2,5-Di-tert-butyl-2,5-diethylpyrrolidine-1-oxyls: Where Is a Reasonable Limit of Sterical Loading for Higher Resistance to Reduction?

The pyrrolidine nitroxides with four bulky alkyl substituents adjacent to the N–O∙ group demonstrate very high resistance to reduction with biogenic antioxidants and enzymatic systems. This makes them valuable molecular tools for studying the structure and functions of biomolecules directly in a living cell and for functional EPR and NMR tomography in vivo. The first example of highly strained pyrrolidine nitroxides with both ethyl and tert-butyl groups at each of the α-carbon atoms of the nitroxide moiety with cis-configuration of the tert-butyl groups was prepared using a three-component domino reaction of tert-leucine and 2,2-dimethylpentan-3-one with dimethyl fumarate with subsequent conversion of the resulting strained pyrrolidine into 1-pyrroline-1-oxide and addition of EtLi. The nitroxide has demonstrated unexpectedly fast reduction with ascorbate, the rate constant k2 = (2.0 ± 0.1) × 10−3 M−1s−1. This effect was explained by destabilization of the planar nitroxide moiety due to repulsion with the two neighboring tert-butyl groups cis to each other.


Introduction
Nitroxides are a broad family of organic free radicals which have been of constantly growing interest to researchers for many decades [1][2][3].The relative simplicity of chemical modification of nitroxide structures resulted in the synthesis of countless numbers of derivatives, allowing for tuning of their chemical and physical properties and spectral parameters for specific applications in various fields of science and technology [4][5][6][7][8][9][10][11][12].Rapid development of the chemistry of nitroxides is facilitating the progress in their applications.For example, the recent findings in synthesis of the nitroxides with enhanced stability to chemical reduction [13][14][15][16] opened up new opportunities for studying the structure and functions of biomolecules directly in a living cell [17][18][19][20] and for the development of new reagents for functional EPR and NMR tomography in vivo [21][22][23][24].The higher stability of these so-called "sterically shielded" nitroxides resulted from introduction of four ethyl groups to the neighboring carbons of the nitroxide moiety instead of methyls, typical for a broad majority of conventionally used nitroxides.This effect has been carefully studied by various authors [25][26][27][28], and it was demonstrated that ring size and steric and electronic effects of the substituents play important roles.However, synthesis of highly sterically loaded structures remains a challenge.
It is known that additional large splittings on the methylene hydrogens of the ethyl (n-alkyl) groups may be observed in the ESR spectra of pyrrolidine radicals with substituents in the 3 and 4 positions of the heterocycle [16,34,35].These splittings were assigned to hyperfine coupling (hfc) with γ-hydrogen in the side chain and result from overlapping of the smaller lobe of the C-H orbital in the α-position of the side alkyl chain and the nonbonding orbital of the nitroxide group [38].This overlapping is efficient when the ethyl group is in the pseudoaxial position of the pyrrolidine ring with the CH2-CH3 bond nearly parallel to the N-O axis.The introduction of a bulky group, such as tert-butyl, makes this conformation unfavorable and leads to the disappearance of the large hfc on the methylene hydrogen of the adjacent ethyl group, which makes the spectrum simpler.
Considering the above-described influence of the tert-butyl group on the properties of nitroxides, it can be assumed that a similar radical with two tert-butyl groups in positions 2 and 5 should be characterized by a higher resistance to reduction and a simple triplet EPR spectrum without large extra splittings.This work describes our efforts to synthesize such a radical and some of its properties and spectral features.To the best of our knowledge, the only communication on synthesis of a stable cyclic nitroxide with two tertbutyl groups at the α-carbons (5) was given at the conference [37], and this information was never published elsewhere.
It is known that additional large splittings on the methylene hydrogens of the ethyl (nalkyl) groups may be observed in the ESR spectra of pyrrolidine radicals with substituents in the 3 and 4 positions of the heterocycle [16,34,35].These splittings were assigned to hyperfine coupling (hfc) with γ-hydrogen in the side chain and result from overlapping of the smaller lobe of the C-H orbital in the α-position of the side alkyl chain and the non-bonding orbital of the nitroxide group [38].This overlapping is efficient when the ethyl group is in the pseudoaxial position of the pyrrolidine ring with the CH 2 -CH 3 bond nearly parallel to the N-O axis.The introduction of a bulky group, such as tert-butyl, makes this conformation unfavorable and leads to the disappearance of the large hfc on the methylene hydrogen of the adjacent ethyl group, which makes the spectrum simpler.
Considering the above-described influence of the tert-butyl group on the properties of nitroxides, it can be assumed that a similar radical with two tert-butyl groups in positions 2 and 5 should be characterized by a higher resistance to reduction and a simple triplet EPR spectrum without large extra splittings.This work describes our efforts to synthesize such a radical and some of its properties and spectral features.To the best of our knowledge, the only communication on synthesis of a stable cyclic nitroxide with two tert-butyl groups at the α-carbons (5) was given at the conference [37], and this information was never published elsewhere.

Results and Discussion
A reaction of amino acids with carbonyl compounds and activated alkenes is known to proceed via oxazolidin-5-one formation with subsequent release of carbon dioxide to give azomethine ylide (Scheme 1).
1,3-Dipolar cycloaddition of alkenes to azomethine ylides can give a set of isomeric pyrrolidines, resulting from the reaction of the "W"-, "S"-or "U"-shaped ylide with the alkene approaching from the upper or lower side [39,40].We have previously observed formation of two diastereoisomeric pyrrolidines in the reactions with symmetric ketones [33,34], which is the maximal number of isomers that can form upon the reaction of two possible configurations of an azomethine ylide with a symmetric trans-alkene (Scheme 2).Introduction of an asymmetric ketone into this reaction was expected to give four isomers due to addition of a new asymmetric center in position 5 of the heterocycle.
1,3-Dipolar cycloaddition of alkenes to azomethine ylides can give a set of isomeric pyrrolidines, resulting from the reaction of the "W"-, "S"-or "U"-shaped ylide with the alkene approaching from the upper or lower side [39,40].We have previously observed formation of two diastereoisomeric pyrrolidines in the reactions with symmetric ketones [33,34], which is the maximal number of isomers that can form upon the reaction of two possible configurations of an azomethine ylide with a symmetric trans-alkene (Scheme 2).Introduction of an asymmetric ketone into this reaction was expected to give four isomers due to addition of a new asymmetric center in position 5 of the heterocycle.According to our previous experience [34], the yield of the target pyrrolidine in the three-component domino reaction of amino acids with ketones and activated alkenes may be sensitive to steric hindrance (volume of the substituents in the reagents); however, the resulting pyrrolidines can be easily separated via extraction with aqueous acidic solution even if the yield is low.After heating tert-leucine (6) and 2,2-dimethylpentan-3-one (7) with dimethyl fumarate in a DMF-toluene mixture in Dean-Stark apparatus for 80 h, a mixture of basic compounds was separated using extraction with an acid, and pure compounds 8, 9 and 10 were isolated using column chromatography (Scheme 3).1,3-Dipolar cycloaddition of alkenes to azomethine ylides can give a set of isomeric pyrrolidines, resulting from the reaction of the "W"-, "S"-or "U"-shaped ylide with the alkene approaching from the upper or lower side [39,40].We have previously observed formation of two diastereoisomeric pyrrolidines in the reactions with symmetric ketones [33,34], which is the maximal number of isomers that can form upon the reaction of two possible configurations of an azomethine ylide with a symmetric trans-alkene (Scheme 2).Introduction of an asymmetric ketone into this reaction was expected to give four isomers due to addition of a new asymmetric center in position 5 of the heterocycle.According to our previous experience [34], the yield of the target pyrrolidine in the three-component domino reaction of amino acids with ketones and activated alkenes may be sensitive to steric hindrance (volume of the substituents in the reagents); however, the resulting pyrrolidines can be easily separated via extraction with aqueous acidic solution even if the yield is low.After heating tert-leucine (6) and 2,2-dimethylpentan-3-one (7) with dimethyl fumarate in a DMF-toluene mixture in Dean-Stark apparatus for 80 h, a mixture of basic compounds was separated using extraction with an acid, and pure compounds 8, 9 and 10 were isolated using column chromatography (Scheme 3).According to our previous experience [34], the yield of the target pyrrolidine in the three-component domino reaction of amino acids with ketones and activated alkenes may be sensitive to steric hindrance (volume of the substituents in the reagents); however, the resulting pyrrolidines can be easily separated via extraction with aqueous acidic solution even if the yield is low.After heating tert-leucine (6) and 2,2-dimethylpentan-3-one (7) with dimethyl fumarate in a DMF-toluene mixture in Dean-Stark apparatus for 80 h, a mixture of basic compounds was separated using extraction with an acid, and pure compounds 8, 9 and 10 were isolated using column chromatography (Scheme 3).The spot of 10 on TLC did not give the characteristic staining with Dragendorff s reagent, but HRMS of 8 and 10 and the element analysis data for 9 corresponded to the same formula, C18H33NO4.The spectra of 8 and 9 showed much similarity to those of previously described diastereomeric pyrrolidines [16,33].In the IR spectra of the 8 and 9 bands, N-H vibrations at 3500-3300 cm −1 were very weak, and C=O vibrations of the ester groups were represented by a single strong band at 1720-1740 cm −1 (see Section 3 and Figures S50 and S51, cf.[33]).Similarly to that described for mono-tert-butyl pyrrolidines [33], one of the isomers (9) showed well-resolved signals of methine protons in the NMR 1 H while they were overlapping in the spectrum of 8. Addition of CF3COOH allowed the spectrum to be recorded with resolved signals of these protons (Figures S3 and S5).The The spot of 10 on TLC did not give the characteristic staining with Dragendorff's reagent, but HRMS of 8 and 10 and the element analysis data for 9 corresponded to the same formula, C 18 H 33 NO 4 .The spectra of 8 and 9 showed much similarity to those of previously described diastereomeric pyrrolidines [16,33].In the IR spectra of the 8 and 9 bands, N-H vibrations at 3500-3300 cm −1 were very weak, and C=O vibrations of the ester groups were represented by a single strong band at 1720-1740 cm −1 (see Section 3 and Figures S50 and S51, cf.[33]).Similarly to that described for mono-tertbutyl pyrrolidines [33], one of the isomers (9) showed well-resolved signals of methine protons in the NMR 1 H while they were overlapping in the spectrum of 8. Addition of CF 3 COOH allowed the spectrum to be recorded with resolved signals of these protons (Figures S3 and S5).The NMR 13 C spectra of 8 and 9 demonstrated much difference, but were both consistent with the pyrrolidine structure (see Section 3 and Figures S4, S6 and S7).
To determine the relative configuration of the asymmetric centers in 8 and 9, 1 H-1 H NOESY correlation spectra were recorded (Figures S36 and S37, Appendix A), which showed that the tert-butyl groups are cis to each other in both isomers 8 and 9. Obviously, formation of these isomers results from addition of the dipolarophil from both sides of the "W"-shaped (with respect to tert-butyls) azomethine ylide plane because a "U" shape is hardly possible for the ylide with such bulky substituents.
In contrast, the IR spectrum of 10 showed a band at 1647 cm −1 , typical for C=N bond vibrations (Figure S52).The 1 H NMR spectrum of 10 (Figure S9) showed the signals of a four-spin system of two methylene and two methine protons, along with the signals of two tert-butyl groups, two methoxy groups and an ethyl group.The NMR 13 C spectrum (Figure S10) showed three signals between 173 and 178 ppm, a signal of a methylene carbon at 32.9 ppm and only two signals of methine carbons at 42.3 and 65.5 ppm.These data support assignment of the acyclic structure to 10. Formation of this compound could result from Michael addition of an azomethine ylide to dimethyl fumarate; for examples of similar reactions, see [41] and reference [2] therein.
The subsequent transformations of 8 were carried out in analogy with our previous syntheses of the sterically shielded nitroxides [16,33,34].The ester groups were reduced with LiAlH 4 to give 11 (Scheme 4).The structure of the new compound was assigned on the basis of IR, NMR 1 H and 13 C spectra and confirmed by the elemental analyses (see Section 3 and Figures S11, S12 and S53).Surprisingly, no trace of conversion was observed upon treatment of 11 with hydrogen peroxide in the presence of sodium tungstate.The desired oxidation to nitrone was achieved using m-chloroperbenzoic acid (mCPBA).To avoid overoxidation to oxoammonium cations with possible affection of the hydroxymethyl groups (cf.[42,43]), the amines were treated with ca.one equivalent of the oxidant.Analysis of the reaction mixtures using TLC showed the spots that give blue staining upon treatment with 10% solution of phosphomolibdic acid in ethanol, typical for hydroxylamines [16].To complete the oxidation, lead dioxide was added to the reaction mixtures after mCPBA was consumed.The resulting nitrone 12 was isolated with the yield 65%.The structure of 12 was proved using NMR 1 H and 13 C spectra and experiments examining correlations 1 H- 13 C HMBC and 1 H-1 H NOESY and supported by elemental analysis data (see Section 3, Figures S13, S14, S38 and S39 and Appendix A).
Earlier, the nitroxides 3a,b were prepared with 50-70% yield via addition of ethyl lithium to corresponding 2-tert-butyl-1-pyrroline-1-oxides [36].This reagent also rapidly reacted with the nitrone 13 to give the nitroxide 1a with 87% yield (Scheme 5).An alternative synthesis of this nitroxide from the same nitrone via addition of ethynylmagnesium bromide with subsequent hydrogenation gave 43%, and it took 72 days for the reaction to complete [34].The structure of 12 was proved using NMR 1 H and 13 C spectra and experiments examining correlations 1 H- 13 C HMBC and 1 H-1 H NOESY and supported by elemental analysis data (see Section 3, Figures S13, S14, S38 and S39 and Appendix A).
Earlier, the nitroxides 3a,b were prepared with 50-70% yield via addition of ethyl lithium to corresponding 2-tert-butyl-1-pyrroline-1-oxides [36].This reagent also rapidly reacted with the nitrone 13 to give the nitroxide 1a with 87% yield (Scheme 5).An alternative synthesis of this nitroxide from the same nitrone via addition of ethynylmagnesium bromide with subsequent hydrogenation gave 43%, and it took 72 days for the reaction to complete [34].
Earlier, the nitroxides 3a,b were prepared with 50-70% yield via addition of ethyl lithium to corresponding 2-tert-butyl-1-pyrroline-1-oxides [36].This reagent also rapidly reacted with the nitrone 13 to give the nitroxide 1a with 87% yield (Scheme 5).An alternative synthesis of this nitroxide from the same nitrone via addition of ethynylmagnesium bromide with subsequent hydrogenation gave 43%, and it took 72 days for the reaction to complete [34].Protected 1-pyrroline-N-oxide 14 was prepared from 12 using a previously described procedure [34] and isolated using column chromatography (Scheme 6), and the structure of this nitrone was confirmed by IR, NMR 1 H and 13 C spectra and the elemental analysis (Figures S15, S16 and S56).Contrary to our expectations, the reaction of 14 with an excess of ethyl lithium did not lead to the formation of a nitroxide radical.A single diamagnetic compound was isolated from the complex mixture of the reaction products.The highresolution mass spectrum of the new compound contained molecular ion [M + ] = 263.2612,which corresponded to the formula C18H33N, i.e., all oxygen atoms were lost.In addition to the signals of tert-butyl and n-alkyl groups, the NMR spectrum 1 H contained multiplets in the high field (0.12 and 0.77 ppm), apparently indicating formation of a cyclopropane ring (Figure S19).The detailed analysis of the spectrum with line shape simulations (Figure S48) allowed us to assign the structure 15 to the compound.Final assignment of the relative configuration of the asymmetric centers was performed on the basis of 1 H-1 H COSY, 1 H- 13 C HSQC, 1 H- 13 C HMBC and 1 H-1 H NOESY correlations (Figures S40-S43 and Appendix A).Scheme 6. Synthesis of 14 and its reaction with EtLi.
Compound 15 could be formed according to Scheme 7. Presumably the nitrone carbon in 14 is too hindered for the addition of EtLi to occur.As a result, metalation proceeds Scheme 5. Addition of EtLi to the nitrone 13.
Protected 1-pyrroline-N-oxide 14 was prepared from 12 using a previously described procedure [34] and isolated using column chromatography (Scheme 6), and the structure of this nitrone was confirmed by IR, NMR 1 H and 13 C spectra and the elemental analysis (Figures S15, S16 and S56).Contrary to our expectations, the reaction of 14 with an excess of ethyl lithium did not lead to the formation of a nitroxide radical.A single diamagnetic compound was isolated from the complex mixture of the reaction products.The highresolution mass spectrum of the new compound contained molecular ion [M + ] = 263.2612,which corresponded to the formula C 18 H 33 N, i.e., all oxygen atoms were lost.In addition to the signals of tert-butyl and n-alkyl groups, the NMR spectrum 1 H contained multiplets in the high field (0.12 and 0.77 ppm), apparently indicating formation of a cyclopropane ring (Figure S19).The detailed analysis of the spectrum with line shape simulations (Figure S48) allowed us to assign the structure 15 to the compound.Final assignment of the relative configuration of the asymmetric centers was performed on the basis of 1 H-1 H COSY, 1 H- 13 C HSQC, 1 H- 13 C HMBC and 1 H- 1

H NOESY correlations (Figures S40-S43 and Appendix A).
lithium to corresponding 2-tert-butyl-1-pyrroline-1-oxides [36].This reagent also rapidly reacted with the nitrone 13 to give the nitroxide 1a with 87% yield (Scheme 5).An alternative synthesis of this nitroxide from the same nitrone via addition of ethynylmagnesium bromide with subsequent hydrogenation gave 43%, and it took 72 days for the reaction to complete [34].Protected 1-pyrroline-N-oxide 14 was prepared from 12 using a previously described procedure [34] and isolated using column chromatography (Scheme 6), and the structure of this nitrone was confirmed by IR, NMR 1 H and 13 C spectra and the elemental analysis (Figures S15, S16 and S56).Contrary to our expectations, the reaction of 14 with an excess of ethyl lithium did not lead to the formation of a nitroxide radical.A single diamagnetic compound was isolated from the complex mixture of the reaction products.The highresolution mass spectrum of the new compound contained molecular ion [M + ] = 263.2612,which corresponded to the formula C18H33N, i.e., all oxygen atoms were lost.In addition to the signals of tert-butyl and n-alkyl groups, the NMR spectrum 1 H contained multiplets in the high field (0.12 and 0.77 ppm), apparently indicating formation of a cyclopropane ring (Figure S19).The detailed analysis of the spectrum with line shape simulations (Figure S48) allowed us to assign the structure 15 to the compound.Final assignment of the relative configuration of the asymmetric centers was performed on the basis of 1 H-1 H COSY, 1 H- 13 C HSQC, 1 H- 13 C HMBC and 1 H-1 H NOESY correlations (Figures S40-S43 and Appendix A).Scheme 6. Synthesis of 14 and its reaction with EtLi.
Compound 15 could be formed according to Scheme 7. Presumably the nitrone carbon in 14 is too hindered for the addition of EtLi to occur.As a result, metalation proceeds Scheme 6. Synthesis of 14 and its reaction with EtLi.
Compound 15 could be formed according to Scheme 7. Presumably the nitrone carbon in 14 is too hindered for the addition of EtLi to occur.As a result, metalation proceeds due to the relatively high acidity of β-hydrogen of the nitrone group.Subsequent nucleophilic substitution leads to cyclopropane ring formation.Earlier, we observed deoxygenation of the nitrone group and nucleophilic substitution of the OTMS group in the reaction of TMS-protected 2-tert-butyl-1-pyrroline-1-oxide with butyllithium [33].
Despite the synthesis of 2,5-di-tert-butylpyrrolidine nitroxide from 12 being unsuccessful, the pyrrolidines 8 and 9 can be converted into less hindered 3-unsubstituted nitrones in analogy to the previously described procedure [35].The major isomer 9 was subjected to oxidation with mCPBA in dichloromethane to give 16 (Scheme 8).The latter was subjected to alkaline hydrolysis in aqueous methanol at ambient temperature.The starting compound reacted completely within 48 h.After acidification of the reaction mixture, the products were extracted and heated to reflux in EtOAc in analogy to the previously described procedure [35].Surprisingly, the two major products (total yield 70%) did not demonstrate an acidic nature.The compounds were isolated using column chromatography with the yields 47 and 23%.NMR, IR and HRMS spectra of both compounds unambiguously corresponded to the structure of methyl esters 17 and 18 of the desired carboxylic acid 19 (Figures S23-S26, S59 and S60).Presumably, the low steric accessibility of the carbon of the ester group at position 4 of the heterocycle prevented its alkaline hydrolysis; however, in an alkaline solution, an inversion of the configuration of the neighboring asymmetric center could occur, which led to the formation of two diastereomers.
due to the relatively high acidity of β-hydrogen of the nitrone group.Subsequent nucleophilic substitution leads to cyclopropane ring formation.Earlier, we observed deoxygenation of the nitrone group and nucleophilic substitution of the OTMS group in the reaction of TMS-protected 2-tert-butyl-1-pyrroline-1-oxide with butyllithium [33].Despite the synthesis of 2,5-di-tert-butylpyrrolidine nitroxide from 12 being unsuccessful, the pyrrolidines 8 and 9 can be converted into less hindered 3-unsubstituted nitrones in analogy to the previously described procedure [35].The major isomer 9 was subjected to oxidation with mCPBA in dichloromethane to give 16 (Scheme 8).The latter was subjected to alkaline hydrolysis in aqueous methanol at ambient temperature.The starting compound reacted completely within 48 h.After acidification of the reaction mixture, the products were extracted and heated to reflux in EtOAc in analogy to the previously described procedure [35].Surprisingly, the two major products (total yield 70%) did not demonstrate an acidic nature.The compounds were isolated using column chromatography with the yields 47 and 23%.NMR, IR and HRMS spectra of both compounds unambiguously corresponded to the structure of methyl esters 17 and 18 of the desired carboxylic acid 19 (Figures S23-S26, S59 and S60).Presumably, the low steric accessibility of the carbon of the ester group at position 4 of the heterocycle prevented its alkaline hydrolysis; however, in an alkaline solution, an inversion of the configuration of the neighboring asymmetric center could occur, which led to the formation of two diastereomers.The minor isomer 21 was prone to rapid tarring when stored at ambient temperature in aerobic conditions.For this reason, the major isomer 20 was used in subsequent syntheses.After protection of the hydroxyl group, the resulting nitrone 22 was treated with a The minor isomer 21 was prone to rapid tarring when stored at ambient temperature in aerobic conditions.For this reason, the major isomer 20 was used in subsequent syntheses.After protection of the hydroxyl group, the resulting nitrone 22 was treated with a 2.5-fold excess of EtLi, affording the nitroxide 23 with 90% yield (77% from 20) (Scheme 9).The resulting nitroxide was isolated as an orange oil.The samples of 23 showed some evidence of slow decomposition at 25 • C. We previously reported on the thermal instability of some 2-tert-butyl-substituted nitroxides [33,44].The structure of the nitroxide was confirmed with the NMR spectra recorded after reduction with Zn in the presence of trifluoroacetic acid in methanol-D 4 .To avoid thermal decomposition of the radical, the mixture was first stirred at −15 • C for 5 min and then heated to reflux, cf.[35].The NMR 1 H spectrum showed singlets of two different tert-butyl groups, well-resolved multiplets of two different ethyl groups and signals of an OCH 2 -CH-CH 2 system (Figure S31).The data of NMR 13 C, IR (Figures S32 and S63) and HRMS spectra and elemental analysis data were in agreement with the assigned structure.The minor isomer 21 was prone to rapid tarring when stored at ambient temperature in aerobic conditions.For this reason, the major isomer 20 was used in subsequent syntheses.After protection of the hydroxyl group, the resulting nitrone 22 was treated with a 2.5-fold excess of EtLi, affording the nitroxide 23 with 90% yield (77% from 20) (Scheme 9).The resulting nitroxide was isolated as an orange oil.The samples of 23 showed some evidence of slow decomposition at 25 °C.We previously reported on the thermal instability of some 2-tert-butyl-substituted nitroxides [33,44].The structure of the nitroxide was confirmed with the NMR spectra recorded after reduction with Zn in the presence of trifluoroacetic acid in methanol-D4.To avoid thermal decomposition of the radical, the mixture was first stirred at −15 °C for 5 min and then heated to reflux, cf.[35].The NMR 1 H spectrum showed singlets of two different tert-butyl groups, well-resolved multiplets of two different ethyl groups and signals of an OCH2-CH-CH2 system (Figure S31).The data of NMR 13 C, IR (Figures S32 and S63) and HRMS spectra and elemental analysis data were in agreement with the assigned structure.Bulky alkyl substituents make 23 insoluble in water, so it was converted into hydrophilic derivative 26 to carry out reduction rate and EPR spectra parameter measurements in conditions similar to previous measurements [16,35,36,45] (Scheme 9).The structure of 26 was confirmed by IR (Figure S65), HRMS TOF (ESI) and NMR 1 H and 13 C spectra recorded after the reduction with Zn/CF3COOH as described above (Figures S33, S34 and  S49).The relative configuration of the asymmetric centers was determined using 1 H-1 H COSY, 1 H- 13 C HSQC, 1 H- 13 C HMBC and 1 H-1 H NOESY correlations (Figures S44-S47 and  Appendix A).Analysis of the correlations showed that the synthesis afforded the nitroxides with cis-configuration of the tert-butyl groups.Formation of this isomer results from the addition of EtLi from the side opposite to the position of the bulky tert-butyl group.
In agreement with our expectations, the EPR spectra of 26 showed broadened triplet with peak-to-peak linewidths, Hp-p = 0.226 mT.This spectrum resembles the spectra of the reduction-resistant nitroxides 3a,b and 4. Thus, introduction of two tert-butyl groups into positions 2 and 5 allowed us to remove the large additional hfc with γ-hydrogens.
Analysis of the kinetics of reduction of nitroxides 1a and 26 with ascorbate in the presence of glutathione gave second-order rate constants (7.0 ± 2) × 10 −5 M −1 s −1 and (2.0 ± 0.1) × 10 −3 M −1 s −1 , correspondingly.While the value of the rate constant for 1a was in line with our previous data for highly strained nitroxides [33,36], the rate constant for 26 was even higher than those for 3-monosubstituted 2,2,5,5-tetraethylpyrrolidin-1-oxyls [35].This result seems paradoxical.However, we earlier noticed that the effect of bulky substituents adjacent to the nitroxide group is associated with relative stabilization or destabilization of a nitroxide and corresponding hydroxylamine [26].Obviously, the repulsion of the oxygen atom with the two neighboring tert-butyl groups makes the planar geometry of the nitroxide group unfavorable, and reduction to pyramidal hydroxylamine makes the molecule less distorted.This example clearly demonstrates that the effect of bulky substit- Bulky alkyl substituents make 23 insoluble in water, so it was converted into hydrophilic derivative 26 to carry out reduction rate and EPR spectra parameter measurements in conditions similar to previous measurements [16,35,36,45] (Scheme 9).The structure of 26 was confirmed by IR (Figure S65), HRMS TOF (ESI) and NMR 1 H and 13 C spectra recorded after the reduction with Zn/CF 3 COOH as described above (Figures S33, S34 and S49).The relative configuration of the asymmetric centers was determined using 1 H-1 H COSY, 1 H- 13 C HSQC, 1 H- 13 C HMBC and 1 H-1 H NOESY correlations (Figures S44-S47 and  Appendix A).Analysis of the correlations showed that the synthesis afforded the nitroxides with cis-configuration of the tert-butyl groups.Formation of this isomer results from the addition of EtLi from the side opposite to the position of the bulky tert-butyl group.
In agreement with our expectations, the EPR spectra of 26 showed broadened triplet with peak-to-peak linewidths, H p-p = 0.226 mT.This spectrum resembles the spectra of the reduction-resistant nitroxides 3a,b and 4. Thus, introduction of two tert-butyl groups into positions 2 and 5 allowed us to remove the large additional hfc with γ-hydrogens.
Analysis of the kinetics of reduction of nitroxides 1a and 26 with ascorbate in the presence of glutathione gave second-order rate constants (7.0 ± 2) × 10 −5 M −1 s −1 and (2.0 ± 0.1) × 10 −3 M −1 s −1 , correspondingly.While the value of the rate constant for 1a was in line with our previous data for highly strained nitroxides [33,36], the rate constant for 26 was even higher than those for 3-monosubstituted 2,2,5,5-tetraethylpyrrolidin-1oxyls [35].This result seems paradoxical.However, we earlier noticed that the effect of bulky substituents adjacent to the nitroxide group is associated with relative stabilization or destabilization of a nitroxide and corresponding hydroxylamine [26].Obviously, the repulsion of the oxygen atom with the two neighboring tert-butyl groups makes the planar geometry of the nitroxide group unfavorable, and reduction to pyramidal hydroxylamine makes the molecule less distorted.This example clearly demonstrates that the effect of bulky substituents is not limited to a decrease in the accessibility of the nitroxide moiety, and distortions and tensions produced by bulky substituents may play an important role for nitroxide stability to reduction.This effect should be taken into account in the molecular design of reduction-resistant nitroxides.

General Information
The IR spectra were recorded on a Bruker Vector 22 FT-IR spectrometer (Bruker, Billerica, MA, USA) in KBr pellets (1:150 ratio) or in neat samples (Figures S50-S65) and are reported in wave numbers (cm −1 ). 1 H NMR spectra were recorded on a Bruker AV 400 (400.134MHz), DRX 500 (500.130MHz) and AV 600 (600.300MHz) spectrometers (Bruker, Billerica, MA, USA). 13C NMR spectrum was recorded on a Bruker AV 400 (100.033MHz), DRX 500 (125.032MHz) and AV 600 (150.075MHz) (Figures S1-S49).All the NMR spectra were acquired for 5-10% solutions in CDCl 3 , (CD 3 ) 2 CO or CD 3 OD at 300 K using the signal of the solvent as a standard.To confirm the structure of stable nitroxides, NMR spectra were recorded of the solutions of corresponding amines prepared via reduction of the nitroxide samples in analogy to the previously described method [35].To avoid thermal decomposition of the radical, the solution of a nitroxide (20-40 mg) in CD 3 OD (0.5 mL) was cooled to −15 • C, and trifluoroacetic acid (0.1 mL) was added dropwise within 5 min, then the mixture was heated to reflux for 5 min and filtered into an NMR tube, cf.[35].HRMS analyses were performed using a High-Resolution Mass Spectrometer DFS (Thermo Electron, Waltham, MA, USA) and Bruker micrOTOF-Q (Bruker, Billerica, MA, USA).
The X-ray diffraction experiments for crystals of 20 and 21 were carried out at 296(2) K on a Bruker KAPPA APEX II diffractometer (graphite-monochromated Mo Kα radiation).Reflection intensities were corrected for absorption by the SADABS program.The structures were solved by direct methods using the SHELXT 2014/5 program [46] and refined by anisotropic (isotropic for all H atoms) with full-matrix least-squares method against the F2 of all reflections by SHELXL2018/3 [46].The positions of the hydrogen were calculated geometrically and refined in a riding model.Crystallographic data for 20 and 21 have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication no.CCDC 2312569, CCDC 2312570.A copy of the data can be obtained free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: +44 122 3336033 or email: deposit@ccdc.cam.ac.uk; internet: www.ccdc.cam.ac.uk, accessed on 1 January 2020).
A solution of propionaldehyde (20 g, 0.356 mol) in dry diethyl ether (70 mL) was added dropwise within 3 h to a solution of t-BuMgCl prepared from 43 g (0.463 mol) of tert-butylchloride and 11 g of magnesium foil in 500 mL of dry diethyl ether upon active stirring.The reaction mixture was then left overnight at room temperature and carefully quenched with water until viscous inorganic sludge formation.The organic solution was separated by decantation, and the sludge was washed repeatedly with portions of diethyl ether.The combined extract was dried with sodium carbonate and concentrated under reduced pressure without heating.The resulting compound contained up to 8% Et 2 O and was then used without purification.Yield of 2,2-dimethylpentan-3-ol was 31.3 g (72%). 1 H NMR spectra (Figure S1) corresponded to the literature data [50].
calculated geometrically and refined in a riding model.Crystallographic data for 20 and 21 have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication no.CCDC 2312569, CCDC 2312570.A copy of the data can be obtained free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: +44 122 3336033 or e-mail: deposit@ccdc.cam.ac.uk; internet: www.ccdc.cam.ac.uk, accessed on 1 January 2020).
A solution of propionaldehyde (20 g, 0.356 mol) in dry diethyl ether (70 mL) was added dropwise within 3 h to a solution of t-BuMgCl prepared from 43 g (0.463 mol) of tert-butylchloride and 11 g of magnesium foil in 500 mL of dry diethyl ether upon active stirring.The reaction mixture was then left overnight at room temperature and carefully quenched with water until viscous inorganic sludge formation.The organic solution was separated by decantation, and the sludge was washed repeatedly with portions of diethyl ether.The combined extract was dried with sodium carbonate and concentrated under reduced pressure without heating.The resulting compound contained up to 8% Et2O and Scheme 10.Synthesis of 2,2-dimethylpentan-3-one (7).

Preparation of Ethyllithium Solution
The EtLi solution was prepared in analogy to the literature procedure [52].Small portions of bromoethane (up to ca. 150 µL) were added to a stirred suspension of finely chopped lithium tape (2 g, 286 mmol) in dry pentane (50 mL) in argon atmosphere until the reaction initiated (which was manifested by self-refluxing and violet coloring).Then, a solution of bromoethane (8 mL, 107 mmol) in dry pentane (15 mL) was added dropwise.The resulting purple suspension was heated to reflux for 1 h, then cooled down to room temperature.The suspension was allowed to settle, and the pentane solution of ethyllithium was siphoned to another vessel through a cannula under argon pressure.Then, dry benzene (50 mL) was added to precipitate, the mixture was stirred for 15 min and the benzene solution was separated as described above.The combined pentane-benzene solution contained 0.7-0.9M EtLi (measured by titration with N-benzylbenzamide [53]).The solution was used immediately after preparation.
3.2.8.Procedure for the Synthesis of (2R(S),3R(S),4R(S),5S(R))-2,2,5-Triethyl-5-tert-butyl-3,4bis(hydroxymethyl)-pyrrolidine-1-oxyl (1a) The ethyllithium solution (11 mL, 7.7-9.9mmol) was added dropwise with stirring to a solution of nitrone 13 (1.34 g, 3.34 mmol) in dry hexane (10 mL) in argon atmosphere.When the reaction was complete (ca.30 min; control by TLC of an aliquot quenched with water, silica gel, eluent ethyl acetate-hexane 2:1, R f = 0.4 for 13), the mixture was carefully quenched with brine, the organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 × 10 mL).The combined organic extract was evaporated in a vacuum, and the residue was dissolved in methanol (30 mL).A 1 mL amount of solution of PPTS (50 mg, 0.2 mmol) in water was added, and the mixture was bubbled with air for 2 days.Then, the solvents were distilled off in vacuum, and the residue was dissolved in ethyl acetate (20 mL), washed with water (3 × 10 mL) and dried over magnesium sulfate.The solvent was distilled off in vacuum, and the residue was triturated with diethyl ether to afford 1a, yield 0.83 g (87%).Yellow crystals, m.p. 106-109 A solution of 17 or 18 (0.98 g, 3.46 mmol) in dry THF (10 mL) was added dropwise to a stirred suspension of LiAlH 4 (0.26 g, 6.92 mmol) in dry THF (10 mL).After addition was complete, the reaction mixture was stirred at reflux for 1 h, then cooled in an ice bath and carefully quenched with 1 mL of 5% aqueous sodium hydroxide and then 1 mL of water.The organic solution was separated, the inorganic mass was washed with diethyl ether (5 mL × 3 times).The combined extract was dried with magnesium sulfate and stirred vigorously with MnO 2 for 2 days for oxidation of the hydroxylamines formed to the corresponding nitrones (TLC control, silica gel, eluent methanol-dichloromethane 1:50, UV detection, R f = 0.8 for the hydroxylamines, 0,2 for 20 or 21).Then, the precipitate of oxidizing agent was filtered off, and the solvent was removed in vacuum.The crude product was purified via column chromatography on silica gel using methanol-dichloromethane 1:50 mixture as eluent and recrystallized from diethyl ether.

EPR Measurements and Kinetics
The EPR spectra were recorded for the 0.2 mM solutions of the nitroxides in 5 mM phosphate-citrate-borate buffer on a Bruker ER-200D-SRC spectrometer in a 50 µL glass capillary for 0.2 mM radical solutions (Figure S70).Spectrometer settings: frequency, 9.87 GHz; microwave power, 5.0 mW; modulation amplitude, 0.05 mT; time constant, 50-100 ms; and conversion time, 5.12 ms.For kinetic measurements in water, stock solutions of nitroxide, ascorbic acid and glutathione in phosphate-citrate-borate buffer (5 mM, pH 7.4) were prepared, and pH was adjusted to 7.4 with NaOH or HCl.All the solutions were deoxygenated with argon, were carefully and quickly mixed in a small tube to attain final concentrations (nitroxide, 0.2-0.4mM; GSH, 5 mM; and ascorbate, 100-300 mM) and were placed into an EPR capillary (50 µL).The capillary was sealed on both sides and

Scheme 2 .
Scheme 2. Formation of pyrrolidines in a three-component reaction of amino acids, carbonyl compounds and activated alkenes.

Scheme 1 .Scheme 1 .
Scheme 1. Formation of pyrrolidines in a three-component reaction of amino acids, carbonyl compounds and activated alkenes.

Scheme 2 .
Scheme 2. Formation of pyrrolidines in a three-component reaction of amino acids, carbonyl compounds and activated alkenes.

Scheme 2 .
Scheme 2. Formation of pyrrolidines in a three-component reaction of amino acids, carbonyl compounds and activated alkenes.

Figure 2 .
Figure 2. The structure of 20 (left) and 21 (right) according to single-crystal X-ray diffraction data.

Scheme 8 .
Scheme 8. Synthesis of 16, alkaline hydrolysis and decarboxylation.Synthesis of 20 and 21.Treatment of 17 and 18 with LiAlH 4 leads to reduction of both the ester group to hydroxymethyl one and the nitrone group to hydroxylamine.Subsequent oxidation with manganese dioxide recovered the nitrone group to give 20 and 21.The data of a single crystal X-ray analysis of these compounds (Figures 2, S66 and S67) showed the relative configuration of the asymmetric centers and confirmed the structure of 17, 18, 20 and 21.The minor isomer 21 was prone to rapid tarring when stored at ambient temperature in aerobic conditions.For this reason, the major isomer 20 was used in subsequent syntheses.After protection of the hydroxyl group, the resulting nitrone 22 was treated with a 2.5-fold excess of EtLi, affording the nitroxide 23 with 90% yield (77% from 20) (Scheme 9).The resulting nitroxide was isolated as an orange oil.The samples of 23 showed some evidence of slow decomposition at 25 • C. We previously reported on the thermal instability of some 2-tert-butyl-substituted nitroxides[33,44].The structure of the nitroxide was confirmed with the NMR spectra recorded after reduction with Zn in the presence of trifluoroacetic acid in methanol-D 4 .To avoid thermal decomposition of the radical, the mixture was first stirred at −15 • C for 5 min and then heated to reflux, cf.[35].The NMR 1 H spectrum showed singlets of two different tert-butyl groups, well-resolved multiplets of two different ethyl groups and signals of an OCH 2 -CH-CH 2 system (FigureS31).The data of NMR13 C,

Scheme 8 .
Scheme 8. Synthesis of 16, alkaline hydrolysis and decarboxylation.Synthesis of 20 and 21.Treatment of 17 and 18 with LiAlH4 leads to reduction of both the ester group to hydroxymethyl one and the nitrone group to hydroxylamine.Subsequent oxidation with manganese dioxide recovered the nitrone group to give 20 and 21.The data of a single crystal X-ray analysis of these compounds (Figures 2, S66 and S67) showed the relative configuration of the asymmetric centers and confirmed the structure of 17, 18, 20 and 21.

Figure 2 .
Figure 2. The structure of 20 (left) and 21 (right) according to single-crystal X-ray diffraction data.