Galvinoxyl radicals: Synthesis of new derivatives, determination of low oxygen contents, and stability studies

Tetrahedron 75 (2019) 2737e2747

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Galvinoxyl radicals: Synthesis of new derivatives, determination of low oxygen contents, and stability studies Lisa Lampp a, Mykhailo Azarkh b, Malte Drescher b, Peter Imming a, * a b

Institute of Pharmacy, Martin Luther University Halle Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle Saale, Germany €tsstraße 10, 78464 Konstanz, Germany Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 February 2019 Received in revised form 23 March 2019 Accepted 26 March 2019 Available online 30 March 2019

Two new derivatives of galvinoxyl (1), a perdeutered (2) and an adamantyl-analog (3) for potential applications as spin probes were synthesized. The synthesis with deuterated educts yielded 2 with 98% D. It exhibited an 18-line EPR spectrum in octanol with narrow peak-to-peak linewidth. The EPR spectrum of 3 was very similar to galvinoxyl, but with differences in the linewidth due to unresolved longrange couplings with adamantyl-protons. Compound 2 showed a higher response to oxygen (4.8 mT/% O2) than 1 (2.8 mT/% O2). The coupling pattern of 2 allowed the determination of oxygen at low levels (0e6%) by a new type of analysis of the EPR pattern. The stability of the radicals strongly depended on the amount of hydrogalvinoxyl, a by-product of the galvinoxyl synthesis, and the solvent type. A molecular mechanism for the stabilization by hydrogalvinoxyl and the influence of solvent type is proposed. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Galvinoxyl radicals EPR Oximetry Mechanism of stabilization

1. Introduction Galvinoxyl or “Coppinger's” radical (1, Fig. 1) was first synthesized by Galvin M. Coppinger 1957 [1]. Since then the synthesis and characteristics of many derivatives of galvinoxyl radical have been published [2e4]. However, only a few derivatives are known with other substituents then tert. butyl in the ortho position. Only analogs with methyl or phenyl groups, but none with other sterically hindered alkyl groups were published [5,6]. While partially deuterated analogs of galvinoxyl radical were prepared [7e9], the fully deuterated galvinoxyl radical has not been reported. Little is known about the stability of galvinoxyl. In the presence of reducing agents like ascorbic acid, glutathione or cysteine, the radical is reduced immediately or within minutes [10]. Galvinoxyl reacts with other short-lived radicals which makes it a useful radical scavenger [11e13]. Coppinger stated that galvinoxyl is stable towards oxygen not only as solid, but also in solution [1]. Later authors found that pure galvinoxyl reacts quite fastly with oxygen, yielding 2,6-di-tert-butyl-1,4-benzoquinone and 3,5-di-tert-butyl4-hydroxybenzaldehyde [14]. Stabilization of the radical against oxygen can be achieved by hydrogalvinoxyl (1H, Fig. 11), an impurity that is formed during the synthesis of galvinoxyl and very

* Corresponding author. E-mail address: [email protected] (P. Imming). https://doi.org/10.1016/j.tet.2019.03.051 0040-4020/© 2019 Elsevier Ltd. All rights reserved.

difficult to remove due to the very similar chromatographic behavior [14]. The mechanism of stabilization is not known. The aim of this study was to prepare a fully deuterated galvinoxyl radical and a galvinoxyl derivative with adamantyl groups instead of tert.-butyl groups and examine their properties (compound 2 and 3, Fig. 1). We further studied the oxygen sensitivity of 1 and 2 to prove their suitability as spin probes for EPR oximetry. In view of applications of the radicals, we gained some insight into of stabilization of galvinoxyl by hydrogalvinoxyl and examined which other aspects influence the stability of galvinoxyl. 2. Materials and Methods 2.1. Synthesis 2.1.1. Materials and general methods for synthesis All chemicals used for synthesis were purchased and used without further purification. All organic solvents were purified before use. Column chromatography was performed on silica gel 60 (70e230 mesh or 230e400 mesh). The purity of all compounds and the progress of the reactions were monitored by thin layer chromatography using silica gel 60 F254 plates (Merck KGaA, Darmstadt, Germany). Visualizations were accomplished with an UV lamp (254 nm) or iodine staining. The Rf values given are uncorrected. 1H and 13C NMR spectra were recorded on an Agilent Technologies VNMRS 400 MHz or a Varian Inova 500 MHz

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Fig. 1. Structure of galvinoxyl (1), hydrogalvinoxyl (1H), perdeuterated galvinoxyl (2) and adamantyl-galvinoxyl (3).

spectrometer. Chemical shifts (d) are reported relative to the residual solvent peak of CDCl3 as internal standard: dH ¼ 7.26 ppm, dC ¼ 77.0 ppm. 13C NMR spectra of the deuterated compounds show more signals than expected. This is a result of coupling between carbon and deuterium atoms and arises also from incomplete exchange of protons which leads to different deuteration patterns. Infrared (IR) spectra were recorded on a Bruker IFS 28 FTIR spectrometer equipped with a Thermo Spectra-Tech attenuated total reflection (ATR) unit with a 20 mm ZnSe-Fresnel crystal. High resolution mass spectra (HRMS) were measured on a LTQ-Orbitrap-XL (ESI source) of Thermo Scientific. Samples were dissolved in chloroform-methanol mixtures. Deuterium incorporation was determined by NMR using hexamethylbenzene as internal standard or by high resolution mass spectrometry. 2.1.2. Synthesis of 2 by H/D exchange The substrate and catalysts (Method A: 5% Pd/C (20 wt%), 5% Pt/ C (20 wt%); Method B: 1% Pd/C (100 wt%), Pt/C (100 wt%)) in D2O were stirred at 180  C in an autoclave under H2 atmosphere (1 atm) for different time periods. The reaction mixture was diluted with DCM or CHCl3 and then filtered to remove the catalysts. The residue was washed several times with DCM or CHCl3. The combined organic layers were washed with water, dried over MgSO4 and were evaporated to dryness. The residue was purified by silica gel column chromatography. 2.1.2.1. 4-(6,3,3,3-D6)Propan-2-yl](2,6-D2)phenyl(D2)methyl)-2,6-bis [2-(D3)methyl(1,1,1,3,3,3-D6)propan-2-yl](D2)phenol (6). Method A: Compound 6 was not obtained. Method B (3 days): After purification on silica gel eluting with heptane/CHCl3 (8/2) compound 6 could be isolated as a pale yellow solid (18%). Rf ¼ 0.40 (heptane/CHCl3 8/2), 1H NMR (400 MHz, CDCl3): d 7.03 (d), 5.03 (d), 3.88e3.77 (m), 1.47e1.32 (m), 13C NMR (126 MHz,CDCl3): d 151.88, 151.86, 135.68, 135.66, 131.92, 125.32, 125.28, 41.03, 40.91, 40.83, 40.70, 34.31, 34.23, 30.33, 30.33, 29.84, 29.68, 29.54, 29.36, 29.25, 29.07. Degree of deuteration (NMR): 53% (for C29H2D42O2, tert. butyl 56%, ortho 5%, methylene bridge 95%). Method B (5 days): After purification on silica gel eluting with heptane/CHCl3 (8/2) compound 6 could be isolated as a pale yellow solid (4%). Rf ¼ 0.40 (heptane/CHCl3 8/2), 1H NMR (400 MHz, CDCl3): d 7.02 (s), 5.02 (s), 3.90e3.78 (m), 1.44e1.21 (m), 13C NMR (126 MHz, CDCl3): d 151.87, 135.65, 135.59, 131.92, 131.84, 131.75, 125.28, 125.17, 124.98, 124.81, 40.85, 40.58, 40.37, 33.67, 33.59, 30.32, 29.85, 29.69, 29.53, 29.38, 29.23, 29.08, 28.96. Degree of deuteration (NMR): 87% (for C29H2D42O2, tert. butyl 90%, ortho 53%, methylene bridge 96%). 2.1.2.2. Bis[2-(D3)methyl(1,1,1,3,3,3-D6)propan-2-yl](D3)phenol (9). Method A: Compound 9 was not obtained. Method B: After purification on silica gel eluting with pentane/DCM (10/0 to 8/2) 9 could be isolated as a colorless liquid. Yield: 28% after 2 d, 18% after 3 d. Degree of deuteration (NMR): 93% D (tert. butyl 95% D, ortho 69% D, para 96% D) independent of reaction time. Rf ¼ 0.38 (heptane/CHCl3 9/1), 1H NMR (500 MHz, CDCl3): d 7.18 (s), 5.18 (d), 1.49e1.38 (m), 13 C NMR (126 MHz, CDCl3): d 153.85, 135.88, 135.81, 124.87, 124.76,

124.68, 124.49, 124.30, 119.39, 119.11, 118.92, 33.65, 33.57, 30.30, 29.66, 29.50, 29.35, 29.20, 29.05, 28.90. 2.1.3. Synthesis of 2 from deuterated educts 2.1.3.1. 4-Bromo-2,6-bis[2-(D 3 )methyl(1,1,1,3,3,3-D 6 )propan-2yl](D2)phenol (11). Compound 10 (1 g, 5.64 mmol) was mixed in a glass pressure vessel with tert. butanol-d10 (2.66 ml, 28.24 mmol) and D2SO4 (150 ml, 2.66 mmol). Vessel was tightly closed and heated at 160  C for 3 h. After cooling to RT mixture was dissolved in 50 ml CHCl3 and washed successively with water and brine. The organic layer was dried over MgSO4 and solvent was evaporated to dryness. The residue was purified with silica gel, eluting with heptane/ethyl acetate (10/0 to 9/1). Compound 11 was isolated, all other fractions containing unreacted or monosubstituted 10 were evaporated and reaction was done again. The reaction was done four times in total to give 500 mg (29% yield) 11 as a white to pale yellow solid: mp 74e76  C, Rf ¼ 0.43 (heptane), 1H NMR (400 MHz, CDCl3): d 7.24 (s), 5.14 (s), 1.37 (s), 13C NMR (101 MHz, CDCl3): d 152.89, 138.06, 129.46e125.73 (m), 112.36, 34.75, 30.09e27.86 (m), HRMS (ESI): calcd. for C14D20BrO [M  H]- 303.196; found 303.196, degree of deuteration 97.7% (ESI-MS). 2.1.3.2. Bis[2-(D3)methyl(1,1,1,3,3,3-D6)propan-2-yl](D3)phenol (9). Compound 11 (456 mg, 1.49 mmol) dissolved in DCM/methanol (55 ml, 2/1) and 5% Pd/C (230 mg, 50 wt% of 11) were stirred under D2 atmosphere (8 bar) at RT for 7 h. Mixture was then filtered to remove the catalyst. The filtered catalyst was washed several times with DCM. The combined organic layers were evaporated to dryness. The residue was purified by column chromatography on silica gel, eluting with heptane/ethyl acetate (10/0.2) to give 340 mg (88% yield) of a colorless liquid: Rf ¼ 0.42 (heptane/ethyl acetate 10/0.2), 1 H NMR (400 MHz, CDCl3): d 7.17 (s), 6.83 (s), 5.17 (s), 1.45e1.38 (m), 13 C NMR (101 MHz, CDCl3): d 153.84, 135.80, 124.72, 124.48, 124.24, 119.38, 119.10, 118.86, 33.55, 29.38, 29.19, 29.00, HRMS (ESI): calcd. for C14D21O [M  H]- 226.292; found 226.296, degree of deuteration 97.7% (ESI-MS). 2.1.3.3. 4-({4-Hydroxy-3,5-bis[2-(D3)methyl(1,1,1,3,3,3-D6)propan-2yl](2,6-D2)phenyl}(D2)methyl)-2,6-bis[2-(D3)methyl(1,1,1,3,3,3-D6) propan-2-yl](D2)phenol (6). Compound 9 (197 mg, 0.867 mmol) and paraformaldehyde-d2 (111 mg, 3.464 mmol) were dissolved in 2 ml isopropanol-d1 and 250 ml D2O under argon atmosphere. Then the mixture was heated to 35  C and 100 ml 40% NaOD in D2O were added in one portion. The mixture was stirred for 40 min. During this time the color of the reaction mixture changed from colorless to dark red. Reaction mixture was then acidified with D2SO4 until the color changed to yellow. Afterwards the mixture was diluted with DCM. The organic layer was separated and the aq. layer was washed several times with DCM. The combined organic layers were dried over MgSO4 and evaporated to dryness. The residue was purified by column chromatography on silica gel eluting with heptane/CHCl3 (8/2) to give 115 mg (57% yield) of white solid: mp 151e152  C, Rf ¼ 0.39 (heptane/CHCl3 8/2), 1H NMR (500 MHz, CDCl3): d 7.03 (s), 5.03 (s), 3.76 (s), 1.38 (s), 13C NMR

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(126 MHz,CDCl3): d 151.87, 135.59, 131.75, 125.17, 124.99, 124.80, 40.56, 40.43, 40.33, 40.27, 33.66, 33.59, 29.84, 29.68, 29.54, 29.38, 29.23, 29.07, 28.94, 28.78, HRMS (ESI): calcd. for C29H1D42O2 [M  H]- 465.591; found 465.599, degree of deuteration 96.0% (ESIMS). 2.1.3.4. [4-({3,5-Bis[2-(D 3 )methyl(1,1,1,3,3,3-D 6 )propan-2-yl]-4oxo(2,6-D2)cyclohexa-2,5-dien-1-ylidene}(D)methyl)-2,6-bis[2-(D3) methyl(1,1,1,3,3,3-D6)propan-2-yl](3,5-D2)phenyl]oxidanyl (2). K3Fe(CN)6 (246 mg, 0.748 mmol) were dissolved under argon in 6.5 ml D2O. 1.3 ml 40% NaOD in D2O and 6 ml benzene-d6 were added. Compound 6 (97 mg, 0.208 mmol) was dissolved in 15 ml benzene-d6 and was added dropwise to the reaction mixture. The reaction mixture was stirred for another 60 min at RT under argon atmosphere. The organic layer was then separated and washed with water until the aq. phase was colorless. The organic layer was dried over MgSO4 and was evaporated to dryness. The dark blue product (97 mg, 100% yield) was used without further purification. Mp 150e153  C, Rf ¼ 0.18 (heptane/ethyl acetate 10/0.2), HRMS (ESI): calcd. for C29HD41O2 [MþH]þ 463.576; found 463.575; degree of deuteration 98% (ESI-MS), IR (ATR): 3619, 3550e3208, 3114, 2929, 2205, 2132, 2088, 2069, 2047, 1655, 1606, 1564, 1529, 1490, 1409, 1319, 1280, 1254, 1209, 1159, 1131, 1061, 1047, 1008, 958, 905, 780, 742 cm1. 2.1.4. Synthesis of 3 2.1.4.1. 2,6-Bis(adamantan-1-yl)-4-bromophenol (15). Compound 12 (1.5 g, 8.67 mmol) was mixed with 1-adamantanol (2.64 g, 17.34 mmol) and 1-bromoadamantane (392 mg, 1.82 mmol) in a glass pressure vessel and for 3 h at 210  C. After cooling to RT the reaction mixture was purified by column chromatography on silica gel, eluting with heptane to give 1.18 g (30% yield) of a white solid: mp > 260  C, Rf ¼ 0.35 (heptane), 1H NMR (400 MHz, CDCl3): d 7.19 (s, 2H), 5.32 (s, 1H), 2.10 (s, 18H), 1.78 (s, 12H), 13C NMR (126 MHz, CDCl3): d 153.58, 138.10, 127.75, 113.26, 41.01, 36.94, 36.86, 28.93, HRMS (ESI): calcd. for C26H33BrO [M]þ 440.171; found 440.170. 2.1.4.2. 2,6-Bis(adamantan-1-yl)phenol (16). 15 (200 mg, 0.453 mmol) was dissolved in 80 ml DCM/methanol (1/1). 5% Pd/C (100 mg, 50 wt%) were added. Mixture was set under H2 atmosphere (2 bar) and was stirred overnight at RT. The reaction mixture was then filtered to remove the catalyst. The residue was washed with DCM. Combined organic layers were evaporated to dryness. The reaction mixture was purified by column chromatography on silica gel, eluting with heptane to give 147 mg (90% yield) of a white solid: mp 255e258  C, Rf ¼ 0.32 (heptane), 1H NMR (400 MHz, CDCl3): d 7.12 (d, J ¼ 7.8 Hz, 2H), 6.87 (t, J ¼ 7.8 Hz, 1H), 5.35 (s, 1H), 2.22e2.04 (m, 18H), 1.80 (s, 12H), 13C NMR (126 MHz, CDCl3): d 154.57, 135.92, 124.71, 119.92, 41.21, 36.94 36.87, 29.05, HRMS (ESI): calcd. for C26H32O [M  H]- 361.254; found 361.252. 2.1.4.3. 2,6-Bis(adamantan-1-yl)-4-{[3,5-bis(adamantan-1-yl)-4hydroxyphenyl]methyl}phenol (17). Compound 16 (295 mg, 0.814 mmol) was suspended in 3.5 ml formic acid. Paraformaldehyde (24 mg, 0.814 mmol) was added. Mixture was stirred and heated to reflux for 2.5 h. After cooling to RT 2 ml water were added and the white precipitate was filtered off. The residue was dissolved in CHCl3 and washed with water until the aqueous layer was neutral. The organic layer was then dried over MgSO4 and was evaporated to dryness. The reaction mixture was purified by column chromatography on silica gel, eluting with heptane/CHCl3 (9/1 to 0/10) to give 250 mg (83% yield) of a white to pale yellow solid: mp  330  C (degradation), Rf ¼ 0.06 (heptane), 1H NMR (500 MHz, CDCl3): d 7.00 (s, 4H), 5.19 (s, 2H), 3.86 (s, 2H), 2.17e2.06 (m, 36H),

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1.79 (s, 24H), 13C NMR (126 MHz, CDCl3): d 152.55, 135.63, 132.05, 125.26, 41.47, 41.34, 37.06, 36.78, 29.09, HRMS (ESI): calcd. for C53H67O2 [M  H]þ 735.514; found 735.515. 2.1.4.4. [2,6-Bis(adamantan-1-yl)-4-{[3,5-bis(adamantan-1-yl)-4oxocyclohexa-2,5-dien-1-ylidene] methyl}phenyl]oxidanyl (3). K3Fe(CN)6 (129 mg, 0.391 mmol) was dissolved in 5 ml water. 1 ml 40% NaOH in water and 15 ml benzene were added. Mixture was set under argon atmosphere. Compound 17 (80 mg, 0.109 mmol) was dissolved in 60 ml benzene and was added dropwise to the reaction mixture. After completely addition of 17 the mixture was stirred for another 60 min during which the color of the mixture turned dark red to brown. The organic layer was then separated, washed with water until the aq. layer was colorless, dried over MgSO4 and was evaporated to give 100 mg (125% yield) of dark violet solid. NMR analysis showed no residual educt or other sideproducts, but a benzene signal (7.36 ppm) which explains the surplus of 25%. The residual benzene could not be removed under vacuum. 3 was used without further purification for the following experiments. Mp  250  C (degradation), Rf ¼ 0.31 (heptane/CHCl3 8/2), HRMS (ESI): calcd. for C53H65O2 [M]þ 733.499; found 733.497, IR (ATR): 3004, 2966, 2898, 2845, 2675, 2655, 1573, 1510, 1453, 1341, 1312, 1253, 1224, 1216, 1204, 1164, 1104, 1024, 983, 908, 827, 751 cm1. 2.2. Characterization 2.2.1. EPR spectroscopy Measurements at defined oxygen contents were conducted in 4 ml glass vials using an EPR spectrometer at 1.3 GHz (Magnettech, Berlin, Germany) equipped with a re-entrant resonator. Measurements were done under ambient conditions without temperature control. General settings were as follows: microwave power 0.42 mW; modulation frequency, 100 kHz; sweep, 0.75e2 mT (depending on linewidth); scan time, 600e1200 s. The modulation amplitude was set so that no line distortions occurred. Stability measurements were done in 50 ml capillaries using an X-band EPR spectrometer at 9.30e9.55 GHz (Miniscope MS 200, Magnettech, Berlin, Germany). Measurements were done under ambient conditions without temperature control. General settings were as follows: microwave power 3.162 mW; modulation frequency, 100 kHz; sweep, 6.76 mT; scan time 60 s. The modulation amplitude was set so that no line distortions occurred. 2.2.2. Measurements at defined oxygen contents For the experiments commercial 1 with about 50% 1H (Sigma Aldrich, determined by HPLC analysis, see supporting information) was used. The amount of 2H in compound 2 could not be determined due to missing pure 2H as reference substance. Samples in toluene (1 ml, 1 mM) were flushed with pure nitrogen at a flow rate of 0.8 l/min for 3 min using septum vials and needles. Samples in octanol (1 ml, 1 mM) were flushed with either pure nitrogen or defined mixtures of oxygen and nitrogen at a flow rate of 0.8 l/min €ger, for 3 min. An anesthesia gas mixer with flow meter tubes (Dra Lübeck, Germany) provided defined gas mixtures. The oxygen content (in %) was measured in the solution directly after the EPR measurements by a needle-type optical oxygen microsensor with temperature control assuming ambient pressure (Type PSt1, PreSens GmbH, Regensburg, Germany). Oxygen sensitivities were determined by plotting the simulated EPR linewidth as a function of oxygen concentration and calculating the slope by linear regression fit. For deuterated galvinoxyl the oxygen sensitivity was also calculated by the ratio of the maximum amplitude and central amplitude.

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2.2.3. Stability measurements All samples were prepared under ambient air and were filled into 50 ml glass capillaries directly before measurement. Capillaries were sealed by melting both sides. The area under the curve (AUC) was calculated by double integration of the spectra. The AUC of the first spectrum measured after preparation of each sample was set as 100%. For ESI-MS measurements samples were stored under air in 4 ml brown glass vials. ESI-MS measurements were recorded on an LCQ classic (Thermo Finnigan, San Jose, California, USA). 2.2.4. Spectral simulations with EasySpin EPR spectra were analyzed and simulated with Matlab using EasySpin [15] for the spectral simulations. In general function “Garlic” for fast and isotropic spectra with either Gaussian or Lorentzian peak-to-peak-linewidth was used for simulation of EPR spectra. The g-factors were not determined and set to 2.0023 for all simulations. 3. Results and discussion 3.1. Synthesis 3.1.1. Synthesis of the perdeuterated galvinoxyl radical (2) We first approached the fully deuterated analog of the galvinoxyl radical by catalytic H/D exchange (Fig. 2) from undeuterated 4,40 -methylenebis(2,6-di-tert-butylphenol) (4), a commercially available, low-priced precursor of galvinoxyl. H/D-exchange in alkyl-substituted aromatic compounds can be achieved by using a mixture of palladium-on-carbon and platinum-on-carbon with D2O as deuterium source at high temperature in the presence of a small amount of H2 gas [16]. The mixture of Pd/C-Pt-C/D2O-H2 has a synergistic effect, wherein Pd/C catalyzes mainly the H/D-exchange reaction at alkyl chains and Pt/C the exchange reaction at aromatic rings. The use of 5% Pd/C (20 wt %) with 5% Pt/C (20 wt %) unexpectedly led to the formation of the cyclohexanone derivative 5. Compound 5 was identified by 13C NMR and IR spectroscopy (for spectra see Supporting Information). The degree of deuteration for compound 5 was not determined. When 1% Pd/C (100 wt %) with 1% Pt/ C (100 wt %) were used, the desired product 6 was isolated albeit with incomplete H/D exchange. After 24 h, almost no H/D exchange had occurred. Proton and 13C NMR revealed that only protons at the methylene bridge were exchanged with high yield. After 3 days, the overall degree of deuteration was about 53% according to NMR analysis. Only 5% H/D exchange occurred at the ortho position, the tert. butyl groups were 56% deuterated, the methylene bridge 95%.

Fig. 2. Deuteration of 4 by H/D exchange reaction. Reagents and Conditions: (I) 5% Pd/ C (20 wt %), 5% Pt/C (20 wt %), D2O, 180  C, H2 (1 atm), 15e24 h. (II) 1% Pd/C (100 wt %), 1% Pt/C (100 wt %), D2O, 180  C, H2 (1 atm), 24 h to 5 d.

ESI-MS analysis also showed there was residual educt with no H/D exchange. Increasing the reaction time to 5 days markedly improved the deuteration (tert. butyl groups 90% D, ortho position 53% D, methylene bridge 96% D), but dropped the yield to 4%. The low deuteration efficiency in ortho position was most likely caused by steric hindrance of the bulky tert. butyl groups. The low yield was a result of degradation and polymerization processes. Therefore we decided to start from an undeuterated precursor with better accessible ortho positions, namely 2,6-di-tert-butylphenol (6, Fig. 3). Use of 5% Pd/C (20 wt %) with 5% Pt/C (20 wt %) again led to the formation of a cyclohexanone derivative (8, spectra see Supporting Information). With 1% Pd/C (100 wt %)/1% Pt/C (100 wt %), the desired product 9 was isolated with 28% yield after 2 days and 18% after 3 days. The overall degree of deuteration was 92% with 69% exchange in the ortho positions. Increasing the reaction time to 3 days did not increase the H/D exchange. Further attempts to improve the exchange in the ortho positions were not successful. Although indeed the degree of deuteration was markedly improved by using compound 7 instead of compound 4, the ortho deuteration was still insufficient for our purposes. Therefore we approached compound 2 by still another pathway, starting with a deuterated benzene nucleus (Fig. 4). First, perdeuterated tert. butyl groups were introduced in the ortho positions of compound 10 by a Friedel-Crafts alkylation with tert. butanol-D10 under acidic conditions. To avoid alkylation in para position, deuterated 4-bromophenol (10) was used as educt. Bromine was removed from intermediate 11 by catalytic reduction. The synthetic steps III and IV followed a literature procedure [17], but using deuterated reactants and reagents. The deuteration degree of compound 6 was found to be 96% (ESI-HRMS). The proton NMR revealed residual protons were located at each position. Compound 1, the fully deuterated galvinoxyl radical, contained a marked amount of hydrogalvinoxyl 2H. The formation of hydrogalvinoxyl during the last synthetic step is also known for undeuterated galvinoxyl radical [14]. Due to its very similar chromatographic behavior, the removal of hydrogalvinoxyl is very difficult and therefore 2 was used without further purification for the EPR experiments. 3.1.2. Synthesis of adamantyl-galvinoxyl (3) The tert. butyl groups of galvinoxyl have only been replaced by phenyl and methyl groups, leading to radicals with complicated EPR spectra due to hyperfine couplings [5,6]. We replaced the tert. butyl groups by bulky adamantyl groups which do not lead to

Fig. 3. Deuteration of 7 by H/D exchange reaction. Reagents and conditions: (I) 5% Pd/C (20 wt %), 5% Pt/C (20 wt %), D2O, 180  C, H2 (1 atm), 15 h to 3 d. (II) 1% Pd/C (100 wt %), 1% Pt/C (100 wt %), D2O, 180  C, H2 (1 atm), 2 de3 d.

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Fig. 4. Synthesis of compound 2 starting from deuterated educts. Reagents and conditions: (I) tert. butanol (D10, 98%), D2SO4 (99.5% D), 160  C, 3 h, glass pressure vessel. (II) 5% Pd/C (50 wt %), D2 (8 bar), DCM/methanol-D1 (2:1), rt, 7 h (III) paraformaldehyde (D2, 99%), isopropanol-D1, NaOD (40%) in D2O, 35  C, argon, 40 min. (IV) K3Fe(CN)6, NaOD (40%) in D2O, benzene-D6, argon, rt, 60 min.

additional hyperfine couplings. While conferring about the same degree of steric hindrance, the more rigid tertiary alkyl moiety of adamantyl with methylene instead of methyl groups in the vicinity of the spin system was expected to help with the analysis of the hyperfine aspects of the EPR spectra. The synthetic pathway is shown in Fig. 5. The most crucial part of the synthesis is the alkylation of the aromatic ring, since due to the steric hindrance twofold substitution with bulky alkyl groups is difficult. To exclude formation of inseparable product mixtures, we started the synthesis again from 4-bromophenol (12). The alkylation was done following a literature procedure [18]: compound 12 was heated to 210  C with a mixture of 1-adamantanol (13) and 1bromoadamantane (14) to give 15 which was converted into compound 16 by reductive debromation with Pd/C as catalyst. Reaction of 16 with paraformaldehyde resulted in the formation of compound 17 with high yield. The synthesis was done under strongly acidic conditions by using formic acid as solvent. This is in contrast to the synthesis of galvinoxyl where the condensation with formaldehyde is done under basic conditions. With the adamantyl derivatives, base-catalyzed condensation of 16 with

Fig. 5. Synthesis of compound 3. Reagents and conditions: (I) 1-adamantanol, 1bromoadamantane, 210  C, 3 h, glass pressure vessel. (II) 5% Pd/C (50 wt %), H2 (5 bar), methanol/DCM, rt, overnight. (III) Paraformaldehyde, formic acid, reflux, 2 h. (IV) K3Fe(CN)6, NaOH (40%) in H2O, benzene, argon, rt, 60 min.

paraformaldehyde led to the formation of a product mixture, perhaps because of additional substitution at the meta positions that are sterically more hindered next to tert. butyl groups. Adamantyl-galvinoxyl 3 was generated from 17 by oxidation with potassium ferricyanide. According to TLC and NMR analysis, 17 was converted completely into radical 3. No residual hydro-adamantylgalvinoxyl 3H (Fig. 12(B)) was found. 3.2. EPR spectra of the galvinoxyl derivatives The EPR spectra of the different galvinoxyl derivatives (c ¼ 1 mM) were recorded in toluene and octanol under anoxic conditions (Fig. 6 and Fig. 8).

Fig. 6. Experimental (black) and simulated (red) EPR spectra of (A) adamantylgalvinoxyl 3 and (B) galvinoxyl 1 (1 mM in toluene under nitrogen atmosphere at room temperature). Spectral parameters were as follows: frequency, 1.3 GHz; microwave power 0.42 mW; sweep time, 600 s; modulation frequency, 100 kHz; sweep, 2 mT; modulation amplitude 0.0045 mT (depending on the linewidth); number of points, 1024.

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Table 1 Simulation parameter of 3 and 1.

3 1

a1 [MHz]

a2 [MHz]

lwpp (Lorentz) [mT]

17.35 17.30

4.20 4.10

90 40

3.2.1. Adamantyl-galvinoxyl (3) The spectrum of commercial galvinoxyl radical 1 shows the common hyperfine coupling pattern of a doublet split into quintets due to the hyperfine coupling with one methine and four aromatic protons. The spectrum of the adamantyl derivative consists also of a doublet (coupling with methine proton) with both peaks split to quintets (coupling with aromatic protons). Though the ratio of intensities within the quintet in both spectra is 1:4:6:4:1, the appearance of the EPR spectra is different. The simulation of the spectra shows that this is caused by an increase of the peak-to-peak linewidth (lwpp) while the coupling constants a1 (coupling with methine proton) and a2 (coupling with aromatic protons) are about the same (Table 1). The hyperfine coupling of the alkyl protons was not resolved in both radicals, but led to line broadening, which was accounted for in the linewidth of the simulations. Adamantyl groups contain three magnetically different types of protons (Fig. 7, g-H, d-H, ε-H)

all contributing to the broadening of the EPR signal, while tert. butyl groups contain only the equivalent of g-protons. The increased linewidth of adamantyl-galvinoxyl results from unresolved longrange hyperfine couplings not only with g-protons, but also with d- and ε-protons of the adamantyl groups. Long-range couplings are based on p-s-spin delocalization and are particularly effective if the 2pz-axis of the aromatic moiety and if the s-bonds linking it with the g- and d-protons lie almost in plane and are arranged in a zigzag line [19]. For d-protons this is called W arrangement [19]. In the adamantyl galvinoxyl 3 (Fig. 7), this zigzag arrangement is fixed in the rigid annulated ring system, and rotation is only possible around the C(a)-C(b)-bond. 3.2.2. Perdeuterated galvinoxyl (2) The EPR spectrum of perdeuterated galvinoxyl radical 2 in toluene (Fig. 8 (A)) shows partly resolved hyperfine structure with four equivalent aromatic deuterium atoms and a single (methine) deuterium atom. The EPR linewidth as obtained from spectral simulations is reduced by 37.5% compared to the undeuterated galvinoxyl radical. The reduced hyperfine coupling constants correlate with the smaller gyromagnetic constant of deuterium compared to hydrogen (Table 2). In octanol, the resolution of the spectrum is increased due to reduced linewidths (Fig. 8 (B)). The difference of the spectra measured in toluene and octanol arises from different solvent properties such as polarity and from differences in specific interactions between the solvent and the radical. The signal pattern is composed of 18 lines with a linewidth of 11 mT which is very narrow. The precursor of the deuterated galvinoxyl (compound 6) contained residual protons at each position, the overall degree of deuteration being 96%. The additional signals of not fully deuterated species are visible in the low- and high-field of the spectrum measured in octanol. 3.3. Oxygen sensitivity of undeuterated and perdeuterated galvinoxyl radical

Fig. 7. 3D structure of alkyl-substituents of (A) adamantyl-galvinoxyl 3 and (B) galvinoxyl radical 1.

Galvinoxyl type radicals react with oxygen to form different degradation products. In the presence of the reduced species (1H and 2H), galvinoxyl radicals are stabilized with the potential to become probes for oxygen determination. Therefore we thoroughly investigated the influence of oxygen on the EPR spectra of galvinoxyl and analogs. Since galvinoxyl type radicals have a high lipophilicity and are not soluble in aq. solutions, it was first necessary to define a solvent which on the one hand dissolves the radicals, but on the other hand is biocompatible for potential in vivo applications. Stability tests in 1-octanol showed no degradation during the course of several hours. Octanol is known to be well tolerated in vivo. To prove the applicability of galvinoxyl radicals as oxygensensitive spin probes, EPR spectra of commercial and deuterated galvinoxyl (1 mM in octanol) were measured at different oxygen levels. For in-vivo applications, oxygen levels between 0 and 10% are of particular interest. 3.3.1. Galvinoxyl 1 The peak-to-peak linewidth of the spectra was extracted from the experimental data by simulating the experimental results with

Fig. 8. Experimental (black) and simulated (red) EPR spectra of perdeuterated galvinoxyl in (A) toluene and (B) octanol (1 mM under nitrogen atmosphere at room temperature). Spectral parameters were as follows: frequency, 1.3 GHz; microwave power 0.42 mW; sweep time, 600e1200 s; modulation frequency, 100 kHz; sweep 0.75 mT; modulation amplitude,  0.0011 mT (depending on linewidth); number of points, 1024.

Table 2 Simulation parameters of perdeuterated galvinoxyl in toluene and octanol.

Toluene Octanol

a1 [MHz]

a2 [MHz]

lwpp (Lorentz) [mT]

2.59 2.59

0.64 0.64

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Matlab using the EasySpin function “Garlic”. For simulation parameters see Materials and Methods and the Supplementary Information. To achieve the best simulation, 0% O2-spectra were simulated using Gaussian line shape. Above 0% oxygen, Lorentzian line shape was used for the simulation. Undeuterated galvinoxyl shows a linear relationship between EPR linewidth (peak-to-peak) and oxygen concentration. The linewidth range (Fig. 9) is comparable with the linewidth of lipophilic chlorinated trityls dissolved in oils (e.g. isopropyl myristate) [20]. The oxygen sensitivity is about 2.8 mT/% O2. The stronger response to oxygen compared to hydrophilic oxygen spin probes (e.g. hydrophilic tertrathia-trityls, 0.5 mT/ % O2 [20]) is a result of the high oxygen solubility in octanol compared to water (1-octanol 1.5 mmol/l, water 0.29 mmol/l oxygen at 20  C, 0.213 bar O2 [21]):at the same oxygen partial pressure octanol dissolves more oxygen and thus a more intense Heisenberg spin exchange and line broadening occurs. In contrast to trityl radicals, galvinoxyl is a commercially available and low-priced spin probe. Due to this and the comparable oxygen sensitivity of chlorinated trityl radicals, galvinoxyl is a reasonable alternative for EPR oximetry applications in lipophilic formulations, esp. at oxygen levels of physiological interest.

3.3.2. Perdeuterated galvinoxyl 2 The EPR spectrum of deuterated galvinoxyl in octanol shows a well resolved hyperfine structure under anoxic conditions. With increasing oxygen concentration (Fig. 10 (A)), the signal is broadened which leads to a complete loss of the hyperfine structure between 5% and 6% oxygen and one broad signal above 6%. The change of oxygen concentration causes both a change of (1) linewidth and (2) line shape. (1) The linewidth of the spectra was determined by simulating the experimental results with Matlab using EasySpin. For simulation parameters, see Materials and Methods and Supplementary Information. The linewidth was calculated as peak-to-peak linewidth on the basis of a Lorentzian model. Fig. 10 (B) shows the linear relationship between linewidth and oxygen concentration. Due to the narrow lines at 0% oxygen, deuterated galvinoxyl exhibits better oxygen sensitivity (4.8 mT/% oxygen) than the undeuterated radical which

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is advantageous for the measurement of small changes in oxygen concentrations. (2) The change of the line shape is directly connected with the change of the linewidth: the broadening leads to a superstition of signals and thus to a change to the amplitudes. Due to that the signal amplitudes can reflect the spin exchange phenomena between the galvinoxyl radical and oxygen. This change of line shape was expressed as the ratio of the maximum amplitude and the amplitude in the center of the spectrum (Fig. 10 (C) and asterisks in Fig. 10 (A)). This calculation provides a reproducible non-linear relationship between line shape and oxygen concentration up to 6% oxygen. The loss of reproducibility above 6% oxygen is associated with the loss of hyperfine structure at this oxygen level. From 1% to 6% oxygen, linearization is possible by taking the logarithm of y-axis (see Supporting Information). This facilitates data analysis. To our knowledge, the calculation of oxygen levels by analyzing the maximum amplitude and the amplitude in the center of the spectrum of a perdeuterated radical has not been reported before. It takes advantage of the narrow and very distinct coupling pattern due to deuterium atoms. So again, deuterated galvinoxyl has a better oxygen sensitivity than fully hydrogenated galvinoxyl. This is advantageous when small changes in oxygen concentrations have to be monitored. 3.4. Stability studies with commercial and adamantyl-galvinoxyl The stability of galvinoxyl radicals depends on different factors. It is known that degradation due to reaction with oxygen is inhibited by hydrogalvinoxyl [14], but to our knowledge this behavior has not been further examined. 3.4.1. Formation of the hydro-analog of galvinoxyl type radicals We found commercial galvinoxyl (Sigma Aldrich) to contain about 50% hydrogalvinoxyl 1H (HPLC analysis, see supporting information). For the synthesis of perdeuterated galvinoxyl we found that the hydro-analog is formed during the last synthetic step as a result of incomplete oxidation of the precursor 6. Formation of hydrogalvinoxyl does not occur during storage of the compound.

Fig. 9. (A) Oxygen calibration curve of commercial galvinoxyl dissolved in octanol (c ¼ 1 mM) (n ¼ 3). (B) EPR spectra of galvinoxyl at different oxygen concentrations. Spectral parameters were as follows: frequency, 1.3 GHz; microwave power 0.42 mW; sweep time, 1200 s; modulation frequency, 100 kHz; sweep, 2 mT; modulation amplitude 0.0045 mT (depending on the linewidth); number of points, 1024.

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Fig. 10. (A) EPR spectra of perdeuterated galvinoxyl 2 (1 mM in octanol) at different oxygen concentrations; parameters see Fig. 8 (B) Oxygen calibration curve of 2 dissolved in octanol (c ¼ 1 mM, n ¼ 3) determined by linewidth change. (C) Oxygen calibration curve of perdeuterated galvinoxyl dissolved in octanol (c ¼ 1 mM, n ¼ 3) determined by change of amplitudes.

This is in accordance with published literature [5]. Increase of the reaction time or of the amount of potassium ferricyanide did not lead to complete formation of 6 to radical 2. Fig. 11 shows possible reaction pathways for the formation of 1 resp. 1H. They are both formed in a multi-step oxidation process starting from the methylenebisphenol 4, and accompanied by rearrangement and disproportionation of intermediates.

Fig. 11. Possible reaction pathway for the formation of galvinoxyl (1) starting from methylenebisphenol (4). Figure adapted from Omelka et al. [5].

3.4.2. Influence of the hydro-analog on the radical stability The presence of 1H next to 1 leads to an inhibition period during which only minor degradation occurs. After this period, the reaction with oxygen causes rapid degradation of the radical. Identifiable degradation products are 2,6-di-tert-butyl-4hydroxybenzaldehyde and 2,6-di-tert-butyl-1,4-benzoquinone [14,22]. The length of the inhibition period is dependent on the hydrogalvinoxyl concentration [14]. We also found this behavior for adamantyl-galvinoxyl 3 in the presence of different amounts of 3H (Fig. 12 (A)). The 3, 3H-mixtures were generated by mixing the radical with different amounts of the methylenebisphenol precursor 17 (Fig. 12 (B)). Compound 3H was formed directly after mixing of both compounds. The samples for the stability tests were prepared under atmospheric air and were measured in sealed 50 ml capillaries to prevent gas exchange with ambient air during the measurements. EPR measurements over different time periods showed intense degradation of the pure radical after 1 h. After 1.8 h, 50% of the EPR

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Fig. 12. (A) Stabilization of 3 (cstart ¼ 1 mM in toluene) by 3H. AUC was calculated by double integration of the EPR spectra. (B) Formation of the hydro-analog (3H) of adamantylgalvinoxyl (2). (C) EPR spectra of 3 with 0% 3H and 50% 3H.

signal of 3 had vanished. With increasing concentration of the hydro-analog 3H the inhibition period until fast degradation was prolonged. The rate of the fast degradation did not change up to about 21% 3H. Above 21% of 3H, the degradation rate was reduced. Fig. 12 (C) shows the EPR spectra of adamantyl-galvinoxyl 3 with and without the presence of 3H. The EPR signal intensity of pure 3 decreased over time while resolution of the hyperfine structure increased. At about 30% decrease of the AUC after 1.75 h, the hyperfine structure was well resolved due to linewidth reduction. This was easily explained by oxygen consumption during the degradation process. This was also observed for the other samples, exemplarily shown for 50% 3H content. However, in contrast to the behavior of pure 3, the signal amplitude slightly increased when 50 or 60 wt% of 3H were present. (Below 50% 3H, this behavior was less distinct.) If this resulted from the fact that reduction of the linewidth compensated the decrease of signal intensity, the signal amplitudes would increase or stay constant. The smaller linewidths suggest that at the same signal intensity (measured as AUC) the oxygen concentration was lower in the sample with the higher amount of 3H. This observation on galvinoxyl stabilization by their hydro-analogs suggests the following mechanistic possibilities:  The hydro-analog may protect the radical by steric shielding and diminish the interaction between radical and oxygen. However, the linewidth of the first spectrum of each sample does not change with increasing concentration of the hydro-analog which should be the case if 3H indeed diminishes the direct interaction between radical and oxygen. It seems more likely that a reaction between hydrogalvinoxyl and oxygen or between hydrogalvinoxyl and galvinoxyl occurred:  The hydro-analog may react directly with oxygen to form either the radical and-or other degradation products. However, ESI-MS analysis of a pure 1H solution (c ¼ 1 mM, toluene) did not show any degradation products resulting from reaction with oxygen. The solution showed a very weak EPR signal under standard conditions for galvinoxyl. Its intensity (measured as AUC) did not increase after bubbling pure oxygen through the solution (10 min, 0.1 l/min). (For the synthesis of 1H see Supplementary Information.) A third possibility seems to be supported best by the data:

 The hydro-analog reactivates galvinoxyl-oxygen adducts like hydro- or endoperoxides by reduction and is oxidized itself to the radical by reaction with these adducts, leading to the observed degradation profile over time. This mechanism is further supported by the behavior of 1 in different solvents (see below).

3.4.3. Stabilization of galvinoxyl radicals by other compounds To shed more light on the degradation and stabilization mechanism, the stabilization of 3 with other substances was investigated. The addition of p-chloranil and benzophenone as bulky aromatic compounds did not result in any stabilization of the radical, supporting the third possibility (v.s. 3.4.2). The addition of phenol and 2,6-di-tert-butylphenol did lead to stabilization, but TLC analysis showed that 3H was formed. The enhanced stability is therefore again a result of the presence of 3H. 3.4.4. Influence of the solvent on radical stability EPR measurements of 1 (stabilized with 1H) in different solvents showed that the stability of galvinoxyl radicals was also solvent dependent (Fig. 13). On the basis of the results shown in Fig. 13 the following conclusions were drawn:  The influence of oxygen solubility in the solvents on stability was negligible. Chloroform and cyclohexane both dissolve 2.4 mM oxygen (0.213 bar oxygen, 20  C) [21], but their ability to stabilize 1 is completely different. The same applies to octanol and butanol. They dissolve oxygen at 1.5 mM and 1.8 mM (0.213 bar oxygen, 25  C) [21].  With decreasing polarity resp. increasing lipophilicity, the stability increases. 1 is stable over 6 h in nonpolar and less polar solvents like heptane, cyclohexane, toluene and octanol, but undergoes degradation in more polar solvents like butanol, chloroform, and dichloromethane. Galvinoxyl solutions in toluene, heptane, octanol, butanol, benzyl alcohol and dichloromethane were analyzed by ESI-MS to see which degradation products were formed. After one day, toluene, butanol, benzyl alcohol and DCM solutions contained no residual 1. Heptane and octanol still contained 1 which was corroborated by EPR spectrometry. After 3 days in heptane, 1 was completely degraded while the octanol solution still showed an

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Fig. 13. Stability of 1 in different solvents (cstart ¼ 1 mM). AUC was calculated by double integration of the EPR spectra.

intense EPR signal. Even after 5 days in octanol, an EPR signal was measurable. Analysis of the degradation products revealed a difference between protic and aprotic solvents: In aprotic solvents, the degradation products were 2,6-di-tert-butyl-4hydroxybenzaldehyde, 2,6-di-tert-butyl-1,4-benzoquinone and different oxygen adducts like the endo- or hydroperoxide. In protic solvents, the main degradation product was the hydro-analog 1H. This was also the main degradation product in octanol after complete loss of the EPR signal. Thus the reaction rate with oxygen seems to be diminished in solvents with increasing lipophilicity, and it was insignificant in protic solvents. The following mechanistic conclusions rationalize this behavior. In lipophilic solvents, the interaction between galvinoxyl radicals and hydro-analog is facilitated as both are not solvated well. They form complexes through polar interactions (e.g. H-bonds, charge transfer), preparing them for mutual interactions (e.g. H-atom and electron transfer). This increases the probability that the radical is formed again by oxidation of hydrogalvinoxyl through H-atom transfer. This actually supports the third theory regarding the stabilization of 3 by 3H, presented in Section 3.4.2. The dependence of the ease of H-atom transfer between phenols and galvinoxyl on the polarity of aprotic solvents was found in earlier studies [23]. Obviously, H-atom transfer is much faster in protic solvents than reaction with oxygen. For the future use of galvinoxyl and analogous radicals the above observations and mechanistic explanations translate into the following practical conclusion. For studies in aqueous solutions including body fluids, galvinoxyls should be packed in a relatively lipophilic medium with neutral pH, adding 50 wt% or more of the hydro-analog. Thus, they can serve as oxygen sensors over a couple of days. Oxygen levels below 6% - that are actually of greater interest physiologically than higher oxygen levels - can be assessed best when determining both line width and line shape as reported above. 4. Conclusion In this study we showed the synthesis and examined the properties of two new galvinoxyl type radicals. Adamantylgalvinoxyl 3 was synthesized with high purity. The adamantyl groups lead to an increased linewidth compared to 1 due to unresolved long range couplings with protons at these groups, whereas the hyperfine coupling with the aromatic protons and methine proton is not influenced. Perdeuterated galvinoxyl 2 was

synthesized with a high degree of deuteration. The EPR spectrum yields a many-line-pattern due to partly resolved hyperfine couplings with deuterium atoms and very small linewidth under anoxic conditions. The oxygen sensitivity of undeuterated and perdeuterated galvinoxyl was examined. Both radicals show good oxygen sensitivity within the physiological range which makes them to potential probes for EPR oximetry. The mechanism of stabilization by hydro-analogs of galvinoxyl-type radicals was examined. Due to the results shown in this study the most reasonable mechanism seems to be a direct reaction of the hydroanalog and radical-oxygen-adducts with can either lead to the reactivation of the radical or to the oxidation of the hydro-analog to the radical. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.tet.2019.03.051. References [1] G.M. Coppinger, A stable phenoxyl radical inert to oxygen, J. Am. Chem. Soc. 79 (1957) 501e502. [2] W. Gierke, W. Harrer, H. Kurreck, J. Reusch, Metallorganische Synthese und EPR-Spektroskopie von Galvinoxyl-Verbindungen, Tetrahedron Lett. 38 (1973) 3681e3684. [3] W. Gierke, W. Harrer, H. Kurreck, J. Reusch, Über Galvinole und Galvinoxyle, II EPR-HFS- und elektronen-spektroskopische Untersuchung von GalvinoxylMehrspinsystemen, Z. Naturforsch. B Chem. Sci. 31 (1976) 965e973. [4] W. Harrer, H. Kurreck, J. Reusch, W. Gierke, Über Galvinole und GalvinoxylMehrspinsystemedI : eine neue metallorganische Synthese von Mono- und Oligo-Galvinolen, Tetrahedron 31 (1975) 625e632.  , J. Pospísil, Radical reactions in the co-ordi[5] L. Omelka, A. Tk a c, L. Jira ckova nation sphere of transition metals. XIIIRadicals derived from 4,40 - and 2,20 Alkylidenebisphenolic Antioxidants, Org. Magn. Reson. 19 (1982) 153e159. [6] I.V. Khudyakov, P.P. Levin, K. De Ionge, K.M. Bekman, Electronic spectra of some phenyl-substituted phenoxyl radicals, Russ. Chem. Bull. 26 (1977) 1525e1527. [7] J. von Gersdorff, B. Kirste, H. Kurreck, Synthesis and EPR and ENDOR investigations of Coppinger's radical with perdeuteriated tert-butyl groups, Magn. Reson. Chem. 24 (1986) 1077e1079. [8] B. Kirste, H. Kurreck, M. Sordo, Syntheses and ENDOR studies of selectively deuterated galvinoxyl radicals. Complete determination of the 13C hyperfine coupling constants of Coppinger's radical, Chem. Ber. 118 (1985) 1782e1797. [9] C. Tsiao, C. Wild, J. Gu, H.C. Dorn, Preparation of the a-[D]galvinoxyl radical, J. Label. Comp. Radiopharm. 24 (1991) 945e948. [10] J. Tsuchiya, T. Yamada, E. Niki, Y. Kamiya, Interaction of galvinoxyl radical with ascorbic acid, cysteine, and glutathione in homogeneous solution and in aqueous dispersions, Bull. Chem. Soc. Jpn. 58 (1985) 326e330. [11] K. Schwarz, G. Bertelsen, L.R. Nissen, P.T. Gardner, M.I. Heinonen, A. Hopia, T. Huynh-Ba, P. Lambelet, D. McPhail, L.H. Skibsted, L. Tijburg, Investigation of

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