Esterified dendritic TAM radicals with very high stability and enhanced oxygen sensitivity

Article abstract of DOI:10.1021/jo301849k

In this work, we have developed a new class of dendritic TAM radicals (TG, TdG, and dTdG) through a convergent method based on the TAM core CT-03 or its deuterated analogue dCT-03 and trifurcated Newkome-type monomer. Among these radicals, dTdG exhibits the best EPR properties with sharpest EPR singlet and highest O₂ sensitivity due to deuteration of both the ester linker groups and the TAM core CT-03. Like the previous dendritic TAM radicals, these new compounds also show extremely high stability toward various reactive species owing to the dendritic encapsulation. The highly charged nature of these molecules resulting from nine carboxylate groups prevents concentration- dependent EPR line broadening at physiological pH. Furthermore, we demonstrate that these TAM radicals can be easily derivatized (e.g., PEGylation) at the nine carboxylate groups and the resulting PEGylated analogue dTdG-PEG completely inhibits the albumin binding, thereby enhancing suitability for in vivo applications. These new dendritic TAM radicals show great potential for in vivo EPR oximetric applications and provide insights on approaches to develop improved and targeted EPR oximetric probes for biomedical applications.

Full text of DOI:10.1021/jo301849k

Article
pubs.acs.org/joc
Esterified Dendritic TAM Radicals with Very High Stability and
Enhanced Oxygen Sensitivity
Yuguang Song, Yangping Liu,* Craig Hemann, Frederick A. Villamena, and Jay L. Zweier*,†
†
,†
†
†,‡
†
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
*
S Supporting Information
ABSTRACT: In this work, we have developed a new class of dendritic TAM radicals
TG, TdG, and dTdG) through a convergent method based on the TAM core CT-03 or
(
its deuterated analogue dCT-03 and trifurcated Newkome-type monomer. Among these
radicals, dTdG exhibits the best EPR properties with sharpest EPR singlet and highest O2
sensitivity due to deuteration of both the ester linker groups and the TAM core CT-03.
Like the previous dendritic TAM radicals, these new compounds also show extremely
high stability toward various reactive species owing to the dendritic encapsulation. The
highly charged nature of these molecules resulting from nine carboxylate groups prevents
concentration-dependent EPR line broadening at physiological pH. Furthermore, we
demonstrate that these TAM radicals can be easily derivatized (e.g., PEGylation) at the
nine carboxylate groups and the resulting PEGylated analogue dTdG−PEG completely
inhibits the albumin binding, thereby enhancing suitability for in vivo applications. These
new dendritic TAM radicals show great potential for in vivo EPR oximetric applications
and provide insights on approaches to develop improved and targeted EPR oximetric probes for biomedical applications.
INTRODUCTION
recently, TAM-based spin labels also have shown great
potential for distance measurements in proteins using pulsed
EPR dipolar spectroscopy.
High stability and water solubility are two important features
of TAM radicals. Current applications of TAM radicals are
mostly based on TAM radicals CT-03 and OX063 (Chart 1).
However, the stability of both radicals is problematic when
exposed to various oxidoreductants. In addition, although CT-
■
Much effort has focused on developing stable free radicals that
are required for a wide variety of applications in functional EPR
spectroscopy and imaging (EPRI) as well as Overhauser-
enhanced magnetic resonance imaging (OMRI). Among the
currently available stable radicals, tetrathiatriarylmethyl (TAM)
and nitroxide (NR) radicals represent two major classes of
water-soluble paramagnetic probes. TAM radicals have great
advantages over NR radicals including high biostability and
long relaxation times that provide narrow line width at
physiological pH and therefore greatly enhance the sensitivity
27
0
3 can be simpler to synthesize than OX063, its relatively high
hydrophobicity and tendency for aggregation at high
concentrations limits its in vivo application. Therefore, new
derivatization strategies for CT-03 are required. Our previous
study demonstrated that dendritic encapsulation of CT-03 can
effectively enhance the stability of the radical and improve their
1
and resolution for EPRI. In addition, the long relaxation times
make TAM radicals easily saturated by radio frequency
irradiation affording enhancement of sensitivity and resolution
for OMRI with less heating of the sample.
19
2
,3
water solubility under various conditions. Unfortunately, the
resulting dendritic TAM radicals DTR1 and DTR2 have
relatively broad EPR signal due to the amide linker, resulting in
relatively low EPR resolution at a given concentration and low
The long
relaxation times of TAM radicals also make them well suited for
4,5
pulsed EPR/EPRI a−n8d hyperpolarized C-13 nuclear magnetic
6
resonance and MRI.
19
O2 sensitivity. We have also showed that the esterified
derivatives of CT-03 have much simpler and sharper EPR
signals owing to the removal of the hyperfine splittings from
TAM radicals belong to partially or fully substituted
derivatives of triphenylmethyl radical, the first free radical
observed in 1900 by Gomberg. Full substitution of each
phenyl ring by alkylthio and carboxylate groups effectively
eliminates the hyperfine splitting from protons and affords a
very sharp EPR singlet. While TAM radicals were first
developed by Nycomed Innovation AB as contrast agents for
OMRI in the late 1990s,
structurally modified to expand their applications and so far
9
15
the amide-N. Partial deuteration of the protons in the ester
28
linker can further narrow the EPR lines of TAM radicals.
Based on the aforementioned results, we herein developed new
dendritic TAM radicals TG, TdG, and dTdG in which the
nondeuterated or deuterated dendrons are conjugated to CT-
1
0−13
these radicals have been
0
3 or its deuterated analogue dCT-03 through an ester linkage
14
15,16
enable measurement of extracellular and intracellular
•
−
17,18
19−23
oxygen (O ) levels, O
generation,
pH,
and
Most
Received: September 5, 2012
Published: January 23, 2013
2
2
24
25,26
glutathione levels as well as total redox status.
©
2013 American Chemical Society
1371
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The Journal of Organic Chemistry
Article
a
Chart 1. Molecular Structure of Dendritic TAM Radicals
TG, by directly deuterating the protons in the ester linkers was
not successful under different conditions (CF COOD/D O,
3
2
K CO /D O−acetone-d (2:1), or K CO /DMSO-d ). There-
2
3
2
6
2
3
6
30
fore, dD1 was synthesized from bromoacetyl bromide-d
2
using the same procedure as for D1 and then reacted with CT-
0
3 or dCT-03 to result in TdG and dTdG, respectively.
Figure 1 shows EPR spectra of the newly synthesized
esterified dendritic TAM radicals TG, TdG, and dTdG under
a
PEG550, poly(ethylene glycol) methyl ether, with average molecular
weight of 550. In the nomenclature of new TAM radicals, the letters
“T”, “d”, and “G” mean “TAM radical”, “deuteration”, and “first
generation dendron”, respectively. For example, dTdG is an esterified
dendritic TAM radical with a deuterated TAM core and the deuterated
dendron.
Figure 1. Experimental EPR spectra and linewidths of esterified
dendritic TAM radicals under anaerobic conditions. For TG, a
simulated spectrum (dotted line) is also shown with the calculated
hyperfine splitting of 83 mG from the six protons of the ester linker
and calculated line width of 85 mG.
(
Chart 1). The O sensitivity and stability toward various
2
oxidoreductants of these radicals were measured, and the
concentration-dependence of their EPR spectra was inves-
tigated. In addition, in order to further improve its
biocompatibility, the PEGylated analogue of dTdG was also
developed.
anaerobic conditions. While both TdG and dTdG have an EPR
singlet, a multiplet signal was observed for TG with a hyperfine
splitting constant of 83 mG due to the interaction of the
unpaired electron with six protons from the three ester groups
through extensive π conjugation along the central carbon, aryl,
and the ester group. As expected, dTdG with a fully deuterated
TAM core and ester linkers has the smallest peak-to-peak line
width with a value of 62 mG under anaerobic conditions as
compared to TG (85 mG) and TdG (94 mG). A slightly
broader line width was observed for TdG than TG under
anaerobic conditions possibly due to the unresolved hyperfine
splittings from the deuterons from the ester groups of TdG.
Owing to its paramagnetic nature, O2 can undergo
Heisenberg spin exchange with exogenous paramagnetic probes
in solution, resulting in broadening of the EPR line. The
magnitude of this broadening is proportional to the O2
concentration, which enables the determination of tissue O2
RESULTS AND DISCUSSION
■
As the first effort, we synthesized the nondeuterated esterified
dendritic TAM radical TG using the procedure shown in
Scheme 1. The trifurcated Newkome-type monomer 1 was first
Scheme 1. Synthesis of Esterified Dendritic TAM Radicals
TG, TdG, and dTdG
concentration. High sensitivity of the EPR signal to O is one of
2
the most important properties of TAM radicals for EPR
oximetry applications. Figure 2 shows the O -dependent line-
2
broadening response of the three TAM radicals. Among these
radicals, dTdG has the highest O sensitivity of 3.9 mG/% O
2
2
with a 130% increase in line width from anaerobic conditions
62 mG) to aerobic conditions (143 mG), while TdG and TG
(
have the O2 sensitivity of 3.5 and 3.3 mG/% O2 with
approximately 90% and 80% increase in line width, respectively,
under the two conditions. Therefore, the use of the fully
obtained by 1,4-addition of tris(hydroxymethyl)aminomethane
deuterated TAM core significantly improves the O sensitivity
2
29
to tert-butyl acrylate according to the reported method. Then,
the monomer 1 was reacted with bromoacetyl bromide in the
presence of K CO to give D1 which was further conjugated
of these radicals. Similar O sensitivity (3.5 mG/%O ) of TdG
2
2
with that of the nondeuterated TAM radical CT-03 (3.6 mG/%
O , Figure 2) indicates that O can still easily diffuse into the
2
3
2
2
with CT-03 to afford TG, followed by deprotection of tert-butyl
ester groups by TFA. This new convergent procedure improves
the synthetic efficiency as compared to the prior divergent
dendrimer core of TdG, resulting in similar Heisenberg
exchange interactions with TdG as compared to CT-03,
although higher generation of TAM dendrimers may have
lower O2 sensitivity due to weaker Heisenberg exchange
1
9
method. Attempts to obtain TdG, the deuterated analogue of
1
372
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The Journal of Organic Chemistry
Article
Figure 3. Concentration effect on the linewidths of TAM radicals
under anaerobic conditions.
Figure 2. Plots of linewidths of (▲) TdG, (●) TG, (◆) CT-03, and
(
■) dTdG as a function of O concentration in PBS (pH 7.4).
2
exerts a significant effect on its line width with a value of 18.8
mG/mM when the concentration goes beyond 0.5 mM. Low
concentration-dependent line broadening observed for these
dendritic radicals could be due to the strong intermolecular
static repulsion, resulting from their highly negative charge.
The presence of carboxylate groups in these new esterified
dendritic TAM radicals allows further conjugation with various
functional molecules to improve their physicochemical proper-
ties. It has been shown that albumin binding could broaden the
EPR lines of paramagnetic probes due to the shortening of their
relaxation times and thus limit their use in EPR oximetry.
Covalent attachment of poly(ethylene glycol) (PEG) to these
probes may overcome this limitation and also enhance their
biocompatibility. Since dTdG has the best EPR properties, it
was chosen to be covalently linked with PEG chains. As shown
in Scheme 2, the PEGylated TAM radical dTdG-PEG was
easily obtained by coupling dTdG with PEG550 (average
molecular weight 550) using EDCI in the presence of HOBt
and DIPEA.
interaction with O . On the other hand, these radicals
consistently afford much higher response of their EPR
2
linewidths to O than the previous dendritic trityl radicals
2
(
<2 mG/% O and 5% line width change) with the amide
2
19
linker. Moreover, there is >12 times sharper EPR line for
dTdG (62 mG) compared to previous dendritic trityl radicals
(780 mG) enabling more than 140-fold enhancement of EPR
sensitivity.
Despite the relatively high stability of TAM radicals such as
CT-03 and OX063, they are still unstable when exposed to
•
−
17−19,31
reactive species such as superoxide (O
)
and
2
32
peroxidase/hydrogen peroxide (H O ), which limit their in
2
2
19
vivo applications. Similar to our previous results, covalent
dendritic encapsulation may increase the stability of TAM
radicals. Therefore, we herein examined the reactivity of TdG
toward oxidants such as hydroxyl radical (HO ), peroxyl
radical, H O , peroxynitrite (ONOO), O , iron(III) (Fe ),
•
•−
3+
2
2
2
and horse radish peroxidase (HRP)/H O as well as reducing
2
2
2
+
agents such as iron(II) (Fe ), glutathione (GSH), and ascorbic
acid. Results showed that TdG has extraordinarily high stability
toward all of these highly reactive species with only less than
Scheme 2. Synthesis of the PEGylated Dendritic TAM
Radical dTdG-PEG (n ≈ 12, PEG 550)
5
% signal decrease after 30-min incubation (Table 1). However,
19,31,32
as observed previously,
problematic, especially when exposed to peroxyl radical, O2
the stability of CT-03 was
•−
,
peroxynitrite, and HRP/H O . Cyclic voltammetric studies
2
2
further revealed that owing to the dendritic encapsulation,
esterified dendritic TAM radical TG is resistant to oxidization
as evidenced by the absence of peak potential up to 1 V but
slightly reducible with a very weak i at −0.77 V (see Figure S1,
pa
Supporting Information). In comparison, the TAM core CT-03
19
is redox active as reported in our previous study.
Aside from the enhanced stability, dendritic TAM radicals
exhibit much less concentration-dependent line broadening.
The intermolecular spin−spin interaction of stable radicals at
high concentrations may induce line broadening and thus limit
their application for EPR oximetry. As shown in Figure 3, the
line width of dTdG did not show any change at concentrations
of up to 10 mM which was much lower than the value used for
in vivo systems. Comparatively, the concentration of dCT-03
Table 1. Percentage of TAM Radicals Remaining after Exposure to Various Reactive Species in PBS
a
ROO•
O2•−
Fe2+
Fe3+
TAM
Asc
99
ONOO−
H O
HO•
HRP
GSH
100
2
2
TdG
99
78
100
97
98
97
99
99
99
100
99
b
b
b
b
b
b
CT-03
97
97
91
none
60
55
95
a
b
In the presence of H O . Data from ref 19. See details in the Experimental Section.
2
2
1
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Figure 4. Binding of TAM radicals (20 μM) with BSA. (A) Effect of the BSA concentration on the EPR spectra of dTdG−PEG and dTdG. (B) Plot
of the EPR signal intensity of TAM radicals as a function of the BSA concentration.
As expected, the solution of dTdG−PEG (20 μM) in PBS
did not show any change in its EPR spectral profile upon
addition of BSA (0−450 μM). However, dTdG exhibits binding
with BSA as verified by the gradual decrease of its EPR peak
intensity as a function of increasing concentration of BSA due
to line broadening. The EPR signal double integration of dTdG
Importantly, the presence of nine free carboxylic acids in these
molecules allows further functionalization to endow new
physiochemical properties. As a proof, dTdG was PEGylated
on the free carboxylic acid groups, and the resulting TAM
radical dTdG−PEG prevents binding to albumin while still
maintaining excellent properties for EPR oximetry. Therefore,
these dendritic TAM radicals show great potential for EPR
oximetric applications and provide a novel strategy for the
development of new TAM radicals with various functions.
(
(
20 μM) remains the same before and after addition of BSA
25−450 μM), excluding its spin quenching by BSA (Figure S2,
Supporting Information). Compared to dTdG, dCT-03 has
even stronger binding with BSA possibly because of the less
bulky nature of the latter or due to the relatively higher
hydrophobicity of dCT-03, making it easier to interact with
BSA than dTdG. As shown in Figure 4, the EPR line width of
EXPERIMENTAL SECTION
■
EPR Measurement. EPR measurements were carried out on
Bruker EMX and EleXsys E580 EPR spectrometers at room
temperature. General instrument settings were as follows: modulation
frequency, 30−100 kHz; microwave frequency, 9.87 GHz; microwave
power, 0.2−5 mW; modulation amplitude, 0.01−0.25 G. Measure-
ments were performed in 50 μL capillary tubes.
dTdG−PEG has good response to O with a value of 3.3 mG/
2
%
O that is slightly lower than that of dTdG (3.9 mG/% O ).
2 2
Of note is that the presence of BSA has no significant influence
on the O sensitivity (3.3 mG/% O ) of dTdG-PEG (Figure 5),
2
2
supporting its use for in vivo biomedical applications.
Oxygen Sensitivity. Oxygen sensitivities of TAM radicals were
evaluated according to our previous method. In brief, aqueous
solutions of each TAM radical (100 μM) were transferred into gas-
permeable Teflon tubes (i.d. = 0.8 mm) that were sealed at both ends.
Sealed samples were placed inside a quartz EPR tube with open ends.
Nitrogen or N /O gas mixtures with varying concentrations of O
2
2
2
were allowed to flow into the EPR tube and after at least 10 min
equilibration changed to the next gas mixture. EPR spectra were
recorded using a model of incremental sweep with the following
acquisition parameters: modulation frequency, 30 kHz; microwave
power, 0.2 mW; modulation amplitude, 0.01 G.
Stability Studies toward Biological Oxidoreductants. Sol-
utions of GSH (1 mM), Asc (1 mM), and H O (1 mM) in PBS buffer
2
2
(
pH 7.4, 50 mM) were used. The Fe(II)−NTA complex was prepared
by dissolving (NH ) Fe(SO ) and NTA with a molar ratio of 1:2 in
4
2
4 2
water under anaerobic conditions. The Fe(III)−NTA solution (Fe/
NTA 1:2) was prepared by slow addition of the appropriate volume of
acidic Fe(III) stock solution into a vigorously stirred solution of NTA
in water. The resulting solution was slowly neutralized to pH 7.4 using
Figure 5. O sensitivity of dTdG−PEG in the presence or absence of
2
BSA (200 μM).
0
.1 M NaOH. Either Fe(II)−NTA or Fe(III)−NTA solution was
•
freshly prepared before use. Hydroxyl radical (HO ) was continuously
generated from the system consisting of Fe(III)−NTA (0.1 mM) and
2 2
CONCLUSIONS
H O (1 mM). Superoxide was generated using the xanthine (X)/
■
xanthine oxidase (XO) system using XO (20 mU/mL) and X (0.4
mM) in the presence of DTPA (0.1 mM). Alkylperoxyl radical was
generated by thermolysis of 2,2′-azobis-2-methylpropanimidamide,
We have developed a new class of esterified dendritic TAM
radicals (TG, TdG, and dTdG) using a convergent method.
These new radicals showed extremely high stability toward
various reactive species and excellent EPR properties. In
particular, the deuterated dTdG has a very sharp EPR singlet
−
dihydrochloride (AAPH, 1 mM) at 37 °C. Peroxynitrite (ONOO )
was generated by decomposition of SIN-1 (1 mM) at 37 °C in the
presence of SOD (50 U/mL). EPR spectra were recorded 30 min after
mixing the TAM radical solution (20 μM) with various oxido-
which is highly O sensitive and concentration-independent.
2
1
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The Journal of Organic Chemistry
Article
reductants. Effect of various reactive species on TAM radicals was
expressed as percentage of TAM radical remaining after exposure to
reactive species for 30 min which was obtained by the double integral
of the EPR signal. Each experiment was conducted three times.
Synthesis of D1. To a solution of the trifurcated Newkome-type
Synthesis of dTdG. The same procedure for the synthesis of TG
was used for the synthesis of dTdG. dCT-03 (32 mg, 31 μmol), dD1,
and K
butyl ester-protected dTdG which was further treated with CF
to afford dTdG as a brown solid (50 mg) with a yield of 75%. Purity: >
CO (17 mg, 120 μmol) in DMSO-d
were used to afford tert-
2
3
6
COOD
3
29
1
monomer 1 (3.5 g, 6.9 mmol) in CHCl (20 mL) was added solid
98% by HPLC (see the Supporting Information). H NMR (D
O): δ
2
3
K CO (1.9 g, 13.8 mmol). The resulting suspension was cooled to 0
2.39 (br, CH CH O, 18H); 3.68 (br, OCH CH and CCH O, 36H).
2
2
2
2
2
2
3
+
°
C, and bromoacetyl bromide (1.7 g, 8.3 mmol) was added dropwise
HRMS (MALDI-TOF, [M + H] , m/z): 2173.205 (measured),
2173.607 (calcd).
with stirring over the period of 0.5 h. The reaction mixture was
allowed to warm to room temperature and stirred for 1 h. The reaction
Synthesis of dTdG-PEG. To a solution of dTdG (30 mg, 13.8
μmol), HOBt (83.8 mg, 621 μmol, 45 equiv), and EDCI (120 mg, 621
mixture was then diluted with 20 mL of CHCl , and 20 mL of water
3
μmol, 45 equiv) in D O (1 mL) and PEG550 (5 mL) was added
was later added. The organic layer was washed with 5% citric acid
solution (w/v, 20 mL) and then water (20 mL), dried over anhydrous
Na SO , and concentrated under vacuum. The resulting residue was
2
DIPEA (90 μL) under N atmosphere. The reaction mixture was
2
stirred at room temperature for 3 days, and then 5 mL of 5% citric acid
solution was added. The resulting solution was dialyzed against water
2
4
purified by column chromatography on silica gel using eluting with
(3 × 1L) with a molecular weight cutoff of 1000D and further purified
hexane/ethyl acetate = 100:5 to 100:10 as eluents to afford D1 as a
1
by column chromatography on Sephadex G-50 using water as an
eluent and then Bio-Beads S-X1 using dichloromethane as an eluent to
give the PEGylated TAM radical dTdG-PEG as a brown solid (48 mg,
50%). HPLC: a broad peak at ∼2.2 min possibly due to the use of the
impure PEG reagent (average MW, 550) and/or the presence of a
colorless oil (3.6 g) with a yield of 83%. H NMR (CDCl , 600 MHz):
3
δ 1.45 (s, (CH ) C, 27H); 2.46 (t, CH CH O, 6H); 3.66 (t,
3
3
2
2
OCH CH , 6H); 3.71 (s, CCH O, 6H); 3.81 (s, BrCH , 2H); 6.86 (s,
2
2
2
2
13
NH). C NMR (CDCl , 600 MHz): 28.3 ((CH ) C); 29.8 (BrCH );
3
3
3
2
3
6.3 (CH CH O); 60.4 (CCH O); 67.3 (CH CH O); 69.0 (CCH O);
2 2 2 2 2 2
small fraction of partially PEGylated analogues (see the Supporting
8
0.7 (C(CH ) ); 165.9 (CONH); 171.1 (COO-t-Bu). HRMS (ESI,
3
3
1
+
79
Information). H NMR (D
2
O): δ ∼2.5 (br, C(O)CH
CH C(O) and CCH
2
2
CH
O, 36H);
2
, 18H);
[
M + Na] , m/z): 648.2352 (D1(Br ), measured), 648.2359 (calcd);
81
3.23 (s, CH
3
O, 27H); 3.48 (br, CH
2
2
650.2335 (D1(Br ), measured), 650.2339 (calcd).
3
.55 (s, OCH CH O, ∼425H). HRMS (MALDI-TOF): a broad peak
2
2
Synthesis of dD1. The same procedure was applied for the
from 6000 to 8000.
synthesis of D1 using the monomer 1 (1.2 g, 2.4 mmol), K CO (0.66
2
3
30
g, 4.8 mmol), bromoacetyl bromide-d2 (0.53 g, 2.6 mmol), and
ASSOCIATED CONTENT
* Supporting Information
CDCl as solvent to afford dD1 as a colorless oil (1.2 g) with a yield of
■
3
1
8
0%. H NMR (CDCl , 600 MHz): δ 1.45 (s, (CH ) C, 27H); 2.46 (t,
S
3
3
3
CH CH O, 6H); 3.66 (t, OCH CH , 6H); 3.71 (s, CCH O, 6H); 6.86
2
2
2
2
2
Cyclic voltammograms, spectroscopic characterization, and
HPLC chromatograms. This material is available free of charge
via the Internet at http://pubs.acs.org.
13
(
(
(
s, NH). C NMR (CDCl , 600 MHz): 28.3 ((CH ) C, BrCD ); 36.3
3 3 3 2
CH CH O); 60.3 (CCH O); 67.3 (CH CH O); 69.0 (CCH O); 80.7
2
2
2
2
2
2
C(CH ) ); 165.8 (CONH); 171.1 (COO-t-Bu). HRMS (ESI, [M +
3
3
+
79
Na] , m/z): 650.2577 (dD1(Br ), measured), 650.2483 (calcd);
(
81
AUTHOR INFORMATION
Corresponding Author
dD1(Br ), measured), 652.2563 (calcd).
■
Synthesis of TG. To a solution of CT-03 (80 mg, 80 μmol) and
D1 (188 mg, 320 μmol) in DMSO was added solid K CO (55 mg,
2
3
*Tel: 614-247-7788. Fax: 614-247-7845. E-mail: (J.L.Z.) jay.
zweier@osumc.edu, (Y.L.) yangping.liu@osumc.edu.
4
2
00 μmol). The reaction mixture was stirred at room temperature for
days under N atmosphere. Then, 20 mL of ethyl acetate and 10 mL
2
Notes
of 5% citric acid solution (w/v) were added, and the two phases were
separated. The organic layer was washed with saturated NaHCO3
solution (10 mL), water (10 mL), and brine (10 mL), dried on
anhydrous Na SO , and concentrated under vacuum. The resulting
The authors declare no competing financial interest.
2
4
ACKNOWLEDGMENTS
■
residue was purified by column chromatography on silica gel eluting
with CH Cl /methanol = 100:0.5 to 100:5 to afford the tert-butyl
This work was supported by NIH Grant Nos. HL38324,
EB0890, and EB4900 (J.L.Z.) and HL81248 (F.A.V.).
2
2
ester-protected form of TG as a brown solid. Thereafter, this brown
solid was treated with TFA (2 mL) overnight. TFA was then removed
using streaming nitrogen, and the residue was dissolved in phosphate
buffer (0.2 M, pH 7.4) and purified by reversed-phase C-18 column
chromatography using water followed by 0−20% methanol in water as
eluents to give the esterified dendritic TAM radical TG as a brown
solid (122 mg, 72%). Purity: 96% by HPLC (see the Supporting
REFERENCES
■
(
1) Ardenkjaer-Larsen, J. H.; Laursen, I.; Leunbach, I.; Ehnholm, G.;
Wistrand, L. G.; Petersson, J. S.; Golman, K. J. Magn. Reson. 1998, 133,
1
−12.
(2) Lurie, D. J.; Li, H. H.; Petryakov, S.; Zweier, J. L. Magn. Reson.
1
Information). H NMR (D O, 600 MHz): δ 2.39 (br, CH CH O,
Med. 2002, 47, 181−186.
2
2
2
1
8H); 3.68 (br, OCH CH and CCH O, 36H). HRMS (MALDI-
(3) Krishna, M. C.; English, S.; Yamada, K.; Yoo, J.; Murugesan, R.;
Devasahayam, N.; Cook, J. A.; Golman, K.; Ardenkjaer-Larsen, J. H.;
Subramanian, S.; Mitchell, J. B. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
2216−2221.
2
2
2
+
TOF, [M] , m/z): 2130.161 (measured), 2130.336 (calcd); ([M +
+
Na] , m/z): 2153.181 (measured), 2153.325 (calcd); ([M + 2Na −
+
H] , m/z): 2175.139 (measured), 2175.307 (calcd); ([M + 3Na −
+
2H] , m/z): 2197.145 (measured), 2197.289 (calcd).
(4) Murugesan, R.; Cook, J. A.; Devasahayam, N.; Afeworki, M.;
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Synthesis of TdG. The same procedure for the synthesis of TG
was applied for the synthesis of TdG. CT-03 (50 mg, 50 μmol), dD1
126 mg, 200 μmol), and K CO (35 mg, 250 μmol) in DMSO-d
6
(
2
3
were used to afford tert-butyl ester-protected TdG which was further
treated with CF COOD to afford TdG as a brown solid (85 mg) with
3
a yield of 80%. Purity: > 98% by HPLC (see the Supporting
1
Information). H NMR (D O, 600 MHz): δ 2.38 (br, CH CH O,
2
2
2
1
8H); 3.67 (br, OCH CH and CCH O, 36H). HRMS (MALDI-
2 2 2
+
TOF, [M] , m/z): 2136.263 (measured), 2136.373 (calcd); ([M +
Na] , m/z): 2159.263 (measured), 2159.362 (calcd); ([M + K] , m/
z): 2175.256 (measured), 2175.336 (calcd).
+
+
1
375
dx.doi.org/10.1021/jo301849k | J. Org. Chem. 2013, 78, 1371−1376

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