Copper-granule-catalyzed microwave-assisted click synthesis of polyphenol dendrimers

Article abstract of DOI:10.1021/jo401603d

Syringaldehyde- and vanillin-based antioxidant dendrimers were synthesized via microwave-assisted alkyne-azide 1,3-dipolar cycloaddition using copper granules as a catalyst. The use of Cu(I) as a catalyst resulted in copper contaminated dendrimers. To produce copper-free antioxidant dendrimers for biological applications, Cu(I) was substituted with copper granules. Copper granules were ineffective at both room temperature and under reflux conditions (<5% yield). However, they were an excellent catalyst when dendrimer synthesis was performed under microwave irradiation, giving yields up to 94% within 8 h. ICP-mass analysis of the antioxidant dendrimers obtained with this method showed virtually no copper contamination (9 ppm), which was the same as the background level. The synthesized antioxidants, free from copper contamination, demonstrated potent radical scavenging with IC₅₀ values of less than 3 μM in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. In comparison, dendrimers synthesized from Cu(I)-catalyzed click chemistry showed a high level of copper contamination (4800 ppm) and no detectable antioxidant activity.

Full text of DOI:10.1021/jo401603d

Article
pubs.acs.org/joc
Copper-Granule-Catalyzed Microwave-Assisted Click Synthesis of
Polyphenol Dendrimers
Choon Young Lee,* Rich Held, Ajit Sharma, Rom Baral, Cyprien Nanah, Dan Dumas, Shannon Jenkins,
Samik Upadhaya, and Wenjun Du*
Department of Chemistry, Central Michigan University, Mount Pleasant, Michigan 48859, United States
S
* Supporting Information
ABSTRACT: Syringaldehyde- and vanillin-based antioxidant
dendrimers were synthesized via microwave-assisted alkyne−
azide 1,3-dipolar cycloaddition using copper granules as a
catalyst. The use of Cu(I) as a catalyst resulted in copper
contaminated dendrimers. To produce copper-free antioxidant
dendrimers for biological applications, Cu(I) was substituted
with copper granules. Copper granules were ineffective at both
room temperature and under reflux conditions (<5% yield).
However, they were an excellent catalyst when dendrimer
synthesis was performed under microwave irradiation, giving yields up to 94% within 8 h. ICP-mass analysis of the antioxidant
dendrimers obtained with this method showed virtually no copper contamination (9 ppm), which was the same as the
background level. The synthesized antioxidants, free from copper contamination, demonstrated potent radical scavenging with
IC50 values of less than 3 μM in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. In comparison, dendrimers synthesized from
Cu(I)-catalyzed click chemistry showed a high level of copper contamination (4800 ppm) and no detectable antioxidant activity.
synthesized syringaldehyde- and vanillin-based dendrimers
using copper iodide or copper sulfate/sodium ascorbate as
the catalyst. However, due to Cu(I) contamination, the
antioxidant dendrimers exhibited no antioxidant activities.
To circumvent the use of Cu(I) catalyst, a variety of copper-
free click reactions have been developed. These alternative
protocols often require high temperature and pressure. In
addition, the regioselectivity between 1,4-disubstitution and
1,5-disubstitution is unpredictable.14,15 Recently, Bertozzi et al.
reported the use of cyclooctyne and its derivatives for copper-
free, orthogonal click reactions. The reaction was highly
efficient even when performed in vivo;16 the relief of the ring
strain is a powerful driving force that links cyclooctyne to azide.
This strategy has also been successfully employed in several
other fields.17−21 Another approach that avoids the use of
Cu(I) is copper nanoparticles,22−26 which are often used in
combination with a ligand, a base,26 and/or ultrasound27,28 to
enhance the rate of the reaction. For example, Yus and co-
workers reported that, when 10 mol % copper nanoparticles
with sizes of 1−5 nm were used in the presence of
triethylamine (TEA), nearly quantitative yields were obtained
within 30 min.26 Furthermore, the reaction also demonstrated
excellent regioselectivity. However, the nanoparticles with small
sizes and, therefore, large surface areas were susceptible to
oxidation, especially when the samples were handled for a
prolonged period. The oxidized products, CuO and/or Cu2O
were ineffective in the catalysis of the click reaction.26 In
INTRODUCTION
■
Syringaldehyde and vanillin are phenolic aldehydes derived
from the breakdown of lignin. Syringaldehyde has various
beneficial biological effects, such as antimicrobial activity,
antioncogenic effects, and antioxidant activity.1 Vanillin has
been reported to possess antimutagenic, antiangiogenetic,
anticolitis, antisickling, and antianalgesic effects.2 Both of
these compounds are weak antioxidants.3 However, when
assembled into dendritic forms, the antioxidant activities are
remarkably improved.4,5
We previously synthesized antioxidant (AO) dendrimers
using “reductive amination”4,5 However, this synthesis method
gave a very low reaction rate and efficiency, especially for
dendritic structures with a high number of branches. For
example, reactions involving four or more simultaneous
reductive aminations gave yields of less than 5% after a two-
week reaction.
Cu(I)-catalyzed azide−alkyne cycloaddition, namely, click
chemistry, has emerged as one of the most orthogonal and
efficient synthesis tools used today. It is especially useful in
large molecule synthesis, such as polymer−polymer coupling,6
antibody−antibody coupling,7 and in dendrimer synthesis.8−11
Despite numerous successes, one of the major limitations of
click chemistry is copper contamination from the use of Cu(I)
as a catalyst. Copper is toxic to certain enzymes. Furthermore,
it has been found that the interaction of antioxidants with
Cu(I) can trigger cascade radical reactions to form a variety of
radicals, resulting in an adverse oxidation process. This
deleterious property of an antioxidant is known as the pro-
oxidant effect.4,5,12,13 In our preliminary studies, we have
Received: July 25, 2013
Published: October 15, 2013
© 2013 American Chemical Society
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comparison, the use of air-stable, large-sized copper metal, such
as copper strips or copper granules (1−5 μm), was ineffective,
giving no product.26 The small surface area of these bulky
catalysts is likely attributed to the limited alkyne−copper
interactions. To use the copper metal (wire) as a catalyst, it had
to be used along with ligand(s) or have an already existing
triazole ring on the reactant(s), which act as a base or ligand.29
Weck and co-workers also reported the use of copper wire as a
catalyst in dendron synthesis, but the copper metal had to be
used along with sodium ascorbate as well as sulfonated
bathophenanthroline as a ligand to drive the reaction.8 In
another study, copper metal was used together with CuSO4 for
the synthesis of small monomeric species under microwave
energy.30 In the reaction, Cu(I) was generated in situ by
comproportionation of Cu(0) and Cu(II). Microwave irradi-
ation helped achieve excellent reaction efficiency.
Scheme 1. Syntheses of Building Blocks 1a and 1b
Microwave energy has been widely employed in organic
syntheses to accelerate reaction rates and to improve reaction
yields.31,32 It has also been used to increase the molecular
weights in polymer syntheses.33,34 Under microwave irradiation,
polar molecules rapidly change their orientations, allowing
functional groups to efficiently interact with larger copper
particles. We hypothesize that the use of microwave energy may
enable bulky copper granules to effectively catalyze the click
synthesis of dendritic antioxidants. Herein, we report a
microwave-assisted copper-granule-catalyzed click reaction for
the synthesis of syringaldehyde- and vanillin-based dendrimers.
The copper level in the synthesized dendrimers and their
antioxidant activities were then discussed.
to reduce the Cu(I) to Cu(0) to facilitate the decontamination.
Unfortunately, none of the methods was successful in complete
removal of the copper. The copper ion contamination of an
antioxidant can be detrimental to its antioxidant activity. It has
been reported that antioxidants can reduce Cu(II) to Cu(I).
When these newly formed ions come into contact with O2
molecules, superoxide radicals are produced,36−38 which can
further react with more antioxidants to produce hydrogen
peroxide in the presence of water. Additional Cu(I) ions
present will ultimately convert the hydrogen peroxide to
hydroxyl radicals, triggering a cascade free radical reaction,
consuming the remaining antioxidants. Because of this reason,
dendrimers synthesized via Cu(I)-catalyzed click chemistry did
not exhibit any antioxidant activities.
Previous reports showed that the use of copper wire
minimizes copper contamination.29,39 Therefore, we substituted
Cu(I) with small copper granules (0.2−0.6 mm diameter) as
the catalyst to synthesize our dendrimers. Unfortunately, even
at elevated temperature (80 °C), the syntheses with copper
granule catalyst only gave poor yields (less than 5%) even after
a two-week reaction. The sluggish reaction and the low yields
are likely the result of the limited accessibility of the copper
catalyst to azide/alkyne functionalities.
Microwave energy has been widely employed to accelerate
reactions and to improve reaction yields in small molecule
organic syntheses.31,32 It has also been employed in polymer
synthesis to increase molecular weights by improving the
accessibility of the reactive functionalities in large polymer
molecules.33,34,40 We hypothesized that, if microwave energy
was applied, the reaction rate and efficiency may improve. To
test the hypothesis, we first evaluated the reaction between 1a
and 3 (Scheme 2) on a small scale. At low microwave energy
(200 W), the reaction was sluggish. Even after reacting for 6 h,
there was little product formed, as monitored by UPLC-ESI.
The reaction efficiency improved with increasing reaction time:
after 7 h, the yield increased to 10%; after 8 h, the yield reached
30% (Figure 1). Encouraged by these results, the microwave
RESULTS AND DISCUSSION
■
Synthesis of Building Blocks and Cores. To synthesize
antioxidant dendrimers via alkyne−azide click chemistry,
alkyne-derived building blocks 1a and 1b were first synthesized
through reductive amination of propargylamine with syringal-
dehyde and vanillin, respectively (Scheme 1; for synthetic
details, please see the Supporting Information).
Next, a triazido core (3) was synthesized with trimethylol-
propane triglycidyl ether (2) and sodium azide (Scheme 2).
Compound 2 contains three epoxide rings, and each can be
opened at two possible positions. Under these reaction
conditions, the azide ion only attacked the less-hindered
carbons of the epoxide rings, as verified by 2D COSY and
HETCOR NMR spectroscopy (Figures S7 and S8 in the
Supporting Information). These results are consistent with
those reported previously.35 To test the versatility of the
copper-granule-/microwave-assisted click synthesis, a second
core (6) was synthesized under similar conditions, using 1,3,5-
triglycidyl isocyanurate as the starting triepoxide (Scheme 3).
Synthesis of G1 Dendrimers. Using building blocks (1a
and 1b) and cores (3 and 6), four novel dendrimers (4a, 4b, 7a,
and 7b) were synthesized via click chemistry (Schemes 2 and
3). The syntheses of dendrimers were initially carried out with
either CuI/DIPEA (in anhydrous THF) or CuSO4/sodium
ascorbate (in DMF-H2O, v/v = 9/1). For each method, the
amounts of copper catalyst used were 0.3, 0.6, and 3 equiv (0.1,
0.2, and 1 equiv for each branch, respectively). Both systems
gave fairly good yields (60−85%). However, the final
dendrimers were found to be contaminated with copper ions.
Various approaches were attempted to decontaminate the
dendrimers, including the use of Sephadex LH-20 (size
exclusion chromatography) and 1 M HCl to displace Cu(I)
from the dendrimer. Sodium borohydride was also used, aiming
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Scheme 2. Syntheses of Dendrimers 4a and 4b
Scheme 3. Syntheses of Dendrimers 7a and 7b
energy was increased to 250 W. The reaction rate was not
significantly different during the first 6 h. However, the yields
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Figure 1. Microwave-assisted synthesis of dendrimer 4a (left) and 7a (right) (the percent yields were determined based on UPLC chromatograms,
as indicated in the Experimental Section).
Table 1. Reaction Conditions and Yields for Cu-Granule-Catalyzed Click Chemistry
a
core
building block
product
time (h)
max temp. (°C)
microwave energy (W)
yield (%)
3
3
6
6
1a
1b
1a
1b
4a
4b
7a
7b
8
8
8
8
77
75
77
73
250
250
250
250
77
78
85
82
a
Based on isolated yields after silica gel column chromatography purification.
were much better after 7 h (45%) and 8 h (84%) (Figure 1). A
further increase in the microwave energy (300 W) did not seem
to provide much improvement (Figure 1).
On the basis of these results, we concluded that 250 W
microwave energy was optimal to “activate” the copper-granule-
catalyzed click reaction and avoid side reactions. Subsequent
large-scale syntheses of syringaldehyde- and vanillin-based
antioxidant dendrimers were performed at 250 W. The results
are summarized in Table 1.
In all cases, there was no product detected by ESI before the
first 6 h of reaction. Because of the large number of reactions
that occur simultaneously on each molecule, it is not surprising
that there is no detectable target product at the early stage of
the reaction. Nevertheless, compared to regular heating, this is a
significant improvement in the synthesis of complex
dendrimers involving multiple alkyne and azide groups. The
use of microwave energy thus provides an efficient and
convenient approach for macromolecule synthesis. Although
the theory behind the microwave effect is controversial, the
generally accepted consensus is that, under microwave
irradiation, polar molecules rapidly change their orientations
according to the microwave frequency. This movement causes
higher molecular friction, resulting in faster and more efficient
reactions.33,34 Furthermore, in the synthesis of macromolecules,
such as dendrimers and polymers, the reactive functional
groups are often wrapped inside the molecules, thereby limiting
their accessibility. Irradiation of molecules with microwave
energy can rapidly change their orientations and increase the
availability of the reactive functionalities, resulting in rapid
reaction rates, high yields, and high molecular weight.40−45
Other factors, such as superheating, and the decrease of bond
dissociation energy by increasing polar bond vibration, may also
be the major contributions for high efficiency of the microwave
reaction.33,34
The formation of copper-contaminated product is a major
dilemma in copper-catalyzed click reactions, especially for the
compounds with functional groups that can chelate metal ions
and used for biological applications. All of our synthesized
dendrimers reported in this study contain many polar
functional groups in the core as well as triazole rings that
may chelate metal ions. Therefore, we analyzed our purified
dendrimers for copper contamination using ICP-MS. The
copper impurity presented in the product synthesized via
copper-granule-catalyzed microwave reaction was only 9.1 ppm.
In comparison, the dendrimer from CuI-catalyzed ‘click’
synthesis contained a very high level of copper (4800 ppm).
As a control, we also measured the copper level in the G1
dendrimer synthesized via reductive amination, which involves
no copper catalyst. Surprisingly, the level of copper
contamination in this dendrimer was 6.2 ppm. Since no copper
was used for its synthesis, this low level of copper may have
come from solvents. Indeed, all solvents (hexane, acetone, ethyl
acetate, and methanol) contained small amounts of copper (4−
31 ppm). On the basis of these results, it can be concluded that
the copper-granule-catalyzed microwave-assisted method does
not contribute to copper contamination. The reference range
for serum copper in humans is about 2 ppm. In pregnant
females, it may be as high as 3 ppm.46 Even if we injected as
much as 1 g of our dendrimer (with 9.1 ppm copper
contamination) into a human being, the serum copper level
will only increase by 0.002 ppm, which is negligible.
To test the versatility of the copper-granule-catalyzed,
microwave-assisted click synthesis, we also evaluated the
reaction between 1a and 6. As expected, similar results were
observed. The maximum yields obtained after 8 h of reaction at
200, 250, and 300 W were 79, 83, and 94%, respectively (Figure
1). It should be noted that, although the reaction under 300 W
microwave energy gave a high yield, we observed the color of
the reaction solution change to dark brown. UPLC analysis also
suggested the formation of byproducts.
Other studies with copper wire as the catalyst showed much
higher copper contamination (ca. 408 and 450 ppm29)
compared to our antioxidant dendrimers (9.1 ppm). We are
uncertain of this discrepancy. We used copper granules that
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were prewashed with 25% NaOH, deionized water, 20%
H2SO4, deionized water, and acetone and were dried with
argon. The resulting bright, pinkish copper granules were used
immediately after washing. In addition, our reaction was
conducted in ultrapure THF (obtained from a solvent
purification system) under argon without any ligand or base
added. Previous reports suggest that copper metal in the click
reaction is an active source of Cu(I).29 The mechanism by
which copper metal is converted to Cu(I) that is required for
click chemistry catalysis is still unclear. It has been postulated
that there is an initial oxidation of the copper metal surface to
Cu(I), which then catalyzes the reaction and, in turn, becomes
Cu(II). The Cu(I) ion is regenerated via comproportionation
of Cu(0) and Cu(II).29,47 In another study, sodium ascorbate
was used to reduce Cu(II), generated from copper wire, to
Cu(I).8 It should be noted that, although we did not add
sodium ascorbate, our reactants (1a and 1b) are also reducing
agents that could reduce Cu(II) to Cu(I). We believe that,
under our conditions and using prewashed copper granules,
only a catalytic amount of copper ions was generated to
catalyze the reaction, thereby limiting copper contamination.
Nonetheless, the detailed reaction mechanism and low level
copper contamination warrants further investigation.
The use of copper granules in click chemistry coupled with
microwave energy offers a simple, convenient, and economical
method for the synthesis of large molecules such as dendrimers.
The results described herein can provide an important
foundation for the syntheses of other antioxidant dendrimers
for biological applications using alkyne−azide click chemistry.
Thus, the use of copper granules as the catalyst allowed us to
solve the long-term problem of product contamination with
copper ions in click chemistry.
EXPERIMENTAL SECTION
■
General Information. Trimethylolpropane triglycidyl ether and
1,3,5-triglycidyl isocyanurate were purchased from commercial sources
and were purified by silica gel flash column chromatography prior to
use (purification methods were described in detail under compound 3
and compound 6 synthesis).
1H NMR spectra were recorded at 500 MHz and 13C NMR spectra
at 125 MHz. Chemical shifts were reported in parts per million (ppm)
with reference to the TMS peak.
ESI mass spectra were obtained using a source capillary voltage of
3000 V, cone voltage of 10 V, and source temperature of 80 °C.
Samples analyzed by flow injection had a desolvation gas temperature
of 250 °C and a gas flow rate of 200 L/h. 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/μL.
The LC-MS contained a diode array detector at 210−400 nm. A
linear gradient from 100% solution A to 80% solution B was used over
4 min at a flow rate of 0.5 mL/min. Solution A consisted of 90% water,
10% acetonitrile, and 0.1% formic acid. Solution B was 100%
acetonitrile with 0.1% formic acid. The injection volume was 3 μL
with a sample concentration of approximately 10 ng/μL. The
conjoined MS unit to the LC had a desolvation gas temperature of
350 °C and a flow rate of 600 L/h. All UPLC samples were filtered
through PTFE syringe filters (0.45 μm).
All microwave-assisted reactions were performed using a microwave
reactor with predetermined reaction parameters. Reaction temper-
atures were recorded with an external surface sensor.
The copper granules (0.2−0.6 mm, ≥99.8%) used in the microwave
reactions were pretreated in the following order: soaked in 25% NaOH
for 20 min, rinsed with deionized water, soaked in 20% H2SO4 for 20
min, rinsed with a copious amount of deionized water, rinsed with
acetone, dried with argon, and used immediately after.
The copper contaminations were measured using an ICP-MS. The
samples (ca. 2 mg) were digested in concentrated HNO3 (2 mL, ICP
grade) for 18 h. The solutions were then diluted to 25 mL using
nanopure water. A standard curve was built using a known
concentration of copper in 4% HNO3 (purchased from a commercial
source), and the levels of copper in each sample were calculated based
on the standard curve.
IC50 values for the antioxidants were determined by DPPH
reduction as previously reported.48 The DPPH reagent and all of
the antioxidants, except for vitamin C, were prepared in methanol
solutions. Vitamin C was in deionized water. The antioxidant sample
(25 μL) was added to 1.2 mL of reagent, and the absorbance was
measured at 517 nm after 1 h. All samples were run in triplicate at
room temperature. The within-run coefficient of variation of the
percent inhibition values was less than 6%.
Small-Scale Synthesis to Optimize Microwave Reaction
Conditions. Dendrimer 4a. Triazide core 3 (0.201 g, 0.466 mmol)
and building block 1a (0.574 g, 1.50 mmol) were dissolved in
anhydrous THF (25 mL). The solution was then split equally into five
35 mL capacity microwave reaction vessels. Additional anhydrous
THF (10 mL) and a stir bar were added to each vessel. Freshly
pretreated Cu(0) granules (0.53 g, 8.41 mmol), as mentioned above,
were added just before each reaction in the microwave reactor, and the
reaction vessel was charged with argon. The microwave reaction was
run at varying conditions. Aliquots (100 μL) were removed at
predetermined time intervals. The aliquots were filtered through a
The final synthesized dendrimers (4a, 4b, 7a, and 7b) free
from Cu(I) contamination were tested for their antioxidant
activities with the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay.
IC50 values of dendrimers 4a, 4b, 7a, and 7b were 2.8, 2.5, 2.2,
and 3.0 μM, respectively. In comparison, the Cu(I)-
contaminated dendrimers synthesized from Cu(I)-catalyzed
click reactions exhibited no detectable antioxidant activities
(Table 2). In addition, our novel dendritic antioxidants showed
Table 2. DPPH Assay Results for Dendrimer Antioxidants
antioxidant
IC50 (μM)
a
a
dendrimer 4a
2.8
dendrimer 4b
2.5
a
dendrimer 7a
dendrimer 7b
2.2
a
3.0
b
dendrimer 7a
quercetin
≫1000
9.0
vitamin C
16.5
a
Synthesized from copper-granule-catalyzed and microwave-assisted
b
click reaction. Synthesized from Cu(I)-catalyzed click reaction.
significantly stronger radical scavenging abilities than the
naturally occurring and popular antioxidants quercetin (IC50
= 9.0 μM) and vitamin C (IC50 = 16.5 μM), under similar
assay conditions.
CONCLUSION
■
The use of solid copper granules in place of Cu(I) was found to
effectively catalyze alkyne−azide click chemistry. Although the
catalyst was ineffective under conventional heating, the rates
and yields were significantly improved when microwave energy
was applied for 8 h. Based on UPLC and NMR analyses, the
purity of each final dendrimer (4a, 4b, 7a, and 7b) was above
98%. Most importantly, the dendrimers synthesized from this
method were virtually copper-free. As a result, these dendrimers
exhibited excellent antioxidant activities. Another advantage
associated with this synthesis strategy is that no additional
ligand or base additives were necessary, thus further simplifying
the click reactions.
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PTFE filter (0.45 μm) to remove any particulates, dried, and diluted in
a solution of water−acetonitrile (9:1) for analysis on UPLC-ESI.
Integrations of UPLC-ESI peaks were compared to a standard curve of
known concentrations to calculate reaction yields.
Dendrimer 7a. Triazide core 6 (0.203 g, 0.523 mmol) and building
block 1a (0.640 g, 1.5 mmol) were dissolved in anhydrous THF (25
mL). The solution was then split equally into five 35 mL capacity
microwave reaction vessels. Other conditions were the same as those
used for dendrimer 4a optimization.
a gradient hexane−acetone mobile phase (5/1 → 2/1). The
compound 3 was dissolved in methanol (5 mL) and further purified
on a 30 g prepacked C18 reverse phase flash chromatography column.
The column was eluted with water (50 mL), followed by a CH3OH−
H2O gradient (2:8, 1 L → 3:7, 1 L) at 15 mL/min. The purity of each
fraction was determined by reverse phase HPLC before combining
identical ones.
Rf = 0.36 (hexane−acetone = 2:1); Yield 40% (5.74 g, clear colorless
1
oily substance); H NMR (500 MHz, CDCl3) δ 3.99−3.88 (m, 3H),
3.53−3.30 (m, 18H), 3.09 (s, 3H), 1.40 (q, J = 7.6 Hz, 2H), 0.85 (t, J
= 7.6 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 72.6, 72.3, 69.4, 53.3,
43.3, 23.3, 7.6; HPLC: a single peak at 22:01 min; HRMS (ESI-TOF)
m/z: [M + H]+ Calcd for C15H30N9O6, 432.2319; found, 432.2324.
Dendrimer 4a. Core 3 (0.12 g, 0.28 mmol), building block 1a (0.36
g, 0.93 mmol), granulated copper metals (1.59 g, 25.2 mmol), and a
stir bar were placed in a 35 mL capacity microwave reaction vessel.
The copper granules were pretreated as mentioned above. Ultrapure
THF (20 mL) was added to the mixture in the reaction vessel. The
reaction vessel was charged with argon, and the microwave reaction
was run with conditions set at a maximum temperature of 85 °C, an
energy of 250 W, a maximum pressure of 250 psi, and a ramping time
of 10 min in power ON mode (reaction is controlled by microwave
energy) for 8 h. Final temperatures reached in the reaction vessel were
77 °C at 250 W. The reaction mixture was filtered through Celite, and
the filtrate was dried under reduced pressure. The resulting oily
substance was resuspended in acetone (5 mL), loaded onto a 40 g
prepacked silica gel column, and purified by flash column
chromatography. The solvent systems were hexane (100 mL),
hexane−ethyl acetate = 1:1 (0.5 L), ethyl acetate (1 L), and ethyl
acetate−methanol = 9:1 (2 L). The purification was repeated using the
hexane (100 mL) and ethyl acetate−methanol = 9:1 (2 L).
Rf = 0.19 (ethyl acetate−methanol = 8:2); Yield 77% (0.34 g,
yellowish amorphous substance); 1H NMR (500 MHz, CDCl3) δ 7.59
(d, J = 1.3 Hz, 3H), 6.61 (s, 12H), 4.56−4.27 (m, 6H), 4.10 (d, J = 2.7
Hz, 3H), 3.82 (s, 36H), 3.72 (s, 6H), 3.52 (s, 12H), 3.42−3.19 (m,
12H), 2.17 (s, 3H), 1.28 (d, J = 7.4 Hz, 2H), 0.77 (t, J = 7.5 Hz, 3H);
13C NMR (126 MHz, CDCl3) δ 146.8, 144.9, 133.5, 129.9, 124.1,
105.4, 72.3, 72.3, 71.5, 68.9, 57.7, 56.1, 52.7, 47.8, 43.2, 22.7, 7.4;
HPLC: single peak at 15.74 min; HRMS (ESI-TOF) m/z: [M + H]+
Calcd for C78H105N12O24, 1593.7365; found, 1593.7345.
Standard curves were made with previously synthesized pure
compounds (4a and 7a) and analyzed in triplicate. The curves
contained three data points of 5, 10, and 17.5 ng/μL and 5, 10, and 15
ng/μL for compounds 4a and 7a, respectively.
Large-Scale Synthesis of Antioxidant Dendrimers. Building
Block 1a (4,4′-((Prop-2-yn-1-ylazanediyl)bis(methylene))bis(2,6-
dimethoxyphenol)). Propargylamine (0.88 g, 16.10 mmol) was
added dropwise to syringaldehyde (3.55 g, 19.50 mmol) dissolved in
1,2-dichloroethane (200 mL). The reaction was stirred for 30 min, and
then sodium triacetoxyborohydride (3.42 g, 16.14 mmol) was added.
The reaction mixture was stirred at rt for 24 h. A second equivalent of
syringaldehyde (3.53 g, 19.40 mmol) was added. After 24 h of stirring,
a second equivalent of sodium triacetoxyborohydride (3.51 g, 16.56
mmol) was added. After 48 h of stirring, the reaction mixture was
washed twice with deionized water. The organic layer was collected
and dried with anhydrous MgSO4. After the solution was filtered, the
filtrate was condensed under reduced pressure. The resulting residue
was redissolved in 5 mL of chloroform and purified by flash column
chromatography (with a 40 g prepacked silica gel cartridge). A
gradient hexane−ethyl acetate (5:1 → 1:1) solvent system was used as
the mobile phase to afford the product as a white crystal (5.5 g, 88%).
Rf = 0.28 (hexane−acetone = 2:1); 1H NMR (500 MHz, CDCl3) δ
6.63 (s, 4H), 5.48 (s, 2H), 3.89 (s, 12H), 3.59 (s, 4H), 3.30 (d, J = 2.4
Hz, 2H), 2.01 (s, 1H); 13C NMR (126 MHz, CDCl3) δ 147.1, 133.9,
129.9, 105.8, 78.8, 73.7, 57.7, 56.5, 41.4; HPLC: single peak at 18.5
min; HRMS (ESI-TOF) m/z: [M + H]+Calcd for C21H26NO6,
388.1760; found, 388.1757.
Building Block 1b (4,4′-((Prop-2-yn-1-ylazanediyl)bis(meth-
ylene))bis(2-methoxyphenol)). Vanillin (3.50 g, 23.02 mmol) was
allowed to react with propargylamine (0.80 g, 14.55 mmol) in THF
(200 mL) overnight at 40−50 °C. After the reaction was cooled to
room temperature, sodium triacetoxyborohydride (3.10 g, 14.62
mmol) was added. From this step and on, heat was no longer used
and the reaction was finished with the same method as building block
1a synthesis.
Dendrimer 4b. To a solution of triazide core 3 (0.200 g, 0.464
mmol) in dry THF (20 mL) were added building block 1b (0.520 g,
1.590 mmol), pretreated Cu(0) granules (2.63 g, 41.7 mmol), and a
stir bar to a 35 mL capacity microwave reaction vessel. The reaction
vessel was flushed with argon, and the microwave reaction was run
with conditions set at a maximum temperature of 85 °C, an energy of
250 W, a maximum pressure of 250 psi, and a ramping time of 10 min
in power ON mode for 8 h. The reaction mixture was filtered through
Celite, and the filtrate was dried under reduced pressure. The reaction
mixture dissolved in 5 mL of chloroform was purified by column
chromatography with a 40 g prepacked silica gel column. The solvent
systems that were used were a hexane−ethyl acetate (1:1, 0.5 L), ethyl
acetate (1 L), ethyl acetate−methanol (9:1, 1 L), and finally ethyl
acetate−methanol (8:2, 1 L). The purification was repeated using the
hexane (100 mL) and ethyl acetate−methanol = 9:1 (2 L).
Rf = 0.52 (hexane−ethyl acetate = 1:1); Yield 57% (2.72 g, light
1
yellow powdery substance); H NMR (500 MHz, CDCl3) δ 6.92 (s,
2H), 6.87−6.84 (m, 4H), 3.86 (s, 6H), 3.59 (s, 4H), 3.26 (d, J = 2.3
Hz, 2H), 2.17 (s, 1H); 13C NMR (126 MHz, CDCl3) δ 146.4, 144.7,
130.4, 121.9, 114.0, 111.5, 78.5, 73.4, 57.1, 55.8, 40.8; HPLC: single
peak at 17.8 min; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for
C19H22NO4, 328.1549; found, 328.1562.
Compound 3 (3,3′-((2-((3-Azido-2-hydroxypropoxy)methyl)-2-
ethylpropane-1,3-diyl)bis(oxy))bis(1-azidopropan-2-ol)). Commer-
cially available technical grade trimethylolpropane triglycidyl ether
(triepoxide 2) was purified on a silica gel column. The column was first
treated with hexane−ethyl acetate (5:1) containing 2% triethylamine
(v/v, 200 mL), followed by hexane (100 mL). Triepoxide 2 was
purified on the column using hexane−ethyl acetate as the mobile
phase (5:1). The purified compound 2 (Rf = 0.35, hexane−acetone =
3:1) was characterized with GC-MS and 1H/13C NMR and
corresponded to literature values.
To compound 2 (10 g, 33.11 mmol) dissolved in DMF (200 mL)
were added NaN3 (7.5 g, 115.38 mmol), NH4Cl (5.5 g, 102.8 mmol),
and deionized water (5 mL). The reaction was run at 50 °C overnight.
On completion of the reaction (revealed by the absence of triepoxide 2
on TLC), the reaction mixture was filtered. The filtrate was dried, and
the resulting oily substance was resuspended in acetone to further
precipitate any remaining solids. The solution was filtered, and the
filtrate was condensed under reduced pressure. The resulting crude
mixture was purified using silica gel flash column chromatography with
Rf = 0.46 (ethyl acetate−methanol = 8:2); Yield 78% (0.51 g, light
1
pinkish amorphous substance); H NMR (500 MHz, d6-acetone) δ
7.90 (s, 3H), 7.04 (d, J = 1.7 Hz, 6H), 6.84 (dd, J = 8.0, 1.8 Hz, 6H),
6.78 (d, J = 8.0 Hz, 6H), 4.59 (dd, J = 14.0, 3.4 Hz, 6H), 4.44 (dd, J =
14.0, 7.2 Hz, 6H), 4.19 (s, 3H), 4.05 (q, J = 7.1 Hz, 3H), 3.82 (s,
18H), 3.69 (s, 6H), 3.48 (s, 12H), 3.38 (d, J = 1.7 Hz, 6H), 3.18 (s,
6H), 1.97 (s, 3H), 1.19 (t, J = 7.1 Hz, 2H), 0.85 (t, J = 7.4 Hz, 3H);
13C NMR (126 MHz, d6-acetone) δ 148.1, 146.3, 145.1, 131.5, 125.2,
122.2, 115.4, 113.0, 73.5, 72.1, 69.8, 57.6, 56.1, 53.7, 48.1, 44.1, 23.4,
8.0; HPLC: 98.9%, major peak at 15.67 min, minor peak at 16.92 min;
HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C72H93N12O18,
1413.6731; found, 1413.6746.
Compound 6 (1,3,5-Tris(3-azido-2-hydroxypropyl)-1,3,5-triazi-
nane-2,4,6-trione). 1,3,5-Triglycidyl isocyanurate (triepoxide 5) is
commercially available as technical grade (∼70% pure). The
11226
dx.doi.org/10.1021/jo401603d | J. Org. Chem. 2013, 78, 11221−11228

The Journal of Organic Chemistry
Article
compound was purified on a silica gel column prior to use. The
column was pretreated with hexane−ethyl acetate (5:1) containing 2%
triethylamine (v/v, 200 mL), followed by hexane (100 mL). Hexane−
ethyl acetate (5:1) was used as the mobile phase. The purified
compound 5 (Rf = 0.66 in hexane−acetone = 1:1) was characterized
peaks at 14.01 and 16.81 min; HRMS (ESI-TOF) m/z: [M + H+]
Calcd for C69H82N15O18, 1408.5962; found, 1408.5952.
ASSOCIATED CONTENT
■
1
by GC-MS and H/13C NMR and corresponded to literature values.
S
* Supporting Information
1H/13C NMR spectroscopy data for all of the synthesized
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Synthesis of compound 6 was carried out by reaction of the purified
triepoxide 5 (5.5 g, 18.52 mmol) with NaN3 (4.21 g, 64.77 mmol) and
NH4Cl (3.00 g, 56.08 mmol) in DMF (200 mL). Deionized water (5
mL) was added as a cosolvent, and the reaction was run overnight at
room temperature. The reaction mixture was filtered after compound
6 was confirmed by ESI. The filtrate was dried under reduced pressure.
The resulting oily substance was resuspended in acetone to further
precipitate any remaining salts. After the filtrate was evaporated on a
rotovap, it was purified on a silica gel column that was pretreated with
a hexane−acetone (4:1) containing 2% triethylamine. The column was
then run with a hexane−acetone gradient solvent system (5:1 → 1:1).
Rf = 0.59 (hexane−acetone = 1:1); Yield 55% (4.3 g, clear colorless
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: lee1cy@cmich.edu.
*E-mail: du1w@cmich.edu.
Notes
The authors declare no competing financial interest.
1
oily substance); H NMR (500 MHz, CDCl3) δ 4.24−3.85 (m, 9H),
3.60−3.36 (m, 9H); 13C NMR (126 MHz, CDCl3) δ 150.1, 68.8, 54.6,
46.4; HPLC: single peak at 14.47 min; HRMS (ESI-TOF) m/z: [M +
H]+ Calcd for C12H19N12O6, 427.1551; found, 427.1557.
ACKNOWLEDGMENTS
■
This work was funded by the National Institutes of Health and
the National Institute of General Medical Sciences (Award
Number 1R15GM087697-01 and 3R15GM087697-01S1). 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.
Dendrimer 7a. Core 6 (0.20 g, 0.47 mmol), building block 1a (0.56
g, 1.45 mmol), copper metal granules (2.67 g, 42.3 mmol, prewashed
as previously mentioned), ultrapure THF (20 mL), and a stir bar were
placed into a 35 mL microwave reaction vessel. The vessel was flushed
with argon. The microwave reactor was set at an energy of 250 W, a
maximum pressure of 250 psi, a maximum temperature of 85 °C, and a
ramping time of 10 min. The reaction was run for 8 h in power ON
mode. The reaction mixture was then filtered through Celite to
remove copper, and the filtrate was condensed under reduced
pressure. The remaining residue was redissolved in acetone (5 mL),
loaded onto a 40 g prepacked silica gel column, and purified using flash
column chromatography. The solvent systems were hexane (0.1 L),
hexane−ethyl acetate = 1:1 (0.3 L), ethyl acetate (0.6 L), and ethyl
acetate−methanol = 9:1 (2 L). The flow rate was 20 mL/min. The
compound was further purified by column chromatography using
hexane (0.1 L) and ethyl acetate−methanol = 9:1 (2 L).
Rf = 0.35 (ethyl acetate−methanol = 8:2); Yield 85% (0.63 g, white
powdery substance); 1H NMR (500 MHz, acetone-d6) δ 7.90 (s, 3H),
7.09 (s, 3H), 6.71 (s, 12H), 4.82 (s, 6H), 4.55 (d, J = 10.2 Hz, 3H),
4.36 (d, J = 9.4 Hz, 6H), 4.06−3.83 (m, 6H), 3.78 (s, 36H), 3.67 (s,
6H), 3.48 (s, 12H); 13C NMR (126 MHz, d6-acetone) δ 148.6, 146.5,
143.3, 133.5, 128.6, 123.2, 104.7, 66.3, 56.0, 54.6, 52.3, 46.2, 45.0;
HPLC: single peak at 14.56 min; HRMS (ESI-TOF) m/z: [M + H]+
Calcd for C75H94N15O24, 1588.6597; found, 1588.6594.
Dendrimer 7b. Triazide core 6 (0.18 g, 0.423 mmol), building
block 1b (0.43 g, 1.315 mmol), prewashed copper granules (2.40 g,
3.81 mmol), anhydrous THF (20 mL), and a stir bar were added to a
35 mL microwave reaction vessel at room temperature. The reaction
vessel was flushed with argon. The microwave reaction was run with
power ON mode at a maximum of 85 °C, 250 psi, and 250 W for 8 h.
The reaction mixture was then filtered through Celite to remove
copper, and the filtrate was removed under reduced pressure. The
resulting residue was redissolved in acetone (5 mL), loaded onto a 40
g prepacked silica gel column, and purified by flash column
chromatography. The solvent systems were hexane (0.2 L), hexane−
ethyl acetate = 1:1 (0.5 L), ethyl acetate (1 L), and ethyl acetate−
MeOH = 9:1 (1 L). The compound was further purified by the
column chromatography using hexane (0.1 L) and ethyl acetate−
methanol = 9:1 (2 L).
Rf= 0.35 (ethyl acetate−methanol = 8:2); Yield 82% (0.49 g, white
powdery substance); 1H NMR (500 MHz, acetone-d6) δ 7.91 (s, 3H),
7.52 (s, 3H), 7.04 (d, J = 1.3 Hz, 6H), 6.87−6.80 (m, 6H), 6.76 (d, J =
8.0 Hz, 6H), 4.85 (s, 6H), 4.59 (d, J = 10.3 Hz, 3H), 4.38 (d, J = 9.2
Hz, 6H), 3.98 (ddd, J = 18.6, 13.6, 5.9 Hz, 6H), 3.82 (s, 18H), 3.66 (s,
6H), 3.47 (s, 12H); 13C NMR (126 MHz, acetone-d6) δ 150.6, 150.6,
148.2, 146.3, 145.3, 131.6, 125.2, 122.2, 115.4, 113.0, 68.4, 57.6, 56.1,
54.3, 48.1, 47.0; HPLC: 98.5%, major peak at 14.56 min, two minor
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