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org/joc

Synthesis of 14N- and 15N-labeled Trityl-nitroxide Biradicals with Strong

Spin-Spin Interaction and Improved Sensitivity to Redox Status and Oxygen
Yangping Liu,† Frederick A. Villamena,†,‡ Yuguang Song,† Jian Sun,† Antal Rockenbauer,§
and Jay L. Zweier*,†
†
Center for Biomedical EPR Spectroscopy and Imaging, The Davis Heart and Lung Research Institute, the
Division of Cardiovascular Medicine, Department of Internal Medicine, and ‡Department of Pharmacology,
College of Medicine, The Ohio State University, Columbus, Ohio 43210, United States, and §Chemical
Research Center, Institute of Structural Chemistry, P.O. Box 17, H-1525 Budapest, Hungary

[email protected]
Received August 26, 2010

Simultaneous evaluation of redox status and oxygenation in biological systems is of great importance for
the understanding of biological functions. Electron paramagnetic resonance (EPR) spectroscopy coupled
with the use of the nitroxide radicals have been an indispensable technique for this application but are still
limited by low oxygen sensitivity and low EPR resolution in part due to the moderately broad EPR triplet
and spin quenching through bioreduction. In this study, we showed that these drawbacks can be overcome
through the use of trityl-nitroxide biradicals allowing for the simultaneous measurement of redox status
and oxygenation. A new trityl-nitroxide biradical TNN14 composed of a pyrrolidinyl-nitroxide and a trityl
and its isotopically labeled 15N analogue TNN15 were synthesized and characterized. Both biradicals
exhibited much stronger spin-spin interaction with J > 400 G compared with that of the previous
synthesized trityl-nitroxide biradicals TN1 (∼160 G) and TN2 (∼52 G) with longer linker chain length.
The enhanced stability of TNN14 was evaluated using ascorbate as reductant, and the effect of different
types of cyclodextrins on its stability in the presence of ascorbate was also investigated. Both biradicals are
sensitive to redox status, and their corresponding trityl-hydroxylamines resulting from the reduction of the
biradicals by ascorbate share the same oxygen sensitivity. Of note is that the 15N-labeled TNN15-H with an
EPR doublet exhibits improved EPR signal amplitude as compared with that of TNN14-H with an EPR
triplet. In addition, cyclic voltammetric studies verify the characteristic electrochemical behaviors of the
trityl-nitroxide biradicals.

Introduction redox assessment because of the reasonable depth of mag-

netic field penetration in animal tissues.1-4 In principle, EPR
Noninvasive measurement and monitoring of molecular
oxygen (O2) or “oximetry” and redox status are of impor-
tance for the understanding of biological functions, and the (1) Berliner, L. J.; Fujii, H.; Wan, X. M.; Lukiewicz, S. J. Magn. Reson.
Med. 1987, 4, 380–384.
pathogenesis of various diseases. Nuclear magnetic reso- (2) Zweier, J. L.; Kuppusamy, P. Proc. Natl. Acad. Sci. U.S.A. 1988, 85,
nance (NMR)- and low field electron paramagnetic reso- 5703–5707.
nance (EPR)-based techniques, coupled with the use of (3) Grucker, D. Prog. Nucl. Magn. Reson. Spectrosc. 2000, 36, 241–270.
(4) Hyodo, F.; Soule, B. P.; Matsumoto, K. I.; Matusmoto, S.; Cook,
exogenous paramagnetic probes, are the most appropriate J. A.; Hyodo, E.; Sowers, A. L.; Krishna, M. C.; Mitchell, J. B. J. Pharm.
approaches for the noninvasive in vivo O2 measurement and Pharmacol. 2008, 60, 1049–1060.

7796 J. Org. Chem. 2010, 75, 7796–7802 Published on Web 10/28/2010 DOI: 10.1021/jo1016844
r 2010 American Chemical Society
Liu et al.
JOC Article
CHART 1. Molecular Strutures of Biradicals and Nitroxide 3-AP

methods (both spectroscopic and imaging) show better superoxide radical anion,24 and pH.18,25 However, their
sensitivity to O2 and redox measurement compared to application as redox probes has been a challenge due to their
NMR methods since the former have a much higher intrinsic inertness to biological reductants such as ascorbate and
sensitivity to exogenous probe concentration. EPR imaging glutathione.16,24
has been widely used to measure oxygenation and redox Recently, we described a novel strategy using trityl-nitr-
status in living tissue and in vivo in mice.5-8 Recently, a oxide biradicals to simultaneously measure redox status and
new MRI-based technique, commonly called Overhauser- oxygenation through EPR spectrocopy.26 As redox and O2
enhanced MRI (OMRI), which combines the sensitivity of probes, these biradicals possess several advantages com-
EPR with the advantage of MRI, has been employed for the pared to nitroxide radicals: (1) Their bioreduction results
measurement of tissue oxygenation and redox status.9-11 in the formation of the trityl-hydroxylamine monoradical
Among the commonly used paramagnetic probes, nitro- whose signal intensity is enhanced, therefore effectively
xide radicals are the most popular, but their application increasing the EPR signal and secondary sensitivity. In
in vivo was greatly limited by their rapid bioreduction and contrast, the EPR signals of simple nitroxides are completely
their relatively low sensitivity to O2. Although the informa- quenched by bioreduction. (2) The resulting trityl radical
tion on the redox status was extracted from the bioreduction from bioreduction has narrow singlet EPR line and high
of the nitroxides in the investigated systems by EPR imaging stability, which can further increase quality and resolution of
or OMRI, this spin quenching mechanism greatly limits the EPR imaging in less time. (3) Introduction of an amide group
imaging resolution. Recent development of extraordinary into the trityl molecule affords a partial overlapping sharp
stable trityl radicals has tremendous benefits in EPR imaging triplet EPR signal that exhibits a higher O2 sensitivity as
and OMRI.12-21 To date, trityl radicals have been utilized to compared with that of the usual trityl radical with a singlet
measure extracellular22 and intracellular17,23 oxygen level, EPR signal or the nitroxide radicals. As part of our continu-
ing efforts in trityl-nitroxide biradical development, we here-
(5) He, G. L.; Shankar, R. A.; Chzhan, M.; Samouilov, A.; Kuppusamy,
in report the synthesis of a new trityl-nitroxide biradical
P.; Zweier, J. L. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4586–4591. TNN14 and its isotopically labeled analogue TNN15 (Chart 1)
(6) Kuppusamy, P.; Li, H. Q.; Ilangovan, G.; Cardounel, A. J.; Zweier, J. L.; with a short linker group. These two biradicals exhibit higher
Yamada, K.; Krishna, M. C.; Mitchell, J. B. Cancer Res. 2002, 62, 307–312.
(7) Elas, M.; Williams, B. B.; Parasca, A.; Mailer, C.; Pelizzari, C. A.; sensitivity for the simultaneous measurement of redox status
Lewis, M. A.; River, J. N.; Karczmar, G. S.; Barth, E. D.; Halpern, H. J. and oxygenation as compared to the prior trityl-nitroxide
Magn. Reson. Med. 2003, 49, 682–691. biradicals with longer linker group. In addition, their electro-
(8) Gallez, B.; Swartz, H. M. NMR Biomed. 2004, 17, 223–225.
(9) Li, H. H.; Deng, Y. M.; He, G. L.; Kuppusamy, P.; Lurie, D. J.; chemical properties were also investigated.
Zweier, J. L. Magn. Reson. Med. 2002, 48, 530–534.
(10) Li, H. H.; He, G. L.; Deng, Y. M.; Kuppusamy, P.; Zweier, J. L. Results and Discussion
Magn. Reson. Med. 2006, 55, 669–675.
(11) Utsumi, H.; Yamada, K.; Ichikawa, K.; Sakai, K.; Kinoshita, Y.;
Matsumoto, S.; Nagai, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1463–1468. The synthesis of the biradical TNN14 and its analogue
(12) Ardenkjaer-Larsen, J. H.; Laursen, I.; Leunbach, I.; Ehnholm, G.; TNN15 is shown in Scheme 1. While the nitroxide radical
Wistrand, L. G.; Petersson, J. S.; Golman, K. J. Magn. Reson. 1998, 133, 1–12. 14
NN (also called 3-amino-PROXYL) is commercially avail-
(13) Reddy, T. J.; Iwama, T.; Halpern, H. J.; Rawal, V. H. J. Org. Chem.
2002, 67, 4635–4639. able, its isotopical analogue 15NN was synthesized using
(14) Xia, S. J.; Villamena, F. A.; Hadad, C. M.; Kuppusamy, P.; Li, Y. B.; 3-carboxy-proxyl (3-CP) as a starting material according to
Zhu, H.; Zweier, J. L. J. Org. Chem. 2006, 71, 7268–7279. a previous procedure.27 3-CaP was obtained by conjugation of
(15) Dhimitruka, I.; Velayutham, M.; Bobko, A. A.; Khramtsov, V. V.;
Villamena, F. A.; Hadad, C. M.; Zweier, J. L. Bioorg. Med. Chem. Lett. 2007, 3-CP with HOSu, followed by amidation using 15NH4Cl.
17, 6801–6805. Subsequent treatment of 3-CaP with sodium hypobromite at
(16) Liu, Y.; Villamena, F. A.; Zweier, J. L. Chem. Commun. 2008, 4336–
4338. 0-70 °C for 3 h affords the 15N-labeled nitroxide, 15NN, with a
(17) Liu, Y.; Villamena, F. A.; Sun, J.; Xu, Y.; Dhimitruka, I.; Zweier, high yield of 52% from 3-CP. The trityl radical CT-03 was
J. L. J. Org. Chem. 2008, 73, 1490–1497. synthesized via the previously reported 4-step method15 and
(18) Dhimitruka, I.; Bobko, A. A.; Hadad, C. M.; Zweier, J. L.; Khramtsov,
V. V. J. Am. Chem. Soc. 2008, 130, 10780–10787. linked with 14NN or 15NN, to afford the biradicals TNN14 or
(19) Driesschaert, B.; Charlier, N.; Gallez, B.; Marchand-Brynaert, J.
Bioorg. Med. Chem. Lett. 2008, 18, 4291–4293.
(20) Dhimitruka, I.; Grigorieva, O.; Zweier, J. L.; Khramtsov, V. V. (24) Rizzi, C.; Samouilov, A.; Kutala, V. K.; Parinandi, N. L.; Zweier,
Bioorg. Med. Chem. Lett. 2010, 132, 3946–3949. J. L.; Kuppusamy, P. Free Radic. Biol. Med. 2003, 35, 1608–1618.
(21) Bobko, A. A.; Dhimitruka, I.; Eubank, T. D.; Marsh, C. B.; Zweier, (25) Bobko, A. A.; Dhimitruka, I.; Zweier, J. L.; Khramtsov, V. V. J. Am.
J. L.; Khramtsov, V. V. Free Radical Biol. Med. 2009, 47, 654–658. Chem. Soc. 2007, 129, 7240–7241.
(22) Kutala, V. K.; Parinandi, N. L.; Pandian, R. P.; Kuppusamy, P. (26) Liu, Y. P.; Villamena, F. A.; Rockenbauer, A.; Zweier, J. L. Chem.
Antioxid. Redox Signaling 2004, 6, 597–603. Commun. 2010, 46, 628–630.
(23) Liu, Y. P.; Villamena, F. A.; Sun, J.; Wang, T. Y.; Zweier, J. L. Free (27) Rozantsev, E. G. Free Nitroxide Radicals; Plenum Press: New York,
Radic. Biol. Med. 2009, 46, 876–883. 1970.

J. Org. Chem. Vol. 75, No. 22, 2010 7797

JOC Article Liu et al.

SCHEME 1. Synthesis of Biradicals TNN14 and TNN15

TNN15, which were characterized by HRMS and IR. The The potential application of the new biradicals as redox
purity of both biradicals was determined by HPLC (>98%) probes was then evaluated using ascorbate, which is the main
and EPR method17 (97 ( 1% for TNN14 and 95 ( 1% for reductant of nitroxides in biological systems. The reduction
TNN15) (see Supporting Information). Interestingly, two close of TNN14 by ascorbate resulted in the formation of the
peaks were observed in their HPLC chromatograms indicating monoradical trityl-hydroxylamine (TNN14-H) and afforded
two components that were further determined to be two isomers an intense partially overlapped EPR triplet signal (Figure 2A
by MS analysis (see Supporting Information). These two and inset). The formation of the signal was accompanied by a
isomers are possibly diastereomers associated with the inherent relatively slow decrease of the biradical signal due to the
chirality of the trityl moiety and C-3 of the nitroxide. transformation of TNN14 into TNN14-H. The unchanged
Figure 1 shows the room-temperature EPR spectra of UV absorbance around 469 nm proved that the trityl moiety
TNN14 and TNN15. Completely identical spectral profiles is stable toward ascorbate (see Supporting Information).
for both biradicals were observed, indicating that the iso- Addition of the oxidant K3Fe(CN)6 resulted in the recovery
topic labeling in the amide group did not affect the magni- of the signal due to TNN14 from TNN14-H (see Supporting
tude of spin-spin interaction between the two radical Information), suggesting the reversibility of the reduction of the
moieties. EPR spectra for both biradicals present a well- nitroxide moiety. Figure 2B shows the effect of the ascorbate
resolved triplet with the lines spaced by ∼7.9 G, slightly less concentration on the production of TNN14-H. Higher concen-
than half the 14N hyperfine splitting of the five-membered trations of ascorbate led to a faster increase in the signal inten-
ring nitroxide 3-AP (16.1 G, Chart 1) in aqueous solution. sity of the singlet peak and vice versa. According to the data
Interestingly, the EPR triplet lines have almost the same line shown in Figure 2B, the second order rate constant (k2) for the
width and amplitude owing to the strong intramolecular TNN14 reduction by ascorbate was determined to be 0.44 (
spin-spin interaction. EPR spectral profile of TNN14 is not 0.07 M-1 s-1. A similar k2 value was observed for the nitroxide
affected at a concentration range of 10-500 μM (see Figure S3 3-AP (Chart. 1) (0.36 ( 0.06 M-1 s-1, see Supporting Informa-
in Supporting Information), further confirming the intramole- tion) which indicates that linkage of the nitroxide to the trityl
cular nature of the spin interaction. Computer simulation28 of does not significantly affect the reactivity of the nitroxide
these spectra gave a J-coupling value of >400 G for TNN14 moiety. Moreover, TNN14 has a much smaller k2 compared
and TNN15 at room temperature, which is much higher than with those of the piperidinyl nitroxide-trityl biradicals TN1
the values of the previously reported biradicals, TN1 (∼160 G) (4.14 ( 0.14 M-1 s-1) and TN2 (3.48 ( 0.09 M-1 s-1).9 This
and TN2 (∼52 G).26 The high J-coupling values for TNN14 result is consistent with the previous work showing the lower
and TNN15 are most likely due to the short distance between rates of reduction for the pyrrolidinyl nitroxide compared to
the NO moiety and the central carbon of the trityl radical the piperidinyl nitroxide due to the conformational flexibility of
compared to those of TN1 and TN2. the latter compared to the former.29
Although the nitroxide bioreduction provides important
information on the redox state in a given biological system,
the bioreduction is often too fast to be monitored. In this
study, the use of five-membered ring nitroxide for the con-
struction of the trityl-nitroxide biradicals effectively slows
their reduction by ascorbate as mentioned above. In order to
further stabilize the biradicals and enhance their in vivo applica-
tion potential, we investigated the effect of three different types of
cyclodextrins (CDs) on the stability of the biradicals on the basis
of the knowledge that CDs can effectively protect nitroxide
radicals30-32 and nitrone spin adducts33-35 from bioreduction.

(28) Rockenbauer, A.; Korecz, L. Appl. Magn. Reson. 1996, 10, 29–43.
(29) Couet, W. R.; Brasch, R. C.; Sosnovsky, G.; Lukszo, J.; Prakash, I.;
FIGURE 1. Room-temperature X-band EPR spectra of TNN14 Gnewuch, C. T.; Tozer, T. N. Tetrahedron 1985, 41, 1165–1172.
and TNN15 in PBS (pH 7.4, 50 mM). Spectra were recorded with (30) Ohara, M.; Hettler, H.; Gauss, D.; Cramer, F. Bioorg. Chem. 1979, 8,
10 mW microwave power and 1 G modulation amplitude. 211–217.

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FIGURE 2. (A) EPR spectrum obtained from the reaction mixture containing TNN14 (50 μM) and ascorbate (4 mM) in PBS (pH 7.4, 50 mM)
after 45 s at room temperature; (v) indicates the increasing signal of the trityl monoradical TNN14-H that resulted from the reduction of the
biradical TNN14; (V) shows the decreasing signal of the biradical TNN14; the inset shows an expanded portion of the spectrum of the trityl
monoradical to better visualize its triplet hyperfine structure. To detect signals from both the biradical and the resulting trityl radical without
any distortion, spectra were recorded with 1 mW microwave power and 0.08 G modulation amplitude. These low values of microwave power
and modulation amplitude are required to prevent broadening or saturation of the trityl radical spectrum. (B) Plots of the TNN14-H
concentration as a function of time at various ascorbate concentrations at room temperature.

FIGURE 3. Effect of various cyclodextrins (2 mM) on the reduc- FIGURE 4. X-band EPR spectra of TNN14-H, TNN15-H, and
tion of TNN14 (50 μM) and ascorbate (4 mM) in PBS. CT03 in PBS (pH 7.4, 50 mM) under anaerobic conditions. Gray
lines show the enlarged (5
) portion of the spectrum. Spectra were
recorded with 0.5 mW microwave power and 0.03 G modulation
As shown in Figure 3, β-CD and its methylated analogue amplitude. These low values were required to prevent distortion of
the very sharp trityl radical spectra detected under anaerobic
(M-β-CD) effectively decrease the reduction of TNN14 by conditions.
ascorbate as evidenced by the slower formation of the trityl
signal intensity in the solution of TNN14 containing ascorbate. offer better strategy for more stable biradicals with longer half-
Comparatively, γ-CD enhances the reaction between TNN14 lives in biological milleiu.
and ascorbate with 148% higher signal intensity in the presence Under anaerobic condition, the partially overlapped EPR
of γ-CD than in the absence of γ-CD. The larger cavity size of triplet signal of TNN14-H (Figure 2A) becomes more pro-
γ-CD compared to β-CD could accommodate both the biradi- nounced (Figure 4, top) with a hyperfine splitting constant
cal, (or at least the nitroxide moiety), and ascorbate, therefore (hfc) of 0.23 G due to reduced Heisenberg exchange between
facilitating their reaction. Although high concentrations of TNN14-H and paramagnetic O2. Comparatively, the trityl-
β-CDs were used to provide protection to biradicals in this hydroxylamine TNN15-H obtained through reduction of
study, the covalent linkage of biradicals with methyl-β-CDs may TNN15 by ascorbate gave a well-resolved doublet with a
hfc of 0.33 G under anaerobic condition (Figure 4). These
additional hyperfine splittings observed from the trityl-hydro-
(31) Ebel, C.; Ingold, K. U.; Michon, J.; Rassat, A. Tetrahedron Lett.
1985, 26, 741–744. xylamines should be due to the amide-N (I=1 for 14N and I=1/
(32) Franchi, P.; Fani, M.; Mezzina, E.; Lucarini, M. Org. Lett. 2008, 10, 2 for 15N) of the linker group, verifying our previous theoretical
1901–1904.
(33) Karoui, H.; Rockenbauer, A.; Pietri, S.; Tordo, P. Chem. Commun.
and experimental results showing long-range radical coupling.26
2002, 3030–3031. Apart from the hyperfine interactions with the main nitrogen
(34) Han, Y. B.; Tuccio, B.; Lauricella, R.; Villamena, F. A. J. Org. Chem. isotope (i.e., 14N for TNN14-H and 15N for TNN15-H), the
2008, 73, 7108–7117.
(35) Han, Y. B.; Liu, Y. P.; Rockenbauer, A.; Zweier, J. L.; Durand, G.; interactions with the 13C (natural abundance, 1.11%) mainly
Villamena, F. A. J. Org. Chem. 2009, 74, 5369–5380. from the carbons of three aromatic groups and the other
J. Org. Chem. Vol. 75, No. 22, 2010 7799
JOC Article Liu et al.

FIGURE 5. (A) EPR spectra of TNN14-H and TNN15-H denoting the spectral ratios Iin and Iout in the presence of 10% O2-90% N2. Spectra
were recorded with 0.5 mW microwave power and 0.03 G modulation amplitude. (B) Plot of Iin/Iout as a function of percent oxygen. The trityl-
hydroxylamines were generated by mixing ascorbate (4 mM) with the corresponding biradical (50 μM).

nitrogen isotope (natural abundance of 15N (0.37%) for

TNN14-H and 14N (1%) for TNN15-H in our experiment)
can be also observed, giving the weak flanking lines as shown in
Figure 4.36 Interestingly, the direct linkage of the amide group
with the trityl moiety can further split these signals from CT-03
into a doublet for TNN15-H or a triplet for TNN14-H (see the
enlarged signals in Figure 4).
It has been demonstrated that the partially overlapped
EPR peaks of the trityl radicals are sensitive to O2 and the
spectral ratio (Iin/Iout) exhibits enhanced sensitivity toward O2,
especially at low pO2, relative to the line width.21,26 It is therefore
reasonable to use the ratio Iin/Iout to evaluate the oxygen
sensitivity of TNN14-H and TNN15-H. Figure 5A shows the
EPR spectra of TNN14-H and TNN15-H, which are denoted by
the spectral intensities, Iin and Iout. As shown in Figure 5B, the FIGURE 6. Cyclic voltammograms of TNN14 (1 mM) and TN1
spectral ratio Iin/Iout is highly sensitive to O2 and both radicals (1 mM) in PBS (20 mM, pH 7.4) containing 0.15 M NaCl. Scan
have almost identical O2 sensitivities with values of 1.55
rate: 100 mV/s.
10-3/%O2 for TNN14-H and 1.53
10-3/%O2 for TNN15-H.
Although the isotopic 15N labeling does not improve the oxygen the above result has showed that the biradical TNN14 can
sensitivity of TNN15-H, TNN15-H has 3/2=1.5 times larger undergo one-electron reduction by ascorbate to the correspond-
EPR signal amplitude than TNN14-H at the same concentra- ing trityl-hydroxylamine. Compared to the redox potential of
tions due to the reduced number of hyperfine lines (Figure 5A). the parent trityl CT-03 [E1/2 (ox)=0.45 V vs Ag/AgCl and E1/2
Therefore, the isotopic 15N labeling of the biradical can greatly (red) =-0.63 V vs Ag/AgCl],16 the trityl moiety in TNN14 is
improve its sensitivity and resolution for simultaneous measure- more difficultly oxidized [E1/2 (ox)=0.515 V vs Ag/AgCl] but
ment of redox status and oxygenation as compared to nitroxides easier to reduce [E1/2 (red)=-0.581 V vs Ag/AgCl] because one
and trityl radicals alone as well as the nonlabeled trityl-nitroxide of carboxylate groups in CT-03 is replaced by a stronger
biradicals, TN1, TN2, and TNN14. electron-withdrawing amide group in the case of TNN14. Cyclic
It has been demonstrated that as neutral radicals, both trityl voltammetric studies were also carried out on the biradical TN1.
and nitroxide radicals can be either reduced or oxidized via a The replacement of the pyrrolidinyl nitroxide in TNN14 with a
one-electron process. It is thus interesting to investigate the piperidinyl nitroxide makes the first oxidation (E1/2 =0.531 V)
electrochemical behavior of the trityl-nitroxide biradicals. In and reduction (E1/2=-0.587 V) slightly more difficult but easier
doing so, cyclic voltammeric studies were carried out on the for the second oxidation (E1/2=0.655 V).
biradical TNN14. As shown in Figure 6, TNN14 undergoes two In summary, the newly synthesized trityl-nitroxide bira-
one-electron reversible oxidations of TNN14 to the correspond- dical TNN14 and its 15N-labeled analogue TNN15 possess
ing trityl cation and oxammonium at E1/2 (ox)=0.515 V vs Ag/ strong intramolecular spin-spin interaction (J > 400 G) due
AgCl (ΔEp=64 mV) and 0.686 V vs Ag/AgCl (ΔEp=61 mV), to the short distance between the two radical centers. Both
respectively. A quasi-reversible one-electron reduction at E1/2 biradicals are sensitive to the variation of the redox status.
(red) = -0.581 V vs Ag/AgCl (ΔEp = 113 mV) was also ob- The use of five-membered ring nitroxide for the construction
served, which was assigned to the reduction of the trityl moiety of the trityl-nitroxide biradicals as well as exogenous addi-
to the corresponding anion. Under our experimental conditions, tion of β-CD and its methylated analogue M-β-CD effec-
the reduction of the nitroxide moiety was not observed, although tively increases the stability of these two biradicals toward
the reductant ascorbate, therefore enhancing their suitability
(36) Bowman, M. K.; Mailer, C.; Halpern, H. J. J. Magn. Reson. 2005, for in vivo application. While both trityl-hydroxylamines
172, 254–267. TNN14-H and TNN15-H resulting from the reduction of the
7800 J. Org. Chem. Vol. 75, No. 22, 2010
Liu et al.
JOC Article
corresponding biradicals show high oxygen sensitivity, the DIPEA (2.49 mL, 14.3 mmol), and N-hydroxysuccinimide
15
N-labeled TNN15-H with its doublet EPR signal exhibits (0.66 g, 5.73 mmol) in DMF (20 mL) was added a solution of
improved EPR signal resolution compared with that of EDCI (1.10 g, 5.73 mmol) in DMF (5 mL) at 0 °C. After
TNN14-H with its EPR triplet. Therefore the new probes addition, the reaction mixture was stirred for 18 h at room
also exhibit enhanced sensitivity for simultaneous measure- temperature. Solvents were removed under vacuum, and the
residue was purified by flash column chromatography using the
ment of redox status and oxygenation compared to either
solvents ethyl acetate/petroleum ether (1:3, 1:2, and then 1:1 v/v) as
nitroxide or trityl radicals alone. eluents. A light yellowish solid (0.71 g) was obtained. Yield: 88%.
IR (cm-1, neat): 2978, 2937, 1810, 1783, 1735, 1464, 1427, 1366,
Experimental Section
1304, 1245, 1200, 1092, 1065, 1047, 994, 958, 920, 899, 813, 735,
EPR Measurements and Simulations. EPR measurements 647. GC-MS: retention time, 5.90 min; [M - C4H4NO3 þ H]þ,
were carried out on a Bruker EMX-X band with an HS 186.11(calculated), 186.00 (measured); [M - C9H15NO3 þ H]þ,
resonator at room temperature. General instrument settings 115.03 (calculated), 114.94 (measured).
were as follows: modulation frequency, 100 kHz; microwave 3-Carbamoyl (15N)-2,2,5,5-Tetramethylpyrrolidin-1-yloxy (3-
frequency, 9.87 GHz; microwave power, 10 mW for biradicals CaP). To a solution of 3-CP-OSu (0.5 g, 1.77 mmol) and K2CO3
and nitroxide radicals and 0.5-2 mW for the trityl-hydroxyla- (0.3 g) in dioxane (5 mL) and water (1 mL) was added solid
15
mines; microwave frequency, 9.87 GHz; modulation amplitude, NH4Cl (99% isotopic purity, 0.106 g, 1.94 mmol). The reac-
1.0 G for biradicals and nitroxide radicals and 0.03-0.08 G for tion mixture was stirred for 22 h at room temperature, filtered,
the trityl-hydroxylamines; receiver gain, (1-10.00)
104; time and evaporated to dryness under vacuum. The residue was
constant, 10.24-40.96 ms; sweep time, 10.49-41.94 s. Measure- redissolved in 10 mL of ethyl acetate. After filtration, the filtrate
ments were performed using 50 μL capillary tubes. was concentrated and separated by flash column chromatogra-
Simulations were carried out using a well-developed EPR phy using 2% methanol in dichlomethane as an eluent. The
simulation program (ROKI\EPR).28 The fitting routine to deter- yellow solid was obtained and recrystallized from hexane and
mine the J values of the trityl-nitroxide biradicals was similar to the ethyl acetate to give the pure 3-Cap (0.31 g, 94%). IR (cm-1,
method described in our previous study.26 Since we could not find neat): 3346, 3195, 2977, 2934, 1668, 1462, 1426, 1365, 1321,
any improvement about the quadratic error between experimental 1283, 1244, 1171, 1154, 1102, 830, 739. GC-MS: retention time,
and calculated spectra when J was larger than 400 G, the J values 7.22 min; [M]þ, 186.13 (calculated), 185.99 (measured).
for TNN14 and TNN15 were suggested to be >400 G. 3-Amino (15N)-2,2,5,5-Tetramethyl-1-pyrrolidin-1-yloxy (15NN).
Oxygen Sensitivity. Oxygen sensitivities of the trityl-hydro- To a solution of sodium hypobromite prepared from 0.36 g of
xylamine TNN14-H and TNN15-H were evaluated according to NaOH (9 mmol), 4 mL of water, and 0.34 g of bromide (2.13
our previous method. In brief, ascorbic acid (4 mM) was added mmol) was added 0.28 g of 3-CaP (1.50 mmol) at 0 °C. The
to a solution of the biradicals (50 μM) in PBS buffer. After 60 reaction mixture was stirred for 2 h at 0 °C, heated to 70 °C, and
min, the solution was transferred into a gas-permeable Teflon kept this temperature for 1 h. The reaction mixture was cooled with
tube (i.d. = 0.8 mm) and was sealed at both ends. The sealed the ice bath, treated with 6 g KOH, and extracted with ether. The
sample was placed inside a quartz EPR tube with open ends. extract was dried on anhydrous Na2SO4, concentrated, and sepa-
Nitrogen or N2/O2 gas mixture with varying concentrations of rated by flash column chromatography using CH2Cl2/CH3OH/
O2 was allowed to bleed into the EPR tube and after about 4 min NH4OH = 10:1:0.05 as an eluent. A yellow solid (0.15 g) was
was changed into another gas mixture. EPR spectra were obtained. Yield: 63%. IR (cm-1, neat): 3483, 3363, 3291, 3198,
recorded using a model of incremental sweep. According to 2972, 2933, 1610, 1463, 1363, 1315, 1254, 1228, 1195, 1164, 1102,
the resulting spectra, the spectral ratio (Iin/Iout) was calculated. 1059, 1024, 895, 834, 763, 673. GC-MS: retention time, 5.13 min;
Cyclic Voltammetry. Cyclic voltammetry was performed on a [M]þ, 158.13 (calculated), 157.99 (measured).
potentiostat and computer-controlled electroanalytical system. TNN14. To a solution of CT-03 (100 mg, 0.1 mmol), HOBt
Electrochemical measurements were carried out in a 10 mL cell (40.5 mg, 0.3 mmol), and BOP (46.4 mg, 0.105 mmol) in dry
equipped with a glassy carbon working electrode (7.07 mm2), a DMF (10 mL) was added DIPEA (90 μL) under N2. The reaction
platinum-wire auxiliary electrode, and a Ag/AgCl reference elec- mixture was stirred at room temperature for 20 min, and then
14
trode. Solutions of biradicals (1 mM) were degassed by bubbling NN (16.5 mg, 0.105 mmol) in 5 mL of DMF was added dropwise.
with the nitrogen gas before the detection. The redox potentials The resulting mixture was continuously stirred for 18 h at room
were calculated according to the relation E = (E ap þ E cp)/2. temperature. Solvent was removed under vacuum, and the residue
Reaction Kinetics of TNN14 with Ascorbate. Various concen- was dissolved in phosphate buffer (0.1 M, pH 7.4) and purified by
trations of ascorbic acid (0.5, 1, 2, and 4 mM) were added to the column chromatography on reverse phase C-18 using water
solution of TNN14 (50 μM) in PBS (50 mM, pH 7.4). Incre- followed by 0-15% acetonitrile in water as eluants to give the
mental EPR spectra were recorded 45 s after mixing. The biradical TNN14 as a green solid (75 mg, 66%). Purity: >98% by
concentration of the trityl-hydroxylamine TNN14-H at each HPLC (see Supporting Information) and 97 ( 1% versus TEM-
time point was obtained by comparing their double integrated POL determined as previously reported.17 IR (cm-1, neat): 3425.1,
signal intensities relative to CT03 as standard. Since the ascorbic 2971, 2933, 1646, 1580, 1454, 1367, 1312, 1237, 1168, 1150, 1113,
acid concentration (0.5, 1, 2, and 4 mM) used was in greater 886, 820, 725, 697. MS ([MþH]þ, m/z): 1139.038 (measured),
excess than the biradical concentration (50 μM), the reaction 1139.071 (calculated); ([M þ Na]þ, m/z): 1161.000 (measured),
kinetics of the biradical with ascorbic acid is a pseudo-first-order 1161.052 (calculated).
reaction. The resulting curves in Figure 2B were fitted with the TNN15. To the solution of CT-03 (63.3 mg, 63.3 μmol), HOBt
equation ln[(C0 - Ct)/C0] = -kobst, where C0 is the initial (25.6 mg, 189.9 μmol) and BOP (29.4 mg, 66.5 μmol) in dry
concentration of TNN14, Ct the concentration of the trityl- DMF (8 mL) was added DIPEA (60 μL) under N2. The reaction
hydroxylamine TNN14-H at each time point, and kobs the mixture was stirred at room temperature for 20 min, and then
observed pseudo-first-order rate constant. Considering kobs = 15
NN (10 mg, 63.3 μmol) in 5 mL of DMF was added dropwise.
k2[Asc], the approximated second-order rate constant k2 was The resulting mixture was continuously stirred for 18 h at room
finally calculated from the slope of the plot of kobs versus [Asc]. temperature. Solvent was removed under vacuum, and the
Synthesis. 1-Oxyl-2,2,5,5-tetramethylpyrrolidinyl-3-carboxy- residue was dissolved in phosphate buffer (0.1 M, pH 7.4) and
lic Acid N-Hydroxysuccinimide Ester (3-CP-OSu). To a solution purified by column chromatography on reverse phase C-18
of 3-carboxy-proxyl (0.53 g, 2.86 mmol), HOBT (1.61 g, 8.60 mmol), using water followed by 0-15% acetonitrile in water as eluants

J. Org. Chem. Vol. 75, No. 22, 2010 7801

JOC Article Liu et al.

to give the biradical TNN15 as a green solid (45 mg, 62%). Acknowledgment. This work was supported by NIH grants
Purity: >98% by HPLC (See Supporting Information) and HL38324, EB0890, EB4900 (J.L.Z.), and HL81248 (F.A.V.).
95 ( 1% versus TEMPOL determined as previously reported.17
IR (cm-1, neat): 3423, 2973, 2918, 1646, 1579, 1455, 1367, 1238, Supporting Information Available: GC-MS, IR, HRMS
1150, 883, 727. MS ([MþH]þ, m/z):1140.044 (measured), 1140.068 spectra and HPLC chromatograms and kinetic studies of the
(calculated); MS ([MþK]þ, m/z): 1177.985 (measured), 1178.024 nitroxide radical 3-AP with ascorbate. This material is available
(calculated). free of charge via the Internet at http://pubs.acs.org.

7802 J. Org. Chem. Vol. 75, No. 22, 2010