4-Dialkylamino-2,5-dihydroimidazol-1-oxyls with Functional Groups at the Position 2 and at the Exocyclic Nitrogen: The pH-Sensitive Spin Labels

Local acidity and electrostatic interactions are associated both with catalytic properties and the adsorption activity of various materials, and with the vital functions of biomolecules. The observation of acid–base equilibria in stable free radicals using EPR spectroscopy represents a convenient method for monitoring pH changes and the investigation of surface electrostatics, the advantages of which are especially evident in opaque and turbid samples and in porous materials such as xerogels. Imidazoline nitroxides are the most commonly used pH-sensitive spin probes and labels due to the high sensitivity of the parameters of the EPR spectra to pH changes, their small size, and their well-developed chemistry. In this work, several new derivatives of 4-(N,N-dialkylamino)-2,5-dihydrioimidazol-1-oxyl, with functional groups suitable for specific binding, were synthesized. The dependence of the parameters of their EPR spectra on pH was studied. Several showed a pKa close to 7.4, following the pH changes in a normal physiological range, and some demonstrated a monotonous change of the hyperfine coupling constant by 0.14 mT upon pH variation by four units.


Introduction
Interfacial phenomena and local protonation effects play an important role in biophysics, biochemistry, and in the chemistry of heterogeneous systems [1]. Catalytic and sorption properties of various materials are dependent on the local acidity and electrostatic interactions inside the pores [2]. Measurements of the local acidity and electrostatic potential of the inner pore surfaces represent a problem of great practical interest. Several methods have been developed for the characterization of the acid-base properties of different surface locations [3].
EPR spectroscopy of ionizable nitroxides is a convenient method for the investigation of the above-mentioned phenomena [2,4,5], and is fully applicable to opaque or turbid materials [5,6]. Nitroxide spin probes are small enough to penetrate directly into the pores and to be adsorbed onto the surface of the material under study. The protonation of basic centers in specially designed spin probes affects the hyperfine coupling A-tensor and gfactor matrix, as well as the rotational dynamics of the nitroxide molecule in the proximity of charged surfaces, and this is reflected in the EPR spectra [7]. An analysis of these data gives information about the acidic centers in the material and the local surface electrostatic potential. Recently, EPR studies using pH-sensitive spin probes were successfully used for the investigation of binary TiO 2 -SiO 2 xerogels [8].
Imidazoline nitroxides are the most commonly used pH-sensitive spin probes and labels due to the high sensitivity of the parameters of the EPR spectra to pH changes, their Imidazoline nitroxides are the most commonly used pH-sensitive spin probes and labels due to the high sensitivity of the parameters of the EPR spectra to pH changes, their small size, and their well-developed chemistry. A large number of pH-sensitive nitroxides of the imidazoline series have been prepared [1,4,9]. Some of them are highly sensitive to pH changes in physiologically important regions. The development of a convenient method for the synthesis of 4-(N,N-dialkylamino)-2,5-dihydrioimidazol-1-oxyls from 4H-imidazole-3-oxides [10] allowed for easy variation of the substituents in position two of the heterocycle to prepare useful spin probes. Examples illustrating the benefits of this strategy include the synthesis of nitroxides with two pKa values showing high sensitivity in a broad range of pH [11,12], e.g., Scheme 1, label 1, and pH-sensitive alkylating spin, labels 2a-c [13][14][15], which were used to prepare the hydrophilic spin probes from glutathione [13,14], thiol-specific pH-sensitive spin, label 3, for site-directed labeling of proteins and lipids [16,17], and siloxane-derived spin, label 4, capable of binding to silica or alumina surfaces [7]. Despite the significant advances in this area, the broad variety of potential research objects produces a request for new pH-sensitive spin labels capable of specific attachment. Here we describe a new set of pH-sensitive imidazoline nitroxides with various functional groups in the side chain. Some of them may find an application in material science or in biophysics. Scheme 1. Structure of the nitroxides 1-4.

Results and Discussion
The high reactivity of 2 in nucleophilic substitution reactions offers easy access to new functional derivatives. Expectedly, 2b readily reacts with sodium azide to produce 5 with a nearly quantitative yield. Nitroxide azides can be used for the spin labeling of acetylene-modified molecules via Huisgen 1,3-dipolar cycloaddition [18,19]. In analogy to the literature [20], a reaction of 5 with tetraisopropyl but-3-yne-1,1-diyldiphosphonate 6 in the presence of Cu(II) salt and ascorbic acid after subsequent re-oxidation produced 7 (Scheme 2).

Results and Discussion
The high reactivity of 2 in nucleophilic substitution reactions offers easy access to new functional derivatives. Expectedly, 2b readily reacts with sodium azide to produce 5 with a nearly quantitative yield. Nitroxide azides can be used for the spin labeling of acetylene-modified molecules via Huisgen 1,3-dipolar cycloaddition [18,19]. In analogy to the literature [20], a reaction of 5 with tetraisopropyl but-3-yne-1,1-diyldiphosphonate 6 in the presence of Cu(II) salt and ascorbic acid after subsequent re-oxidation produced 7 (Scheme 2).
The addition of nitroxides with a terminal acetylene group to azide-modified biomolecules, e.g., nucleic acids, is another way to use the Huisgen-click reaction for spin labeling. Terminal acetylenes can also be attached via Pd-catalyzed coupling [21]. A spin label with a terminal acetylene group was prepared from 8 in two steps (Scheme 3). The oxidation of benzyl alcohol 8 with the activated manganese dioxide in methanol smoothly led to the formation of the corresponding aldehyde 9, with the nitroxyl group and the amidine moiety being unaffected. Alternatively, 8 can be oxidized to the aldehyde 9 with 1-oxo-2,2,6,6-tetramethylpiperidinium chloride 10 with similar yield. The aldehyde 9 readily reacts with Bestmann-Ohira reagent to produce 11 with a yield of 84% [22]. The structure of 11 was confirmed with X-ray analysis data ( Figure S1).

Scheme 2. Synthesis of the nitroxide 7.
The addition of nitroxides with a terminal acetylene group to azide-modified biomolecules, e.g., nucleic acids, is another way to use the Huisgen-click reaction for spin labeling. Terminal acetylenes can also be attached via Pd-catalyzed coupling [21]. A spin label with a terminal acetylene group was prepared from 8 in two steps (Scheme 3). The oxidation of benzyl alcohol 8 with the activated manganese dioxide in methanol smoothly led to the formation of the corresponding aldehyde 9, with the nitroxyl group and the amidine moiety being unaffected. Alternatively, 8 can be oxidized to the aldehyde 9 with 1-oxo-2,2,6,6-tetramethylpiperidinium chloride 10 with similar yield. The aldehyde 9 readily reacts with Bestmann-Ohira reagent to produce 11 with a yield of 84% [22]. The structure of 11 was confirmed with X-ray analysis data ( Figure S1).

Scheme 3. Synthesis of the nitroxide 11.
Unless the attachment of a pH-sensitive nitroxide to a primary amino group is successfully performed via alkylation with 2b [7], the acylation reaction is more selective, allowing for the binding of a single nitroxide to the site. Carboxylic acids can be easily prepared from 8. Here we used a convenient one-pot process, where the reaction of 8 with an oxoammonium salt 10 was followed by the Lindgren-Kraus-Pinnick procedure [23] (Scheme 4). The carboxylic acid 12 was isolated with a 95% yield. To activate carboxylic group for acylation, the nitroxide 12 was treated with SOCl2 in the presence of pyridine. The chloroanhydride formed readily reacted with ethanol to give ester 14. The reaction of 13 with N-hydroxysuccinimide (NHS) produced spin label 15. The addition of nitroxides with a terminal acetylene group to azide-modified biomolecules, e.g., nucleic acids, is another way to use the Huisgen-click reaction for spin labeling. Terminal acetylenes can also be attached via Pd-catalyzed coupling [21]. A spin label with a terminal acetylene group was prepared from 8 in two steps (Scheme 3). The oxidation of benzyl alcohol 8 with the activated manganese dioxide in methanol smoothly led to the formation of the corresponding aldehyde 9, with the nitroxyl group and the amidine moiety being unaffected. Alternatively, 8 can be oxidized to the aldehyde 9 with 1-oxo-2,2,6,6-tetramethylpiperidinium chloride 10 with similar yield. The aldehyde 9 readily reacts with Bestmann-Ohira reagent to produce 11 with a yield of 84% [22]. The structure of 11 was confirmed with X-ray analysis data ( Figure S1).

Scheme 3. Synthesis of the nitroxide 11.
Unless the attachment of a pH-sensitive nitroxide to a primary amino group is successfully performed via alkylation with 2b [7], the acylation reaction is more selective, allowing for the binding of a single nitroxide to the site. Carboxylic acids can be easily prepared from 8. Here we used a convenient one-pot process, where the reaction of 8 with an oxoammonium salt 10 was followed by the Lindgren-Kraus-Pinnick procedure [23] (Scheme 4). The carboxylic acid 12 was isolated with a 95% yield. To activate carboxylic group for acylation, the nitroxide 12 was treated with SOCl2 in the presence of pyridine. The chloroanhydride formed readily reacted with ethanol to give ester 14. The reaction of 13 with N-hydroxysuccinimide (NHS) produced spin label 15. Unless the attachment of a pH-sensitive nitroxide to a primary amino group is successfully performed via alkylation with 2b [7], the acylation reaction is more selective, allowing for the binding of a single nitroxide to the site. Carboxylic acids can be easily prepared from 8. Here we used a convenient one-pot process, where the reaction of 8 with an oxoammonium salt 10 was followed by the Lindgren-Kraus-Pinnick procedure [23] (Scheme 4). The carboxylic acid 12 was isolated with a 95% yield. To activate carboxylic group for acylation, the nitroxide 12 was treated with SOCl 2 in the presence of pyridine. The chloroanhydride formed readily reacted with ethanol to give ester 14. The reaction of 13 with N-hydroxysuccinimide (NHS) produced spin label 15.
Another nitroxide with a carboxylic group on a longer spacer was prepared in one step from 8 via acylation with succinic anhydride (Scheme 5).
The EPR spectra of nitroxides 7, 11, 12, 15, and 16 are strongly pH-dependent with ∆a N > 0.1 mT, and a pK a between 6 and 6.7 ( Table 1). The titration curves demonstrate optimal sensitivity in slightly acidic media [14,15], but the sensitivity is not optimal in the normal physiological range of 7.35-7.45 [24]. Similar structures without aromatic substituents are known to show higher pK a values [11].
To prepare 2-functionalized nitroxides with a pK a above 7, the reaction of 4H-imidazol-3-oxide 17 with Grignard reagents was used (Scheme 6). The treatment of 17 with alkenylmagnesium bromides produced nitroxides with a terminal ethylene bond 18a,b. The hydroboration of 18a,b with 9-BBN, followed by oxidation with hydrogen peroxide, was performed using the protocol developed by Hideg for 2-allyl pyrrolidine nitroxides [25]. The reaction produced alcohols 19a,b, which were then treated with carbonyldiimidazole (CDI) to give 20a,b. To demonstrate the feasibility of the carbonylimidazole pH-sensitive spin labels for binding to primary amino groups, 20b was allowed to react with N,Ndiethyl-1,3-diaminopropane. Another nitroxide with a carboxylic group on a longer spacer was prepared in one step from 8 via acylation with succinic anhydride (Scheme 5).

Scheme 5.
A reaction of 8 with succinic anhydride.
The EPR spectra of nitroxides 7, 11, 12, 15, and 16 are strongly pH-dependent with aN > 0.1 mT, and a pKa between 6 and 6.7 ( Table 1). The titration curves demonstrate optimal sensitivity in slightly acidic media [14,15], but the sensitivity is not optimal in the normal physiological range of 7.35-7.45 [24]. Similar structures without aromatic substituents are known to show higher pKa values [11].  [25]. The reaction produced alcohols 19a,b, which were then treated with carbonyldi idazole (CDI) to give 20a,b. To demonstrate the feasibility of the carbonylimidaz pH-sensitive spin labels for binding to primary amino groups, 20b was allowed to r with N,N-diethyl-1,3-diaminopropane. The nitroxide 21 was isolated with a 60% yield. The oxidative cleavage of the term double carbon-carbon bond in 18b with osmium tetraoxide-oxone system yielded boxylic acid 22. Scheme 6. Synthesis of spin labels and spin probes from 17.
The addition of 2-(1,3-dioxolan-2-yl) ethylmagnesium bromide to 17 is another c venient way to create 2-functionalized pH-sensitive spin labels. The reaction produ nitroxide 23 with a high yield. The dioxolane protection group in 23 was readily remo under relatively mild conditions to give the corresponding aldehyde 24, which can be Scheme 6. Synthesis of spin labels and spin probes from 17.
The nitroxide 21 was isolated with a 60% yield. The oxidative cleavage of the terminal double carbon-carbon bond in 18b with osmium tetraoxide-oxone system yielded carboxylic acid 22.
The addition of 2-(1,3-dioxolan-2-yl) ethylmagnesium bromide to 17 is another convenient way to create 2-functionalized pH-sensitive spin labels. The reaction produced nitroxide 23 with a high yield. The dioxolane protection group in 23 was readily removed under relatively mild conditions to give the corresponding aldehyde 24, which can be either oxidized to carboxylic acid 25 using the Lindgren-Kraus-Pinnick procedure, or reduced with sodium borohydride to 19a. The sequence 17 → 23 → 24 → 19a gives a remarkably higher yield of the target nitroxide than the addition of allylmagnesium bromide with subsequent hydroboration. A titration of the nitroxides 20, 22, and 25 showed that they may be valuable spin labels and probes with high sensitivities to changes of pH within the physiological range (see Table 1, Figure 1 and Supplementary Materials). ther oxidized to carboxylic acid 25 using the Lindgren-Kraus-Pinnick procedure, or reduced with sodium borohydride to 19a. The sequence 17  23  24  19a gives a remarkably higher yield of the target nitroxide than the addition of allylmagnesium bromide with subsequent hydroboration. A titration of the nitroxides 20, 22, and 25 showed that they may be valuable spin labels and probes with high sensitivities to changes of pH within the physiological range (see Table 1, Figure 1 and Supplementary Materials). An investigation of the surfaces of many inorganic and organo-inorganic materials (catalysts, sorbents, etc.) requires nitroxides with a high sensitivity to acidity changes within a broad range of pH. A good example of such a spin probe is two-pKa nitroxide 1, which was successfully used in numerous studies [8,[26][27][28][29][30][31]. A covalent attachment of sim-  An investigation of the surfaces of many inorganic and organo-inorganic materials (catalysts, sorbents, etc.) requires nitroxides with a high sensitivity to acidity changes within a broad range of pH. A good example of such a spin probe is two-pK a nitroxide 1, which was successfully used in numerous studies [8,[26][27][28][29][30][31]. A covalent attachment of similar nitroxides to the surface of a catalyst or a sorbent may provide a useful method for studies of the near-surface layer in these materials. Here we designed analogs of 1 with a functional group in a substituent at the exocyclic nitrogen atom of the amidine moiety.
N-(4-(1,3-dioxolan-2-yl)benzyl)-N-methylamine 26 was prepared in two steps from tereftaldicarboxaldehyde 27 (Scheme 7). An investigation of the surfaces of many inorganic and organo-inorganic materials (catalysts, sorbents, etc.) requires nitroxides with a high sensitivity to acidity changes within a broad range of pH. A good example of such a spin probe is two-pKa nitroxide 1, which was successfully used in numerous studies [8,[26][27][28][29][30][31]. A covalent attachment of similar nitroxides to the surface of a catalyst or a sorbent may provide a useful method for studies of the near-surface layer in these materials. Here we designed analogs of 1 with a functional group in a substituent at the exocyclic nitrogen atom of the amidine moiety.

Scheme 7. Synthesis of N-(4-(1,3-dioxolan-2-yl)benzyl)-N-methylamine (26).
A reaction of the 5-cyano-4H-imidazole-3-oxide 29 with 26 resulted in cyanide substitution with the formation of 30, and the latter was treated with an excess of ethylmagnesium bromide (Scheme 8). The nitroxide 31 was isolated after a quenching of the reaction mixture with water and oxidation. To hydrolyze the dioxolane ring, 31 was heated to reflux in 0.5 M aqueous HCl. The resulting aldehyde 32 was reduced with sodium borohydride to the corresponding alcohol 34, or oxidized with sodium chlorite to carboxylic acid 33 as described above for 23. Similarly to 11, the nitroxide 33 was converted into succinimidyl ester 35 via a reaction of in situ generated chloroanhydride with NHS.
A reaction of the 5-cyano-4H-imidazole-3-oxide 29 with 26 resulted in cyanide substitution with the formation of 30, and the latter was treated with an excess of ethylmagnesium bromide (Scheme 8). The nitroxide 31 was isolated after a quenching of the reaction mixture with water and oxidation. To hydrolyze the dioxolane ring, 31 was heated to reflux in 0.5 M aqueous HCl. The resulting aldehyde 32 was reduced with sodium borohydride to the corresponding alcohol 34, or oxidized with sodium chlorite to carboxylic acid 33 as described above for 23. Similarly to 11, the nitroxide 33 was converted into succinimidyl ester 35 via a reaction of in situ generated chloroanhydride with NHS. Titration of the nitroxides 32-35 showed a gradual monotonous increase of HFC on the nitroxide nitrogen atom by ca. 0.14 mT upon a pH change from 1.5 to 5.5 (see Figure  2, Table 2, and Supplementary Materials). The shape of the titration curve perfectly corresponded to a two-step acid-base equilibrium, and fitting with the Henderson-Hassellbalch function (Equation (2), see experimental part) gave two pKa values for each ni-Scheme 8. Synthesis of two-pK a nitroxides. Structure of the nitroxide 36. Titration of the nitroxides 32-35 showed a gradual monotonous increase of HFC on the nitroxide nitrogen atom by ca. 0.14 mT upon a pH change from 1.5 to 5.5 (see Figure 2, Table 2, and Supplementary Materials). The shape of the titration curve perfectly corresponded to a two-step acid-base equilibrium, and fitting with the Henderson-Hassellbalch function (Equation (2), see experimental part) gave two pK a values for each nitroxide (Table 1), corresponding to the sequential protonation of the basic centers, amidine group and pyridine nitrogen. Scheme 8. Synthesis of two-pKa nitroxides. Structure of the nitroxide 36.
Titration of the nitroxides 32-35 showed a gradual monotonous increase of HFC on the nitroxide nitrogen atom by ca. 0.14 mT upon a pH change from 1.5 to 5.5 (see Figure  2, Table 2, and Supplementary Materials). The shape of the titration curve perfectly corresponded to a two-step acid-base equilibrium, and fitting with the Henderson-Hassellbalch function (Equation (2), see experimental part) gave two pKa values for each nitroxide (Table 1), corresponding to the sequential protonation of the basic centers, amidine group and pyridine nitrogen.   In accordance with the general concept of basicity, the pK a value of the amidine fragment should be higher than that of the pyridine one. However, according to the simulation, the protonation of the center with a more acidic pK a is accompanied by a change in the hyperfine constant by 0.077-0.092 mT, which is typical of the amidine group in 4-amino-2,5-dihydroimidazol-1-oxyls, while the higher pK a (4.73-4.89) corresponds to a smaller change in the hyperfine constant (0.05-0.063 mT), which may correspond to pyridine moiety protonation. Moreover, the basic pK a showed minor dependence on the nature of the substituent at the exocyclic nitrogen, while the acidic pK a varies from 2.58 for 34 to 2.19 for 35. Meanwhile, a comparison of 34 and 35 shows that an increase in the electron-withdrawing character of the substituent at the exocyclic nitrogen leads to an increase of ∆a N in the more acidic region, and a decrease of that correspondent to higher pK a . A comparison of the titration data for 1 and 36 [11] gives similar results. Data in the literature show that pK a values for 4-amino-2,5-dihydroimidazol-1-oxyls are strongly dependent on substituents at exocyclic nitrogen and can go below four [32,33]. Thus, it is obvious that the pyridine nitrogen and amidine group in 31-35 have similar basicity and the contribution of different monoprotonated forms is varying depending on the electronic effect of the substituents at the exocyclic nitrogen.

Conclusions
In this paper feasibility of the approach to synthesis of pH-sensitive spin labels of 4amino-2,5-dihydrioimidazol-1-oxyl series was once again demonstrated. We have showed that various functional groups can be easily placed in the substituents both in position 2 and to exocyclic nitrogen to make the spin probe suitable for a specific purpose. The potential of this synthetic scheme is still far from exhaustion.
The X-ray diffraction experiment was carried out on a Bruker KAPPA APEX II (Bruker, Billerica, MA, USA) diffractometer (graphite-monochromated Mo Kα radiation). Reflection intensities were corrected for absorption by SADABS-2016 program [38]. The structure of compound 11 was solved by direct methods using the SHELXT-2014 program [39] and refined by anisotropic (isotropic for all H atoms) full-matrix least-squares method against F 2 of all reflections by SHELXL-2018 [40]. The positions of the hydrogen atoms were calculated geometrically and refined in riding model. One of the geminal ethyl groups is disordered due to thermal motion at approximate ratio 3:2. Crystallographic data for 11 have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 2124865. Copy of the data can be obtained, free of charge, by 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 29 November 2021)). The details are shown in Supplementary Materials.