Effect of electron donating groups on polyphenol-based antioxidant dendrimers

Article abstract of DOI:10.1016/j.biochi.2015.02.001

Numerous studies have reported the beneficial effects of antioxidants in human diseases. Among their biological effects, a majority of antioxidants scavenge reactive radicals in the body, thereby reducing oxidative stress that is associated with the patho

Full text of DOI:10.1016/j.biochi.2015.02.001

Accepted Manuscript
Effect of Electron Donating Groups on Polyphenol-based Antioxidant Dendrimers
Choon Young Lee, Cyprien Nanah, Rich Held, Amanda Clark, Uyen Huynh, Marina
Pressnall, Rebecca L. Uzarski, John McCraken, Ajit Sharma
PII:
S0300-9084(15)00028-0
10.1016/j.biochi.2015.02.001
BIOCHI 4640
DOI:
Reference:
To appear in: Biochimie
Received Date: 29 December 2014
Accepted Date: 1 February 2015
Please cite this article as: C.Y. Lee, C. Nanah, R. Held, A. Clark, U. Huynh, M. Pressnall, R.L. Uzarski,
J. McCraken, A. Sharma, Effect of Electron Donating Groups on Polyphenol-based Antioxidant
Dendrimers, Biochimie (2015), doi: 10.1016/j.biochi.2015.02.001.
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Effect of Electron Donating Groups on Polyphenol-based Antioxidant Dendrimers
Choon Young Lee,*1 Cyprien Nanah1, Rich Held1, Amanda Clark1, Uyen Huynh1, Marina
Pressnall1, Rebecca L. Uzarski2, John McCraken3 and Ajit Sharma†1
1Department of Chemistry, Central Michigan University, Mount Pleasant, MI 48859, USA
2 Department of Biology, Central Michigan University, Mount Pleasant, MI 48859, USA
3 Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA
Corresponding authors: C. Y. Lee*, lee1cy@cmich.edu, 989-774-3289; A. Sharma†,
sharm1a@cmich.edu, 989-774-3303, Department of Chemistry, Central Michigan University
Abstract
Numerous studies have reported the beneficial effects of antioxidants in human diseases.
Among their biological effects, a majority of antioxidants scavenge reactive radicals in the body,
thereby reducing oxidative stress that is associated with the pathogenesis of many diseases.
Antioxidant dendrimers are a new class of potent antioxidant compounds reported recently. In
this study, six polyphenol-based antioxidant dendrimers with or without electron donating groups
(methoxy group) were synthesized in order to elucidate the influence of electron donating groups
(EDG) on their antioxidant activities. Syringaldehyde (2 ortho methoxy groups), vanillin (1
ortho methoxy group), and 4-hydroxybenzaldehyde (0 methoxy group) were derivatized with
propargylamine to form building blocks for the dendrimers. All the six dendrimers contain
polyether cores, which were synthesized by attaching pentaerythritol and methyl α-D-
glucopyranoside to in-house prepared spacer units. To prepare generation 1 antioxidant
dendrimers, microwave energy and granulated metallic copper catalyst were used to link the
cores and building blocks together via alkyne-azide 1,3-cycloaddition click chemistry. These
reaction conditions resulted in high yields of the target dendrimers that were free from copper
contamination. Based on DPPH antioxidant assay, antioxidant dendrimers decorated with
syringaldehyde and vanillin exhibited over 70- and 170-fold increase in antioxidant activity
compared to syringaldehyde and vanillin, respectively. The antioxidant activity of dendrimers
increased with increasing number of EDG groups. Similar results were obtained when the
dendrimers were used to protect DNA and human LDL against organic carbon and nitrogen-
based free radicals. In addition, the antioxidant dendrimers did not show any pro-oxidant activity
on DNA in the presence of physiological amounts of copper. Although the dendrimers showed
potent antioxidant activities against carbon and nitrogen free radicals, EPR and DNA protection
studies revealed lack of effectiveness of these dendrimers against hydroxyl radicals. The
dendrimers were not cytotoxic to CHO-K1 cells.
Keywords: Polyphenol, dendrimer, antioxidant, pro-oxidant, electron donating group
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1. Introduction
Free radicals are produced during metabolism and inflammation. These highly reactive
species are neutralized by antioxidant enzymes or endogenous/dietary antioxidants. Within cells,
antioxidant enzymes such as glutathione peroxidase, superoxide dismutase and catalase play an
important role in preventing oxidation of biomolecules. However, in extracellular fluids, key
antioxidants include proteins that bind metal ions such as ceruloplasmin, transferrin and albumin
and exogenous small molecules such as vitamins C and E and dietary polyphenols [1]. Excess
free radicals are often associated with oxidative stress, which has been implicated in various
diseases such as cancer, cardiovascular and neurological disorders [2,3,4]. Antioxidants work in
many different ways such as quenching singlet oxygen, binding metal ions, and scavenging free
radicals [1]. The majority of antioxidants including polyphenols scavenge free radicals. In this
process, the antioxidant transfers hydrogen atom (HAT) or electron (ET) to neutralize the free
radical [5,6], thereby preventing chain reactions such as lipid peroxidation. Other antioxidants
prevent free radical formation by chelating transition metal ions such as Fe2+ and Cu+, which are
known to generate free radicals by reaction with hydrogen peroxide [7,8]. Unfortunately, many
antioxidants are associated with deleterious pro-oxidant effects during which transition metal
ions, such as Cu2+ and Fe3+ are reduced by the antioxidants to lower oxidation states (Cu+ and
Fe2+) [9,10,11,12]. The reduced metal ions subsequently react with H2O2 to form powerful
hydroxyl radicals, which are capable of damaging cellular components including proteins, lipids,
and nucleic acids.
There are a variety of free radicals with different aqueous solubilities and atomic make-
ups that are found in hydrophilic or lipophilic compartments of the human body. Therefore, it is
not surprising that a given antioxidant is unable to quench all types of free radicals. A desirable
antioxidant should show strong radical scavenging but poor pro-oxidant activities. To achieve
this goal of designing an ideal antioxidant, we reported synthesis of a new class of antioxidants
termed as antioxidant dendrimers [13,14]. The surface of the antioxidant dendrimer is composed
of free radical scavenging moieties such as phenolic groups while the core of the dendrimer
contains groups that sequester metal ions. Our initial success with antioxidant dendrimers
exhibiting potent antioxidant activities and reduced pro-oxidant effects inspired us to synthesize
and evaluate other dendritic antioxidants with different cores. The construction of a library of
such novel compounds will help us understand their structure-activity relationships. In this study
we have synthesized dendrimers with varying amounts of surface electron donating groups
(EDG) in order to evaluate the influence of EDG on their antioxidant properties. A pair of
dendrimers with polyether cores was prepared from syringaldehyde (two methoxy groups),
another pair from vanillin (one methoxy group) and a third pair from 4-hydroxybenzaldehyde
(no methoxy group). In the syringaldehyde and vanillin dendrimers, the methoxy groups were
ortho to the hydroxyl group. Natural and synthetic phenolic compounds with EDG at ortho or
para position, were reported to be better antioxidants than those with meta-EDG [15,16].
Synthesis was performed via microwave-assisted 1,3-dipolar cycloaddition utilizing copper
metal as a catalyst. The antioxidant ability of these dendrimers to scavenge organic carbon and
nitrogen radicals as well as hydroxyl radicals was examined by DPPH assay, DNA and
lipoprotein protection studies, and electron spin resonance. Their pro-oxidant effects on copper-
mediated DNA damage and cell toxicity were also evaluated.
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2. Materials and Methods
Syringaldehyde, vanillin, 4-hydroxybenzaldehyde, pentaerythritol (97%), methyl-α-D-
glucopyranoside, sodium ascorbate, quercetin, sodium triacetoxyborohydride (Na(OAc)3BH),
1,1-diphenyl-2-picrylhydrazyl (DPPH), Fat Red 7B, phosphate-buffered saline (PBS), potassium
persulfate, glacial acetic acid, sodium acetate and methanol were purchased from Sigma Aldrich
and were used without further purification. 2,2'-Azobis(2-amidinopropane) dihydrochloride
(AAPH) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Human low-density
lipoprotein (LDL) was obtained from Kalen Biomedical (Montgomery Village, MD, USA). The
lipoprotein solution (protein = 5 mg/mL) contained 154 mM NaCl and 0.34 mM EDTA.
1H-NMR spectra were recorded with a Varian Mercury spectrometer operating at 500
MHz. 13C-NMR spectra were recorded using a Varian Mercury spectrometer operating at 125
MHz.
ESI mass spectra were obtained using a Waters LCT Premier XE mass spectrometer.
The source capillary voltage was 3000 V, cone voltage was 10 V and source temperature was
80 °C. Samples analyzed by flow injection had a desolvation gas temperature of 250 °C and a
gas flow rate of 200 L/hr. The mobile phase was water (50%)-acetonitrile (50%) containing 0.1%
formic acid and its flow rate was 50 µL/min. The injection volume was 10 µL with a sample
concentration of approximately 10 ng/uL.
Hitachi HPLC, consisting of L-7200 autosampler, L-7100 pump, L-7400 UV detector and
D-7000 interface, was used to analyze the purity of the final dendrimers by reversed phase
chromatography. Mobile phase was an acetonitrile-H2O gradient system (5→95 % acetonitrile)
with 0.1% trifluoroacetic acid. The sample was detected at 214 nm. Flow rate was 1
mL/min. Separation was performed on a Varian C18 RP column.
All spectrophotometric data were obtained using Perkin Elmer UV/Vis spectrometer
(Lambda 20) and Molecular Devices Corp. Spectra Max (M2e).
The microwave used for the click chemistry was a CEM brand Discover SP v 2.15.
2.1. General procedures for the synthesis of building block
Building block 1a and 1b were synthesized as previously described [17].
2.1.1. Building block 1c
To synthesize the building block, 250 mL of distilled THF was added to a 500 mL round
bottom flask. 4-Hydroxybenzaldehyde (3.56g, 29.18 mmol) was then dissolved into the THF.
Propargylamine (3.27 g, 59.4 mmol) was then added to the solution dropwise via a syringe. The
solution was then heated at 40–45 °C. Sodium tri-acetoxyborohydride (Na(OAc) BH) (6.10 g,
3
28.8 mmol) was then added to the reaction mixture after the reaction was cooled to room
temperature. After 24 h, a second equivalent of 4-hydroxybenzaldehyde (3.56g, 29.18 mmol)
was added to the reaction mixture without heating. After 36 h the last equivalent of reducing
agent was added (Na(OAc)
days.
3
BH) (6.10 g, 28.8 mmol) and the reaction was left to proceed for two
The reaction mixture was then filtered under reduced pressure and the filtrate was dried
on the rotovap. The resulting residue was re-dissolved in 200 mL chloroform and then washed
with 50 mL of water three times. The aqueous layer was extracted with 50 mL of methylene
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chloride twice. The combined organic layer was dried over magnesium sulfate. After evaporating
the organic layer on a rotovap, the residue was purified using flash column chromatography
equipped with a 40 g pre-packed silica gel column using a hexane-ethyl acetate gradient solvent
system (6:1→3:1).
Yield 74% (11.73 g); Rf = 0.25 in hexane–ethyl acetate (2:1); 1H NMR (500 MHz,
CDCl3) δ 6.95 (d, J = 8.5 Hz, 4H), 6.78 (d, J = 8.5 Hz, 4H), 5.02 (s, 2H), 3.58 (s, 4H), 3.22 (d, J
= 2.3 Hz, 2H), 2.26 (s, 1H); 13C NMR (126 MHz, CDCl3) δ 191.1, 161.7, 154.9, 132.1, 78.9,
73.5, 57.0, 41.0; HRMS (ESI-TOF) m/z: Calcd for C17H18NO2 [M+H]+ 268.34, Found 268.53
2.2. Synthesis of compound 4
Sodium azide (5.886 g, 15.77 mmol) and 2-(2-chloroethoxy) ethanol (11.28 g, 90.55
mmol) were dissolved in a mixture of DMF (200 mL) and water (5 mL). The reaction was stirred
for 48 h at 70–80 °C. After the reaction was complete, the reaction mixture was filtered and the
filtrate was dried at 50 °C under reduced pressure. The resulting residue was re-suspended in
acetone, followed by vacuum filtration. The filtrate was dried on the rotovap and the residue
(27.86 g, 212.7 mmol) was then dissolved in 200 mL of chloroform. Triethylamine (21.05 g, 208
mmol) was added to the reaction vessel at 0 °C. Para-toluenesulfonyl chloride (25 g, 131 mmol)
dissolved in chloroform was added dropwise. Reaction was stirred for 24 h and then washed with
deionized water. Chloroform layer was dried with MgSO4. After filtering off MgSO4, chloroform
was removed under reduced pressure. Before loading the dried oily residue on a 40 g pre-packed
silica gel column, the column was pre-treated with 250 mL of hexane-ethyl acetate (3:1) solvent
system containing triethylamine (3%, V/V), followed by hexane (100 mL).
Then, the aliquot of dried oily reaction mixture (5 mL) was loaded on the column and purified
with flash column chromatography using a hexane-ethyl acetate gradient system, 1:0 (100 mL),
5:1 (600 mL), and 3:1 (800 mL).
Yield 61%; Rf= 0.44 in hexane–acetone (2:1); 1H NMR (300 MHz, CDCl3) δ 3.33 (t, J=
4.80 Hz, 2H), 3.60 (t, J= 4.81 Hz, 2H), 3.69 (t, J= 4.65 Hz, 2H), 4.16 (t, J= 4.52 Hz, 2H), 7.34
(d, J= 9.01 Hz, 2H), 7.80 (d, J= 8.70 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 22.2, 50.3, 69.2,
69.5, 70.2, 128.2, 130.8, 134.0, 146.1; HRMS(ESI-TOF) m/z: Calcd for C11H15N3O4SNa
[M+Na+] 308.31, Found 308.21.
2.3. Synthesis of compound 5a (1,15-diazido-8,8-bis((2-(2-azidoethoxy)ethoxy)methyl)-
3,6,10,13-tetraoxapentadecane)
Pentaerythritol (1.055 g, 7.70 mmol) was dissolved in anhydrous DMF (80 mL). NaH
(0.80 g, 33.33 mmol) in powder form was added portion by portion to the reaction under argon.
The reaction was stirred for 30 min and then compound 4 (8.843 g, 31.04 mmol) was added
dropwise. After reaction was complete, the reaction mixture was filtered and the filtrate was
dried at 50 °C under reduced pressure. The resulting residue was re-dissolved in chloroform and
washed with water three times. The chloroform layer was dried with MgSO4. After chloroform
was removed, the oily residue was purified using silica gel column chromatography in a gradient
hexane-ethyl acetate system (10:1→2:1).
Yield 48% (2.170 g); Rf= 0.172 in hexane–acetone (2:1); 1H NMR (300 MHz, CDCl3) δ
3.70 – 3.65 (m, 8H), 3.64 – 3.60 (m, 8H), 3.57 (ddd, J = 5.8, 3.5, 1.1 Hz, 8H), 3.46 (s, 8H), 3.41
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– 3.34 (m, 8H); 13C NMR (75 MHz, CDCl3) δ 71.3, 70.7, 70.3, 70.2, 51.0, 45.8; HRMS (ESI-
TOF) m/z: Calcd for C21H41N12O8 [M+H]+ 589.62, Found 589.23.
2.4. Synthesis of compound 5b ((2R,3R,4S,5R,6R)-3,4,5-tris(2-(2-azidoethoxy)ethoxy)-2-((2-(2-
azidoethoxy)ethoxy)methyl)-6-methoxytetrahydro-2H-pyran)
Methyl α-D-glucopyranoside (4.00 g, 20.60 mmol) was dissolved in anhydrous DMF
(300 mL). NaH (2.00 g, 83.30 mmol) was added under argon and the reaction mixture was
stirred for 30 min. Compound 4 was added and the reaction mixture was stirred for 48 h. The
reaction mixture was filtered. The DMF filtrate was removed under reduced pressure at 50 °C.
The residue was re-dissolved in chloroform and washed with water three times. Chloroform layer
was dried with MgSO4. After filtering off MgSO4, the chloroform filtrate was dried under
reduced pressure. The resulting yellow oily residue was purified with silica-based flash column
chromatography using a gradient hexane-acetone solvent system (10:1→3:1).
Yield 38% (5.062 g); Rf = 0.35 in hexane–acetone (1:1) system; 1H NMR (500 MHz,
CDCl3) δ 4.80 (d, J = 3.5 Hz, 1H), 4.09 – 3.94 (m, 2H), 3.91 (dd, J = 10.4, 5.0 Hz, 1H), 3.88 –
3.82 (m, 1H), 3.82 – 3.74 (m, 2H), 3.72 (m, 8H), 3.65 (qdd, J = 10.2, 4.0, 2.8 Hz, 16H), 3.38 (dt,
J = 8.3, 3.3 Hz, 16H); 13C NMR (126 MHz, CDCl3) δ 97.6, 81.8, 80.5, 77.5, 72.0, 71.7, 70.4,
70.2, 69.7, 54.6, 50.4; HRMS (ESI-TOF) m/z: Calcd for C23H42N12O10Na [M+Na]+ 669.65,
Found 669.44.
2.5. General procedures for the formation of dendrimers 6a-c and 7a-c
Core 5a (0.2174 g, 0.370 mmol) and building block 1a (0.6475 g, 1.673 mol) were
dissolved in ultrapure THF (20 mL) in a microwave reactor vessel. Granulated copper (1.5 g,
0.2–0.6 mm grain size, 99.8%) was pre-treated in the order of 25% aqueous NaOH (30 min), de-
ionized H2O, 20% aqueous H2SO4 (30 min) and rinsed with copious amount of water, followed
by acetone. Immediately after they were dried with argon, they were added to the microwave
reactor vessel. The reaction was set at a maximum temperature of 85 °C, pressure of 250 psi, and
microwave energy of 150 W (1 h), 200 W (1 h), and 250 W (5 h). The maximum temperature
reached was 71 °C, 79 °C, and 83 °C, respectively. The reaction was filtered to remove the
copper spheres. The filtrate was condensed on the rotary evaporator. The reaction mixture was
purified with flash chromatography with a 30 g pre-packed silica gel column. The solvent
systems were hexane-ethyl acetate (1:1), ethyl acetate, ethyl acetate-methanol (9:1), and ethyl
acetate-methanol (8:2).
2.5.1. Compound 6a
Yield 67% (0.531 g); Rf= 0.760 in methanol–ethyl acetate (8:2) system; 1H NMR (500
MHz, CDCl3) δ 7.54 (s, 4H), 6.64 (s, 12H), 4.92 (s, 8H), 4.46 (t, J = 5.2 Hz, 8H), 3.84 (s, 48H),
3.82 – 3.73 (m, 16H), 3.56 (s, 8H), 3.46 (dd, J = 5.9, 4.0 Hz, 16H), 3.35 (dd, J = 5.7, 3.9 Hz,
8H), 3.21 (s, 8H); 13C NMR (126 MHz, CDCl3) δ 147.2, 147.1, 133.8, 130.0, 123.8, 105.6, 70.8,
70.4, 69.8, 69.6, 57.7, 56.3, 50.9, 50.3, 47.8, 45.3; HRMS (ESI-TOF) m/z: Calcd for
C105H142N16O32 [M+2H]+21069.66, Found 1070.22.
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2.5.2. Compound 6b
Yield 50 %; Rf= 0.56 in ethyl acetate–methanol (4:1); 1H NMR (500 MHz, acetone-d6) δ
7.86 (s, 4H), 7.05 (d, J = 1.7 Hz, 8H), 6.85 (dd, J = 8.0, 1.8 Hz, 8H), 6.78 (d, J = 8.0 Hz, 8H),
4.94 (s, 8H), 4.52 (t, J = 5.3 Hz, 8H), 3.88 – 3.84 (m, 8H), 3.83 (s, 24H), 3.70 (s, 8H), 3.50 (s,
16H), 3.38 (dd, J = 5.5, 3.9 Hz, 8H), 3.31 (s, 8H), 3.23 (s, 8H); 13C NMR (126 MHz, acetone-d6)
δ 148.2, 146.4, 145.2, 131.6, 124.6, 122.2, 115.4, 113.0, 71.7, 70.8, 70.2, 70.2, 57.6, 56.2, 50.7,
48.2, 46.2; HRMS (ESI-TOF) m/z: Calcd for C97H125N16O24[M+H]+ 1899.12, Found 1899.20.
2.5.3. Compound 6c
Yield 73%; Rf = 0.77 in ethyl acetate–methanol (4:1); 1H NMR (500 MHz, acetone-d6) δ
7.85 (s, 4H), 7.23 (d, J = 8.5 Hz, 12H), 6.80 (d, J = 8.5 Hz, 12H), 4.93 (s, 8H), 4.52 (t, J = 5.2
Hz, 8H), 3.85 (t, J = 5.2 Hz, 8H), 3.67 (s, 8H), 3.46 (s, 16H), 3.38 (dd, J = 5.5, 3.7 Hz, 8H), 3.32
(s, 8H), 3.24 (s, 8H); 13C NMR (126 MHz, acetone-d6) δ 157.2, 145.3, 130.9, 130.8, 124.6,
115.8, 71.7, 70.8, 70.1, 57.3, 50.8, 48.1, 46.2; HRMS (ESI-TOF) m/z: Calcd for C89H109N16O16
[M+H]+ 1658.91, Found 1658.15.
2.5.4. Compound 7a
Yield 59%; Rf = 0.27 in ethyl acetate–methanol (7:3); 1H NMR (500 MHz, CDCl3) δ 7.58
– 7.42 (m, 4H), 6.64 (s, 16H), 4.56 – 4.37 (m, 2H), 3.94 – 3.82 (m, 51H), 3.82 – 3.69 (m, 32H),
3.55 (s, 16H), 3.52 – 3.41 (m, 2H), 3.33 – 3.27 (m, 1H), 3.25 (s, 8H), 3.16 – 3.07 (m, 2H); 13C
NMR (126 MHz, CDCl3) δ 147.1, 145.0, 133.7, 130.1, 123.7, 105.6, 97.7, 82.1, 80.8, 78.0, 72.0,
71.8, 70.8, 69.6, 57.8, 56.4, 55.1, 53.9, 50.1, 48.0; HRMS (ESI-TOF) m/z: Calcd for
C107H144N16O34 [M+2H]+2 1098.68, Found 1099.17.
2.5.5. Compound 7b
Yield 43%; Rf = 0.33 in ethyl acetate–methanol (4:1); 1H NMR (500 MHz, acetone-d6) δ
7.98 – 7.77 (m, 4H), 7.06 (s, 8H), 6.86 (d, J = 7.9 Hz, 8H), 6.78 (dd, J = 8.0, 1.0 Hz, 8H), 4.71 –
4.41 (m, 2H), 3.90 – 3.75 (m, 40H), 3.71 (d, J = 4.0 Hz, 3H), 3.65 – 3.43 (m, 24H), 3.37-3.42
(m, 1H), 3.31 (s, 16H), 3.20 (s, 2H), 3.12 – 3.00 (m, 2H); 13C NMR (126 MHz, CD3OD) δ 148.2,
146.3, 145.1, 131.6, 124.6, 122.2, 115.4, 113.1, 98.5, 82.9, 81.5, 78.7, 72.6, 72.4, 71.4, 70.0,
57.6, 56.2, 55.0, 50.6, 49.6, 48.2; HRMS (ESI-TOF) m/z: Calcd for C99H127N16O26 [M+H]+
1957.15, Found 1957.17.
2.5.6. Compound 7c
Yield 76%; Rf = 0.67 in ethyl acetate–methanol (4:1); 1H NMR (500 MHz, acetone-d6) δ
7.95 – 7.78 (m, 4H), 7.23 (d, J = 7.2 Hz, 16H), 6.81 (d, J = 8.5 Hz, 16H), 4.93 (s, 8H), 4.63 –
4.40 (m, 2H), 3.91 – 3.74 (m, 16H), 3.74 – 3.64 (m, 3H), 3.62 – 3.57 (m, 16H), 3.55 – 3.49 (m,
16H), 3.47 (s, 8H), 3.33 (s, 16H), 3.19 (s, 2H), 3.14 – 3.06 (m, 2H); 13C NMR (126 MHz,
CD3OD) δ 157.2, 145.3, 145.3, 130.8, 124.7, 115.8, 98.4, 82.8, 81.4, 78.6, 72.6, 72.4, 71.2, 70.1,
57.2, 55.1, 50.7, 49.7, 48.1; HRMS (ESI-TOF) m/z: Calcd for C91H111N16O18[M + H]+ 1716.94,
Found 1716.05.
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2.6. DPPH assay
Reduction of DPPH radical was determined for dendrimers as previously reported [18].
DPPH and antioxidants were dissolved in methanol. Antioxidant sample (10 µL) was added to
1.0 mL of DPPH reagent and the absorbance was monitored at 515 nm after one hour. All
samples were run in triplicates at room temperature. The within-run coefficient of variation of
the % inhibition values was less than 6%.
2.7. LDL oxidation-electrophoresis
Low-density lipoprotein was incubated with 20 mM AAPH in PBS and 70 µM
antioxidant (made in methanol) at 37 ºC for 21 h. The mixture was then subjected to
electrophoresis on 1% agarose gels (Helena Labs, Beaumont, TX) using the Ciba Corning
Clinical Electrophoresis System. The gels were stained with Fat Red 7B.
2.8. Antioxidant effects on DNA
DNA electrophoresis was performed on pBR 322 after 4 h incubation at 37 ºC in the
presence of 10 mM AAPH in PBS and various concentrations (12-90 µM) of antioxidants (made
in methanol).
2.9. Hydroxyl radical (OH•) assay by Electron Spin Resonance (ESR) spectrometer
Hydroxyl radicals were generated by reaction between FeSO4 (final concentration 0.25 mM) and
H2O2 (final concentration 0.25 mM) in PBS and in the presence of spin trap agent 5,5-Dimethyl-1-
pyrroline N-oxide (DMPO; final concentration 63 mM) and antioxidant (final concentration, 16 µM in
acetonitrile) [19,20]. The DMPO-OH 1:2:2:1 signal was detected with Bruker ESR spectrometer at 5
min. The antioxidant potency was determined from suppression of the DMPO-OH adduct signal.
2.10. Pro-oxidant effects on DNA
DNA (pBR 322) was incubated with 10 µM CuCl2 in the presence of 12-90 µM
antioxidants (made in methanol) at 37 ºC for 1 h. DNA damage was monitored by agarose
electrophoresis.
2.11. Cell Viability Assay
Chinese Hamster Ovary (CHO-K1) cells were cultured in F-12K medium supplemented
with 10% fetal bovine serum in a 5% CO2 humidified incubator. The cells (2.5 X 105/ml) were
seeded in 100 µL volumes in 96-well culture plates and incubated with either 100 µL solvent
(cell culture grade DMSO, 0.15%) or dendrimer for 1, 3, or 5 days. The dendrimer
concentrations tested include 1000, 100, or 10 nM of dendrimers 6a and 7a and 250, 25, and 2.5
nM dendrimers 6b, 6c, 7b, and 7c. Dendrimer concentrations selected for this study were
determined by their solubility in DMSO. Two hours before termination of the experiment, 20 µL
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of a 5 mg/ml 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution in
0.01 M phosphate buffered saline was added to each well. The culture plates were centrifuged at
450 g for 10 minutes and supernatant was removed from each well. The resultant formazan
crystals were dissolved in 100 µL DMSO. Absorbance was measured using a Biolog microplate
reader (Biotek Instruments) at dual wavelengths, 590 and 650 nm. Percent control response was
calculated as follows:
(Absorbance of treatment /Absorbance of control) X 100
All experiments were performed in triplicate and repeated. The data were analyzed using Systat
12 for Windows. Multiple groups were compared using a one-way analysis of variance and a
Tukey test for mean separation. A p value < 0.05 was considered statistically significant.
3. Results and Discussion
3.1. Synthesis
Dendrimers were synthesized using copper metal catalyzed alkyne-azide1,3-dipolar
cycloaddition, click chemistry. In order to perform the click reactions, alkyne-derivatized
building blocks and azide-derivatized core were first synthesized. Alkyne derivatives 1a, 1b,
and 1c were prepared by reacting propargylamine with syringaldehyde, vanillin and 4-
hydroxybenzaldehyde via reductive amination, respectively.
Since our goal was to synthesize potent dendritic antioxidants with metal ion chelation
capability, the interior branches were designed to contain metal binding sites. Polyether units are
well known for binding metal ions as exemplified by 18-crown-6-ether that coordinates
potassium and 12-crown-4-ether that chelates sodium. We hypothesized that incorporation of
multiple ether units and nitrogen atoms within the dendrimers would be beneficial for binding
copper and iron ions, which are involved pro-oxidant effects. Compound 3 is the branch that
constitutes the interior of the dendrimer and provides ethylene glycolic ether units (Scheme 2).
This compound was synthesized using 2-(2-chloroethoxy) ethanol (2) and sodium azide (NaN3).
The reaction was carried out in DMF and H2O as a co-solvent (40:1) at 70-85 °C. The use of
high temperatures (>100 °C) resulted in formation of several unknown side products and gave a
poor yield (<10%). The compound was an oily substance that was soluble in water and
chloroform but not in dichloromethane. In order to attach 3 to the polyol core, its hydroxyl
terminal was tosylated (compound 4) in chloroform.
Core compound 5a with four extended arms carrying ethylene glycolic ether units and
azide group on each terminal, was formed by reacting pentaerythritol (PETN) with compound 4.
Similarly, core 5b was synthesized from methyl α-D-glucopyranoside (pyranoside). Pyranoside
with hydroxyl groups in a staggered conformation was selected to reduce the steric hindrance
between branches and ultimately between surface building blocks. The extended arms with
multiple ethylene glycol units were incorporated to enhance the polarity of dendrimer’s interior
compartment as well as its metal binding capacity.
To synthesize six generation 1 (G1) antioxidant dendrimers, in-house prepared building
blocks (compound 1a-c) were attached to the PETN-based core (compound 5a) and pyranoside-
based core (compound 5b) via our previously reported granulated copper metal-assisted 1,3-
dipolar cycloaddition [17].
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Alkyne-azide 1,3-dipolar cycloaddition was pioneered by Huisgen but produces a
mixture of 1,4-disubstituted and 1,5-disubstituted 1,2,3-triazoles. In comparison, the same
alkyne-azide 1,3-dipolar cycloaddition but, using Cu(I) as a catalyst, produces 1,4-disubstituted
1,2,3-triazole as a sole product in high yield and is called Sharpless click chemistry. Due to the
sterospecificity and high efficiency, the Sharpless click reaction has been widely adopted in
modern organic synthesis. We also initially attempted dendrimer synthesis using Cu(I) in the
form of copper iodide (CuI) or copper sulfate/ascorbate. However, the dendrimer products we
obtained were contaminated with copper ions (detected by ESI). Although the copper
contaminated antioxidant dendrimers could be isolated, they could not be used as antioxidants
due to the presence of toxic copper. Therefore, alternative copper free-click chemistry methods
were attempted. Although the use of highly strained cyclooctyne ring derivative and azide is
reported to be highly efficient, the ring might decrease solubility of the target dendrimers in
biologically compatible solvents. We carried out the reaction with copper metal spheres (0.2-
0.6mm, 99.8%) in place of Cu(I). To enhance the reaction rate and yield, microwave energy was
applied to the reaction. Microwave energy-assisted click reaction produced the target dendrimers
(6a, 6b, 6c, 7a, 7b, and 7c) in very good yields (50-76%) in a relatively short reaction time (7 h).
Based on HPLC analysis, the purified fraction consists of mostly the target compound (>98%)
with a minor amount of defective species such as missing phenol rings.
3.2. Free radical scavenging
An important source of cell damage is from highly reactive chemical species called free
radicals such as hydroxyl radicals and organic radicals. Free radicals are formed as a result of
normal metabolism. Their high chemical reactivity leads to damage of any cellular structures in
their vicinity. Oxidative damage to membranes, proteins and DNA are especially important in
impairing cell function, accelerating aging and causing cell injury. The ability of the synthesized
dendrimers to scavenge nitrogen-based free-radicals was evaluated by the DPPH assay.
Syringaldehyde derivatized dendrimers (6a and 7a) had similar potency (IC50 mean of three
experiments = 3.1 µM and 3.3 µM, respectively; SD = 1 µM at 60 min). The vanillin derivatized
dendrimers (6b and 7b) had IC50 value of 5.4 µM and 5.7 µM. In comparison, 4-
hydroxybenzaldehyde derivatized dendrimers (6c and 7c) had no detectable DPPH quenching
activity under the same assay conditions. These results clearly show that the antioxidant activity
increases with increasing number of EDG groups. The lack of antioxidant activity of 4-
hydroxybenzaldehyde dendrimer suggests the importance of the ortho-electron donating groups.
Under identical experimental conditions, average IC50 value for naturally occurring antioxidants
quercetin and vitamin C were 16 µM and 36 µM while the starting materials used for syntheses
of the antioxidants displayed negligible DPPH activity. IC50 values of syringaldehyde and
vanillin were 1.8 mM and 7.4 mM, respectively and were significantly higher than their
corresponding dendrimers. The extremely weak antioxidant activities of syringaldehyde and
vanillin may be partly due to the presence of aldehyde groups, which are electron-withdrawing.
During synthesis of antioxidant dendrimers, these aldehyde groups are converted into electron
donating benzylic moieties, resulting in formation of phenol rings with benzylic group and
methoxy group at para and ortho position to the OH, respectively. The syringaldehyde dendrimer
6a (IC50 = 3.1 µM) showed 580-fold decrease IC50 value compared to syringaldehyde (IC50= 1.8
mM) while vanillin dendrimer 6b (IC50= 5.4 µM) showed 1370-fold decrease compared to
vanillin (IC50= 7.4 mM). Even if we take into account that dendrimers 6a and 6b contain eight
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syringaldehyde and vanillin units, IC50 values of the dendrimers are still 73-fold and 171-fold
lower than the IC50 values of syringaldehyde and vanillin, respectively. This extremely high
antioxidant potency of dendrimers may be due to two factors. One is the replacement of electron
withdrawing aldehyde with electron donating benzylic groups. The other may be a “dendritic
effect”, whereby multiple phenolic units located within the same dendrimer molecule interact
with one another to enhance its antioxidant properties. The dendritic effect of dendrimers is well
known and involves the changes in their intrinsic physico-chemical properties with alterations in
their size, shape, architecture, surface chemistry, flexibility and elemental composition [21].
These results clearly establish that formation of powerful macromolecular antioxidants from
weak antioxidants is possible if they are appropriately assembled into dendritic architecture.
Scavenging of carbon radicals (AAPH) by the dendritic compounds was evaluated
indirectly by monitoring the damage to two biomolecules, human low-density lipoproteins
(LDL) and plasmid DNA (pBR 322) [22]. LDL was used as a model lipid for evaluating the
ability of the dendrimers as chain-breaking antioxidants for lipids. When LDL is exposed to free
radicals, both protein and lipid components of LDL may be oxidized. Since the integrity of
multiple components may be affected during LDL damage by free radicals, the assessment of
lipoprotein oxidation is not straightforward. The electrophoretic migration pattern of oxidized
LDL is one of the best methods to evaluate the extent of lipoprotein oxidation [23]. We have
successfully used a similar strategy for other antioxidants [13,14]. LDL was incubated with 20
mM AAPH and 70 µM dendrimers at 37 ºC for 21 h. The lipoproteins were then subjected to
agarose electrophoresis. As shown in Figure 1, native LDL showed as a sharp band (negative
control, lane 1). Under the electrophoresis conditions, the LDL particles have a net negative
charge that enables them to migrate towards the positive pole (anode). In comparison, LDL
incubated with AAPH radical without any antioxidant (positive control) showed a lightly stained
broad band, indicating that the sample is populated by heterogeneous species that were formed as
a result of lipoprotein oxidation (lane 2, Fig. 1). The darkest part of this band even migrated
faster than the native LDL, suggesting that some of the species in oxidized LDL had a higher
negative charge density than native LDL. Under similar conditions, lipoprotein samples
incubated with dendrimers 6a, 6b, 7a, or 7b showed similar migration rate and band intensity to
that of native LDL, implying that the dendrimers protected LDL against AAPH free radical
damage (lanes 3-6, Fig. 1). In contrast, LDL incubated with vitamin C showed a faster-moving
smeared band (lane 7), similar to the positive control (lane 2), implying that vitamin C could not
rescue most of the LDL under these conditions. Quercetin (lane 8) also showed a broad band like
vitamin C; however, there was a sharper, more intense band with increased charge density. These
results suggest that quercetin showed some protection of LDL, especially when compared to
vitamin C. Nevertheless, its protective effect was far less than dendrimers 6a, 6b, 7a, and 7b.
Under similar conditions, dendrimers derivatized with 4-hydroxybenzaldehyde (6c and 7c) were
completely ineffective in protecting LDL (data not shown). These results once again confirm that
the antioxidant properties increase with increasing number of EDGs.
The ability of the antioxidant dendrimers to protect DNA against AAPH-derived carbon
radicals was also evaluated. In these experiments plasmid DNA (pBR 322) was incubated with
AAPH (final concentration, 10 mM) at 37 ºC for 4 h with antioxidants (final concentrations, 12-
90 µM). The extent of DNA damage was evaluated by agarose electrophoresis [24]. As shown in
Figure 2, native DNA was mostly in its supercoiled (SC) form (lane 1, Fig. 2). In the presence of
AAPH, the DNA was transformed almost entirely into its open circular (OC) form (lane 2, Fig.
2). Vitamin C did not protect DNA at all concentrations (12-90 µM) tested. The DNA incubated
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with AAPH and vitamin C showed not only the OC form but also the more damaged linear form
(lanes 3-6, Fig. 2). Quercetin showed a slight protection of DNA at all concentrations but it was
noticeable that the intensity of SC bands became weaker as the concentration of quercetin
decreases. In comparison, the native SC bands of DNA incubated with syringaldehyde
derivatized dendrimers 6a and 7a were much more intense than those of quercetin or vitamin C.
Interestingly, the amount of DNA protected at all concentrations were the same; the increase in
the concentrations of antioxidant did not result in enhanced protection. This might be due to the
limited aqueous solubility of the antioxidant dendrimers, which limits antioxidant activity at
higher concentrations. Vanillin-surfaced dendrimers (6b and 7b) showed slightly less effective
protection than syringaldehyde derivatized dendrimers (6a and 7a). In case of 4-
hydroxybenzaldehyde derivatized dendrimers, the PETN core-based dendrimer (6c) showed a
minor amount of protection at 90, 45, and 23 µM but negligible protection at 12 µM. In
comparison, pyranoside-core based dendrimer 7c did not show any protection. It should be noted
that the PETN- and pyranoside-derivatized cores (5a and 5b) by themselves did not have
antioxidant effects (data not shown). Overall, in each dendrimer family, syringaldehyde
derivatized dendrimers showed superior DNA protective effects against the AAPH radical
induced damage compared to vanillin derivatized dendrimers, followed by 4-
hydroxybenzaldehyde-derivatized dendrimers. These DNA protection assay results also
emphasize the importance of number of electron donating group(s).
Similar studies were performed to determine the ability of dendritic antioxidants to
protect DNA against damage by hydroxyl radicals. In these experiments, pBR 322 was incubated
at 37 ºC for 1 h with a hydroxyl radical generating system (Fenton reaction) composed of ferrous
ions and hydrogen peroxide (final concentrations 0.06 µM ferrous ions and 22 µM hydrogen
peroxide) in 2-(N-morpholino)ethane sulfonic acid (MES) buffer, pH 6.0. The damaged OC form
of DNA was obtained in all cases (with or without antioxidants; data not shown), suggesting that
the antioxidants do not protect against hydroxyl radicals. ESR was also used to study hydroxyl
radical scavenging by the dendrimers. Hydroxyl radicals, generated via Fenton reaction (Fe2+/
H2O2) were reacted with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a nitrone spin-trap agent in
the absence (control) and presence of antioxidant. The DMPO-OH 1:2:2:1 signal suppression by
the antioxidant derivatized dendrimers was similar to control (methanol without antioxidants;
data not shown). The inability of the relatively hydrophobic dendritic antioxidants to scavenge
hydroxyl radicals may be due to the polar nature of the hydroxyl radicals compared to organic
carbon or nitrogen radicals.
3.3. Pro-oxidant effect
Many naturally available antioxidants including quercetin and vitamin C have been
reported to exhibit pro-oxidant property in the presence of transition metals ions (e.g., Cu2+ and
Fe3+) [25]. This antioxidant-induced production of free radicals may damage biomolecules such
as DNA and cause oxidative stress. The damaging effects on DNA can pose mutagenic and
carcinogenic health hazards [9-12]. Quercetin is one of the most abundantly obtained flavonoids
from dietary source and produces many beneficial health effects. However, it has been reported
to be associated with pro-oxidant effects [26]. Since our antioxidants were developed for
potential use in biological applications, we evaluated their pro-oxidant activity compared to
vitamin C and quercetin. This was performed by co-incubation of plasmid DNA (pBR 322) with
physiological amounts of Cu2+ ion (~10 µM) and antioxidants at 90, 45, 23, 12 uM at 37 °C for 1
11

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h. Figure 3 shows an agarose gel obtained with vitamin C and quercetin (A) and dendrimers (B-
D). In each panel, lane 1 is native DNA (negative control), lane 2 is native DNA incubated with
Cu2+ (positive control), lanes 3-6, native DNA incubated with Cu2+ and 90, 45, 23, and 12 µM
antioxidant, respectively, and lanes 7-10, native DNA incubated with Cu2+ and 90, 45, 23, and 12
µM antioxidant, respectively. Native DNA was mostly in the supercoiled form but contained
some open circular form (lane 1). DNA incubated with Cu(II) ions also displayed similar gels,
indicating that Cu(II) ion by itself does not damage DNA under these conditions (lane 2). As
shown in Figure 3A, vitamin C showed severe pro-oxidant effect. It generated the OC form at all
concentrations tested (lanes 3-6, 90, 45, 23, and 12 µM of vitamin C, respectively). High
concentrations even gave highly damaged linear DNA. Compared to vitamin C, quercetin
showed less pro-oxidant activity under these conditions. The intensity of SC band was very
similar to that of OC band at all concentrations (Fig. 3A, lanes 7-10; 90, 45, 23, and 12 µM
quercetin, respectively). Unlike the natural antioxidants, neither dendrimers 6a (lanes 3-6) nor 6b
(lanes 7-10) caused any damage to DNA, indicating that the dendrimers do not show pro-oxidant
effects under these conditions (Fig. 3B). Dendrimers 6c (lanes 3-6) and 7b (lanes 7-10) in Figure
3C and dendrimers 7c (lanes 3-6) and 7a (lanes 7-10) in Figure 3D also gave similar gel patterns
with no pro-oxidant effects. The PETN (5a) and pyranoside-derivatized core (5b) themselves did
not show any pro-oxidant effect on DNA under these conditions (data not shown). In summary,
all of the synthesized dendrimers did not produce the deleterious pro-oxidant effects regardless
of surface functionality. The lack of pro-oxidant effects displayed by our dendrimers is probably
due in part to their copper ion chelating capability. Copper chelation by dendrimers containing
multiple oxygen and nitrogen atoms is well-known. For example, polyamidoamine dendrimers
with multiple amide and tert-amino groups have been reported to possess strong copper chelation
properties [27]. Another evidence of copper chelation by our dendrimers was observed during
their click synthesis catalyzed by copper ions instead of granulated copper metal, which led to
strongly bound copper ions (detected by mass spectrometry) that were difficult to decontaminate.
These results demonstrate that chelation of copper ions by antioxidants can markedly reduce new
free radical formation. This is especially important in the chromatin region where one copper ion
is present per kilobase of DNA [28]. In addition, the phenols with bulky functional group(s) on
the surface of the dendrimers did not allow copper ions to bind for subsequent reduction. The
lack of pro-oxidant properties of 4-hydroxybenzaldehyde-derivatized dendrimers (6c and 7c) was
probably due to their weak reducing properties. As shown by the DPPH data and other
antioxidant activity tests, it is evident that these dendrimers (6c and 7c) do not quench radicals,
which means that they are not able to reduce copper ions to sustain the pro-oxidant activity.
These results indicate that 4-hydroxybenzaldehyde-derivatized dendrimers (6c and 7c) are
neither antioxidants nor pro-oxidants.
3.4. Cell testing
CHO-K1 cell cytotoxicity following exposure to dendrimers was evaluated using the 3-
(4,5-di-methylthizol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) conversion assay [29].
Exposure of cells (2.5 X 105/ml) to dendrimer 6a and 7a (1000 – 10 nM) and 6b, 6c, 7b, and 7c
(250 – 2.5 nM) caused no statistically significant change in cell viability for up to 5 days (Fig. 4).
This lack of toxicity results offer hope for the potential use of these novel compounds in
biological systems.
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4. Conclusion
The aim of this study was to evaluate effect of EDGs on the antioxidant behavior of
antioxidant dendrimers. We synthesized antioxidant dendrimers using PETN and pyranoside as
the cores and di-ethylene glycolic unit as spacers to help chelate metal ions. Syringaldehyde,
vanillin, and 4-hydroxybenzaldehyde were used as surface building blocks to furnish antioxidant
properties. The difference between the various building blocks is the number of electron
donating groups (methoxy groups) at ortho position with respect to the hydroxyl group on the
phenol ring. Based on the antioxidant activity tests, syringaldehyde-based dendrimers (with two
methoxy groups on each phenol ring) showed the best antioxidant activities, followed by
vanillin-derivatized dendrimers (one methoxy on each ring). 4-Hydroxybenzaldehyde derivatized
dendrimers (no methoxy group) showed virtually no antioxidant activity. The antioxidant activity
testing data provide important information on the structure-activity relationship of the
antioxidant dendrimers. Our results of this study clearly indicate that the existence of electron
donating group ortho (possibly para as well) to phenolic OH group is more important than
simply having multiple phenol rings. Although these dendrimers were effective in scavenging
organic radicals, they did not show any effects in quenching polar hydroxyl radicals. These
antioxidant dendrimers did not show any pro-oxidant side effects nor were they cytotoxic.
Conflict of Interest
The authors declare that there is no conflict of interest.
Acknowledgements
This work was supported by Award Number 1R15GM087697-01 and 3R15GM087697-
01S1 from the National Institute of General Medical Sciences. The content is solely the
responsibility of the authors and does not necessarily represent the official views of the National
Institute of General Medical Sciences or the National Institutes of Health.
List of abbreviations
AAPH: 2,2'-azobis(2-amidinopropane) dihydrochloride
CDCl3: Deuterated Chloroform
CHO-K1: Chinese hamster ovary cells
CuCl2: Cupric chloride
DMSO: Dimethylsulfoxide
DMF: Dimethylformamide
DMPO: 5,5-Dimethyl-1-pyrroline N-oxide
DMPO-OH: 5,5-Dimethyl-1-pyrroline N-oxide spin trap and hydroxyl radical adduct
DNA: Deoxyribonucleic acid
DPPH: 2,2-diphenyl-1-picrylhydrazyl
ESI-TOF: Electrospray ionization-time of flight
ESR: Electron spin resonance spectrometer
ET: Electron transfer
FeSO4: Ferrous sulfate
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HAT: Hydrogen atom transfer
H2O2: Hydrogen peroxide
LDL: Low density lipoprotein
MgSO4: Magnesium sulfate
MTT: 3-(4,5-Di-methylthizol-2-yl)-2,5-diphenyltetrazolium bromide
NaH: Sodium hydride
Na(OAc)3BH: Sodium triacetoxyborohydride
NaN3: Sodium azide
OC: Open circular form of DNA
PAGE: Polyacrylamide gel electrophoresis
pBR 322: Plasmid DNA
PBS: Phosphate-buffered saline
PETN: pentaerythritol
TEA: Triethylamine
SC: Supercoiled form of DNA
V/V: Volume to volume
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Figure and Scheme Legends
Scheme 1. Building block synthesis.
Scheme 2. Polyol-based core synthesis (a) NaN3, DMF/H2O= 40:1, 70 – 80 °C; (b) CHCl3, Tosyl
chloride, TEA, 0 °C; (c) NaH, anhydrous DMF
Scheme 3. Dendrimer synthesis
Fig. 1. Effect of antioxidants on LDL oxidation; lane 1 (native LDL); lanes 2 (AAPH-oxidized
LDL with no antioxidant), lane 3-8 (6a, 6b, 7a, 7b, vitamin C, and quercetin at 70 µM,
respectively).
Fig. 2. Protection against AAPH-induced DNA oxidation by antioxidants.
Lane 1(native DNA); lane 2 (AAPH-oxidized DNA, no antioxidant); lanes 3-6 (AAPH-oxidized
DNA with 90, 45, 23, 12 µM syringaldehyde-derivatized dendrimers (6a, 7a), respectively);
lanes 7-10 (AAPH-oxidized DNA with 90, 45, 23, 12 µM vanillin-derivatized dendrimers (6b,
7b), respectively); lanes 11-14 (AAPH-oxidized DNA with 90, 45, 23, 12 µM 4-HBA-
derivatized dendrimers (6c, 7c), respectively).
Fig. 3. Pro-oxidant effects of antioxidants on DNA (pBR 322)
(A) Lane 1 (native DNA), lane 2 (native DNA + Cu2+ ion), vitamin C (lanes 3–6 contain 90, 45,
23, and 12 µM, respectively) and quercetin (lanes 7–10 contain 90, 45, 23, and 12 µM,
respectively)
(B) Same as A, but with dendrimers 6a (lanes 3–6) and 6b (lanes 7–10)
(C) Same as A, but with dendrimers 6c (lanes 3–6) and 7b (lanes 7–10)
(D) Same as A, but with dendrimers 7c (lanes 3–6) and 7a (lanes 7–10).
Fig. 4. Comparative effects of dendrimers on CHO-K1 cell viability. CHO-K1 cells were
incubated with DMSO control, or dendrimer 6a or 7a (1000 nM), 6b, 6c, 7b, or 7c (250 nM) for
1, 3, or 5 days. Cytotoxicity was measured using the MTT conversion assay. Control values are
represented as 100% viability. Data represents mean ± SEM. A p < 0.05 was considered
statistically significant.
18

ACCEPTED MANUSCRIPT
Anode (+)
1
2
3
4
5
6
7
8

ACCEPTED MANUSCRIPT
SC
Linear
OC
Vit. C
Quercetin
6a
6b
7b
6c
7c
7a
1
2
3 4 5
6
7 8 9 10 11 12 13 14

ACCEPTED MANUSCRIPT
SC
Linear
OC
(A)
(B)
(C)
(D)
1
2 3
4
5
6 7
8
9 10

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
O
H
O
N
OH
O
O
O
OH
O
O
(Syringaldehyde)
HO
1a
O
H
O
O
N
OH
Na(OAc)3BH,
THF, 45 o
OH
(Vanillin)
C
NH2
O
HO
1b
O
H
N
OH
OH
(4-hydroxybenzaldehyde)
HO
1c

ACCEPTED MANUSCRIPT
N3
O
O
O
O
N3
OH
HO
N3
O
O
OH
O
HO
O
(Pentaerythritol)
(c)
5a
N3
O
O
O
S
O
(b)
N3
(a)
O
O
N3
HO
Cl
HO
N3
OH
O
O
O
4
2
3
O
HO
OH
O
O
O
OH
O
(D-Gluco-pyranoside)
O
O
O
N3
(c)
N3
O
5b
N3

ACCEPTED MANUSCRIPT
1a
1b
1c
Microwave energy
(150-250 W)
1a
1b
1c
Microwave energy
(150-250 W)
7a
7b
7c
6a
6b
6c
7 h, 250 psi, 85 o
C
7 h, 250 psi, 85 o
C
5a
5b
O
HO
HO
HO
O
O
OH
O
OH
O
OH
N
N
N
O
N
N
N
N
N
N
N
N
N
HO
HO
O
O
HO
O
O
O
O
OH
O
OH
O
O
OH
N
O
N
N
O
N
N
O
N
N
N
N
O
N
N
N
N
O
O
O
N
O
O
N
N
O
O
N
N
N
O
O
O
N
O
N
O
N
O
N
N
O
HO
O
HO
O
HO
O
O
O
O
O
N
O
OH
O
OH
OH
N
N
N
N
N
N
O
N
N
HO
N
HO
N
O
HO
N
O
O
O
O
OH
OH
OH
6a
6b
6c
HO
O
HO
HO
O
O
O
O
O
OH
N
OH
OH
N
N
N
N
N
N
N
O
N
N
N
N
OH
O
O
OH
O
OH
O
O
O
O
O
HO
O
O
O
HO
O
O
O
O
O
HO
O
O
O
O
O
O
N
O
O
N
N
N
O
N
N
O
O
N
O
O
O
N
N
O
N
N
N
O
N
N
N
O
N
N
O
N
N
O
N
N
N
O
N
N
OH
O
OH
O
HO
OH
O
O
O
HO
N
HO
N
N
N
N
N
N
N
O
N
N
O
OH
N
N
OH
O
OH
O
HO
HO
O
HO
O
7c
7a
7b

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
Formation of powerful polyphenolic macromolecular antioxidants by assembling various
weak phenolic antioxidants into dendritic architecture.
Dendritic antioxidants showed strong free radical scavenging due to multiple phenolic
units and no pro-oxidant effects due to their metal chelation properties.
Dendritic antioxidants with more electron donating groups showed stronger antioxidant
effects
Effective against carbon and nitrogen-based radicals but not hydroxyl radicals.