This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 12527–12529 12527

Cite this: Chem. Commun., 2011, 47, 12527–12529

Indirect 1H NMR characterization of H2@C60 nitroxide derivatives and

their nuclear spin relaxationw

Yongjun Li,aXuegong Lei,

aXia Li,

aRonald G. Lawler,

bYasujiro Murata,

cKoichi Komatsu

d

and Nicholas J. Turro*a

Received 18th August 2011, Accepted 5th October 2011

DOI: 10.1039/c1cc15149e

1H NMR of two H2@C60 nitroxide derivatives has been

characterized indirectly by reducing to their corresponding

hydroxylamines. Nuclear spin relaxation of the endohedral H2

and external protons of the H2@C60 nitroxide and its corresponding

hydroxylamine were measured and analyzed. The observed spectra

are consistent with negligible scalar coupling between the unpaired

electron and the endo-H2. An unexpectedly large bimolecular

relaxivity induced in the hydroxylamine by the corresponding

nitroxide can be explained by rapid hydrogen atom transfer between

the two species.

Nitroxides are stable free radicals that have been widely used

as spin labels in biological systems.1 Recently, research on

dual probe systems in which nitroxide radicals are linked to

fluorophores has attracted intense interest.2 Among them,

fullerene C60 derivatives covalently linked to a nitroxide

radical are of particular interest.3 The interaction between

C60 triplet and the radical makes it a good candidate for

chemically induced dynamic electron polarization (CIDEP)

investigated by time-resolved electron paramagnetic resonance

(EPR) spectroscopy.4,5 C60 nitroxide derivatives are commonly

characterized by mass spectroscopy and/or elemental analysis.3

Due to the paramagnetic nature of the nitroxide radical, it is

usually not possible to obtain detailed structural information by

NMR spectroscopy. It is well-known that paramagnetic nitroxide

radicals can be reduced to their corresponding diamagnetic

hydroxylamines in which NMR information can be obtained.6

However, there have been no reports applying the method

to C60 nitroxide derivatives likely due to the overlapped

NMR peaks.

We report that NMR structural information of C60 nitroxide

derivatives can be easily obtained by in situ reduction to their

corresponding hydroxylamines. We chose hydrazobenzene7

as the reducing reagent because its 1H NMR and the oxidized

product—azobenzene—are in the aromatic region, and do not

interfere with the reduced C60 nitroxide derivatives.

We chose two C60 nitroxide derivatives, 1 and 2, as shown in

Fig. 1 to demonstrate NMR characterization by reducing

them to the hydroxylamines by hydrazobenzene. Two corres-

ponding H2@C60 nitroxide derivatives, H2@1 and H2@2,

have also been synthesized. Measurements on mixtures of

the nitroxide and hydroxylamine exhibit a bimolecular relax-

ivity (R1) contribution to spin relaxation times of the external

and endohedral protons in the hydroxylamine. H2@3 was

used as a control compound.

The synthesis of 1 and 2 has been previously reported.3,8

Analogously, H2@C60 was used as the starting material for the

synthesis of H2@1, H2@2 and H2@3. The 1H NMR spectra of

1 and 2 in CDCl3 are shown in Fig. S1a (ESIw) and Fig. 2a,

respectively. Because of paramagnetic broadening of the nitroxide,

NMR signals corresponding to the protons of the functional

groups are not detected. Note that peaks at 0.9, 1.3 and 2.2 ppm

are probably from impurities from plasticizers or silicone grease as

C60 has a strong tendency to retain these materials.9

The progress of the reduction is readily followed by

monitoring the EPR spectrum of the nitroxide (Fig. S2, ESIw).In the presence of excess hydrazobenzene, CDCl3 solutions of

1 and 2 are completely reduced within 5 minutes. 1H NMR

spectra were taken (32 scans, B5 min) following the addition

and shown in Fig. S1b (ESIw) for reduced 1 and Fig. 2b for

reduced 2. Due to the rather symmetric structure of 1, a singlet

appears at 2.1 ppm from the four equivalent methyl groups,

which is consistent with the 1H NMR spectrum of its

synthetic precursor 3 (Fig. S1c, ESIw). Note that two peaks at

7.5 and 7.9 ppm are from azobenzene—the oxidative product of

hydrazobenzene. For 2, a set of new NMR peaks appear

between 1 and 5 ppm. The assignment of the NMR signals is

Fig. 1 H2@C60 nitroxide derivatives.

aDepartment of Chemistry, Columbia University, New York,NY 10027, USA. E-mail: njt3@columbia.edu

bDepartment of Chemistry, Brown University, Providence,Rhode Island 02912, USA

c Institute for Chemical Research, Kyoto University, Kyoto 611-0011,Japan

dDepartment of Environmental and Biological Chemistry,Fukui University of Technology, Gakuen, Fukui 910-8505, Japan

w Electronic supplementary information (ESI) available: 1H NMRspectra of 1, reduction profiles monitored by EPR, and analysis ofcontact shift. See DOI: 10.1039/c1cc15149e

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12528 Chem. Commun., 2011, 47, 12527–12529 This journal is c The Royal Society of Chemistry 2011

straightforward. Two singlets at 1.38 and 1.77 ppm (c) are

from two nonequivalent methyl groups on the piperidine ring.

Two doublets (b) at 2.50 (J=13Hz) and 2.65 ppm (J=13Hz)

are from the two pairs of nonequivalent geminal methylene

protons on the piperidine ring. The NMR of reduced 2 indicates

that the piperidine ring adopts a chair conformation with a

plane of symmetry axis through the spiro carbon and the N–O

group. The methylene protons of the pyrrolidine ring attached

to C60 appear as a doublet at 4.87 ppm (J = 10 Hz) due to

coupling with the N–H proton. A triplet at 3.2 ppm (J=10Hz)

is assigned to the N–H proton of the pyrrolidine ring. The

integration of each peak matches the structure. A peak at

4.4 ppm is assigned to the N–OH proton that is broadened

possibly due to slow exchange with the larger H2O signal.

By performing the reduction on H2@1 and H2@2 it is

possible to use the NMR signal from the endo-H2 to simulta-

neously monitor the nitroxide and the hydroxylamine in

partially reduced mixtures. First, we used excess hydrazo-

benzene to completely reduce the nitroxides. A single narrow

peak at �4.51 ppm for H2@1 and �4.39 ppm for H2@2

corresponding to the endohedral H2 of the reduced form

appears. It has been known that the hydroxylamine is gradually

oxidized to the nitroxide in air.10 We monitored this process by1H NMR, showing that a broader peak corresponding to the

nitroxide gradually increases in intensity with time (B5 days).

The linewidth of the endohedral H2 peak at �4.42 ppm of

H2@1 is much broader than that of the endohedral H2 peak

at �4.35 ppm of H2@2, indicating that the paramagnetic effect

by the radical in H2@1 is stronger due to the shorter distance

between the radical center and the endohedral H2 as confirmed

by earlier T1 measurements11 on H2@1 and H2@2.

The gradual conversion of the hydroxylamine to the nitroxide

enables us to compare simultaneously the NMR properties of

the nitroxide with those of the corresponding diamagnetic

hydroxylamine (Fig. 3).12 By carrying out T1 measurements on

mixtures of H2@1 and its hydroxylamine we were also able to

demonstrate a bimolecular contribution to the relaxation time,

or relaxivity, R1, of protons in the hydroxylamine. Results of

relaxivity measurements of the outside methyl protons and

inside H2 of the H2@1 reduced form demonstrate the increase

in 1/T1 with increase of the nitroxide concentration (Fig. 4).

Somewhat surprisingly the values of R1 are almost identical for

the exo (587 � 9 M�1s�1) and endo-protons (689 � 76 M�1s�1).

As a second example of the relaxivity we measured R1 for the

protons in the amine precursor H2@3 with nitroxide 1 (Fig. 5).

The corresponding values of R1 for the exo and endo-protons

are 156 � 11 M�1s�1 and 105 � 18 M�1s�1, confirming the

similarity of access of both types of protons to the external

paramagnet.

The relaxivity of the endo-H2 in H2@3 is comparable to

that observed for H2@C60 with TEMPO,13 which is itself

Fig. 2 1H NMR in CDCl3 solutions. (a) 2; (b) 2 after reduction

by hydrazobenzene; new peaks labeled with red dots; peaks from

azobenzene labeled with blue dots; hydrazobenzene peaks labeled with

green dots; (c) expanded spectrum of (b) between 2–5 ppm.

Fig. 3 1H NMR of endohedral H2 in CDCl3 solutions (a) H2@1 and

the corresponding hydroxylamine; (b) H2@2 and the corresponding

hydroxylamine at [c] = 3.34 mM.

Fig. 4 Relaxation rates of the corresponding hydroxylamine induced

by H2@1 in CDCl3.

Fig. 5 Relaxation rates of the H2@3 induced by 1 in CDCl3.

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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 12527–12529 12529

somewhat larger than expected on the basis of the diffusion

coefficients and most likely distance of closest approach of the

radical and fullerene. It is therefore surprising to find that the

relaxivities of both types of protons in the hydroxylamine of

H2@1 are increased relative to H2@3 by nearly a factor of

five, putting them outside the range of values attainable by

reasonable estimates of diffusion coefficients and distances of

approach. We offer as an explanation for the accelerated

relaxivity an additional contribution to the observed relaxation

rate from the limitation of the lifetime of H2 in the hydroxylamine,

in the slow exchange limit,14 due to exchange between the radical

and hydroxylamine by hydrogen atom transfer (HAT)

*RNOH + RNO = *RNO + RNOH

where the star (*) simply indicates molecules containing the

nucleus of interest that would be affected by the HAT process.

This reaction has been shown to occur with other hydroxyl-

amine/nitroxide pairs15 and may be sufficiently fast under some

conditions to produce line broadening in the hydroxylamine.16

Although analogous broadening of the RNOH peaks was

too small to be measured, the effect on T1 may be described in

terms of a classic chemical exchange between two sites with

different T1’s. In this case the T1 of the peak of interest,

RNOH, is ca. 8 times as long as that of the RNO peak,

corresponding to 1/T1 values for the endo-H2 of 6.5 s�1 and

50 s�1, respectively. The corresponding 1/T1 value for the

methyl group of RNOH is 1.4 s�1, and that for the RNO

methyl group is presumably much larger because of the

expected much larger dipolar and scalar interactions between

the methyl protons and unpaired electron in H2@1. This is

consistent with the fact that the corresponding peak is too

broad to be seen by high resolution NMR (Fig. S1a, ESIw).Under these conditions, following the inversion pulse of the

T1 measurement procedure, the RNOH peak exchanges

magnetization with an almost fully relaxed RNO peak. It is

easily shown14 that in this case the recovery of the RNOH

peak is essentially exponential with an effective relaxation rate

given by

1/T1 = 1/T10 + R1,dd [RNO] + kHAT[RNO]

where 1/T10 is the relaxation rate when the concentration

[RNO] is zero; R1,dd, is the intermolecular dipolar contribution

that may be estimated to be the value for H2@3.

Using the above method, the resulting second order rate

constants, kHAT, for the endo and exo-protons of the H2@1

hydroxylamine, obtained by subtracting the corresponding

values of R1 for H2@3 from those for H2@1, are estimated

to be 584 � 78 and 431 � 14 M�1s�1, respectively (Fig. 4 and 5;

error estimate 1 standard deviation). The values are statistically

equal, as expected for protons in the same exchanging species.

The values themselves are near the upper range of values for

such HAT reactions15 and are similar to those obtained when

an intermediate complex has been postulated.16 The presence of

a complex in the present case would also be consistent with the

unusually large dipolar contribution to the relaxivity between

nitroxides and endofullerenes.13 Such complexation is commonly

invoked to explain enhanced relaxivity between paramagnetic

metal ions and diamagnet substrates.17 In the present case,

however, it is not supported by a model calculation13 of the

potential energy between a nitroxide and a C60 molecule in

the gas phase which predicted a purely repulsive interaction as

the molecules approach each other. Further theoretical, and

refined experimental study of both the uni- and bimolecular

relaxation processes involved here would clearly be desirable.

In summary, we have demonstrated that 1H NMR structural

information of C60 nitroxide derivatives are obtained indirectly

by reduction in situ to the corresponding hydroxylamine by

hydrazobenzene. The quantitative reduction reaction enables us

to identify and assign all NMR peaks. Due to simplicity and

convenience, the present method would be very useful for

characterization of newly synthesized C60 nitroxide derivatives.

We have also demonstrated the use of the endo-H2 signal from

the H2@fullerene to simultaneously monitor and study the

relaxation of mixtures of the nitroxides and hydroxylamines.

The authors thank the National Science Foundation for its

generous support through Grant CHE 07-17518.

Notes and references

1 P. P. Borbat, A. J. Costa-Filho, K. A. Earle, J. K. Moscicki andJ. H. Freed, Science, 2001, 291, 266.

2 J. P. Blinco, K. E. Fairfull-Smith, B. J. Morrow and S. E. Bottle,Aust. J. Chem., 2011, 64, 373.

3 F. Arena, F. Bullo, F. Conti, C. Corvaja, M. Maggini, M. Pratoand G. Scorrano, J. Am. Chem. Soc., 1997, 119, 789.

4 C. Corvaja, M. Maggini, M. Prato, G. Scorrano and M. Venzin,J. Am. Chem. Soc., 1995, 117, 8857.

5 E. Sartori, A. Toffoletti, C. Corvaja and L. Garlaschelli, J. Phys.Chem. A, 2001, 105, 10776.

6 T. D. Lee and J. F. W. Keana, J. Org. Chem., 1975, 40, 3145.7 A. D. Malievskii and A. B. Shapiro, Kinet. Catal., 2005, 46, 472.8 M. Mazzoni, L. Franco, A. Ferrarini, C. Corvaja, G. Zordan,G. Scorrano and M. Maggini, Liq. Cryst., 2002, 29, 203.

9 J. Nossal, R. K. Saini, L. B. Alemany, M. Meier and W. E. Billups,Eur. J. Org. Chem., 2001, 4167.

10 A. A. Bobko, I. A. Kirilyuk, I. A. Grigor’ev, J. L. Zweier andV. V. Khramtsov, Free Radical Biol. Med., 2007, 42, 404.

11 Y. Li, X. Lei, R. G. Lawler, Y. Murata, K. Komatsu andN. J. Turro, J. Phys. Chem. Lett., 2010, 1, 2135.

12 Preliminary analysis (ESIw) of the shifts and widths of the endo H2

peaks in Fig. 3 indicates that there is no detectable contactinteraction between the protons and unpaired electron and a negligiblecontribution of scalar relaxation to the nitroxide linewidths. Theobserved chemical shift difference between the endo-H2 in the nitroxideand hydroxylamine must therefore arise primarily from differences inshielding of the nuclei in the slightly different electronic environments.

13 E. Sartori, M. Ruzzi, N. J. Turro, K. Komatsu, Y. Murata,R. G. Lawler and A. L. Buchachenko, J. Am. Chem. Soc., 2008,130, 2221.

14 J. Schotland and J. S. Leigh, J. Magn. Reson., 1983, 51, 48.15 A. Wu, E. A. Mader, A. Datta, D. A. Hrovat, W. T. Borden and

J. M. Mayer, J. Am. Chem. Soc., 2009, 131, 11985.16 R. W. Kreilick and S. I. Weissman, J. Am. Chem. Soc., 1966,

88, 2645.17 L. Banci, I. Bertini and C. Luchinat, Nuclear and Electronic

Relaxation, VCH, Weinheim, Germany, 1991.

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