Structural Equilibrium in New Nitroxide-Capped Cyclodextrins: CW

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Structural Equilibrium in New NitroxideCapped Cyclodextrins: CW and Pulse EPR Study Olesya Krumkacheva, Matvey V. Fedin, Dmitriy Polovyanenko, Laszlo Jicsinszky, Sylvain R. A. Marque, and Elena G. Bagryanskaya J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp404173j • Publication Date (Web): 14 Jun 2013 Downloaded from http://pubs.acs.org on June 19, 2013

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The Journal of Physical Chemistry

Structural Equilibrium in New Nitroxide-Capped Cyclodextrins: CW and Pulse EPR Study

Olesya A. Krumkachevaa,b, Matvey V. Fedina, Dmitry N. Polovyanenkoc, Laszlo Jicsinszkyd, Sylvain R. A. Marquee and Elena G. Bagryanskayaa,c*

a

International Tomography Center SB RAS, Institutskaya 3a, Novosibirsk 630090, Russia b

c

Novosibirsk State University, Pirogova str. 2, Novosibirsk 630090, Russia

N.N.Voroztsov Novosibirsk Institute of Organic Chemistry SB RAS, Pr. Lavrentjeva 9, Novosibirsk 630090, Russia d

e

Cyclolab Ltd., P.O. Box 435, H-1525 Budapest, Hungary

Aix-Marseille Université, CNRS-ICR UMR 7273, case 521, Avenue Escadrille NormandieNiemen, 13397 Marseille cedex 20, France

ACS Paragon Plus Environment

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ABSTRACT Design of the new spin-labeled cyclodextrins (CD) can significantly extend the functionality of nitroxides. A series of new complexes based on fully-methylated cyclodextrin (TRIMEB) covalently bound to the piperidine, pyrroline, pyrrolidine, and pH-sensitive imidazoline type nitroxides has been synthesized and studied using pulse and continuous wave Electron Paramagnetic Resonance (EPR). The influence of the radical and linker properties on the structure of complexes formed has been investigated. Using Electron Spin Echo Envelope Modulation (ESEEM) technique we have analyzed quantitatively the accessibility of radicals to solvent molecules in studied complexes depending on the structure and length of the linkers. In all studied systems we observed different types of equilibria between conformations with radical fragment being outside the TRIMEB cavity and radical fragment capping the cavity of TRIMEB. The observed guest-induced shift of equilibrium toward the complex with radical capping TRIMEB cavity was explained by a change of macrocyclic configuration of TRIMEB. Complex with the -NH-CO- linker has been found most perspective for the applications requiring close location of nitroxide to the inclusion complex of TRIMEB. Using continuous wave EPR we have shown that the pH-sensitive radical covalently bound to TRIMEB maintains its pH-sensitivity, but this complexation does not reduce radical reduction rate in the reaction with ascorbic acid.


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INTRODUCTION Supramolecular design of new functional systems is one of the perspective fields in current chemistry, biology and nanotechnology.1-3 The applications of such supramolecular complexes can be broadened by the development of new synthetic approaches where the properties of wellknown host molecules are enhanced by the new functions of molecules attached to them.4-6 Recently a series of new compounds based on cyclodextrins (CDs) has been developed owing to the high efficiency of supramolecular complex formation of CD with many hydrophobic molecules.7-9 One of the new directions of research in this field is the synthesis of CDs covalently bound to nitroxides (NCDs). 10-15 These systems can be considered as potential probes to study supramolecular assemblies of CDs with biomolecules,14 as reduction agents in aqueous solutions, as polarizing agents for DNP signal enhancement in NMR studies of proteins16, and as efficient and selective fluorescence quenchers.17 Moreover, complex formation of cyclodextrins with spin traps is used to increase the lifetimes of spin adducts formed in reactions with shortlived radicals (superoxide,18-23 thyil,12 etc.). NCDs are known to exhibit equilibrium between different structural forms of two types: type (B - A) (Scheme 1, model 1), and type (B - D) (Scheme 1, model 2).10, 13 In case of (B - A) type equilibrium is observed between the complex with radical fragment outside the cavity and radical fragment capping the cavity of CD. In case of (B - D) type, the structure of stronglycoupled complex is characterized by nitroxide fragment being deeply immersed into the cavity of CD. The scope of applications of NCDs depends on the equilibrium type they exhibit. On one hand, the lifetime of spin adducts that form complexes with deeply-included radical is significantly larger compared to the lifetime of free adducts12, 19, 21. On the other hand, systems with deeply-included nitroxide cannot be applied when the formation of complex with the other


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molecule is required. Therefore, understanding of the equilibrium type in the complex and determination of key parameters influencing this equilibrium are interesting and topical tasks. The major factor determining the equilibrium type in aqueous solution is the structure of nitroxide and covalent linker between the nitroxide and CD, namely its rigidity/mobility, length and composition. Scheme 1. Structural equilibrium in NCDs

The nitroxide moiety is shown by yellow color, TRIMEB – red color

Magnetic resonance techniques (NMR, EPR) are often used to investigate structure and properties of supramolecular complexes of CDs with nitroxides10, 13-15, 24-25. Recently Chechik et.al. proposed to use Electron Spin Echo Envelope Modulation (ESEEM) for investigation of the equilibrium type in spin-labeled CDs.26 ESEEM, one of the pulse EPR techniques, allows one to study weak hyperfine interactions (HFI) between unpaired electron and surrounding nuclear spins. In this approach the experiments are carried in the deuterated water, and interaction of nitroxide with surrounding 2H nuclei is monitored by ESEEM.27-29 It was shown26, that the formation of complex with deeply-immersed radical is allowed only if the covalent linker between CD and radical is long and flexible enough. Using Double Electron-Electron


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Resonance (DEER) it was demonstrated, that the formation of intramolecular complexes is preferred compared to the formation of intermolecular complexes between spin-labeled CDs. Often the additional experiments with competitors (compounds with high binding constant with CDs) are also used to verify the structural forms of NCDs13, 26. Sometimes the methylated CDs (where OH groups are substituted by CH3 groups) are used to increase the complex solubility in water30-31; in such cases the variation of CD structure may also influence the equilibrium type observed, due to the alteration of macrocycle conformation. Generally glucose residues of CDs are considered as fairly “rigid’’ building blocks: shape and the 4С1 chair conformation (Scheme 2) of individual glucose units in β-CD are close, no matter what guest is included within the cavity32. However, this is not always true for fully methylated β-CDs (TRIMEB), which can adopt unusual macrocyclic conformation due to the absence of geometric strains imposed by intramolecular hydrogen bonds.33 For instance, the conformational change of TRIMEB macrocycle to that with the one 0S2 – twist boat glucose unit (Scheme 2) was observed in the crystal structure of TRIMEB/m-Iodophenol complex.34 This guest-induced conformational change of a pyranose ring of TRIMEB affects the cavity shape and rim size,33-34 and this should be taken into account in the experiments with competitors. Scheme 2. Conformations of TRIMEB glucose unit

In this work we have synthesized and investigated new complexes of TRIMEB covalentlybound to the piperidine, pyrroline, pyrrolidine, and imidazoline type nitroxides using linkers of


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different length and rigidity (Scheme 3). The work was aimed at the investigation of influences of the radicals and linkers properties on the structure of complexes formed. We used ESEEM and CW EPR techniques and studied the complexation in the presence/absence of competitor 1Adamantanemethanol (AM), which exhibits high equilibrium constants with CDs (k ≈ 5×1025×104 M-1).31, 35 Moreover, we have studied the CD influence on the functional properties of pHsensitive imidazoline nitroxide and its stability against the reduction by ascorbic acid. Scheme 3. Chemical structure of studied compounds.

EXPERIMENTAL SECTION Materials. Synthesis.

Solvents and reactants, model nitroxides PCA1 and PCA2, 1-

adamantanemethanol (AM) and 2,2,5,5-Tetramethyl-3-pyrrolin-1-oxyl-3-carboxylic acid Nhydroxysuccinimide ester were purchased from Aldrich and Acros and used as received.


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Cyclodextrins were provided by Cyclolab. Intermediates (TEMPO1 – TEMPO4) and the subsequent CD compounds (NCD1 – NCD5) as well as model compounds AMP1- AMP2 were prepared using the procedures described previously10 and identified using EPR and High Resolution Mass spectrometry (HRMS-ESI, 400 µL of dichloromethane, and then, diluted to 1/105 or 1/106 3 mM ammonium acetate in MeOH, ESI+). pH-sensitive nitroxide ATI36-37 was kindly provided by Dr. Igor A. Kirilyuk (NIOCH SB RAS). Deuterated water D2O (98 %) and deuterated dimethylsulphoxide-d6 (DMSO, 98 %) were produced by Isotope. EPR analyses were performed on Bruker ELEXSYS E580 machine and mass analysis on QStar Elite (Applied Biosystem SCIEX) equipped with atmospheric pressure ionization source (API). Sample was ionized in positive electrospray mode (electrospray tension (ISV): 5500V, exit tension: 10V, nebulizating gas pressure (air): 10 psi. Experiments were performed as triplicates with double internal standard. General procedure for the preparation of succinimide derivatives. Triethylamine (1.2 equiv) was added dropwise to a solution of nitroxide (200 mg, 1 equiv) and disuccinimidyl carbonate (304 mg, 1.4 equiv) in anhydrous acetonitrile (6 mL) under a nitrogen atmosphere. After stirring the mixture for overnight at room temperature, the solvent was removed. The residue was dissolved in methylene chloride (20 mL) and washed with distilled water (8 mL) and with a saturated NaHCO3 solution (8 mL). The organic phase was then washed with brine (8 mL) and dried over Na2SO4. Solvent was removed to afford the crude product which was used directly in the next step without further purification. The reaction was monitored by TLC (CH2Cl2/Et2O 2:3). For TEMPO2: C14H22N3O5 M = 312.1529, m/z 300.2016 [M+NH4]+. For TEMPO4: C14H21N2O6 M = 313.1400, m/z 314.1472 [M+H]+. For AMP2: C13H20N3O5 M = 298.1403, m/z 299.14.76 [M+H]+.


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General procedure for the preparation of TEMPO1,3, and AMP1. A solution of crude nitroxide succinimide carbonate or carbamate (100 mg, 0.32 mM, 1 eq.) and triethylamine (32 mg, 0.32 mM, 2 eq.) in dichloromethane (2 mL) was added dropwise to a solution of isopropylamine (38 mg, 0.64 mM, 2 eq.) and triethylamine (32 mg, 0.32 mM, 1 eq.) in anhydrous dichloromethane (3 mL) under nitrogen. The solution was then stirred for 26 hours and washed with water (10 mL). The organic phase was washed with a saturated aqueous NaHCO3 solution (2 × 10 mL) followed by brine (10 mL) and dried with anhydrous MgSO4. Distillation under reduced pressure yielded carbonate or carbamate nitroxides in more than 90% and needed no more purification. For TEMPO1: C13H26N3O2 M = 256.2025, m/z 257.2098 [M+H]+. For TEMPO3: C13H25N2O3 M = 257.1865, m/z 258.1938 [M+H]+. For AMP1: C12H24N3O2 M = 242.1869, m/z 243.1934 [M+H]+. General procedure for the preparation of NCD. Under nitrogen, triethylamine (110 µL, 1.2 equiv) was added to a vigorously stirred solution of monoamino-6-monodeoxy-permethyl-β-CD hydrochloride (1 g, 1.1 equiv) in dichloromethane (14 mL). A solution of the above-described crude nitroxide succinimide carbonate or carbamate (200 mg, 1 eq.) in dichloromethane (8 mL) was then added, followed by a second equivalent of triethylamine. After 2 h, the organic phase was washed with water (10 mL) and with a saturated NaHCO3 solution (10 mL). The organic phase was then washed with brine (10 mL) and dried over Na2SO4. After filtration, the solvent was distilled under reduced pressure. Then the crude was purified by chromatography by plate, and extracted with CH2Cl2. The solvent was then removed under vacuum to afford a solid in 3035% yields. The reaction was monitored by TLC (Rf = 0.5) and compounds were purified using the mixture CH2Cl2/Et2O 2:3. For NCD1: C721H128N3O36 M = 1610.8278, m/z 814.9344 [M+H+NH4]+. For NCD2: C72H127N2O37 M = 1611.8118, m/z 823.9397 [M+2NH4]2+. For


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NCD3: C71H126N3O36 M = 1596.8121, m/z 807.9299 [M+2NH4]2+. For NCD4: C71H123N2O36 M = 1579.7856, m/z 799.4133 [M+H+NH4]+. For NCD5: C70H123N4O36 M = 1595.7917, m/z 807.4164 [M+H+NH4]2+. ESEEM Measurements. To obtain glassy solutions at low temperatures all samples were prepared using a mixture of solvents D2O/DMSO-d6 in volume proportion 8:2. Nitroxide concentration in all ESEEM experiments was equal to 5×10-4 M. Concentration of the competitor molecules 1-adamantanemethanol (AM) was 5×10-3 М. TRIMEB has a weaker binding constant with adamantane derivatives (0.6×103 M-1) compared to unmodified β-CD (3.8×104 M-1).35 Nevertheless, this binding constant is high enough to use AM as a competitor in our experiment. ESEEM experiments were carried out at 50 K at X-band (9 GHz) using commercial Bruker ELEXSYS E580 EPR spectrometer. All measurements were done at the magnetic field corresponding to the electron spin echo (ESE) maximum. Three-pulse ESE sequence π/2-τ-π/2T-π/2-τ-echo with the four-step phase cycling was used; π/2-pulse duration was 16 ns. The τdelay of 210 ns was optimized to achieve the maximum modulation depth due to the 2Н nuclei. Typical time-domain dependence of the ESE intensity is shown in Figure 1. The background was removed by division followed by renormalization. The amplitude of modulation K(2H) was defined as the ratio between the extrapolated echo intensity between the first two maxima and at the first minimum (K(2H)=a/(a+b) , Figure1e).

This treatment is more sensitive to the

contribution of residual protons in comparison to the Fourier transform of the ESEEM traces.26 CW EPR and Kinetics Measurements. All CW EPR experiments were performed at room temperature at X-band (9 GHz) using commercial Bruker EMX EPR spectrometer using 50 µl quartz capillaries with an inner diameter ID = 0.8 mm. Typical experimental EPR settings were: sweep width - 100 G; microwave power - 6.377; modulation frequency - 100 kHz; modulation


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amplitude - 0.5 G; time constant - 40.96 ms; sweep time - 41.94 s; number of points - 1024; harmonic – first; number of scans – 4. EPR characterization of NCDs was performed in water/DMSO (8:2) mixture in order to compare EPR and ESEEM results. Nitroxide concentration was equal to 5×10-4 M. Concentration of the competitor molecules 1-adamantanemethanol (AM) was 5×10-3 М. The solution of NCD5 (0.1 мМ) in water was used to study the dependence of CW EPR on pH of the solutions. The pH of the solutions was monitored using a pH-meter equipped with a Sigma-Aldrich micro pH combination electrode. NaOH and HCl (final concentration N-O•, leading to a change in the g-factor and decrease of the HFI constant.42 Figure 3 shows the dependence of aN value for NCD5 on pH of aqueous solution. HFI constant was measured using central and lowfield components of the nitroxide EPR spectrum. It was found that the NCD5 complex shows high pH-sensitivity of the 14N HFI constant with ∆aN = 0.078 mT. The obtained titration curve is described by the Henderson-Hasselbalch equation38-39:

a N ( pH ) = a RH + +

a R − a RH + 1 + 10 pK a − pH

,

where a RH + = 1.487 mT is the splitting in the EPR spectrum for purely protonated radical form (i.e. when the further decrease of pH value has no longer any effect on it); aR =1.565 mT is the splitting in the EPR spectrum for purely deprotonated radical. This allowed us to obtain the value pKa = 2.89 for the NCD5.


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Figure 3. (a) X-band EPR spectra of the nitroxide NCD5 at pH = 5.3 (black line) and pH = 1.2 (blue line). (b) pH induced changes of HFI constant aN for NCD5 in aqueous solution. aN was measured as the distance between the zero-crossing points of the low-field and central lines of EPR spectrum of radical. (c) decay of EPR signal of 0.1 mM NCD5 in the presence of 1 mM ascorbate at pH = 6.8. We investigated the persistence of the radical fragment in NCD5 complex against the reduction by ascorbic acid. Time dependence of the CW EPR signal of 0.1 mM NCD5 was


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measured after addition of 1 mM of ascorbic acid at physiological pH = 6.8 (Figure 3). The nitroxide decay kinetics in the presence of ascorbate is shown in Figure 3; the measured rate constant equals to 26 s-1M-1.

DISCUSSION Based on the analysis of experimental data for investigated NCDs, we obtained several parameters (tcorr, aN and K(2H)) affording conclusions on the equilibrium types for each case. The rotational correlation time tcorr describes the dynamics of radicals, and we associated its change with the shift to stronger/weaker bound complex of nitroxide with TRIMEB (increase/decrease of tcorr, respectively).43-44 The change of

14

N HFI constant aN also indirectly indicates the

modification of nitroxide location relative to TRIMEB cavity. The formation of noncovalent complex between nitroxide and TRIMEB, as we expected, leads to slight decrease of aN value due to decrease of local polarity.24,

44-46

The amplitude of modulation K(2H) obtained from

ESEEM data is highly sensitive to the location of the nitroxide with respect to TRIMEB, and its decrease indicates the immersion of radical into a TRIMEB cavity. We verified that K(2H) value is sensitive to the local (5 Å) have no influence on the modulation amplitude. It means that nitroxide in NCD experiences different solvent effect compared to free nitroxide depending on its position (outside/close/inside) with respect to the cavity. In order to determine the equilibrium type for NCDs we simultaneously analyzed parameters tcorr, aN and K(2H) and compared them with those obtained for free nitroxide analogues and for NCDs in the presence of competitor AM. Based on these results, studied NCDs can be assigned to 4 types of complexation which is shown qualitatively in Scheme 4:


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Type I (NCD2). For this NCD the K(2H), Rk and aN values are smaller than those for free nitroxides; the addition of AM increases the K(2H) and aN values, and at the same time reduces slightly tcorr, implying that the radical has been partially included in the cavity of TRIMEB before its displacement by AM (Scheme 3). Type II (NCD5 at pH = 7.7 and pH = 1.3. In this case the addition of AM does not influence noticeably the ESEEM and CW parameters, and K(2H) values are very close to those reported for free nitroxides. It means that the radical fragment is mainly located outside the TRIMEB cavity (Scheme 3), what is indirectly confirmed by high value of the tumbling rate for NCD5. Type III (NCD4). For this NCD we observed significant decrease of the accessibility of radical to deuterons compared to that for free radical in D2O RK = 81% (e.g. for TEMPO completely complexated with β-CD RK=75%26), however, upon addition of AM the K(2H) value did not change noticeably. The low value of K(2H) implies that the nitroxide moiety is shielded from solvent, and the absence of effects of the competitor points out that the nitroxide moiety is not included into the cavity. Thus, for the shorter linker (NCD4, one amido function bound nitroxide and CD) the nitroxide is maintained in the methoxy crown of TRIMEB cavity, although it is not hosted by the cavity (Scheme 3). The decrease of the tumbling rate for NCD4 upon complex formation with AM will be discussed below. Type IV (NCD1, NCD3). For these NCDs the K(2H) values are very close to those reported for the free nitroxides, but at the same time covalent attachment of nitroxide to TRIMEB significantly affects dynamics of radicals and decreases HFI constant (for NCD3). This indicates the equilibrium between the complex with radical fragment outside the cavity and radical fragment capping TRIMEB cavity (Scheme 3), however, the location of nitroxide with respect to TRIMEB cavity cannot be determined more precisely using this approach.


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NCD1 and NCD3 include a urea-like linker RNHC(=O)NHR with different types of nitroxides (5- and 6-membered rings of different rigidity), whereas NCD2 able to be partly included into the TRIMEB cavity has a carbamate-like linker RNHC(=O)OR. In case of amide function, the nitrogen lone pair is conjugated with the carbonyl function leading to a drastically restricted rotation around the C(=O)—N bond, whereas for the C(=O)—O bond such restriction is lower. Since the nitroxide moiety should pass through the methoxy crown of the CD rim to be hosted by the cavity, the linker should be flexible enough to take the most suitable conformation for inclusion. Therefore one reasonably assumes that the NCD2 forms stronger bound complex compared to NCD1 and NCD3. NCD5 has the same urea-like linker as NCD1 and NCD3, but in this case the conjugation includes also imine nitrogen of the nitroxide moiety increasing the rigidity of the linker and hindering the complex formation between nitroxide and TRIMEB cavity.

Scheme 4. Structural equilibrium in studied NCDs before (left) and after (right) complex formation with AM. Note, that the lengths of arrows are not proportional to the corresponding rate constants and reflect the structural equilibria only qualitatively. The nitroxide moiety is shown by yellow, TRIMEB – red, AM –blue color


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Taking into account the high RK values for NCD1 and NCD3 (98% and 97%, correspondingly) one would expect the ESEEM and CW EPR parameters to be unaffected by complex formation with AM. Interestingly, the addition of AM significantly reduces the tumbling rate of nitroxide and accessibility of radicals to deuterons (to 93% and 82% for NCD1 and NCD3, respectively), implying an equilibrium shift toward complex with radical capping TRIMEB cavity (Scheme 3). This is a rather unusual phenomenon, which has never been observed in the study of spinlabelled β-CDs.14-15, 26 As was mention above, TRIMEB, in contrast to β-CD, has a quite flexible macrocycle and exhibits the conformational variations: one glucose unit changes between 4C1 and 0S2-twist boat form.33-34 This conversion results in more elliptical cavity and nonequivalent tilting of glucose unit disordering thereby methoxy crown of the primary (narrow) ring of TRIMEB and increasing the accessibility to TRIMEB cavity.33 We assume that most probably this modification of TRIMEB macrocyclic configuration affects the equilibrium for NCDs and changes the nitroxide location with respect to TRIMEB cavity, as was observed for NCD1 and NCD3. The decomposition of CW EPR spectra of NCD3 into two components having different correlation times seems to support this assumption: tcorr= 0.54 ns corresponds to the NCD3/AM complex containing TRIMEB with all glucose units in 4C1 form and is the same as for free NCD3; tcorr = 1.6 ns corresponds to the dominant (80%) NCD3/AM complex, with one glucose unit of TRIMEB in 0S2 form. These guest induced distribution of macrocyclic configurations is very close to those reported previously for the mono-altro-β-cyclodextrin upon inclusion of adamantanecarboxylate.47-48 Note, that in principle it is also possible that AM creates a microhydrophobic environment suitable for nitroxide accommodation by a host-induced guestguest hydrophobic effect, which would suggest an alternative explanation of the above


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observations. However, this effect has not been previously observed for nitroxides bound to unmodified (not methylated) β-CDs26, and therefore is unlikely to play an important role in TRIMEB studied in this work. Under this assumption, it is necessary to assume modification of TRIMEB macrocyclic configuration upon complex formation with AM for all studied NCDs. For NCD2, the increase of the K(2H) and slight reduction of tcorr observed in the presence of AM may result from two processes: displacement of partly included nitroxide moiety from the TRIMEB cavity and modification of TRIMEB macrocyclic configuration. In case of NCD4, owing to the short linker the nitroxide is located very close to TRIMEB cavity, therefore the change of TRIMEB macrocyclic configuration upon complex formation with AM practically does not affect the Rk value, although a significant increase of tcorr value is observed. In contrast, radical fragment in NCD5 is mainly located outside the TRIMEB cavity due to rather rigid linker, and thus its EPR and ESEEM parameters are absolutely insensitive to conformational changes of TRIMEB. Note, that obtained value pKa=2.89 for NCD5 is lower than pKa=6.1 found for ATI36. (The comparison of NCD5 with similar N-acyl equivalent was not possible, since the latter compound is unstable; therefore we compare pKa values of NCD5 and ATI). The pKa for NCD5 and ATI are associated with reversible protonation of amidine group (Scheme 3) and depend on the electronic nature of the substituent. Replacement of the one hydrogen atom at the amino nitrogen position by electronegative carbonyl group decreases the basicity of amidine and, as a result, the pKa value for NCD5 shifts to more acidic region compared to ATI. The measured reduction rate constant for NCD5 equals to 26 s-1M-1 and does not differ noticeably from reduction constant for ATI36 22.5 s-1M-1. This result agrees well with the data obtained from the ESEEM analysis. As was mentioned above, nitroxide in NCD5 does not


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interact with TRIMEB and is located outside the TRIMEB cavity. For this structure of the complex, one would expect TRIMEB to have no influence on the reduction rate constant, as is observed in experiment. It is probably the structure of linker that prohibits the TRIMEB influence on the reduction constant of pH-sensitive NCD5. To optimize the stability of pHsensitive radicals attached to TRIMEB one should use another structure of linker allowing for the deep immersion of radical into the cavity of cyclodextrin.

CONCLUSIONS We have synthesized and characterized using EPR the series of piperidine (NCD1, NCD2), pyrroline (NCD4), pyrrolidine (NCD3), and pH-sensitive imidazoline (NCD5) type nitroxides attached to TRIMEB. Using ESEEM and continuous wave EPR we determined the equilibrium type for each complex and performed quantitative estimations of the accessibility of nitroxide to the solvent molecules. In all studied systems we observed the equilibria between conformations with radical fragment being outside the CD cavity and radical fragment capping the cavity of CD, in some cases with a predominance of one of the forms. Although the linker between radical fragment and TRIMEB exhibits various rigidities for the studied complexes, it is not long enough for the formation of complex with nitroxide deeply included into the TRIMEB cavity. The accessibility of nitroxide to the solvent molecules is determined by the structure and length of the linker between radical and TRIMEB. In complexes NCD1 and NCD3 the nitroxide and TRIMEB are connected via urea-like rigid –NH-CO-NH- bridge, therefore these complexes exhibit high accessibility of radical fragment to the solvent molecules. The structure of nitroxide in NCD2 is the same as in NCD1, but the linkers with TRIMEB are different: bridging by more flexible –NH-CO-O- fragment in NCD2 leads to the partial inclusion of nitroxide moiety into


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TRIMEB cavity and decrease of accessibility to solvent molecules. In the case of NCD4, the nitroxide is located close to TRIMEB due to the short and rigid linker, resulting in the lowest accessibility to solvent molecules. Upon complex formation of NCD1 and NCD3 with AM we observed the shift of equilibrium toward complex with radical predominantly capping TRIMEB cavity. We believe that this stems from flexible TRIMEB macrocycle which exhibits conformational variations and shape modifications. Our observation suggests that the nitroxides covalently bound to TRIMEB are good potential probes for studying guest-induced changes of macrocyclic configuration using EPR, and thus may complement the other approaches in this field. Using continuous wave EPR we have shown that covalent bonding of CD with pH-sensitive radical in NCD5 does not interfere with the pH-sensitivity at pKa=2.89. On the other hand, unfortunately, it does not decrease the reduction rate of the radical by ascorbic acid. This correlates with the ESEEM data showing high accessibility values to solvent (and hence to ascorbic acid) in both protonated and deprotonated forms. Finally, perhaps the most useful enhancement of functional properties was observed in complex NCD4. Among the five studied systems, NCD4 is most perspective because (1) it efficiently forms complexes with other molecules in the bulk, and (2) it has nitroxide with the proper linker located closely to the inclusion complex.

AUTHOR INFORMATION Corresponding Author *e-mail: [email protected]

ACKNOWLEDGEMENTS


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The authors are thankful to Dr. Vitaly G. Kiselev (ICKC SB RAS) for DFT geometry optimizations of free nitroxide radicals and Dr. Igor A. Kirilyuk (NIOCH SB RAS) for providing pH-sensitive nitroxide ATI and for the fruitful discussions. This work was supported by the Russian Foundation for Basic Research (12-03-33010, 12-04-01435, 12-03-01023), and The Ministry of Education and Science of Russian Federation (projects 8436 and 8456).

ASSOCIATED CONTENT Supporting Information Supporting information contains detailed description of CW EPR spectra simulation for NCDs and free nitroxide analogues. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES 1. Lehn, J. M. Perspectives in Supramolecular Chemistry - from Molecular Recognition Towards Molecular Information-Processing and Self-Organization. Angew Chem Int Edit 1990, 29 (11), 1304-1319. 2. Aida, T.; Meijer, E. W.; Stupp, S. I. Functional Supramolecular Polymers. Science 2012, 335 (6070), 813-817. 3. Moulin, E.; Cormos, G.; Giuseppone, N. Dynamic combinatorial chemistry as a tool for the design of functional materials and devices. Chem Soc Rev 2012, 41 (3), 1031-1049. 4. Nguyen, T. D.; Liu, Y.; Saha, S.; Leung, K. C. F.; Stoddart, J. F.; Zink, J. I. Design and optimization of molecular nanovalves based on redox-switchable bistable rotaxanes. J Am Chem Soc 2007, 129 (3), 626-634. 5. de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Signaling recognition events with fluorescent sensors and switches. Chem Rev 1997, 97 (5), 1515-1566. 6. Engeldinger, E.; Armspach, D.; Matt, D. Capped cyclodextrins. Chem Rev 2003, 103 (11), 4147-4173. 7. Bohm, I.; Kreth, S. K.; Ritter, H.; Branscheid, R.; Kolb, U. Switchable Supramolecular Crosslinking of Cyclodextrin-Modified Hyperbranched Polyethylenimine via Anthraquinone Dyes. Macromol Chem Physic 2012, 213 (2), 243-248. 8. Harada, A. Supramolecular polymers based on cyclodextrins. J Polym Sci Pol Chem 2006, 44 (17), 5113-5119. 9. Aguilera-Sigalat, J.; Casas-Solvas, J. M.; Morant-Minana, M. C.; Vargas-Berenguel, A.; Galian, R. E.; Perez-Prieto, J. Quantum dot/cyclodextrin supramolecular systems based on efficient molecular recognition and their use for sensing. Chem Commun 2012, 48 (20), 25732575.


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 38

10. Bardelang, D.; Rockenbauer, A.; Jicsinszky, L.; Finet, J.-P.; Karoui, H.; Lambert, S.; Marque, S. R. A.; Tordo, P. Nitroxide Bound β-Cyclodextrin: Is There an Inclusion Complex? J. Org. Chem. 2006, 71 (20), 7657-7667. 11. Chechik, V.; Ionita, G. Bis spin-labelled cyclodextrins. New J Chem 2007, 31 (10), 17261729. 12. Polovyanenko, D. N.; Marque, S. R. A.; Lambert, S.; Jicsinszky, L.; Plyusnin, V. F.; Bagryanskaya, E. G. Electron Paramagnetic Resonance Spin Trapping of Glutathiyl Radicals by PBN in the Presence of Cyclodextrins and by PBN Attached to beta-Cyclodextrin. J Phys Chem B 2008, 112 (41), 13157-13162. 13. Bagryanskaya, E. G.; Bardelang, D.; Chenesseau, S.; Finet, J. P.; Jicsinszky, L.; Karoui, H.; Marque, S. R. A.; Mobius, K.; Polovyanenko, D.; Savitsky, A.; Tordo, P. EPR, NMR, and Thermodynamic Evidences for Forced Nuclear Spin-Electron Spin Interactions in the Case of 1Phenyl-2-Methylpropyl-1,1-Dimethyl-2-Nitroxide (TIPNO) Attached to Permethylated betaCyclodextrin. Appl Magn Reson 2009, 36 (2-4), 181-194. 14. Ionita, G.; Chechik, V. Spin-labelled cyclodextrins as hosts for large supramolecular assemblies. Org Biomol Chem 2005, 3 (17), 3096-3098. 15. Chechik, V.; Ionita, G. Supramolecular complexes of spin-labelled cyclodextrins. Org Biomol Chem 2006, 4 (18), 3505-3510. 16. Grucker, D.; Guiberteau, T.; Eclancher, B.; Chambron, J.; Chiarelli, R.; Rassat, A.; Subra, G.; Gallez, B. Dynamic Nuclear-Polarization with Nitroxides Dissolved in BiologicalFluids. J Magn Reson Ser B 1995, 106 (2), 101-109. 17. Krumkacheva, O.; Tanabe, M.; Yamauchi, S.; Fedin, M.; Marque, S. R. A.; Bagryanskaya, E. Time-Resolved and Pulse EPR Study of Triplet States of Alkylketones in betaCyclodextrin. Appl Magn Reson 2012, 42 (1), 29-40. 18. Bardelang, D.; Charles, L.; Finet, J. P.; Jicsinszky, L.; Karoui, H.; Marque, S. R. A.; Monnier, V.; Rockenbauer, A.; Rosas, R.; Tordo, P. alpha-phenyl-N-tert-butylnitrone-type derivatives bound to beta-cyclodextrins: Syntheses, thermokinetics of self-inclusion and application to superoxide spin-trapping. Chem-Eur J 2007, 13 (33), 9344-9354. 19. Karoui, H.; Tordo, P. ESR-spin trapping in the presence of cyclodextrins. Detection of PBN-superoxide spin adduct. Tetrahedron Lett 2004, 45 (5), 1043-1045. 20. Hardy, M.; Bardelang, D.; Karoui, H.; Rockenbauer, A.; Finet, J. P.; Jicsinszky, L.; Rosas, R.; Ouari, O.; Tordo, P. Improving the Trapping of Superoxide Radical with a betaCyclodextrin-5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) Conjugate. Chem-Eur J 2009, 15 (42), 11114-11118. 21. Karoui, H.; Rockenbauer, A.; Pietri, S.; Tordo, P. Spin trapping of superoxide in the presence of beta-cyclodextrins. Chem Commun 2002, (24), 3030-3031. 22. Han, Y. B.; Liu, Y. P.; Rockenbauer, A.; Zweier, J. L.; Durand, G.; Villamena, F. A. Lipophilic beta-Cyclodextrin Cyclic-Nitrone Conjugate: Synthesis and Spin Trapping Studies. J Org Chem 2009, 74 (15), 5369-5380. 23. Han, Y. B.; Tuccio, B.; Lauricella, R.; Villamena, F. A. Improved spin trapping properties by beta-cyclodextrin-cyclic nitrone conjugate. J Org Chem 2008, 73 (18), 7108-7117. 24. Franchi, P.; Lucarini, M.; Pedulli, G. F. Use of nitroxide radicals to investigate supramolecular entities. Curr Org Chem 2004, 8 (18), 1831-1849. 25. Franchi, P.; Fani, M.; Mezzina, E.; Lucarini, M. Increasing the persistency of stable freeradicals: Synthesis and characterization of a nitroxide based [1]rotaxane. Org Lett 2008, 10 (10), 1901-1904.


28

Page 29 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


26. Ionita, G.; Florent, M.; Goldfarb, D.; Chechik, V. Studying Supramolecular Assemblies by ESEEM Spectroscopy: Inclusion Complexes of Cyclodextrins. J Phys Chem B 2009, 113 (17), 5781-5787. 27. Schweiger, A.; Jeschke, G. Principles of Pulse Electron Paramagnetic Resonance Spectroscopy. Oxford University Press: 2001; p 572 pp. 28. Erilov, D. A.; Bartucci, R.; Guzzi, R.; Shubin, A. A.; Maryasov, A. G.; Marsh, D.; Dzuba, S. A.; Sportelli, L. Water concentration profiles in membranes measured by ESEEM of spin-labeled lipids. J Phys Chem B 2005, 109 (24), 12003-12013. 29. Milov, A. D.; Samoilova, R. I.; Shubin, A. A.; Grishin, Y. A.; Dzuba, S. A. ESEEM Measurements of Local Water Concentration in D(2)O-Containing Spin-Labeled Systems. Appl Magn Reson 2008, 35 (1), 73-94. 30. Uekama, K.; Hirayama, F.; Irie, T. Cyclodextrin drug carrier systems. Chem Rev 1998, 98 (5), 2045-2076. 31. Rekharsky, M. V.; Inoue, Y. Complexation thermodynamics of cyclodextrins. Chem Rev 1998, 98 (5), 1875-1917. 32. Saenger, W. R.; Jacob, J.; Gessler, K.; Steiner, T.; Hoffmann, D.; Sanbe, H.; Koizumi, K.; Smith, S. M.; Takaha, T. Structures of the common cyclodextrins and their larger analogues Beyond the doughnut. Chem Rev 1998, 98 (5), 1787-1802. 33. Li, W. S.; Wang, S. C.; Hwang, T. S.; Chao, I. Substituent Effect on the Structural Behavior of Modified Cyclodextrin: A Molecular Dynamics Study on Methylated beta-CDs. J Phys Chem B 2012, 116 (11), 3477-3489. 34. Harata, K.; Hirayama, F.; Arima, H.; Uekama, K.; Miyaji, T. Crystal structure of heptakis(2,3,6-tri-O-methyl)-[small beta]-cyclodextrin complexes with m-iodophenol and 4biphenylacetic acid. Guest-induced conformational change of a pyranose ring. Journal of the Chemical Society, Perkin Transactions 2 1992, 0 (7), 1159-1166. 35. Wenz, G. Influence of intramolecular hydrogen bonds on the binding potential of methylated β-cyclodextrin derivatives. Beilstein Journal of Organic Chemistry 2012, 8, 18901895. 36. Kirilyuk, I. A.; Bobko, A. A.; Grigorev, I. A.; Khramtsov, V. V. Synthesis of the tetraethyl substituted pH-sensitive nitroxides of imidazole series with enhanced stability towards reduction. Org Biomol Chem 2004, 2 (7), 1025-1030. 37. Kirilyuk, I. A.; Shevelev, T. G.; Morozov, D. A.; Khromovskih, E. L.; Skuridin, N. G.; Khramtsov, V. V.; Grigor'ev, I. A. Grignard reagent addition to 5-alkylamino-4H-imidazole 3oxides: Synthesis of new pH-sensitive spin probes. Synthesis-Stuttgart 2003, (6), 871-878. 38. Henderson, L. J. Concerning the Relationship between the Strength of Acids and their Capacity to Preserve Neutrality. Am. J. Physiol. 1908, 21, 173-9. 39. Khramtsov, V. V. Biological imaging and spectroscopy of pH. Curr Org Chem 2005, 9 (9), 909-923. 40. Stoll, S.; Schweiger, A. Easyspin: Simulating CW ESR spectra. Bio Magn Re 2007, 27, 299-321. 41. Stoll, S.; Schweiger, A. EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 2006, 178 (1), 42-55. 42. Khramtsov, V. V.; Grigor'ev, I. A.; Foster, M. A.; Lurie, D. J.; Nicholson, I. Biological applications of spin pH probes. Cell Mol Biol 2000, 46 (8), 1361-1374. 43. Martinie, J.; Michon, J.; Rassat, A. Nitroxides. LXX. Electron spin resonance study of cyclodextrin inclusion compounds. J. Am. Chem. Soc. 1975, 97 (7), 1818-23.


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44. Ionita, G.; Caragheorgheopol, A.; Caldararu, H.; Jones, L.; Chechik, V. Inclusion complexes of cyclodextrins with nitroxide-based spin probes in aqueous solutions. Org Biomol Chem 2009, 7 (3), 598-602. 45. Lucarini, M.; Luppi, B.; Pedulli, G. F.; Roberts, B. P. Dynamic aspects of cyclodextrin host - Guest inclusion as studied by an EPR spin-probe technique. Chem-Eur J 1999, 5 (7), 2048-2054. 46. Owenius, R.; Engstrom, M.; Lindgren, M.; Huber, M. Influence of solvent polarity and hydrogen bonding on the EPR parameters of a nitroxide spin label studied by 9-GHz and 95GHz EPR spectroscopy and DFT calculations. J Phys Chem A 2001, 105 (49), 10967-10977. 47. Fujita, K.; Chen, W. H.; Yuan, D. Q.; Nogami, Y.; Koga, T.; Fujioka, T.; Mihashi, K.; Immel, S.; Lichtenthaler, F. W. Molecular modeling of saccharides, part 20 - Guest-induced conformational change in a flexible host: mono-altro-beta-cyclodextrin. Tetrahedron-Asymmetr 1999, 10 (9), 1689-1696. 48. Fujita, K.; Fukudome, M.; Yuan, D. Q. Flexible cyclooligosaccharides: Guest-binding and regio-selective modification. J Incl Phenom Macro 2002, 44 (1-4), 323-328.

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Structural Equilibrium in New Nitroxide-Capped Cyclodextrins: CW

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