Development of microwave-assisted reactions for PAMAM dendrimer synthesis

Article abstract of DOI:10.1002/ejoc.201101717

PAMAM dendrimers up to G1 have been synthesized by the divergent method taking advantage of microwave irradiation and using the CoolMate^TM accessory to avoid high temperatures and long reaction times, which usually lead to defects in the dendrimer structure. Two tools have been successfully used to determine the purity of the products in addition to NMR spectroscopy, namely MALDI-TOF mass spectrometry and capillary-zone electrophoresis. These techniques show that the dendrimers were obtained in good yields, high purity, and with near-perfect regioselectivity. Molecular engineering on a nanoscale level to produce PAMAM dendrimers that can be used as drugs requires the use of technologies that lead to highly defined products. PAMAM synthesis can be assisted by microwave irradiation at low temperature to reduce the reaction time significantly while reducing the occurrence of defects in the PAMAM skeleton.

Full text of DOI:10.1002/ejoc.201101717

FULL PAPER
DOI: 10.1002/ejoc.201101717
Development of Microwave-Assisted Reactions for PAMAM Dendrimer
Synthesis
[
a]
[a,b]
[c]
[a]
Javier López-Andarias, Javier Guerra,*
Gregorio Castañeda, Sonia Merino,
Valentín Ceña,*[ and Prado Sánchez-Verdú*
b,d]
[a]
Keywords: Dendrimers / Synthetic methods / Materials science / Microwave chemistry
PAMAM dendrimers up to G1 have been synthesized by the
used to determine the purity of the products in addition to
NMR spectroscopy, namely MALDI-TOF mass spectrometry
and capillary-zone electrophoresis. These techniques show
that the dendrimers were obtained in good yields, high pu-
rity, and with near-perfect regioselectivity.
divergent method taking advantage of microwave irradiation
TM
and using the CoolMate
accessory to avoid high tempera-
tures and long reaction times, which usually lead to defects
in the dendrimer structure. Two tools have been successfully
Introduction
PAMAM dendrimers starts with a 1,4-Michael addition re-
action between the central core [usually ammonia or ethyl-
enediamine (EDA)] and methyl acrylate. The resultant
product with three or four ester groups is then fully amid-
ated by using an excess of ethylenediamine to give a mole-
cule with three or four reactive amine groups. High-genera-
tion dendrimers can be obtained by repeating the Michael
addition and amidation reactions in an iterative fashion.
The whole ensemble of properties will be driven by the
Dendrimers are monodisperse macromolecules with a
regular and highly branched three-dimensional architecture
and surface functionality. These systems are prepared by
repetition of a given set of reactions using either diver-
[
1–3]
[4–12]
gent
or convergent strategies.
Nowadays, dendri-
mers are used in fields ranging from materials and synthetic
chemistry to biological and physical chemistry as a result
of their unique features such as the precise control of size
and shape, uncommon physical properties, and the place-
ment of numerous functional groups on the periphery and/
choice of core, the number of repetitive branches, and the
[17]
functional groups on the periphery.
The last feature is
controlled by the amine derivative used in the final amid-
ation reaction. PAMAM dendrimers with hydroxy, amine,
and acid groups at the surface of the molecule are commer-
[
13]
or core.
However, a significant limitation in the synthesis of large
dendrimers is that quantitative reactions are required. Con-
sequently, the types of dendritic structures that are commer-
cially available, and therefore widely used, are extremely
limited. Poly(amidoamine) (PAMAM) dendrimers are cer-
tainly the most widely used type of dendrimer. These mate-
rials are based on a dendritic mixed structure that contains
tertiary alkylamines as branching points and secondary
®
cially available (Dendritech , Inc., MI).
Unfortunately, structural deviations are inevitable during
the synthesis of the ethylenediamine-core PAMAM dendri-
[18]
mers, as reported by other researchers. Thus, the mono-
dispersity of commercially available PAMAM dendrimers
[19,20]
was recently found to be low.
The purity of commercial
PAMAM dendrimers has been well studied and it has been
clearly shown that their purity is far from ideal, even for
low generation dendrimers (G1 or G1.5).
amides as chain extension points, and they are synthesized
by a divergent approach.[
14–16]
Typically, the synthesis of
Dendrimer purification has always been a problematic
[
21]
[
a] Departamento de Química Inorgánica, Orgánica y Bioquímica, issue and recent work
has also highlighted the need for
Facultad de Química, Universidad de Castilla-La Mancha,
better synthetic methodologies, purification, and characteri-
1
3071 Ciudad Real, Spain
Fax: +34-926295318
zation tools that will allow the application of dendrimers in
E-mail: mariaprado.sanchez@uclm.es
b] NanoDrugs, S.L., Paseo de la Innovación 1,
Campus Universitario, 02006 Albacete, Spain
[22–24]
sensitive fields such as nanomedicine.
In fact, poly-
[
[
amide-based dendrimers are of particular interest because
c] Departamento de Química Analítica, Facultad de Química, they are based on a peptide-like amide backbone and have
Universidad de Castilla-La Mancha,
3071 Ciudad Real, Spain
[25–29]
proven low toxicities and non-immunogenicities.
1
[d] Unidad Asociada Neurodeath, Farmacología, Universidad de
Nevertheless, the aforementioned PAMAM dendrimers
have to be prepared in tedious multistep syntheses and this
is clearly a limiting factor for most applications. The con-
ventional synthesis of PAMAM requires long reaction
Castilla-La Mancha, Campus Universitario,
0
2006 Albacete, Spain
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101717.
Eur. J. Org. Chem. 2012, 2331–2337
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2331

J. Guerra, V. Ceña, P. Sánchez-Verdú et al.
FULL PAPER
times and new technology is needed to accelerate the syn- temperatures (293 K). Thus, G0 and G1 PAMAM synthesis
thetic methodology. Even the synthesis of the different can be assisted by microwave irradiation to reduce the reac-
building blocks necessary for the assembly and construction tion time significantly. The synthesized dendrimers were an-
of asymmetrical PAMAM dendrimers in a convergent man- alyzed by NMR spectroscopy, MALDI-TOF MS, and cap-
ner demands long reaction times and numerous reac- illary-zone electrophoresis. These techniques showed that
[
30,31]
tions.
Furthermore, better control is required to reduce the dendrimers were obtained in good yields, high purity,
the occurrence of defects in the PAMAM skeleton.
and with near-perfect regioselectivity.
Since 1986, microwave chemistry has experienced ex-
ponential growth, both in industry and academia, as an ef-
ficient heating method widely used in chemistry.[32] Micro-
wave irradiation is a well-known non-invasive and clean
Results and Discussion
processing tool that has been exploited to activate or accel-
Although, as reported before, PAMAM dendrimers are
erate chemical reactions.[ Such activation can provide an commercially available, there is a lack of homogeneity and
33]
[
19,20]
extended reaction range with lower temperatures and/or re- monodispersity.
Therefore improved synthetic proto-
duced reaction times, which can in turn obviate unwanted cols were developed to achieve the low dispersity required
[
22]
side-reactions and products due to thermal effects and lead for biological applications. The synthetic strategy for PA-
to accrued cost savings.[
34,35]
This technology has recently MAM dendrimers is shown in Scheme 1. The intermediate
been used for the grafting of PAMAM dendrons onto the ester dendrimers (G-0.5 and G0.5) are not depicted.
[
36]
surface of silica leading to reduced reaction times relative
to classical conditions.[
G0 and G1 PAMAM dendrimers were synthesized by the
standard divergent strategy using EDA as the core.
37]
[14,16,41]
In this study microwave technology was used to synthe- This methodology involves the typical stepwise and iterative
size G0 and G1 PAMAM dendrimers with the aim of re- two-step reaction sequences mentioned above: i. The
ducing the reaction time and, consequently, minimizing the Michael addition of primary amines to methyl acrylate to
possible byproducts that might form during the synthetic produce methyl ester terminal groups in half-generations
sequence. PAMAM dendrimers are known to be sensitive to and ii. the amidation of the resulting methyl ester groups
heat and undergo retro-Michael addition reactions at high with a large excess of EDA (full generations). Attempts to
[
38]
temperature.
In this work we took advantage of three synthesize PAMAM dendrimers according to literature
key facts: i. The use of a monomode microwave reactor is methods led to the products but extremely long reaction
[
38,42,43]
associated with a more efficient and reproducible heating times were required.
Our improved synthesis is less
mechanism as the microwave irradiation is guided directly time-consuming and the product purity meets the standard
to the inner region of the reaction mixture; ii. the use of an achieved by using an organic method.
optical fiber to measure the temperature in the reaction;
the choice of method to measure this parameter has proven cules such as methanol leads to an increase in the reaction
The absorption of microwave irradiation by polar mole-
[
33,44]
critical for obtaining real and reproducible results when temperature.
In an effort to avoid the high tempera-
compared with the conventional heating of reactions under tures that could result from microwave irradiation, we used
[
39]
TM
TM
microwave conditions; iii. the use of the CoolMate
cessory
ac- the CoolMate
accessory. This consists of a reactor
[
40]
allows the use of microwave technology at low equipped with a jacketed low-temperature vessel sur-
Scheme 1. Reagents and conditions: (a) Methyl acrylate/ethylenediamine (10:1), methanol (1 mL), MW irradiation (10 W): 2ϫ45 min, T
338 K; (b) G-0.5 dendrimer/ethylenediamine (1:10), methanol (1.35 mL), MW irradiation (40 W): 6ϫ45 min, T = 293 K; (c) G0 den-
drimer/methyl acrylate (1:100), methanol (1 mL), MW irradiation (10 W): 5ϫ45 min, T = 338 K. (d) G0.5 dendrimer/ethylenediamine
1:330), methanol (1 mL), MW irradiation (40 W): 17ϫ45 min, T = 293 K.
=
(
2332
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 2331–2337

PAMAM Dendrimer Synthesis
rounded by a microwave-transparent cooling medium and reaction while maintaining low temperatures. The tempera-
chilling technology to keep the bulk temperature low (193– ture measured by using the optical fiber controls the irradi-
[
40]
338 K).
This accessory therefore enables sub-ambient ation power by means of CEM software. The two plots in
temperature chemistry to be performed in microwave sys- Figure S2 in the Supporting Information show the corre-
tems. Thus, the thermal degradation of materials in tem- lation between temperature and power in a cycle of 45 min.
perature-sensitive reactions may be prevented when micro-
wave energy is applied to a reaction mixture.
One of the main problems in the field of dendrimers con-
cerns the determination of the degree of purity. Even in the
Four methods were chosen for our synthesis and these case of low-generation dendrimers (as in our case), 1
H
are summarized as follows: i. Method I, classical round- NMR spectroscopy has proven to be an unsuitable tool for
bottomed flask at 293 K, ii. method II, microwaves as the this purpose as the different side-products that could form
TM
energy source in conjunction with the CoolMate
access- in the synthetic procedure or incomplete dendrimers display
ory to keep the temperature at 293 K, iii. method III, meth- NMR signals in similar regions as the pure target dendri-
anol at reflux at 338 K, and iv. method IV, methanol at re- mers. This drawback, combined with signal degeneracy and
flux with microwaves as the energy source. A known and the broadness of signals due to different fluxional processes,
proportional amount of p-tert-butylphenol with respect to makes NMR spectroscopy a technique that it is not suitable
[
19]
the limiting reactant was used as an internal standard to alone for characterizing these macromolecules. Two tools
1
follow the progress of the reaction by H NMR spec- have been successfully used to complement the information
troscopy (see Figure S1 in the Supporting Information as obtained by NMR, namely MALDI-TOF MS and electro-
[
45]
an example).
phoresis.
The mass spectra of full PAMAM dendrimers G0 and
The results obtained in the PAMAM dendrimer synthesis
following the methods described above are summarized in G1, synthesized by Method II, are shown in Figure 1. Sin-
Table 1. Shorter reaction times are clearly achieved by using gle peaks and the corresponding signals with the exact mass
microwave irradiation. In particular, the amidation reaction plus the mass of Na or K atoms reveal the high degree of
is accelerated and this effect is indicative of an improvement purity achieved with our procedure. MALDI-TOF MS is a
in the first step of this reaction, which involves a nucleo- very useful technique as it helps to determine the presence
philic substitution. This reaction is an illustrative example of different dendrimers and side-products in the reactions.
of a bimolecular reaction that proceeds from neutral reac- Although this technique is very sensitive, the information
tants to a charged transition state and the finding is consis- that it provides will depend on the ionization efficacy, the
tent with similar results in this type of reaction, as reviewed susceptibility of the target compounds to absorb at the laser
by Perreux and Loupy.[
44]
wavelength, the choice of matrix, and other instrumental
parameters. This technique is therefore appropriate for
Table 1. Reaction times needed to complete the different synthetic
steps.
PAMAM Method I Method II
dendrimer
Method III Method IV
G-0.5
G0
G0.5
G1
270 min
180 min
270 min
405 min
12 h 45 min
90 min
n.a.
225 min
n.a.
90 min
n.a.
225 min
n.a.
[
a]
[b]
4–5 d
2 d
[
a]
4–5 d
[
a] See ref.[38,42,43]. [b] n.a.: not applicable.
It was also observed that the amidation reaction is the
critical step in the synthetic sequence. Strict control of the
temperature is needed in this reaction and high tempera-
tures unequivocally lead to dendrimer degradation. Thus,
a substantial modification of the reaction temperature is
required to obtain highly pure products. In the synthesis of
full generation dendrimers with ethylenediamine, special
care has to be taken that the temperature does not rise
above 293 K. Reactions that were not controlled in terms
of temperature gave high levels of byproducts, as deter-
mined by electrophoresis (see below). Therefore the meth-
ods that we finally considered appropriate in terms of short
reaction time and degree of purity were as follows:
Method III or IV for the 1,4-Michael addition and
Method II for the amidation reactions.
The use of microwave heating in conjunction with the
Figure 1. MALDI-TOF spectra of (a) G0 and (b) G1 PAMAM
dendrimers, both synthesized by method II. Intensity is expressed
TM
CoolMate
accessory (Method II) is an approach that
takes advantage of a more efficient way of accelerating the in arbitrary units.
Eur. J. Org. Chem. 2012, 2331–2337
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2333

J. Guerra, V. Ceña, P. Sánchez-Verdú et al.
FULL PAPER
structural identification but its main drawback is that it simple approach, the separation of ions is proportional to
cannot provide quantitative data. In our case, it is expected the charge-to-mass ratio. The electrophoretic behavior is
that the high level of structural similarity between the pure also influenced by the pH as this determines the final
products and the side-products will lead to similar behavior charge-to-mass ratio.
with this analytical technique.
The analytical methodology was similar that used by
It is critical in the approach described here to remove Tomalia[46] and Ebber[47] and their co-workers, with the
any excess methyl acrylate in the 1,4-Michael addition reac- main difference being the use of hydrodynamic injection to
tions and excess ethylenediamine after amidation as the introduce the sample in our case instead of electrokinetic
presence of these reactants will give rise to dendrimers of injection. Our procedure involved the introduction of all
lower generations with respect to the desired product. The components of the sample in the proportion in which they
MALDI-TOF mass spectrum recorded after the synthesis are present, irrespective of the ionic strength of the solution
of the G1 PAMAM dendrimer is shown in Figure S3 in the and their electrophoretic mobilities. In electrokinetic injec-
Supporting Information. This spectrum clearly reveals the tion the quantity loaded is dependent on the electrophoretic
presence of the G0 dendrimer, which results from the inef- mobility of solutes. Discrimination occurs for ionic species
ficient removal of the aforementioned reactants.
because the more mobile ions are loaded to a greater extent
The electropherograms of the different synthesized den- than the less mobile ions. As a result, sample loading is
drimers are shown in Figure 2 (a–d). The presence of the dependent on the electro-osmotic flow, sample concentra-
main peaks in these plots indicates that our synthetic pro- tion, and sample mobility. Variations in conductivity, which
cedures (method III or IV for the 1,4-Michael addition and could be due to matrix effects, result in differences in volt-
method II for the amidation reactions) were highly regiose- age drop and quantity loaded. These phenomena generally
lective. However, we also noticed the presence of minor im- make electrokinetic injection less reproducible than the
purities, which could be a result of intramolecular cycliza- hydrodynamic counterpart and could provide an undesir-
tion, dimerization, or retro-Michael reactions. This obser- able lack of information in the study of purity and stability
[
46,47]
vation is consistent with the results obtained by others.
In any case, capillary-zone electrophoresis (CZE) analyses
of the compounds of interest.
To enhance the sensitivity and resolution, an easy on-
are useful for discriminating those methods that involve the capillary sample concentration was performed just after
use of high temperatures in the amidation reaction (Fig- sample injection by dissolving the sample in low conductiv-
ure S4).
ity buffer [1:10 (v/v) phosphate running buffer/water]. This
CZE is based on the displacement of charged molecules method is based on field strength differences between the
dissolved in a buffer at a known pH under the influence of sample zone and the running buffer and it is the simplest
an electric field. The separation of compounds depends on way to perform sample stacking after application of a sepa-
the electro-osmotic flow and electrophoretic mobility. In a ration voltage.
Figure 2. Electropherograms of G-0.5 (a), G0 (b), G0.5 (c), and G1 (d) PAMAM dendrimers. Ester-terminated dendrimers (G-0.5 and
G0.5) were synthesized by method IV and amine-terminated dendrimers were obtained by method II. Intensity is expressed in arbitrary
units.
2334
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PAMAM Dendrimer Synthesis
The half-generation dendrimers terminated with ester generations by using this strategy for tailored applications.
groups were analyzed at neutral pH (7.2) because, at this CZE and MS were used to determine the purity of the den-
pH value, the strength of the phosphate buffer was higher drimers synthesized. The potential of CZE to follow the
(pKa = 7.2). The full-generation dendrimers were com- ester degradation under neutral buffer conditions has also
pletely ionized at pH 2.7 and, at this pH, the electro-os- been demonstrated and this could be of interest in bio-
motic flow was practically suppressed, which led to a better logical applications.
degree of separation.
As reported by Ebber et al.,[ we found evidence for the
47]
hydrolysis of the ester-terminated dendrimers in the pres- Experimental Section
ence of phosphate buffer at neutral pH (Figure 3). This hy-
Materials and Methods: Ethylenediamine, methanol, p-tert-but-
drolysis is based on the presence of esterases and proteases
ylphenol, toluene, deuterated methanol, deuterated chloroform, so-
of different cells that can exist in the buffer media and the
dium phosphate monobasic, sodium phosphate dibasic (Sigma–Al-
rapid rate of this hydrolytic process is apparent in Figure 3.
drich), and methyl acrylate (99%, stabilized with 200 ppm MEHQ)
were used as received. All experiments were performed in air unless
specified otherwise.
Buffer solutions were prepared with 18 MΩcm Milli-Q deionized
water (Millipore) by dissolving the appropriate amount of sodium
phosphate monobasic and sodium phosphate dibasic (0.02 m phos-
phate buffer for pH 7.2 and 0.1 m phosphate buffer for pH 2.7).
These solutions were filtered before use through 25 mm syringe-
driven filters (0.4 μm pore, Millipore).
Characterization: NMR spectra were recorded with a Varian Unity
1
Inova NMR spectrometer operating at 499.7722 MHz for
H
1
3
NMR and at 125.678 MHz for C NMR. The spectrometer was
equipped with a gradient amplifier and a four-nucleus 5 mm
H{ N P} high-field indirect detection probe. All 1D H experi-
1
15 31
1
ments and 2D experiments (COSY, HMBC, HSQC) were per-
formed at 298 K using standard pulse sequences from the Varian
library. Deuterated methanol (CD
and deuterated chloroform (CDCl
3
OD, 99.98%, Sigma–Aldrich)
, Sigma–Aldrich) were used as
3
solvents to perform the corresponding experiments. A known and
proportional amount of p-tert-butylphenol with respect to the lim-
iting reactant was used as an internal standard to follow the pro-
gress of the reaction.
MALDI-TOF mass spectra were measured with a Bruker Autoflex
II TOF/TOF spectrometer (Bremen, Germany) by using 2Ј,4Ј,6Ј-
trihydroxyacetophenone (THAP) as the matrix. Samples co-crys-
tallized with the matrix on the probe were ionized with a nitrogen
laser pulse (337 nm) and accelerated under 20 kV with time-delayed
extraction before entering the time-of-flight mass spectrometer.
Matrix (10 mg/mL) and sample (1 mg/mL) were separately dis-
solved in methanol and mixed in a matrix/sample ratio ranging
from 100:1 to 50:1. Typically, a 5 μL mixture of matrix and sample
was applied to a MALDI-TOF MS probe and air-dried. MALDI-
TOF MS in positive reflector mode was used for all dendrimers.
Figure 3. Time evolution observed by CZE of (a) G-0.5 and
b) G0.5 PAMAM dendrimers dissolved in 0.02 m phosphate buffer
at pH 7.2. Color code: black: 0 min; red: 150 min; green: 280 min;
blue: 260 min. Intensity is expressed in arbitrary units.
(
Conclusions
MW irradiation leads to a reduction in reaction times in
the syntheses of G0 and G1 PAMAM dendrimers in com-
parison with classical conditions. The amidation reaction is External calibration was performed by using Peptide Calibration
Standard II (covered mass range: 700–3200 Da), Protein Cali-
the critical step in the synthetic sequence. Strict control of
bration Standard I (covered mass range: 5000–17500 Da) and Pro-
the reaction temperature is required. Reactions were per-
formed under microwave irradiation with a Discover™
CEM monomode system coupled with the CoolMate™ ac-
cessory, which allows the use of microwaves at low tempera-
tures. Application of this methodology allowed us to
tein Calibration Standard II (covered mass range: 20000–
70000 Da) from Care (Bruker). The applied peak (m/z determi-
nation) detection method was the threshold centroid at 50% height
of the peak maximum.
Capillary-zone electrophoretic (CZE) analyses were performed
with a Beckman P/ACE 5510 instrument (Analytical Chemistry
Department, UCLM) coupled to a data acquisition system. The
system was controlled with a Dell Dimension P133V card and data
treatment was carried out with P/ACE station software. The sepa-
ration was performed with uncoated fused-silica capillaries, 47 cm
achieve
days
a reduction in reaction time from several
[
38,42,43,48]
to approximately 13 h for the amidation step
to give the G1 PAMAM dendrimer in good yield and high
purity. It was also demonstrated that an increase in the tem-
perature of the 1,4-Michael addition reduced the time re-
quired without altering the regioselectivity of the reaction. (40 cm effective length)ϫ75 μm internal diameter in a cartridge
We are currently working towards the synthesis of higher with a detection window (100ϫ800 μm). UV absorption was mea-
Eur. J. Org. Chem. 2012, 2331–2337
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2335

J. Guerra, V. Ceña, P. Sánchez-Verdú et al.
FULL PAPER
sured at 212 nm. pH measurements were carried out with a Crison
pH meter (2002 model) with glass electrodes.
225 min (5ϫ45 min) at 318 K. A condenser was fitted to the micro-
wave reactor flask. The solvent was removed under vacuum along
with the excess methyl acrylate. The rotary evaporator bath tem-
CZE Procedure: Half-generations of dendrimers were analyzed in
perature was maintained at 313 K. The product was a colorless oil
0.02 m phosphate buffer (pH 7.2) and for full-generation dendri-
1
(
2
1
(
yieldϾ 0.232 g, 95%). H NMR (500 MHz, CDCl
3
, Me
CONH), 2.44 (t, J = 7.5 Hz,
COOMe), 2.49 (s, 4 H, NCH CH N), 2.54
CH N), 2.76 (t, J = 7.5 Hz,
COOMe, NCH CH CONH), 3.27 (dt, 8 H,
N), 3.67 (s, 24 H, COOCH
): δ = 32.8, 32.9, 37.4, 49.5, 50.0, 51.9, 52.5, 53.2,
4
Si): δ =
mers, 0.1 m phosphate buffer (pH 2.7) was used. In general, the
.36 (t, J = 5 Hz, 8 H, CH
6 H, CH CH
2 2
CH
method of analysis was similar to the procedure described by Tom-
[
46]
[47]
2
2
2
2
alia
capillary was rinsed with 0.1 m NaOH for 20 min, Milli-Q water
10 min) and phosphate buffer (10 min, pH 2.7 or 7.2, depending
and Ebber
and their co-workers. Prior to first use, the
t, J = 5 Hz, 8 H, CONHCH
2
2
2
4 H, NCH
2
CH
2
2
2
(
1
3
1
CONHCH
(
2
CH
2
3
) ppm. C{ H} NMR
on the sample). The capillary was rinsed between samples with
Milli-Q water (1 min) and phosphate buffer (2 min) to avoid ad-
sorption processes on the capillary inner surface. Samples were in-
troduced by means of hydrodynamic injection (5 s). For full-genera-
tion dendrimers, a voltage of 30 kV was applied and in the case of
half-generation dendrimers the voltage was 10 kV at 293 K.
125 MHz, CDCl
3
1
+
72.5, 173.2 ppm. MS (MALDI-TOF): m/z (%) = 1206.1 (100) [M
+
+
+
H] , 1228.2 (63) [M + Na] , 1244.4 (16) [M + K]
.
PAMAM Dendrimer Synthesis (G1): A solution of G0.5 PAMAM
dendrimer (0.10 g, 0.09 mmol) and ethylenediamine (1.798 g, 2 mL,
3
4
0 mmol) in methanol (1 mL) was irradiated at 40 W for 12 h and
The samples for analysis were prepared by dissolving dendrimer
TM
5 min (17ϫ45 min) at 293 K. The Discover
CoolMate access-
(4 mg) in low conductivity buffer [4 mL; 1:10 (v/v) running buffer/
ory was coupled to the microwave reactor. The solvent was re-
moved under vacuum and an azeotropic mixture of toluene/meth-
anol (10 mL, 9:1, v/v) was added to the crude mixture to remove
the excess ethylenediamine (three times) by rotary evaporation.
Finally, methanol (10 mL) was added and then removed under
vacuum. The rotary evaporator bath temperature was maintained
at 313 K. The product was a yellowish oil (yieldϾ 0.113 g, 95%).
milliQ-water] and were applied immediately after dissolution.
Dendrimer Synthesis: Reactions were performed under microwave
TM
irradiation in a Discover
CEM monomode reactor with an IR
temperature controller (for 1,4-Michael reactions). In the amid-
TM
ation reactions, the Discover
CoolMate accessory was coupled
to the microwave reactor. In this case, an optical fiber was used to
control the temperature.
1
H NMR (500 MHz, CD OD, Me Si): δ = 2.36 (t, J = 7.5 Hz,
3
4
2
(
4 H, NCH
t, J = 5 Hz, 4 H, CONHCH
CONHCH CH NH ), 2.74 (t, J = 7.5 Hz, 24 H, NHCH
NHCH CH N), 2.80 (t, J = 7.5 Hz, 24 H, NCH CH CONH), 3.25
t, J = 7.5 Hz, 24 H, CONHCH CH ) ppm. C{ H} NMR
125 MHz, CD OD): δ = 33.3, 33.4, 37.2, 41.5, 49.7, 49.8, 49.9,
1.0, 52.1, 173.3, 173.8 ppm. MS (MALDI-TOF): m/z (%) = 1430.0
2
CH
2
CONH), 2.54 (s, 4 H, NCH
CH N), 2.59 (t, J = 5 Hz, 8 H,
CH NH
2 2
CH N), 2.56
PAMAM Dendrimer Synthesis (G-0.5): A solution of ethylenedi-
amine (0.13 g, 2.11 mmol) and methyl acrylate (1.82 g, 21.1 mmol)
in methanol (1 mL) was irradiated at 10 W for 90 min (2ϫ45 min)
at 318 K. A condenser was fitted to the microwave reactor flask.
The solvent was removed under vacuum and an azeotropic mixture
of toluene/methanol (10 mL, 9:1, v/v) was added to the crude mix-
ture to remove the excess ethylenediamine (three times) by rotary
evaporation. Finally, methanol (10 mL) was added and then re-
moved under vacuum. The rotary evaporator bath temperature was
2
2
2
2
2
2
2
2
,
2
2
2
2
3
1
1
(
(
5
2
2
3
+
+
+
(
50) [M + H] , 1452.0 (100) [M + Na] , 1468.0 (15) [M + K]
.
Supporting Information (see footnote on the first page of this arti-
cle): NMR spectra of the G0 PAMAM dendrimer synthesized by
using method II, measurements of temperature and power during
an irradiation cycle, MALDI-TOF MS for a mixture of G0 and
maintained at 313 K. The product was a colorless oil (yieldϾ
1
0
=
.840 g, 95%). H NMR (500 MHz, CDCl
3
, Me
COOMe), 2.29 (s, 4 H, NCH
CH COO), 3.46 (s, 12 H, COOCH
C{ H} NMR (125 MHz, CDCl ): δ = 32.6, 49.8, 51.4, 52.3,
72.9 ppm. MS (MALDI-TOF): m/z (%) = 405.0 (100) [M + H] ,
4
Si): δ = 2.24 (t, J
7.5 Hz, 8 H, CH
t, J = 5 Hz, 8 H, NCH
2
CH
2
2
CH N), 2.56
2
(
2
2
3
) ppm. G1 PAMAM dendrimers, CZE plots of different synthetic method-
1
3
1
3
ologies for the G0 PAMAM dendrimer.
+
1
4
+
+
27.0 (10) [M + Na] , 443.0 (2) [M + K]
.
Acknowledgments
PAMAM Dendrimer Synthesis (G0): A solution of G-0.5 PAMAM
dendrimer (0.10 g, 0.24 mmol) and ethylenediamine (1.50 g,
We acknowledge the Spanish Ministerio de Ciencia e Innovación
2
2
4.8 mmol) in methanol (1.35 mL) was irradiated at 40 W for
(
MICINN) (BFU2011-30161-C02-02) and the Junta de Comunid-
TM
70 min (6ϫ45 min) at 293 K. The Discover
CoolMate access-
ades de Castilla-La Mancha (JCCM) (project PCI08-0033). This
work has been supported, in part, by grants to V. C. from the
Fondo de Investigaciones Sanitarias (PI081434) and from the Con-
sejería de Educación of the JCCM (PII1I09-0163-4002 and
POII10-0274-3182). J. G. is a recipient of a Torres-Quevedo re-
search contract funded by the MICINN (Spain) and NanoDrugs
S. L. The authors also are grateful to Dr. M. Antonia Herrero
ory was coupled to the microwave reactor. The solvent was re-
moved under vacuum and an azeotropic mixture of toluene/meth-
anol (10 mL, 9:1, v/v) was added to the crude mixture to remove
the excess ethylenediamine (three times) by rotary evaporation.
Finally, methanol (10 mL) was added and then removed under vac-
uum. The rotary evaporator bath temperature was maintained at
1
3
13 K. The product was a yellowish oil (yieldϾ 0.118 g, 95%). H
(
UCLM) and Dr. Joaquín C. García-Martínez (UCLM) for their
NMR (500 MHz, CD
3
OD, Me
CONH), 2.54 (s, 4 H, NCH
.5 Hz, 8 H, NHCH CH NH ), 2.76 (t, J = 7.5 Hz, 8 H,
CH CONH), 3.24 (t, J = 7.5 Hz, 8 H, CONHCH CH
OD): δ = 33.3, 41.6, 43.4,
9.9, 51.0, 173.8 ppm. MS (MALDI-TOF): m/z (%) = 518.6 (100)
4
Si): δ = 2.36 (t, J = 7.5 Hz,
help in the MALDI-TOF experiments. We also acknowledge the
research team of Prof. A. de la Hoz (UCLM) for helpful advice and
for providing the facilities to run the experiments under microwave
conditions.
8
7
H, NCH
2
CH
2
2
CH N), 2.71 (t, J =
2
2
2
2
NCH
ppm. 13C{ H} NMR (125 MHz, CD
2
2
1
2
2
)
3
4
+
+
+
[
M + H] , 540.5 (35) [M + Na] , 556.3 (6) [M + K]
.
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2336
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 2331–2337

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Received: November 29, 2011
Published Online: March 2, 2012
Eur. J. Org. Chem. 2012, 2331–2337
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2337