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Timestamp: 2019-04-26 06:31:27+00:00

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The synthesis of novel dibenzo-24-crown ether substituted nitroxides and their use as spin probes for the detection of cation guests by EPR are reported. Formation of a host–guest complex between the proposed spin probes and several cations, both organic and inorganic, was evidenced by a significant change in the value of the benzylic and nitrogen EPR hyperfine splittings upon complexation. This favorable feature provided a reliable EPR sensor that is able to selectively distinguish different cationic guests.
Crown ethers, discovered more than 50 years ago by the Nobel Prize winner Charles Pedersen,1 are cyclic organic compounds composed of several repeating oligo(ethylene oxide) ether units that are known to bind different cationic species.
The combined use of nitroxide4 spin labels and electron paramagnetic resonance (EPR) spectroscopy represents a complementary and useful tool for the characterization of supramolecular assemblies.5 Thus, a possible approach for the investigation of crown ethers as cation complexing agents is through the EPR spectral studies of nitroxide spin-labeled crown ethers. Actually, examples of spin-labelled crown ethers have been reported in the past. However, mono-spin labelled crown ethers were found to be rather poor EPR reporting agents for alkali metal cations.6 A remarkable exception was represented by the spin-labelled monoazacrown reported by Sosnovsky et al.7 In this case, the participation of the sodium cation in the molecular structure of the spin-labelled complex was established by observing the coupling of the unpaired electrons with Na+ nuclei by EPR. However, interactions with other cations were not described.
Di- or tri-spin labelled crown ethers have also been reported in the literature.8 Although modulation of the spin–spin contact generally reported the binding of K+ cations, this interaction did not permit to differentiate the complexation of diverse cationic guests.
Very recently, two examples of rotaxanes incorporated with a spin-labelled crown ether have also been studied.9,10 In these systems, the shuttling process of the paramagnetic wheel along a dumbbell composed of dialkylammonium and 4,4′-bipyridinium recognition sites by EPR spectroscopy was demonstrated.
In particular, the formation of a supramolecular assembly with this class of nitroxide radicals by a complexing agent normally produces significant differences in the resonance fields of MI(2Hβ) EPR spectral lines in solution compared to the uncomplexed radical. These large spectral changes can arise both/or from the change in the value of the nitrogen hyperfine splittings, aN, induced by the different polar environments of the complexing hosts, and the strong variation in the coupling constants of benzylic protons, a2Hβ, due to conformational changes occurring upon complexation.11 This shows that a sizeable change in the value of the benzylic and nitrogen EPR hyperfine splittings upon complexation was generally observed depending on the nature of the complexed cationic guests.
Scheme 2 summarizes the synthetic steps to prepare the spin probes 2 and 4. In particular, amine 1 obtained by the condensation of formyl-DB24C812 with tert-butyl amine and reduction with NaBH4 was successively oxidized directly inside an EPR tube to the corresponding nitroxide 2 upon the addition of 3-chloroperbenzoic acid (MCPBA). Similarly, probe 4 was produced by the oxidation of the nitroxide precursor 3 using 2-amino-2-methyl-1-propanol as the starting amine (see the Experimental section).
A good EPR spectrum of nitroxide 2 was obtained upon oxidation of amine 1 (1.0 mm) with MCPBA (1.0 mm) in CH2Cl2 (DCM) at 298 K (see Fig. 1a).
Fig. 1 EPR spectra of 2 in CH2Cl2 at 298 K: (a) in the absence of Na+; (b) in the presence of 7 mM Na+; (c) in Na+ saturated solution.
The spectrum was easily interpreted on the basis of the coupling of the unpaired electron with the nitrogen and with the two benzylic protons (see Table 1).
The host properties of 2 towards cations which are known to be selectively complexed by crown ether derivatives were then investigated by EPR. Alkaline metal M+ iodide or picrate salts are practically insoluble in CH2Cl2. However complete solubilisation of metal salts was observed after the addition of an excess of the starting amine 1. This clearly suggests that host 1 maintains the complexation ability of crown ethers towards alkaline cations M+.
When the oxidant, MCPBA, is added to these complexes, the corresponding EPR spectrum shows additional signals (Fig. 1b, M+ = Na+; Fig. 2b, M+ = K+) assigned to the complexed radical 2@M+ in equilibrium with the free nitroxide 2 (Scheme 3).
Fig. 2 EPR spectra of 2 in CH2Cl2 at 298 K: (a) in the absence of K+; (b) in the presence of 7 mM K+; (c) in K+ saturated solution.
When the concentration of metal cations was increased until metal salt precipitation was obtained, the spectrum of the complexed radical 2@M+ became dominant (Fig. 1c, M+ = Na+; Fig. 2c, M+ = K+), allowing the easy measurement of the spectroscopic parameters of the complexed nitroxide.
This behaviour is shared by Na+, K+, and Cs+ cations, while Li+, known to be poorly complexed by large crown ethers,13 did not lead to any observable change in the EPR spectrum of the free nitroxide (see Table 1).
Table 1 shows that the value of aN does not change significantly, and that of a(2Hβ) varies considerably upon complexation, giving rise to significant differences in the resonance fields of MI(2Hβ) = ±1 lines of the complexed and free species.
In particular, a(2Hβ) increases inversely with cation size, being 8.47 G in the presence of Cs+ and rising monotonically to 8.73 G in the presence of Na+ (8.06 G is the value of the nitroxide in the absence of the alkali-metal cation). The observation that nitrogen splitting does not change significantly strongly suggests that the variation observed in the benzylic coupling value is not due to a general medium effect, that is, a change in the polarity of the environment surrounding the probe, but is caused by a specific interaction with M+. According to the Heller–McConnell equation, the value of the hyperfine splitting constant for β-protons in alkyl nitroxides is mainly determined by the dihedral angle between the symmetry axis of the 2pz-orbital of nitrogen and the N–C–Hβ plane.14 The change in the geometry of crown ether upon formation of the complex with the metal cation (2@M+) is also responsible for the variation in the geometry adopted around the benzylic bond, giving rise to a substantial change in the values of β-H couplings.
Similar results were also obtained with probe 4 and the corresponding EPR parameters are reported in Table 2.
We checked the possibility of obtaining quantitative information on the complexation of metal cations by recording the EPR spectra of 2 in ACN, a solvent in which alkaline metal cation iodides are soluble.
In eqn (1), 0 is the total amount of nitroxide, aEPR represents the apparent value of a2H measured from the EPR spectrum, and afree and acomplex are the coupling constants of benzylic protons in the free and complexed nitroxides, respectively. Fig. 3 (circle symbols) presents the dependence of the degree of complexation on K+ concentrations calculated by substituting in eqn (1), the aEPR values of radical 2 measured in ACN at 298 K.
This sigmoid dependence, however, could not be well-modelled by assuming a value for the association constant, Ka, equal to that reported in the literature in ACN for the unlabelled DB24C8 (Ka = 6900 M−1)15 and by admitting a simple standard 1 : 1 binding model as that reported in Scheme 3.
As mentioned in the Introduction section, nitroxide probes like 2 and 4, having hydrogen atoms in β-positions, are characterized by a reduced lifetime when compared to sterically hindered nitroxides.4 This behaviour is largely determined by their ability to disproportionate into the corresponding hydroxylamine and nitrone (see Scheme 1).
A steady-state concentration of nitroxide is, thus, observed inside the EPR tube when the rates of formation and destruction are approximately equal.† In our case, this condition is reached few minutes after mixing the reagents in the oxidation step. The amount of nitroxide measured from the EPR spectrum does not generally exceed 3% of the starting amine. Under these conditions, the diamagnetic species containing the crown ether unit (CEdia), i.e. the starting amine 1 and the disproportion products 2a and 2b, represent the larger fraction of the metal receptor present in solution and cannot be ignored. As a consequence, the concentration of M+ available for complexation (M+eff) by a paramagnetic probe is much lower than that initially dissolved in solution (M+0) and its quantity inversely depends on the amount of diamagnetic crown ether derivatives actually present in solution. This was definitively proved by recording a series of EPR spectra of 2 in the presence of a constant amount of M+ and by varying the initial concentration of the amine precursor and the oxidant.
As an example, Fig. 4 (circle symbols) presents the dependence of the degree of complexation of 2 on the amount of host 1 measured in the presence of two different concentrations of Na+. For each sample, the quantity of peracid used in the oxidation step was optimized to maintain nearly constant the relative amount of nitroxide and the starting amine (0/0 ≈ 60).
As shown in Fig. 3 and 4, the dependence of the complexation degree of 2 on M+ (Fig. 3) or the starting amine (Fig. 4) was remarkably modelled by solving simultaneously the reported equilibria and assuming for all labelled crown ether derivatives a Ka value very similar to that reported in the literature for the unsubstituted DB24C8.
Fig. 5a also presents the EPR spectrum of 1 recorded in CH2Cl2 after the addition of dibenzylammonium cation (Bn2NH2+). Once again the EPR spectroscopy parameters (see Table 1) differ from those recorded in CH2Cl2, indicating the formation of the complex 2@Bn2NH2+.
Fig. 5 EPR spectra of 2 recorded in CH2Cl2 at 298 K in the presence of: (a) Bn2NH2+; (b) 5H3+; (c) 5H3+ and 0.5 eq. of DIPEA; (d) 5H3+ and an excess of DIPEA.
Because the EPR read-out discriminates between different cationic guests, we evaluated the EPR behaviour of radical probes 2 or 4 in a pseudorotaxane containing both dialkylammonium and 4,4′-bipyridinium (viologen) recognition sites in the thread (5H3+, Scheme 4). Again, evidence for the formation of the pseudorotaxane in CH2Cl2 was obtained by observing complete solubilisation of 5H3+, which is almost completely insoluble in chlorinated hydrocarbons, after the addition of the host.
Fig. 5b shows the EPR spectrum of the pseudorotaxane 2@5H3+ recorded in CH2Cl2 at 298 K, after the oxidation of 1@5H3+ with MCPBA. The values of aN = 15.42 G and a2H = 8.59 G (see Table 1) are close to those observed for the 2@Bn2NH2+ complex. According to the literature data,16 these data suggest that the macrocycle predominantly encircles the ammonium group.
Treatment of 2@5H3+ in CH2Cl2 with 0.5 eq. of the non-nucleophilic diisopropylethylamine (iPr2EtN, DIPEA), which is strong enough to deprotonate the NH2+ centre, led to the appearance of a new radical species (Fig. 5c). This new signal is characterized by very different coupling constants (aN = 15.54 G and a2H = 9.94 G) and was attributed to a new complex in which the ring is shifted from the ammonium site to the viologen unit (see Scheme 4).16 The simultaneous presence of both signals indicates that the exchange between the protonated and deprotonated forms of the pseudorotaxane must be significantly slower than the EPR timescale.
Addition of increasing amounts of iPr2EtN to 2@5H3+ produced a noticeable increase in the intensity of this new signal, which became predominant in the presence of an excess amount of base (Fig. 5d). Finally, the addition of trifluoroacetic acid after addition of iPr2EtN caused the recovery of the initial EPR spectrum as a consequence of the return of the macrocycle to the ammonium site.
In summary, we have developed novel radical probes that feature a crown ether moiety. EPR coupling constants of benzylic protons are affected by cationic binding, which leads to distinguish between different cationic guests. The amount of cations in the order of mM concentrations can easily be detected by this method. The general good agreement with quantitative data previously determined suggests that EPR can be usefully employed to study complexation by crown ethers even in supramolecular systems of higher complexity when traditional methods based on NMR or fluorescence cannot be applied.
Compound 1. A stirred mixture of formyl-DB24C8 (250 mg, 0.52 mmol) and tert-butylamine (82 μL, 0.788 mmol, 1.5 eq.) in dry methanol (19 mL) was refluxed for 18 h. Formation of the imine intermediate was observed by TLC (CH2Cl2/MeOH 9 : 1). The mixture was then cooled to room temperature and NaBH4 (78 mg, 2.1 mmol, 4 eq.) was added in small portions. The mixture was stirred at rt for 5 h and quenched with 1 M HCl (3 mL). Methanol was evaporated under vacuum, and the residue was suspended in 1 M NaOH (10 mL) and stirred for 15 min. The aqueous layer was extracted with CH2Cl2 (4 × 10 mL), washed with water and brine (40 mL each) and dried over MgSO4. The solvent was removed in vacuo to obtain a yellowish solid. The crude product was purified by silica gel chromatography (eluent CH2Cl2/MeOH/NH4OH 25% 90 : 10 : 1) to obtain an off-white sticky solid (176 mg, 63%). 1H-NMR (400 MHz, CDCl3) δ 1.18 (s, 9 H), 3.65 (s, 2 H), 3.82 (s, 8 H), 3.88–3.91 (m, 8 H), 4.09–4.18 (m, 8 H), 6.77–6.95 (m, 7 H); 13C NMR (101 MHz, CD2Cl2) δ 149.64, 149.43, 148.56, 121.94, 115.43, 114.87, 114.61, 71.57, 70.38, 69.83, 54.00, 47.05, 28.88. ESI-MS (m/z): 534.2 [M + H]+, 556 [M + Na]+.
Compound 3. Formyl-DB24C8 (250 mg, 0.52 mmol, 1 eq.) was reacted with 2-amino-2-methyl-1-propanol (49 mg, 0.54 mmol, 1.05 eq.) in methanol (19 mL) under reflux conditions for 18 h. Formation of the imine intermediate was observed by TLC (CH2Cl2/MeOH 9 : 1). The mixture was then cooled to room temperature and NaBH4 (78 mg, 2.1 mmol, 4 eq.) was added to the reaction mixture in small portions. The mixture was stirred at rt for 5 h and the reaction was quenched with 1 M HCl (3 mL). Methanol was evaporated under vacuum and the residue was suspended in 1 M NaOH (10 mL), stirred for 15 min, and the aqueous layer was extracted with CH2Cl2 (4 × 10 mL), washed with water and brine (40 mL each) and dried with MgSO4. The solvent was removed in vacuo to obtain a yellowish solid. The crude product was purified by silica gel chromatography (eluent CH2Cl2/MeOH 9 : 1 and then CH2Cl2/MeOH/NH4OH 90 : 10 : 1) to obtain a white sticky solid (160 mg, 55%). 1H-NMR (400 MHz, CD2Cl2) δ 1.11 (s, 6 H), 3.28 (s, 2 H), 3.60 (s, 2 H), 3.76 (s, 8 H), 3.83–3.86 (m, 8 H), 4.09–4.14 (m, 8 H), 6.81–6.89 (m, 7 H); 13C NMR (101 MHz, CD2Cl2) δ 149.64, 148.52, 134.98, 121.93, 121.32, 114.86, 71.59, 70.40, 69.82, 68.81, 54.00, 46.50, 24.42. ESI-MS (m/z) 550 [M + H]+, 572 [M + Na]+.
3,5-Di-tert-butylbenzyl-4-[(4,4′-(methylbipyridinium)methyl)]benzylammonium tris(hexafluorophosphate) (5H3+). 3,5-Di-tert-butylbenzyl-4-[(4,4′-pyridylpyridinium)methyl]benzylammonium bis(hexafluorophosphate)16 (50 mg, 0.065 mmol) was dissolved in CH3CN (5 mL) and then methyl iodide (12 μL, 0.194 mmol, 3 eq.) was added. The mixture was heated under reflux conditions for 3 d. The solvent was removed in vacuo and the crude mixture was subjected to column chromatography (eluent CH2Cl2/MeOH 9 : 1 then 7 : 1 and finally MeOH/H2O/NH4Cl 2M 7 : 2.5 : 0.5). Methanol was removed in vacuo and the solution was then treated with NH4PF6 aqueous solution. The precipitate was filtered and washed with water to afford the title compound as a white solid (34 mg, 56%). 1H-NMR (400 MHz, (CD3)2CO) δ 1.30 (s, 18 H), 4.62 (s, 2 H), 4.69 (s, 2 H), 4.73 (s, 3 H), 6.22 (s, 2 H), 7.45 (s, 2 H), 7.56 (s, 1 H), 7.71–7.77 (m, 4H), 8.77 (m, 4 H), 9.35 (m, 2 H), 9.45 (m, 2 H). 13C NMR (101 MHz, (CD3)2CO) δ = 152.54, 151.59, 147.77, 146.92, 135.56, 133.76, 132.21, 131.21, 130.68, 128.48, 127.75, 125.08, 124.37, 65.09, 53.25, 51.92, 35.50, 31.59, 29.84. ESI-MS (m/z) 784 [M − PF6]+.
Nitroxides 2 and 4 are generated by mixing a solution of the corresponding amine (4–30 mM) with a solution of 3-chloroperbenzoic acid (Aldrich, technical grade, 0.5–5 mM). Then, aliquots from a concentrated cation solution are added to the solution of nitroxide to yield the required concentrations. Samples are then transferred to capillary tubes (1 mm i.d.), and the EPR spectrum is immediately recorded. Radical concentrations were measured with respect to a solution of DPPH of known concentration using the signal from a ruby crystal as the internal standard.
This work was supported by the University of Bologna. We acknowledge Dr Andrea Gualandi for providing a sample of formyl-DB24C8.
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† It must be considered that nitroxide can also be re-formed by the oxidation of hydroxylamine 2b.
‡ Because diamagnetic crown ether derivatives are in large excess when compared to nitroxide 2, the amount of M+ complexed by 2 has been omitted in eqn (2).

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