Efficient and rapid divergent synthesis of ethylene oxide-containing dendrimers through catalyst-free click chemistry

Article abstract of DOI:10.1021/ma300648r

Third and fourth generation ethylene oxide-containing dendrimers containing triazole units were prepared divergently through metal-free Huisgen 1,3-dipolar cycloaddition (click) reaction between activated disubstituted alkyne and terminal azide groups. Th

Full text of DOI:10.1021/ma300648r

Article
pubs.acs.org/Macromolecules
Efficient and Rapid Divergent Synthesis of Ethylene Oxide-
Containing Dendrimers through Catalyst-Free Click Chemistry
Mathieu Arseneault, Isabelle Levesque, and Jean-Franco̧ is Morin*
Dep
Queb
́
artement de chimie and Centre de Recherche sur les Mater
́ ́ ́ ́
iaux Avances (CERMA), 1045 Ave de la Medecine, Universite Laval,
́
ec, Canada G1V 0A6
S
* Supporting Information
ABSTRACT: Third and fourth generation ethylene oxide-
containing dendrimers containing triazole units were prepared
divergently through metal-free Huisgen 1,3-dipolar cyclo-
addition (“click”) reaction between activated disubstituted
alkyne and terminal azide groups. The growth of the
dendrimers was achieved in high yield with minimal
purification effort and without the participation of toxic
metal or excess of reagents. Preliminary in vitro testing on
different cell lines indicates that this series of dendrimers is
very promising for bio-related applications.
becomes very attractive. The extensive use of EO in bio-related
areas comes from, among other things, its very good solubility
in aqueous media, chemical stability in physiological environ-
ments, low toxicity, and a prolonged circulation period in the
human body due to its nonimmunogenicity.12 However, EO
does not offer good chemical versatility because only a few EO-
based building blocks with two or more reactive or functional
ends are available. To circumvent this lack of chemical
versatility, chemists often used EO moieties to decorate the
surface of nanoparticles,13 quantum dots,14 dendrimers,15 and
biomolecules16 since these strategies require EO with only one
reactive end. With the exception of linear EO polymers and
oligomers, more complex molecular architectures such as
dendrons and dendrimers in which the EO moiety is part of the
molecular scaffold are thus rather limited.
Recently, Gnanou et al. have developed practical approaches
to prepare functional high-generation dendrimer-like molecules
based on EOs with narrow size distribution (PDI ≤1.5)
through the divergent anionic ring-opening polymerization of
ethylene oxide.17−19 They have demonstrated that those
structures show interesting properties in terms of water
solubility, possibility of functionalization, and pH responsive-
ness.20 Using a different approach, Veronese et al. have
prepared low-generation EO-based dendrons through an
amidation reaction for targeted drug delivery applications.21
Almost at the same time, Hildgen et al. reported on the
synthesis of monodisperse EO-based dendrimers (up to the
third generation) through a combination of convergent and
divergent synthetic steps, also for drug delivery purposes.22,23
INTRODUCTION
■
Dendrimer materials are now considered to be one of the most
important and promising classes of macromolecules for
applications ranging from electronics to nanomedicine.1,2 For
bio-related applications, many water-soluble and biocompatible
dendrimers were developed over the past few decades including
poly(amidoamine) (PAMAM),3 glycerol,4 and ethylene oxide
(EO)-based dendrimers5 to name but a few. When properly
decorated with active molecules, those particular dendrimeric
structures were found to be very useful in various biological
appplications such as drug delivery,6 boron neutron capture
therapy (BNCT),7 and magnetic resonance imaging (MRI).8,9
However, even the most promising classes of dendrimers suffer
from serious drawbacks that could limit their penetration in
real-world applications. For example, poly(amidoamine)
(PAMAM) dendrimers, one of the most studied families of
dendrimers, have significant disadvantages, the most important
being their thermal and chemical instability. In fact, PAMAM
dendrimers can undergo retro-Michael reactions, resulting in
loss of branches and the creation of secondary amines that can
further undergo an intramolecular transamidation reaction.10,11
Moreover, the hydrolysis (or solvolysis) of terminal ester
functionalities into carboxylic acids makes the dendrimer
termini unreactive toward ethylenediamine, thus impeding the
dendrimer growth to higher generations.3 Finally, a large excess
of reagent has to be used at each step in order to avoid the
occurrence of side reactions, including intramolecular bridging
and intermolecular cross-linking.3 Since most of these draw-
backs are inherent to the PAMAM chemical structure, the
preparation of a new family of readily accessible dendrimers
circumventing all these negative aspects is desired.
In this context, the development of more stable and water-
soluble dendrimers made from readily accessible biocompatible
building blocks such as glycerol and ethylene oxide (EO)
Received: March 29, 2012
Revised: April 13, 2012
Published: April 18, 2012
© 2012 American Chemical Society
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Figure 1. Examples of third and fourth EO-containing dendrimers grown by metal-free click chemistry.
Although the properties of those EO-based dendrimers are
very promising, their preparation involves either toxic metal
and chemicals, harsh conditions, long reaction times, or excess
of chemical reagents resulting in chemical wastes. Thus, an
efficient and clean preparative method for monodisperse
dendrimers for bio-related applications still represents an
important cornerstone that has not been totally reached yet.
The search for the “perfect” conditions to grow dendrimers
represents a demanding challenge because the synthetic
strategy must be very efficient in order to avoid structural
defects, free of side reactions and byproducts to ensure
dendrimer monodispersity and conducted without the
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4H, J = 6.7 Hz). 13C NMR: 151.75, 75.08, 72.00, 68.07, 65.74, 2.36. IR
(ATR): 1037, 1109, 1232, 1714, 2869, 2958 ν cm−1. HRMS (ESI+):
calcd for C12H16I2O6: 509.9036; found: 532.8963 (M + Na+).
participation of toxic metal. The latter requirement is of utmost
importance since polar dendrimers such as PAMAM are known
to trap metal ions very efficiently, as shown by us24 and
others.25 Herein we report the divergent synthesis of third and
fourth generation EO-containing dendrimers (Figure 1), using
sequential azidation/metal-free 1,3-dipolar cycloaddition (click)
reactions.26−28 These dendrimers and the reactions used to
prepare them have carefully been designed for biorelated
applications. First, the dendritic structures include 1,2,3-triazole
heterocycles, which are known for their promising pharmaco-
logical properties.29 Second, the ester groups directly attached
to the triazole units should allow the dendrimers to be
decomposed in vivo through enzymatic hydrolysis reactions to
create low molecular weight units that could be easily
assimilated by the human body.11 Third, the presence of either
azide or iodide termini provides us with versatile dendrimers in
terms of surface decoration. Finally, the synthetic strategy we
developed is relatively simple, high yielding, and free of toxic
metal and reagent.
Synthesis of X-EO2-G1-I. Following the above procedure for
iodo-terminated generations, α,α′-diazido-p-xylene (0.184 g, 0.980
mmol, 1 equiv) was used with compound 3 (1.00 g, 1.96 mmol, 2
equiv). No purification was needed. The reaction gave a viscous dark
yellow oil. Yield is over 98% (1.184 g, 0.980 mmol). 1H NMR (CDCl3,
400 MHz): 7.29 (s, 4H), 5.81 (s, 4H), 4.51 (t, 4H, J = 4.7 Hz), 4.46 (t,
4H, J = 4.6 Hz), 3.83 (t, 4H, J = 4.7 Hz), 3.75 (m, 12H), 3.23 (dt, 8H,
J = 5.2 Hz). 13C NMR: 160.10, 158.17, 140.71, 134.96, 129.89, 128.96,
72.12, 71.94, 68.54, 68.12, 65.73, 64.97, 53.72. IR (ATR): 1056, 1117,
1195, 1264, 1358, 1551, 1724, 2869, 2955 ν cm−1. HRMS (ESI+):
calcd for C32H40I4N6O12: 1207.8883; found: 1208.8925 (M + H+).
Synthesis of X-EO2-G1-N3. Following the above procedure for
azido-terminated generations, X-EO2-G1-I (0.800 g, 0.660 mmol, 1
equiv) was used with NaN3 (0.34 g, 5.29 mmol, 8 equiv) and DMF.
No further purification was needed. The reaction gave a viscous bright
1
yellow oil. Yield is 97% (0.560 g, 0.64 mmol). H NMR (CDCl3, 400
MHz): 7.28 (s, 4H), 5.78 (s, 4H), 4.51 (t, 4H, J = 4.6 Hz), 4.46 (t, 4H,
J = 4.6 Hz), 3.83 (t, 4H, J = 4.8 Hz), 3.74 (t, 4H, J = 4.8 Hz), 3.70 (t,
4H, J = 4.9 Hz), 3.64 (t, 4H, J = 4.9 Hz), 3.36 (dt, 8H, J = 5.2 Hz). 13C
NMR: 160.10, 158.20, 140.60, 134.95, 129.93, 128.84, 70.28, 70.28,
68.95, 68.56, 65.69, 64.93, 53.58, 50.79, 50.36. IR (ATR): 1058, 1122,
1197, 1464, 1727, 2095, 2872, 2923, 2952 (broad band) ν cm−1.
HRMS (ESI+): calcd for C32H40N18O12: 868.3073; found: 869.3238
(M + H+).
EXPERIMENTAL SECTION
■
Materials. All solvents (ACS grade) were distilled and put through
a Vac Atmosphere (Hawthorne, CA) solvent purification system. All
the reagents were purchased from either Sigma-Aldrich Co., TCI
America, or Oakwood Products and used as received. All equivalents
are molar. 2-(2-Iodoethoxy)ethanol (compound 2)30 and tetra-
ethylene glycol mono(p-toluenesulfonate) (compound 7)31 were
synthesized according published procedures.
Synthesis of X-EO2-G2-I. Following the above procedure for
iodo-terminated generations, X-EO2-G1-N3 (0.535 g, 0.616 mmol, 1
equiv) was used with compound 3 (1.26 g, 2.46 mmol, 4 equiv). No
purification was needed. The reaction gave a viscous yellow oil. Yield is
over 98% (1.795, 0.616 mmol). 1H NMR (CDCl3, 400 MHz): 7.28 (s,
4H), 5.78 (s, 4H), 4.81 (dt, 8H, J = 5 Hz), 4.51 (m, 16H,), 4.38 (m,
8H), 3.78 (m, 44H), 3.65 (t, 4H), 3.25 (m, 16H). 13C NMR: 160.06,
160.04, 159.91, 158.50, 158.46, 158.10, 140.34, 139.92, 139.72, 135.01,
131.96, 131.43, 130.06, 128.89, 72.11, 72.09, 72.07, 71.87, 71.84,
69.61, 69.35, 69.09, 68.59, 68.54, 68.20, 68.13, 65.77, 65.68, 65.51,
64.87, 64.80, 64.74, 52.69, 50.39, 50.34, 3.17, 3.09, 3.07, 3.05. IR
(ATR): 1059, 1116, 1198, 1269, 1462, 1552, 1724, 2872, 2956 ν cm−1.
HRMS (ESI+): calcd for C80H104N18I8O36: 2907.9218; found:
Characterization. SEC analysis was performed using a Jordi DBV
1
500A in THF. H and 13C NMR spectra were recorded on a Varian
AS400 apparatus in appropriate deuterated solvent solution at 298 K.
Chemical shifts were reported as values (ppm) relative to internal
tetramethylsilane. High resolution mass spectrometry (HRMS) was
performed on an Agilent model 62-10 MS-TOF.
General Procedure for Iodo- or Tosylate-Terminated
Generations. In a round-bottom flask, either 2, 4, or 8 equiv of
compound 3 or 8 were added to 1 equiv of the azido compound
moieties. A minimal amount of chloroform was added (typically 2 mL/
g of substrate). The reaction was started in an ice bath at 0 °C for 2 h
and stirred for 1 h, allowing the mixture to reach room temperature.
The mixture was then stirred at 60 °C overnight. The reaction was
allowed to cool down at 25 °C, and the chloroform was then
evaporated. The compound was used without any further purification.
General Procedure for Azido-Terminated Generations. The
iodo- or tosylate-terminated dendrimer was dissolved in DMF (0.1
M), and 2 equival of NaN3 per iodine terminals was added to the
solution. The reaction was then stirred and heated at 70 °C for 24 h.
The mixture was then allowed to cool down to 25 °C and extracted
with AcOEt/“half-brine”. “Half-brine” is brine that has been diluted by
an equal volume of distilled water. We found that this was a good
compromise to obtain good phase separation while eliminating DMF.
This was done because standard ether/water extractions failed to
dissolve the dendrimer at any given generation. The aqueous layer was
then washed with AcOEt, and the organic layers were combined. They
were then washed 10 times with “half-brine”. The organic layer was
then dried and concentrated.
Synthesis of Compound 3. In a round-bottom flask fitted with a
Dean−Stark apparatus, acetylenedicarboxylic acid (3.83 g, 33.6 mmol,
1 equiv) was dissolved in benzene (66 mL). To the solution, p-
toluenesulfonic acid (0.638 g, 3.36 mmol, 0.1 equiv) and 2-(2-
iodoethoxy)ethanol (16.0 g, 73.8 mmol, 2.2 equiv) were added. The
mixture was refluxed for 24 h and was then allowed to cool down at
room temperature. Diethyl ether and NaHCO3 (1.41 g, 16.8 mmol,
0.5 equiv) were then added and stirred for 1 min. The purification was
achieved through column chromatography (silica gel, DCM and
hexanes 4:1 as the eluent). Yield is 75% (12.7 g, 24.9 mmol). 1H NMR
(CDCl3, 400 MHz): 4.40 (t, 4H, J = 4.6 Hz), 3.75 (m, 8H), 3.26 (t,
+
2926.9660 (M + NH4 ).
Synthesis of X-EO2-G2-N3. Following the above procedure for
azido-terminated generations, X-EO2-G2-I (0.50 g, 0.172 mmol, 1
equiv) was used with NaN3 (0.179 g, 2.75 mmol, 16 equiv) and DMF.
No further purification was needed. The reaction gave a viscous bright
yellow oil. Yield is 72% (0.278 g, 0.125 mmol). 1H NMR (CDCl3, 400
MHz): 7.28 (s, 4H), 5.78 (s, 4H), 4.81 (dt, 8H, J = 5 Hz), 4.51 (m,
16H,), 4.38 (m, 8H), 3.83 (m, 44H), 3.68 (m, 4H), 3.39 (m, 16H).
13C NMR: 159.99, 159.98, 159.86, 158.38, 158.34, 157.99, 140.17,
139.75, 139.56, 134.97, 131.84, 131.35, 130.06, 128.70, 70.12 70.02,
69.95, 69.45, 69.20, 68.90, 68.83, 68.51, 68.46, 68,41, 65.63, 65.57,
65.44, 64.75, 64.69, 64.58, 53.52, 50.73, 50.71, 50.19. IR (ATR): 960,
1062, 1125, 1203, 1273, 1462, 1554, 1730, 2102, 2874, 2923, 2955 ν
cm−1. HRMS (ESI+): calcd for C80H104N42O36: 2228.7598; found:
+
2247.7653 (M + NH4 ).
Synthesis of X-EO2-G3-I. Following the above procedure for
iodo-terminated generations, X-EO2-G2-N3 (0.278 g, 0.125 mmol, 1
equiv) was used with compound 3 (0.509 g, 0.998 mmol, 8 equiv). No
purification was needed. The reaction gave a viscous dark yellow oil.
1
Yield is over 98% (0.787 g, 0.125 mmol). H NMR (CDCl3, 400
MHz): 7.29 (s, 4H), 5.78 (s, 4H), 4.81 (m, 22H), 4.51 (m, 30H,), 4.40
(m, 26H), 3.79 (m, 114H), 3.25 (m, 32H). 13C NMR: image available
below. IR (ATR): 1060, 1102, 1269, 1461, 1552, 1723, 2872, 2956
(broad band) ν cm−1. HRMS (ESI+): calcd for C176H232I16N42O84:
+
6307.9888; found: 3172.9986 (m/z = M2+ + NH4 ).
Synthesis of EO3-EO2-G1-I. Following the above procedure for
iodo-terminated generations, 1,2-bis(2-azidoethoxy)ethane (5) (0.587
g, 2.94 mmol, 1 equiv) was used with compound 3 (3.00 g, 5.88 mmol,
2 equiv). No purification was needed. The reaction gave a viscous
1
amber oil. Yield is over 98% (3.59 g, 2.94 mmol). H NMR (CDCl3,
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400 MHz): 4.81 (t, 4H, J = 5.3 Hz), 4.23 (m, 8H), 3.79 (m, 20H),
3.44 (s, 4H), 3.26 (t, 8H, J = 6.5 Hz). 13C NMR: 160.15, 158.50,
139.84, 131.74, 72.12, 71.93, 70.66, 69.49, 68.58, 68.21, 65.68, 64.83,
50.43. IR (ATR): 1061, 1102, 1198, 1270, 1462, 1553, 1725, 2870,
2954 ν cm−1. HRMS (ESI+): calcd for C30H44I4N6O14: 1219.9094;
found: 1220.9173 (M + H+).
Synthesis of EO3-EO2-G1-N3. Following the above procedure for
azido-terminated generations, EO3-EO2-G1-I (0.705 g, 0.577 mmol, 1
equiv) was used with NaN3 (0.300 g, 4.62 mmol, 8 equiv) and DMF.
No further purification was needed. The reaction gave a viscous bright
yellow oil. Yield is 98% (0.500 g, 0.566 mmol). 1H NMR (CDCl3, 400
MHz): 4.79 (t, 4H, J = 5.1 Hz), 4.53 (t, 8H, J = 4.5 Hz), 3.84 (qt, 8H,
J = 4.9 Hz), 3.77 (t, 4H, J = 5.1, 3.70 (qt, 8H, J = 4.9 Hz), 3.43 (s, 4H),
3.50 (t, 8H, J = 4.8 Hz). 13C NMR: 160.16, 158.49, 139.82, 131.73,
70.60, 70.31, 70.20, 69.46, 69.01, 68.68, 65.61, 64.83, 50.89, 50.35. IR
(ATR): 1062, 1112, 1200, 1273, 1463, 1728, 2096, 2870, 2922, (broad
band) ν cm−1. HRMS (ESI+): calcd for C32H40N18O12: 880.3284;
found: 881.3745 (M + H+).
Synthesis of EO3-EO2-G2-I. Following the above procedure for
iodo-terminated generations, EO3-EO2-G1-N3 (0.300 g, 0.341 mmol,
1 equiv) was used with compound 3 (0.695 g, 1.36 mmol, 4 equiv).
No purification was needed. The reaction gave a viscous amber oil.
Yield is over 98% (0.965, 0.341 mmol). 1H NMR (CDCl3, 400 MHz):
4.84 (q, 8H, J = 4.7 Hz), 4.74 (t, 4H, J = 4.9 Hz), 4.51 (m, 16H), 4.42
(q, 8H, J = 4.3 Hz), 3.90 (q, 8H, J = 4.3 Hz), 3.80 (m, 20H), 3.60 (m,
24H), 3.42 (s, 4H), 3.396 (m, 16H). 13C NMR: 72.11, 72.08, 71.89,
71.87, 68.55, 68.22, 68.14, 65.79, 65.71, 64.80, 64.60, 64.57, 64.74,
50.41, 3.04. IR (ATR): 1061, 1103, 1198, 1272, 1462, 1553, 1726,
2872, 2956 ν cm−1. HRMS (ESI+): calcd for C78H108I8N18O38:
2919.9429; found: 1461.4744 (m/z = M2+ + 2H+).
Synthesis of EO3-EO2-G2-N3. Following the above procedure for
azido-terminated generations, EO3-EO2-G2-I (4.00 g, 1.369 mmol, 1
equiv) was used with NaN3 (1.424 g, 21.91 mmol, 16 equiv) and
DMF. No further purification was needed. The reaction gave a viscous
amber yellow oil. Yield is 92% (2.82 g, 1.26 mmol). 1H NMR (CDCl3,
400 MHz): 4.84 (q, 8H, J = 4.7 Hz), 4.74 (t, 4H, J = 4.9 Hz), 4.51 (m,
16H), 4.42 (q, 8H, J = 4.3 Hz), 3.90 (q, 8H, J = 4.3 Hz), 3.80 (m,
20H), 3.60 (m, 24H), 3.42 (s, 4H), 3.39 (m, 16H). 13C NMR: 160.11,
160,08, 159.99, 158.52, 158.49, 158.29, 139.98, 139.72, 139.65, 131.94,
131.75, 131.32, 70.49 70.30, 70.21, 70.13, 69.69, 69.39, 69.27, 69.07,
68.99, 68.69, 68.63, 65.72, 65.65, 65.36, 65.03, 64.87, 64.80, 64.59,
50.89, 50.87, 50.85, 50.45, 50.33, 50.31. IR (ATR): 1061, 1115, 1199,
1273, 1461, 1553, 1727, 2098, 2874, 2905, 2953 ν cm−1. HRMS
(ESI+): calcd for C78H108N42O38: 2240.7810; found: 1121.8963 (m/z
= M2+ + H+).
(ATR): 1061, 1114, 1273, 1461, 1553, 1726, 2098, 2974, 2915, 2954 ν
cm−1. HRMS (ESI+): calcd for C174H236N90O86: 4961.6860; found:
+
1672.5885 (m/z = M3+ + NH4 + H+).
Synthesis of EO3-EO2-G4-COOH. Following the above proce-
dure for iodo-terminated generations, EO3-EO2-G3-N3 (1.000 g,
0.201 mmol, 1 equiv) was used with acetylenedicarboxylic acid (0.735
g, 6.45 mmol, 32 equiv). No purification was needed. The reaction
gave a viscous dark amber oil insoluble in most solvent. Yield is over
1
98% (1.735 g, 0.201 mmol). H NMR (D2O, 400 MHz): 4.91−5.22
(m), 4.41−4.15 (m), 3.99−3.59 (m). Image available below. IR
(ATR): 1063, 1117, 1210, 1278, 1370, 1455 1551, 1627, 1724, 2878,
2957, 3400 ν cm−1. MS data could not be obtained due to the
limitations of our apparatus.
Preparation of EO3-EO2-G4-COO−Na+ Salt for Cytotoxicity
Assays. EO3-EO2-G4-COOH was mixed with deionized water
(1.735 g in 25 mL) and NaHCO3 (1 equiv per −COOH group) was
added. The mixture was vigorously stirred until all dendrimer was
dissolved. To remove any smaller species, dialysis was performed in
nanopure water. Dialysis tubing was left to soak in nanopure water for
30 min to remove most of its preservative. The tube was then clamped
at the bottom, and five small glass beads (prewashed with nanopure
water) were added. Typically, 10 mL of the dendrimer solution
mentioned above was used. The tube was clamped at its top and put in
a 1 L beaker filled with nanopure water with gentle magnetic stirring
for 24 h. Nanopure water was replaced two more times, each at 24 h
intervals. The tube was then emptied, and the water was evaporated
under reduced pressure. A pale yellow powder was obtained.
Synthesis of Compound 8. In a round-bottom flask fitted with a
Dean−Stark apparatus, acetylenedicarboxylic acid (0.708 g, 6.22
mmol, 1 equiv) was dissolved in benzene (22 mL). To the solution,
p-toluenesulfonic acid (0.118 g, 0.622, 0.1 equiv) and Ts-tetraglycol
(4.77 g, 13.691 mmol, 2.2 equiv) were added. The mixture was
refluxed for 24 h and was then allowed to cool down at room
temperature. Diethyl ether and NaHCO3 (0.261 g, 3.112 mmol, 0.5
equiv) were then added and stirred for 1 min before being
concentrated. The purification was achieved through column
chromatography (silica gel, AcOEt and hexanes 4:1 as the eluent).
Yield is 47% (2.27 g, 2.93 mmol). 1H NMR (CDCl3, 400 MHz): 7.79
(t, 4H, J = 4.1 Hz), 7.34 (t, 4H, J = 4.1 Hz), 4.37 (t, 4H, J = 4.7 Hz),
4.16 (t, 4H, J = 4.8 Hz), 3.73 (t, 4H, J = 4.6 Hz), 3.69 (t, 4H, J = 4.8
Hz), 3.63 (bs, 8H), 3.59 (s, 8H), 2.45 (s, 6H). 13C NMR: 151.90,
145.09, 133.13, 130.08, 128.20, 75.11, 70.94, 70.87, 70.83, 70.77,
69.52, 68.89, 68.63, 66.09, 21.89. IR (ATR): 916, 1095, 1174, 1251,
1352, 1451, 1598, 1720, 2872, 2949 ν cm−1. HRMS (ESI+): calcd for
+
C34H46O16S2: 774.2227; found: 792.2985 (M + NH4 ).
Synthesis of EO3-EO4-G1-Ts. Following the above procedure for
iodo-terminated generations, 1,2-bis(2-azidoethoxy)ethane (5) (0.08
g, 0.400 mmol, 1 equiv) was used with compound 8 (0.619 g, 0.800
mmol, 2 equiv). No purification was needed. The reaction gave a
viscous yellow oil. Yield is over 98% (0.699 g, 0.400 mmol). 1H NMR
(CDCl3, 400 MHz): 7.79 (d, 8H, J = 7.8 Hz), 7.34 (d, 8H, J = 7.8 Hz),
4.37 (t, 8H, J = 4.6 Hz), 4.16 (t, 8H, J = 4.8 Hz), 3.63 (m, 60H), 2.44
(d, 12H, J = 3.0 Hz). 13C NMR: 160.24, 158.50, 145.06, 139.84,
133.14, 130.08, 128.21, 70.97, 70.96, 70.87, 70.82, 70.77, 70.70, 70.57,
69.53, 69.52, 69.47, 68.96, 68.91, 68.90, 68.87 68.64, 65.79, 64.95,
50.39, 21.90 IR (ATR): 919, 1096, 1175, 1352,1453, 1731, 2872, 2919,
2952, 3010 (broad band) ν cm−1. HRMS (ESI+): calcd for
Synthesis of EO3-EO2-G3-I. Following the above procedure for
iodo-terminated generations, EO3-EO2-G2-N3 (0.598 g, 0.267 mmol,
1 equiv) was used with compound 3 (1.088 g, 2.13 mmol, 8 equiv).
No purification was needed. The reaction gave a viscous amber oil.
1
Yield is over 98% (1.686 g, 0.267 mmol). H NMR (CDCl3, 400
MHz): 4.80 (m, 26H), 4.50 (m, 30H), 4.40 (m, 28H), 3.90 (m, 22H,),
3.78 (m, 98H), 3.26 (m, 32H). 13C NMR: 160.09, 159.93, 158.54,
158.45, 158.31, 139.56, 70.12 72.04, 71.85, 69.60, 69.26, 69.06, 68.55,
68.20, 68.14, 67.97, 67.68, 65.85, 65.80, 65.71, 65.47, 65.40, 65.38,
64.88, 64.80, 64.55, 50.36, 3.26. IR (ATR): 1062, 1107, 1200, 1268,
1462, 1553, 1725, 2873, 2918, 2957 (broad band) ν cm−1. HRMS
(ESI+): calcd for C174H236I16N42O86: 6320.0100' found: 2125.3672
+
+
(m/z = M3+ + NH4 + H+).
C74H104N6O34S4: 1748.5476; found: 1766.585 (M + NH4 ).
Synthesis of EO3-EO4-G1-N3. Following the above procedure for
azido-terminated generations, EO3-EO4-G1-Ts (0.7423 g, 0.424
mmol, 1 equiv) was used with NaN3 (0.179 g, 3.395 mmol, 8
equiv) and DMF. No further purification was needed. The reaction
gave a viscous bright yellow oil. Yield is 63% (0.330 g, 0.267 mmol).
1H NMR (CDCl3, 400 MHz): 4.79 (t, 4H, J = 4.9 Hz), 4.50 (t, 8H, J =
4.6 Hz), 3.80 (m, 12H), 3.66 (m, 40H,), 3.42 (s, 4H), 3.38 (m, 8H).
13C NMR: 160.25, 158.52, 139.87, 131.77, 70.90, 70.84, 70.74, 70.61,
70.29, 70.27, 69.50, 68.97, 68.64, 65.78, 64.97, 50.90, 50.88, 32.10. IR
(ATR): 1063, 1104, 1201, 1274, 1462, 1552, 1729, 2098, 2869 νcm−1.
HRMS (ESI+): calcd for C46H76N18O22: 1232.5382; found: 1250.5932
Synthesis of EO3-EO2-G3-N3. Following the above procedure for
azido-terminated generations, EO3-EO2-G3-I (4.00 g, 0.63 mmol, 1
equiv) was used with NaN3 (1.424 g, 21.91 mmol, 16 equiv) and
DMF. No further purification was needed. The reaction gave a viscous
amber yellow oil. Yield is 86% (2.70 g, 0.54 mmol). 1H NMR (CDCl3,
400 MHz): 4.80 (m, 26H), 4.52 (m, 34H), 4.39 (m, 22H) 3.89 (d,
24H, J = 4.4 Hz), 3.82 (m, 38H), 3.70 (m, 56H), 3.39 (m, 36H).). 13C
NMR: 160.10, 160.08, 160.07, 160.00, 159.93, 159.92, 158.51, 158.50,
158.44, 158.28, 139.97, 139.76, 139.70, 139.67, 139.63, 139.57, 131.99,
131.96, 131.85, 131.74, 131.39, 131.37, 131.23, 70.46, 70.28, 70.18,
70.12, 69.60, 69.56, 69.35, 69.23, 69.04, 68.97, 68.66, 68.60, 68.72,
65.65, 65.43, 64.99, 64.86, 64.79, 64.63, 64.59, 50.87, 50.41, 50.32. IR
+
(M + NH4 ).
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Scheme 1. Synthesis of X-EO2-G3-I
Synthesis of EO3-EO4-G2-Ts. Following the above procedure for
iodo-terminated generations, EO3-EO4-G1-N3 (0.313 g, 0.254 mmol,
1 equiv) was used with compound 8 (0.787 g, 1.015 mmol, 4 equiv).
No purification was needed. The reaction gave a viscous yellow oil.
Yield is over 98% (1.1 g, 0.254 mmol). 1H NMR (CDCl3, 400 MHz):
7.77 (d, 12H, J = 8.1 Hz), 7.33 (d, 12H, J = 8.1 Hz), 4.78 (m, 12H),
4.48 (m, 24H,), 4.14 (m, 16H), 3.78 (m, 34H), 3.58 (m, 122H), 3.41
(s, 4H), 2.42 (m, 24H). 13C NMR: 160.24, 159.49, 145.07, 133.18,
130.09, 128.21, 70.99, 70.96, 70.95, 70.91, 70.87, 70.86, 70.83, 70.81,
70.79, 70.76, 70.71, 70.65, 70.61, 70.57, 69.54, 69.52, 69.48, 69.46,
68.96, 68.93, 68.91, 68.88, 68.66, 68,61, 65.76, 65.74, 65.72, 64.94,
64.92, 50.41, 50.39, 50.38, 21.89. IR (ATR): 917, 1012, 1064, 1096,
1174, 1274, 1351, 1456, 1552, 1598 1729, 2871, 2920, 2954 ν cm−1.
HRMS (ESI+): calcd for C182H260N18O86S8: 4329.4291; found:
1084.1100 (m/z = 4 + 2H+).
Synthesis of EO3-EO4-G2-N3. Following the above procedure for
azido-terminated generations, EO3-EO4-G2-Ts (0.625 g, 0.144 mmol,
1 equiv) was used with NaN3 (0.150 g, 2.31 mmol, 16 equiv) and
DMF. No further purification was needed. The reaction gave a viscous
bright yellow oil. Yield is 92% (0.437 g, 0.132 mmol). 1H NMR
(CDCl3, 400 MHz): 4.67 (m, 8H), 4.36 (m, 16H), 3.67 (m, 40H),
3.51 (m, 96H), 3.39 (m, 22H), 3.29 (s, 4H), 3.24 (t, 16H, J = 4.8 Hz).
13C NMR: 160.10, 158.37, 158.36, 139.69, 139.67, 139.60, 131.66,
131.64, 70.75, 70.73, 70.69, 70.67, 70.60, 70.55, 70.51, 70.47, 70.44,
70.13, 70.11, 69.48, 69.42, 69.34, 69.20, 68.84, 68.81, 68.53, 68.49,
65.64, 64.84, 64.81, 63.71, 50.75, 53.73, 53.31, 50.29, 21.09. IR (ATR):
940, 1063, 1102, 1201, 1274, 1462, 1552, 1729, 2100, 2869, 2915
(broad band) ν cm−1. HRMS (ESI+): calcd for C126H204N42O62:
+
3297.4101; found: 1666.8335 (m/z = 2 + NH4 ).
Synthesis of EO3-EO4-G3-Ts. Following the above procedure for
iodo-terminated generations, EO3-EO4-G2-N3 (0.350 g, 0.106 mmol,
1 equiv) was used with compound 8 (0.657 g, 0.848 mmol, 8 equiv).
No purification was needed. The reaction gave a viscous yellow oil.
1
Yield is over 98% (1.007 g, 0.106 mmol). H NMR (CDCl3, 400
MHz): 7,77 (d, 32H, J = 7,6 Hz); 7.33 (d, 4H, J = 7.9 Hz), 4.80 (m,
24H), 4.48 (m, 24H), 4.14 (m, 36H), 3.83 (m), 3.79 (m), 3.61 (m),
3.56 (br s), 3.51 (br s), 3.41 (br s), 2.42 (s, 48H) all peaks from 3.83
to 3.41 integrated for 348H combined. 13C NMR: 160.23, 158.48,
145.08, 133.17, 130.09, 128.20, 70.94 70.86, 70.82 70.80, 70.76, 70.70,
70.63, 69.54, 68.94, 68.89, 68.87, 68.65, 65.72, 65.44, 64.75, 64.94,
64.92, 64.88, 50.40, 21.87. IR (ATR): 916, 1096, 1175, 1275, 1351,
1453, 1522, 1729, 2096, 2871 ν cm−1.
RESULTS AND DISCUSSION
■
The synthetic strategy for the synthesis of EO-containing
dendrimers is depicted in Scheme 1. For our initial attempt,
very short EO chains (−[CH2CH2O]−, named EO2 there-
after) were used. A phenyl-containing core obtained from a
xylene derivative (named X thereafter) was chosen to enable
the monitoring of the dendrimer growth using thin layer
chromatography and size-exclusion chromatography (SEC,
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UV/vis detector). In order to conduct metal-free click
chemistry without using harsh heating conditions, an activated
alkyne bearing at least one electron-deficient unit must be
used.32,33 Since internal alkyne is needed for the preparation of
dendrimers using a divergent approach, we chose to start with
the commercially available acetylenedicarboxylic acid. Dehaen
et al. have shown that this building block is reactive enough to
attach multiple dendrons on a highly functionalized core
without the participation of copper or other metal catalysts.34
Thus, acetylenedicarboxylic acid was coupled with 2-(2-
iodoethoxy)ethanol (compound 2) using p-toluenesulfonic
acid in benzene using a Dean−Stark apparatus as developed
by Finn et al. to provide the compound 3 in 75% yield.35 It is
noteworthy that the commercially available 2-(2-chloroethoxy)-
ethanol was transformed into its iodo analogue in order to
enable further azidation reaction. In fact, the reaction
conditions needed to quantitatively displace the chlorine
atom by the azide on our substrates were too harsh and led
to the partial decomposition of our dendrimers as observed by
1H NMR. The replacement of the chlorine atom by a better
leaving group toward SN2 reactions allowed us to conduct the
azidation reactions at a lower temperature for a shorter period
of time without any trace of decomposition.
The key step and major feature of our dendrimer lie in the
convenient character of the branching click reaction. Inspired
by Brook et al.,26 we mixed neat compounds 3 and 1,4-
bis(azidomethyl)benzene36 in the appropriate 1:2 molar ratio
and stirred for 24 h at room temperature with no other
precaution. Initially, no change was observed, but on a larger
scale, we noticed the mixture turned yellow and thickened. The
1H NMR spectrum showed all the expected shifts with faint
traces of both starting materials (less than 2%), but the HRMS
showed a very clean spectrum with nothing but the desired
product. In large quantities (>2 g scale), an intense exothermic
reaction lasting a few seconds was observed, bringing the
reactants to their boiling point and, therefore, to the
decomposition of the starting materials and the desired
product. This exothermic reaction can be considered as
empirical proof of the high efficiency of the click reaction
with these specific moieties. After a series of optimizations that
were monitored by 1H NMR and ESI MS, we chose to start the
reaction with a minimum amount of chloroform at 0 °C under
stirring for about an hour before the mixture was heated to 60
°C for 16 h. The addition of a small amount of chloroform or
dichloromethane was necessary to decrease the viscosity and
increase the homogeneity of the reaction mixture. Also, the
gentle heating was necessary to bring the reaction to
completion. Indeed, small amounts (up to 10%) of unreacted
starting materials were still present when the reaction was
started in an ice bath instead of at room temperature when no
further heat was applied. Following these optimized conditions,
we obtained the desired X-EO2-G1-I in 98% yield using this
simple protocol. This reaction can also be achieved on
multigrams scale with the same result.
1
Figure 2. H NMR spectra in the terminal methylene region during
the growth of X-EO2-G3-I.
terminal methylene groups are very dependent on the nature of
the heteroatom attached. Thus, the signals coming from
iodomethylene (3.25 ppm) and azidomethylene (3.37 ppm)
never overlap and it is very clear whether or not the click or the
SN2 reaction ( I→ N3) has been brought to completion, even at
higher generations. This analysis can also be applied to detect if
there is any unreacted diester synthon (compound 3)
remaining in the mixture. Thus, careful examination of the
3.10−3.50 ppm region suggests that all the starting materials
were consumed after each click reaction on any generation,
meaning that potential impurities are present at a concentration
below 2% (vide infra). These clean conversions are quite
surprising considering that no purification except usual workup
(to remove the DMF after the iodo conversion to azide) was
performed all along the divergent growth. This is a tremendous
advantage compared to other divergently grown dendrimers.1
With the aforementioned optimized reactions, X-EO2-G2-I, X-
EO2-G2-N3, and X-EO2-G3-I were readily obtained in similar
yields and excellent purity with great ease.
Size-exclusion chromatography (SEC) analysis was per-
formed in THF for X-EO2-G1-I, X-EO2-G2-I, and X-EO2-
G3-I, and the results are shown in Figure 3. As expected, all the
dendrimers show a very sharp and well-resolved peak.
However, unlike what was observed by H NMR analysis,
very small amounts of the previous generation dendrimer can
1
The second step in the iterative process to build the
dendrimer involved the replacement of all the terminal iodo
atoms into azides. This reaction was performed at 70 °C in
DMF as a solvent for 16 h to provide X-EO2-G1-N3 in 97%
yield. 1H NMR (Figure 2) easily assessed the complete
conversion of iodo into azides since both EO chains on any
given generation are not exactly symmetrical toward the triazole
branching point, meaning that each methylene unit is well
resolved. Furthermore, the chemical shifts of the protons of the
Figure 3. SEC traces for X-EO2-G1-I, X-EO2-G2-I, and X-EO2-G3-I
(UV/vis detector, THF as solvent).
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Scheme 2. Synthesis of X-EO2-G3-I
be found in X-EO2-G2-I and X-EO2-G3-I (Figure 3). A
quantitative analysis of those peaks confirms that those lower
generation dendrimers are present in very low concentrations
(<2%), which is still very low for unpurified materials. One can
assume that the preparation of such dendrimers on larger scale
would be beneficial regarding dendrimers purity since the
amount of reagents can be better controlled. Studies in this
regard are currently underway since biological applications
required very pure materials. Nevertheless, analytically pure
samples can be obtained by purification using preparative SEC.
In order to extend the scope of our strategy, we prepared
analogous dendrimers with structural differences. Initially, we
replaced the xylene core by a small ethylene oxide moiety (see
Supporting Information). As expected, the reaction yields and
the purity of the dendrimers were also very good. A more
significant change was to extend the length of the ethylene
oxide moieties in the dendrimers. As with any dendritic
architecture, branch length dictates, in part, how densely a
dendrimer will pack on itself.37 Denser packing may lead to
steric hindrance, which in turn results in a restricted growth
passed a certain generation. Furthermore, we hypothesized that
longer EO branches could enhance the hydrophobicity of the
dendrimers. Thus, tetraethylene glycol was selected to
investigate these issues. Since unsymmetrical tetraethylene
glycol derivatives are not commercially available, a dissymmet-
rization reaction was performed directly on tetraethylene glycol.
Following Bauer et al.,31 we introduced a tosyl group to one
end of the tetraglycol (Scheme 2) to yield compound 7 in a
59% yield. The above-mentioned esterification method using
Dean−Stark apparatus was used to couple compound 7 to
acetylenedicarboxylic acid in a 47% yield. The click reactions
and the conversions of tosylate to azido groups proceeded very
efficiently until the third generation was reached. At that point,
IR analysis showed some unreacted azide groups and SEC
analysis (see Supporting Information) showed remaining
monomer 8 and dendrimer precursors in an estimated 5%
ratio. Trying to push the reaction toward completion, 2% molar
excess monomer was added and the reaction was heated again
for 72 h. Unfortunately, no change was observed. Simple
geometric optimization indicated that dendrimer termini lie
relatively closer to each other, thus harming the azide reactivity
toward disubstituted alkynes. This new series of dendrimers is
probably packed much more densely than its shorter-chain
counterpart. In light of this result, we turned back to the EO2-
EO2-Gn series to optimize biocompatibility. A fourth
generation dendrimer bearing 32 carboxylate groups (EO2-
EO2-G4-COO−Na+, Figure 1) was synthesized following the
iterative strategy we developed. As expected, EO2-EO2-G4-
COO−Na+ is very soluble in water. NMR and MS analysis
failed to acertain its completion, but IR showed a clean
spectrum, free of any azide group, even at the fourth generation
(see Supporting Information). The choice of carboxylate as a
terminal decoration was motivated by previous evidence with a
PAMAM dendrimer that anionic dendrimers are far less toxic
than their cationic versions.38
A proliferation assessment on two cell lines (PANC-1 and
Ovcar-3) with concentrations ranging from 0.1 to 100 μM was
performed alongside a positive control of doxorubicine. In all
cases, compound EO2-EO2-G4-COO−Na+ showed no toxicity,
as cell proliferation was never impeded upon treatment with
this dendrimer (see Supporting Information). These prelimi-
nary results confirm that the EO-containing dendrimers
presented here did not present any toxicological activity as
expected and should be suitable for biomedical applications.
Detailed biological evaluation of this new class of dendrimers is
now underway.
CONCLUSION
■
In summary, we have prepared EO-containing dendrimers
containing triazole units using a relatively simple and efficient
divergent method. No toxic metal and excess of reagents were
required to obtain nearly defect free dendrimers in high yield.
The hydrophilic nature of these dendrimers and the absence of
metal during the synthesis make these dendrimers very
promising candidates for bio-related applications. Preliminary
in vitro proliferation testing on different cell lines shows that the
anionic version of this series of dendrimers is not toxic for
ovarian and pancreatic cells. This opens the way to the use of
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(20) Feng, X.; Taton, D.; Borsali, R.; Chaikof, E. L.; Gnanou, Y. J.
Am. Chem. Soc. 2006, 128, 11551−11562.
this readily prepared dendrimeric scaffold for bio-related
applications. Dendrimers with functional and bioactive termini
are under preparation.
(21) Berna, M.; Dalzoppo, D.; Pasut, G.; Manunta, M.; Izzo, L.;
Jones, A. T.; Duncan, R.; Veronese, F. M. Biomacromolecules 2006, 7,
146−153.
ASSOCIATED CONTENT
■
(22) Dhanikula, R. S.; Hildgen, P. Bioconjugate Chem. 2006, 17, 29−
41.
(23) Dhanikula, R. S.; Hildgen, P. Biomaterials 2007, 28, 3140−3152.
(24) Arseneault, M.; Dufour, P.; Levesque, I.; Morin, J.-F. Polym.
Chem. 2011, 2, 2293−2298.
(25) Zhang, Q.; Sha, Y.; Wang, J.-H. Molecules 2010, 15, 2962−2971.
(26) Metal-free click chemistry was used by Prof. Mike Brook
(McMaster University, Canada) to prepare a completely different
dendrimer with other building blocks. No report on this from the
Brook group has been published yet.
S
* Supporting Information
Experimental procedures and characterization data for all the
new compounds; SEC traces and toxicological assays results for
EO3-EO2-G4-COO−Na+. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: jean-francois.morin@chm.ulaval.ca.
(27) Qin, A.; Jim, C. K. W.; Lu, W.; Lam, J. W. Y.; Haussler, M.;
̈
Dong, Y.; Sung, H. H. Y.; Williams, I. D.; Wong, G. K. L.; Tang, B. Z.
Macromolecules 2007, 40, 2308−2317.
Notes
The authors declare no competing financial interest.
(28) Li, H.; Wang, J.; Sun, J. Z.; Hu, R.; Qin, A.; Tang, B. Z. Polym.
Chem. 2012, 3, 1075−1083.
(29) Kharb, R.; Sharma, P. C.; Yar, M. S. J. Enzyme Inhib. Med. Chem.
2011, 26, 1−21.
ACKNOWLEDGMENTS
■
The authors thank the National Science and Engineering
Council of Canada (NSERC) for financial support and Dr.
Donald Poirier and Lucie Carolle Kenmogne from CRCHUQ
for toxicological assays. Mathieu Arseneault thanks the NSERC
for a PhD scholarship. We are also grateful for professional
support from CQMF and CERMA.
(30) Saha, S.; Johansson, E.; Flood, A. H.; Tseng, H. R.; Zink, J. I.;
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(35) Kislukhin, A. A.; Higginson, C. J.; Finn, M. G. Org. Lett. 2011,
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