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Faculty of Physical SciencesUniversity of Iceland

2016

Faculty of Physical SciencesUniversity of Iceland

2016

Syntheses of Biradicals for SurfaceStudies by DNP ssNMR

Snædís Björgvinsdóttir

SYNTHESES OF BIRADICALS FOR SURFACE STUDIESBY DNP SSNMR

Snædís Björgvinsdóttir

90 ECTS thesis submitted in partial fulfillment of aMagister Scientiarum degree in Chemistry

AdvisorSnorri Þór Sigurðsson

Faculty RepresentativeGuðmundur G. Haraldsson

Faculty of Physical SciencesSchool of Engineering and Natural Sciences

University of IcelandReykjavik, January 2016

Syntheses of Biradicals for Surface Studies by DNP ssNMR90 ECTS thesis submitted in partial fulfillment of a M.Sc. degree in Chemistry

Copyright c© 2016 Snædís BjörgvinsdóttirAll rights reserved

Faculty of Physical SciencesSchool of Engineering and Natural SciencesUniversity of IcelandVR-II, Hjarðarhagi 2-6107, ReykjavikIceland

Telephone: 525 4000

Bibliographic information:Snædís Björgvinsdóttir, 2016, Syntheses of Biradicals for Surface Studies by DNP ssNMR,M.Sc. thesis, Faculty of Physical Sciences, University of Iceland.

Printing: Háskólaprent, Fálkagata 2, 107 ReykjavíkReykjavik, Iceland, January 2016

AbstractDynamic nuclear polarization (DNP) is a method that can be used to increase signal intensi-ties in nuclear magnetic resonance (NMR) spectroscopy. This is usually attained by dopingthe sample of interest with a paramagnetic polarizing agent, such as a stable organic radical.The high electron polarization of the radical is then transferred to the nuclei of the sampleby irradiating electron-nuclear transitions, resulting in increased NMR signal intensities. Inthis work, two different types of biradical nitroxide spin labels for solid-state DNP NMR ex-periments are introduced. A novel sulfhydryl-specific spin label for proteins was developed,synthesized and covalently attached to proteins. Four biradicals for immobilization on sur-faces of materials have also been prepared. These radicals have different functional groupsand the various methods used for coupling them to different surfaces are described. Finally,radicals that might be useful for studying surfaces of nanoparticles are reported. Evaluationof the DNP properties of all radicals is in progress.

ÚtdrátturMögnun á kjarnskautun (dynamic nuclear polarization, DNP) er tækni sem hægt er að notatil að magna upp merki í kjarnsegulgreiningu (nuclear magnetic resonance, NMR). Láganæmni NMR má rekja til lágrar skautunar segulvirkra kjarna, en næmnina er hægt að aukameð því að nýta háa skautun stakra (óparaðra) rafeinda. Þetta er oft gert með því að bætastöðugri lífrænni stakeind í sýnið sem á að rannsaka og geisla það með örbylgjum. Þannig erhá skautun stakeindarinnar færð yfir á kjarnann sem verið er að mæla, sem leiðir til sterkarimerkja í NMR. Í þessari ritgerð eru tvær mismunandi gerðir af tvístakeindum fyrir DNP íföstum efnum kynntar. Hönnun og smíði á cysteine-sértæku spunamerki fyrir prótein erugerð skil, sem og festingu þess á tvö mismunandi prótein. Fjórar mismunandi tvístakeindirsem hægt er að tjóðra við yfirborð efna voru smíðaðar. Þessar stakeindir hafa mismunandivirknihópa og eru festar á yfirborð með mismunandi aðferðum. Tvístakeind sem gæti nýstvið rannsóknir á yfirborðum nanóagna var líka smíðuð. Greiningar á eiginleikum tvístakein-danna til mögnunar á kjarnskautun standa ný yfir.

Contents

List of Figures vii

List of Schemes vii

Abbreviations xi

Acknowledgements xiii

1 Introduction 11.1 Dynamic Nuclear Polarization . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1.1 Polarization Transfer Mechanisms . . . . . . . . . . . . . . . . . . 21.1.2 Biradical Polarizing Agents for CE DNP . . . . . . . . . . . . . . 4

1.2 Conjugation of Radicals to Surfaces . . . . . . . . . . . . . . . . . . . . . 51.2.1 Site-directed Spin Labeling of Proteins . . . . . . . . . . . . . . . 51.2.2 Spin Labeling of Mesoporous Silica Materials . . . . . . . . . . . . 6

1.3 Objective of Thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2 Biradicals for Protein Labeling 92.1 Synthesis of Biradical Spin Label 5 . . . . . . . . . . . . . . . . . . . . . . 102.2 Deuterated TOTAPOL Spin Label . . . . . . . . . . . . . . . . . . . . . . 112.3 Spin Labeling of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.3.1 Spin Labeling of Papain . . . . . . . . . . . . . . . . . . . . . . . 122.3.2 Spin Labeling of αB-Crystallin . . . . . . . . . . . . . . . . . . . 14

2.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3 Biradicals for Immobilization on Surfaces of Materials 193.1 bTUreaG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.1.1 Immobilization of bTUreaG on Mesoporous Silica . . . . . . . . . 203.1.2 Determination of Radical Content on Silica Surface . . . . . . . . . 213.1.3 Preliminary DNP Results . . . . . . . . . . . . . . . . . . . . . . . 23

3.2 bTUrea with Amine Linker . . . . . . . . . . . . . . . . . . . . . . . . . . 233.3 bTUrea for Click Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . 25

v

3.4 Incorporation into Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . 26

4 Conclusion and Future Outlook 29

5 Experimental 31

Bibliography 69

vi

List of Figures

1.1 Monoradical polarizing agents . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Energy level diagram of the solid effect . . . . . . . . . . . . . . . . . . . 31.3 Energy level diagram of the cross effect . . . . . . . . . . . . . . . . . . . 41.4 Biradicals BTnE and bTbk . . . . . . . . . . . . . . . . . . . . . . . . . . 51.5 Radical functionalized mesostructured silica material . . . . . . . . . . . . 7

2.1 Biradical polarizing agent TOTAPOL. . . . . . . . . . . . . . . . . . . . . 92.2 EPR spectra of biradical spin label 5 and MTSSL . . . . . . . . . . . . . . 112.3 A ribbon and stick representations of papain . . . . . . . . . . . . . . . . . 122.4 EPR spectra of spin labeled papain . . . . . . . . . . . . . . . . . . . . . . 132.5 Molecular models of spin labeled papain . . . . . . . . . . . . . . . . . . . 142.6 EPR spectra of spin labeled αB-crystallin . . . . . . . . . . . . . . . . . . 152.7 ssNMR spectra of αB-crystallin . . . . . . . . . . . . . . . . . . . . . . . 152.8 DNP NMR spectra of αB-crystallin . . . . . . . . . . . . . . . . . . . . . 162.9 Biradicals for site-specific attachment to proteins. . . . . . . . . . . . . . . 17

3.1 Biradical polarizing agent bTUrea. . . . . . . . . . . . . . . . . . . . . . . 193.2 EPR spectra of free and immobilized bTUreaG . . . . . . . . . . . . . . . 223.3 DNP NMR spectra of radical functionalized material . . . . . . . . . . . . 233.4 Structures of HDA and dppb . . . . . . . . . . . . . . . . . . . . . . . . . 263.5 bTUrea derivatives for incorporation into nanoparticles . . . . . . . . . . . 26

4.1 bTUrea derivatives with ethyl groups . . . . . . . . . . . . . . . . . . . . . 29

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List of Schemes

1.1 Incorporation of MTSSL into protein . . . . . . . . . . . . . . . . . . . . . 6

2.1 Incorporation of biradical spin label 5 into a protein. . . . . . . . . . . . . 102.2 Synthesis of biradical spin label 5 . . . . . . . . . . . . . . . . . . . . . . 102.3 Synthesis of a deuterated biradical spin label . . . . . . . . . . . . . . . . . 112.4 Reaction of Ellman’s reagent with thiol . . . . . . . . . . . . . . . . . . . 13

3.1 Total synthesis of bTUreaG. . . . . . . . . . . . . . . . . . . . . . . . . . 203.2 Synthesis of amine functionalized SBA-15 material. . . . . . . . . . . . . . 213.3 Immobilization of bTUreaG on mesoporous silica . . . . . . . . . . . . . . 213.4 Potential immobilization of spin label 19 on mesoporous silica . . . . . . . 243.5 Synthesis of an azidoamine. . . . . . . . . . . . . . . . . . . . . . . . . . 243.6 Synthesis of a bTUrea derivative with amine linker. . . . . . . . . . . . . . 243.7 Immobilization of compound 21 on mesoporous silica . . . . . . . . . . . . 253.8 Synthesis of an alkyne derivative of bTUrea. . . . . . . . . . . . . . . . . . 253.9 Immobilization of spin label 18 on mesoporous silica . . . . . . . . . . . . 263.10 Synthesis of a bTUrea phosphine derivative. . . . . . . . . . . . . . . . . . 273.11 Synthesis of a bTUrea derivative with a long amine linker. . . . . . . . . . 27

ix

Abbreviations

APTES (3-aminopropyl)triethoxysilaneBDPA 1,3-bisdiphenylene-2-phenylallylbTbk bis-TEMPO-bisketalBTnE bis-TEMPO-n-ethylene oxidebTUrea bis-TEMPO-ureaCE cross effectCES carboxyethylsilanetriolCP cross polarizationCW continuous waveDABCO 1,4-diazabicyclo[2.2.2]octaneDMF dimethylformamideDNP dynamic nuclear polarizationdppb 1,4-bis(diphenylphosphino)butaneEPR electron paramagnetic resonanceHDA hexadecylamineMAS magic angle spinningMTS (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonateMS mass spectrometryNMR nuclear magnetic resonancePRE paramagnetic relaxation enhancementSBA Santa Barbara amorphousSDSL site-directed spin labelingSE solid effectTCE 1,1,2,2-tetrachloroethaneTLC thin layer chromatographyTEOS tetraethyl orthosilicateTEMPO 2,2,6,6-Tetramethylpiperidine 1-oxylTOTAPOL 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-olUV/VIS ultraviolet-visible

xi

Acknowledgements

First of all, I would like to thank my advisor Prof. Snorri Þór Sigurðsson for his guidanceand support during my studies. I also want to thank members of the Sigurdsson group forall their help, in particular Anil Jagtap who has been an endless source of useful informationin the lab. Thanks to collaborators Prof. Hartmut Oschkinat, Johanna Münkemer and othermembers of the Oschkinat group at the FMP in Berlin, as well as to Dr. Torsten Gutmannand the Buntkowsky group at the Technical University of Darmstadt. Finally, thanks to allthe people in the chemistry department who helped me out over the course of this project.

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1 IntroductionNuclear magnetic resonance (NMR) spectroscopy is a method that uses the magnetic prop-erties of atomic nuclei to provide information about their chemical environment. This an-alytical technique is extensively used to obtain information on atomic scale structure anddynamics of various chemical systems, both in liquid and solid state. Over the past fewdecades, magic angle spinning (MAS) NMR spectroscopy has become prevalent for stud-ies on solid samples, both in materials science1 and biological systems2 such as membraneproteins3 and amyloid fibrils.4, 5

NMR is disadvantaged by low sensitivity, due to relatively small spin population dif-ferences at thermal equilibrium. This limits the applicability of the method, especially whendetecting low-γ nuclei such as 13C and 15N. Therefore, high sample concentrations of NMRactive nuclei or long acquisition times are needed to provide adequate results.

Dynamic nuclear polarization (DNP) can increase the sensitivity of NMR by severalorders of magnitude by increasing spin population differences before recording the NMRspectra. The idea of DNP was introduced in the 1950s,6, 7 but technical limitations preventedit from becoming widely applicable until high frequency microwave sources and cryogenicMAS probes became available.8, 9 DNP is now routinely used for MAS NMR experimentsat high magnetic fields and low temperatures and the physics of polarization transfer in theseexperiments will be accounted for in the following section. DNP can also be utilized in bothliquid10 and dissolution11 NMR applications, but these will not be discussed in detail here.

1.1 Dynamic Nuclear Polarization

DNP increases the sensitivity of NMR when the high spin polarization of electrons is trans-ferred from paramagnetic centres to sample nuclei. This is usually attained by doping thesample of interest with a paramagnetic polarizing agent, such as a stable organic radical. Spinpolarization from the unpaired electron of the radical is then transferred to nearby protonsby irradiating electron-nuclear transitions with microwaves. Subsequently, spin diffusiondistributes the polarization to other protons of the sample. If other nuclei than 1H are to beinvestigated, cross-polarization (CP) can be used to transfer the polarization from protons tothose nuclei, for example 13C and 15N. Theoretically, DNP can increase NMR signal inten-sities by the ratio of electronic and nuclear Larmor frequencies, this corresponds to as much

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as a factor of ∼660 for 1H spins and ∼2600 for and 13C spins.12

There are several mechanisms possible for polarization transfer in DNP, namely theOverhauser effect, the solid effect, the cross effect and thermal mixing. Since current MASDNP experiments are typically based on the solid effect (SE) or, more commonly, the crosseffect (CE), only these mechanisms will be discussed further.13

1.1.1 Polarization Transfer Mechanisms

The dominant process for polarization transfer is determined by the electron paramagneticresonance (EPR) characteristics of the radical used as a polarizing agent, as well as by thenuclei of the sample molecule. The most important EPR characteristics are the relative mag-nitudes of the homogeneous (δ ) and inhomogeneous (∆) EPR linewidths, and the nuclearLarmor frequency (ω0I). The homogeneous linewidth, δ , represents the width of a sin-gle EPR transition but the inhomogeneous linewidth, ∆, stands for the width of the entireEPR spectrum. The Larmor frequency, ω0I , is the characteristic frequency of precession ofmagnetic moments and depends on the strength of the external magnetic field and nuclearisotope.13, 14

The solid effect. The SE is a process where one electron and one nucleus interact.It is dominant when the polarizing agent being used has homogeneous and inhomogeneouslinewidths that are both smaller than the nuclear Larmor frequency, ω0I > δ ,∆.14 This istypically the case for radicals with narrow EPR spectra, such as the monoradicals trityl andBDPA (Figure 1.1).

Figure 1.1. Monoradical polarizing agents. The trityl-type radical CT-03 and BDPA arecarbon-centred, while TEMPO is a nitroxide radical.

Figure 1.2a shows an energy level diagram of solid effect DNP. At thermal equilibriumquantum populations are controlled by the Boltzmann distribution and lower energy statesare more populated. Mixing of states leads to forbidden zero quantum or double quantumtransitions becoming partially allowed and when microwave irradiation is applied to these

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transitions, it leads to either positive or negative enhancement of nuclear polarization (Figure1.2b).13

(a) (b)

Figure 1.2. (a) Energy level diagram showing solid effect DNP. (b) 1H DNP enhance-ment of sulfonated BDPA plotted as a function of magnetic field. Figure taken from Niet al.13

The cross effect. The CE mechanism is a three spin process which uses two electronsand one nucleus and is dominant when the polarizing agent used has an inhomogeneouslinewidth that is larger and a homogeneous linewidth that is smaller than the nuclear Larmorfrequency (∆ > ω0I > δ ).14 The energy level diagram on Figure 1.3 describes DNP with theCE. Microwave irradiation is used to excite one electron which then leads to excitation ofthe dipole-dipole coupled electron (zero or double quantum transition). The third spin in thesystem, the nuclear spin is excited if the difference between the Larmor frequencies of thetwo electrons is equal to the nuclear Larmor frequency; |ω0S1−ω0S2|= ω0I . This means thatan ideal radical for CE has an EPR spectrum with two narrow lines separated by the nuclearLarmor frequency, however radicals with broad EPR linewidth, such as nitroxides (Figure1.1), also meet the requirements. CE becomes more efficient at high magnetic fields andis often the choice for high field experiments because the mechanism is based on allowedtransitions.13–16

Although monoradicals with broad EPR linewidth are able to satisfy the conditionsneeded for CE DNP, the efficiency of the mechanism can be improved with biradicals. Thisis because the process involves coupling of two electrons and when monoradicals are used,only part of the radicals will satisfy the frequency matching condition.17, 18 The next sectiondescribes in more detail the advantages of using biradicals as polarizing agents in CE DNP.

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Figure 1.3. Energy level diagram showing cross effect DNP. The EPR spectrum of anideal radical for CE is in the middle. Figure taken from Ni et al.13

1.1.2 Biradical Polarizing Agents for CE DNP

Biradicals were developed to improve the electron-electron interaction in CE polarizationtransfer, the idea being that control over dipole-dipole coupling and orientation of the rad-icals could lead to better signal enhancements. Biradical polarizing agents usually consistof two TEMPO moieties linked together and their efficiency is controlled by the distanceand orientation between the two radicals.17, 19, 20 It can also be noted that with increasedefficiency of electron-electron coupling, lower concentrations of spins are needed, whichreduces paramagnetic effects such as signal broadening.16, 17

The biradical BTnE (bis-TEMPO-n-ethylene oxide, Figure 1.4) was designed to in-vestigate the effect of dipolar coupling between electrons on DNP efficiency. The n in thename denotes the number of ethylene glycol units and results indicate that shorter linkers,with stronger dipolar coupling, give better enhancements.17 The rigid biradical bTbk (bis-TEMPO-bisketal, Figure 1.4) was designed to lock the relative orientation between the twonitroxide moieties so that optimal CE DNP enhancement could be obtained. bTbk has thetwo TEMPOs at a favourable 90◦ angle and gives larger enhancements than the flexibleBT2E, which has a similar electron-electron distance.18, 19 Furthermore, electron relaxationproperties have also been shown to affect DNP enhancements, with longer relaxation times(PyPol, Figure 1.4) providing larger enhancements.21

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Figure 1.4. Biradical polarizing agents BTnE (n=2-4), bTbk and PyPol.

In addition to factors related to the structure of the polarizing agent, the conditions atwhich the DNP NMR experiments are conducted, such as magnetic field and temperature,can affect the efficiency of polarization. Since these aspects are not directly related to radicaldesign, they will not be discussed here in detail.

DNP enhancement, ε , is used to evaluate the polarization efficiency of radicals. It isdefined as ε = Ion/Ioff, where Ion and Ioff are intensities of NMR signals recorded with andwithout microwave irradiation. This definition is, however, not an ideal way to evaluate theefficiency of polarizing agents, since the presence of radicals combined with sample spinningcan depolarize the sample nuclei. This depolarization is radical dependant and makes the Ioff

smaller than when no radical is present in the sample.22

1.2 Conjugation of Radicals to Surfaces

CE DNP experiments are conventionally performed using biradicals and homogeneouslyfrozen solutions (glass) to get an even distribution of polarizing agents at optimal concen-trations. In this thesis, the possibility of covalently attaching biradicals to macromoleculesand then using the resulting radical functionalized compounds for DNP NMR studies, is ex-plored. This section describes two different types of systems that can be investigated using acombination of spin labeling and DNP NMR. Section 1.2.1 describes site specific labeling ofproteins and Section 1.2.2 describes immobilization of radicals on the surface of mesoporoussilica materials.

1.2.1 Site-directed Spin Labeling of Proteins

Paramagnetic tags can be introduced at specific sites of proteins using site-directed spinlabeling (SDSL). Most commonly this involves cysteine residues in the protein of interestbeing modified with thiol specific nitroxide spin labels, such as MTSSL23 ((1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate spin label, Scheme 1.1). These cys-teine groups can either be native to the protein, or they can be introduced by mutation. Afterlabeling, the unpaired electrons of the nitroxide make the protein suitable for EPR spec-

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troscopy. This is useful because nitroxides, like other spin labels, are sensitive to their localenvironment and when attached to a macromolecule, their EPR lineshape gets affected bysteric restraints and shifting in the molecule surrounding the spin. This makes SDSL com-bined with EPR a useful method for investigating structure and dynamics of proteins.24, 25

Scheme 1.1. Incorporation of the nitroxide spin label MTTSL into a protein using athiol-specific reaction.

Paramagnetic tags have also been used to get information about proteins with NMRspectroscopy by utilizing paramagnetic relaxation enhancements (PRE). Unpaired electronsof the spin labels couple strongly to the surrounding nuclei through hyperfine interactions,which causes paramagnetic shifts or relaxation enhancements of signals. These signal changesdepend on the distance between the nuclei and unpaired electrons, making PRE values de-termined by experiments capable of deducing nuclear-electron distances of up to 2 nm.26

1.2.2 Spin Labeling of Mesoporous Silica Materials

Solid-state NMR is routinely used to investigate inorganic and hybrid materials, as it candirectly measure both bulk and surface functionalities.27 These experiments are, however,as was rationalized before, limited by low sensitivity. The concentration of NMR activenuclei is often so low that it takes hours or days to obtain spectra with decent signal-to-noiseratios. The sensitivity of surface signals in these kind of experiments, typically performed athigh field and low temperature, experiments can be improved significantly with DNP.1, 27, 28

The radical polarizing agents are, in the case of mesoporous materials, introduced to thevicinity of surfaces by impregnating the materials with enough radical solution to fill thepores without diluting the sample. After irradiating with microwaves to polarize protons,cross-polarization can be used to selectively polarize nuclei of 13C, 15N or 29Si, either on thesurface or on molecules immobilized on the surface.28

DNP surface-enhanced NMR spectroscopy is a useful method for characterizing ma-terials and as with experiments on biological samples, the properties of the polarizing agentand the solvent system are important factors. As an example, both mono- and biradicalsin aqueous solutions have been used successfully for surface studies of mesoporous silicafunctionalized with phenol,27 but for systems that are not compatible with water, bTbk andorganic solvents have been used.28

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Mesoporous silica materials with radicals covalently attached have been reported pre-viously29, 30 and in 2013, Gajan et al. showed that hybrid silica materials functionalizedwith radicals (Figure 1.5) could be used as polarization matrices in solid-state DNP NMRexperiments. These matrices have organic radicals covalently bound to the surface and areadvantageous because they eliminate the need for glass formers and the insoluble polariza-tion matrices can be separated from the analyte by filtration. Additionally, since the matrix isinsoluble, solubility of the radical immobilized on it is irrelevant and radicals that are poorlysoluble in water can be used to polarize aqueous solutions.31

Figure 1.5. Mesostructured hybrid silica material with radical polarizing agents cova-lently bound to the surface. Figure taken from Gajan et al.31

1.3 Objective of Thesis

This thesis describes the design and synthesis of biradical spin labels that can be conjugatedto different types of surfaces. As discussed before, the structure of the radical is importantfor efficient polarization transfer and in this work, known biradical polarizing agents weremodified so that they can be covalently and selectively attached to proteins and surfaces.

The synthesis and application of a radical that can be covalently attached to a proteinis described in Chapter 2 and Chapter 3 accounts for the design and synthesis of biradicalsthat can be coupled to various materials with different surface functionalities.

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2 Biradicals for Protein LabelingSite-directed spin labeling with monoradicals is routinely used for structural investigationsof proteins, making use of both EPR spectroscopy and PRE effects in NMR, as was coveredin Section 1.2.1. By using biradicals as spin labels it is possible that, in addition to theaforementioned methods, DNP NMR can be used to gain even more information on thestructure and dynamics of proteins than when monoradical labels are used. This refers inparticular to the possibility of generating localized signal enhancements in ssNMR.

TOTAPOL (1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol, Figure 2.1) is an ex-ample of a biradical commonly used as a polarizing agent in MAS NMR experiments. Itconsists of two TEMPO radicals linked with a tether containing both secondary amine andhydroxyl groups, which function to make the biradical water-soluble. Solubility of TO-TAPOL in aqueous solutions is important because DNP NMR experiments on biologicalsamples are typically carried out on frozen samples of the analyte and polarizing agent in aglass forming mixture of glycerol, D2O and H2O. Furthermore, the short tether constrainsthe two unpaired electrons at a favourable distance which, along with the flexibility providingappropriate orientations of the nitroxides, results in very good signal enhancements.32

Figure 2.1. Biradical polarizing agent TOTAPOL.

This applicability of TOTAPOL to biological conditions is the reason it was chosen tobe derived into a protein spin label. Since paramagnetic tags are commonly attached to pro-teins with SDSL using sulfhydryl specific reactions, it seemed logical to modify TOTAPOLwith a methanethiosulfonate group. The target molecule would then react with proteins in asite-specific manner, as shown in Scheme 2.1. Synthesis of spin label 5 will be described inSection 2.1 and synthesis of a partially deuterated version of it is accounted for in Section2.2. SDSL of proteins with the biradical spin label and NMR experiments on labeled proteinare the subject of Section 2.3.

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Scheme 2.1. Incorporation of biradical spin label 5 into a protein.

2.1 Synthesis of Biradical Spin Label 5

The three step synthesis of a TOTAPOL derivative suitable for site directed spin labelingis shown in Scheme 2.2. First, 4-(N-methylamino)-TEMPO (1) and 4-(2,3-epoxypropoxy)-TEMPO (2) were coupled together to make TOTAPOL derivative 3. Compound 3 has amethyl group on the nitrogen atom in the linker between the two TEMPO moieties, whichwas considered necessary to avoid intramolecular reactions after the hydroxyl group has beenconverted into a good leaving group. Compound 3 was reacted with methanesulfonyl chlo-ride, resulting in compound 4, which has chlorine instead of a hydroxyl group. This reactionwas actually expected to yield a mesylate, but since chlorine is also a good leaving group, theunanticipated result did not cause much trouble. In the final step of the synthesis, compound4 was reacted with sodium methanethiosulfonate, resulting in the target compound 5. Allcompounds were characterized with a combination of NMR, EPR and mass spectroscopy(MS).

Scheme 2.2. Synthesis of biradical spin label 5.

The EPR spectrum of biradical spin label 5 (Figure 2.2a) looks very similar to thespectrum of TOTAPOL.32 It shows five lines because of strong exchange interaction betweenthe two unpaired electrons in addition to hyperfine couplings.17 In comparison, the spectraof monoradical MTSSL (Figure 2.2b) shows only three lines, corresponding to hyperfine

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coupling between the electron and the nitrogen atom.

(a) Biradical spin label 5 (b) MTSSL

Figure 2.2. EPR spectra of biradical spin label 5 (left) and MTSSL (right). The spectrawere recorded at 25 ◦C.

2.2 Deuterated TOTAPOL Spin Label

As was briefly mentioned in Section 1.1.2, longer electron relaxation times can increasethe polarization efficiency of radicals, because polarization transfer depends on saturationof an EPR transition. By replacing the protons of the methyl groups at the Cα carbons withdeuterons, the relaxation time of the radical is increased.21 Results from Hartmut Oschkinat’sgroup at FMP in Berlin (unpublished) showed that deuteration at specific sites of TOTAPOLincreases enhancement of proline NMR signals around 75% compared to non-deuterated TO-TAPOL under the same experimental conditions.33 Since synthesis of a deuterated analogueof biradical spin label 5 seemed relatively straight-forward (Scheme 2.3), it was conducted,as its properties might prove useful in later DNP NMR experiments.

Scheme 2.3. Synthesis of biradical 10, a deuterated analogue of spin label 5.

The synthetic route of compound 10 was nearly identical to the one used to make5. The compounds are structurally the same except 10 has deuterons instead of protons on

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the methyl groups α to the nitroxide moiety. Deuterated analogues of 4-(N-methylamino)-TEMPO (6) and 4-(2,3-epoxypropoxy)-TEMPO (7), were synthesized from deuterated oxo-TEMPO, which was made according to references.33 Compounds 6 and 7 were coupledtogether to make biradical compound 8. The hydroxyl group of radical 8 was then convertedto methanethiosulfonate in a two step process to yield 10. All compounds were confirmedwith MS.

2.3 Spin Labeling of Proteins

2.3.1 Spin Labeling of Papain

A protein labeling reaction was carried out in order to confirm that the biradical spin label5 could be used for site-specific attachment to cysteine residues. Papain (Figure 2.3), anenzyme made up of 212 amino acid residues, was chosen as a model compound because itcontains only one free sulfhydryl group, cys25. The enzyme actually contains 7 cysteines,but 6 of them form disulfide bridges and are therefore not available for spin labeling.34

Figure 2.3. A ribbon and stick representations of papain. The free sulfhydryl group islocated at the active site of the enzyme.

As a control sample, papain was reacted with MTSSL (Scheme 1.1) following a knownreaction protocol.35 A solution of the radical in DMF was added to the activated protein ina buffer solution and the mixture stirred at room temperature overnight. EPR spectroscopywas used to confirm successful binding of MTSSL to papain (Figure 2.4), as the spectrumobtained resembles the spectra in references.23, 35 The same reaction procedure was then

12

used to attach biradical spin label 5 to papain. The resulting EPR spectrum (Figure 2.4) isbroader than the spectra of the corresponding free radical, and indicates successful bindingto the protein. The broadening of the spectra arise from reduced averaging of the EPRspectrum due to restricted mobility and rotation of the biradical spin label. In contrast,smaller linewidths are seen when mobility of the nitroxide is not restricted, as is the case forfree spin labels.

Figure 2.4. EPR spectra of papain labeled with MTSSL (black) and papain labeled withbiradical 5 (red). The spectra were recorded at 25 ◦C.

To determine the efficiency of the spin labeling reaction, the concentration of freesulfhydryl groups present on the protein after labeling was determined using Ellman’s reagent(DTNB).36 The reagent reacts with thiols, as shown in Scheme 2.4, and releases TNB whichhas a yellow color in alkaline conditions. The reaction happens fast and allows for the quan-tification of thiols using a UV/VIS spectrometer. Ellman’s reagent was reacted with papain,labeled with either MTSSL or biradical spin label 5 and the concentration of free sulfhydrylgroups calculated. Despite of inconsistency in absolute concentration values, the results in-dicated considerably more binding (10-20% more) in the case of MTSSL than biradical spinlabel 5. This accounts, at least in part, for the high signal to noise ratio in the EPR spectrumof papain labeled with radical 5.

Scheme 2.4. Reaction of Ellman’s reagent (DTNB) with thiol.

Molecular modelling was used to give insight into whether the lower extent of spinlabeling with 5 might be caused by steric reasons, since the free sulfhydryl group of papainis located at the active site of the enzyme and the TOTAPOL spin label is considerably biggerthan MTSSL. Figure 2.5 shows that MTSSL fits in the groove of the protein with room tospare, but the TOTAPOL label looks as if it does not fit comfortably at this specific site. This

13

might be one of the reasons why the TOTAPOL spin label is less reactive towards papainthan MTSSL.

Figure 2.5. Molecular models of spin labeled papain. MTSSL (left) and biradical spinlabel 5 (right) attached to cys25 at the active site of the enzyme.

2.3.2 Spin Labeling of αB-Crystallin

αB-crystallin is a heat shock protein that is highly dynamic and naturally exists in oligomersof 12-50 subunits. Both the C- and N-termini of the protein are flexible and contributeto the inherent heterogeneity of the oligomers.37 Since the dynamics of the termini arenot yet understood, it was considered possible that SDSL might be able to provide somenew information about their movement, more specifically if they are binding to the samehydrophobic groove. By utilizing a biradical for the labeling, DNP NMR experiments couldbe used, in addition to localized PRE effects, to gather information on the dynamics of theprotein.

In collaboration with Prof. Hartmut Oschkinat and his research group at the FMPin Berlin, αB-crystallin was labeled with biradical spin label 5. Since αB-crystallin doesnot have any cysteine residues in its native form, two mutants of the protein were prepared,P160C and A4C. The P160C mutant has residue 160, on the C-terminus, as a cysteine insteadof proline and the A4C has residue 4, on the N-terminus, as a cysteine instead of alanine.In both cases the replaced amino acid was located between two isoleucine residues. Thespin labeling reaction was carried out in a similar way as described in the previous section.First, reducing agent dithiothreitol (DDT), which reduces disulfide bonds, was removed fromsolution with a desalting spin column and then one equivalent of the spin label in a smallamount of DMF was added. The protein and biradical were mixed at room temperature for30 min and the labeled protein then eluted through a size-exclusion column again to removeany unreacted spin label.

14

Continuous wave X-band EPR measurements, carried out by Dr. Enrica Bourdignonat the Free University in Berlin, were used to confirm that the spin label had attached tothe protein to some extent. Figure 3.2 shows the spectra of biradical labeled αB-crystallinmutant P160C (a) and as a comparison αB-crystallin labeled with the well known mono-radical MTSSL (b). Both spectra are broader than spectra of the corresponding free radical,indicating binding.

(a) αB-crystallin labeled with MTSSL (b) αB-crystallin labeled with 5

Figure 2.6. EPR spectra of αB-crystallin mutant P160C labeled with MTSSL (left) andbiradical spin label 5 (right). The spectra were recorded at 25◦C.

The PRE effects were investigated using both solution and solid state NMR. Spectrawere recorded for the biradical labeled P160C mutant. Liquid state spectra provided infor-mation on flexible parts of the protein and solid state spectra on the rigid parts. Significantrelaxation enhancements were observed in both cases. Carbon-carbon correlated solid-statespectra of αB-crystallin (Figure 2.7) show the difference between the signals acquired forunlabeled protein and the signals for monoradical and biradical labeled protein.

Figure 2.7. C-C correlated ssNMR spectra of αB-crystallin recorded at 700 MHz. Blueis unlabeled protein, red is MTSSL labeled and green is labeled with biradical 5.

15

These kind of spectra show direct couplings between carbon atoms and are routinelyused to obtain structural information about biomolecules. When the monoradical and biradi-cal effects are compared, more signals have vanished in the case of biradical which illustratesthat it has stronger PRE effects than the monoradical spin label. PRE effects are currentlybeing evaluated by Johanna Münkemer at the FMP in Berlin.

Preliminary DNP experiments were performed on the P160C sample of αB-crystallinlabeled with biradical 5. Two spectra were recorded on a 400 MHz DNP NMR spectrometer,at 100 K and 200 K (Figure 2.8). At 100 K, a 1.2 fold enhancement was obtained, but at200 K the enhancement was tenfold. A good isotope labeling scheme is needed to limit spindiffusion and get decent, hopefully localized, enhancements. Lower concentration of spinlabel is also needed to limit line broadening due to PRE.

(a)

(b)

Figure 2.8. DNP NMR spectra of αB-crystallin. (a) Spectra recorded at 100 K withmicrowaves (red) and without microwaves (blue), ε=1.2. (b) Spectra recorded at 200 Kwith microwaves (blue) and without microwaves (red), ε ∼10.

16

2.4 Summary

The possibility of attaching a biradical to a biomolecule had not been reported when thesynthesis of spin label 5 was carried out. In 2015, two different biradical spin labels (Figure2.9) were introduced,38, 39 both derived from biradicals commonly used for polarization ofbiological samples. AMUPol-MTSSL is, as the name suggests, derived from AMUPol40

and ToSMTSL is a derivative of TOTAPOL.32 These biradicals were used for SDSL ofbiomolecules and shown to be able to provide DNP enhancements in addition to localizedPRE effects, thus broadening the scope of SDSL of proteins. They did, however, not generatelocalized DNP enhancements.

Figure 2.9. Biradicals for site-specific attachment to proteins.

17

3 Biradicals for Immobilization onSurfaces of Materials

bTUrea18 (Figure 3.1) is a biradical that consists of two TEMPO moieties connected with aurea linker. The short urea tether leads to strong dipolar couplings between the two unpairedelectrons and in frozen solutions, the relative orientation between the two TEMPOs is con-strained at an almost perpendicular angle. These qualities are very attractive in biradicals,but since bTUrea is poorly soluble in aqueous media, it has not been routinely used in DNPNMR experiments.18

Figure 3.1. Biradical polarizing agent bTUrea.

This section describes the synthesis of several bTUrea derivatives and their applicabil-ity to surface studies using DNP. Section 3.1 describes the synthesis of a previously knownbiradical, bTUreaG, and how it can be used to functionalize mesoporous silica materials.Sections 3.2 and 3.3 show the syntheses of new bTUrea derivatives that can be covalentlyattached to materials using different types of coupling chemistry. Finally, Section 3.4 intro-duces a radical that might be useful for surface studies of nanoparticles.

3.1 bTUreaG

Synthesis of bTUreaG, a derivative of bTUrea was conducted in a way very similar to thereported procedure,41 the only difference being the protecting group for the carboxylic acid.Scheme 3.1 shows the total synthesis of bTUreaG. First, 6-aminohexanoic acid was treatedwith SOCl2 in MeOH to form methyl ester 11, which protects the carboxylic acid moiety insubsequent reaction steps. This methyl ester marks the difference from the reported protocol,where the carboxylic acid was protected with an ethyl ester. Compound 11 was then used forreductive amination of oxo-TEMPO, yielding 4-amino-TEMPO derivative 12. Compound12 and 4-amino-TEMPO were reacted with triphosgene in the presence of Et3N to give

19

bTUrea derivative 13, which was then hydrolysed to the target compound, bTUreaG. Thecompounds were characterized with a combination of NMR and EPR spectroscopy and massspectrometry.

Scheme 3.1. Total synthesis of bTUreaG.

bTUreaG can be immobilized on the surface of a mesoporous silica material function-alized with amine groups and the resulting material can then be used to polarize samples inDNP NMR experiments, as was mentioned in Section 1.2.2. In the following Sections (3.1.1and 3.1.2), preparation and characterization of radical functionalized materials is described.Preliminary DNP experiments have also been conducted on the materials, and those resultsare presented in Section 3.1.3. This work was carried out in collaboration with Prof. GerdBuntkowsky and his group at the Technical University of Darmstadt.

3.1.1 Immobilization of bTUreaG on Mesoporous Silica

Three different types of mesoporous silica materials were obtained from Prof. Gerd Bun-tkowsky’s group. These were amine-functionalized and carboxylate-functionalized materi-als, as well as material containing both amine and carboxylic acid functionalities. The ma-terials were made by co-condensating tetraethyl orthosilicate (TEOS) with (3-aminopropyl)-triethoxysilane (APTES) and/or carboxyethylsilanetriol sodium salt (CES), as is shown inScheme 3.2. The resulting mesoporous materials, that were prepared in the presence of astructure-directing agent, have hexagonal structures and well defined pore sizes of up to 30nm.42 These kind of materials are called SBA-15, where SBA stands for Santa Barbaraamorphous. Co-condensation was considered appropriate because the resulting productshave a better distribution of functional groups compared to when organic groups are post-grafted onto unfunctionalized materials.31 The synthesis of material SBA-15-NH2 is shownin Scheme 3.2. The APTES:TEOS ratio used was 1:9, which corresponds to approximately

20

1.4 mmol of amine per gram of the resulting material. Similarly, when the carboxylic func-tionalized material (SBA-15-COOH) was made, the CES:TEOS ratio was also 1:9 and whenSBA-15-NH2-COOH was synthesized, the APTES:CES:TEOS ratio was 1:1:8.

Scheme 3.2. Synthesis of amine functionalized SBA-15 material from APTES andTEOS. The reaction was performed by Jiquan Liu in Darmstadt.

The amine-functionalized SBA-15-NH2 was used to make a series of samples withdifferent radical content on the surface, by coupling different amounts of radicals to thesilica. Six portions of ca. 200 mg SBA-15-NH2 were reacted with bTUreaG, in initial ratiosvarying from 1:7 - 1:200 (radical:amine). The amine on the silica surface and the acid linkerof the spin label form an amide bond, immobilizing the biradical on the mesoporous silica(Scheme 3.3).

Scheme 3.3. Immobilization of bTUreaG on the surface of a functionalized hybridsilica material.

Even though the resulting radical functionalized material is the same as had been re-ported previously by Gajan et al.,31 the approach taken to make it is slightly different. Here,a constant concentration of amines was reacted with different amounts of radical, in contrastto synthesizing mesoporous silica materials with different concentrations of amines on thesurface, and then reacting it with 1 equivalent of radical.

3.1.2 Determination of Radical Content on Silica Surface

The amount of radical attached to each sample of the mesoporous material was determinedby continuous wave (CW) EPR spectroscopy. First, bTUreaG was dissolved in tetrachloro-ethane (TCE) and a series of dilutions made for the concentration range 0.1-3.0 mM. EPR

21

spectra of the samples were then recorded (Figure 3.2) and doubly integrated to create acalibration curve for spin counting. The EPR spectrum of radical bTUreaG is similar to thespectrum of bTUrea18 but the spectra of the immobilized radicals have significantly broaderlines, due to restricted motion.

(a) bTUreaG (b) bTUreaG immobilized on SBA-15

Figure 3.2. EPR spectra of free and immobilized bTUreaG, recorded at 25◦C.

EPR spectrum of each silica sample was recorded in TCE and integrated so that the rad-ical content of the samples could be determined from the aforementioned calibration curve.The sample preparation used is not ideal because the silica materials are insoluble and donot form homogeneous solutions, which are usually used in spin counting experiments. Theresults do however show the radical concentration dropping proportionally with the radi-cal/amine ratio before reaction (Table 3.1). The most dilute samples had the spectra with thesmallest amplitudes, and that might mean the coverages calculated are not accurate enough.More accurate results might be acquired if the silica samples are prepared by impregnatingthe materials with enough TCE to fill the pores without diluting the sample, but the experi-mental procedure is not straight-forward.

Sample Ratio before reaction Calculated coverage

SB-II-120 1:7 6 %SB-II-115 1:10 3 %SB-II-116 1:20 2 %SB-II-117 1:50 1 %SB-II-118 1:100 <1 %SB-II-119 1:200 <1 %

Table 3.1. Results of spin counting experiments. The ratio before reaction shows theamount of radical that was stirred with the silica sample. The calculated coverage isdetermined from EPR experiments and gives the radical content of the sample after re-action.

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3.1.3 Preliminary DNP Results

DNP NMR experiments on the radical functionalized materials were performed by Dr. TorstenGutmann at the Technical University of Darmstadt. The sample which had the lowest amountof radical reacted to it, 1:200 (SB-II-119, Table3.1), showed the biggest enhancements andthose results are presented here. For these reasons, an additional silica sample with evenlower radical concentration on the surface was prepared, but it has not yet been evaluated.

1H, 13C and 29Si spectra were recorded of the material, impregnated with H2O:D2O90:10, with and without microwave irradiation. Figure 3.3 shows 13C and 29Si NMR ofthe radical functionalized material, where CP was used to transfer polarization from protonsto the nuclei being detected. The enhancement factors obtained are ε=3.12 and ε=2.89,respectively.

(a) 29Si DNP ssNMR (b) 13C DNP ssNMR

Figure 3.3. DNP NMR spectra of radical functionalized material. Cross polarizationwas used to transfer polarization from 1H to 29Si and 13C nuclei. Black is without mi-crowaves, red is with microwaves.

3.2 bTUrea with Amine Linker

The previous section (3.1) described the already reported spin label bTUreaG31 and howit can be immobilized on the surface of an amine functionalized hybrid silica material. Forsome applications it may be advantageous to use silica covered with carboxylates. By havinga carboxylic functionality on the silica surface and an amine linker on the biradical, a similartype of radical containing silica material could be acquired (Scheme 3.4). This way of react-ing the radical with carboxylic acid moieties could be feasible if, for example, an analyte isto be immobilized on the surface with coupling to amine.

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Scheme 3.4. Potential immobilization of spin label 19 on the surface of a carboxylicfunctionalized material.

Azidoamine 16 is a precursor for the synthesis of target molecule 19. It was madefrom a diol by first converting the hydroxyl groups into good leaving groups with sulfonylchloride, tosyl chloride in this case. Tosylate 14 was then turned into a diazide, 15, andsubsequently partially reduced to afford azidoamine 16.

Scheme 3.5. Synthesis of an azidoamine.

The three step synthesis of a bTUrea derived amine 19 is shown in Scheme 3.6. Thefirst step was reductive amination of oxo-TEMPO with azidoamine linker 16, resulting inmonoradical 17. Radical 17 was then linked to 4-amino-TEMPO with a urea moiety in orderto make biradical 18, which was then reduced in the presence of triphenylphosphine to affordtarget compound 19. The compounds were analyzed with a combination of NMR and EPRspectroscopy and mass spectrometry but biradical 19 has not yet been coupled to a surface.

Scheme 3.6. Synthesis of a bTUrea derivative with amine linker.

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3.3 bTUrea for Click Reactions

Should an amide bond not be the ideal linkage between the radical and the surface, thereare other possible ways of immobilizing radicals on mesostructured silica materials. Oneoption is using click chemistry, in particular utilizing the 1,3-dipolar Huisgen cycloadditionbetween an azide and an alkyne. This would require a radical with either an azide or alkynefunctional group, and a surface with the opposite. Scheme 3.7 shows how a derivative ofbTUrea with an alkyne linker, 21, could be coupled to a surface with an azide linker throughclick chemistry. The azide functionalized surface can be synthesized with co-condensationin a way similar to how the SBA-15-NH2 and SBA-15-COOH were made.

Scheme 3.7. An alkyne derivative of bTUrea could be attached to a surface with an azidelinker using click chemistry.

The synthesis of compound 21, a bTUrea with an alkyne linker, is shown in Scheme3.8. Oxo-TEMPO was reacted with pentynamine in the presence of NaCNBH3 and theresulting compound 20 was then reacted with 4-amino-TEMPO and triphosgene to formtarget compound 21. This bTUrea derivative had actually been reported before, by Sauvéeet al. in 201441 but in their case they clicked the radical to a polyethylene glycol chain andnot to a surface. The compounds were, as before, characterized with a combination of NMR,EPR and mass spectrometry.

Scheme 3.8. Synthesis of an alkyne derivative of bTUrea.

In contrast, a bTUrea with an azide linker could be immobilized on a surface withalkyne groups. Compound 18, which is an intermediate in the synthesis of compound 19,would react as shown in Scheme 3.9.

25

Scheme 3.9. Potential immobilization of spin label 18 on the surface of an alkyne func-tionalized material.

3.4 Incorporation into Nanoparticles

Hexadecylamine (HDA, Figure 3.4) is known to stabilize nanoparticles in solution43 and thesame is true for phosphine compounds such as 1,4-bis(diphenylphosphino)butane (dppb).44

Figure 3.4. Structures of HDA and dppb.

By functionalizing radicals with either long amine linkers or phosphine linkers, itmight be possible to use radicals to stabilize the nanoparticles and DNP could then be usedto get structural information that would otherwise not be attainable. By varying the ratioof a mixture of the designed radical and HDA/dppb, optimal concentrations for DNP NMRcould be found. Figure 3.5 shows two different types of bTUrea derivatives that are possiblecandidates for nanoparticle stabilization. Both biradicals are functionalized with relativelylong alkyl chains that have either a terminal amine or a diphenylphosphine group.

Figure 3.5. bTUrea derivatives for incorporation into nanoparticles. n = 10 or 14

Scheme 3.10 shows the total synthesis of compound 24, a derivative of bTUrea thathas been functionalized with a diphenylphosphine linker. The first step is the conversionof bromoheptanitrile into 7-(diphenylphosphino)heptanenitrile and then how it was turnedinto amine 22. Reductive amination was then performed on oxo-TEMPO and the product,23, subsequently coupled to 4-amino-TEMPO, in a way similar to what has been shown forthe other bTUrea derivatives. The ability of this radical to stabilize nanoparticles and itspolarization efficiency will be evaluated at the Technical University of Darmstadt.

26

Scheme 3.10. Synthesis of a bTUrea phosphine derivative.

Biradical 27 (Scheme 3.11), which has either 12 or 16 carbon atoms in the alkyl chain,would be synthesized using the same route as when making compound 19. Compounds16b and 16c were made from the corresponding diols, through tosylate and azide (as shownbefore in Scheme 3.5). The synthesis of 27 has been started but not finished. They arehowever expected to have the same chemical properties as in analogous reactions with ashorter azidoamine. The azidoamines 16b and 16c are expected to form compound 25 whenreacted with oxo-TEMPO and NaCNBH3 under acidic conditions. Monoradical 25 couldthen be reacted with triphosgene and 4-amino-TEMPO to form compound 26, which wouldthen be reduced to compound 27, a biradical that might be able to stabilize nanoparticles.

Scheme 3.11. Synthesis of a bTUrea derivative with a long amine linker. Compounds25, 26 and 27 have not yet been synthesized.

27

4 Conclusion and Future OutlookA novel biradical spin label for proteins, 5, has been developed and synthesized, as well as itspartially deuterated analogue, 10. Spin label 5 has been successfully incorporated into twodifferent proteins and used in NMR experiments, where its PRE effects have been evaluated.Further experiments will be conducted, hopefully revealing whether or not the spin label isuseful in generating localized DNP signal enhancements.

Four bTUrea-like biradicals for covalent attachment to materials were prepared andthe methods used for coupling them to surfaces were described. In addition, one of theradicals (bTUreaG) was immobilized on the surface of mesoporous silica and preliminaryDNP experiments carried out. The other radicals (18, 19 and 21) have not yet been coupled tosurfaces, but their polarizing efficiency will be evaluated after doing so. Radicals that mightbe useful for studying surfaces of nanoparticles are also reported. The design and synthesisof radical 24, which has diphenylphospine group, is accounted for. The properties of theradical, both as a stabilizing agent and as a polarizing agent, will be tested in Darmstadt.

After the properties of the compounds reported here have been investigated sufficiently,new insight might be gained into how the radicals can be modified to get the best possibleresults, especially in terms of polarization efficiency. As an example, increasing the electronrelaxation times is expected to have an effect on DNP efficiency. Modifications can be madeat the position α to the nitroxide groups, similar to when deuterons were used for compound10. It could therefore be of interest to replace the methyl groups of the previously describedbTUrea derivatives with larger groups in order to increase the electron relaxation time andinduce more efficient polarization. Figure 4.1 shows bTUrea derivatives with ethyl groupsin the α position. In addition, these compounds are more resistant to reduction, which mightprove useful if drastic conditions are used when preparing the materials. Even bulkier groups,such as cyclohexyl, could also be used, but that requires a greater synthetic effort.

Figure 4.1. bTUrea derivatives with ethyl groups in the position α to nitroxide.

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5 Experimental

General

Chemicals were purchased primarily from Sigma-Aldrich Chemical Company, and wereused without further purification. TLC was carried out using glass plates pre-coated withsilica gel (Kieselgel 60 F254, 0.2 mm, Merck). Visualisation was by UV light and/or io-dine unless stated otherwise. Silica gel was purchased from Silicycle, and used for columnchromatography. 1H, 13C and 31P NMR spectra were recorded on a Bruker Avance 400 spec-trometer, using deuterated solvents as internal standards. EPR spectra were recorded on aMiniScope MS200, at room temperature unless stated otherwise. Radicals were reduced forNMR measurements by adding 1.1 equivalent of phenylhydrazine to the sample solution.

DNP experimental

All DNP enhanced solid-state NMR experiments in Darmstadt were performed on a BrukerAvance III 400 spectrometer system equipped with an AscendTM 400 DNP magnet, witha low-temperature triple resonance 1H/X/Y probe, and a 9.7 Tesla Bruker gyrotron systemcorresponding to a microwave frequency of 263 GHz. Samples were measured at a spinningrate of 8 kHz at nominally 110 K. Spectra were recorded at 9.4 T according to a frequencyof 400.02 MHz for 1H, 100.59 MHz for 13C and 79.47 MHz for 29Si. Build up curves for1H, 13C and 29Si were measured employing saturation recovery experiments with an initial90◦ pulse train to saturate the z-magnetization, followed by a delay time before employing a90◦ detection pulse.

Cross polarization (CP) experiments were performed with contact times of 2 ms for13C and 3 ms for 29Si employing a recycle delay according to 1.3×T1 of the protons. Dur-ing data acquisition dipolar interactions to protons were decoupled employing either tppmdecoupling with a 20◦ phase jump45 or spinal64.46 13C MAS and 29Si MAS direct polariza-tion experiments were performed employing a saturation recovery pulse sequence utilizingdifferent delay times according to the former calculations of the built up times.

31

4-(N-methylamino)-TEMPO (1). 4-oxo-TEMPO (1.01 g, 5.93 mmol) and methylaminehydrochloride (2.36 g, 34.95 mmol) were dissolved in MeOH (40 mL). NaCNBH3 (0.28 g,4.46 mmol) was added and subsequently 5.5 N HCl in MeOH to adjust the pH of the reactionmixture to ca. 6. The resulting solution was stirred at 24 ◦C for 48 h, concentrated underreduced pressure and diluted with water (10 mL). The reaction mixture was extracted withCH2Cl2 (5 x 15 mL) while keeping the aqueous phase alkaline (pH ca. 10) with 1 N aqueousNaOH. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concen-trated to obtain the crude product, which was purified with column chromatography using agradient elution (MeOH:CH2Cl2:NH3, 3:97:0 to 5:93:2), resulting in 1 (950 mg, 5.13 mmol,87% yield) as a dark red oil.

Notebook reference: SB-I-22.

TLC: 10% MeOH in CH2Cl2, silica gel.

NMR: 1H and 13C recorded in CDCl3 after reduction of radical.

MS: calculated 185.1654 for C10H21N2O, found 186.1727±1.4 (M+H).

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1-(TEMPO-4-oxy)-3-(TEMPO-4-methylamino)propan-2-ol or N-methyl-TOTAPOL (3).4-(2,3-epoxy-propoxy)-TEMPO (970 mg, 4.25 mmol) and LiClO4 (440 mg, 4.14 mmol)were stirred in anhydrous CH3CN (8 mL) under inert atmosphere. 4-(N-methylamino)-TEMPO (750 mg, 4.05 mmol), dissolved in CH3CN (3 mL), was added to the reactionmixture, which was then stirred at 24 ◦C for 24 h. The reaction mixture was concentratedunder reduced pressure and diluted with water (10 mL) before extracting with EtOAc (3 x 15mL). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concen-trated to obtain the crude product, which was purified with column chromatography usinga gradient elution (MeOH:CH2Cl2, 0:100 to 5:95), resulting in 3 (1.38 g, 3.34 mmol, 82%yield) as a brown/orange viscous solid.

Notebook reference: SB-I-31.


NMR: 1H and 13C recorded in CDCl3 after reduction of radicals.

MS: calculated 413.3254 for C22H43N3O4, found 414.3465±1.3 (M+H).

33

1-(TEMPO-4-oxy)-3-(TEMPO-4-methylamino)-2-chloropropan (4). TOTAPOL deriva-tive 3 (155 mg, 0.375 mmol) was dissolved in CH2Cl2 (4.5 mL) and Et3N (58 µL, 0.416mmol) added to the solution. The reaction mixture was cooled to 0 ◦C and methanesulfonylchloride (32 µL, 0.413 mmol) added. Stirring was continued at 0 ◦C for 1 h and then for24 h at 24 ◦C. The reaction mixture was combined with water (15 mL) and then extractedwith CH2Cl2 (3 x 10 mL). The organic layer was washed with brine, dried over anhydrousNa2SO4 and concentrated under reduced pressure. The crude product, an orange oil, wasdried in high vacuum and used for the next reaction without purification.

Notebook reference: SB-II-11.


NMR: Not recorded.

MS: calculated 431.2915 for C22H42ClN3O3, found 432.2987±1.2 (M+H).

34

TOTAPOL spin label 5. To a stirred solution of TOTAPOL derivative 4 (501 mg, 1.16mmol) in acetone:water (2:1, 15 mL), NaSSO2Me (186 mg, 1.36 mmol) was added. The re-action mixture was stirred at 40 ◦C for 30 min, the acetone was removed under reduced pres-sure and the product extracted with CH2Cl2 (3 x 10 mL). The organic layer was washed withbrine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crudeproduct was purified with column chromatography using gradient elution (MeOH:CH2Cl2,0:100 to 1:99) giving 5 (347 mg, 0.683 mmol, 59%) as an orange coloured viscous solid.

Notebook reference: SB-II-18


NMR: 1H and 13C recorded in CDCl3 after reduction of radicals.

MS: Calculated 507.2801 for C23H45N3O5S2, found 530.2693±2.9 (M+Na).

35

Deuterated 4-(N-methylamino)-TEMPO (6). Deuterated 4-oxo-TEMPO (195 mg, 1.07mmol) and methylamine hydrochloride (745 mg, 11.03 mmol) were dissolved in MeOH (7mL). NaCNBH3 (86 mg, 1.37 mmol) was added and subsequently 5.5 N HCl in MeOH toadjust the pH of the reaction mixture to ca. 6. The resulting solution was stirred at 24 ◦Cfor 48 h, concentrated under reduced pressure and basified with aqueous NaOH until pH ca.10. This aqueous solution was extracted with CH2Cl2 (3 x 5 mL) while keeping the aque-ous phase alkaline with 1 N aqueous NaOH. The organic layer was washed with brine, driedover anhydrous Na2SO4 and concentrated under reduced pressure to obtain 6 (186 mg, 0.942mmol) as an orange solid in 57% yield.



MS: calculated 197.24 for C10H9D12N2O, found 198.25 (M+H).

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Deuterated 1-(TEMPO-4-oxy)-3-(TEMPO-4-methylamino)propan-2-ol or deuteratedN-methyl-TOTAPOL (8). Deuterated 4-(2,3-epoxy-propoxy)-TEMPO (7, 160 mg, 0.67mmol) and LiClO4 (80 mg, 0.75 mmol) were stirred in CH3CN (5 mL) and the resultingsolution added to deuterated 4-(N-methylamino)-TEMPO (6, 130 mg, 0.66 mmol). The re-action mixture was stirred at 24 ◦C for 72 h, concentrated under reduced pressure and purifiedwith column chromatography using a gradient elution (MeOH:CH2Cl2, 3:97 to 5:95), giving8 (228 mg, 0.52 mmol, 79%) as an orange viscous solid.



MS: calculated 437.4760 for C22H19D24N3O4, found 438.4833±1.8 (M+H).

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Deuterated 1-(TEMPO-4-oxy)-3-(TEMPO-4-methylamino)-2-chloropropan (9). To asolution of deuterated TOTAPOL derivative 8 (130 mg, 0.297 mmol) in anhydrous CH2Cl2(4 mL), Et3N (50 µL, 0.358 mmol) was added. The reaction mixture was cooled to 0 ◦C andmethanesulfonyl chloride (30 µL, 0.388 mmol) added. Stirring was continued at 0 ◦C for 1h and then for 24 h at 24 ◦C. Water (10 mL) was added to the reaction mixture, which wasthen extracted with CH2Cl2 (3 x 10 mL). The organic layer was washed with brine, driedover anhydrous Na2SO4 and concentrated under reduced pressure. The crude product wasused for the next reaction without purification.



MS: calculated 455.4421 for C22H18D24ClN3O3, found 456.4494±2.0 (M+H).

38

Deuterated TOTAPOL spin label (10). To a stirred solution of compound 9 (130 mg, 0.28mmol) in acetone:water (2:1, 3 mL), NaSSO2Me (65 mg, 0.48 mmol) was added. The re-action mixture was stirred at 40 ◦C for 30 min, the acetone was removed under reducedpressure and the product extracted with CH2Cl2 (3 x 10 mL). The organic layer was washedwith brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. Thecrude product was purified with prep TLC (MeOH:CH2Cl2, 4:96) giving 10 (77 mg, 0.14mmol, 50%) as an orange viscous solid.



MS: Calculated 531.4307 for C23H22D24N3O5S2, found 532.4380±0.6 (M+H).

39

Spin labeling of papain with TOTAPOL spin label 5. Papain was activated by adding 30µL of 1.0M DDT solution to 285 µL of 24 mg/mL papain in 900 µL of papain buffer (50mM Na2HPO4, 1 mM EDTA, pH 6.5). The solution was allowed to stand for 30 min beforesize exlusion purification. The stationary phase was Sephadex G-25 and the elution buffer20 mM Na2HPO4, 100 mM NaCl, pH 7.0. Absorbance at 280 nm was used to monitor con-centration of the eluted enzyme. εpapain = 50000 M−1cm−1.

20 mM stock solution of TOTAPOL spin label 5 was prepared by adding 5.10 mg of spinlabel (0.01 mmol) to 0.5mL of DMF. 1 mL of 0.086 mM papain solution and 4.3 µL ofbiradical spin label (5) solution were stirred at 24 ◦C overnight. 1 mL of papain solution wasstirred with 4.3 µL of MTSSL solution as a control sample. Excess spin label was removedon a Sephadex G-25 column, monitored with UV/VIS spectrometer. Samples were washedand concentrated in centrifugal filters before recording EPR spectra at 24 ◦C.


40

4-[(6-Methoxy-6-oxohexyl)amino]-TEMPO (12). Oxo-TEMPO (209 mg, 1.23 mmol),methyl 6-aminohexanoate hydrochloride 11 (189 mg, 1.05 mmol) and NaBH3CN (108 mg,1.72 mmol) were dissolved in MeOH (12 mL). HCl in MeOH was then added to make thepH of the solution ca. 5. The reaction mixture was stirred overnight at 24 ◦C, concentratedunder reduced pressure and then diluted with CH2Cl2. The organic phase was washed with asolution of aqueous NaHCO3. The aqueous phase was extracted twice with CH2Cl2 and thenthe combined organic phases were washed with brine and dried over anhydrous Na2SO4. Thecrude product was purified with prep TLC (3:97, MeOH:CH2Cl2), giving compound 12 (61mg, 0.216 mmol, 50%).


TLC: CH2Cl2 + few drops MeOH, silica gel.

NMR: 1H NMR recorded in CDCl3 after reduction of radical.


41

bTUreaG methyl ester (13). Compound 12 (150 mg, 0.50 mmol), 4-amino-TEMPO (82mg, 0.48 mmol) and Et3N (240 µL, 1.72 mmol) were stirred in CH2Cl2 (7 mL) at 24 ◦.Triphosgene (65 mg, 0.22 mmol) in CH2Cl2 (1 mL) was added to the reaction mixture, whichwas then stirred for 24 h. The reaction mixture was combined with aqueous NaHCO3 (10mL) and then extracted with CH2Cl2 (2 x 10 mL). The organic phase was washed with brine,dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude productwas purified with column chromatography using a gradient elution (EtOAc:Pet ether, 15:85to 20:80) yielding 13 (60 mg, 0.12 mmol, 24%) as an orange compound.


TLC: 50% EtOAc in Pet. ether, silica gel.

MS: calculated 496.3625 for C26H48N4O5, found 519.3517±1.4 (M+Na).

42

bTUreaG. Compound 12 (220 mg, 0.44 mmol) was dissolved in EtOH:H2O (8:2, 10 mL)and NaOH (92 mg, 2.3 mmol) added while stirring at 24 ◦C. The reaction mixture was heatedto 70 ◦C and stirred for 6 h. The EtOH was removed under reduced pressure and pH of theremaining aqueous phase adjusted to ca. 3 with aqueous HCl solution. The aqueous phasewas extracted with CH2Cl2, saturated with NaCl and extracted twice with MeOH:CH2Cl2(10:90, 5 mL). The combined organic layers were washed with brine, dried over anhydrousNa2SO4 and concentrated under reduced pressure. The crude product was purified with prepTLC (MeOH:CH2Cl2, 4:96), resulting in bTUreaG (124 mg, 0.257 mol, 58%) as an orangesolid.



NMR: 1H NMR recorded in CDCl3.


43

Hexane-1,6-diyl bis(4-methylbenzenesulfonate) (n=4, 14). DABCO (1672 mg, 14.90 mmol),hexane-1,6-diol (819 mg, 6.93 mmol) and CH2Cl2 (20 mL) were stirred at 0 ◦C. TsCl (2840mg, 14.90 mmol) was added, which made the clear solution go white, and stirring was con-tinued for 30 min at 0 ◦C and then at 24 ◦C for 3 h. Water (10 mL) was added to the reactionmixture, which was then extracted with CH2Cl2 (3 x 10 mL). The organic phase was washedwith brine, dried over Na2SO4 and concentrated. The crude was dried in high vacuum, yield-ing compound 16 (2900 mg, 6.80 mmol, 98%) as a white solid.

Notebook reference: SB-II-144.TLC: 30% EtOAc in Pet. ether, silica gel.NMR: 1H and 13C NMR recorded in CDCl3.

Dodecane-1,12-diyl bis(4-methylbenzenesulfonate) (n=10, 14b). Same procedure as above.DABCO (638 mg, 5.69 mmol), dodecane-1,12-diol (504 mg, 2.49 mmol), TsCl (1060 mg,5.56 mmol) and CH2Cl2 (10 mL). Yielded compound 16b as a white solid (1.034g, 2.025mmol, 82%).

Notebook reference: SB-II-145.TLC: 30% EtOAc in Pet. ether, silica gel.

Hexadecane-1,16-diyl bis(4-methylbenzenesulfonate) (n=14, 14c). Same procedure asabove. DABCO (179 mg, 1.60 mmol), hexadecane-1,16-diol (190 mg, 0.74 mmol), TsCl(312 mg, 1.64 mmol) and CH2Cl2 (5 mL). Yielded compound 16c as a yellowish solid inquantitative yield.


44

1,6-Diazidohexane (n=4, 15). Tosylate 14 (920 mg, 2.16 mmol) was dissolved in CH3CN(25 mL) and NaN3 (370 mg, 5.69 mmol) added. The reaction mixture was refluxed at 80◦C for 24 h, concentrated under reduced pressure and then diluted with H2O (10 mL). Thereaction mixture was extracted with CH2Cl2 (3 x 10 mL), dried over Na2SO4, concentratedand then dried in high vacuum, yielding compound 15 (326 mg, 1.94 mmol, 90%). Used fornext reaction step without further purification.

Notebook reference: SB-II-147.TLC: 50% EtOAc in Pet. ether, silica gel.NMR: 1H and 13C NMR recorded in CDCl3.

1,12-Diazidododecane (n=10, 15b). Same procedure as above. Tosylate 14b (283 mg, 0.55mmol), NaN3 (95 mg, 1.46 mmol) and CH3CN (7 mL). Product 15b was obtained as a liquidin quantitative yield.


1,16-Diazidohexadecane (n=14, 15c). Same procedure as above. Tosylate 14c (292 mg,0.515 mmol), NaN3 (84 mg, 1.29 mmol) and CH3CN (7 mL). Product 15c was obtained asa liquid in quantitative yield.


45

6-Azidohexan-1-amine (n=4, 16) Diazidoalkane 15 (1180 mg, 7.02 mmol) was dissolvedin a Et2O:EtOAc mixture (5 mL: 5mL) and aqueous HCl added (1M, 7.5 mL). The reactionmixture was cooled and PPh3 (1837 mg, 7.00 mmol) added in small portions over 20 min.The resulting solution was stirred at 24 ◦C for 3 h and then extracted with Et2O (2 x 10 mL).The aqueous phase was basified with NaOH until pH ca. 12 and then extracted again withEt2O (3 x 10 mL). The organic layer was dried over Na2SO4 and concentrated under reducedpressure. The crude was purified with column chromatography using a gradient elution (5:95MeOH:CH2Cl2 and then 10:2:88 MeOH:NH3:CH2Cl2) to yield compound 16 (236 mg, 1.66mmol, 23%).

Notebook reference: SB-II-193.TLC: 10:90 MeOH:CH2Cl2 + NH3.NMR: NMR: 1H and 13C NMR recorded in CDCl3.

12-Azidododecan-1-amine (n=10, 16b). Same procedure as above. Diazidoalkane 15b(1728 mg, 6.85 mmol), Et2O:EtOAc mixture (5 mL: 5mL), aqueous HCl (1M, 7.5 mL) andPPh3 (1617 mg, 6.17 mmol). Purified with column chromatography using a gradient elution(10:90 MeOH:CH2Cl2 and then 10:2:88 MeOH:NH3:CH2Cl2) to yield compound 16b.

Notebook reference: SB-II-194.TLC: 10:90 MeOH:CH2Cl2 + NH3.

16-Azidohexadecan-1-amine (n=14, 16c). Same procedure as above. Diazidoalkane 15c(160 mg, 0.515 mmol), Et2O:EtOAc mixture (0.5 mL: 0.5mL), aqueous HCl (1M, 0.75 mL)and PPh3 (121 mg, 0.463 mmol). Purified with prep TLC (10:90 MeOH:CH2Cl2 + NH3) toyield compound 16c (27 mg, 0.119 mmol, 23%).

Notebook reference: SB-II-195.TLC: 10:90 MeOH:CH2Cl2 + NH3.

46

4-[(6-Azido)amino]-TEMPO (17). Oxo-TEMPO (145 mg, 0.85 mmol), 6-azidohexan-1-amine 16 (115 mg, 0.81 mmol) and NaBH3CN (87 mg, 1.38 mmol) were dissolved in MeOH(10 mL). HCl in MeOH was added to make the pH of the solution ca. 5. The reaction mixturewas stirred for 48 h at 24 ◦C and then at 40 ◦C for 6 h, concentrated under reduced pressureand then diluted with CH2Cl2. The organic phase was washed with a solution of aqueousNaHCO3 (10 mL). The aqueous phase was extracted with CH2Cl2 (2 x 10 mL) and then thecombined organic phases were washed with brine and dried over anhydrous Na2SO4. Thecrude product was purified with prep TLC (80:20 EtOAc:Pet. ether + NH3), yielding com-pound 17 (76 mg, 0.256 mmol, 32%).


TLC: 80% EtOAc in Pet. ether + NH3, silica gel.

NMR: 1H recorded in CDCl3 after reduction of radical.

MS: calculated 296.2450 for C15H30N5O, found 297.2573±1.8 (M+H).

47

Compound 18. Compound 17 (80 mg, 0.270 mmol), 4-amino-TEMPO (48 mg, 0.280 mmol)and Et3N (160 µL, 1.147 mmol) were stirred in CH2Cl2 (9 mL). Triphosgene (34 mg, 0.115mmol) in CH2Cl2 (1 mL) was added to the reaction mixture, which was then stirred for 24h at 24 ◦C. The reaction mixture was combined with aqueous NaHCO3 (10 mL) and thenextracted with CH2Cl2 (2 x 10 mL). The organic phase was washed with brine, dried overanhydrous Na2SO4 and concentrated under reduced pressure. The crude product was pu-rified with prep TLC (EtOAc:Pet ether, 80:20) to obtain pure compound 18 (40 mg, 0.081mmol, 30% yield).



NMR: 1H NMR recorded in CDCl3 after reduction of radical.


48

Compound 19. Compound 18 (30 mg, 0.061 mmol) and PPh3 (87 mg, 0.33 mmol) weredissolved in a mixture of THF (3 mL) and H2O (0.3 mL). The reaction mixture was stirredfor 24 h at 24 ◦C. The solvent was evaporated under reduced pressure and the crude dilutedwith EtOAc (10 mL). The organic phase was then washed with 1 M aqueous HCl solutionand extracted with EtOAc (2 x 5 mL) to remove any PPh3 and OPPh3. The aqueous phasewas basified with 1 M NaOH solution and then extracted with CH2Cl2 (2 x 5 mL). The or-ganic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated underreduced pressure to obtain the sufficiently pure product 19 (14 mg, 0.030 mmol, 49%) as ayellow viscous solid.


TLC: 80% EtOAc in Pet ether, silica gel.

NMR: not recorded.

MS: calculated 467.3835 for C25H49N5O3, found 469.3908±5 (M+H).

49

4-(Pent-4-yn-1-ylamino)-TEMPO (20). Oxo-TEMPO (296 mg, 1.74 mmol), pentynamine(120 µL, 1.24 mmol) and NaBH3CN (155 mg, 2.47 mmol) were dissolved in MeOH (18mL). HCl in MeOH was then added to make the pH of the solution ca. 5. The reactionmixture was stirred overnight at 24 ◦C, under inert atmosphere and away from light. Itwas then concentrated under reduced pressure and diluted with CH2Cl2. The organic phasewas washed with a solution of aqueous NaHCO3. The aqueous phase was extracted twicewith CH2Cl2 and then the combined organic phases were washed with brine and dried overanhydrous Na2SO4. The crude product was purified with column chromatography using agradient elution (EtOAc:Pet ether, 40:60 to 100:0), yielding orange compound 20 (172 mg,0.72 mmol, 58%).



NMR: 1H and 13C NMR recorded in CDCl3 after reduction of radical.

50

Compound 21. Compound 20 (54 mg, 0.23 mmol), 4-amino-TEMPO (45 mg, 0.26 mmol)and Et3N (90 µL, 0.65 mmol) were stirred in CH2Cl2 (4 mL) at 24 ◦C. Triphosgene (23 mg,0.08 mmol) in CH2Cl2 (1 mL) was added to the reaction mixture, which was then stirred for24 h. The reaction mixture was combined with aqueous NaHCO3 (10 mL) and then extractedwith CH2Cl2 (3 x 10 mL). The organic phase was washed with brine, dried over anhydrousNa2SO4 and concentrated under reduced pressure. The crude product was purified with prepTLC (EtOAc:Pet ether, 80:20) yielding 21 (32 mg, 0.074 mmol, 32%).



NMR: 1H and 13C NMR recorded in CDCl3 after reduction of radical.


51

7-(Diphenylphosphino)heptanenitrile. HPPh2 (0.4 mL, 2.30 mmol) in THF (2 mL) wasadded to a suspension of NaH (98 mg, 4.08 mmol) in THF (5 mL) and the reaction mix-ture refluxed for 1 h at 70 ◦C and under argon. It was then allowed to cool to 50 ◦C and7-bromoheptanitrile (393 mg, 2.07 mmol) in THF (1 mL) added. Stirring was continued at50 ◦C for 24 h and then water was added to quench the reaction. The mixture was extractedwith EtOAc (3 x 10 mL) and the organic phase washed with brine, dried over Na2SO4 andconcentrated under reduced pressure. The product compound (564 mg, 1.91 mmol, 92%)was used for next reaction step without further purification.



NMR: 1H, 13C and 31P NMR recorded in CDCl3.

52

7-(Diphenylphosphino)heptan-1-amine (22). 7-(Diphenylphosphino)heptanenitrile (47 mg,0.16 mmol) in THF (1 mL) was added to a solution of LiAlH4 in THF (1 M, 300 µL, 0.04mol), while cooling and under inert conditions. After refluxing for 24 h at 70 ◦C, the reactionmixture was quenched with saturated aqueous solution of Na2SO4 and then filtered throughcelite. The reaction mixture was extracted with EtOAc and the organic layer then washedwith brine, dried over Na2SO4 and concentrated under reduced pressure. The crude productwas purified with prep TLC (10:88:2 MeOH:CH2Cl2:NH3) to give pure product 22 (47 mg,0.157 mmol, 16 %).


TLC: 10% MeOH in CH2Cl2 + NH3, silica gel.

NMR: 1H, 13C and 31P NMR recorded in CDCl3.

53

Compound 23. Oxo-TEMPO (302 mg, 1.77 mmol), amine 22 (515 mg, 1.72 mmol) andNaBH3CN (156 mg, 2.48 mmol) were dissolved in MeOH (20 mL). HCl in MeOH was thenadded to make the pH of the solution ca. 5. The reaction mixture was stirred overnight at 50◦C, concentrated under reduced pressure and then diluted with CH2Cl2. The organic phasewas washed with a solution of aqueous NaHCO3. The aqueous phase was extracted twicewith CH2Cl2 and then the combined organic phases were washed with brine and dried overanhydrous Na2SO4. The crude product was purified with prep TLC (2:98, MeOH:CH2Cl2 +NH3), yielding compound 23 (195 mg, 0.43 mmol, 24%).


TLC: 5% MeOH in CH2Cl2 + NH3, silica gel.

NMR: 1H and 31P NMR recorded in CDCl3.

MS: calculated 453.3035 for C28H42N2OP, found 454.3108±0.6 (M+H).

54

Compound 24. Compound 23 (23 mg, 0.051 mmol), 4-amino-TEMPO (10 mg, 0.058 mmol)and Et3N (25 µL, 0.179 mmol) were stirred in CH2Cl2 (1 mL) at 24 ◦C. Triphosgene (6.5mg, 0.022 mmol) in CH2Cl2 (0.5 mL) was added to the reaction mixture, which was thenstirred for 24 h. The reaction mixture was combined with aqueous NaHCO3 (5 mL) and thenextracted with CH2Cl2 (2 x 5 mL). The organic phase was washed with brine, dried over an-hydrous Na2SO4 and concentrated under reduced pressure. The crude product was purifiedwith prep TLC (EtOAc:Pet ether, 80:20) yielding compound 24 (11 mg, 0.015 mmol, 30%).



NMR: not recorded.

MS: calculated 650.4325 for C38H59N4O3P, found 651.4398±0 (M+H).

55

�� Figure 5.1. 1H NMR spectrum of reduced 4-(N-Methylamino)-TEMPO (1).�� Figure 5.2. 13C NMR spectrum of reduced 4-(N-Methylamino)-TEMPO (1).

56

�� Figure 5.3. 1H NMR spectrum of reduced N-Methyl-TOTAPOL (3).�� Figure 5.4. 13C NMR spectrum of reduced N-Methyl-TOTAPOL (3).

57

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Figure 5.5. 1H NMR spectrum of reduced compound 12.

Figure 5.6. 1H NMR spectrum of biradical bTUreaG.

58

n=4

Figure 5.7. 1H NMR spectrum of compound 14.

n=4

Figure 5.8. 13C NMR spectrum of compound 14.

59

n=4


n=4


60

n=4


n=4


61

n=10

Figure 5.13. 1H NMR spectrum of compound 16b.

n=14

Figure 5.14. 1H NMR spectrum of compound 16c.

62



63


Figure 5.18. 13C NMR spectrum of reduced compound 20.

64


Figure 5.20. 13C NMR spectrum of reduced compound 21.

65

Figure 5.21. 1H NMR spectrum of 7-(diphenylphosphino)heptanenitrile.

Figure 5.22. 13C NMR spectrum of 7-(diphenylphosphino)heptanenitrile.

66


Figure 5.24. 31P NMR spectrum of compound 22.

67


Figure 5.26. 31P NMR spectrum of compound 23.

68

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Syntheses of Biradicals for Surface Studies by DNP ssNMR › bitstream › 1946 › 23582 › 1 › MS_snaedis.pdf · magnetic moments and depends on the strength of the external