Synthesis and Properties of (1R(S),5R(S),7R(S),8R(S))-1,8-Bis(hydroxymethyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl: Reduction-Resistant Spin Labels with High Spin Relaxation Times

Abstract

Site-directed spin labeling followed by investigation using Electron Paramagnetic Resonance spectroscopy is a rapidly expanding powerful biophysical technique to study structure, local dynamics and functions of biomolecules using pulsed EPR techniques and nitroxides are the most widely used spin labels. Modern trends of this method include measurements directly inside a living cell, as well as measurements without deep freezing (below 70 K), which provide information that is more consistent with the behavior of the molecules under study in natural conditions. Such studies require nitroxides, which are resistant to the action of biogenic reductants and have high spin relaxation (dephasing) times, T_m. (1R(S),5R(S),7R(S),8R(S))-1,8-bis(hydroxymethyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl is a unique nitroxide that combines these features. We have developed a convenient method for the synthesis of this radical and studied the ways of its functionalization. Promising spin labels have been obtained, the parameters of their spin relaxation T₁ and T_m have been measured, and the kinetics of reduction with ascorbate have been studied.

1. Introduction

Site-directed spin labeling coupled to Electron Paramagnetic Resonance spectroscopy (SDSL–EPR) is a rapidly expanding powerful biophysical technique to study biomolecules in physiologically relevant environments [1,2,3,4,5,6]. These methods are based on site-specific introductions of paramagnetic labels (unpaired electrons) into biomolecules of interest with subsequent investigation using various EPR techniques. The latter give inter-spin distances, solvent accessibility, the polarity of its immediate environment, and the dynamics of the labeled region, which are complimentary to information obtained by other methods of structural biology, such as NMR, X-ray crystallography, and cryo-electron microscopy. This helps to identify biologically important conformations of large biomolecules, especially membrane and intrinsically disordered proteins. Nitroxides are the most widely used spin labels [7]. Unlike other spin labels, they can be used both to study local dynamics in biomolecules at physiological temperatures and to measure inter-spin distances [5]. Examples of specific applications of nitroxide spin labels to structural biology studies of protein can be found in hundreds of publications [2].

The best way to obtain correct information about the native structure and functions of biomolecules implies their study in natural conditions—in a living cell. Currently, a set of evidence has been accumulated for the difference in protein structure and function in model and natural conditions [8,9,10]. For this reason, SDSL–EPR experiments in living cells attract more and more attention. A serious obstacle to the use of nitroxide spin labels in living cells is the rapid reduction of the nitroxide into diamagnetic compounds by low molecular weight reductants and enzymatic systems, typically found within cells [11]. Resistance of nitroxides to bioreduction can be strongly improved via introduction of several bulky alkyl (larger than methyl) substituents to the α-carbon atom of the nitroxide group [12]. A number of reduction-resistant spin labels for in-cell application have been developed based on tetraethyl nitroxides [13,14,15,16,17,18,19].

Several EPR methods have been developed for inter-spin distance measurement [20]. All of them are dependent on electron spin relaxation parameters of spin labels [2,21,22]. In Pulsed Electron–Electron Double Resonance (PELDOR or DEER) technique, which is currently the most popular approach for distance measurements, the maximal distance one can measure and the precision of the distance distribution are determined by the phase memory time (T_m) of the spin label [22]. The T_m parameter is temperature-dependent and must be as high as possible. To achieve optimal performance using conventional tetramethyl or tetraethyl spin labels, the measurements must be carried out at 40–65 K, because higher temperature rotation of the alkyl groups leads to a decrease in T_m. This rotation is impossible in nitroxides with spirocyclic moieties at α-carbons of nitroxide group, and these spirocyclic spin labels can be used for measurements at much higher temperatures (125 K) and even at room temperature [23]. Regretfully, the spirocyclic spin labels demonstrate much lower resistance to bioreduction compared to tetraethyl nitroxides [24].

According to expert estimates, labels that can eliminate the need for data acquisition at cryogenic temperatures and labels that can enter and survive in the cellular environment are of current and future interest [2]. The attempts to improve reduction resistance of spirocyclic nitroxides have been a subject of recent research [25]. The literature data on the rate constants of chemical reduction of representative dispirocyclic nitroxides and tetraethyl nitroxides are listed in the Figure 1. The data demonstrate that reduction resistance of the nitroxide 1 by far exceeds those of other spirocyclic nitroxides. The nitroxide group in 1 is stabilized with two spiro-(2-hydroxymethyl)cyclopentane moieties with the hydroxymethyl groups directed towards N-O^•. In addition, this nitroxide showed the highest longitudinal relaxation time T₁ among a broad set of various nitroxides [26]. This parameter is very important for distance measurement using Saturation Recovery (SR) method, another pulsed EPR technique widely used in structural biology [2,21].

The nitroxide 1 was prepared from 3,4-di-tert-butoxypyrroline 1-oxide [31] via repetitive sequence of procedures: pent-4-en-1-ylmagnesium bromide addition, intramolecular 1,3-dipolar cycloaddition, isoxazolidine ring opening and oxidation [27,30]. The literature protocol implies multi-step synthesis with low overall yield (ca. 5%) from commercially available L-tartaric acid, making 1 unfavorable object for further chemical transformations [27,30].

Recent advances in condensation, cyclization, and dipolar cycloaddition cascade chemistry [32] allowed us to develop convenient and scalable protocol for synthesis of dispirocyclic nitroxide 5, a 3,4-unsubstituted analog of 1, with overall yield 43% from commercially available 4-chlorobutyryl chloride (6). Chemical properties of the nitroxide 5 were studied and several dispirocyclic spin labels were prepared. The new nitroxides showed improved resistance to reduction compared to 1. The spin relaxation times T₁ and T_m were measured for some of the nitroxides.

2. Results and Discussion

2.1. Synthesis

The (1R(S),8R(S))-6-oxa-5-azatricyclo[6.3.0.01,5]undecane (8) (6aR(S),9aR(S))-hexahydro-1H,6H-cyclopenta[c]pyrrolo[1,2-b]isoxazole) was prepared in two steps as enantiomeric mixture according to the literature protocol [32] (Scheme 1). This allowed us to avoid multi-step synthesis.

Subsequent oxidative isoxazolidine ring opening with m-cloroperoxybenzoic acid (m-CPBA) afforded aldonitrone 9. The reaction was carried out in dry dichlorometane (DCM) using dry reagent to prevent formation of hydroxamic acid. The structure of 9 was confirmed with IR, UV, ¹H, and ¹³C NMR (see Section 3 and Figure S1, S24, S31 and S32) and X-ray diffraction data (Figure 2).

We have previously published a set of procedures to convert 1-pyrroline 1-oxides into pyrrolidine nitroxides with spiro-(2-hydroxymethyl)cyclopentane moiety [30]. Here, we used trimethylsilyl protection of the hydroxy group in order to avoid unproductive consumption of the organometallic reagent (Scheme 2). The crude silylated nitrone 10 was treated with 1.5-fold excess of pent-4-en-1-ylmagnesium bromide to give hydroxylamine 11 and the latter was in situ oxidized with air oxygen in presence of Cu²⁺.

The nitrone 12 easily undergoes intramolecular 1,3-dipolar cycloaddition affording single product. The best yield was achieved upon heating to reflux in toluene in presence of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). Intramolecular cycloaddition in 2-pent-4-en-1-ylpyrroline 1-oxides was reported to give a single regioisomer [27,30,33]. Similarly, the ¹H and ¹³C NMR spectra and ¹H-¹H and ¹H-¹³C correlations revealed the signals of two different CH-CH₂O moieties (see Section 3 and Figures S35, S36 and S64–S67), confirming formation of (hexahydro-1H-spiro[cyclopenta[c]pyrrolo[1,2-b]isoxazole-3,1′-cyclopentane]-2′-yl) methanol system. However, one could not exclude the possibility of C=C bond approaching the plane of the nitrone group from different sides with the formation of cis- and trans-isomers relative to the position of the hydroxymethyl group. To assign a structure to the cycloadduct ¹H-¹H NOESY NMR spectra were recorded (Figure S67). ¹H-¹H NOESY correlation showed a cross-peak between protons of ⁶CH₂ and ¹⁵CH₂ groups, while no cross-peak was observed between ¹¹CH₂ and ⁶CH₂ protons (Figure 3). Apparently, the hydroxymethyl group prevents C=C from approaching the nitrone group, and the formation of a cycloadduct is possible only through the transition state 15 (Scheme 2).

Reductive scission of the isoxazolidine ring N-O bond with Zn-AcOH system afforded 14, which was isolated as a colorless crystalline solid. Half of the signal set was observed in ¹H and ¹³C NMR spectra of 14, indicating symmetrical structure. Single-crystal X-ray diffraction data of this compound showed C₂-symmetry of the molecule, which confirms our previous assignment of structure to 13. Thus, despite the absence of bulky substituents at positions 3 and 4 of the pyrroline ring, the intramolecular 1,3-dipolar cycloaddition reaction in 12 proceeds stereospecifically.

Previously, we reported that oxidation of 2,2-disubstituted 1-azaspiro[4.4]nonan-6-ylmethanols to nitroxides with m-CPBA may be accompanied with conversion of hydroxymethyl group into aldehyde, and acylation of the hydroxy group(s) before oxidation increases the nitroxide yield [30]. Following this procedure, 14 was heated with acetic anhydride (Ac₂O) to give 16, which was then oxidized with m-CPBA (Scheme 3). The reaction afforded two nitroxides. The main product 17 was isolated as yellow crystals with 86% yield. The structure of 17 was confirmed by single-crystal X-ray diffraction data (Figure 2).

The structure of the minor product 18 was assigned on the basis of ¹H and ¹³C NMR, and ¹H-¹H COSY, ¹H-¹³C HSQC, and ¹H-¹³C HMBC spectra were acquired after reduction of the nitroxide to corresponding amine with Zn in presence of trifluoroacetic acid according to the earlier described procedure [18] (see Section 3 and Figures S41, S42 and S68–S70) and confirmed with high-resolution mass-spectrum and IR spectral data (Figure S17). We have previously observed the formation of similar dehydrogenated nitroxide upon oxidation of (2,2-dimethyl-1-azaspiro[4.4]nonan-6-yl)methyl acetate, and proposed a mechanism implying proton abstraction from intermediate oxoammonium cation [30].

Nitroxide 17 was then subjected to alkaline hydrolysis to give 5 with nearly quantitative yield. The structure of 5 was confirmed by single-crystal X-ray diffraction data (Figure 4). The overall yield of this nitroxide starting from commercially available 4-chlorobutyryl chloride and 5-bromo-1-pentene exceeds 40%, which makes it an attractive material for the synthesis of spin labels.

Modification of the hydroxymethyl groups seems to be the simplest way to functional derivatives capable of binding to biomolecules. In our recent study, we demonstrated that activation of the hydroxyl group to nucleophilic substitution in 1-unsubstituted pyrrolidines and corresponding alkoxyamines, acyloxyamines, or nitroxides with spiro-(2-hydroxymethyl)cyclopentane moiety always leads to cyclization, which may be followed by rearrangement [34]. Here, we studied the oxidation of hydroxymethyl groups to carboxylic ones, and the alkylation/acylation of hydroxyl groups.

Attempts to oxidize the hydroxymethyl groups in 5 using TEMPO—Sodium chlorite system [35] were unsuccessful, leading to strong tarring. This may occur due to the formation of an oxoammonium cation, which can cause oxidative transformations in the side chains (cf. [30] and above). Therefore, we used the diamagnetic precursor 14 to prepare the desired nitroxide with carboxylate groups. Direct oxidation of 14 with Jones reagent afforded crude 20 as a colorless viscous oil (Scheme 4). The NMR spectra showed half of the signal set, showing that the compound remained a racemic mixture (no other diastereomers formed). The pure crystalline sample was isolated via column chromatography and crystallization from methanol-ethyl acetate mixture 50:1, and the structure was confirmed by single-crystal X-ray diffraction data (Figure 4).

Attempts to oxidize of 20 either with m-CPBA or H₂O₂/Na₂WO₄ were not successful, so the crude 20 was dissolved in methanol saturated with HCl and diester 21 was isolated. Oxidation of 21 with m-CPBA afforded 22, which was isolated as a yellow crystalline solid. The ¹H NMR spectrum acquired after reduction of freshly prepared nitroxide with Zn/CF₃COOH showed half of the signal set with significant downfield shift (due to protonation) as compared to spectrum of 21. The structure of 22 was confirmed by single-crystal X-ray diffraction data (Figure 4). Alkaline hydrolysis of 22 gave an inseparable mixture of structurally related compounds with total yield 42% (Scheme 5). The element analysis of the mixture corresponded to the formula C₁₄H₂₀NO₅, which allowed us to assume that the product was a mixture of isomers. The ¹³C NMR spectrum of the mixture after reduction with Zn/CF₃COOH corresponded to a mixture of three isomers, two symmetric and one asymmetric dicarboxylic acids (Figure S51). This picture corresponds to a mixture of three isomers, two symmetric, and one asymmetric dicarboxylic acids. The ratio of the isomers was estimated using integrals of the signals of methine hydrogens at 3.02–3.30 ppm in ¹H NMR spectrum.

Inversion of the asymmetric center adjacent to the ester group may result from C-H acidity. TLC analysis of samples of 22 after long-term storage showed the emergence of two compounds with close R_f, probably the isomers. The formation of these compounds accelerates in the presence of a base or LiI; however, these reactions were accompanied by tarring. Presumably, in alkaline solution, isomerization proceeds faster than hydrolysis, resulting in nearly statistical ratio of isomers.

It was shown that nitroxide group can decrease pK_a of the acidic center in two σ-bond distance by 2.5 orders of magnitude compared to corresponding methoxyamine derivative [36]. Thus, reduction of the nitroxide group may slow down the isomerization. To verify this hypothesis, the freshly prepared nitroxide 22 was reduced to corresponding hydroxylamine with ascorbic acid in oxygen-free conditions, and then, potassium hydroxide was added (Scheme 6). After the hydrolysis was complete, the products were oxidized with air oxygen, acidified, and extracted. Dicarboxylic acid 23a was a major component of the resulting mixture and it was isolated with the yield 45%. The structure of 23a was confirmed by single-crystal X-ray diffraction data (Figure 5).

Inversion of the asymmetric center at the ester group in 22 leads to a loss of configuration that provides higher hindrance to the nitroxide group. Despite succeeding in isolating pure 23a, it is obvious that activated esters capable of binding to biomolecules, which could be prepared from 23a, will behave similarly to 22. Thus, the nitroxides with spiro-(2-carboxy)cyclopentane moieties are not optimal for spin labeling.

Exploring the possible ways to functional derivatives of 5, we tried several alkylation and acylation reactions. A reaction of nitroxide alcohols with carbonyldiimidazole (CDI) was used for binding to primary amino groups [37,38]. A reaction of 5 with CDI afforded the nitroxide 26 with an excellent yield (Scheme 7). The structure of 26 was confirmed by single-crystal X-ray diffraction data (Figure 5). A reaction of 26 with N,N-dimethyl-1,3-diaminopropane gave 27 with 55% yield.

Another carbamate derivative 28 was prepared via treatment of 5 with methyl 3-isocyanatopropionate with quantitative yield. After alkaline hydrolysis of the ester groups corresponding dicarboxylic acid 29 was isolated. The structure of the nitroxides 27, 28, and 29 were confirmed by element analyses, and IR spectra data and ¹H and ¹³C NMR spectra were acquired after reduction with Zn/CF₃COOH, which showed half of the signal set (See Supporting Information).

Recently, copper-catalyzed azide-alkyne cycloaddition (CuAAC) was successfully used for the site-directed spin labeling of the protein in vivo [39]. The spin labels capable of binding azides were prepared via alkylation of 5 with propargyl bromide (Scheme 8). Two nitroxides 30 and 31 were isolated from the reaction mixture. The proposed structures were confirmed with IR spectra, HRMS, and element analysis data. The ability of the spin label 31 to bind to azide-containing biomolecules was demonstrated by the reaction with 2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl azide in analogy to the literature protocols [40,41,42]. The reaction of enantiomerically pure galactose derivative with racemic mixture 31 expectedly gave a mixture of diastereomers 32, which was not separated. The structure of 32 was confirmed by element analysis, and IR spectral data and ¹H and ¹³C NMR spectra were acquired after reduction with Zn/CF₃COOH (See SI). The NMR spectra of two diastereomers are very close: only few signals of carbon atoms of the spirocyclic system differ and it is impossible to assign them to a specific diastereomer. Ammonolysis of 32, in analogy with the literature procedure [42], afforded spin-labelled galactose 33, which was isolated as a glassy solid. To acquire ¹H and ¹³C NMR spectra, the nitroxide sample was reduced with Zn in presence of formic and oxalic acids mixture. Similarly to the above-mentioned for 32, most of the signals of the two diastereomers were not resolved, and very few slightly differed in chemical shift (see Section 3 and Figures S60 and S61).

2.2. CW EPR Spectra

CW EPR spectra of nitroxides 5 and 33 are shown in Figure 6 and their parameters obtained from simulations of experimental spectra using EasySpin software are given in

Table 1. Values of the line width (Gaussian shape, ω_g, and Lorentzian shape, ω_l,); nitrogen hfc constants, a_N, in water and toluene, mT.

Nitroxide	ωg (mT) (±0.02)	ωl (mT) (±0.002)	aN (H2O) (mT), (±0.05)	aN (Toluene) (mT), (±0.05)
	0.23	0.016	1.52	1.44
	0.23	0.019	1.52
	0.10	0.005	1.62	1.42
	0.21	0.076	1.61

. For comparison spectra of 3-carboxy-Proxyl (34) and 2,2,5,5-tetramethyl-3-(((1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)pyrrolidine-1-oxyl (35) (Table 1) (see Section 3 for synthesis and Figure S21 and S63 for spectra). Measurements were carried out in aqueous solutions, and in addition for 5 and 34, the spectra were recorded in toluene (33 and 35 are poorly soluble in toluene, partition coefficients octanol/water (K_p) for 5, 29, and 33 were 11, 6, and 0.1, respectively (see Section 3 and Figure S73 for details). Broadening of the spectral lines is a typical feature of spirocyclic nitroxides (cf. [28,43]). This broadening is caused by the contribution of hyperfine interactions of an unpaired electron with hydrogen nuclei on substituents. In the spectra of 34 and 35 at room temperature, the rotation of methyl groups leads to averaging of hyperfine interactions.

2.3. Electron Spin Relaxation Time

Electron spin relaxation times were measured for four radicals 5, 33, 34, and 35 at two temperatures of 80 K and 120 K in water−glycerol solution (1:1); the data are shown on Figure 7 and are listed in

Table 2. Electron spin echo dephasing time (T_m), electron relaxation time (T₁), and n-parameter in water−glycerol (1:1) solution for investigated radicals at 80 and 120 K.

Nitroxide	80 K			120 K
	T1, ms (±0.01)	Tm, µs (±0.1)	n (±0.03)	T1, ms (±0.01)	Tm, µs (±0.1)	n (±0.03)
5	0.94	3.0	1.70	0.34	2.8	1.67
33	1.01	3.9	1.97	0.34	3.9	2.1
34	0.51	3.1	1.70	0.20	0.9	1.0
35	0.53	3.4	1.97	0.22	1.1	1.0

. In Figure 7a, the non-exponential decay of spin echo for both radicals is clearly visible. A similar effect was described by Eaton and coworkers [22,24,26] for the phase memory time of the spin echo of nitroxyl radicals with spirocyclohexyl moieties—the positions 2 and 6 of the piperidine ring. It was shown that the observed spin echo decay curves in a water–glycerole solution (1:1) are described by the following formula: I = I₀ × exp(−(t/T₂)ⁿ, where n > 1, and are determined by the nuclear spin diffusion of solvent protons [22]. This effect has been studied in detail by G. Jeschke et al. in recent years [44,45]. An increase in the contribution to the spin−spin relaxation mechanism of various dynamic processes, such as the rotation of methyl groups, leads to a decrease in the value of n to 1. As expected, spirocyclic nitroxides 5 and 33 showed a less pronounced dependence of relaxation time T_m on temperature than radicals with methyl substituents 34 and 35. An increase in temperature from 80 to 120 K did not lead to a change in the time T_m for 33 within the measurement accuracy, while for radical 35 it decreased more than 3-fold from 3.4 to 1.1 μs. This effect is caused by the manifestation of an additional mechanism of electron spin relaxation due to faster rotation of methyl groups with increasing temperature. It can be seen from Figure 7b that, already at 120 K, an exponential decay of spin echo signal is observed for 35 (n ≈ 2.1), whereas for 33 there is no additional contribution to relaxation and n ≈ 2 is retained due to the absence of methyl substituents.

Curiously, binding to galactose does not cause large changes in T₁, but in all cases leads to a significant increase in T_m. This effect may be associated with another mechanism of electron spin relaxation due to modulation of hfi and g-tensor anisotropy by libration motion of nitroxide molecule [46,47]. The influence of the solvent matrix to the libration motion of nitroxide ring was discussed previously and it was shown that the attachment of long chains to nitroxide ring can affect the libration motion.

2.4. Reduction Rate Constants

The kinetics of reduction of nitroxides 5, 29, and 33 with ascorbate was studied. The reaction was carried out under argon using large excess of ascorbate in presence of glutathione to suppress possible reverse processes [48] and followed by EPR. The second-order rate constants were calculated from exponential decay of the nitroxide signal (see Section 3 and Figure S71 and S72). The resulting values (k_red, M⁻¹s⁻¹) for 5, 29, and 33 were as follows: (3.2 ± 0.2) × 10⁻³, (1.40 ± 0.06) × 10⁻³ and (2.70 ± 0.06) × 10⁻³, respectively. All the new radicals demonstrate higher resistance to reduction as compared to 1 (Figure 1). Interestingly, removal of electron-withdrawing tert-butoxy groups produces only minor effect on the reduction rate (cf. k_red for 1 and 5). Presumably, the electron effect of t-BuO-groups is compensated by their influence on the conformation of spirocyclic fragments in analogy to [25]. An increase in steric demand of the substituents adjacent to nitroxide group is known to stabilize the radicals against reduction, enlargement of all neighboring substituents being most efficient [49]. Comparison of the reduction rates for 5, 33, and 29 shows similar effect, with 29 being the most resistant to reduction. Presumably, an increase in substituent size in the positions 1 and 8 of the 6-azadispiro[4.1.4.2]tridecane-6-oxyl system stabilize the nitroxide due to proximity of the bulky groups to the radical center. One can expect much higher stabilization effect upon binding of similar nitroxides to large biomolecules.

3. Materials and Methods

3.1. General

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 (see the Supplementary Information in this article pp. 6–17, Figures S1–S23) and are reported in wave numbers (cm⁻¹). UV spectra were acquired on a HP Agilent 8453 spectrometer (Agilent Technologies, Santa Clara, CA, USA) in ethanol solutions (concentration ~10⁻⁴) (see the Supplementary Information in this article pp. 18–21, Figures S24–S30). ¹H NMR spectra were recorded on a Bruker AV 300 (300.132 MHz), AV 400(400.134 MHz), DRX 500 (500.130 MHz), and Bruker AV 600 (600.300 MHz) spectrometers (Bruker, Billerica, MA, USA). ¹³C NMR spectra were recorded on a Bruker AV 300 (75.467 MHz), AV 400 (100.614 MHz), DRX 500 (125.758 MHz), and Bruker AV 600 (150.945 MHz) spectrometers (see the Supplementary Information in this article pp. 19–42, Figures S31–S70). All the NMR spectra were acquired for 5–10% solutions in CDCl₃ or CD₃OD at 300 K using the signal of the solvent as a standard. NMR spectra of nitroxides for analysis and structure assignment were recorded after reduction with Zn in CD₃OD-CF₃COOH (or CD₃OD-HCOOH-HOOCCOOH mixture) at 65 °C as described in [19] or with Zn and ND₄Cl in CD₃OD at 5 °C. Atoms numbering are shown in figures placed on spectra in SI. HRMS analyses were performed using a High-Resolution Mass Spectrometer DFS (Thermo Electron, Waltham, MA, USA).

Reactions were monitored by TLC on precoated TLC sheets ALUGRAM Xtra SIL G/UV₂₅₄ (Macherey-Nagel GmbH & Co. KG, Düren, Germany) using UV light 254 nm, 1% aqueous permanganate, 10% solution of phosphomolybdic acid in ethanol and Dragendorff’s reagent as visualizing agents. Kieselgel 60 (Macherey-Nagel GmbH & Co. KG) or neutral alumina were utilized as an adsorbent for column chromatography.

The X-ray diffraction experiments were carried out on a Bruker KAPPA APEX II diffractometer (graphite-monochromated Mo Kα radiation). Reflection intensities were corrected for absorption by SADABS2016/2 program [50] except of 14, 17, 20 treated by SADABS2008/1 version [50]. The structures were solved by direct methods using the SHELXS-97 (Sheldrick, 2008) program [51] of 14, 17, 20, and SHELXT 2014/5 (Sheldrick, 2014) [52] for the rest ones. All compounds were refined by anisotropic (isotropic for all H atoms) full-matrix least-squares method against F² of all reflections by SHELXL2018/3 [53]. The positions of the hydrogen atoms were calculated geometrically and refined in riding model except of hydrogenes in OH and NH-groups of 14 and 23a localized from difference map and refined independently with restriction of bond lengths. The presence of two hydrogen atoms on N1 in 20 was proved from the electron density difference map. The asymmetric units of 17, 22, and 26 include a half of molecule, the same one of 5 and 14 consists of three and two molecules, respectively. Note that cyclopentane and azalidine cycles in 22 and 23 are statistically disordered due to conformational mobility in approximate ratio 3:1 and 7:1, respectively. The imidazole cycles of 26 are also disordered due to rotational mobility in approximate ratio 1:1. For experimental details see Table S1.

Crystallographic data for 5, 9, 14, 17, 20, 22, 23a, and 26 have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 2269369–2269376. Copy of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: +44-1223336033 or e-mail: [email protected]; internet: www.ccdc.cam.ac.uk).

3.2. EPR

CW EPR spectra were recorded at X-band frequencies (~9.4 GHz) on a commercial Bruker spectrometer, Elexsys E 540 (Bruker Corporation, Billerica, MA, USA). Electron spin resonance spectra were recorded with the following settings: frequency, 9.25 GHz; microwave power, 2.0 mW; modulation amplitude, 0.02–0.10 mT; time constant, 40.96 ms; and conversion time, 20.8 ms. Simulations of solution electron spin resonance lines were carried out in the EasySpin software (5.2.35), which is available at http://www.easyspin.org (accessed on 18 May 2023).

Electron spin relaxation times were measured using home-made pulse EPR spectrometers equipped with a flow helium cryostat and temperature control system [54]. T_m was measured using a two-pulse electron spin echo (ESE) sequence; T₁ was measured by an inversion recovery technique with inversion π-pulse and detection of a two-pulse ESE sequence. The π-pulse length was 40 ns.

3.3. Kinetic Measurements and Partition Coefficients

For kinetic measurements, stock solutions were prepared in phosphate-citrate-borate buffer (0.5 mM of each): (1) 1 M solution of ascorbic acid and (2) 5 mM solution of glutathione (GSH). Nitroxides were dissolved in the same buffer and diluted to a concentration of 0.2–0.3 mM. The pH was adjusted to 7.5 with NaOH and the solutions were deoxygenated via bubbling with argon. The solutions carefully and quickly mixed in appropriate proportions in a small tube and placed into EPR capillary (50 μL). Oxygen-free conditions were kept permanently. Capillaries were sealed and placed into EPR resonator. EPR experiments were performed on CW EPR X-band spectrometer Bruker ER-200D (9.87 GHz). Spectra were recorded in oxygen-free conditions using the following settings: microwave power 5 mW, modulations amplitude 0.1 or 0.2 mT; time constant 100 ms; conversion time 50.12 ms. The decay of amplitude of low field component of the EPR spectrum was followed in kinetics measurements. Kinetics of decay were fitted with monoexponential function to calculate the first order rate constants. The kinetics measurements were performed at ascorbate concentrations of 100, 200, and 300 mM. The calculated first reaction constants were plotted versus ascorbate concentration; data were fitted with linear dependence. The slope corresponds to second order reaction (See Figures S71 and S72).

For partition coefficients measurements nitroxides were dissolved in 1 mL of distilled water in plastic tube. At this step, the EPR signal was measured as a zero point. Then, the fraction of octanol was added to this solution and shacked for a few minutes to achieve the stationary distribution of radical in the mixture. The tube was shortly centrifuged to separate the octanol and water fractions. The aliquot of radical solution (water phase) was carefully taken with capillary from the bottom of the tube and new EPR spectrum was recorded with the same settings. This procedure was repeated three times for three different octanol contents. The inverse normalized decrease in the EPR signal was plotted versus octanol/water ratio and the slope of the linear fit was used to determine the partition coefficient (see Figure S73).

3.4. Synthesis

2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosyl azide [40], 8 [32], and 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (36) [55] were prepared according to the literature procedures.

((1R(S),5R(S),7R(S),8R(S))-1,8-Bis(hydroxymethyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl (5). Aqueous 10% solution of NaOH (8 mL) was added to a solution of nitroxide 17 (0.335 g, 0.99 mmol) in MeOH (8 mL), and allowed to stand at room temperature until the reaction was complete (up to 12 h). The progress of the reaction was monitored by TLC (silica gel, hexane:Et₂O = 1:2, visualization with UV-254). The solution was evaporated under reduced pressure. The residue was dissolved in the saturated solution of NaCl (10 mL) and extracted with Et₂O (4 × 10 mL). The extract was dried with Na₂SO₄. After evaporation of the solvent, the crude residue was purified via column chromatography (silica gel, EtOAc) and recrystallized from Et₂O to give 5 as yellow crystals. Yield: 0.236 g (94%). m.p. 75–77 °C. UV-Vis (λ_max (nm)/lg(ε)): 240 (3.24). IR (KBr, cm⁻¹): 3276 (O-H). Analysis: found C 65.85, H 9.90, N 5.45; calcd. for C₁₄H₂₄NO₃ C 66.11, H 9.51, N 5.51. HRMS (EI/DFS) m/z [M⁺]: found: 254.1748; calcd. for C₁₄H₂₄NO₃: 254.1751.

[(5R(S),6R(S))-1-Oxido-1-azaspiro[4.4]non-1-en-6-yl]methanol (9). The solution of MCPBA (20.29 g; 117.6 mmol) in dry CH₂Cl₂ (200 mL) was added portion-wise within 1 h to the solution of 8 (18.0 g; 117.6 mmol) in dry CH₂Cl₂ (100 mL) under argon at −50 °C. The mixture was allowed to warm up to room temperature (TLC control on silica gel, CHCl₃:CH₃OH = 10:1, R_f = 0.4, visualization with UV-254 and Dragendorff’s reagent). Then the mixture was concentrated in a vacuum, and the residue was separated via column chromatography (Al₂O₃, CH₂Cl₂) to give 9 as colorless crystals, m.p. 67–68 °C (from Et₂O). Yield 15.9 g (80%). UV-Vis (λ_max (nm)/lg(ε)): 232 (3.91). IR (KBr, cm⁻¹): 3222 (O-H), 3074.1 (H-C=), 1593 (C=N). Analysis: found C 63.90, H 8.44, N 8.27; calcd. for C₉H₁₄NO C 63.88, H 8.93, N 8.28. HRMS (EI/DFS) m/z [M-17] ⁺: found: 152.1069; calcd. for C₉H₁₄NO: 152.1070. ¹H NMR (400 MHz, CDCl₃, δ): 1.55 (dddd, J_d1 = 3.9, J_d2 = 8.1, J_d3 = 10.2, J_d4 = 16.5, 1H ⁷CH₂); 1.69 (ddd, J_d1 = 2.8, J_d2 = 8.1, J_d3 = 13.1, 1H ⁶CH₂); 1.78 (dddd, J_d1 = 3.6, J_d2 = 3.9, J_d3 = 8.1, J_d4 = 12.6, 1H ⁸CH₂); 1.88 (ddd, J_d1 = 7.3, J_d2 = 7.9, J_d3 = 12.6, 1H ⁸CH₂); 1.93 (ddddd, J_d1 = 2.8, J_d2 = 3.6, J_d3 = 7.9, J_d4 = 8.5 J_d5 = 16.5, 1H ⁷CH₂); 2.01 (dddd, J_d1 = 3.7, J_d2 = 5.5, J_d3 = 7.3, J_d4 = 8.1, 1H ⁹CH); 2.13 (ddd, J_d1 = 6.7, J_d2 = 8.9, J_d3 = 13.0, 1H ⁴CH₂); 2.19 (ddd, J_d1 = 4.9, J_d2 = 8.3, J_d3 = 13.0, 1H ⁴CH₂); 2.52 (ddd, J_d1 = 8.5, J_d2 = 10.2, J_d3 = 13.1, 1H ⁶CH₂); 2.53 (dddd, J_d1 = 2.6, J_d2 = 4.9, J_d3 = 8.9, J_d4 = 18.6, 1H ³CH₂); 2.63 (dddd, J_d1 = 2.6, J_d2 = 6.7, J_d3 = 8.3, J_d4 = 18.6, 1H ³CH₂); 3.62 (ddd, J_d1 = 5.5, J_d2 = 5.8, J_d3 = 13.7, 1H ¹⁰CH₂); 3.65 (ddd, J_d1 = 3.7, J_d2 = 7.4, J_d3 = 13.7, 1H ¹⁰CH₂); 4.81 (dd, J_d1 = 5.8, J_d2 = 7.4, 1H OH); 6.99 (dd, J_d1 = 2.6, J_d2 = 2.6, 1H ²CH=). ¹³C NMR (125 MHz, CDCl₃, δ): 22.87, 25.34, 27.60, 35.55, 36.61 (³CH₂, ⁴CH₂, ⁶CH₂, ⁷CH₂, ⁸CH₂), 51.22 (⁹CH), 61.84 (¹⁰CH₂), 84.39 (⁵C), 137.33 (²CH=N).

[(5R(S),6R(S))-1-Oxido-2-pent-4-en-1-yl-1-azaspiro[4.4]non-1-en-6-yl]methanol (12). To the cooled-on-the-ice-bath solution of triethylamine (5.95 g; 59.0 mmol) and nitrone 9 (7.66 g; 45.3 mmol) in dry THF (40 mL), the solution of Me₃SiCl (5.88 g, 54.4 mmol) in dry THF (15 mL) was added dropwise. The mixture was stirred at room temperature until the reaction complete (TLC control on silica gel, CHCl₃:CH₃OH = 10:1, R_f = 0.75, visualization with UV-254). The reaction mixture was concentrated in a vacuum. The crude mixture was diluted with dry Et₂O (40 mL) and insoluble precipitate was filtered off. The resulting solution contained (5R,6R)-6-(((trimethylsilyl)oxy)methyl)-1-azaspiro[4.4]non-1-ene 1-oxide (10), ¹H NMR (400 MHz, CDCl₃, δ): 0.04 (s, 9H)(¹¹CH₃, ¹²CH₃, ¹³CH₃); 1.50–1.64 (m, 1H); 1.69–1.82 (m, 3H); 1.88–2.00 (m, 1H); 2.00–2.11 (m, 1H); 2.14–2.24 (m, 1H); 2.31–2.46 (m, 2H); 2.48–22.70 (m, 2H); 2.67 (d, J_d = 7.0, 2H ¹⁰CH₂); 6.75 (t, J_t = 2.5, 1H ²CH=). The solution of crude 10 without further purification was added dropwise to the solution of pent-4-en-1-ylmagnesium bromide which was prepared via slow addition of a 5-bromopentene-1 (10.13 g, 68 mmol) and Et₂O (15 mL) mixture to a suspension of Mg chips (1.74 g, 72.5 mmol) in dry Et₂O (15 mL). The reaction mixture was stirred for 2 h, quenched with water (10 mL), and filtered. The filtrate was concentrated in vacuum and the residue was dissolved in MeOH (80 mL). Then, the solution of CuSO₄·5 H₂O (10 mg) in 1 mL of NH₃ (aq.) was added to the mixture, and the air was bubbled until the solution turned dark blue (TLC control on silica gel, CHCl₃:CH₃OH = 15:1, R_f = 0.4, visualization with UV-254, KMnO₄ in water). Methanol was evaporated in a vacuum. Then, the mixture was diluted with brine (20 mL) with the addition of 25% aqueous NH₃ (5 mL) and was extracted with CHCl₃ (3 × 20 mL). The organic phase was evaporated in a vacuum, and the residue was separated via column chromatography on silica gel (CHCl₃:CH₃OH = 15:1) to give 12 as colorless oil. Yield: 9.13 g (85%). UV-Vis (λ_max (nm)/lg(ε)): 236 (3.97). IR (neat, cm⁻¹): 3310 (O-H), 3076 (H-C=), 1641 (C=C), 1600 (C=N). Analysis: found C 70.90, H 9.77, N 5.78; calcd. for C₁₄H₂₃NO₂ C 70.85, H 9.77, N 5.90. HRMS (EI/DFS) m/z [M⁺]: found: 237.1722; calcd. for C₁₄H₂₃NO₂: 237.1723. ¹H NMR (400 MHz, CDCl₃, δ): 1.46–1.56 (m, 1H); 1.56–1.66 (m, 3H); 1.70–1.81 (m, 1H); 1.83–2.10 (m, 7H); 2.37–2.67 (m, 5H); 3.52 (ddd, J_d1 = 2.7, J_d2 = 4.6, J_d3 = 12.2, 1H ¹⁰CH₂); 3.58 (ddd, J_d1 = 6.1, J_d2 = 8.0, J_d3 = 12.2, 1H ¹⁰CH₂); 4.93 (tdd, J_t = 1.1, J_d1 = 1.9, J_d2 = 10.2, 1H ^15bCH); 4.98 (tdd, J_t = 1.6, J_d1 = 1.9, J_d2 = 17.1, 1H ^15aCH); 5.36 (dd, J_d1 = 4.6, J_d2 = 8.0, 1H OH); 5.74 (tdd, J_t = 6.7, J_d1 = 10.2, J_d2 = 17.1, 1H ¹⁴CH=). ¹³C NMR (100 MHz, CDCl₃, δ): 22.72, 24.19, 26.79, 27.16, 28.41, 33.63, 33.92, 36.09 (³CH₂, ⁴CH₂, ⁶CH₂, ⁷CH₂, ⁸CH₂, ¹¹CH₂, ¹²CH₂, ¹³CH₂), 50.99 (⁹CH), 61.87 (¹⁰CH₂), 84.45 (⁵C), 115.62 (¹⁵CH₂=), 137.52 (¹⁴CH=), 151.46 (²C=N).

((1R(S),2R(S),6a′R(S),9a′R(S))-Hexahydro-6′H-spiro[cyclopentan-1,3′-cyclopenta[c]pyrrolo[1,2-b]isoxazol]-2-yl)methanol (13). A solution of 12 (0.823 g; 3.42 mmol) and TEMPO (2–3 mg) in toluene (10 mL) was bubbled with Ar and the mixture was heated at 100 °C for 48 h. The resulting solution was concentrated in vacuum, and the residue was separated via column chromatography on silica gel (hexane:EtOAc = 1:1) to give 13 as colorless oil. Yield: 0.75 g (91%). IR (neat, cm⁻¹): 3330 (O-H). Analysis: found C 71.15, H 9.78 N 5.65; calcd. for C₁₄H₂₃NO₂ C 70.85, H 9.77, N 5.90. HRMS (EI/DFS) m/z [M⁺]: found: 237.1720; calcd. for C₁₄H₂₃NO₂: 237.1723. ¹H NMR (600 MHz, CDCl₃, δ): 1.13–1.20 (m, 1H ⁸CH₂); 1.36–1.43 (m, 2H)(one from ⁶CH₂ and one from ⁷CH₂); 1.44–1.50 (m, 1H ¹³CH₂); 1.51–1.61 (m, 3H)(one from ⁴CH₂, one from ¹¹CH₂ and one from ¹²CH₂); 1.61–1.71 (m, 4H one from ⁴CH₂, one from ⁷CH₂, one from ⁸CH₂ and one from ¹²CH₂); 1.73–1.79 (m, 1H ¹³CH₂); 1.79–1.84 (m, 1H ⁹CH); 1.84 (ddd, J_d1 = 6.5, J_d2 = 9.3, J_d3 = 12.9, 1H ³CH₂); 1.92 (ddd, J_d1 = 5.0, J_d2 = 6.8, J_d3 = 12.9, 1H ³CH₂); 1.98–2.03 (m, 1H ¹¹CH₂); 2.10–2.18 (m, 1H ⁶CH₂); 2.45 (ddd, J_d1 = 3.3, J_d2 = 7.0, J_d3 = 12.1, 1H ¹⁴CH); 3.36 (dd, J_d1 = 3.9, J_d2 = 11.4, 1H ¹⁰CH₂); 3.50 (dd, J_d1 = 3.3, J_d2 = 8.5, 1H ¹⁵CH₂); 3.66 (dd, J_d1 = 10.0, J_d2 = 11.4, 1H ¹⁰CH₂); 3.81 (dd, J_d1 = 7.0, J_d2 = 8.5, 1H)(¹⁵CH₂); 5.40–5.75 (br. s, 1H OH). ¹³C NMR (150 MHz, CDCl₃, δ): 21.43 (⁷CH₂), 25.76 (¹²CH₂), 25.98 (⁸CH₂), 30.83 (⁶CH₂), 32.58 (¹³CH₂), 34.40 (³CH₂), 38.56 (⁴CH₂), 39.44 (¹¹CH₂), 48.16 (⁹CH), 55.01 (¹⁴CH), 65.49 (¹⁰CH₂), 76.30 (¹⁵CH₂), 80.07 (⁵C), 83.41 (²C).

(1R(S),5R(S),7R(S),8R(S))-6-Azadispiro[4.1.4.2]tridecane-1,8-diyldimethanol (14). Zn powder (1.08 g, 16.62 mmol) was added in one portion to a warm (60 °C) stirred solution containing 13 (0.394 g, 1.66 mmol), EtOH (3 mL), 10 M AcOH (10 mL), and EDTA disodium salt (2.4 g). The reaction mixture was stirred at 60 °C for 1 h and then cooled down to room temperature. The mixture was basified to pH 10 with NaOH solution and extracted with EtOAc (4 × 15 mL). The organic extract was dried with Na₂CO₃. After evaporation of the solvent, the crude residue was purified via recrystallisation (hexane:EtOAc = 10:1) to give 14 as colorless crystals. Yield: 0.393 g (99%). M.p. 73–77 °C. IR (KBr, cm⁻¹): 3288 (OH, N-H). Analysis: found C 70.39, H 10.28, N 5.87; calcd. for C₁₄H₂₅NO₂ C 70.25, H 10.53, N 5.85. HRMS (EI/DFS) m/z [M⁺]: found: 239.1883; calcd. for C₁₄H₂₅NO₂: 239.1880. ¹H NMR (400 MHz, CDCl₃, δ): 1.33–1.43 (m, 2H); 1.43–1.55 (m, 4H); 1.55–1.63 (m, 2H); 1.63–1.81 (m, 10H); 3.59 (dd, J_d1 = 4.4, J_d2 = 11.0, 2H) (one from ¹⁰CH₂ and one from ¹⁵CH₂); 3.63 (dd, J_d1 = 7.0, J_d2 = 11.0, 2H one from ¹⁰CH₂ and one from ¹⁵CH₂); 4.16–4.54 (br. s, 2H OH). ¹³C NMR (100 MHz, CDCl₃, δ): 21.49, 26.53, 37.69, 39.28 (³CH₂ and ⁴CH₂, ⁶CH₂ and ¹¹CH₂, ⁷CH₂ and ¹²CH₂, ⁸CH₂ and ¹³CH₂), 47.88 (⁹CH and ¹⁴CH), 64.01 (¹⁰CH₂ and ¹⁵CH₂), 72.32 (²C and ⁵C).

((1R(S),5R(S),7R(S),8R(S))-6-Azadispiro[4.1.4.2]tridecane-1,8-diyl)bis(methylene) diacetate (16). Acetic anhydride (11.73 g; 115 mmol) was added to a solution of 14 (2.75 g; 11.5 mmol) in dry chloroform (40 mL) and the mixture heated under reflux until the reaction was complete (TLC control on silica gel, CHCl₃:CH₃OH = 10:1, R_f = 0.75, visualization with Dragendorff’s reagent). The reaction mixture was washed with a saturated solution of Na₂CO₃ and dried with Na₂CO₃. After evaporation of the solvent, the crude residue was separated via column chromatography (silica gel, hexane:EtOAc = 2:1) to give 16 as colorless crystals. Yield: 3.60 g (97%). M.p. 36–37 °C. IR (KBr, cm⁻¹): 1739 (C=O). Analysis: found C 66.83, H 9.02, N 4.30; calcd. for C₁₈H₂₉NO₄ C 66.84, H 9.04, N 4.33. HRMS (EI/DFS) m/z [M⁺]: found: 323.2088; calcd. for C₁₈H₂₉NO₄: 323.2088. ¹H NMR (500 MHz, CDCl₃, δ): 1.34–1.44 (m, 2H); 1.44–1.53 (m, 4H); 1.53–1.67 (m, 6H); 1.69–1.80 (m, 4H); 1.83–1.92 (m, 2H); 1.99 (s, 6H ¹⁷CH₃ and ¹⁹CH₃); 3.94 (dd, J_d1 = 7.4, J_d2 = 11.0, 2H one from ¹⁰CH₂ and one from ¹⁵CH₂); 4.17 (dd, J_d1 = 6.1, J_d2 = 11.0, 2H one from ¹⁰CH₂ and one from ¹⁵CH₂). ¹³C NMR (125 MHz, CDCl₃, δ): 21.14 (¹⁷CH₃ и ¹⁹CH₃), 21.16, 27.85, 37.78, 40.99 (³CH₂ and ⁴CH₂, ⁶CH₂ and ¹¹CH₂, ⁷CH₂ and ¹²CH₂, ⁸CH₂ and ¹³CH₂), 46.48 (⁹CH and ¹⁴CH), 66.09 (¹⁰CH₂ and ¹⁵CH₂), 70.88 (²C and ⁵C), 171.18 (¹⁶C=O and ¹⁸C=O).

((1R(S),5R(S),7R(S),8R(S))-1,8-Bis(acetyloxymethyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl (17). A solution of 16 (3.49 g, 10.8 mmol) in dry CHCl₃ (30 mL) was cooled to ca. -50 °C with liquid nitrogen and MCPBA (2.8 g, 16.2 mmol) was added upon stirring. The mixture was allowed to warm up to room temperature (TLC control on silica gel, hexane:EtOAc = 3:1, R_f = 0.6, visualization with UV-254 and Dragendorff’s reagent). The reaction mixture was washed with a saturated solution of Na₂CO₃, water, and dried with Na₂SO₄. After evaporation of the solvent, the crude residue was purified via column chromatography (silica gel, hexane:EtOAc = 4:1) and recrystallized from hexane to give 17 as yellow crystals. Yield: 3.14 g (86%). m.p. 70.8–70.9 °C. UV-Vis (λ_max (nm)/lg(ε)): 241 (3.18). IR (KBr, cm⁻¹): 1720 (C=O), 1243 (C-OAc). Analysis: found C 63.59, H 8.94, N 4.14; calcd. for C₁₈H₂₈NO₅ C 63.88, H 8.34, N 4.30. HRMS (EI/DFS) m/z [M⁺]: found: 338.1960; calcd. for C₁₈H₂₈NO₅: 338.1958

(4R(S),5S(R),7R(S),8R(S))-4,8-Bis[(acetyloxy)methyl]-6-azoniadispiro[4.1.4.2]tridec-1-ene-6-oxyl (18). Yield: 0182 g (5%). Yellow oil. UV-Vis (λ_max (nm)/lg(ε)): 241 (3.17). IR (neat, cm⁻¹): 3054 (H-C=), 1739 (C=O), 1618 (C=C). HRMS (EI/DFS) m/z [M⁺]: found: 336.1802; calcd. for C₁₈H₂₆NO₅: 336.1806. To confirm the structure 18 was reduced with Zn-CF₃COOH to give (4R(S),5S(R),7R(S),8R(S))-4,8-Bis[(acetyloxy)methyl]-6-azadispiro[4.1.4.2]tridec-1-ene trifluoroacetate (19). ¹H NMR (400 MHz, CD₃OD + CF₃COOH, δ: 1.60–1.80 (m, 3H); 1.81–2.21 (m, 7H); 2.01 and 2.04 (s, both 3H ¹⁷CH₃ and ¹⁹CH₃); 2.28–2.43 (m, 2H ¹⁴CH and one from ⁸CH₂); 2.52–2.62 (m, 1H ⁸CH₂); 2.69–2.78 (m, 1H ⁹CH); 4.13–4.16 (m, 2H ¹⁵CH₂); 4.32 (dd, J_d1 = 8.1, J_d2 = 12.0, 1H ¹⁰CH₂); 4.39 (dd, J_d1 = 6.3, J_d2 = 12.0, 1H ¹⁰CH₂); 5.93–5.96 (m, 1H ⁶CH=); 6.14–6.18 (m, 1H ⁷CH=). ¹³C NMR (100 MHz, CD₃OD + CF₃COOH, δ): 20.70 and 20.77 (¹⁷CH₃ and ¹⁹CH₃), 27.31 (¹³CH₂), 35.53 (⁸CH₂), 20.90, 36.23, 37.03, 37.17 (³CH₂, ⁴CH₂, ¹¹CH₂, ¹²CH₂), 46.41 (⁹CH), 47.37 (¹⁴CH), 63.82 (¹⁰CH₂), 64.74 (¹⁵CH₂), 77.16 (²C), 81.66 (⁵C), 132.27 (⁶CH=), 137.88 (⁷CH=), 172.23 and 172.46 (¹⁶C=O and ¹⁸C=O).

Dimethyl (1R(S),5R(S),7R(S),8R(S))-6-azadispiro[4.1.4.2]tridecane-1,8-dicarboxylate (21). CrO₃ (2.16 g, 21.6 mmol) was dissolved in H₂SO₄ (4.31 g, 41.1 mmol) and H₂O (33 mL). The resulting mixture was added dropwise to a stirring cooled in an ice bath solution of 14 (1.0 g, 4.18 mmol) in acetone (20 mL). The mixture was stirred at room temperature until the reaction was complete (20 h. TLC control on silica gel, CH₃OH:EtOAc = 1:2, visualization with I_2(vap.₎). Acetone was evaporated and Ba(OH)₂ water solution was added to the residue to pH = 6. The precipitate formed was filtered off, washed with water and EtOH. The filtrate was evaporated to dryness under reduced pressure and extra pumped out on a high vacuum pump to give crude (1R(S),5R(S),7R(S),8R(S))-6-azadispiro[4.1.4.2]tridecane-1,8-dicarboxylic acid (20). A small portion of 20 was isolated using column chromatography on silica gel, eluted with gradient from EtOAc:MeOH = 4:1 to pure MeOH. The pure fractions were collected, evaporated, triturated in the mixture CHCl₃:CCl₄ = 1:2 and crystallized from the mixture MeOH:EtOAc = 50:1. M.p. 190.6–191.9 °C. IR (KBr, cm⁻¹): 3423, 2765, 2700, 2588, 2514, 2468 (O-H, N-H), 1654 (C=O). Analysis: found C 62.91, H 7.89, N 5.31; calcd. for C₁₄H₂₁NO₄ C 62.90, H 7.92, N 5.24. ¹H NMR (400 MHz, CD₃OD, δ): 1.77–1.97 (m, 6H); 1.97–2.10 (m, 4H); 2.15–2.35 (m, 6H); 2.88 (t, J_t = 9.0, 2H ⁹CH and ¹⁴CH). ¹³C NMR (75 MHz, CD₃OD, δ): 20.77, 28.50, 34.88, 36.45 (⁴CH₂ and ³CH₂, ⁶CH₂ and ¹¹CH₂, ⁷CH₂ and ¹²CH₂, ⁸CH₂ and ¹³CH₂), 50.20 (⁹CH and ¹⁴CH), 74.64 (²C and ⁵C), 177.89 (¹⁰COOH and ¹⁵COOH). The crude 20 was dissolved in methanol (50 mL), saturated with gaseous HCl and left for 5 days at room temperature to complete the reaction (TLC control on silica gel, CH₃OH:EtOAc = 1:5, visualization with Dragendorff’s reagent). The methanol was distilled off in vacuum and brine (20 mL) was added to the residue, the mixture was basified to pH 10–11 with Na₂CO₃ solution and extracted with EtOAc (3 × 20 mL). The organic extract was dried with Na₂SO₄. After evaporation of the solvent, the crude residue was purified via column chromatography (silica gel, hexane:EtOAc = 4:1) to give 21 as colorless oil. Yield: 0.778 g (63%). IR (neat, cm⁻¹): 1729 (C=O). Analysis: found C 64.90, H 8.76, N 4.81; calcd. for C₁₆H₂₅NO₄ C 65.06, H 8.53, N 4.74. HRMS (EI/DFS) m/z [M⁺]: found: 295.1777; calcd. for C₁₆H₂₅NO₄: 295.1778. ¹H NMR (300 MHz, CDCl₃, δ): 1.37–1.51 (m, 4H); 1.60–1.73 (m, 6H); 1.73–1.82 (m, 2H); 1.83–2.01 (m, 4H); 2.57 (dd, J_d1 = 7.5 J_d2 = 8.5, 2H ⁹CH and ¹⁴CH); 3.61 (s, 6H ¹⁶CH₃ and ¹⁷CH₃). ¹³C NMR (75 MHz, CDCl₃, δ): 22.24, 27.52, 37.59, 41.02 (³CH₂ and ⁴CH₂, ⁶CH₂ and ¹¹CH₂, ⁷CH₂ and ¹²CH₂, ⁸CH₂ and ¹³CH₂), 51.09 (⁹CH and ¹⁴CH), 53.63 (¹⁶CH₃ and ¹⁷CH₃), 72.50 (²C and ⁵C), 175.03 (¹⁰C=O and ¹⁵C=O).

(1R(S),5R(S),7R(S),8R(S))-1,8-Bis(methoxycarbonyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl (22). A solution of 21 (2.98 g, 10.8 mmol) in dry CHCl₃ (30 mL) was cooled in liquid nitrogen to ca. −50 °C and MCPBA (2.17 g, 12.6 mmol) was added upon stirring. The mixture was allowed to warm up to room temperature (TLC control on silica gel, hexane:EtOAc = 3:1, R_f = 0.4, visualization with UV-254 and Dragendorff’s reagent). The reaction mixture was washed with a saturated solution of Na₂CO₃, water and dried with Na₂SO₄. After evaporation of the solvent, the crude residue was purified via column chromatography (silica gel, hexane:EtOAc = 5:1) and recrystallized from hexane:Et₂O = (10:1) mixture to give 22 as yellow crystals. Yield: 2.6 g (83%). M.p. 84.4–88.8 °C. UV-Vis (λ_max (nm)/lg(ε)): 241 (3.26). IR (KBr, cm⁻¹): 1731 (C=O). Analysis: found C 61.88, H 7.72, N 4.58; calcd. for C₁₆H₂₄NO₅ C 61.92, H 7.79, N 4.51. HRMS (EI/DFS) m/z [M⁺]: found: 310.1649; calcd. for C₁₆H₂₄NO₅: 310.1646. ¹H NMR (300 MHz, CD₃OD + CF₃COOH, δ): 1.80–2.00 (m, 6H); 2.00–2.37 (m, 10H); 3.15 (dd, J_d1 = 7.1 J_d2 = 8.6, 2H ⁹CH and ¹⁴CH); 3.81 (s, 6H ¹⁶CH₃ and ¹⁷CH₃).

1,8-Dicarboxy-6-azadispiro[4.1.4.2]tridecane-6-oxyl (23a–c) (mixture of isomers). A solution of KOH (10%) in water:methanol = 1:1mixture (8 mL) was added to a solution of 22 (0.11 g, 0.32 mmol) in MeOH (5 mL) and allowed to stand at r.t. until the reaction was complete (up to 24 h). The progress of the reaction was monitored by TLC (silica gel, EtOAc:AcOH = 100:1, visualization with UV-254). The solution was evaporated under reduced pressure. The residue was dissolved in water, the pH was adjusted to 3 with NaHSO₄ 10% solution and the mixture was extracted with EtOAc (3 × 8 mL). The extract was dried with Na₂SO₄ and evaporated to give 23a-c as a yellow powder with 1:2:1 ratio of isomers according to ¹H NMR after reduction with Zn/CF₃COOH (see Figure S50).

(1R(S),5R(S),7R(S),8R(S))-1,8-Dicarboxy-6-azadispiro[4.1.4.2]tridecane-6-oxyl (23a). A solution of ascorbic acid (0.109 g, 6.21 mmol) in water (3 mL) was stirred under argon and pH was adjusted to ca. 5 with Na₂CO₃. Then, a solution of 22 (0.175 g, 5.65 mmol) in methanol (2 mL) was added and the mixture was stirred for 1.5 h. The resulting mixture was extracted with Et₂O (3 × 1 mL) under argon via injecting, mixing, and taking the upper layer with a syringe. The extract was placed into separate vial and ether was removed in the stream of argon. A solution of KOH (10%) in water–methanol mixture 1:1, (8 mL) was added and the solution was allowed to stand at room temperature for 48 h under argon. Then, air was bubbled through the reaction mixture for 8 h. The pH was adjusted to 3 with NaHSO₄ 10% solution and the mixture was extracted with EtOAc (3 × 2 mL). The extract was dried with Na₂SO₄ and concentrated in vacuum; the residue was separated via column chromatography (silica gel, hexane:EtOAc:AcOH = 10:100:1) to give pure 23a as yellow crystals. Total yield: 0.072 g (45%). M.p. 152–153 °C. UV-Vis (λ_max (nm)/lg(ε)): 239 (3.23). IR (KBr, cm⁻¹): 3400, 3078, 2669, 2570 (O-H), 1704 (C=O). Analysis: found C 59.58, H 6.90, N 4.87; calcd. for C₁₄H₂₀NO₅ C 59.56, H 7.14, N 4.96. HRMS (EI/DFS) m/z [M⁺]: found: 282.1333; calcd. for C₁₄H₂₀NO₅: 282.1336. ¹H NMR (500 MHz, CD₃OD + CF₃COOH, δ): 1.81–1.96 (m, 6H); 2.04–2.14 (m, 4H); 2.16–2.23 (m, 2H); 2.23–2.32 (m, 4H); 3.06 (t, J_t = 8.3, 2H ⁹CH and ¹⁴CH). ¹³C NMR (125 MHz, CD₃OD + CF₃COOD, δ): 21.79, 29.38, 36.08, 37.28 (³CH₂ and ⁴CH₂, ⁶CH₂ and ¹¹CH₂, ⁷CH₂ and ¹²CH₂, ⁸CH₂ and ¹³CH₂), 50.46 (⁹CH and ¹⁴CH), 76.22 (²C and ⁵C), 178.53 (¹⁰C=O and ¹⁵C=O).

(1R(S),5R(S),7R(S),8R(S))-1,8-Bis(((1H-imidazol-1-ylcarbonyl)oxy)methyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl (26). The solution of CDI (0.359 g, 2.216 mmol) in dry THF (3 mL) was added dropwise to the solution of 5 (0.268 g; 1.055 mmol) in dry THF (4 mL). The mixture was stirred at room temperature until the reaction was complete (about 12 h. TLC control on silica gel, hexane:Et₂O = 1:2, visualization with UV-254). The reaction mixture was diluted with dry Et₂O and cooled in a refrigerator for 2 h. The precipitate formed was filtered off, washed with dry Et₂O, dried in air, and recrystallized from CH₂Cl₂. Yellow crystals. Yield: 0.448 g (96%). M.p. 128.5–128.8 °C. UV-Vis (λ_max (nm)/lg(ε)): 230 (3.95). IR (KBr, cm⁻¹): 3149, 3130, 3112, 3101 (H-C=), 1766 (C=O). Analysis: found C 60.01, H 5.99, N 15.87; calcd. for C₂₂H₂₈N₅O₅ C 59.72, H 6.38, N 15.83. HRMS (EI/DFS) m/z [M⁺]: found: 442.2087; calcd. for C₂₂H₂₈N₅O₅: 442.2085.

(1R(S),5R(S),7R(S),8R(S))-1,8-Bis[(([{3-(dimethylamino)propyl}amino]carbonyl)oxy)methyl]-6-azadispiro[4.1.4.2]tridecane-6-oxyl (27). N,N-Dimethylpropane-1,3-diamine (0.217 g, 2.12 mmol) was added to the solution of 26 (0.313 g; 0.708 mmol) in dry CH₂Cl₂ (6 mL). The mixture was stirred at room temperature until the reaction was complete (about 100 h. TLC control on silica gel, hexane:Et₂O = 1:2, visualization with UV-254). The reaction mixture was evaporated in a vacuum, and the residue was separated via column chromatography on Al₂O₃ (CH₂Cl₂:CH₃OH = 10:1) to give 27 as yellow oil. Yield: 0.199 g (55%). IR (neat, cm⁻¹): 3332 (N-H), 1714, 1699 (C=O). Analysis: found C 61.00, H 9.47, N 13.91; calcd. for C₂₆H₄₈N₅O₅ C 61.15, H 9.70, N 13.71. HRMS (EI/DFS) m/z [M⁺]: found: 510.3648; calcd. for C₂₆H₄₈N₅O₅: 510.3650. ¹H NMR (500 MHz, CD₃OD + CF₃COOD, δ): 1.70–1.84 (m, 4H); 1.85–2.03 (m, 10H); 2.03–2.15 (m, 4H); 2.16–2.26 (m, 2H); 2.36–2.43 (m, 2H ⁹CH and ¹⁴CH); 2.89 (s, 12H ²⁰CH₃, ²¹CH₃, ²⁶CH₃, ²⁷CH₃); 3.13–3.19 (m, 4H ¹⁷CH₂ and ²³CH₂); 3.23 (t, J_t = 6.5, 4H ¹⁹CH₂ and ²⁵CH₂); 4.22 (dd, J_d1 = 5.4 J_d2 = 11.9, 2H one from ¹⁰CH₂ and one from ¹⁵CH₂); 4.28 (dd J_d1 = 6.3 J_d2 = 11.9, 2H one from ¹⁰CH₂ and one from ¹⁵CH₂). ¹³C NMR (125 MHz, CD₃OD + CF₃COOD, δ): 20.92, 26.19, 27.33, 36.41, 37.22, 38.61 (³CH₂ and ⁴CH₂, ⁶CH₂ and ¹¹CH₂, ⁷CH₂ and ¹²CH₂, ⁸CH₂ and ¹³CH₂, ¹⁷CH₂ and ²³CH₂, ¹⁸CH₂ and ²⁴CH₂), 43.42 and 43.47 (²⁰CH₃, ²¹CH₃, ²⁶CH₃, ²⁷CH₃), 47.68 (⁹CH and ¹⁴CH), 56.55 (¹⁹CH₂ and ²⁵CH₂), 65.26 (¹⁰CH₂ and ¹⁵CH₂), 77.62 (²C and ⁵C), 158.75 (¹⁶C=O and ²²C=O).

1R(S),5R(S),7R(S),8R(S)-1,8-Bis[(([{3-methoxy-3-oxopropyl}amino]carbonyl)oxy)methyl]-6-azadispiro[4.1.4.2]tridecane-6-oxyl (28). Methyl 3-isocyanatopropanoate (0.754 g, 5.846 mmol) was added to the solution of 5 (0.675 g; 2.567 mmol) in dry THF (8 mL). The mixture was heated to reflux until the reaction was complete (ca. 72 h, TLC control on silica gel, hexane:Et₂O = 1:5, visualization with UV-254). The reaction mixture was evaporated in a vacuum, and the residue was purified via column chromatography (silica gel, Et₂O) to give 28 as yellow crystals. Yield: 1.35 g (99%). M.p.: 74.2–80.1 °C. IR (neat, cm⁻¹): 3349 (N-H), 1735, 1720, (C=O). Analysis: found C 56.10, H 7.50, N 8.09; calcd. for C₂₄H₃₈N₃O₉ C 56.24, H 7.47, N 8.20. ¹H NMR (500 MHz, CD₃OD + CF₃COOD, δ): 1.70–1.85 (m, 4H); 1.87–2.23 (m, 12H); 2.36–2.44 (m, 2H ⁹CH and ¹⁴CH); 2.56 (t, J_t = 6.5, 4H ¹⁸CH₂ and ²³CH₂); 3.42 (t, J_t = 6.5, 4H ¹⁷CH₂ and ²²CH₂); 3.68 (s, 6H ²⁰CH₃ and ²⁵CH₃); 4.21–4.30 (m, 4H ¹⁰CH₂ and ¹⁵CH₂). ¹³C NMR (125 MHz, CD₃OD + CF₃COOD, δ): 20.91, 27.41, 34.98, 36.58, 36.86, 37.76 (³CH₂ and ⁴CH₂, ⁶CH₂ and ¹¹CH₂, ⁷CH₂ and ¹²CH₂, ⁸CH₂ and ¹³CH₂, ¹⁷CH₂ and ²²CH₂, ¹⁸CH₂ and ²³CH₂), 47.86 (⁹CH and ¹⁴CH), 52.21 (²⁰CH₃ and ²⁵CH₃), 65.35 (¹⁰CH₂ and ¹⁵CH₂), 77.37 (²C and ⁵C), 158.50 (¹⁶C=O and ²¹C=O), 173.90 (¹⁹C=O and ²⁴C=O).

1R(S),5R(S),7R(S),8R(S)-1,8-Bis[(([{2-carboxyethyl}amino]carbonyl)oxy)methyl]-6-azadispiro[4.1.4.2]tridecane-6-oxyl (29). An aqueous solution of NaOH (1%, 100 mL) was added to a solution of nitroxide 28 (1.21 g, 2.36 mmol) in MeOH (100 mL), and the mixture was allowed to stand at room temperature until the reaction was complete (ca. 24 h). The progress of the reaction was monitored by TLC (silica gel, EtOAc, visualization with UV-254). The methanol was distilled off under reduced pressure and the pH was adjusted to 4 with the saturated solution of NaHSO₄ and extracted with EtOAc (5 × 15 mL). The organic phase was concentrated in a vacuum, and the residue was separated via column chromatography on silica gel (EtOAc:AcOH = 50:1) to give 29 as yellow crystals. Yield: 1.05 g (92%). M.p.: 116–119 °C. IR (KBr, cm⁻¹): 3369 (N-H, O-H), 1718 (C=O). Analysis: found C 54.10, H 6.87, N 8.63; calcd. for C₂₂H₃₄N₃O₉ C 54.54, H 1.07, N 8.67. ¹H NMR (400 MHz, CD₃OD + CF₃COOD, δ): 1.60–1.78 (m, 4H); 1.78–2.15 (m, 12H); 2.28–2.37 (m, 2H ⁹CH and ¹⁴CH); 2.47 (t, J_t = 6.5, 4H ¹⁸CH₂ and ²²CH₂); 3.34 (t, J_t = 6.5, 4H ¹⁷CH₂ and ²¹CH₂); 4.19 (d, J_d = 5.5, 4H ¹⁰CH₂ and ¹⁵CH₂). ¹³C NMR (150 MHz, CD₃OD + CF₃COOD, δ): 20.96, 27.47, 34.99, 36.63, 36.99, 37.83 (³CH₂ and ⁴CH₂, ⁶CH₂ and ¹¹CH₂, ⁷CH₂ and ¹²CH₂, ⁸CH₂ and ¹³CH₂, ¹⁷CH₂ and ²¹CH₂, ¹⁸CH₂ and ²²CH₂), 47.88 (⁹CH and ¹⁴CH), 65.36 (¹⁰CH₂ and ¹⁵CH₂), 77.47 (²C and ⁵C), 158.45 (¹⁶C=O and ²⁰C=O), 175.37 (¹⁹COOH and ²³COOH).

(1R(S),5R(S),7R(S),8R(S))-1,8-Bis[(prop-2-yn-1-yloxy)methyl]-6-azadispiro[4.1.4.2]tridecane-6-oxyl (30) and (1R(S),5R(S),7R(S),8R(S))-1-(Hydroxymethyl)-8-[(prop-2-yn-1-yloxy)methyl]-6-azadispiro[4.1.4.2]tridecane-6-oxyl (31). To the solution of 5 (1.015 g; 3.99 mmol) in dry THF (16 mL), the NaH (50% suspension in oil, 0.228 g, 5.99 mmol) was added. The mixture was stirred at room temperature for 1 h. Then, the solution of propargyl bromide in toluene (80%, 0.713 g; 5.99 mmol) was added. The mixture was stirred at room temperature until the reaction was complete (about 12 h. TLC control on silica gel, hexane:Et₂O = 3:4, visualization with UV-254). The mixture was diluted with AcOH (pH = 5–6) and Et₂O (15 mL) and washed with brine (2 × 15 mL). The organic phase was separated, and after evaporation of the solvent, the crude residue was separated using column chromatography on silica gel (hexane:Et₂O from 3:1 to 1:4) to give 30 and 31. 30: yellow crystals, yield 30 0.475 g (36%), m.p.: 54.1–54.9 °C. UV-Vis (λ_max (nm)/lg(ε)): 240 (3.26). IR (KBr, cm⁻¹): 3293, 3251 (H-C≡), 2113 (C≡C). Analysis: found C 73.11, H 8.96, N 4.27; calcd. for C₂₀H₂₈NO₃ C 73.69, H 8.54, N 4.24. HRMS (EI/DFS) m/z [M⁺]: found: 330.2062; calcd. for C₂₀H₂₈NO₃: 330.2064. 31: yellow oil, yield 31 0.467 g (40%). UV-Vis (λ_max (nm)/lg(ε)): 232 (3.17). IR (neat, cm⁻¹): 3421 (O-H), 3305, 3253 (H-C≡), 2111 (C≡C). Analysis: found C 69.60, H 8.64, N 4.58; calcd. C 69.83, H 8.96, N 4.79. HRMS (EI/DFS) m/z [M⁺]: found: 292.1907; calcd. for C₁₇H₂₆NO₃: 292.1902.

(1R(S),5R(S),7R(S),8R(S))-1-(Hydroxymethyl-8-({(1-[2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl]-1H-1,2,3-triazol-4-yl)methoxy}methyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl (32). The solution of 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl azide (0.344 g, 0.921 mmol) in EtOH (4 mL) was added dropwise to the solution of 31 (0.269 g; 0.921 mmol) in EtOH (2 mL). A solution of CuSO₄ in H₂O (10%, 300 μL, 0.19 mmol) was mixed with a solution of ascorbic acid (190 mg, 1.08 mmol) in water (2 mL) and immediately poured into the above solution of azide and alkyne. The mixture was stirred at room temperature until the reaction was complete (ca. 12 h, TLC control on silica gel, hexane:Et₂O = 1:2, visualization with UV-254). EtOH was distilled off in vacuum, brine (8 mL) was added to the residue and the mixture was extracted with CHCl₃ (2 × 15 mL). The organic phase was evaporated in a vacuum, and the residue was separated via column chromatography on silica gel (CHCl₃:CH₃OH = 20:1) to give 32 as yellow glassy solid. Yield: 10.21 g (68%). IR (KBr, cm⁻¹): 3434 (O-H), 3145 (H-C=), 1755 (C=O), 1226 (C-OAc). Analysis: found C 56.07, H 6.80, N 8.07; calcd. for C₃₁H₄₅N₄O₁₂ C 55.93, H 6.81, N 8.42. ¹H NMR (400 MHz, CD₃OD + CF₃COOD, δ): 1.55–2.25 (m, 17H); 1.87, 1.97, 2.01, 2.20 (s, all 3H ²⁹CH₃, ³⁰CH₃, ³¹CH₃, ³²CH₃); 2.27–2.37 (m, 1H); 3.71 (ddd, J_d1 = 4.2 J_d2 = 7.3 J_d3 = 10.3, 1H ¹⁰CH₂ or ¹⁵CH₂); 3.81 (ddd, J_d1 = 6.6 J_d2 = 6.8 J_d3 = 11.5, 1H ¹⁰CH₂ or ¹⁵CH₂); 3.86 (ddd, J_d1 = 3.6 J_d2 = 10.2 J_d3 = 10.3, 1H ¹⁰CH₂ or ¹⁵CH₂); 3.94 (ddd, J_d1 = 0.9 J_d2 = 3.5 J_d3 = 11.5, 1H ¹⁰CH₂ or ¹⁵CH₂); 4.15 (ddd, J_d1 = 0.7 J_d2 = 7.0 J_d3 = 11.4, 1H); 4.24 (dd, J_d1 = 5.6 J_d2 = 11.4, 1H); 4.50 (tdd, J_t = 0.9 J_d1 = 6.0 J_d2 = 6.9, 1H); 4.71 (m, 2H); 5.44 (ddd, J_d1 = 1.0 J_d2 = 3.4 J_d3 = 10.3, 1H); 5.56–5.63 (m, 2H); 6.11 (dd, J_d1 = 1.9 J_d2 = 9.1, 1H ¹⁹CH); 8.29 (d, J_d = 5.4, 1H ¹⁸CH=). ¹³C NMR (100 MHz, CD₃OD + CF₃COOD, δ): 20.15, 20.37, 20.41, 20.46 (²⁹CH₃, ³⁰CH₃, ³¹CH₃, ³²CH₃), 21.16, 21.51, 26.22, 26.55, 35.77, 35.81, 36.00, 36.86, 36.96, 37.70 (³CH₂, ⁴CH₂, ⁶CH₂, ⁷CH₂, ⁸CH₂, ¹¹CH₂, ¹²CH₂, ¹³CH₂), 46.32 and 47.31 (⁹CH and ¹⁴CH), 62.12, 62.59, [64.74 and 64.82], [70.64 and 70.72], [77.76 and 77.87], [78.81 and 78.82], 68.58, 69.75, 72.10, 74.99 (²⁰CH, ²¹CH, ²²CH, ²³CH), 87.09 (¹⁹CH), 124.41 (¹⁸CH=), 145.23 (¹⁷C), 170.67, 171.36, 171.89, 172.15 (²⁵C=O, ²⁶C=O, ²⁷C=O, ²⁸C=O).

(1R(S),5R(S),7R(S),8R(S))-1-(Hydroxymethyl)-8-({(1-[β-d-galactopyranosyl]-1H-1,2,3-triazol-4-yl)methoxy}methyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl (33). Nitroxide 32 (0.26 g; 0.39 mmol) was dissolved in saturated solution of NH₃ in MeOH (5 mL) and allowed to stand at r.t. until the reaction was complete (up to 72 h). The progress of the reaction was monitored by TLC (silica gel, CHCl₃:MeOH = 7:1, visualization with UV-254). The solution was evaporated under reduced pressure and the residue was separated using column chromatography on silica gel (SiO₂, CHCl₃:CH₃OH from 3:1 to 2:1) to give 33 as yellow glassy solid. Yield: 0.154 g (79%). IR (neat, cm⁻¹): 3440 (O-H). Analysis: found C 55.87, H 7.80, N 11.07; calcd. for C₂₃H₃₇N₄O₈ C 55.52, H 7.50, N 11.26. ¹H NMR (500 MHz, CD₃OD + CF₃COOD, δ): 1.53–1.72 (m, 4H); 1.74–2.08 (m, 9H); 2.09–2.23 (m, 4H); 2.31–2.38 (m, 1H); 3.75–3.84 (m, 5H); 3.88–3.96 (m, 3H); 4.04–4.08 (m, 1H); 4.17–4.23 (m, 1H); 4.73 (d, J_d = 12.5, 1H ¹⁶CH₂); 4.73 (d, J_d = 12.5, 1H ¹⁶CH₂); 5.65 (d, J_d = 9.2, 1H ¹⁹CH); 8.29–8.31 (m, 1H ¹⁸CH=). ¹³C NMR (125 MHz, CD₃OD + CF₃COOD, δ): [21.20 and 21.22], 21.42, 26.15, 26.54, 35.73, [35.94 and 35.97], 36.88, [37.57 and 37.59] (³CH₂, ⁴CH₂, ⁶CH₂, ⁷CH₂, ⁸CH₂, ¹¹CH₂, ¹²CH₂, ¹³CH₂), 46.23 and 47.19 (⁹CH and ¹⁴CH), 62.14, 62.30, 64.82, 70.89 (¹⁰CH₂, ¹⁵CH₂, ¹⁶CH₂, ²⁴CH₂), 77.78 and 78.75 (²C и ⁵C), 70.22, 71.33, 75.10, 79.70 (²⁰CH, ²¹CH, ²²CH, ²³CH), 89.95 (¹⁹CH), [124.26 and 124.31] (¹⁸CH), [144.75 and 144.78] (¹⁷C).

2,2,5,5-Tetramethyl-3-(((1-((β-d-galactopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)pyrrolidine-1-oxyl (35) was prepared according to the Scheme 9.

2,2,5,5-Tetramethyl-3-((prop-2-ynyloxy)methyl)pyrrolidine-1-oxyl (37). Sodium hydride (50% in oil, 170 mg, 3.54 mmol) was added portion-wise under argon to a stirred solution of 36 (580 mg, 3.37 mmol) in dry THF (5 mL). The mixture was stirred at room temperature under argon for 1 h; then, the solution of propargyl bromide in toluene (80%, 0.5 g, 4.2 mmol) was added. The mixture was stirred at room temperature overnight, quenched with HOAc (0.5 mL), diluted with brine (5 mL), the resulting nitroxide was extracted with ether:hexane = 1:1 mixture and the extract was dried with MgSO₄. The solution was concentrated in vacuum and the residue was separated using column chromatography on silica gel (CH₂Cl₂) to give 37 as a yellow oil. Yield 0.45 g (64%). IR (KBr, cm⁻¹): 3289, 3232 (H-C≡), 2112 (C≡C). Analysis: found C 68.46, H 9.55, N 6.67; calcd. for C₁₂H₂₀NO₂ C 68.54, H 9.59, N 6.66. HRMS (EI/DFS) m/z [M⁺]: found: 210.1488; calcd. for C₁₂H₂₀NO₂: 210.1489.

2,2,5,5-Tetramethyl-3-(((1-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)pyrrolidine-1-oxyl (38). 2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosyl azide (0.570 g, 1.53 mmol) and 2,2,5,5-tetramethyl-3-((prop-2-ynyloxy)methyl)pyrrolidine 1-oxyl (330 mg, 1.57 mmol) were dissolved in a mixture of EtOH (5 mL) and H₂O (1 mL) at 40 °C. A solution of CuSO₄ in H₂O (10%, 300 μL, 0.19 mmol) was mixed with a solution of ascorbic acid (100 mg, 0.57 mmol) in water (1 mL) and immediately poured into the above solution of azide and alkyne. The mixture was left overnight; then, MnO₂ (0.5 g, 5.7 mmol) was added, the mixture was stirred for 0.5 h, the precipitate was filtered off, washed with EtOH and CHCl₃, the combined filtrates were concentrated in vacuum and the residue was separated using column chromatography on silica gel (CHCl₃ + 2% MeOH), and fractions were evaporated in vacuum and triturated with dry diethyl ether to give 38 as a yellow glassy solid, which was dried in high vacuum. Yield 0.79 g (89%). IR (KBr, cm⁻¹): 3143 (H-C=), 1755 (C=O), 1227 (C-OAc). Analysis: found C 53.11, H 6.96, N 6.27; calcd. for C₂₆H₃₉N₄O₁₁ C 53.51, H 6.74, N 9.60. ¹H NMR (300 MHz, CD₃OD + CF₃COOH, δ): 1.34, 1.48, 1.51, 1.53 (each s, 3H)(¹CH₃, ²CH₃, ³CH₃, ⁴CH₃); 1.89, 2.01, 2.04, 2.24 (each s, 3H ⁵CH₃, ⁶CH₃, ⁷CH₃, ⁸CH₃); 1.88 (m, 1H) and 2.09 (m, 1H) (⁹CH₂); 2.58 (ddd, J_d1 = 6.7 J_d2 = 6.6 J_d3 = 12.4, 1H ¹⁰CH); 3.54 (m, 2H ¹¹CH₂); 4.64 (m, 2H ¹²CH₂); 4.21 (m, 2H ¹⁹CH₂); 4.35 (t, J = 6.4, 1H ¹⁸CH); 5.38 (dd, J_d1 = 3.1 J_d2 = 10.2, 1H ¹⁶CH); 5.57 (m, 2H ¹⁴CH and ¹⁵CH); 6.0 (m, 1H ¹⁷CH); 8.07 (m, 1H ¹³CH).

2,2,5,5-Tetramethyl-3-(((1-(β-d-galactopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)pyrrolidine-1-oxyl (35). The nitroxide 38 (0.6 g, 1.03 mmol) was dissolved in the saturated solution of NH₃ in dry methanol (25 mL). The solution was left overnight; then, evaporated in vacuum and the residue was separated using column chromatography on silica gel (CHCl₃ + 20% MeOH). The pure fractions were collected, evaporated in vacuum and the residue was triturated with diethyl ether and dried in high vacuum to give 35 as light-yellow glassy powder. Yield 0.32 g (77%). IR (KBr, cm⁻¹): 3410 (O-H), 3153 (H-C=). Analysis: found C 52.11, H 7.96, N 13.27; calcd. for C₁₈H₃₁N₄O₇ C 52.04, H 7.52, N 13.49. ¹H NMR (300 MHz, CD₃OD, reduced with Zn/NH₄Cl at 5 °C, and then, filtered and acidified with few drops of CF₃COOH, a mixture of invertamers and diastereomers, δ): 1.2–1.6 (m, 12H CH₃ groups); 1.88 (m), 1.99 (m), 2.11 (m), 2.30 (m), 2.46 (m) and 2.65 (m), (total 3H pyrrolidine ring, two invertamers); 3.63 (m, 2H OCH₂-); 4.66 (m, 2H CH₂-C=); 3.7–3.84 (m, 3H), 3.89 (m, 1H), 4.03 (m, 1H), 4.18 (m, 1H), 5.62 (d, J = 8.8 Hz) (Galactose); 8.28 (s, 1H CH=).

4. Conclusions

In this work, we described a new family of dispirocyclic nitroxides that show very attractive properties for SDSL/EPR studies in biological media and in cells. The unique feature of these radicals is the combination of high resistance to reduction with improved spin relaxation characteristics typical of nitroxides with two spirocyclic moieties adjacent to N-O^• group. These nitroxides can be prepared from commercially available chemicals using a set of simple procedures with an overall yield over 40%. The resulting dispirocyclic core contains two hydroxymethyl groups near the radical center, which opens up the possibility of synthesizing mono- and bifunctional spin labels. It is important that binding to large biomolecules may further increase the resistance to reduction due to proximity of the functional groups to nitroxide moiety.