7 The parahydrogen fueled NMR RASER

As with the other heteronuclei, also the 15N polarization is lower than the 1H polarization of 15N-pyridine. Its spectrum is also symmetric with respect to the center, but not antisymmetric. These peaks of the nitrogen coupling to the five protons of 15N-pyridine are depicted with a negative phase. Note, that both spectra are recorded at 41.7 kHz. Therefore, the magnetic field strength in the case of the 15N experiment is about ten times higher than in the 1H experiment, due to the lower gyromagnetic ratio of 15N compared to 1H.

Figure 71. 1H and 15N SABRE of 15N-pyridine at 41.7 kHz: a) 1H FID detected during 20 ml/min at 15 4.1 bar p-H2 and b) N FID at = 20 ml/min and 5.2 bar p-H2. For a) one amplifier was 𝑉𝑉̇ = turned to be in an optimal detector range. In (c) and (d) the corresponding spectra are ̇ 𝑉𝑉 15 depicted. The sample contained 2 µl N-pyridine and 1.17 mg IMes in 600 µl methanol-d4. In Figure 72, SABRE experiments with 15N-acetonitrile are depicted: In (a), the FID after a 90° rf-pulse in a 1H experiment is shown. A longer FID was acquired, as the signal does not decay to zero but evolves into a 1H RASER, which will be discussed in the next subsection. This cutoff also creates small Gibbs artifacts291 in the spectrum obtained through a Fourier transformation at a jump discontinuity of the signal. The starting 15N-acetonitrile RASER is shown in the supplement (see Figure S 21). To reset the continuous MT magnetization of the

RASER, a crusher gradient was applied Consecutively, MZ is read out with a 90° rf-pulse. The corresponding 1H spectrum is shown in (d) and features an antiphase doublet with a

J-coupling constant of 3JNH = 1.74 Hz. The phase difference can be explained by a higher spin order. This assumption is supported by looking at the FID in (a), which starts at zero. The negative peak is a bit larger due to the underlying starting RASER process. Two different 15N experiments are shown in (b) and (c). They are also detected at 41.7 kHz and thus at a ten times higher magnetic field than the 1H experiments due to the lower gyromagnetic ration of 15N. In Figure 72b, the 15N experiment has been conducted in an analogous way as all experiments with the heteronuclei before. The corresponding spectrum in (e) features a quartet as a result of the coupling from 15N to the three protons of 15N-acetonitrile. 7 The parahydrogen fueled NMR RASER 123

Interestingly, the nature of its intensities (−1; −2; +2; +1) suggests not only two-spin order but partly even 4 spin order, which has been simulated and theoretically elaborated in the supplement of Suefke et al.58 In (c) however, the FID of an alternative 15N experiment is shown. The polarization transfer is carried out in a magnetic evolution field, until the sample is shuttled into the field the detection field B0, which is kept the same as in (b). Following the theory of LACs, as introduced in section 2.5 of this work, a very low magnetic field enables an efficient spin order transfer to 15N. This approach, introduced by Theis et al. as

SABRE-SHEATH35, was applied in various studies.33, 158, 164 To obtain such a low Bevo, a magnetic shielding from the earth’s magnetic field is necessary. A small coil within a µ-metal shielded box creates the small magnetic field that is required for the efficient spin order transfer. Details on this magnetic shielding and the corresponding experimental procedures are part of the methods subsection 3.1.2. Although there are losses e.g. due to relaxation during the transfer into B0, the detected polarization is higher than in the experiment shown in (b). This is also reflected in the spectrum (f), where the SABRE-SHEATH polarization is more than two times higher than in the prior 15N experiment. Furthermore, in the case of SHEATH, the whole polarization is of negative, dipolar spin order and therefore all lines are shown in an emissive way. Unfortunately, the polarization was not sufficient for a 15N based RASER. Nonetheless, the proton RASER of 15N-acetonitrile has interesting characteristics and therefore will be the subject to the following subsection.

Figure 72. 1H and 15N SABRE and SABRE SHEATH of 15N-acetonitrile at 41.7 kHz: The sample contained 15 1 5 µl N-acetonitrile, 1.17 mg IMes and 0.4 µl pyridine-d5 in 600 µl methanol-d4. a) H FID 15 detected during 20 ml/min at 3.8 bar p-H2. b) and c) N FID at = 20 ml/min and 6.0 bar

p-H2. In (a) one amplifier was turned off to be in an optimal detector range. For the SABRE- 𝑉𝑉̇ = 𝑉𝑉̇ SHEATH experiment (c), the sample was kept for 20 s in a 2 µT field and then shuttled within 10 s into the detection field. In (d), (e), and (f) the corresponding spectra are depicted. 124 7 The parahydrogen fueled NMR RASER

7.3.4 Two-spin ordered RASER

The 1H RASER of 15N-acetonitrile differs from the previous ones with respect to the underlying spin order. It is based on heteronuclear two-spin order of 15N and 1H. During the stationary, single mode 1H RASER, MT can be observed as shown before. With a crusher gradient, followed by a 90° rf-pulse, the underlying MZ magnetization during the stationary RASER was read out. Its spectrum is an antiphase doublet as shown in Figure 72d. The three regimes of such an experiment are shown in Figure 73: The starting condition (I) is a stationary, single mode RASER. In this case, a precession around the z-axis, against B0, with the amplitude MT is observed. In Figure 73, this is shown over a time period of 14 s. The corresponding spectrum consists of a single line, the RASER mode. During the next regime (II), which is kept very short (< 1 s), a crusher gradient is applied. It dephases MT and only the MZ part remains. The regime ends with a 90° rf-pulse, which flips the magnetization in MZ into the transverse plane. Now the decay of the underlying MZ is recorded, analogous to a canonical NMR experiment (III).

Figure 73. 1H RASER of 15N-acetonitrile at 41.7 kHz based on heteronuclear two-spin order: a) Three different regimes in one experiment: (I) Stationary RASER, (II) short crusher gradient followed by a 90° rf-pulse and (III) FID into the stationary RASER again. b) Sections from

(a) over 14 s intervals for regimes (I) and (III). c) Vector representation of the MT and MZ

components in all three regimes during a constant B0: (I) Stationary RASER oscillation with

MT and MZ . (II) Residual MZ+ and MZ after the crusher gradient destroyed the MT

component.− −(III) Evolution of the MZ components− after their flip into MT by a 90° rf-pulse. d) Fourier transformed spectra of (b): A single RASER line for regime (I) and an antisymmetric pair of J-coupled lines for (III). Compared to other experiments in this chapter, the difference between the major tics in (d) equals 20 a.u. One amplifier (6-fold amplification) was turned off to be in an optimal detector range. The sample contained 15 1.17 mg IMes, 5 µl N-acetonitrile and 0.4 µl pyridine-d5 in 600 µl methanol-d4, was

supplied with 20 ml/min p-H2 at 3.8 bar.

𝑉𝑉̇ = 7 The parahydrogen fueled NMR RASER 125

The FID starts at a minimum (around zero) after the rf-pulse. This hints, that longitudinal two-spin order and/or higher even spin orders are present, which was confirmed by theoretical considerations and simulations.58 The spectrum features two lines, separated by

3JNH = 1.74 Hz, where one is positive and one is negative. The latter is RASER active (see Figure 73d) and negative, as RASER operation requires population inversion. Its intensity is lower than its positive counterpart in the doublet, as it was oscillating during the stationary RASER until the crusher gradient was applied. Thus, the RASER state shows a weaker signal in the spectrum as only its MZ part is read out. The FID was Fourier transformed over a period of 14 s. During the detection, there is still constant SABRE pumping and therefore polarization build-up. Thus, the RASER state does not decay down to zero, but into its constant RASER amplitude under invariant conditions. This is also reflected in the spectrum, which therefore differs depending on the chosen time window.

7.3.5 Multi-mode RASER

Finally, different conditions for multi-mode RASER operation were found. To coexist, the involved modes need to surpass the RASER threshold. Additionally, at the chosen ultra-low field conditions in the strong coupling regime, the RASER lines in the spectrum need to be far enough apart (>0.2 Hz), that they do not collapse.287 Two cases, to separate multiple transitions far enough from each other, are presented in this subsection: By a large enough chemical shift difference and large enough J-couplings. The former case is demonstrated with 3-picoline, while for the latter 15N-pyridine is used. In subsection 7.3.2, Figure 68a illustrates the single mode RASER of 3-picoline at 41.7 kHz. At 166.7 kHz, the chemical shift difference of ~6 ppm between the aromatic protons and the

CH3 group reaches ~1 Hz, which was sufficient for multi-mode RASER operation. Now, at least two modes, that are far enough separated, surpass the RASER threshold, which gives a multi-mode RASER: After the initial nonlinear dynamics, shown in Figure 74a, the RASER evolves into a different stationary state than in the previous experiments. Giant RASER pulses in a repeating pattern are observed, whose maximum amplitude surpasses all previous RASER experiments (see Figure 74c). The spectrum, obtained by a Fourier transformation over 10 s of such a RASER, gives a multiplet of equidistant lines (see Figure 74b, d). Such a pattern is the analogous of a frequency comb when Fourier transformed and one of many effects known from physics, when two different frequencies reach their limit of separability. An analogous phenomenon with picosecond pulsations in a mode-locked LASER was demonstrated by T. Hänsch to obtain ultra-high resolution in optical spectroscopy of hydrogen.292 126 7 The parahydrogen fueled NMR RASER

Figure 74. Multi-mode 1H RASER of 3-picoline at 166.7 kHz: a) Starting RASER, b) RASER in the stationary state over 10 s c) stationary RASER signal over 4 min and d) spectrum consisting of multiple equidistant lines corresponding to (b). To reduce artifacts, a hamming window is laid over the RASER signal in (b) prior to the Fourier transform. Giant RASER bursts were observed in this multi-mode case. One amplifier was turned off and the preamplification was halved to be in an optimal detector range. This reduces the detected signal and therefore also the RASER amplitude by a factor of twelve. The sample contained 900 µl

methanol-d4 and 1.76 mg IMes, 1.5 µl 3-picoline and was detected with 40 ml/min p-H2 at 3.7 bar. 𝑉𝑉̇ = The substrate 15N-pyridine was already discussed in the context of heteronuclei in subsection 7.3.3. For multi-mode operation, the modes are far enough separated from each other, due to the large coupling constants of 15N to the protons, as can be seen in the 1H spectrum in Figure 71c. Thus, in contrast to the case of 3-picoline, no high magnetic field was necessary for multi-mode operation and experiments were kept at B0 = 1 mT (41.7 kHz). Initially, the polarization was not high enough to bring more than one mode over the RASER threshold and evolve into a stationary, multi-mode state. Thus, the built-up polarization was increased by a higher p-H2 flow rate. In this way stored p-H2 is provided more quickly to be exchanged with the hydrogen from the solution and then at the catalytic center, but also the mixing is increased due to the bubbling itself. Thus, with these overlapping phenomena, the polarization increase on the individual SABRE pumped modes is not linear with respect to the higher volume flow. A flow rate of 110 ml/min increases the polarization up to a level, where two modes surpass the RASER threshold and are observed simultaneously. Such a RASER and the corresponding spectrum are shown in Figure 75a, c. This mode was sustained over 20 min.58 By increasing the flow rate even further, more and more modes could be observed. A flow rate of 230 ml/min resulted in a “beat pattern”, which stems from more than two modes. A short sequence of this case is depicted in Figure 75b. The Fourier transform of this RASER shows, that this pattern originates from four modes oscillating 7 The parahydrogen fueled NMR RASER 127 simultaneously (see Figure 75d). Although the beat pattern looks chaotic, it is pseudo chaotic and its shape can be reproduced by the frequencies and intensities taken from the spectrum in Figure 75d.58

Figure 75. Multi-mode 1H RASER of 15N-pyridine at 41.7 kHz: Stationary two-mode (a) and four-mode (b) RASER over 10 s. The corresponding RASER modes are depicted in spectrum (c) and (d): To reduce artifacts, a hamming window is laid over the RASER signal in (a) and (b) prior

to the Fourier transform. The sample contained 900 µl methanol-d4 and 1.76 mg IMes, 15 1.5 µl N-pyridine and was detected with a) 110 ml/min p-H2 at 3.7 bar and b) 230

ml/min p-H2 at 2.4 bar. 𝑉𝑉̇ = 𝑉𝑉̇ = In Figure 76, this spectrum with its four RASER modes is compared to a 1H NMR spectrum from a SABRE experiment with 15N-pyridine. The latter was recorded after a short crusher gradient (τcrusher = 2 s) applied during the multi-mode RASER. The spectrum is antisymmetric, as it is based on heteronuclear two-spin order of 15N and 1H, which was elaborated for 15N-actonitrile in the previous subsection 7.3.4. Some of the resonances in the negative half of the spectrum are sharper. Their lifetime is prolonged by the interaction of the negative polarization, the source for the RASER activity continuously built-up by SABRE, with the resonator. Comparing this spectrum with the RASER spectrum from Figure 75d, the RASER active resonances can be identified. Combined with simulations, they were assigned to the individual RASER active spin quantum states.58 These are depicted in Figure 76 for modes – . The modes are ordered in the sequence in which they surpass the RASER threshold① and④ appear in the spectrum with increasing p-H2 supply. The first three modes of oscillation are a RASER of the ortho-protons, while the fourth line is associated to the meta protons, assigned as in the SABRE spectrum 15N-pyridine. For the RASER frequency of each mode, all other ④coupling partners need to be considered. For mode , both ortho- protons are not only chemically but also magnetically equivalent and indistinguishable.① The 128 7 The parahydrogen fueled NMR RASER same applies for the meta-protons of mode . With the spin quantum states identified,

J-couplings, or sums of J-couplings, can be dete④rmined from the RASER spectrum.

Figure 76. SABRE polarized 1H spectrum of 15N-pyridine compared to the 1H multi-mode RASER spectrum from Figure 75d. ( – ) The four RASER active modes with their spin quantum states 1) – 4). The SABRE spectrum is recorded during the stationary RASER ① ④ conditions after a crusher gradient and 90° rf-pulse. To decrease the impact of the underlying constant polarization build-up by SABRE during the experiment, the Fourier transform was applied over 7.5 s. The RASER spectrum is depicted with an offset of –10 a.u. The chosen time window for it was 10 s as a compromise between resolution and field

fluctuations. The sample contained 900 µl methanol-d4 and 1.76 mg IMes, 1.5 µl 15 N-pyridine and was detected with 230 ml/min p-H2 at 2.4 bar.

𝑉𝑉̇ = 7.3.6 Implications of the parahydrogen fueled multi-mode RASER

The parahydrogen fueled RASER shows analogies in its characteristics to known LASERs and MASERs including the initial nonlinear dynamics. The obvious differences are the polarization source and the frequency of operation, but the main difference lies in another field: The self-organized multi-mode operation dictated by the molecular spin states. This is contrary to all RASER, MASERs and LASERs observed before, where the resonator determines the frequency of the possible modes of operation. For the parahydrogen pumped RASER, the relaxation 1/T2* and the pumping rates 1/τrd are much slower (0.1 – 1 s–1) compared to the damping rate κM of the LC resonator ( 103 s–1). Therefore, the order parameters for the coherent emission process of the parahydrogen≳ fueled NMR RASER are the slowly varying spin states (MT, MZ). Therefore, the chemical system including the molecular spin states determines the RASER modes. The SABRE pumped spin eigenstates of 3-picoline collapse into a single mode RASER at 41.7 kHz, but form a multi-mode RASER at 166.7 kHz, with a 7 The parahydrogen fueled NMR RASER 129

RASER spectrum of equidistant lines comparable to a frequency comb.292 While active mode locking inducing an optical frequency comb is the base for optical spectroscopy with ultra- high resolution, the observed frequency comb in the parahydrogen fueled RASER is a self- organized process associated with various nonlinear phenomena such as frequency shift effects and period doubling.287 The experiments with 15N-pyridine reported here are even more astonishing. Multiple NMR lines from the NMR spectrum, i.e. spin eigenstates of the molecule, could be brought above the threshold to emit constant radiation simultaneously (see Figure 75). This has drastic consequences: As described in the sensitivity section 1.1, the resolution of an NMR experiment is limited by the SNR and the linewidth connected with the relaxation times of the individual components among other parameters. Therefore, many high precision measurements increase the number of scans to yield a sufficiently high SNR. This can be very time consuming, as the SNR only increases with the squareroot of the number of scans. While high levels of polarization can already be achieved with the SABRE approach leading to very high SNR, RASER experiments even allow for continuous signal detection and therefore a smaller linewidth surpassing the T2 limit. The detection time in a RASER experiment can be chosen freely, i.e. without any need for signal averaging of decaying FIDs. The precision of frequency measurements and therefore the sensitivity is ultimately limited by the Cramer- Rao lower bound293. This limiting equation gives an increase for the precision of frequency 3/2 1/2 determination with the measurement time Tm of Tm for the RASER instead of Tm as in canonical NMR experiments.58 Therefore, spin quantum states of 15N labelled pyridine, correlating to J-couplings, could be resolved with unprecedented precision. Within a detection time of minutes, the resolution was in the sub-mHz regime. Distant dipolar fields diminish the precision of frequency measurements in Zeeman MASERs based on nuclear spin population inversion with strong nonlinear effects.294 A RASER based on two-spin order, as introduced here, partly self-compensates with respect to distant dipolar fields and allows for an even higher precision of frequency determination.58

7.4 Conclusion and outlook

This chapter featured the discovery of a parahydrogen fueled RASER.58 As LASERs and MASERs, it is based on population inversion. A 1H NMR RASER is realized with different SABRE pumped molecules such as pyridine, acetonitrile, 3-picoline and 3-fluoropyridine. The evolution of the magnetization during the initial nonlinear dynamics of these RASERs was elaborated. As known from LASERs, MASERs and RASERs, it evolves into a stationary state after initial RASER bursts. In addition, the influence of heteronuclei in the strong coupling regime was studied and their SABRE spectra in this regime are examined. This included labelled, SABRE active substances as the natural abundance of 13C (1.1%) and 15N (0.4 %) is 130 7 The parahydrogen fueled NMR RASER very low. Unfortunately, up to now the built-up of negative SABRE polarization on these more sensitive heteronuclei was not sufficient for RASER activity, yet. Nonetheless, their introduction into different molecules as a strong coupling partner also impacts the proton RASER. For example, the 1H RASER of 15N-acetonitrile is based on heteronuclear two-spin order. In this case, the evolution of the individual components of the magnetization was studied. Based on the different single mode RASER experiments, complicated but exceptional multi-mode RASERs were examined. In a stationary multi-mode state, several resonances coherently oscillate simultaneously. Thus, in contrast to known LASERs and MASERs, the individual modes feature structural information about the involved SABRE pumped substances as they are spin eigenstates of these molecules. This fundamental difference enabled high precision measurements of molecular coupling parameters.58 When based on two-spin order, it even self-compensates the created distant dipolar fields, which plague other MASERs, LASERs and RASERs based on population inversion.294 The RASER combines the narrow bandwidth of the LASER with the structure elucidation of NMR. These properties constitute the parahydrogen fueled RASER as a powerful tool, with most of its potential yet to discover. It envisions manifold applications in different fields of science and technology. As the first two-spin ordered self-oscillating system, it is particularly interesting for fundamental physics. The possibility of high precision measurements allows for the development of precise magnetic field and rotation sensors or coherent narrow- band amplifiers. Such an extraordinarily sensitive structure elucidation technology is not only interesting for e.g. reaction monitoring in chemistry, but even ranges into the life sciences. 8 Summary 131

8 Summary

Parahydrogen based hyperpolarization can drastically boost NMR signals and merges many disciplines from science and technology. Especially, the relatively new SABRE approach is promising as it enables easy, affordable and repetitive hyperpolarization of suitable target molecules. Despite many successful efforts towards broad applicability, especially in the context of biocompatibility have already been taken, it is still a growing topic with vast untapped potential. To advance and broaden this field, four hitherto not accomplished steps were chosen for this work: Heterogeneous catalysts allow for convenient recovery from reaction mixtures and reuse compared to the often toxic homogeneous catalysts. For this purpose, homogeneous PHIP catalysts were anchored on Al2O3 using a heteropolyacid as a linker. This modular system, known for efficient and enantioselective hydrogenations at negligible leaching, is evaluated for its potential in parahydrogen experiments. The activation, experimental procedures and operating conditions for the immobilized catalysts were optimized in a model system and extended to various exemplary substrates and catalysts. Additionally, a suitable choice of the nature and particle size of the support material turned out to be essential. Only PHIP mechanisms based on p-H2 addition and replacement in target substrates were observed so far. Thus, experiments with alternate immobilization approaches with a focus on heterogeneous SABRE and water based systems were part of a sidetrack. Moving towards biomedical applications, water and L-histidine were hyperpolarized with parahydrogen for the first time. The impact of various experimental conditions on the water polarization was studied using a water soluble SABRE catalyst. A special focus is laid on the polarization transfer field, crucial for the polarization of both water and histidine. Based on these results, the underlying spin order transfer mechanisms are elaborated. In addition, a setup for hyperpolarization during continuous flow was introduced. A membrane reactor at its core brought the parahydrogen into solution. Important experimental parameters such as pressure and flow rate of this system are evaluated in proof of principle experiments under continuous flow SABRE over several hours. Finally, a parahydrogen fueled RASER has been discovered by us. Based on the protons of organic molecules and continuously fed by SABRE pumping, this system emits coherent radiation similar to LASERs and MASERs. Its initial nonlinear dynamics is studied by simulations and experiments with various substrates. The impact of heteronuclei such as 13C and 15N on proton RASERs is elaborated. The first self-oscillating system based heteronuclear two-spin order is presented, which as a result self-compensates distant dipolar fields. Most excitingly, its multi-mode operation, based on spin eigenstates of RASER active molecules, envisions precise chemical structure elucidation and therefore applications not only in the fundamental physics, but in various fields of science and technology. 132 9 Supplement: A modular immobilization system

9 Supplement: A modular immobilization system

The supplement for this chapter is divided into two parts. First, an excursus with experiments containing other immobilization approaches is presented (9.1). Then, further measurements belonging with the modular immobilization system are discussed (9.2).

9.1 Other immobilization approaches – an excursus

Other immobilization approaches were discussed as described in the introduction of chapter 4. For that purpose, Rh nanoparticles physisorbed on a glass plate and iridium catalysts with phosphine ligands including immobilization of the homogeneous catalyst by coordination to a PPh3 group included in a polystyrene polymer were synthesized. The synthesis and results are shown below.

9.1.1 Rh nanoparticles on a glass slide

Rh nanoparticles were synthesized in situ on a glass slide to obtain a heterogeneous PHIP catalyst, which can be implemented in a microfluidic reactor to obtain a continuous hyperpolarization setup. The immobilization system was evaluated by application in a continuous hyperpolarization setup with styrene as a target substrate and a 1 T NMR-spectrometer for detection.

1) Methods: A glass slide fitting into a microreactor for continuous measurements is coated in situ with a thin, homogeneous rhodium layer. Therefore a thin film of [Rh(nbd)2]BF4 (Bis-nbd) solved in tetrahydrofurane (THF) is dripped onto a glass slide. Under a reductive hydrogen atmosphere and subsequent heating to remove the organic ligands, rhodium nanoparticles (Rh-NP) are grafted onto the surface of the glass slide (Figure S 1a). This coated glass slide can be implemented into a 3D printed microfluidic system shown in Figure S 1b.

9 Supplement: A modular immobilization system 133

Figure S 1. a) Synthesis of Rh-NP from Bis-nbd on a glass slide and b) microfluidic reactor including a glass slide with adsorbed Rh-NP prior to application excluding (top) and a rhodium coated glass slide (bottom).

2) Results In a first step Rh-NP were formed on glass plates attached by physisorption as described in the methods paragraph above. These catalysts successfully showed hydrogenation and hyperpolarization of unsaturated substrates like styrene with p-H2. The glass plates could also be implemented into a membrane reactor module as shown in Figure S 1b. Under continuous flow conditions, hydrogenation could be observed in form of the aliphatic signal from ethylbenzene, but no significant hyperpolarization (Figure S 2a). Unfortunately the catalyst adsorbed to the glass slide was subject to high leaching especially at higher flow rates. This means that the catalyst is washed out over time. The reactor and the glass plate after such an experiment are shown in Figure S 2b. The leaching not only prevents long time applications but also the application in biological systems as the toxic metal catalyst is released into the solution. Therefore other immobilization approaches with stronger binding of the homogeneous catalyst to the support were applied and discussed in the following subsection.

134 9 Supplement: A modular immobilization system

Figure S 2. Experiment with Rh-nanoparticles in a microfluidic reactor module: a) Spectrum taken

during continuous hyperpolarization experiments with methanol-d4 and styrene using detected at RT and 1 T. The peaks of the saturated ethyl group of ethylbenzene are framed by a dashed line. b) The microfluidic reactor excluding (top) and including (bottom) the rhodium coated glass slide after application in a continuous measurement.

9.1.2 Iridium catalysts with phosphine ligands

Prior to the strongly electron donating carbene ligands such as IMes, different phosphine ligands were used in hyperpolarization catalysts. As also common in hydrogen related chemistry, the hydrogenative PHIP14 as well as SABRE12 were initially presented using two famous catalysts with phosphine ligands: In the case of h-PHIP it was Wilkinson’s catalyst295

149 [RhCl(PPh3)3] and for SABRE it was Crabtree’s catalyst , already introduced as PCy3. This excursus briefly describes experiments with such ligands ranging from water soluble ligands to a polymer containing phosphine groups which itself is heterogeneous.

1) Methods: Immobilization of IMes on triphenylphosphine polymer bound

The immobilization method on a PPh3 that is incorporated in a polymer has been published by Wilkinson et al. in 2004 using Crabtree’s catalyst.260 In this case a PPh3-group of polymer acts as an additional ligand and the phosphorus atom binds to the metal center of the catalyst precursor. The activation of the homogeneous IMes catalyst precursor the combined activation and immobilization on PPh3PB of IMes are displayed in Figure S 3. The polymer bound catalyst is from here on referred to as IMes@PPh3PB. The synthesis of IMes as well as its preparation for shake-and-drop experiments is described in the methods section 3.2. The activation takes place during the shake-and-drop experiments in the NMR tube. To obtain the IMes@PPh3PB, 50 mg of the IMes precursor and 24.8 mg of the diphenylphosphinated copolymer of styrene and divinylbenzene with a particle size of 100−200 mesh polymer were stirred in 7.5 ml DCM. For activation, 18 µl py were added and

H2 was bubbled through the solution under stirring overnight. A second batch contained 9 Supplement: A modular immobilization system 135

100.5 mg IMes, 99.3 mg PPh3PB and 36 µl pyridine in 15 ml DCM. The DCM was removed with a syringe and the solution washed six times with EtOH. After drying under vacuum a light yellow-brownish fine grained solid was obtained. a) + py, H N N 2 N N Mes Mes COD Mes Mes py H Ir Ir Cl MeOH py H Cl py

+ b) N N Mes N N Mes py, H2 Mes Mes PPh PB 3 py Ir H Ir COD H Cl py Cl P Ph DCM Ph

Figure S 3. activation of the IMes and the IMes@PPh3PB precursors: a) Formation of the SABRE active homogeneous IMes(b) species from IMes and b) formation of a heterogeneous SABRE

active catalyst with the IMes@PPh3PB. Additionally homogeneous iridium phosphine catalysts were synthesized. A catalyst mixture of two different precursors is obtained when using 2.5 equivalents of the ligand

PPh3 (Figure S 4a). The ligand is added to a solution of 20 mg [Ir(OMe)(1,5-COD)]2 in 7.5 ml MeOH giving a cherry red solution. While bubbling hydrogen through the solution for three hours turns more transparent and grey. Finally the solvent is removed using a vacuum pump. To obtain a water soluble phosphine catalyst with iridium as the transition metal center, the polar PPh2PhSO3 (ptppds) phosphine ligand was used (ptppds = 3-(diphenylphosphino)benzenesulfonate). After an equivalent procedure compared to the

PPh3 ligand using PPh2PhSO3Na and subsequent drying a grey solid is obtained. (see Figure S 4b). 136 9 Supplement: A modular immobilization system

a) 2.5 eq PPh 3 PPh3 O L H py, H2 Ir Ir Ir COD S H O P Ph MeOH Ph

Ir-PPh3

ptppds b) L H 2.5 eq PPh2PhSO3Na Ir O py, H S H Ir Ir COD 2 O P Ph Ph MeOH SO3

Ir-ptppds Figure S 4. Synthesis of iridium hydride complexes where different isomers are created. Thus L can either be a third phosphine ligand or a second solvent molecule S. They were synthesized

in methanol with (a) PPh3 ligands to obtain hydrophobic complexes and (b) ptppds ligands to create polar, water soluble complexes.

2) Results

A polystyrene polytriphenylphoshine polymer (PPh3PB) has been chosen as a support.

The PPh3 groups allow for coordination of metal precursors such as iridium or rhodium. In these cases the polymer acts as the support and ligand at the same time consisting of multiple catalytic centers. The synthesis and activation of IMes@PPh3PB and the activation of the homogeneous IMes with parahydrogen are shown in in the methods paragraph (Figure S 3). The homogeneous analogues to the polymer bound species,

[Ir(Imes)(py)2H2PPh3]Cl where the single PPh3 ligand is coordinated trans to the IMes ligand, 151 is a SABRE active catalyst for various substrates. Compared to the catalyst without PPh3, the detected polarization is nearly one order of magnitude lower for pyridine as a substrate.47, 151

The synthesized IMes@PPh3PB allowed for hyperpolarization with p-H2. This catalyst containing the IMes ligand was found to be SABRE active (see Figure S 5a). The spectrum shows the solvent MeOH (σ = 4.2 ppm), ortho-hydrogen (σ ≈ 4 ppm) and the hyperpolarized pyridine (σ ≈ 6.5 - 8.2 ppm). The observed enhancement of pyridine was smaller than for its homogeneous counterpart (SEF < 10-fold). There are different reasons for the diminished detected polarization. The altered chemical structure changes the polarization transfer activity at the catalytic center from p-H2 to pyridine. The polymer makes the catalyst less mobile and therefore pyridine and parahydrogen have to diffuse to the transition metal center. Furthermore in the case of these immobilized catalysts, relaxation effects take place 9 Supplement: A modular immobilization system 137 and the magnetic susceptibility difference at the polymer surface is high. Thus the polarization decreases significantly when adding more polymeric support without catalyst bound to it to the solution. This effect is shown in Figure S 5b, where polymeric support material was sequentially added to a SABRE active solution of the homogeneous IMes(b)Cl catalyst.

Figure S 5. Impact of polymeric support on the hyperpolarization: Shake-and-drop experiments with

p-H2 at RT and 1 T: (a) 18.2 µl pyridine with 4.0 mg [Ir(H)2IMes(py)2]Cl@PPh3PB as a

heterogeneous SABRE active catalyst in 182 µl methanol-d4: Hyperpolarized spectrum (bottom) and a thermal reference spectrum (top) of pyridine. (b) effect of the polymeric support on the detected polarization: 40 µl pyridine and 5 mg IMes catalyst precursor in

400 µl methanol-d4. The absolute intensities of the pyridine signals are added.

The more polymeric material msupport was added, the more the polarization Isignal is diminished. The magnitude of this polarization loss due to the heterogeneous material is illustrated by a first order exponential fit (blue) following equation (28), with a loss factor

support = 134 and the initial intensity I0 = 2 as fit parameters:

τ = (28) 𝑚𝑚support − I0 𝐼𝐼signal τsupport ∙ 𝑒𝑒 Nevertheless some hyperpolarization has been detected, which is already significant, as a heterogeneous SABRE catalysts are rare and of similar effectivity. Unfortunately at least a part of the observed polarization may not be caused by a heterogeneous catalysts. Minimal leaching of the homogeneous material could be responsible for the observed polarization as these homogeneous catalysts are so efficient, that they even allow for trace analysis. A further experiment with 4 mg of the second batch of the synthesized heterogeneous catalyst still showed hyperpolarization with enhancement about 2 in methanol (not in DCM though), but also broad lines in the spectrum due to the heterogeneous polymeric support material in the tube during the detection (Figure S 6a). In this case, when measuring the liquid methanol phase of the solution in a subsequent experiment, hyperpolarization of the contained pyridine using p-H2 could be achieved (Figure S 6b, SEF 10), but no

≈ 138 9 Supplement: A modular immobilization system hyperpolarization was observed when probing the residual solid in DCM. This indicates, that there are traces of SABRE active material in the liquid phase. Either small polymer chains from this batch or homogeneous catalyst that remained from leaching or insufficient washing of the polymeric material could be the reasons for the detected polarization.

Figure S 6. SABRE with a polymer bound catalyst: Shake-and-drop hyperpolarization experiments

with 6 bar p-H2 (top, grey) and thermally polarized reference spectra (bottom, black)

detected at 1 T and RT. a) 4.5 mg [Ir(H)2IMes(py)2]Cl@polymer as a heterogeneous SABRE

active catalyst with 20 µl pyridine in 200 µl methanol-d4. b) liquid phase of (a) was used for another hyperpolarization experiment indicating possible catalyst leaching.

When adding the activated catalyst [Ir(IMes)(py)2H2]Cl@PPh3PB to a solution of water and butylacrylate, no hyperpolarization and also no hydrogenation could be observed. But after one week the solid material nearly completely dissolved and turned from colorless to orange and after adding p-H2, both mechanisms typically observed when using unsaturated terminal hydrocarbons to be polarized with p-H2 could be observed (Figure S 7): pr-PHIP showing the unsaturated CHCH2 group of the unaltered substrate butylacrylate

(σ = 4.75 – 6.25 ppm) and h-PHIP in form of the terminal CH2CH3 group of butylpropionate

(σ = 0 – 2 ppm) resulting from hydrogenated butylacrylate with p-H2 (SEF ~ 10-20). In this case no leaching could be observed or detected in high field NMR measurements or subsequent shake-and-drop experiments with the liquid phase. An explanation for the hyperpolarization in this case could be that the active catalyst could have been chemically altered with time. One possibility could be iridium phosphine based catalysts, whose homogeneous analogues are known to be active. 9 Supplement: A modular immobilization system 139

Figure S 7. h-PHIP and pr-PHIP with a polymer bound catalyst: Shake-and-drop experiment with p-H2

at 1 T and RT: 35 µl pyridine, 25 µl butylacrylate, 4.2 mg [Ir(H)2IMes(py)2]Cl@polymer 200

µl D2O thermally polarized reference spectrum measured in DCM (bottom) a) hyperpolarized spectrum (grey upper spectrum) and thermal reference spectrum (black lower part) Thermally polarized spectrum enlarged by a factor of 10 b) enlarged section of the hyperpolarized spectrum of a) (framed in a) by a dashed line) to display the hydrogen exchange with parahydrogen at the terminal protons of butylacrylate (pr- PHIP). Figure S 8 shows that homogeneous phosphine catalysts are active in various solvents such as toluene (a) and water (b). The hyperpolarized triplet and quartet group (σ ≈ 0.5 – 2.5 ppm) can be attributed to the CH2CH3 group after hydrogenation of the double bond with p-H2 (h-PHIP). Additionally the pr-PHIP mechanism in form of the hyperpolarized CHCH2 substrate is observed for butylacrylate in toluene (σ ≈ 4.5 – 5.5 ppm). While toluene represents the group of organic solvents, relevant in many chemical studies (Figure S 8a), water as the medium of life is of special interest for biomedical applications (Figure S 8b).

Figure S 8. PHIP experiments in toluene and water: Shake-and-drop s with 6.1 bar p-H2 using

homogeneous iridium based phosphine catalysts at 1 T: a) 1 Ir-PPh3 and 20 µl

butylacrylate in 200 µl toluene-d8 at 52°C b) 1 mg Ir-ptppds and 20 µl

hydroxybutylacrylate in 200 µl D2O at 65°C. 140 9 Supplement: A modular immobilization system

Although this immobilization approach seemed to be promising, the polarization was quite low compared to the homogeneous analogues and the relatively small polymer (particle size of 100 200 mesh) needs to be held in place for continuous hyperpolarization setups. Additionally minimal leaching could not be excluded as concentrations which are − already a challenge for typical NMR experiments, are not negligible for hyperpolarization experiments.

9.2 Further measurements with the modular immobilization system

In this chapter, further results from hyperpolarization experiments with the Augustine system are presented. First the effect of varied reaction conditions is shown for styrene and methylacrylate as substrates instead of the model substrate butylacrylate from main text. Secondly hyperpolarization experiments with two additional anchored catalysts are shown:

IMes@Al2O3 and DuPhos@Al2O3. Finally further hyperpolarization experiments at high field are presented including ALTADENA conditions.

9.2.1 Methylacrylate and styrene

Methylacrylate was hyperpolarized with the Augustine system under similar conditions as the substrate butylacrylate from the main text. It shows similar trends for the varied parameters, but the experiments at each condition were not repeated so some points may contain a significant error. The detected polarization increases with increasing p-H2 pressure or temperature (see Figure S 9).

1 Figure S 9. H NMR hyperpolarization experiments with methylacrylate at different p-H2 pressures and temperatures of a pre-heating bath: The of Normalized 1H NMR Signal intensity of the

CH2CH3 group of methylpropionate resulting from hydrogenation of methylacrylate with

p-H2 depending on a) the pressure and b) the temperature. One sample contained 10 µl

methylacrylate and 1 mg BINAP@Al2O3 (particle size: 125 – 250 µm) in 300 µl methanol-d4

at 55°C and 6 bar p-H2 pressure. 9 Supplement: A modular immobilization system 141

Also for both substrates, methylacrylate and styrene, an optimum particle size of the support at different temperatures can be found between enhanced relaxation and lower catalyst loading for large particles and arising susceptibility issues at a low particle size (see Figure S 10).

Figure S 10. 1H NMR hyperpolarization experiments with methylacrylate and styrene at different 1 particle sizes of the support material: Normalized H NMR Signal intensity of the CH2CH3 group of methylpropionate (hollow circles) and ethylbenzene (black triangles) resulting

from hydrogenation of methylacrylate and styrene with p-H2 depending on the particle size of the support at a) 25°C and b) 55°C. One sample contained 10 µl butylacrylate or

styrene and 1 mg BINAP@Al2O3 in 300 µl methanol-d4 at 7 bar p-H2 pressure.

9.2.2 [Ir(COD)(IMes)]Cl@Al2O3 and [Rh(COD)(Duphos)][BF4]@Al2O3

Also IMes and Duphos were immobilized with the Augustine system and tested in hyperpolarization experiments with p-H2. Both catalysts were activated prior to the experiment, indicated by a loss of their respective yellow and orange color, and successfully probed with butylacrylate as a substrate (see Figure S 11). Note that these experiments were carried out at RT. Thus, the polarization is lower and the spectra are shown with an enlargement factor of eight compared to the ones in the main text in 4.4.3. The lower polarization can be attributed to the lower p-H2 pressure in a) and lower temperature in b).

142 9 Supplement: A modular immobilization system

Figure S 11. 1H NMR spectra from hyperpolarization experiments with the anchored homogeneous catalysts IMes and DuPhos: The homogeneous catalyst precursors that were anchored

on PTA@Al2O3 are shown below the spectra. All spectra are enlarged by a factor of eight compared to the main text. In a) the sample contained 4.3 mg of the IMes, anchored on

neutral alumina, along with 20 µl butylacrylate in 200 µl methanol-d4 at RT and 3 bar p-H2

while b) contained 1 mg DuPhos@Al2O3 and 10 µl butylacrylate in 390 µl methanol-d4 at

RT and 7 bar p-H2.

9.2.3 Further hyperpolarization experiments at high field

Prior to the hyperpolarization experiment at high field shown in the main text (Figure 46), two more spectra were recorded with the same sample. The first one is the ALTADENA spectrum recorded directly after the sample was dropped into the magnet (see Figure S 12). If features strong susceptibility artefacts including significant line broadening from the suspended solid material leads in the NMR tube. The region from 0.75 to 2.55 ppm containing the hyperpolarized peaks is magnified and shown in an inset. The first PASADENA scan taken 36 s after the ALTADENA experiment above and before the periodical scans every 45 s is shown in a similar way in Figure S 13. At this point there is no significant superposition with the thermal signal from butylpropionate yet as in the experiment shown in the main text. 9 Supplement: A modular immobilization system 143

Figure S 12. 1H NMR hyperpolarization experiment at 300 MHz under ALTADENA conditions with 6

bar p-H2 at 60°C. The sample was shaken for 10 s dropped into the spectrometer and directly recorded. This spectrum features strong line broadening due to the solid material suspended in the NMR tube after shaking. The sample contained 5 µl butylacrylate along

with 2.3 mg dppb@Al2O3 catalyst (particle size: 80-125 µm) in 500 µl methanol-d4 in a young type tube and was activated prior to the experiment. The resonances from the

CH2CH3 group of butylpropionate (molecular structure shown in the figure) resulting from

hydrogenation of butylacrylate with p-H2 are highlighted in the inset.

144 9 Supplement: A modular immobilization system

Figure S 13. 1H NMR hyperpolarization experiment at 300 MHz under PASADENA conditions recorded

directly after the ALTADENA experimentat 60°C: The sample prepared with 6 bar p-H2, shaken for 10 s dropped into the spectrometer and directly recorded as shown in Figure S 12. This spectrum was recorded 36 s after the spectrum above. The sample contained 5

µl butylacrylate along with 2.3 mg dppb@Al2O3 catalyst (particle size: 80-125 µm) in 500

µl methanol-d4 in a young type tube and was activated prior to the experiment. The

resonances from the CH2CH3 group of butylpropionate (molecular structure shown in the

figure) resulting from hydrogenation of butylacrylate with p-H2 are highlighted in the inset. Supplement: Hyperpolarizing water with parahydrogen 145

Supplement: Hyperpolarizing water with parahydrogen

The chemical shift of water is temperature dependent.258 The hyperpolarization experiments in the main text (chapter 5) included a pre-heating bath to control the temperature of the sample. In Figure S 14 the chemical shift dependence of HDO on this temperature corresponding to the results in Figure 50b is shown. The chemical shift of HDO decreases linearly from 4.95 to 4.50 with increasing temperature between 22.5 °C and 95 °C.

Figure S 14. Corresponding chemical shifts of the water peaks (HDO) shown in Figure 50b depending on the temperature the sample was exposed to prior to detection. As a reference, normal water is shown in a dashed line taken from literature.258 The negative polarization of water in the hyperpolarization experiments depends on the polarization transfer field the sample was exposed to. Figure S 15 shows the hydride region from -14 to –28 ppm corresponding to Figure 51 from the main text. Compared to Figure 51, the spectra here are depicted 16-fold enlarged for a better visualization. In contrast to the water signal, the polarization of two groups of asymmetric hydrides seems to be independent from the polarization transfer field.

Figure S 15. Hydride region of the 1H NMR spectra of the hyperpolarization experiments of water at

different Bevo corresponding to Figure 51 in the main text. For better visualization this hydride region is depicted 16-fold enlarged compared to the spectra in the main text.

146 Supplement: Hyperpolarizing water with parahydrogen

15 The signal of hyperpolarized N3-histidine is significantly lower than the one for HDO as discussed in the main text. Therefore an enlarged 1H NMR spectrum of Figure 52a is shown 15 in Figure S 16 to highlight the signal of the polarized N3histidine proton (red) and the two groups of asymmetric hydrides (yellow). The hyperpolarized HDO (blue) and HD (green) signals are also shown.

Figure S 16. Enlarged 1H NMR spectrum of Figure 52a containing several hyperpolarized species 15 highlighted in different colors: hyperpolarized N3-histidine (red, 7.24 ppm), HD gas (green, 4.65 ppm), HDO (blue, 4.6 ppm) and the corresponding hydrides (yellow, -21.6 and -28.9 ppm). The hyperpolarization experiment was conducted under the standard conditions defined in the methods section of chapter 5 but with triply 15N labeled histidine instead of ordinary unlabeled histidine.

15 The polarization of water as well as N3-histidine depends on Bevo as shown in Figure 53 of the main text. The corresponding hydride region is shown in Figure S 17 and is 16-fold enlarged for visualization. The detected polarization of both groups of the hydride signals at -21.6 and -28.9 ppm in these hyperpolarization experiments is independent from Bevo.

Figure S 17. Hydride region of the 1H NMR spectra of the hyperpolarization experiments of water and 15 N3-histidine at different Bevo corresponding to Figure 53 in the main text. For better visualization the spectra are depicted enlarged 16-fold compared to the spectra shown in the main text containing the hyperpolarized water and histidine signals. Supplement: The parahydrogen fueled NMR RASER 147

Supplement: The parahydrogen fueled NMR RASER

Simulations of α and PZ based on equations (21) and (22) for pyridine and acetonitrile are shown in Figure 67 of the main text. They are compared to experimental data and the simulation parameters are set accordingly. Although the frequency with which the RASER bursts reoccur, fits to the experimental data, they decay faster into the equilibrium state of the RASER with constant α and PZ for both pyridine and acetonitrile. As discussed in 7.3.2, one reason for this may be a longer T1 in the measurements than estimated for the simulations. In Figure S 18, simulations with the same parameters as in Figure 67 are shown, but neglecting T1 by skipping the term –MZ(t)/T1. This results in a pronounced initial nonlinear dynamics for both pyridine and acetonitrile. Due to the higher pumping rate 1/τP and higher M0 far above the RASER threshold, pyridine shows longer relaxation oscillations than acetonitrile.

1 Figure S 18. Simulated α (blue) and PZ (red) for a H RASER experiment with a) pyridine and b) acetonitrile. The simulation parameter are equivalent to Figure 67 except for the impact

of T1 relaxation by the term –MZ(t)/T1, which is neglected.

In Figure 67 of the main text, a short crusher gradient (τcrusher < 1 s) is applied to reset α and demonstrate the initial nonlinear dynamics. A longer crusher pulse leads to a continuous decoherence of the MT magnetization and therefore inhibits the build-up of α.

Thus, the built-up, negative PZ polarization by the SABRE pumping is not diminished by α and keeps growing to a higher value. If the crusher gradient is now turned off, the high PZ leads to a large α build-up and with it a pronounced initial nonlinear dynamics. This can be shown by simulations, as it is included in the prediction of the mathematical model introduced in subsection 7.3.2 in form of equations (21) and (22). In Figure S 19 simulations for a) pyridine and b) acetonitrile with a starting value of α = 0 are shown. Just after 30 s, a α of 10-6 is given, which then starts to grow. This is analogous to a crusher gradient over these 30 s. This rising α now shows a very strong backaction onto PZ and a pronounced initial nonlinear dynamics is visible. 148 Supplement: The parahydrogen fueled NMR RASER

Figure S 19. Simulated α and PZ for a crusher pulse length of 30 s for a) pyridine and b) acetonitrile. The other simulation parameter are equivalent to Figure 67. The simulations are line with the experimental results. As an example, five different crusher pulse lengths are applied to a 1H acetonitrile RASER (see Figure S 20). Starting from a crusher pulse length of 30 s (τ30) until a very short crusher pulse (τ<1). The latter resulted in the measurement shown in Figure 67b from the main text.

Figure S 20. Different crusher pulse length in a 1H acetonitrile RASER experiment. The last part (from 480 s to 560 s) is shown in Figure 67b of the main text. In Figure S 21 the (starting) RASER of SABRE pumped a) 13C- and b) 15N-acetonitrile is shown. The varied coupling network by the additional couplings to 13C or 15N decreases the polarization build-up on the protons as it splits up the respective energy levels.

Figure S 21. Starting 1H RASER of SABRE pumped 13C-acetonitrile and 15N-acetonitrile at 41.7 kHz: The 13 sample contained a) 1.76 mg IMes, 7.5 µl C-acetonitrile, 3 µl pyridine-d5 in 900 µl 15 methanol-d4 and b) 1.17 mg IMes, 5 µl N-acetonitrile and 0.4 µl pyridine-d5 in 600 µl

methanol-d4. It was detected with a) 80 ml/min at 3.0 bar and b) 20 ml/min at

2.8 bar p-H2. 𝑉𝑉̇ = 𝑉𝑉̇ = References 149

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160 Eidesstattliche Erklärung

Eidesstattliche Erklärung

Sören Lehmkuhl erklärt hiermit, dass diese Dissertation und die darin dargelegten Inhalte die eigenen sind und selbstständig, als Ergebnis der eigenen originären Forschung, generiert wurden. Hiermit erkläre ich an Eides statt 1. Diese Arbeit wurde vollständig oder größtenteils in der Phase als Doktorand dieser Fakultät und Universität angefertigt; 2. Sofern irgendein Bestandteil dieser Dissertation zuvor für einen akademischen Abschluss oder eine andere Qualifikation an dieser oder einer anderen Institution verwendet wurde, wurde dies klar angezeigt; 3. Wenn immer andere eigene oder Veröffentlichungen Dritter herangezogen wurden, wurden diese klar benannt; 4. Wenn aus anderen eigenen- oder Veröffentlichungen Dritter zitiert wurde, wurde stets die Quelle hierfür angegeben. Diese Dissertation ist vollständig meine eigene Arbeit, mit der Ausnahme solcher Zitate; 5. Alle wesentlichen Quellen von Unterstützung wurden benannt; 6. Wenn immer ein Teil dieser Dissertation auf der Zusammenarbeit mit anderen basiert, wurde von mir klar gekennzeichnet, was von anderen und was von mir selbst erarbeitet wurde; 7. Ein Teil oder Teile dieser Arbeit wurden zuvor veröffentlicht und zwar in:

Suefke, M., Lehmkuhl, S., Liebisch, A., Blümich, B., Appelt, S., 2017. Para-hydrogen raser delivers sub-millihertz resolution in nuclear magnetic resonance. Nature Physics 13, 568-572.

Lehmkuhl, S., Emondts, M., Schubert, L., Spannring, P., Klankermayer, J., Blümich, B., Schleker, P.P.M., 2017. Hyperpolarizing Water with Parahydrogen. Chemphyschem 18, 2426-2429.

Lehmkuhl, S., Wiese, M., Schubert, L., Held, M., Küppers, M., Wessling, M., Blümich, B., 2018. Continuous hyperpolarization with parahydrogen in a membrane reactor. J Magn Reson 291, 8-13.

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