Synthesis and biological testing of aminoxyls designed for long-term retention by living cells

Article abstract of DOI:10.1039/b415586f

Owing to recent advances in electron paramagnetic resonance (EPR) imaging methodologies, it is now potentially possible to track and image, in real time in vivo, cells that had been tagged with aminoxyl spin probes. We had previously reported that living cells can accumulate 3-carboxy-2,2,5,5-tetramethyl-1- pyrrolidinyloxyl to high (millimolar) intracellular concentrations through passive incubation with the corresponding acetoxymethyl (AM) ester. In the present study, we show that under physiological conditions aminoxyl is rapidly extruded by cells through an organic anion transport mechanism, resulting in an intracellular exponential lifetime (t_1/e or τ) of just 9.84 min at 37°C. Through successive rational structural modifications, we arrived at (2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-ylmethyl)amine-N,N-diacetic acid, which can still be accumulated by cells to high intracellular concentrations, but which, with an intracellular exponential lifetime of τ = 114 min, is well retained by cells for long periods of time, where one expects 14% retention even after 5 h. These results suggest that it should be feasible to use EPR imaging to perform in vivo tracking of populations of cells that have accumulated high intracellular levels of aminoxyls.

Full text of DOI:10.1039/b415586f

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A R T I C L E
Synthesis and biological testing of aminoxyls designed for long-term
retention by living cells
Gerald M. Rosen,a,b Scott R. Burks,b,c Mark J. Kohrb,c and Joseph P. Y. Kao*b,c
a Department of Pharmaceutical Sciences and the Center for Very Low Frequency EPR Imaging
for In Vivo Physiology, University of Maryland School of Pharmacy, Baltimore, MD 21201,
USA
b Medical Biotechnology Center, University of Maryland Biotechnology Institute,
725 West Lombard Street, Baltimore, MD 21201, USA. E-mail: jkao@umaryland.edu;
Fax: 410-706-8184; Tel: 410-706-4167
c Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201,
USA
Received 8th October 2004, Accepted 8th December 2004
First published as an Advance Article on the web 19th January 2005
Owing to recent advances in electron paramagnetic resonance (EPR) imaging methodologies, it is now
potentially possible to track and image, in real time in vivo, cells that had been tagged with aminoxyl spin probes. We
had previously reported that living cells can accumulate 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [1] to high
(millimolar) intracellular concentrations through passive incubation with the corresponding acetoxymethyl (AM) ester
[2]. In the present study, we show that under physiological conditions aminoxyl [1] is rapidly extruded by cells through
an organic anion transport mechanism, resulting in an intracellular exponential lifetime (t1/e or s) of just 9.84 min
at 37 ◦C. Through successive rational structural modifications, we arrived at (2,2,5,5-tetramethylpyrrolidin-1-oxyl-
3-ylmethyl)amine-N,N-diacetic acid [10], which can still be accumulated by cells to high intracellular concentrations,
but which, with an intracellular exponential lifetime of s = 114 min, is well retained by cells for long periods of time,
where one expects 14% retention even after 5 h. These results suggest that it should be feasible to use EPR imaging
to perform in vivo tracking of populations of cells that have accumulated high intracellular levels of aminoxyls.
Introduction
The movement of specific populations of cells through the
body occurs in normal physiology as well as pathophysiology.
Examples include the “homing” of lymphocytes to specific
tissues and organs, and the spread of metastatic cancer cells from
the original malignant lesion to distant sites. The ability to track
and visualize such cell movements within the body would be
useful clinically and scientifically. With the development of low-
frequency electron paramagnetic resonance (EPR) spectroscopy
and imaging1 and the ability to detect paramagnetic species in
Scheme 1 Esterase-assisted loading of aminoxyl [1] into a living cell.
situ, in vivo and in real time,2 tracking and imaging of cells within
a living animal is now a real possibility. Before such studies can
be undertaken, however, we must develop spin probes that can be
loaded into cells at sufficiently high concentrations as to enable
detection of the cells by low-frequency EPR spectroscopy. This
is not a trivial task. The most common spin probes are the
aminoxyls, which, until very recently, have not been successfully
loaded into living cells at high concentrations. Moreover,
aminoxyls, depending on their structure, can be susceptible to
destruction by bioreduction.3 Therefore, a successful spin probe
that can be used to track living cells must 1) be readily loaded
into cells at high concentration and 2) be retained intracellularly
over long periods of time. We have recently demonstrated
esterase-assisted accumulation of aminoxyl [1] to millimolar
concentrations in living lymphocytes after incubation with the
corresponding acetoxymethyl (AM) ester [2]4 (Scheme 1). In this
paper, we report the synthesis and biological testing of new spin
probes that meet the criteria given above.
Jurkat lymphocytes with aminoxyl [1] by incubation with the
◦
AM ester [2] at 23 C, essentially as described previously4 (see
Experimental section for details). We found that at 37 ◦C the
amount of aminoxyl [1] retained intracellularly declined rapidly
with time (circles in Fig. 1). The time course of decline was
well-fitted by a single exponential, with a time constant (or
lifetime) of s = 9.84
0.06 min, or equivalently the half-life
was t1/2 = 0.693 × s = 6.82 0.04 min. The rule of thumb that
an exponential process is essentially (97%) complete after 5 half-
lives implies that after 30–35 min very little aminoxyl [1] would
be retained by the cells, as verified by the data at 40 and 80 min.
We also estimated the concentration of aminoxyl [1] loaded into
the lymphocytes. In three independent experiments, immediately
after incubation with AM ester [2], the average intracellular
concentration of aminoxyl [1] was found to be 2.27 0.12 mM
(see Experimental section for details). This confirmed what we
have demonstrated previously4, namely that through incubation
with the AM ester [2] cells can accumulate aminoxyl [1] to high
concentrations intracellularly.
Results and discussion
To elucidate what process might underlie the rapid loss of
aminoxyl [1] from the cells at 37 ◦C we performed a parallel
experiment wherein lymphocytes loaded with aminoxyl [1] were
incubated for 80 min at 23 ◦C rather than at 37 ◦C. Intracellular
retention of aminoxyl [1] was markedly improved at 23 ◦C, with
Once loaded into cells, a spin probe must be well retained by
the cells at physiological temperature in order to be useful for
cell tracking studies in vivo. As a starting point, we examined
the cellular retention of aminoxyl [1] (Scheme 1). We loaded
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 6 4 5 – 6 4 8
6 4 5
©

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Scheme 2 Reagents and conditions: (a) 1-(ethoxycarbonylmethyl)-
piperazine, CH2Cl2; (b) i. KOH–CH3OH, ii. BrCH2O2CCH3, (CH3)2SO.
Scheme
3
Esterase-catalyzed conversion of AM ester [5] into
mixed-charge aminoxyl [6].
Fig. 1 Intracellular retention of aminoxyl [1] under different condi-
tions: Cells lose aminoxyl [1] rapidly at 37 ◦C, but the rate of loss is greatly
reduced at a lower temperature and in the presence of probenecid, an
inhibitor of organic anion transport. Jurkat lymphocytes were allowed
to accumulate aminoxyl [1] by incubation with the acetoxymethyl ester
[2] as described in the Experimental section. Replicate suspensions of
loaded lymphocytes in Hanks’ Balanced Salt Solution (HBSS) were
maintained at 37 ◦C for varying durations of time. Thereafter, the lym-
phocytes were separated from the HBSS by centrifugation. The amount
of aminoxyl [1] in the cells and in the supernatant HBSS were assayed by
EPR to permit calculation of the fraction of label retained intracellularly
(see Experimental section for details). Circles (᭺) are data obtained at
37 ◦C in HBSS; diamonds (᭛) are data obtained at 37 ◦C in HBSS
containing 10 mM probenecid. The solid curves are least-squares fits to
a single exponential of the form y = y0 + Ae−t/s, where s is the exponential
time constant, or lifetime, and is related to the half-life by t1/2 = 0.693s.
Plus signs (+) represent data collected at 23 ◦C in HBSS and the dashed
line is a single-exponential fit of the data. EPR spectrometer settings are
detailed in the Experimental section.
Replicate suspensions of lymphocytes loaded with aminoxyl
[6] ◦by incubation with the AM ester [5] were maintained at
37 C for different periods of time. Thereafter, the amounts of
aminoxyl [6] retained by the cells and extruded into the extracel-
lular solution were assayed by EPR spectroscopy. As shown in
Fig. 2, aminoxyl [6] (+) was much better retained than aminoxyl
[1] (᭺). Least-squares analysis of the data yielded an exponential
time constant of s = 30.0 1.2 min (t1/2 = 20.8 0.8 min) at
37 ◦C for aminoxyl [6]; a 3-fold improvement over aminoxyl
[1]. By reference to a standard calibration curve, the average
initial intracellular concentration of aminoxyl [6] was estimated
to be 1.41 mM. Thus, introduction of mixed-charge character
enhanced intracellular retention significantly without adversely
affecting the ability of cells to accumulate the spin probe.
◦
◦
31% retention at 80 min as compared with 0% at 37 C (+ in
Fig. 1). This implies s ≈ 69 min (t1/2 ≈ 48 min) at 23 C for a
single-exponential time course (dashed line in Fig. 1). That is,
loss of spin label was ∼7-fold slower for a 14 ◦C decrease from
37 ◦C to 23 ◦C. Such a strong temperature dependence suggests
that the loss of aminoxyl [1] could be mediated by a biological
transport process.5 Numerous studies suggest that cellular trans-
porters of organic anions are ubiquitously expressed in the body.
Organic anion transport has been observed in diverse cell types
including neuronal cell lines,6 pancreatic islet cells,7 fibroblasts,8
cardiac myocytes,9 cells of the kidney tubules,10 as well as cells
of the immune system.11 Probenecid and sulfinpyrazone are
effective pharmacological inhibitors of such transport processes
11b,12
that extrude organic anions from cells.
We tested the effect
of probenecid and found that inclusion of 10 mM probenecid
in the Jurkat cell suspension greatly improved the ability of
the lymphocytes to retain aminoxyl [1] (diamonds in Fig. 1).
Nonlinear least-squares fit of the data yielded an exponential
time constant of s = 104 12 min (t1/2 = 72 9 min). Thus, the
anion transport inhibitor slowed the loss of aminoxyl [1] at 37 ◦C
by more than 10-fold. This result suggests that loss of the spin
probe from Jurkat lymphocytes is mediated by organic anion
transporters that actively extrude the anionic aminoxyl [1].
An organic anion extrusion mechanism suggests a potential
approach to its circumvention. Cells retain the natural amino
acids L-glutamate and L-aspartate, both of which bear a net
negative charge at physiological pH but also contain a positively-
charged group owing to the presence of the a-amino substituent.
We investigated whether introducing such “mixed-charge” char-
acter into a xenobiotic molecule such as an aminoxyl could
improve cellular retention. To test this we synthesized aminoxyl
[5] from the known aminoxyl [3] (Scheme 2). Cleavage of [5]
by cellular esterases is expected to release the mixed-charge
aminoxyl [6] (Scheme 3).
Fig. 2 Comparison of retention of aminoxyls [1], [6] and [10] by
Jurkat lymphocytes at 37 ◦C. Suspensions of Jurkat lymphocytes loaded
with aminoxyls were treated as in Fig. 1. Data for aminoxyl [1] (open
circles, ᭺) are reproduced from Fig. 1 for ease of visual comparison.
Data for aminoxyls [6] and [10] are represented by plus signs (+) and
filled circles (᭹), respectively. Solid curves are least-squares fits to the
single-exponential function, y = y0 + Ae−t/s, where s is the exponential
time constant, or lifetime, and is related to the half-life by t1/2 = 0.693s.
EPR spectrometer settings are detailed in the Experimental section.
We next investigated whether a mixed-charge polyionic
aminoxyl with increased net ionic charge would show further
improved retention by cells. We synthesized aminoxyl [9] from
the known aminoxyl [7] (Scheme 4). Esterase cleavage of [9]
is expected to generate the mixed-charge aminoxyl [10] in the
intracellular environment (Scheme 5).
When we examined lymphocytes that had been loaded by
incubation with the AM ester [9], we found that intracellular
retention of aminoxyl [10] was remarkably long-lived (᭹, Fig. 2).
Nonlinear least-squares analysis yielded an exponential time
constant of s = 113.8 6.9 min (t1/2 = 78.9 4.8 min) at 37 ◦C
6 4 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 6 4 5 – 6 4 8

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To a CH3OH (10 cm3) solution of [4-(2,2,5,5-tetramethyl-
pyrrolidin-1-oxyl-3-carbonyl)-piperazin-1-yl]acetic acid ethyl
ester [4] (0.5 g, 1.47 mmol) was added KOH (82 mg, 1.47 mmol).
This reaction mixture was heated to ∼50 ◦C for 3 h; disap-
pearance of starting material was monitored by TLC (silica gel
plates, CHCl3–acetone, 1 : 1). After evaporating the solvent, the
residue was taken up in H2O (10 cm3) and extracted with CH2Cl2
(three times). The aqueous solution was evaporated to dryness,
and then DMSO (1 cm3) and CH2Cl2 (10 cm3) were introduced.
This mixture was stirred at room temperature for 5 min, at
which point bromomethyl acetate (0.22 g, 0.14 cm3, 1.47 mmol;
Aldrich, Milwaukee, WI) was added. The reaction was stirred at
room temperature for 2 h. An ice–water mixture was then added
and the organic solution was removed. The aqueous solution
was extracted with CH2Cl2 (three times). The organic solution
was dried over anhydrous Na2SO4, and evaporated to dryness.
Any remaining DMSO was removed under a high vacuum.
The residual oil was purified by flash column chromatography
on silica gel 60 (230–400 mesh, CHCl3–acetone, 9 : 1). The
product solidified upon addition of hexane. Recrystallization
from hexane–benzene yielded [4-(2,2,5,5-tetramethylpyrrolidin-
1-oxyl-3-carbonyl)piperazin-1-yl]acetic acid acetoxymethyl es-
ter [5] (0.37 g; 65%) as a yellow solid; mp 71–72 ◦C; (found: C,
56.32; H, 7.99; N, 10.85. Calc. for C18H30N3O6: C, 56.25; H, 7.81;
N, 10.94); mmax(CHCl3)/cm−1: 1765 and 1641 (CO).
Scheme
4
Reagents and conditions: (a) EtO2CCH2Br, K2CO3,
(CH3)2SO–CH3CN; (b) i. KOH–CH3OH, ii. BrCH2O2CCH3, (CH3)2SO.
Scheme
mixed-charge aminoxyl [10].
5
Esterase-catalyzed conversion of AM ester [9] into
for aminoxyl [10]. This represents a close to 4-fold improvement
over aminoxyl [6] and nearly 12-fold improvement over aminoxyl
[1]. These results indicate that at physiological temperature cells
can retain significant amounts of spin label even after 5 hours.
We also examined how effectively incubation with the AM ester
loaded aminoxyl [10] into cells. In two replicate measurements,
we determined that the initial average concentration of [10] in
loaded Jurkat lymphocytes was high at 2.1 0.7 mM.
In summary, by introducing rational structural modifica-
tions, we have successfully made aminoxyl spin probes that
simultaneously exhibit two desirable characteristics: 1) they can
be accumulated to high intracellular concentrations through
passive incubation of cells with the acetoxymethyl ester and 2)
once loaded into cells, they are retained intracellularly for long
periods of time under physiological conditions.
(2,2,5,5-Tetramethylpyrrolidin-1-oxyl-3-ylmethyl)amine-N,N-
diacetic acid diethyl ester [8]
To a solution of 3-methylamino-2,2,5,5-tetramethyl-1-pyrro-
lidinyloxyl [7] (prepared as described in the literature14), (1.4 g,
8.1 mmol) in DMSO (2 cm3) and CH3CN (2 cm3) was
added anhydrous K2CO3 (3.39 g, 24.5 mmol). After stirring
for 5 min, ethyl bromoacetate (2.73 g, 1.81 cm3, 16.2 mmol)
was added and the reaction mixture was stirred at room
temperature for 3 h, at which point H2O was added. The
solution was washed with CH2Cl2 (three times). The organic
solution was dried over anhydrous Na2SO4 and evaporated
to dryness. The residual oil was purified by flash column
chromatography on silica gel 60 (230–400 mesh, CHCl3–
acetone, 48 : 2) to yield (2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-
ylmethyl)amine-N,N-diacetic acid diethyl ester [8] (1.9 g, 70%)
as a thick orange oil; m/z (FAB) 344.2320 (M+ + H. C17H32N2O5
requires 344.2311); mmax(CHCl3)/cm−1: 1733 (CO).
Experimental
Reagents
All chemical reagents and solvents were obtained from commer-
cial vendors and used without further purification. Media and
biochemicals for cell culture were from Biosource International
(Rockville, MD). Probenecid was from Sigma-Aldrich (St.
Louis, MO). IR spectra were recorded on a FT-IR spectrometer
(Perkin-Elmer, Norwalk, CT) in CHCl3. Melting points were
obtained on a Thomas Hoover capillary melting point apparatus
and were corrected.
(2,2,5,5-Tetramethylpyrrolidin-1-oxyl-3-ylmethyl)amine-N,N-
diacetic acid diacetoxymethyl ester [9]
[4-(2,2,5,5-Tetramethylpyrrolidin-1-oxyl-3-carbonyl)piperazin-
1-yl]acetic acid acetoxymethyl ester [5]
To a CH3OH (10 cm3) solution of (2,2,5,5-tetramethylpyrrolidin-
1-oxyl-3-ylmethyl)amine-N,N-diacetic acid diethyl ester [8]
(0.6 g, 1.7 mmol) was added KOH (200 mg, 3.6 mmol). The
mixture was heated to ∼50 ◦C for 3 h; disappearance of
starting material was monitored by TLC (silica gel plates, ether).
After evaporating the solvent, the residue was taken up in
H2O (10 cm3) and extracted with CH2Cl2 (three times). The
aqueous solution was evaporated to dryness then DMSO (1 cm3)
and CH3CN (2 cm3) were added. This mixture was stirred
at room temperature for 5 min, at which point bromomethyl
acetate (0.55 g, 0.35 cm3, 3.6 mmol; Aldrich, Milwaukee, WI)
was added. The reaction was stirred at room temperature for
3 h. Water (10 cm3) and then CH2Cl2 (20 cm3) were added
and the organic solution was removed. The aqueous solution
was further extracted with additional CH2Cl2 (three times).
The organic solution was dried over anhydrous Na2SO4 and
evaporated to dryness. Any remaining DMSO was removed
under a high vacuum. The residual oil was purified by flash
chromatography on silica gel 60 (230–400 mesh, CHCl3–ethyl
acetate, 45 : 5) to yield (2,2,5,5-tetramethylpyrrolidin-1-oxyl-
3-ylmethyl)amine-N,N-diacetic acid diacetoxymethyl ester [9]
(0.44 g, 60%) as a thick orange oil; m/z (FAB) 432.2095
To a CH2Cl2 (10 cm3) solution of 3-[[(2,5-dioxo-1-pyrrolidinyl)-
oxy]carbonyl]-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [3] (pre-
pared as described in the literature13) (0.79 g, 2.79 mmol) was
added 1-(ethoxycarbonylmethyl)piperazine (0.48 g, 2.79 mmol;
Lancaster Synthesis Inc., Windham, NH). The reaction mixture
was stirred overnight and evaporated to dryness, resulting
in an oil which was then taken up in ice cold dilute HCl
(5%) and extracted with CHCl3 (three times). The remaining
aqueous solution was made basic with Na2CO3 and extracted
with CHCl3 (three times). The organic phase was dried over
anhydrous Na2SO4 and evaporated to dryness. The resid-
ual oil was purified by flash column chromatography using
silica gel 60 (230–400 mesh, CHCl3–acetone, 9 : 1). The
product solidified upon addition of hexane. Recrystallization
from boiling hexane with dropwise addition of ethyl ether
afforded [4-(2,2,5,5-tetramethyl-pyrrolidin-1-oxyl-3-carbonyl)-
piperazin-1-yl]acetic acid ethyl ester [4] (0.71 g; 75%) as a yellow
solid; mp 108–109 ◦C; (found: C, 60.02; H, 8.98; N, 12.25. Calc.
for C17H30N3O4: C, 60.00; H, 8.82; N, 12.35); mmax(CHCl3)/cm−1:
1739 and 1640 (CO).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 6 4 5 – 6 4 8
6 4 7

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(M+ + H. C19H32N2O9 requires 432.2108); mmax(CHCl3)/cm−1:
was measured and plotted against the aminoxyl concentration
to construct a standard calibration curve. From the EPR
spectrum of the clear lysate of the cells that had been incubated
with AM ester, the middle field peak height was measured
and compared with the calibration curve, which allowed the
aminoxyl concentration in the lysate to be determined. Knowing
that the original suspension contained 1.8 × 107 cells cm−3 in
400 lL HBSS, and that 7.65 × 10−13 L is the average Jurkat
cell volume,15 the average intracellular aminoxyl concentration
in intact cells could be estimated by straightforward volumetric
calculation.
1759 (CO).
EPR spectroscopy
For EPR spectroscopy, each sample was added to a quartz flat
cell, which was introduced into the cavity of the spectrometer
(model E-109, Varian Associates, Palo Alto, CA). The quartz
cell was open to the atmosphere to allow free equilibration
with air. EPR spectra were recorded at room temperature with
the following instrument settings: microwave power, 20 mW;
field set, 3340 G; sweep width, 100 G; modulation frequency,
100 kHz; modulation amplitude, 0.5 G; response time, 0.5 s;
sweep, 12.5 G min−1. Receiver gain ranged from 1 × 103 to 1 ×
104 for the lymphocyte experiments and 2 × 102 to 1 × 104 when
constructing calibration curves. The hyperfine splitting constant
of the aminoxyls is AN = 14.9 G.
Data analysis
All data analysis, graphing and nonlinear least-squares curve
fitting were performed through Origin software (OriginLab
Corp, Northampton, MA). Reported values are mean
standard error; standard errors of curve-fitting parameters are
obtained from the nonlinear least-squares error matrix after v2
minimization.
Loading of lymphocytes with aminoxyls
Jurkat lymphocytes (gift of Dr Alfredo Garzino Demo) were
suspended in bicarbonate buffered RPMI 1640 medium, sup-
plemented with 100 unit cm−3 penicillin and 100 lg cm−3
streptomycin, that also contained 40 lM of the acetoxymethyl
(AM) ester of an aminoxyl and 0.0015% w/v Pluronic F-127
surfactant (BASF Corp., Washington, NJ). The cell suspension
(2.2 × 106 cells cm−3) was incubated at 23 ◦C for 70 min whilst
being gently agitated by a rocker. After incubation, the cell
suspension was centrifuged for 3.5 min at 1000 rpm, and the
cell pellet was resuspended and centrifuged two more times
in RPMI 1640 medium that contained 10% v/v fetal bovine
serum but no AM ester. The final cell pellet was resuspended
in Hanksꢀ Balanced Salt Solution (HBSS) at a density of 3.6 ×
106 cells cm−3.
Acknowledgements
This research was supported in part by grants from the
U.S. National Institutes of Health, EB-2034 (G. M. R.) and
GM-56481 (J. P. Y. K.).
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Agents Chemother., 1999, 43, 2457–2462.
Estimation of intracellular concentration of aminoxyls in
lymphocytes incubated with the AM esters of aminoxyls
A Jurkat lymphocyte suspension at the same cell density as all
samples used in the experiments (4.5 × 107 cells cm−3 HBSS)
that had not been incubated with AM ester was pulse-sonicated
(60 s, 20 ◦C) to ensure cell lysis. The lysate was clarified by
centrifugationat 15 300rpmfor 6minto sedimentcellular debris.
Standard solutions of the Na+ salt of an aminoxyl in the clear cell
lysate were made by serial dilution to bracket the concentration
range of 10–1000 lM. An EPR spectrum was acquired for
each standard solution; the height of the middle field peak
6 4 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 6 4 5 – 6 4 8