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Polyradical PROXYL/TEMPO-Derived Amides: Synthesis,Physicochemical Studies, DFT Calculations, andAntimicrobial Activity**Patrik Poprac, Peter Poliak, Miroslav Kavala, Zuzana Barbierikov#, Michal Zalibera,Marek Fronc, Ľubom&r Švorc, Zuzana Vihonsk#, Petra Olejn&kov#, Karol Lušpai,Vladim&r Lukeš, Vlasta Brezov#, and Peter Szolcs#nyi*[a]

Dedicated to the memories of Professors Stanislav Biskupič and František Považanec

Introduction

Persistent nitroxide radicals, which possess an unpaired elec-

tron on a sterically hindered NO group, can be easily synthe-

sized, manipulated, and derivatized; thus intensive effort hasbeen focused on the preparation of new 2,2,6,6-tetramethyl-1-

piperidinyloxy (TEMPO) and/or 2,2,5,5-tetramethyl-1-pyrrolidi-nyloxy (PROXYL) derivatives with diverse potential applications

in chemistry, biology, and medicine.[1–6] Stable nitroxides areused as spin probes or spin labels in studies on membranes orcells,[7, 8] as contrast agents for magnetic resonance imaging,[4, 9]

as scavengers with distinct reactivity against reactive free radi-cals,[5, 10] as redox mediators in dye-sensitized solar cells,[11] and

as mediators in radical polymerizations.[12] In comparison withmononitroxides, di- and polynitroxide scaffolds reveal im-

proved potential as paramagnetic polarizing agents in dynamicnuclear polarization (DNP) to enhance the sensitivity of NMR

spectroscopy experiments.[13–16] Recent investigations into the

suitable design of dinitroxide biradicals applicable for DNP inbiological samples confirm the distinct impact of the mutual

orientation of nitroxide moieties on NMR signal enhance-

ment.[15]

We have systematically designed and synthesized a series ofdinitroxide amides possessing TEMPO or PROXYL units con-

nected by various bridges, along with TEMPO-containing tetra-and hexaradicals (Scheme 1), to explore the relationship be-

tween their structures and properties. A comprehensive ap-proach is essential to document and explain the properties

and behavior of polynitroxide molecules in diverse systems to

select compounds that meet the requirements of particular ap-plications. Therefore, the aim of this study is to provide a com-

plex assessment of amidic polynitroxide derivatives by employ-ing multiple techniques, including synthesis, X-ray characteriza-

tion, and a detailed study of the paramagnetic character byEPR spectroscopy complemented with theoretical calculations.

Additionally, cyclic voltammetry (CV) is used to describe theredox properties of the investigated polynitroxides, and theirinhibitory activities against selected bacteria yeasts and fila-

mentous fungi are assessed in biological assays.

Results and Discussion

Synthesis

The preparation of dinitroxides 1P–6P and 1T–6T relies on thestandard amidation reaction of commercially available dichlor-ides of aliphatic 14–17 and/or aromatic dicarboxylic acids 18or 19 with 4-amino-TEMPO 20 and/or 3-amino-PROXYL 21under basic conditions. All amidic biradicals were obtained as

A series of polynitroxide amides possessing 2,2,5,5-tetrameth-yl-1-pyrrolidinyloxy (PROXYL) and/or 2,2,6,6-tetramethyl-1-pi-

peridinyloxy (TEMPO) units connected through various bridges

were synthesized and their properties were analyzed. EPRspectroscopy provided detailed insight into their paramagnetic

character and related properties. A thorough examination ofthe EPR spectra of dinitroxides in organic solvents provided

valuable information on the intramolecular motions, thermody-namics, and spin-exchange mechanisms. Analysis of low-tem-

perature X- and Q-band EPR spectra of the dissolved dinitrox-ides provided spin–spin distances that were comparable with

the theoretical values obtained by DFT. Cyclic voltammetry in-

vestigations revealed (quasi)reversible electrochemical behaviorfor PROXYL-derived biradicals, whereas significant loss of the

reversibility was found for TEMPO-containing bi- and polyradi-cals. The inhibitory activities of the nitroxides against model

bacteria, yeasts, and filamentous fungi were assessed.

[a] P. Poprac, Dr. P. Poliak, Dr. M. Kavala, Dr. Z. Barbierikov#, Dr. M. Zalibera,Dr. M. Fronc, Assoc. Prof. Ľ. Švorc, Z. Vihonsk#, Assoc. Prof. P. Olejn&kov#,Dr. K. Lušpai, Prof. V. Lukeš, Prof. V. Brezov#, Assoc. Prof. P. Szolcs#nyiFaculty of Chemical and Food TechnologySlovak University of Technology in BratislavaRadlinsk8ho 9, SK-812 37 Bratislava (Slovakia)E-mail : [email protected]

[**] DFT=density functional theory, PROXYL=2,2,5,5-tetramethyl-1-pyrrolidiny-loxy, TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxy.

Supporting information for this article can be found under:https://doi.org/10.1002/cplu.201700343.

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high-purity solids (CHN analysis) after flash column liquid chro-

matography (Scheme 2 and Table 1).

The molecular structures of 1T and 10T are shown in Fig-ures 1 and 2, respectively. The piperidine rings in both com-pounds exhibit chair-like conformations, in correlation withprevious X-ray analysis of 1T, which provided evidence of thetrans conformation of its oxamide bridge, with hydrogenbonds between NH and CO groups.[17] In contrast to a previousstudy,[17] our crystal structure of 1T reveals that the moleculesare linked into a dimeric chain through a intermolecular hydro-

gen bond between O4 and H@N2 (interatomic distance of2.240(2) a), with distinct conformations of two molecules in a

dimer (Figure 1). The two conformations differ by 1808 rotationof both piperidine rings around the N@C bond (N4@C13, N4*@C13*, N2@C3, and N2*@C3* in Figure 1). The midpoint distanceof the N@O bond of the individual 1T conformers evaluated

from the X-ray structure is slightly different: 13.152 and

12.774 a, respectively. When likened to pristine TEMPO radical,all N@O distances are shortened: N1@O1 1.283(1) a, N3@O3

Scheme 1. Overview of di-, tri-, tetra-, and hexaradicals investigated herein(P=PROXYL, T=TEMPO).

Scheme 2. General reaction scheme for the preparation of dinitroxides 1P–6P and 1T–6T (for details, see Table 1).

Figure 1. Molecular structure of dinitroxide 1T and its intermolecular hydro-gen bond (distances in a). Ellipsoids are shown at the 50% probability level.

Table 1. Reaction conditions for the preparation of dinitroxides 1P–6Pand 1T–6T through the procedure outlined in Scheme 2.

Substrate Amine(equiv)

Reagents, conditions Product(yield [%])[a]

20 (2)Et3N (2.5 equiv), CH2Cl20 8C to RT, 16 h

1T (68)

21 (2) Et3N (2.5 equiv), CH2Cl20 8C to RT, 10 h

1P (72)

20 (2)pyridine (2.5 equiv), THF0 8C to RT, 6 h

2T (79)

21 (2) DIPEA (2 equiv), THF0 8C to RT, 6 h

2P (74)


3T (46)

21 (2) Et3N (2.2 equiv), CH2Cl20 8C to RT, 18 h

3P (25)


4T (63)

21 (2) DIPEA (2.05 equiv), CH2Cl20 8C to RT, 23 h

4P (57)

20 (2.2)Et3N (3 equiv), 1,4-dioxaneRT to 80 8C, 2 h

5T (74)

21 (2) Et3N (2.5 equiv), tolueneRT, 20 h

5P (89)

20 (2.2)

Et3N (3 equiv), 1,4-dioxaneRT to 80 8C, 2 h

6T (79)21 (2) 6P (77)

[a] Yield of isolated product. DIPEA=N,N-diisopropylethylamine.

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1.282(1) a for 1T and N2@O2 1.288(0) a, N3@O3 1.282(0) a for10T, in comparison to 1.294(4) a for TEMPO.[18] Crystallographicdata from single-crystal X-ray analysis of dinitroxide 1T aresummarized in Table S1 in the Supporting Information.

The amidic trinitroxide 9T was prepared from the corre-sponding triacyl chloride 22 with 4-amino-TEMPO 20 in thepresence of base. However, a similar preparation of tetranitrox-

ide 10T from bis-TEMPO-amine 23 and fumaryl chloride (17)proved troublesome and, after extensive experimentation,

product 10T was finally obtained in only 23% yield. Eventually,the reaction of bis-TEMPO-amine 23 with isophthaloyl dichlor-ide (18) yielded the corresponding tetranitroxide 11T in an un-optimized yield of 49% (Scheme 3).

The structure of tetranitroxide 10T was confirmed by X-rayanalysis of single crystals obtained by slow evaporation of asolution of 10T in a mixture of CHCl3/isooctane (Figure 2). The

crystallographic data are summarized in Table S2 in the Sup-porting Information.

Finally, tetraradical 12T was obtained as the major productduring the attempted preparation of hexaradical 13T throughthe reaction of a threefold excess of 23 with 22 (Scheme 4).

EPR spectroscopy

Owing to the presence of two unpaired electrons in the dinitr-oxide radicals, EPR spectroscopy is an outstanding tool for the

characterization of the intramolecular electron spin exchange

and conformational dynamics.[13, 19–26] Analysis of a dinitroxideEPR spectrum can provide valuable information on intramolec-

ular movements, thermodynamics, and spin exchange mecha-nism.[24–29]

The spin Hamiltonian for a nitroxide biradical dissolved in asolvent with low viscosity can be constructed as shown inEquation (1) because the anisotropic hyperfine and Zeeman in-

teractions are completely averaged to zero.[26,28]

Ĥ ¼gð1ÞmBBSð1Þz þ gð2ÞmBBSð2Þz þ Að1ÞIð1Þz Sð1ÞzþAð2ÞIð2Þz Sð2Þz @ J~Sð1Þ~Sð2Þ

ð1Þ

Superscripts 1 and 2 denote different nitroxide fragments;gð1ÞmBBS

ð1Þz þ gð2ÞmBBSð2Þz represents the Zeeman coupling be-

tween the unpaired electron spin and the magnetic field, B,

oriented along the z axis (mB is the Bohr magneton and g(1) and

g(2) are the isotropic g factors); and Að1ÞIð1Þz Sð1Þz þ Að2ÞIð2Þz Sð2Þz de-

scribe the hyperfine interactions of the electron spins withmagnetically active nuclei (one 14N nucleus characterized with

hyperfine coupling constant AN(1) or AN

(2) is considered in the vi-

cinity of one unpaired electron for a dinitroxide). The last com-ponent, J~Sð1Þ~Sð2Þ, represents the exchange coupling betweentwo electron spins, in which J is the exchange coupling con-stant.[26, 28] In the case of tri- and tetranitroxide radicals, three

(J12, J13, J23) or six (J12, J13, J14, J23, J24, J34) exchange couplings, re-spectively, define the spin exchange.[30,31] The structure of the

Scheme 3. Preparation of trinitroxide 9T and tetranitroxides 10T and 11T.FLC= flash column liquid chromatography, DCM=dichloromethane.

Scheme 4. Preparation of tetranitroxide 12T and hexanitroxide 13T.

Figure 2. Molecular structure of tetranitroxide 10T, with 25% probabilitythermal displacement ellipsoids.


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nitroxide fragments, along with the type of connecting bridge,has a substantial effect not only on the J value, but also on the

mechanism of the spin exchange.[22–26,32–34]

Furthermore, the EPR spectra of dinitroxides in solution sen-

sitively reflect the conformational dynamics of nitroxide frag-ments because the appearance of the exchange interaction de-

pends on the distance between the radical fragments andtheir mutual orientation.[28] For biradicals with flexible, long-chain bridges, the EPR spectra consist of alternately broadened

lines and exhibit a temperature dependence that can be inter-preted by using a three-conformation model.[20,21, 29,35] Thismodel includes spin exchange between three dinitroxide con-formations, namely, “elongated” conformation A, which is char-acterized by JA=0 and lifetime tA, and two other “loop” con-formations, B and C, with a combined lifetime of tB+C, inwhich the nitroxide centers are close to each other in a “cage”

of solvent molecules, with JB=0 and JC@AN.[22,28,29,35] Thus, rele-

vant experimental EPR spectra represent a superposition of

two signals: one corresponding to conformation A and theother to the mixed contribution from conformations B andC.[22,35]

The experimental EPR spectra of dinitroxides 1P–6P and 1T–6T measured in CH2Cl2 at 295 K are summarized in Figure 3. A

rather short and rigid bridge between two PROXYL units in di-nitroxide 1P limits spin exchange by through-space interac-tions. Under the given experimental conditions, the lines in theEPR spectrum match the state well with the exchange cou-

pling J&0.2AN,[32] and the spin-Hamiltonian parameters eluci-dated from the simulation (Table 2) are fully compatible with

this assignment.

For derivatives 2P and 3P, elongation of the connectingbridge results in enhanced flexibility, and the experimental

spectra in CH2Cl2 consist of alternately broadened lines, whichprovide evidence of conformational dynamics with a rapid spinexchange (Figure 3). Exchange broadening of EPR lines is a

function of teff, which represents a complex combination ofthe modulation parameters, and its value corresponds to the

longest lifetime of the preferred conformation under given ex-perimental conditions.[29] Consequently, we monitored changes

in the liquid-phase EPR spectra caused by a temperature in-

crease in CH2Cl2, DMSO, and toluene, which is a solvent inerttowards specific solvent–solute interactions,[22] and with suita-

ble freezing and boiling points.[36] The EPR line shapes of dinitr-oxides 3P and 2P showed a strong temperature dependencein all solvents used. Figures 4a and 5a illustrate the changes inthe EPR spectra of 3P and 2P, respectively, in toluene at various

Figure 3. Experimental EPR spectra (magnetic field sweep, SW=6 mT) of di-nitroxides in CH2Cl2 (c=0.1 mm) obtained at 295 K under argon: a) 1P–6P ;b) 1T–6T. The spin-Hamiltonian parameters elucidated from the simulationsare summarized in Table 2.

Table 2. Spin-Hamiltonian parameters of di-, tri-, and tetranitroxides elu-cidated from simulations of experimental EPR spectra measured in CH2Cl2at 295 K.

Nitroxide AN[mT]

Ai [mT] j J j [MHz][b] g

1P 1.441[a] – &8 2.0058[a]

4P 1.447 – &6 2.0058

6P 1.449 0.530 (13C); 0.503 (13C);0.429 (13C); 0.519 (13C);0.883 (13C); 0.871 (13C)

&0 2.0059

1T 1.578[a] – &2 2.0062[a]

2T 1.581[a] – &24 2.0061[a]

4T 1.579 0.040 (6H); 0.008 (6H);0.050 (2H); 0.040 (2H)

&0 2.0061

5T 1.577 0.042 (6H); 0.001 (6H);0.053 (2H); 0.032 (2H)

&0 2.0061

6T 1.576 0.048 (6H); 0.001 (6H);0.035 (2H); 0.033 (2H)

&0 2.0061

9T 1.588[a] – &2; &1; &1 2.0061[a]

11T 1.576[a] – >500; >482;&0; &0; &0; &0

2.0063[a]

12T 1.602[a] – >490; >420;&0; &0; &0; &0

2.0062[a]

[a] Averaged values. [b] 1 mT=28.0 MHz (g=2); 10@4 cm@1=3 MHz.


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temperatures; these spectra reveal a trend typical for the pro-

posed three-conformation model, with a fast transition be-tween conformations B and C.[22,28, 29] Analysis of the tempera-ture-dependent line-width alteration in the EPR spectra of di-nitroxides 2P and 3P was performed (a detailed description ofdata analysis is summarized in Scheme S1 in the Supporting In-formation).

For illustration, the Arrhenius plot of ln (ANteff), along with Javfunction versus reciprocal temperature, is depicted for dinitrox-ide 3P in Figure 4. The parameters (e, t0) describing the transi-tion from conformation B (JB=0) to conformation C (JC@AN)were evaluated for derivatives 2P and 3P in three different sol-vents (CH2Cl2, DMSO, and toluene); the values obtained are

summarized in Table 2. Our data showed a good correlationwith the parameters reported previously for dinitroxides with

analogous conformational dynamics.[22,28, 29] The enhanced in-terconversion between conformations B and C upon increasingtemperature may be explained by the rotational movement ofnitroxide rings.[22] The rotational diffusion motion of the nitro-

xide units in dinitroxides was described recently by usingDebye–Stokes–Einstein law, and a linear dependence of a mi-croscopic parameter (ANteff)

@1 on the Stokes parameter T/h wasfound.[35] An analogous approach was also applied herein, andthe linear dependence of (ANteff)

@1 on T/h obtained for dinitr-oxide 3P in toluene demonstrated a good correlation of themicroscopic behavior inside the solvent cage with that of the

macroscopic parameter T/h (Figure 4c).Quantum chemical calculations of 1P–4P indicated the pres-

ence of various model conformations. From the conformationalanalysis, the most energetically preferred conformations, withrespect to the arrangement of the connecting bridge and the

mutual orientations of the lateral moieties, are depicted in Fig-ure S1 in the Supporting Information. For 1P, only one stableconformation is predicted and no electron spin–spin interac-tion is present. This structure possesses C2 symmetry and thedistance between nitrogen atoms of NO groups is 10.64 a

(Table 3). In the case of 2P and 3P, the longest N···N distance is8.57 a for conformation 2P(II) and 13.14 a for conformation3P(I). Both are “elongated” conformations (A), similar to themost stable conformation, 2P(I), with a N···N distance of 8.22 a.

Figure 4. a) EPR spectra of dinitroxide 3P measured at various temperaturesin toluene under argon. b) Plot of ln (aNteff) as a function of reciprocal tem-perature. c) Plot of (aNteff)

@1 as a function of the Stokes parameter, T/h.d) Plot of Jav as a function of reciprocal temperature. Toluene viscosity datawere taken from Ref. [36]. A detailed description of data analysis is summar-ized in Scheme S1 in the Supporting Information.

Figure 5. EPR spectra of dinitroxides measured at various temperatures intoluene (c=0.1 mm) under argon: a) 2P and b) 2T.


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On the other hand, the shortest distance was 7.97 a for confor-mation 2P(IV) and 9.20 a for 3P(III). Additionally, the moststable conformations of 2P are stabilized by an intramolecularhydrogen bond between the nitrogen and oxygen atoms ofamidic groups to form a highly favored six-membered ring.Molecule 4P exhibits three stable conformers with N···N distan-ces of 11.79–13.10 a. According to the Boltzmann distributionand calculated DE values, the population of 4P(I) at 295 K is99.7%. At 373 K, the theoretical population of 4P(II) rises from0.2 to 0.7%. The intramolecular j J j value is close to zero formost of the conformations. For the J value to be observable,

both the short spin–spin distance and their appropriate orien-tation are crucial. For example, the theoretical j J j value of3P(III) is approximately 526 MHz (corresponding to conforma-tion C), whereas for the other 3P conformations no interactionis found. Although the gas-phase population of 3P(III), accord-ing to the Boltzmann distribution, is less than 0.02% below330 K, the effect of solvent may significantly alter the stability

of given conformations. For 2P, a value of almost j J j =0 is pre-dicted for the optimal geometry. However, this value can theo-

retically vary between 0 and 2000 MHz through full rotation ofthe PROXYL group with respect to the plane of the amide

group. This rotation can be induced by higher temperatures.

Conformational changes also affect the Fermi contact coupling

constants (AN) of the NO group nitrogen atoms, and theirvalues vary by up to 0.1 mT. Upon comparing the values withthe experimental results, they appear to be underestimated byup to 0.53 mT. For 2P, the AN values vary from 1.008 to1.045 mT. For both conformations, the molecule is asymmetricand different values of the nitrogen coupling constants for the

individual NO groups are calculated.Although the highest AN value of 1.025 mT was obtained for

3P(VI), structure 3P(I) exhibited the lowest value of 0.916 mT.The predicted J values and hyperfine coupling constants, to-gether with energetics and NO group distances, are summar-

ized in Table 3. For derivatives 2P and 3P, results of the theo-retical calculations correlate well with the presence of the

most stable elongated conformation A and two loop confor-mations B and C proposed by the analysis of experimental EPRspectra (Figure 3).

In the absence of precise structural data from XRD analysis,partial information about the dinitroxide geometry can be ob-

tained from solid-state EPR data. For a dilute solution of the di-nitroxide in a frozen glass, the scalar parameters of the spin

Table 3. The DFT/B3LYP/6-31+G(d,p)-calculated relative gas-phase electronic energies with respect to the most stable conformer (DE), interatomic distan-ces of the NO group nitrogen atoms (rN···N), and DFT/B3LYP/EPR-II-calculated spin-Hamiltonian parameters (AN, J) of the most stable conformations.

Nitroxide Conformation DE [kJmol@1] rN···N [a] AN1 [mT] AN2 [mT] J [MHz]

1P I 0.0 10.64 0.918 0.918 0

2P I 0.0 8.22 0.914 1.009 0II 7.6 8.57 1.045 1.012 66III 7.7 8.20 1.027 1.008 @66IV 15.4 7.97 0.921 1.010 @66

3P I 0.0 13.14 0.916 0.919 0II 14.4 11.26 1.022 1.008 0III 24.3 9.20 0.927 0.917 @526IV 30.0 12.76 0.921 0.920 0V 30.2 12.75 0.919 0.921 0VI 39.4 11.44 1.025 1.025 @66

4P I 0.0 13.10 0.917 0.917 0II 15.4 11.79 0.917 0.923 0III 18.0 12.40 0.917 0.915 0

1T I 0.0 11.99 1.242 1.242 0II 18.7 11.49 1.252 1.252 66III 27.3 10.68 1.240 1.242 0

2T I 0.0 10.61 1.236 1.243 @66II 0.3 10.68 1.233 1.244 @66III 20.1 10.62 1.253 1.251 0

3T I 0.0 10.30 1.244 1.234 @66II 6.5 14.52 1.239 1.239 0III 10.4 9.06 1.242 1.249 0IV 21.5 11.40 1.196 1.235 @66V 74.9 6.50 1.189 1.236 1513

4T I 0.0 14.47 1.242 1.242 0II 20.2 13.87 1.249 1.249 66III 25.3 13.51 1.255 1.245 0IV 26.2 11.81 1.247 1.237 0


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Hamiltonian in Equation (1) transform into tensor quantities[Eq. (2)][37]

Ĥ ¼mB~Bgð1Þ~̂Sð1Þ þ mB~Bgð2Þ~̂Sð2Þ þ ~̂Sð1ÞAð1Þ~̂Ið1Þ

þ~̂Sð2ÞAð2Þ~̂Ið2Þ @ J~̂Sð1Þ~̂Sð2Þ þ~̂Sð1ÞDd~̂Sð2Þð2Þ

and the Hamiltonian is extended by an additional term thatrepresents the magnetic electron dipole–dipole interaction~̂Sð1ÞDd~̂S

ð2Þ, which is characterized by a traceless dipolar tensor

Dd.[38] In principle, the spin Hamiltonian of Equation (2) con-tains 25 independent parameters to describe the electronic

and geometric structure of a nitroxide biradical (see the Sup-porting Information for details). Fortunately, for dinitroxidesconsisting of identical monoradical units, the g-tensor principalvalues can be considered identical, and the g and A tensors inthe individual units can be considered coplanar. Moreover, theg- and A-tensor principal values should not deviate considera-

bly from the values known for the monoradicals. Informationon the molecular geometry can then be compiled from themagnitude of J, the mutual orientation of the two g tensors(described by the a, b, and g Euler angles), their orientationwith respect to the dipolar vector re–e (defined by h, x angles),

and from the magnitude of the dipolar coupling Dd. However,this still leaves seven parameters to be extracted from the ex-

perimental spectra, and data from a single-frequency experi-

ment would not provide enough confidence in the estimatedconstrains. For dinitroxides 1P–3P and 1T–3T, we have thus re-corded the spectra in frozen glass at the X- and Q-band fre-quencies. We have also not attempted a blind search of the

geometrical parameters, but used the DFT-optimized geome-tries (see above) as a starting point. For 1P, DFT calculationspredicted a single conformer with negligible electron ex-

change between the two subunits. The X- and Q-band EPRspectra calculated based on this geometry showed reasonable

correspondence with the experimental data (Figure 6 andTable 4). Additionally, the electron–electron distance estimated

from Dd=33 MHz by using a point dipole approximation,11.6 a, is in good agreement with the predicted distance be-

tween the centers of the N@O bonds of 11.67 a, which roughlyreflects the unpaired electron spin density location. The 1P(I)

geometry, although predicted from gas-phase calculations, isthus in agreement with the 1P geometry in the frozen solu-tion.

The EPR spectra of dinitroxides 2P and 3P measured in aglassy matrix are quite different; this reflects the structure of

the bridge between two PROXYL units (Figure S3 in the Sup-porting Information). Although the spectra of 2P show re-solved contributions from the electron–electron dipolar inter-action, for derivative 3P we observe a broad singlet (g=2.0060, DB&1.4 mT) at both X- and Q-band frequencies andno transitions at half-field (DMS=2), which suggests that dinitr-oxide 3P possesses a rather small dipolar coupling.[33] Alterna-tively, distinct interactions were not detected, owing to fastspin relaxation caused by strong spin–spin exchange as a

result of the spatial proximity of nitroxide groups in a frozen

Figure 6. a) X- (&9.46 GHz) and b) Q-band (&34.06 GHz) EPR spectra of di-nitroxides 1P and 1T obtained in toluene/tetrahydrofuran (9:1; v/v) frozensolvent glass. Experimental temperatures are indicated in the figure. Coloredlines represent the calculated spectra obtained by using the spin-Hamiltoni-an parameters listed in Table 4.

Table 4. Spin-Hamiltonian parameters of the dinitroxides 1P and 1T used to calculate the X- and Q-band spectra in frozen solution. Electron–electron dis-tances were estimated as the distance between the centers of the N@O bonds.

Nitroxide g AN [mT] j J j [MHz] jDd j [a] [MHz] Euler angles[b] [8] Method, (rNN+ rOO)/2 [a]

1P

gxx 2.0092 Axx 4.3 0:2 33:2 a, b, g (@54, 179, @126):10 X-ray, –gyy 2.0062 Ayy 4.8 h, x (31, @88) :10 DFT, 11.67gzz 2.0022 Azz 33.0 EPR,

[c] 11.6:0.4

1Tgxx 2.0102 Axx 5.9 2:2 25:2 a, b, g (95, 180, 93) :10 X-ray,[d] 13.152, 12.774gyy 2.0065 Ayy 4.4 h, x (0, 94) :10 DFT,[e] 13.27, 12.77gzz 2.0026 Azz 34.8 EPR,

[c] 12.8:0.4[a] Dd represents the electron dipole–dipole coupling and is related to the dipolar splitting parameter D used in the description of molecular triplet statesby Dd=2D/3. [b] Euler angles (a, b, g and h, x) of the rotational transformation of the g(1) principal axis systems to that of g(2) and Dd, correspondingly, aregiven in zyz convention. [c] Estimated by using the point dipole approximation Dd (MHz)&51920 (r [a])@3. [d] From the X-ray structure of the 1T monocrys-tal (Figure 1). [e] From conformations 1T(I) and 1T(II).


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solvent glass.[39] The observation of a resolved EPR spectrum inthe tenfold diluted frozen solution of 3P (Figure S3 in the Sup-porting Information) suggests an intermolecular character ofthe nitroxide group contacts. Collapse of the anisotropic spec-

tral features into a single line thus likely results from partial ag-gregation of the 3P molecules in the glass, and subsequentonset of intermolecular exchange.

The sets of variable-temperature EPR spectra of 2P and 3P(Figures 5a and 4a, respectively) in solution suggest that the

most stable conformer (dominant at low temperatures) repre-sents elongated conformation A (JA=0). However, we werenot able to find reasonable agreement between the EPR spec-tra calculated based on the DFT-predicted geometries of the

individual conformers for 2P(I–IV) (Table 3) and the experimen-tal data. The flexible 2P dinitroxide might thus generate a mix-ture of conformations, even in a slowly frozen solvent glass.

Replacement of the @CH2@CH2@ fragment in the bridge ofderivative 3P by @CH=CH@ in dinitroxide 4P substantially hin-dered the formation of loop conformations (Table 3 and Fig-ure S1 in the Supporting Information), and the EPR spectrum

monitored in CH2Cl2 at 295 K (Figure 3) was attributed to asingle conformation with an exchange coupling of J&6 MHz(Table 2), upon considering the indirect through-bond spin ex-

change mechanism. The set of EPR spectra measured for 4P inDMSO over the temperature range 303–373 K revealed only a

slight increase in exchange coupling upon increasing the tem-perature (Figure S4 in the Supporting Information). This is also

fully compatible with the trend observed in the variable-tem-perature EPR spectra of rigid dinitroxide 1P in DMSO (Figure S4in the Supporting Information). Consequently, conformer 4Pcan be considered as rigid as 1P, and the lower line width ob-served in its EPR spectra could originate from a lower contribu-

tion of the electron–electron dipolar interaction to electronspin relaxation (Figure S4 in the Supporting Information).

The connecting bridges in dinitroxides 5P and 6P are 1,3-and 1,4-disubstituted benzene diamide, respectively. Although

the EPR spectra of 5P demonstrated, especially at higher tem-peratures, characteristic features of conformation dynamics insolution, which was compatible with the three-conformation

model, dinitroxide 6P, which was measured in CH2Cl2, toluene,or DMSO at temperatures up to 373 K exhibited an almost un-

changed three-line EPR spectrum that was assigned to a rigidconformer with J&0 (Figure 3 and Figure S3 in the SupportingInformation). We propose that in the meta-substituted dinitrox-ide 5P the rotational motion of the amidic-PROXYL moieties athigher temperatures facilitates the formation of conformations

B and C, and their enhanced interconversion results in thechanges to the EPR spectra (Figure S4 in the Supporting Infor-

mation). Previously, ortho- and para-substituted dinitroxide–phthalate esters containing two TEMPO units were investigat-

ed, and the lack of exchange interaction in the para isomer(J=0) was attributed to the unsuitable orientation of the nitro-xide groups, with an average distance of approximately

13 a.[40]

Despite identical connecting bridges between nitroxide moi-

eties, the EPR spectra of dinitroxides 1T–6T containing twoTEMPO units revealed decreased exchange couplings

(Figure 3), most probably owing to enhanced steric demandand different space orientation of the six-membered rings. Thespin distribution on the NO group is influenced by the degreeof pyramidalization of the nitrogen atom and by the polarityof the surrounding medium.[8] Whereas the NO group is planarfor five-membered nitroxides, with an out-of-plane angle of a

&08, for six-membered nitroxide it is pyramidal (a&10–208).[2, 8] Crystallographic studies of pyrroline-type radicals pre-viously confirmed the planar structure of the radical ring,

whereas the piperidine-type radicals have been characterizedby a chair-like conformation.[2,8]

The EPR spectra of dinitroxides 1T and 4T–6T, measured inCH2Cl2 at 295 K, represent three-line signals (Figure 3), which

reflects a negligible exchange interaction between two TEMPOunits (J=0). Analogous spectra of noninteracting dinitroxides

were obtained, even at higher temperatures, in all solvents

used. The EPR spectra of dinitroxide 1T, along with its DNP effi-ciency, were studied previously, and the previously published

three-line EPR spectrum measured in CH2Cl2 at 293 K (AN=1.58 mT, J

liquid toluene demonstrated line-width alteration coupled withthe conformational dynamics, because the separation of the

in-between lines was more evident with increasing tempera-ture (Figure 7a); this matched previous results well.[42] Analo-

gous spectra were measured previously for two 4-amino-TEMPO units linked by two to six methylene groups, and the

conformation dynamics was interpreted by using the three-conformation model.[28] Similar behavior was monitored for de-rivative 3T in toluene, CH2Cl2, or DMSO (Table 5). Analysis ofthe EPR spectra (Scheme S1 in the Supporting Information) ob-tained in all solvents provided values of ANteff

solution.[30,40,44] The experimental EPR spectra of trinitroxides9T, 11T, and 12T measured in CH2Cl2 at 295 K, along with theirsimulations, are shown in Figure 8, and the correspondingspin-Hamiltonian parameters are summarized in Table 2. In cor-

relation with previous results,[45] a three-line signal was ob-tained for trinitroxide 9T, which provided evidence of the lackof spin exchange between TEMPO units (J12= J13= J23&0),

most probably owing to steric hindrance and unsuitablemutual orientation of NO groups. Analogous EPR spectra were

monitored previously for tris(2,2,6,6-tetramethyl-1-piperidiny-

loxy) trimesate (TMNO) in trichlorobenzene at room tempera-ture; however, if these were measured over the temperature

range 400–470 K, conformational interconversion between dif-ferent radical conformations was observed.[30]

A sharp triplet (AN=1.570 mT) dominates the RT EPR spec-trum of tetranitroxide 10T in CH2Cl2 and DMSO, with broad-ened lines at positions AN/4 (Figure 8). Such spectra are com-patible with the major contribution of the J modulation to thetransverse electron relaxation time in polynitroxides.[46] The

lines of the EPR spectrum arising from polyradicals, in which all14N nuclei have the same spin state, do not have hyperfine

fluctuations and are not broadened by J modulation. Conse-quently, two extreme spectral lines (MI= :4) and one compo-nent of the central line (MI=0) remain relatively sharp, where-

as the other lines are broadened to varying degrees.[46] The in-fluence of temperature on the EPR spectrum of 10T providesinformation about the flexibility of molecule and the impact ofJ modulation.[46] Considering the unlimited very rapid electron–

electron spin exchange between the four nitroxide units uponincreasing the temperature, the spectrum of 10T should have

evolved towards a nine-line signal, with a line separation ofAN/4 and relative intensities of 1:4:10:16:19:16:10:4:1.

[32,44, 46]

However, the variable-temperature EPR spectra of tetranitrox-ide 10T measured in DMSO or toluene (Figure S6 in the Sup-porting Information) demonstrated that, under the given ex-perimental conditions, rapid spin exchange between four ni-

troxide units was not achieved, owing to the limited flexibilityof the specific connecting bridge in 10T tetranitroxide.

Tetranitroxides 11T and 12T revealed similar five-line EPRspectra in CH2Cl2 at 295 K (Figure 8) ; this demonstrated strongspin exchange between two nitroxide groups located at the @N(TEMPO)2 unit (J12 and J34) and a negligible interaction be-tween NO groups situated at different nitrogen atoms (J13, J14,

J23, and J24). This attribution is supported by J=0 found for di-nitroxide analogue 5T, with two TEMPO units at meta positionsin benzene diamide (Table 2).

The EPR spectrum of hexanitroxide 13T obtained in CH2Cl2at 295 K reveals slight similarities with the spectrum of 10T re-corded under analogous conditions, rather than with theabove-described five-line EPR spectra of tetranitroxides 11Tand 12T. A rather sharp three-line signal is recognizable in thespectrum, with significantly broadened in-between lines, as a

result of the major contribution of J modulation to the trans-

verse electron relaxation time in polynitroxides.[46]

Cyclic voltammetry (CV)

The redox potentials of polyradicals were determined by CV ata scan rate of 100 mVs@1 in phosphate-buffered saline (PBS,pH 7.0), with a paraffin-impregnated graphite electrode

(PIGE).[47] In general, nitroxides are readily electrochemicallyoxidized to the corresponding N-oxoammonium cations.[48] CV

results for 1P/T–6P/T (Figure S7 in the Supporting Information)reveal that all of the studied compounds undergo a one-elec-

tron transfer process, with determined half-wave potentials(E1/2) in the range of 570–645 mV versus a Ag/AgCl reference

electrode (Table 6).

While all PROXYL-derived biradicals, except 5P, showed (qua-si)reversible electrochemical behavior, as reflected by approxi-mately equal anodic and cathodic peak current values (Ipa/Ipc&1.0–1.5; Table 6), most of the TEMPO-containing biradicals(2T–5T) exhibited current intensities sufficiently far from equal(Ipa/Ipc&1.8–2.7, Table 6), which indicated a significant loss in ki-netic reversibility.[49]

In comparison with biradicals, all studied TEMPO-derived tri-,tetra-, and hexaradicals 9T–13T provided high values of anodicpotentials (Epa&715–758 mV). However, these polynitroxidesexhibited completely irreversible CV results (Figure S7 in the

Supporting Information).

Antimicrobial assays in vitro

Although rare, there are known examples of various TEMPO

derivatives with interesting bioactivity, including antifungal[50]

and anti-inflammatory[51] properties. Recently, we have shown

that, unlike PROXYL radicals, mono-/dinitroxyl amides contain-ing the TEMPO moiety exhibit potentially useful antimicrobial

Figure 8. Experimental EPR spectra (black line, SW=8 mT) of polynitroxides9T–13T in CH2Cl2 (c=0.1 mm) obtained at 295 K under an argon atmos-phere, along with their simulations (green line). The spin-Hamiltonian pa-rameters elucidated from the simulations are summarized in Table 2.


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activities.[52] Notably, piperidinyl radicals significantly inhibitedthe growth of Staphylococcus sp. Moreover, the more electron

withdrawing a substituent present on the piperidine ring was,the stronger the antibacterial effect of the respective nitroxide

against Staphylococcus epidermidis.[52]

Thus, we have tested the newly prepared dinitroxides 1P/T–6P/T and polynitroxides 9T, 10T and 12T, 13T for their in vitroantimicrobial activity (Table 7) by using bacteria (Staphylococ-cus aureus, S. epidermidis, Proteus sp.), yeast (Candida albicans,

Candida parapsilosis), and filamentous fungi (Fusarium culmo-rum, Botrytis cinerea, Aspergillus fumigatus).

Similarly, as we have shown previously,[52] the best inhibitionwas observed in the case of S. epidermidis. Notably, the most

active TEMPO tetraradicals 10T and 12T and hexaradical 13Talso inhibited the growth of S. aureus. On the other hand, di-

nitroxides 1P/T–5P/T were only able to inhibit Gram-positive(G+) bacteria, reaching maximum 75% inhibition (Table 7).Only dinitroxide derivatives 6P/T (at a concentration of1 mmoldm@3) were able to inhibit the growth of S. epidermidisup to 100%, but the growth of S. aureus was inhibited onlyvery weakly (17% for 6P). Considering the inhibitory effect onall model microbes, the best overall activity was exhibited bytetranitroxide 10T. This compound inhibited both G+ (Staphy-lococcus sp.) and Gram-negative model bacteria (Proteus sp.)

Naturally, the more sensitive G+-bacteria were inhibited at thehighest levels (the concentration of the derivative inhibiting

the growth of bacteria by 50% (MIC50)=0.05 mmoldm@3 for

S. epidermidis ; MIC50=0.2 mmoldm@3 for S. aureus). The

growth of Proteus sp. was also significantly inhibited by 10T(50% activity at c=0.9 mmoldm@3).

Taking into account the inhibitory potential of derivatives on

fungal cells, only weak antifungal activity was observed. Simi-larly, as found in the case of bacterial cells, higher activity was

measured for polynitroxides 10T and 13T. A notable inhibitoryeffect was evidenced on the yeast C. parapsilosis, in compari-son with C. albicans (Table 8). Tetraradical 10T has shown thehighest activity among filamentous fungi on F. culmorum(MIC50 value was observed at a concentration of 1 mmoldm

@3).Dinitroxides 3T and 4P/T–6P/T inhibited the growth of F. cul-morum by approximately 20–32%, whereas the other model

filamentous fungi were basically uninhibited by these deriva-

tives (Table 7).Finally, the most active polynitroxides 9T, 10T, and 13T were

tested for their potential mutagenic activity. Based on ourdata, it can be concluded that the newly synthesized deriva-

tives have not increased the number of revertants of Salmonel-la typhimurium TA 98 or TA 100. Thus, they do not induce

Table 7. The inhibitory effect [%] of di- and polynitroxides[a] on the growth of bacteria, yeasts, and filamentous fungi.[b]

Nitroxide Bacteria Yeasts Filamentous fungiS. a. S. e. P. s. C. a. C. p. F. c. B. c. A. f.

1P 24 50 13 0 15 20 0 02P 0 20 0 0 10 5 0 104P 0 75 0 0 0 16 0 05P 0 71 0 0 0 17 0 116P 17 100 11 0 14 24 0 101T 15 0 0 0 0 0 0 02T 11 65 0 0 0 17 13 103T 63 62 0 0 11 20 0 104T 20 72 0 0 0 32 0 115T 67 85 0 0 66 32 15 106T 0 100 33 23 51 0 13 09T 37 100 18 38 36 0 10 010T 100 100 60 34 66 50 27 1512T 75 67 32 0 0 22 0 1113T 100 100 100 50 79 22 0 13

[a] Growth inhibition determined at a concentration of 1 mmoldm@3 of the tested compound. [b] S. a.=S. aureus, S. e.=S. epidermidis, P. s.=Proteus sp. , C.a.=C. albicans, C. p.=C. parapsilosis, F. c.=F. culmorum, B. c.=B. cinerea, A. f.=A. fumigatus.

Table 6. Experimental redox potentials (versus Ag/AgCl reference elec-trode) and current responses of nitroxide/N-oxoammonium cation redoxcouples.

Cmpd Epa[a]

[mV]Epc

[b]

[mV]E1/2

[c]

[mV]DE[d]

[mV]Ipa/Ipc

1P 679 611 645 68 1.02P 683 575 629 107 1.33P 658 568 613 90 1.14P 686 598 642 88 1.35P 672 568 620 104 2.26P 670 580 625 90 1.51T 636 504 570 132 1.12T 666 540 603 126 1.93T 640 542 591 98 1.84T 665 531 598 134 2.75T 663 545 604 118 2.16T 658 528 593 130 1.19T 730 – – – –10T 758 – – – –11T 755 – – – –12T 715 – – – –13T 730 – – – –

[a] Epa=anodic peak potential. [b] Epc=cathodic peak potential. [c] E1/2=(Epa++Epc)/2. [d] DE=Epa@Epc. [e] Ipa=anodic peak current, Ipc=cathodicpeak current.


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point and frameshift mutations at any of the tested concentra-

tions and are considered to be nonmutagenic.

Conclusion

A series of dinitroxide amides possessing PROXYL or TEMPO

units (1P/T–6P/T) connected by various bridges, along withTEMPO-containing tri- (9T), tetra- (10T–12T), and hexaradicals(13T), were synthesized and their properties were analyzedthrough multiple experimental approaches, including single-crystal X-ray structure assessment for dinitroxide 1T and tetra-nitroxide 10T.

The results revealed a substantial effect of the nitroxide het-

erocyclic ring structure, as well as the character of the spacer

on the exchange coupling constant. Although a rather shortand rigid bridge between two PROXYL units in dinitroxide 1Plimited the spin exchange by through-space interactions, elon-gation of the connecting bridge in derivatives 2P and 3P re-sulted in enhanced flexibility, and the experimental EPR spectrain CH2Cl2, DMSO, and toluene consisted of alternately broad-

ened lines, which provided evidence of conformation dynamicswith rapid spin exchange. In the TEMPO-derived dinitroxide

series, such behavior was evidenced only for dinitroxide 3T,most probably owing to the increased steric demand and dif-ferent space orientation of the six-membered rings. The intra-molecular dynamics of 2P, 3P, and 3T, which were evaluated byusing a three-conformation model, correlated well with the

theoretical energy and space orientation of individual dinitrox-ide conformers evaluated by DFT calculations. Analysis of low-

temperature X- and Q-band EPR spectra of the dissolved dinitr-oxides 1P and 1T provided spin–spin distances comparablewith those of the theoretical values.

Cyclic voltammetry investigations revealed (quasi)reversibleelectrochemical behavior for PROXYL-derived biradicals. For di-

nitroxides 1P/T–6P/T, the one-electron transfer process wascharacterized by half-wave potentials in the range of 570–

645 mV versus Ag/AgCl. The anodic redox potentials of poly-radicals 9T–13T gave high values (Epa&715–758 mV versus Ag/AgCl) and the electron-transfer process was coupled with a sig-nificant loss of kinetic reversibility.

Assessment of the inhibitory activities of studied di- and pol-ynitroxides against model bacteria (S. aureus, S. epidermidis,

Proteus sp.), yeasts (C. albicans, C. parapsilosis), and filamentousfungi (F. culmorum, B. cinerea, A. fumigatus) was conducted. The

best inhibition was observed in the case of S. epidermidis. No-

tably, the most active TEMPO tetraradicals 10T and 12T andhexaradical 13T also inhibited the growth of S. aureus. On theother hand, dinitroxides 1P/T–5P/T were only able to inhibitG+ bacteria, reaching a maximum of 75% inhibition. Although

dinitroxide derivatives 6P/T were able to inhibit the growth ofS. epidermidis up to 100%, their effect on the growth of

S. aureus was very weak (17% for 6P). Rather weak growth in-hibition of yeast and/or filamentous fungi was obtained formost of the tested nitroxides, which revealed again the highest

antifungal activity for polynitroxides 10T and 13T. No mutagen-ic function of tetra- and hexanitroxides with highest antibacte-

rial activity was found.

Experimental Section

General

All solvents of p.a. purity were dried over 4 a molecular sieves. Allother reagents were purchased and used without further purifica-tion. TLC was performed on aluminum plates precoated with0.2 mm silica gel 60 F254. FLC was performed on Kieselgel 60 (40–63 mm). IR spectra were recorded on a FTIR spectrometer as filmson a diamond sampler (attenuated total reflectance (ATR)). Meltingpoints were determined on capillary apparatus and are uncorrect-ed. LC-MS analyses were performed on an instrument equippedwith a multimode MS detector by using the MM ESI/APCI ioniza-tion method (column: Zorbax SB C-8 12.5V2.1 mm, particle size5 mm, eluent: water/MeOH with 0.1% HCO2H, gradient 0–100%MeOH for 2.5 min, flow 1.5 mLmin@1). HRMS spectra were recordedon a TOF-Q instrument and evaluated by using Compass DataAnal-ysis 4.0 software. Elemental analyses were performed at the De-partment of Inorganic Chemistry, Slovak University of Technologyin Bratislava, Slovakia. All synthetic procedures, with full analyticaldata of prepared compounds, can be found in the Supporting In-formation.

EPR spectroscopy

Stock solutions of all di- and polynitroxides were prepared in anhy-drous dichloromethane (Merck, SeccoSolv, max. 0.004% H2O),DMSO (Merck, SeccoSolv, max. 0.025% H2O), or toluene (Merck,SeccoSolv, max. 0.005% H2O). EPR spectra were measured by usingdilute solutions (c=0.1 mm) to avoid intermolecular interaction ef-fects. The prepared solutions were carefully saturated with argonand immediately transferred to a small quartz flat cell (WG 808-Q,Wilmad-LabGlass, USA) optimized for the TE102 cavity (Bruker, Ger-many). The X-band spectra were recorded by using EMX Plus orEMX EPR spectrometers (Bruker, Germany). The effect of tempera-ture on the EPR spectra of di- and polynitroxides in solution wasmonitored in CH2Cl2 over the temperature range 190–295 K, inDMSO over the temperature range 298–373 K, and in toluene overthe temperature range 210–360 K. A Bruker temperature control

Table 8. Evaluation of MIC50 values [mmoldm@3] of di- and polynitroxides

on the growth of bacteria and yeasts.[a]

Nitroxide Bacteria YeastsS. a. S. e. P. s. C. a. C. p.

1P N 1 N N N2P N N N N N4P N 0.3 N N N5P N 0.4 N N N6P N 0.4 N N N1T N N N N N2T N 0.2 N N N3T 0.8 0.6 N N N4T N 0.4 N N N5T 0.5 0.1 N N 0.86T N 0.2 N N 19T N 0.05 N N N10T 0.2 0.05 0.9 N 0.812T 0.8 0.7 N N N13T 0.3 0.2 0.5 1 0.7

[a] N=not active. S. a.=S. aureus, S. e.=S. epidermidis, P. s.=Proteus sp. ,C. a.=C. albicans, C. p.=C. parapsilosis.


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unit ER 4111 VT was used to adjust the temperature. The g valueswere determined by using a built-in magnetometer. Typical EPRspectrometer settings were as follows: microwave frequency:9.428 GHz; microwave power: 1.053 or 10.53 mW; center field:335.8 mT; sweep width: 6–10 mT; gain: 5V103 ; modulation ampli-tude: 0.02–0.05 mT; scan: 42–82 s; time constant: 10.24 ms;number of scans: 5. The EPR spectra of selected dinitroxides wereadditionally measured at 77 K in toluene/tetrahydrofuran (9:1; v/v)glassy solvent matrix by using a PS 100-X (Adani, Belarus) X-bandspectrometer. The Q-band EPR spectra of selected dinitroxides intoluene/tetrahydrofuran (9:1; v/v) solvent mixture were recordedat 295 and 110 K by using an EMX Plus EPR spectrometer equippedwith an ER 5106 QT resonator. The experimental EPR spectra wereanalyzed by using the Bruker software WinEPR. The simulatedspectra of di- and polynitroxides were calculated with the EasySpintoolbox[53] or Winsim2002 software.[54]

Theoretical calculations

The gas-phase geometries of the studied molecules in the elec-tronic ground state were optimized by using DFT employingBecke’s three-parameter hybrid functional with the Lee, Yang, andParr correlation functional (B3LYP).[55] The energy cutoff used forthe geometry optimization was 4V10@3 kJmol@1 and the final root-mean-square energy gradient was below 0.04 kJmol@1a@1. All geo-metries were optimized by using the 6-31+G(d,p) basis set.[56] Onthe basis of optimized B3LYP geometries, the isotropic Fermi con-tact couplings were calculated in the EPR-II basis set.[57] A spin con-tamination value of up to 1.01 was found for the studied biradicalstructures. The intramolecular exchange coupling energies, J, werecalculated according to the formalism of Yamaguchi and co-work-ers [Eq. (3)] ,[58]

J ¼ ðES @ ETÞs2h iT@ s2h iS ð3Þ

in which ES and ET are the electronic B3LYP energies of the singletand triplet states, respectively, and hS2iS and hS2iT denote the aver-age spin square values for those spin states, respectively. The nu-merical accuracy for these energies was within 10@8 hartree (&66 MHz). Numerical integration of the DFT functional was per-formed by using a default fine integration grid. All quantum chem-ical calculations were performed by using the Gaussian 09 programpackage.[59] The Molekel program package was used for the visuali-zation of the obtained theoretical results.[60] Structural coordinatesare available on request.

Cyclic voltammetry (CV)

Chemicals : Stock solutions of all compounds (c=1V10@3 molL@1)in H2O/MeOH (9/1, v/v) were used for the preparation of workingsolutions (1V10@4 molL@1) for electrochemical measurements bydilution with PBS at pH 7 as a supporting electrolyte. The latterwas prepared in the usual way by mixing an appropriate amountof NaH2PO4·H2O) and Na2HPO4·7H2O. All chemicals (analytical-re-agent grade) were used without further purification. Aqueous solu-tions were prepared with double-distilled deionized water with aresistivity above 18 MWcm.

Electrochemical measurements : The CV measurements were per-formed by using an AUTOLAB PGSTAT-302N (Metrohm AutolabB.V. , The Netherlands) potentiostat/galvanostat equipped with aUSB electrochemical interface connected to a three-electrode

single-compartment glass cell and personal computer for data stor-age and processing. NOVA 1.9 software was employed for elabora-tion and evaluation of all CV measurements. The glass electro-chemical cell consisted of Ag/AgCl (3 molL@1 KCl) and Pt wire asreference and counter electrodes, respectively. A PIGE with a diam-eter of 5 mm was used as the working electrode. The PIGE waspolished with aluminum oxide (grain size=0.3 mm) and rinsed withdeionized water to obtain the fresh electrode surface before eachexperiment. The pH value of PBS was monitored with a pH meter(Model 215; Denver Instrument, USA) with combined a glass elec-trode, which was regularly calibrated with standard buffer solu-tions. All half-wave potentials (E1/2) were given versus the Ag/AgCl(3 molL@1 KCl) reference electrode at an ambient temperature of(25:1) 8C. The supporting electrolyte (20 mL) containing an appro-priate amount of studied compound was added to the glass elec-trochemical cell. Before each measurement, ultrapure N2 (O2<2 ppm) was used to degas the solutions (10 min) and provide aninert atmosphere inside the electrochemical cell. CV measurementswere recorded over a potential range from @1 to +1 V with theuse of optimized instrumental parameters as follows: scan rate of0.1 Vs@1, step potential of 0.005 V, and interval time of 0.05 s. Atthe beginning, the current response for a blank (PBS at pH 7 with-out the presence of any studied nitroxide) was measured to checkthe electrochemical background of the system. Subsequently, CVmeasurements of each studied species were performed fivefold(n=5), and the average scan was considered for the evaluation ofE1/2 and construction of CV graphs.

X-ray analyses

X-ray data collection was performed on an Oxford Diffraction Gem-ini R four circle diffractometer equipped with a Ruby charge-cou-pled device (CCD) detector and sealed-tube sources, by using MoKaradiation at 100(1) K for 1T and at room temperature for 10T. Datareduction was achieved by using the CrysAlis 1.171.36.24a pro-gram.[61] Crystal structures were refined by using the SHELXL pro-gram.[62] The DIAMOND program was used for molecular graph-ics.[63]

Biological assays

Antimicrobial assay in vitro : Antibacterial activity of polynitrox-ides on Firmicutes S. epidermidis CCM 7221, S. aureus CCM 3953,and g-Proteobacteria Proteus sp. CCM 1779 (Czech Collection of Mi-croorganisms, Masaryk University, Brno, Czech Republic), as well asantifungal activity on model yeast (C. albicans SC 5314, C. parapsi-losis ATCC 22019), were evaluated by the microdilution methodrecommended by EUCAST.[64]

Antifungal activity on filamentous fungi F. culmorum CCM F-21,B. cinerea CCM F-16 (Czech Collection of Microorganisms, MasarykUniversity, Brno, Czech Republic), and Aspergillus fumigatus (Collec-tion of Microorganisms of Department of Biochemistry and Micro-biology, Faculty of Chemical and Food Technology STU, Bratislava,Slovakia) was studied by the macrodilution method.[65]

Subcultures of microorganisms were prepared separately in Petridishes containing appropriate agar medium and incubated at 37 8Cfor 24 h for bacteria or 48 h for model yeasts, and at 25 8C for 96 hfor filamentous fungi. Assessment of antibacterial and antifungalactivities was expressed as MIC50. Values were derived from thetoxicity curves. Chromatographically pure compounds were dis-solved in DMSO (Sigma Aldrich), their final concentrations neverexceeded 1.0 vol% in control or treated samples.


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Ames test : Assessment of potential mutagenicity was performedby using a classical plate incorporation method,[66] without meta-bolic activation, by using S. typhimurium TA 98 and TA100. As apositive control, the mutagen 3-(5-nitro-2-furyl)acrylic acid (NFAA)was used. A positive response was defined as a reproducible two-fold increase of revertans with a dose–response relationship.

Acknowledgements

This study was supported by the Science and Technology Assis-

tance Agency of the Slovak Republic under contract nos. APVV-0797-11, APVV-0282-10, APVV-15-0053, and APVV-15–0079; the

Grant Agency of the Slovak Republic (VEGA 1/0489/16, VEGA

1/0041/15, VEGA 1/0594/16 and VEGA 1/0871/16) ; and with thesupport of the Ministry of Education, Science, Research and Sport

of the Slovak Republic within the Research and Development Op-erational Programme for the project “University Science Park of

STU Bratislava”, ITMS 26240220084, co-funded by the EuropeanRegional Development Fund.

Conflict of interest

The authors declare no conflict of interest.

Keywords: biological activity · density functional calculations ·electrochemistry · nitroxides · radicals

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Manuscript received: August 8, 2017

Revised manuscript received: October 18, 2017Accepted manuscript online: October 20, 2017


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