High-field EPR study of persistent substituted trityl radicals

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Dipicolinic acid and derivatives (PyMTA and PyMDPDA)

Accordingly, we decided to combine a pyridine ring with carboxylate moieties. The already mentioned dipicolinic acid (DPA 1, see pp. 37 – 40) is an example, and increasing the denticity leads to derivatives known as pyridinedimethylenenitrilo-tetraacetate (PyMTA 66). The structure of these ligands is depicted in Figure 49. Functionalized DPA72,73,74 and PyMTA21,34,39 have already been used with gadolinium as spin labels for PELDOR distance measurements. This strengthened our intent to investigate their use with MnII.
The synthesis of PyMTA has been achieved starting from pyridine 2,6-dimethanol 63. Bromination with hydrobromic acid afforded 2,6-dibromomethylpyridine 64 in modest yield. Double nucleophilic substitution with ethyl iminodiacetate gave Et-PyMTA 65 in good yield, and a final saponification with lithine followed by anion exchange with an Amberlite resin led to the formation of PyMTA 66 in quantitative yield.132 The corresponding MnII complex 67 was prepared using MnCl2 at controlled pH for elucidation of the coordination sphere using X-ray crystallography (Scheme 14). However, no crystals could be obtained, so the coordination sphere depicted in Scheme 14 is hypothetical and based on the corresponding GdIII-PyMTA complex without the coordinated water molecule, even if numerous related MnII complexes include a water molecule in their coordination sphere.

LINKERS AND GRAFTING OF LIGANDS

We have a set of ligands for MnII to be used as paramagnetic centers for the PELDOR method. We now need to graft two of them on a central linker to obtain bis-MnII systems and to test them in PELDOR. This central spacer has to comply with the specifications of a “molecular rod”. It must be rigid (in order to obtain a constrained distance between the two paramagnetic centers), easily incrementable (to have access to a broad range of distances) and must possess functional groups allowing straightforward coupling to the magnetic centers. Moreover, these two groups should be either identical (to incorporate the same paramagnetic center) or different (to obtain dissymmetrical platforms containing, for instance, a MnII complex and a stable radical, or two different MnII complexes). Readily crystallizable rods are desirable to determine the distance from X-ray crystallography for comparison with the distances obtained with the PELDOR methodology. To this aim, DFT calculations will also be employed. Finally, the ideal linker should also be water-soluble, so that the PELDOR experiment could be performed in water in order to be as close as possible to the conditions that will be used in future biological applications.

Synthesis of the oligo(piperidine) linker

To fulfill these requirements, we turned our attention to a quite recently described oligo(piperidine) linker.158 This spacer consists of an oligomeric backbone of piperidines, which adopt a chair conformation, both in solution (as shown by NMR experiments) and in the solid state (as revealed by the crystal structure depicted in Figure 64). Furthermore, this rod is water-soluble and suitable for asymmetric platforms, bearing a keto group at one side and an amino group at the other side. The elongation methodology is based on iterative reductive aminations (Scheme 22).158 First, 4-piperidone 100 was protected with a benzyl (Bn) or a carboxybenzyl (Cbz) group to give the corresponding N-protected piperidones (101 and 102, respectively) in good to high yield. The Cbz and the Bn groups have been chosen because they can be removed by hydrogenation: in the first case, we followed the literature conditions, but in the second case, the Bn group was used because it can also be removed using 1-chloroethyl chloroformate,159 which could be useful if hydrogenation-sensitive moieties are also present.
Next, reductive amination with 4-piperidone ethylene ketal 103 in the presence of NaBH(OAc)3 and AcOH afforded the orthogonally protected bis(piperidines) 104 and 105 (with a Bn or a Cbz group, respectively) in good yields. Optimization was needed for this step, as the literature conditions158 (no AcOH) afforded the product in non reproducible yields (30 to 60%) with a slow conversion, even when a large excess of NaBH(OAc)3 was employed. In our hands, the use of AcOH160 gave consistent good yields and conversions.
The orthogonally protected compound 105 was then either hydrogenated under pressure to give the free amine 106 in nearly quantitative yield, or treated with HCl to regenerate the ketone 107 by removal of the ketal group (Scheme 22). This step was problematic, as the conditions described158 (37% aq. HCl for 25 min) led to poor yields (around 15%), presumably due to degradation. The use of more diluted HCl (6 M) improved the yield to 36%. With higher dilutions of cold HCl (4M), smooth deprotection of the ketone was obtained with excellent yields (> 90%), albeit in one week. This procedure was also successful in the synthesis of ketone 108, but the Bn group could not be removed using 1-chloroethyl chloroformate in DCM and then refluxing the mixture with MeOH,159 maybe because of the cleavage of the other C-N bond.
Lastly, a second reductive amination between 106 and 107 with NaBH3CN in the presence of Ti(OiPr)4160 afforded the orthogonally protected tetra(piperidine) 109 in low yield. Again, the original conditions158 (NaBH(OAc)3) did not allow for the formation of the desired product, even when AcOH was added, or when freshly distilled DCE was used. Replacing NaBH3CN for NaBH4 led to poorer yields. The difficulty to perform reductive aminations on bis(piperidines) 106 and 107 was confirmed by the fact that no reaction took place between 107 and piperazine, while the coupling between amine 106 and cyclohexanone led to the isolation of the adduct with only 15% yield using NaBH(OAc)3 with AcOH (Scheme 22).

Dissymmetric version

Targeting a dissymmetric version of this linker, we exploited the different reactivity of the bromo and the iodo group on 1-bromo-4-iodobenzene 132 to sequentially introduce Boc-Pip 127 and Bn-Pip 128.174 The Boc and the Bn protecting groups being orthogonal, it would permit the sequential introduction of two different paramagnetic moieties. The replacement of bromo for iodo moities should not be detrimental to the efficiency of the Hartwing-Buchwald coupling, as special conditions have been developed in this case: the addition of 18-crown-6 (18C6) activates NaOtBu by increasing the solvation of Na+,175 allowing efficient couplings on aryl iodides.
Indeed, a coupling with Boc-Pip 127 in the same conditions as above afforded bromoarene 133, but with a low yield (24%). As described by Buchwald,175 adding 18C6 enhanced considerably the yield to 57%. We found that dibenzo-18C6 gave better results (91%), which could be traced to its superior affinity for Na+. It is worth noting that Ullmann-like versions of this coupling176 (CuI/ethylene glycol/K3PO4 or CuI/proline/K3PO4) did not afford the desired product. Good yields were also obtained when Bn-Pip 128 was used to give the expected bromoarene 134 (Scheme 39).
Optimization of the next coupling step was also necessary. The coupling between bromoarene 133 and Bn-Pip 128 in the original Buchwald conditions afforded equimolar amounts of the desired product 135 and recovered 133. The conversion was not complete even after extended reflux time and could not be improved using other classical (Pd(OAc)2/BINAP/Cs2CO3) conditions. Low yields were also observed when bromoarene 134 was coupled with Boc-Pip 127 to give compound 135. We solved this issue using 2-dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl (XPhos177) in combination with Pd2(dba)3 and Cs2CO3, which afforded the orthogonally protected linker BocPhPipPhBn 135 in 66% yield. The global yield of this Pd/Pd method is higher than the Cu/Pd literature method (Scheme 39).

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2 with DPA, PyMTA and PyMDPDA derivatives

Despite this lack of reactivity, we still tried to couple pyridine-based ligands to the phenylpiperazine
linkers. No reaction was observed between Et-pBrPyMTA 70 and Bn-Pip 128 (Pd2(dba)3/BINAP/NaOtBu or Pd(OAc)2/XPhos/NaOtBu). The coupling between Et-pBrPyMTA 70 and asymmetric BocPipPhPip 137 was attempted several times (Pd2(dba)3/BINAP/NaOtBu, Pd2(dba)3/XPhos/ NaOtBu, Pd(OAc)2/BINAP/NaOtBu, Pd(OAc)2/BINAP/Cs2CO3 or Pd(OAc)2/P(tBu)3/Cs2CO3) but when the reactants seemed to be consumed, only intractable mixtures were obtained. The use of EtpBrPyMDPDA 87 instead of Et-pBrPyMTA 70 did not improve the results. Finally, when phenylpiperazine linker 132 was reacted with Et-pBrPyMTA 70, diester 9 or triflate 79, no desired product could be isolated (Scheme 45).

Oligo(phenylene-ethynylene) linker

We have also used oligo(phenylene-ethynylene)s181 (OPE) as a third type of spacers. These shape-persistent, fully conjugated, stiff nanowires consist of alternating phenyl-acetylene repeat unit. PELDOR measurements at X-band on these linkers grafted with two nitroxides at both ends have led to narrow distance distributions16,17,182 confirming their rigidity. Systems containing more than two nitroxides have also been constructed using OPE linkers.41,42 These linkers are often derivatized with alkyl chains to enhance their solubility in organic solvents. We have chosen small poly(ethylene)glycol (PEG) chains, as systems incorporating two charged MnII complexes at both ends in combination with PEG chains should provide sufficient water solubility.

Symmetric version

Retrosynthetically, a symmetric OPE linker could be obtained by a coupling reaction between a central diiodinated building block equipped with PEG chains 154 and a para-substituted ethynylbenzene, also substituted with PEG chains, that could be obtained by desymmetrization of the central diiodinated building block 154. This strategy is analogous as the construction of the phenyl-piperazine linker, but Sonogashira couplings would be employed instead of Hartwig-Buchwald couplings (Scheme 47).
Scheme 47: Retrosynthetic analysis of an OPE linker with PEG chains on each benzene ring The synthesis of key intermediate 154 was achieved in three steps. Tosylation of diethylene glycol monomethyl ether 151 gave tosylate 152 in good yield.183 This compound was then allowed to react with hydroquinone (p-dihydroxybenzene) in a double Williamson reaction to afford Ph(OPEG)2 153 in a similar yield.184 Diiodination of this compound using iodine in conjunction with KIO3 in AcOH led to the formation of Ph(OPEG)2I2 154 in good yield after recrystallization.185 A scale-up of this protocol gave us an easy access to nearly 30 g of building block 154 (Scheme 48).

Table of contents :

List of figures
List of schemes
List of tables
General introduction
Introduction: theory, literature review and aim of the project
1. Theoretical background
1.1 EPR: introduction and scope of application
1.2 Theoretical background
1.2.1 Magnetic moment of the electron
1.2.2 Interaction between a paramagnetic center and a magnetic field
1.2.3 Continuous-wave EPR
1.2.4 The case of a real EPR spectrum
1.3 Spin Hamiltonian for MnII
1.4 Continuous-wave high-field EPR
1.4.1 Design of the J-band cw-HFEPR spectrometer
1.4.2 Influence of the high-field on the EPR spectrum
1.4.2.1 The case of MnII
1.4.2.2 The case of TEMPO
1.5 Pulsed EPR
1.5.1 Introduction and main pulse sequences
1.5.2 The PELDOR pulse sequence
1.5.2.1 Theory
1.5.2.2 Pulse sequence
1.5.2.3 Optimization of parameters
1.5.2.3.1 Concentration
1.5.2.3.2 Pulses
1.5.2.3.3 Temperature
1.5.2.3.4 Solvent
1.5.2.4 High-spin metals vs nitroxide spin labels
1.5.2.5 MnII vs GdIII
2. Pulsed EPR measurements involving metals
2.1 Low-spin metals
2.1.1 CuII complexes
2.1.1.1 CuII-CuII distance measurements
2.1.1.2 CuII-nitroxide distance measurements
2.1.2 Fe-S clusters
2.1.3 Mn-tyrosyl measurements in the S2 state of PSII
2.2 High-spin GdIII complexes
2.2.1 PELDOR distance measurements between two GdIII complexes
2.2.1.1 Gd-Gd measurements on rigid models compounds
2.2.1.2 Gd-Gd measurements on biological objects
2.2.1.2.1 In vitro PELDOR
2.2.1.2.1.1 Gd-DPA-based tags
2.2.1.2.1.2 Gd-DO3A and Gd-DOTA-based tags
2.2.1.2.2 In-cell PELDOR
2.2.1.2.2.1 Gd-PyMTA tags
2.2.1.2.2.2 Gd-DOTA-based tags
2.2.1.3 Gd-nitroxide measurements
2.2.1.3.1 With Gd-Tpy or Gd-DTPA labels
2.2.1.3.2 With Gd-DOTA labels
2.2.2 RIDME
2.3 MnII complexes
2.3.1 Mn-Mn distance measurements
2.3.2 Mn-nitroxide measurements
3. Aim of the project
Chapter I – Synthesis of platforms with a constrained distance between two MnII complexes
1. Ligand screening
1.1 Bis(imino)pyridines
1.2 Terpyridines
1.3 Dipicolinic acid and derivatives (PyMTA and PyMDPDA)
1.4 PCTA and PCMA
1.5 DOTA and DO3A
2. Linkers and grafting of ligands
2.1 Oligo(piperidine) linker
2.1.1 Synthesis of the oligo(piperidine) linker
2.1.2 Coupling with ligands
2.1.2.1 Grafting of BImPs
2.1.2.2 Grafting of Tpys
2.2 Phenyl-piperazine linker
2.2.1 Symmetric version
2.2.2 Dissymmetric version
2.2.3 Elongation
2.2.4 Couplings with ligands
2.2.4.1 with terpyridines
2.2.4.2 with DPA, PyMTA and PyMDPDA derivatives
2.2.4.3 with DOTA derivatives
2.3 Oligo(phenylene-ethynylene) linker
2.3.1 Symmetric version
2.3.2 Dissymmetric version
2.3.3 Elongation
2.3.4 Coupling with ligands
2.3.4.1 with terpyridines
2.3.4.2 DPA and PyMTA derivatives
2.3.4.3 DO3A and DOTA derivatives
2.4 Polyprolines
3. Bis(nitroxides)
3.1 Platforms with a phenyl-piperazine linker
3.2 Platforms with an OPE linker
4. Dissymmetric platforms
4.1 With the phenyl-piperazine linker
4.2 With the OPE linker
Conclusion
Chapter II – PELDOR distance measurements between two high-spin MnII centers 
1. General sample preparation for PELDOR measurements
2. Measurements on the bis-Mn-Tpy platforms 
3. Measurements on the polyproline bis-dota platforms
3.1 Measurement of the ZFS interaction of Mn-DOTA
3.2 PELDOR results
3.3 Analysis of the pseudo-secular contribution
4. Measurements using rigid platforms
4.1 Phenyl-piperazine bis-DOTA platforms 149 and
4.2 OPE bis-DOTA platforms 202, 203 and
4.3 OPE bis-TEMPO platform
Conclusion
Chapter III – High-field EPR study of persistent substituted trityl radicals
1. Persistent trityl radicals
1.1 Origins of persistent trityl radicals
1.2 Reported synthesis and applications of PTM radicals
1.2.1 Synthesis
1.2.2 Applications
1.3 Reported synthesis and applications of TAM radicals
1.3.1 Synthesis
1.3.2 Applications
2. Towards TAM/PTM – MnII-DOTA model systems
2.1 Synthesis of PTMTE and PTMTC
2.2 Attempted couplings of PTM and TAM radicals
3. Accurate measurement of the g-anisotropy of substituted trityl radicals
3.1 Design of the experimental setup
3.2 Results and discussion
Conclusion
General conclusion and perspectives
Annexes
1. Glassing agents
2. PELDOR measurement process
3. Data Analysis
4. Final products
5. List of publications
Experimental part
1. Synthesis
2. ITC
3. CD
4. EPR
5. Computational methods
6. X-ray crystallography
References

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