Room-temperature electron spin relaxation of nitroxides immobilized in trehalose: Effect of substituents adjacent to NO-group

Article abstract of DOI:10.1016/j.jmr.2016.02.014

Trehalose has been recently promoted as efficient immobilizer of biomolecules for room-temperature EPR studies, including distance measurements between attached nitroxide spin labels. Generally, the structure of nitroxide influences the electron spin relaxation times, being crucial parameters for room-temperature pulse EPR measurements. Therefore, in this work we investigated a series of nitroxides with different substituents adjacent to NO-moiety including spirocyclohexane, spirocyclopentane, tetraethyl and tetramethyl groups. Electron spin relaxation times (T₁, T_m) of these radicals immobilized in trehalose were measured at room temperature at X- and Q-bands (9/34 GHz). In addition, a comparison was made with the corresponding relaxation times in nitroxide-labeled DNA immobilized in trehalose. In all cases phase memory times T_m were close to 700 ns and did not essentially depend on structure of substituents. Comparison of temperature dependences of T_m at T = 80-300 K shows that the benefit of spirocyclohexane substituents well-known at medium temperatures (～100-180 K) becomes negligible at 300 K. Therefore, unless there are specific interactions between spin labels and biomolecules, the room-temperature value of T_m in trehalose is weakly dependent on the structure of substituents adjacent to NO-moiety of nitroxide. The issues of specific interactions and stability of nitroxide labels in biological media might be more important for room temperature pulsed dipolar EPR than differences in intrinsic spin relaxation of radicals.

Full text of DOI:10.1016/j.jmr.2016.02.014

Accepted Manuscript
Room-Temperature Electron Spin Relaxation of Nitroxides Immobilized in
Trehalose: Effect of Substituents Adjacent to NO-group
Andrey A. Kuzhelev, Rodion K. Strizhakov, Olesya A. Krumkacheva, Yuliya
F. Polienko, Denis A. Morozov, Georgiy Yu. Shevelev, Dmitrii V. Pyshnyi, Igor
A. Kirilyuk, Matvey V. Fedin, Elena G. Bagryanskaya
PII:
S1090-7807(16)00117-8
DOI:
http://dx.doi.org/10.1016/j.jmr.2016.02.014
YJMRE 5835
Reference:
Journal of Magnetic Resonance
To appear in:
Received Date:
Revised Date:
4 November 2015
4 February 2016
Please cite this article as: A.A. Kuzhelev, R.K. Strizhakov, O.A. Krumkacheva, Y.F. Polienko, D.A. Morozov, G.Y.
Shevelev, D.V. Pyshnyi, I.A. Kirilyuk, M.V. Fedin, E.G. Bagryanskaya, Room-Temperature Electron Spin
Relaxation of Nitroxides Immobilized in Trehalose: Effect of Substituents Adjacent to NO-group, Journal of
Magnetic Resonance (2016), doi: http://dx.doi.org/10.1016/j.jmr.2016.02.014
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Room-Temperature Electron Spin Relaxation of Nitroxides Immobilized in
Trehalose: Effect of Substituents Adjacent to NO-group
Andrey A. Kuzhelev,1,3 Rodion K. Strizhakov,1,2 Olesya A. Krumkacheva,2,3 Yuliya F. Polienko,1,3
Denis A. Morozov,1,3 Georgiy Yu. Shevelev,3,4 Dmitrii V. Pyshnyi,4 Igor A. Kirilyuk,1,3 Matvey V.
Fedin,2,3,* Elena G. Bagryanskaya1,3,*
1N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev ave. 9, Novosibirsk,
630090, Russia; egbagryanskaya@nioch.nsc.ru
2International Tomography Center SB RAS, Institutskaya str. 3a, Novosibirsk, 630090, Russia;
mfedin@tomo.nsc.ru
3Novosibirsk State University, Pirogova str. 2, Novosibirsk, 630090, Russia
4Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk 630090, Russia
Abstract
Trehalose has been recently promoted as efficient immobilizer of biomolecules for room-
temperature EPR studies, including distance measurements between attached nitroxide spin labels.
Generally, the structure of nitroxide influences the electron spin relaxation times, being crucial
parameters for room-temperature pulse EPR measurements. Therefore, in this work we investigated
a series of nitroxides with different substituents adjacent to NO-moiety including spirocyclohexane,
cyclopentane, tetraethyl and tetramethyl groups. Electron spin relaxation times (T1, Tm) of these
radicals immobilized in trehalose were measured at room temperature at X- and Q-bands (9/34
GHz). In addition, a comparison was made with the corresponding relaxation times in nitroxide-
labeled DNA immobilized in trehalose. In all cases phase memory times Tm were close to 700 ns
and did not essentially depend on structure of substituents. Comparison of temperature dependences
of Tm at T=80-300 K shows that the benefit of spirocyclohexane substituents well-known at medium
temperatures (~100-180 K) becomes negligible at 300 K, because the conformational mobility of
these groups becomes sufficiently high. Therefore, unless there are specific interactions between
spin labels and biomolecules, the room-temperature value of Tm in trehalose is weakly dependent on
the structure of substituents adjacent to NO-moiety of nitroxide. The issues of specific interactions
and stability of nitroxide labels in biological media might be more important for room temperature
pulsed dipolar EPR than differences in intrinsic spin relaxation of radicals.
1

I. Introduction
Pulsed dipolar Electron Paramagnetic Resonance (EPR) spectroscopy is nowadays being
extensively used for nanoscale distance measurements in biomolecules, including large enough
complexes of biological significance [1-7]. Although most of the experiments are usually done at
cryogenic temperatures in frozen solutions, several recent advances opened up possibilities for
application of the method at room/physiological temperatures as well [8-10]. Pulsed dipolar EPR
spectroscopy, largely represented by Double Electron-Electron Resonance (DEER or PELDOR)
[11-13], Double Quantum Coherence (DQC) [14,15] and a few other methods [16-20], relies on the
measurement of dipole-dipole interaction between two spin labels. The accuracy of the
measurement and accessible range of distances depend on the electron spin dephasing (or phase
memory) time (Tm) of spin label.
Short Tm of most common nitroxide spin labels was a major complication for application of
pulsed dipolar EPR at room temperatures (RT) until recently. The problem was partly remedied by
implementation of new triarylmethyl (TAM) based spin labels allowing measurement of interspin
distances up to ~4.6 nm [9]. In parallel, it has been demonstrated that the use of spirocyclohexane-
substituted nitroxides in conjunction with immobilization in glassy trehalose allows measurement of
interspin distances ~3.2 nm at RT [10]. Although Tm of TAM-based labels is still superior compared
to nitroxides, there are certain advantages of using nitroxides for short-distance measurements at
RT, since their spin-labeling strategies are much better developed and their availability is at the
moment much broader.
The first RT distance measurement in nitroxide-labeled protein relied on two important
advances [10]. First, spirocyclohexane-substituted nitroxide was used instead of more common
tetramethyl-substituted one; the authors notice that DEER measurements were only possible for the
former one due to noticeably longer Tm time [10,21,22]. Second, immobilization in trehalose was
implemented to prevent averaging of dipolar interaction by motion of spin-labeled protein.
Trehalose recently attracted huge attention as immobilizing medium due to its cryoprotective
properties and ability to mimic water solution by creating hydrogen bonding networks [23-25].
Therefore, the use of trehalose for immobilization and advanced nitroxides for spin labeling appears
to be a promising approach for RT distance measurements.
In this work we investigated a series of nitroxides with different substituents adjacent to
NO-moiety (spirocyclohexane, spirocyclopentane, tetraethyl and tetramethyl groups) in order to
find structure/property relations and establish further routes of optimization for RT distance
measurements. Surprisingly, the dependence of Tm on structure of substituents adjacent to NO-
moiety was not significant at RT. This was further elaborated by studying temperature dependence
of Tm and allowed more general conclusions and recommendations for future studies.
2

II. Results and Discussion
II.1. Structure of studied nitroxides
Figure 1 shows chemical structures of studied nitroxides of piperidine (2a,b), pirroline (1, 2c,d),
pyrrolidine (3), imidazoline (4b, 5b, 6b) and imidazolidine (4a, 5a, 6a) series with different bulky
substituents adjacent to NO-moiety including tetramethyl (1), tetraethyl (4a,b), bis-
spirocyclohexane (2a-d), bis-spirocyclopentane (3), diethyl and spirocyclohexane (5a,b), diethyl
and spirocyclopentane (6a,b) fragments.
Figure 1. Structure of studied nitroxides.
3

The variety of these radicals allows us to investigate electron spin relaxation depending on type of
radical as well as on the structure of bulky substituents. If the rotational motion of radical is
hindered (as is the case in glassy trehalose), the primary relaxation mechanisms are (i) modulation
of 14N hyperfine interaction (HFI) anisotropy of NO group by librational motion, and (ii)
modulation of HFI with other nuclei of radical by rotation of corresponding groups (e.g. rotation of
tetramethyl groups in 1). We considered both symmetric and asymmetric structures with combined
cyclic/acyclic substituents. Cyclopentane and cyclohexane rings differ by rigidity and thus might
induce contribution to Tm in different extent. In addition, Tm might depend on the amount of
remaining water in the vicinity of NO group. In this regard, compound 3 represents radical with
hydrophilic OH-groups, for which one expects the largest concentration of H2O molecules close to
NO moiety. Compound 2d represents radical attached to glutathione (GSH), as a simple example of
attachment to a small biomolecule.
II.2. Immobilization in trehalose evidenced by CW EPR
All studied radicals were prepared in glassy trehalose as described in Experimental Section below.
In order to demonstrate that the immobilization is achieved we have measured CW EPR spectra of
all samples at RT and simulated them using solid-state powder pattern. Four representative spectra
for different groups of radicals are shown in Figure 2, whereas the rest of the data for other radicals
is given in Supporting Information (SI). Spectral shapes correspond to the essentially immobile
radical, i.e. the rotational motion is strongly suppressed. At the same time, simulations using solid
state powder pattern do not allow good enough agreement in every case (Figure 2 and SI),
indicating that the librational motions are clearly present. This follows from suppression of Azz
components compared to theoretical predictions, as well as by generally better agreement with
simulations at 80 K compared to 300 K, since librations are more intensive at higher temperature
(see SI for details).
4

Figure 2. X-band CW EPR spectra of studied samples at room temperature (black) and corresponding
simulations (red).
II.3. Relaxation times of free nitroxides at RT
As described in Experimental Section below, we used two-step drying procedure: mild drying (10–3
bar for 2 hours) and extensive drying (3.510–9 bar for 24 hours after mild drying). Relaxation times
at RT were measured after each step (see Table 1). We have noticed that Tm is quite sensitive to the
extent of drying, although not for every radical. At the same time, T1 values were found similar after
each step of drying. It has been shown that the concentration of remaining water in glassy trehalose
has a significant influence on the EPR spectra and low-temperature relaxation rates [23, 24].
Evacuation at 10-3 bar results in concentration of water in trehalose being 7% [10]. Unfortunately,
we could not determine the concentration of water remaining after evacuation at 3.510–9 bar, but
obviously it could only decrease compared to evacuation at 10-3 bar. As a result, one expects better
immobilization of radical and further suppression of relaxation mechanisms. Still, Table 1 shows
that extensive evacuation changes Tm relaxation times by no more than ~15% for most of the
radicals, with the exception for radical 3 exhibiting ~22% change of Tm. Indeed, one would expect
that the OH-groups in the structure of 3 should lead to the largest amount of H2O molecules
remaining in the vicinity of NO group.
5

Table 1. Electron spin relaxation times Tm and T1 measured at X-band for studied radicals in dried trehalose at RT. The
results are given for two drying conditions, indicated by different pressure during evacuation and described in more
detail in the text. Accuracy of Tm and T1 measurements is approx. 40 ns and 1 s, respectively.
Tm, ns Tm, ns
Radical 10–3 bar
3.5·10–9 bar
T1, s
1
625
660
680
740
610
575
550
650*
570
630
610
630
735
750
730
800
630
730
680
650*
640
740
655
730
12
17
17
21
22
30
18
14
18
16
23
16
2a
2b
2c
2d
3
4a
4b
5a
5b
6a
6b
*strong deuterium modulation allows rough estimation (80 ns) only
Despite these relatively small effects, evidently, the Tm values of all radicals immobilized in
trehalose are roughly 700 ns and do not demonstrate pronounced dependence on the structure of
substituents adjacent to NO-moiety. At the same time, T1 varies depending on structure between 12
s for 1 and 30 s for 3. The Tm relaxation curves are slightly better described by biexponential
decay with approximately equal weights and characteristic times ~400 and ~900 ns, however for
simplicity of comparison Table 1 reports the result of monoexponential analysis (for rigorous
biexponential treatment see Supporting Information). Note also that Tm noticeably depends on the
spectral position (see Supporting Information); all values reported in Table 1 were obtained at the
absolute maximum of the spectrum. In fact, all other spectral positions demonstrate shorter Tm
values, as is quite typical for relaxation induced by librations of nitroxide [26,27,28].
No noticeable dependence of Tm on magnetic field was found during X- and Q-band
measurements. For instance, for two representative radicals 1 and 2c the values of Tm 72040 ns
and 74040 ns were obtained at Q-band at the spectral maximum, respectively. Obviously, these
values are similar to those reported in Table 1 within experimental accuracy.
Note that similarity of Tm values found for common tetramethyl-substituted nitroxide and
spirocyclohexane-substituted one disagrees with the results of Ref.10, where noticeable
improvement was reported for spirocyclohexane-substituted spin label (920 ns vs. 550 ns for
tetramethyl-substituted one) in glassy trehalose at RT. In order to understand the reasons for these
differences and underlying relaxation mechanisms we investigated temperature dependence of Tm in
glassy trehalose for these two types of radicals.
6

II.4. Temperature dependence of Tm for free nitroxides
Figure 3 shows temperature dependences of Tm and T1 for two representative radicals 1 and 2c in
trehalose. The shapes of Tm vs. T dependence (Fig.3a) are generally similar compared to the trends
observed in frozen solutions (see e.g. Refs.21,29). Tetramethyl-substituted radical demonstrates
drastic acceleration of phase relaxation (1/Tm) at T>100 K as rapid rotation of methyl groups
becomes thermally-activated; at T>140 K this rotation becomes fast enough for averaging the HFI
anisotropy, leading to some decrease of phase relaxation; finally, at T>220 K librational motion of
NO group governs phase relaxation and its rate continues to increase with the temperature [30,31].
Contrary to tetramethyl-substituted radical, no bell-like shape is observed for spirocyclohexane-
substituted radical, and only an increase of 1/Tm due to librations of NO group begins above ~170
K. Temperature dependence of 1/T1 is similar for both radicals and generally similar to that
obtained in frozen solutions.
Figure 3. Temperature dependence of Tm (a) and T1 (b) for 1 and 2c in trehalose measured at X-band.
Note, remarkably, at room temperature the difference in 1/Tm between free 2c and 1
becomes very small. At the same time, based on the study of spin-labeled protein, Ref.10 suggests
7

that there is a noticeable benefit of using spirocyclohexane-substituted spin labels compared to
common tetramethyl-substituted ones. This disagreement can be reconciled assuming, very
generally, that there are specific interactions of spin labels with the protein studied in Ref.10, and
such interactions are different for the two types of labels. To elaborate on this hypothesis, at least to
some extent, we have also compared two types of spin labels attached to oligonucleotide and
immobilized in trehalose.
II.5. Relaxation times of nitroxides attached to DNA at RT
We have synthesized three DNA duplexes spin-labeled by pairs of tetramethyl- or
spirocyclohexane-substituted nitroxides (Fig.4). Note that similar DNA duplexes were investigated
in our previous work [32] (see this reference for structural details). The sample preparation
approach used for free radicals turned out to be damaging for the structure of duplex, which partly
“frayed” at its ends during water drying from trehalose [33-35]. This clearly follows from
DEER/PELDOR data differing in trehalose compared to water solution [32] (more details in
Supporting Information). However, for the purpose of the present work, such systems are still
useful for investigation of spin relaxation of label attached to oligonucleotide.
Table 2 shows room-temperature relaxation data for three spin-labeled DNA systems in
trehalose. The spin labels used are most close to 2c and 1, studied in trehalose vs. temperature in
previous section. Remarkably, Tm value of spirocyclohexane-substituted label increased compared
to 2c radical (Table 1), whereas Tm of tetramethyl-substituted label remained close to that of 1. In
addition, we verified the effect of linker on Tm of tetramethyl-substituted label and found that more
rigid NH linker leads to prolongation of phase relaxation. Both observations imply that the
interaction with biomolecule is important (as was also found by us recently in Ref.32), and this
interaction depends on the type of radical and linker used and, of course, strongly depends on the
particular biomolecule and attachment site/strategy. In our experiment with spin-labeled DNA in
trehalose, perhaps spirocyclohexane-substituted label is better immobilized compared to free 2c in
trehalose. The same happens to tetramethyl-substituted label, but only using a shorter, more rigid
linker. We cannot provide an exhaustive explanation why such a noticeable difference in Tm of
spirocyclohexane- and tetramethyl-substituted labels (920 vs. 550 ns) was found in Ref.10.
Lengthening of Tm in spirocyclohexane-substituted label [10] compared to our results on free 2-type
radicals in trehalose can possibly be ascribed to interaction of spirocyclohexyl groups with protein
hindering the NO librations. Opposite trend (shortening of Tm) observed for 1-type label [10]
compared to corresponding radical studied by us here implies higher mobility of label compared to
free radical in glassy trehalose. Regardless the exact reasons, our results clearly demonstrate that the
8

attachment to biomolecule (not the intrinsic relaxation property) is responsible for the difference in
Tm observed for two types of labels in Ref.10.
Note also that despite Tm of spirocyclohexane-substituted label attached to DNA is slightly
superior compared to that of tetramethyl-substituted one, T1 is ca. two times longer in the former
case, hindering faster sequence repetition rate and prolonging the accumulation time. In many
situations this might compensate the benefit of longer Tm for DEER measurements. At the same
time, long T1 might be useful for relaxation enhancement or saturation-recovery methods. In this
sense spirocyclopentane-substituted radicals (especially 3) are superior in comparison with
spirocyclohexane-substituted ones.
Table 2. Relaxation times measured for spin-labeled single/double-stranded DNA in trehalose at 298 K at X-band. The
samples were degassed at 3.5·10–9 bar for 24 h.
Tm / ns
850
720
T1 / s
25
13
D1-pip-spNIT/spNIT
D1-pip-NIT/NIT
D1-NH-NIT/NIT
800
13
Figure 4. Structures of spin-labeled DNA duplexes.
III. Conclusions
Room-temperature pulse EPR distance measurements in biomolecules, being a long-standing goal
since a few decades, are nowadays becoming more and more practical, owing to new types of spin
labels used and/or new approaches of their immobilization, in particular using trehalose [10]. Being
9

motivated by a recent work of Eaton and colls.[10], we investigated a series of nitroxides with
different substituents adjacent to NO-moiety as potential labels for room-temperature distance
measurements. Surprisingly, we did not find sensible advantages in room-temperature relaxation
properties of spirocyclohexane-substituted nitroxides immobilized in trehalose. At the same time,
some differences in relaxation behavior between spirocyclohexane- and tetramethyl-substituted
nitroxides arise when spin label is attached to biomolecule (in our case to 10-mer oligonucleotide).
Therefore, our study shows that predicting room-temperature Tm values of spin-labeled
biomolecules in glassy trehalose is more complicated than it was believed. In particular, specific
interactions with biomolecule and spin labeling strategy might be more crucial than intrinsic
relaxation properties of nitroxide. Another point to be considered for practical use is the stability of
nitroxides in biological media, and tetraethyl-substituted radicals might be superior in this regard
compared to tetramethyl- and spirocyclohexane-substituted ones [7, 29, 36]. Certainly, more studies
are encouraged to provide systematization of various factors influencing ultimate Tm values and
finding routes for long-range distance measurements at RT using nitroxides.
IV. Experimental Section
IV.1. Synthesis
We have synthesized 12 nitroxides 1, 2a-d, 3, 4a,b, 5a,b and 6a,b shown in Figure 1 below. The
nitroxides 1 [37], 2a [38], 2c [29], 2d [29], 3 [39], 4a [40], 4b [41], 5a [42], 5b [43] were prepared
according to the literature protocols. Reagent grade MnO2 (for catalysis) was used for oxidation.
The IR spectra were acquired on an FT-IR spectrometer in KBr pellets (the concentration was
1
13
0.25%; the pellet thickness was 1 mm). The H and C NMR spectra were recorded on for 5-10%
solutions at 300 K, using the signal of the solvent as the standard.
1,4,14,17-Tetraoxa-9-aza-21-oxotetraspiro[4.2.1.2.4.2.3.2]tetracos-9-yl-9-oxyl (2b) [44]
was prepared in two steps from acetonine and 1,4-cyclohexanedione monoethylene acetal (Scheme
1) in analogy with the general methods described in Refs.44,45.
10

Scheme 1. Synthesis of 2b.
1,4,14,17-Tetraoxa-9-azatetraspiro[4.2.1.2.413.210.38.25]tetracosan-21-one
(8).
A
mixture of acetonin (7) monohydrate (4.5 g, 29 mmol), NH4Cl (1.6 g, 29 mmol), 1,4-cyclohexanedione
monoethylene acetal (27.2 g,174.3 mmol) and DMSO (20 mL) was stirred for 48 h at 60 C under argon. The
mixture was poured into water (200 mL) and acidified with CH3COOH to pH about 6-5 and extracted with
EtOAc (10 mL  5). The water phase was basified to pH 10 using Na2CO3 and extracted with EtOAc (10 mL
 5). The extract was dried with Na2CO3 and the solvent was evaporated under reduced pressure. The residue
was purified using column chromatography on silica gel (eluent chloroform) and crystallized from ether to
give 8 (3.5 g, 35%). Mp 190-191 °C; νmax (KBr)/cm-1 3327, 3016, 2981, 2941, 2885, 2864, 1701, 1519, 1454,
1444, 1425, 1367, 1342, 1294, 1255, 1207, 1178, 1164, 1103, 1085, 1035, 1001, 981, 948, 933, 889, 862,
800, 769, 742, 665, 626, 607, 538, 486,435; 1H NMR (300 MHz; CDCl3) H (ppm) 1.57-1.69 (12H, m), 1.86
(4H, m), 2.29 (4H, s), 3.89 (8H, t, 3JH,H = 2.1 Hz); 13C NMR (75 MHz; CDCl3) C (ppm) 30.75 (CH2), 37.47
(CH2), 52.47 (C), 56.02 (CH2), 64.03 (CH2),64.05 (CH2), 108.04 (C), 210.27 (C–O). The spectral data are
very close to those reported in Ref.44.
1,4,14,17-Tetraoxa-9-aza-21-oxotetraspiro[4.2.1.2.4.2.3.2]tetracos-9-yl-9-oxyl
(2b)
was
synthesized according modified published procedure [44]. A solution of Na2WO4×2 H2O (0.3 g, 0.9 mmol)
and EDTA disodium salt (0.3 g, 0.89 mmol) in water (5 ml) was poured into a solution of amine 8 (3 g, 8.5
mmol) in methanol (25 ml). Then 30% hydrogen peroxide (5 ml) was added and the solution was stored in a
dark place at room temperature (20 °C) for 10 days. The methanol was distilled off under reduced pressure,
the residue was extracted with chloroform, the chloroform solution was washed with 5% solution of NaHSO4
and dried with Na2CO3. The solvent was distilled off under reduced pressure, the product was separated by
column chromatography with CHCl3 and recrystallized from hexane/ EtOAc to afford compound 2b as an
orange needles (2.2 g, 69%). mp 182-183 °C; max (KBr)/cm-1 2987, 2974, 2951, 2933, 2885, 2866, 1726,
1716, 1454, 1367, 1325, 1305, 1267, 1234, 1213, 1172, 1161, 1107, 1093, 1060, 1037, 1001, 979, 939, 898,
825, 754, 677, 661, 640, 528, 514, 482, 439.
The nitroxides 6a and 6b were prepared according to the Scheme 2.
Scheme 2. Synthesis of 6a,b.
11

2,2-Diethyl-3-methyl-1,4-diazaspiro[4,4]non-3-en-1-ol (10).
A
suspension of 3-
hydroxyamino-3-ethylpentan-2-one hydrochloride 9 [40] (5 g, 27.5 mmol) and ammonium acetate
(7.7 g, 100 mmol) in a mixture of cyclopentanone (10 mL, 93 mmol) and methanol (8 mL) was
stirred under argon for 24 h. The resulting solution was poured into water (20 mL), basified with
NaHCO3, and extracted with diethyl ether (3 × 10 mL). The ether layers were thoroughly washed
with water (5 × 10 mL) and dried over Na2CO3. The ether was removed under reduced pressure, the
residue was triturated with hexane at -5 °C, and the crystalline precipitate of 10 was filtered off and
washed with hexane, yielding 3.8 g, 65%, of colorless crystals, mp 111-115° C (hexane) (Found: C,
68.24; H, 10.55; N, 13.25. Calcd for C12H22N2O: C, 68.53; H, 10.54; N, 13.32); νmax (KBr)/cm-1
2957, 2874, 2845, 1649, 1466, 1452, 1433, 1387, 1375, 1254, 1213, 1121, 1103, 1040, 1009, 986,
941, and 868; δH (500 MHz; CDCl3) 0.80 (6 H, t, J = 7 Hz, 2 × CH3, Et), 1,51 and 1.65 (each 2H,
dq, Jd = 15 Hz, Jq = 7 Hz, CH2, Et ), 1.58, 1.66, 1.74 and 2.11 (each 2H, m, (CH2)4), 1.85 (3 H, s,
CH3C=N), and 5.44 (1 H, br s, OH); δC (125 MHz; CDCl3) 9.1 (CH3, Et), 16.9 (CH3-C=N), 27.0
(CH2, Et), 24.6 and 36.2 ((CH2)4), 78.0 (C5), 100.3 (C2), and 171.5 (C=N).
2,2-Diethyl-3-methyl-1,4-diazaspiro[4,4]non-3-en-1-oxyl (6b). Manganese dioxide (2 g,
23 mmol) was added to a stirred solution of 10 (1 g, 4.8 mmol) in chloroform (20 mL). The
suspension was stirred for 0.5 h, manganese oxides were filtered off, and filtrate was evaporated
under reduced pressure to leave orange liquid, which was purified by column chromatography on
silica gel, eluent diethyl ether:hexane 1:20, to yield nitroxide 6b (0.90 g, 90%), orange liquid,
(Found: C, 69.09, H, 10.09; N, 13.28. Calcd for C12H21N2O: C, 68.86; H, 10.11; N, 13.38); νmax
(neat)/cm-1 2968, 2939, 2878, 1638, 1456, 1431, 1383, 1325, 1258, 1209, 1140, 1126, 1030, 1022,
988, 939 and 540.
2,2-Diethyl-3,4-dimethyl-1,4-diazaspiro[4,4]nonan-1-oxyl (6a). Dimethyl sulfate (0.5 g, 4
mmol) was added to a solution of 6b (0.5 g, 2.4 mmol) in dry diethyl ether (3 mL). The solution
was allowed to stand for 0.5 h at 25 °C and filtered, then diethyl ether was removed under reduced
pressure. The residue was heated to 50 °C for 30 min under reduced pressure and then triturated
with dry diethyl ether to form a highly hygroscopic yellow crystalline precipitate of quaternary salt.
The precipitate was filtered off (without drying), washed with dry diethyl ether, and immediately
dissolved in ethanol (5 mL). NaBH4 (150 mg, 4 mmol) was added to the solution and the mixture
was stirred for 1 h. Ethanol was removed under reduced pressure, and the residue was dissolved in
water (2 mL) and extracted with diethyl ether (3 × 2 mL). The extract was dried over Na2CO3, then
diethyl ether was removed under reduced pressure to leave a yellow oil, which was purified by
column chromatography on silica gel, eluent diethyl ether:hexane 1:20, to yield the nitroxide 6b
(410 mg, 75%) as a yellow oil (Found: C, 69.22; H, 11.28; N, 12.43. Calcd for C13H25N2O: C,
12

68.98; H, 11.58; N, 12.38); νmax (neat)/cm-1 2966, 2941, 2874, 2845, 2791, 1458, 1433, 1383, 1321,
1252, 1230, 1167, 1123, 1093, 1066, 1038, 995, 949, 923, 885, 849, and 507.
Synthesis of spin-labeled DNA. Synthesis of DNA spin-labeled by nitroxide 1 using NH-
and piperazine type linkers is described in Ref.32. Synthesis of DNA spin-labeled by nitroxide 2c
using piperazine linker was done analogously [32], where N-hydroxysuccinimidic ether of nitroxide
2c was used for spin-labeling.
IV.2. Sample preparation
All studied nitroxides were prepared in glassy trehalose (purchased from Sigma Aldrich and used as
received) by means of lyophilization. Water solution of radical and 0.8 M trehalose in 1.5 ml
Eppendorf tube was shock-frozen in liquid nitrogen, quickly transferred to the dessiccator and dried
under vacuum for two hours. The concentration of radicals was adjusted to avoid spin-spin
interactions between neighboring radicals in the dried sample (see Supporting Information). Below
0.05 mM (in initial solution) the dependence of Tm on concentration became insignificant; therefore
such concentration was used as a tradeoff between reasonable signal intensity and small influence
on Tm.
The samples were placed into quartz tubes (OD 3.8 mm, ID 2.8 mm for X-band and OD 2.8
mm, ID 1.8 mm for Q-band) and dried in two steps in order to investigate the influence of
dehydration on relaxation times obtained. First samples were evacuated using forepump (10–3 bar)
for two hours, as mentioned above. For deeper dehydration, samples were further evacuated at RT
using turbomolecular pump (3.510–9 bar) for 24 hours. The splitter was connected directly to the
head of the TMP. Commercial vacuummeter (Pfeiffer vacuum DPG 101) was connected to one of
the outputs of the splitter. The sample tube was connected to the other output of the splitter via
vacuum hose. Such design insured that the pressure measured by vacuummeter is closely the same
as that in the sample tube, and test experiments evidenced that any leakage was directly detected by
vacuummeter.
IV.3. EPR measurements
Continuous wave (CW) EPR measurements were carried out using commercial X-band
spectrometer Bruker EMX. The CW EPR spectra were simulated using EasySpin [46]. Pulse EPR
experiments were carried out using commercial X/Q-band Bruker Elexsys E580 spectrometer
equipped with an Oxford flow helium cryostat and temperature control system. ER 4118X-MD5W
and ER 5106QT resonators were used for X- and Q-band measurements, respectively. Tm was
measured using a two-pulse electron spin echo (ESE) sequence; T1 was measured using inversion-
13

recovery technique with inversion π-pulse and detecting two-pulse ESE sequence. Unless indicated
otherwise, the -pulse lengths were 20 and 100 ns at X/Q-band, respectively.
Acknowledgement
This work was supported by Russian Science Foundation (No. 14-14-00922). Synthesis of 2b was
performed with the support of RFBR (No. 15-03-04980).
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TOC graphic
16

Highlights
Being motivated by a recent work of Eaton and colls.[ Biophys. J. 108 (2015) 1213-1219], we
investigated a series of nitroxides with different substituents adjacent to NO-moiety including
spirocyclohexane, cyclopentane, tetraethyl and tetramethyl groups as potential labels for room-
temperature distance measurements. Electron spin relaxation times (T1, Tm) were measured at
room temperature at X- and Q-bands (9/34 GHz) and a comparison was made with the
corresponding relaxation times in nitroxide-labeled DNA immobilized in trehalose. Surprisingly,
we did not find sensible advantages in room-temperature relaxation properties of
spirocyclohexane-substituted nitroxides immobilized in trehalose. Therefore, unless there are
specific interactions between spin labels and biomolecules, the room-temperature value of Tm in
trehalose is weakly dependent on the structure of substituents adjacent to NO-moiety of
nitroxide. The issues of specific interactions and stability of nitroxide labels in biological media
might be more important for room temperature pulsed dipolar EPR than differences in intrinsic
spin relaxation of radicals.
.