Multicomponent host-guest chemistry of carboxylic acid and phosphonic acid based guests with dendritic hosts: An NMR study

Article abstract of DOI:10.1021/ja052074m

A new way to analyze supramolecular dendritic architectures is reported by making use of ¹³C NMR and ³¹P NMR. Two ethylene glycol guest molecules have been synthesized containing a ¹³C labeled carboxylic acid headgroup (2)

Full text of DOI:10.1021/ja052074m

Published on Web 06/23/2005
Multicomponent Host-Guest Chemistry of Carboxylic Acid
and Phosphonic Acid Based Guests with Dendritic Hosts: An
NMR Study
Maarten A. C. Broeren,† Bas F. M. de Waal,† Marcel H. P. van Genderen,†
H. M. H. F. Sanders,†,‡ George Fytas,‡ and E. W. Meijer*,†
Contribution from the Laboratory of Macromolecular and Organic Chemistry, EindhoVen
UniVersity of Technology. P.O. Box 513, 5600 MB EindhoVen, The Netherlands, and Department
of Materials Science and Technology, UniVersity of Crete and IESL/FORTH, P.O. Box 1527,
71110 Heraklion, Greece
Received April 1, 2005; E-mail: e.w.meijer@tue.nl
Abstract: A new way to analyze supramolecular dendritic architectures is reported by making use of 13C
NMR and 31P NMR. Two ethylene glycol guest molecules have been synthesized containing a 13C labeled
carboxylic acid headgroup (2) and a phosphonic acid headgroup (3). The binding of these guests to urea-
adamantyl modified poly(propylene imine) dendrimers has been investigated with 13C NMR and 31P NMR
next to 1D and 2D 1H NMR techniques. Different amounts of guest 2 have been added to fifth generation
dendrimer 1e, and the observed chemical shift values in 13C NMR were fitted to a model that assumes 1:1
binding between guest and binding site. An association constant of 400 ( 95 M-1 is obtained for guest 2
with 41 binding sites per dendrimer. When different amounts of phosphonic acid guest 3 are added to
dendrimer 1e, two different signals are observed in 31P NMR. Deconvolution gives the fractions of free and
bound guest, resulting in an association constant of (4 ( 3) × 104 M-1 and 61 ( 1 binding sites. A statistical
analysis shows that guest 2 forms a “polydisperse supramolecular aggregate”, while guest 3 is able to
form a “monodisperse supramolecular aggregate” when the amount of guest is high enough. The NMR
results are compared with dynamic light scattering experiments, and a remarkable agreement is found.
Phosphonic acid guest 3 is able to exchange with guest 2, which is in agreement with the obtained
association constants, and shows that these techniques can be used to analyze multicomponent dendritic
aggregates.
Introduction
employed to obtain supramolecular dendritic architectures. As
the strength of these interactions depends on external factors
Dendrimers are multivalent, well-defined, and highly branched
macromolecules that tend to form a globular shape in solution.1,2
The stepwise synthesis of dendrimers allows for a large degree
of control over the molecular architecture and has enabled or-
ganic chemists for over 20 years to create dendrimers with dif-
ferent morphologies and properties.3-8 Next to covalent modi-
fication, several noncovalent interactions such as hydrogen bond-
ing,9,10 electrostatic interactions,11,12 hydrophobic interactions,13-15
π-π interactions,16,17 or metal coordination18-20 have been
such as temperature, solvent polarity, or pH, an adaptive system
is obtained that can be designed to change under specific stimuli.
Moreover, decorating the periphery of dendrimers in a nonco-
valent fashion is particularly interesting as many biological
processes occur via supramolecular interactions at surfaces.
Recently, our group developed a methodology to modify the
periphery of dendrimers in a noncovalent manner.21-29 Poly-
(propylene imine) dendrimers modified with urea-adamantyl end
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† Eindhoven University of Technology.
‡ University of Crete and IESL/FORTH.
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J. AM. CHEM. SOC. 2005, 127, 10334-10343
10.1021/ja052074m CCC: $30.25 © 2005 American Chemical Society

31P/13C-NMR Analysis of Dendritic Architectures
A R T I C L E S
groups can bind ureido-acetic acid guest molecules via elec-
trostatic interactions and hydrogen bonding. The electrostatic
interactions occur through an acid-base interaction between
the carboxylic acid functionality of the guest and a tertiary amine
of the dendrimer. Hydrogen bonding occurs between the urea-
groups of the guest and host and should direct complexation to
the periphery of the dendrimer. A major increase in binding
strength can be achieved by increasing the acid strength of the
guest molecules. This has been observed both in the gas phase25
and in chloroform,26,30 when the carboxylic acid group was
replaced with a phosphonic or sulfonic acid group.
dependent on the pH. In water, the 13C chemical shift of
carboxylic acids can shift over 5 ppm downfield when the acid
is deprotonated and the carboxylate salt is formed.49-53 For
phosphonic acids, both upfield and downfield shifts in 31P NMR
have been reported upon deprotonation.54-57 This inspired us
to use 13C NMR and 31P NMR as a tool to investigate the
binding of ureido-acetic acid and ureido-phosphonic acid type
guests to urea-adamantyl dendrimers in chloroform. To make
analysis easier and to prevent long measurement times, a 13C
label has been incorporated in the carbonyl group of the acid
functionality of the ureido-acetic acid guest molecules.
For solubility reasons, guest 2 and 3 were synthesized and
used. Complexation experiments of both guests have been
performed to several dendritic hosts (1a-1e) (Scheme 1), and
the experimental results in case of host 1e were fitted to get an
idea of the binding stoichiometry and the association constant.
Exchange experiments have been performed in which both guest
molecules were simultaneously added to the dendrimer host, to
investigate the difference in binding strength between guest 2
and 3 in more detail.
Although mass spectrometry and NMR spectroscopy have
given useful information about the binding of these types of
guests to dendrimers, specific information about the number of
guests that bind to the host, the binding strength between guest
and dendritic host and the exchange kinetics between free and
bound guest in solution remains difficult to obtain. Overlap of
the signals in 1H NMR of guest and host hampers the analysis
of several complexes. Ideally, we would like to have a general
analytical methodology to investigate the binding of any guest
to the dendrimer in any solvent. In this report, we present the
results obtained by making use of 13C NMR and 31P NMR next
Results
1
to H NMR to investigate the guest-host interactions of 13C
Covalent Synthesis. The starting material for the 13C label
in guest 2 was the commercially available 1-13C-glycine
(Scheme 2). Reaction with benzyl alcohol in toluene in the
presence of p-toluenesulfonic acid resulted in the benzyl ester
of 1-13C-glycine as the p-toluenesulfonic acid salt (4). A Dean-
Stark setup was used to azeotropically remove the water that is
formed during the reaction. As a starting material for the
oligoethylene glycol part of guest 2, 3,4,5-tri(tetraethyleneoxy)-
benzoic acid was used, which was synthesized according to a
described procedure.58 This acid was reacted with ethylchloro-
formate in THF with triethylamine to afford the mixed
anhydride. This mixture was added to a solution of sodium azide
in water, and extractions with dichloromethane resulted in the
acyl azide derivative. The acyl azide could subsequently be
converted to the isocyanate 7 via the Curtius rearrangement in
labeled carboxylic and 31P phosphonic acid containing guest
molecules in more detail. The principle of the methodology is
investigated in detail for the fifth generation dendrimer.
Designing the System. In an apolar aprotic solvent like
chloroform, an acid (HA) and a base (B) are usually in
equilibrium with a tight ion pair. The formed anion (A-) and
cation (BH+) are in close proximity of each other due to
electrostatic attraction and hydrogen bonding (eq 1), and it is
generally believed that no free ions are present in solution.31-48
It is known that both the 13C chemical shift of carboxylic
acids and the 31P chemical shift of phosphonic acids are strongly
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A R T I C L E S
Scheme 1
Broeren et al.
refluxing toluene. The isocyanate was reacted with 4, after
triethylamine was added to liberate the free amine. Ester 8 could
be purified using column chromatography. Finally the benzyl
ester was converted to free acid 2 by catalytic hydrogenolysis
with H2 gas and Pd/C as a catalyst. Benzyl ester 8 has to be
treated carefully. A first attempt to purify 8 by a basic workup
with 1 M NaOH (aq) resulted in hydantoin 9. When a saturated
NaHCO3 (aq) is used, this side reaction can be prevented.
Though initially unwanted, hydantoin 9 was purified and
analyzed and has been used for several control experiments.
Phosphonic acid guest 3 was synthesized from phthalimide
derivative 5, which has been synthesized according to an adapted
literature procedure.59 Treatment with hydrazine resulted in
amine 6, which was immediately used for reaction with
isocyanate 7. Product 10 could be purified using column
chromatography, and treatment with TFA to remove the tert-
butyl groups resulted in pure 3. All compounds were analyzed
guest 2 and 3 to dendrimer 1e (S7, S8) are in agreement with
previously reported results.
If guest and host are bound, they should be in close proximity
to each other. This has been investigated with 1H-1H-NOESY
NMR spectroscopy. 1H-1H-NOESY spectra of 1e+232 and
1e+332 (Figure 1b and 1d) show cross-peaks between the
oligoethylene glycol tails of the guests (in all spectra indicated
as 2) and the adamantyl groups (signal 3) of the dendrimer,
indicating that these protons are close to each other through
space. Also a weak cross-peak is observed between the aromatic
protons of guest 2 and 3 (signal 1) and the adamantyl signals
of 1e, which is an indication that the headgroup of the guest is
in close proximity to the periphery of the host and is responsible
for complexation, as has been investigated in earlier work.27
1
This is also confirmed by H-1H-NOESY spectra that have
been recorded for 1e+932 and 1e+1032. In both cases no
intermolecular NOE contacts were observed between the den-
drimer and the guests, showing that no complexation occurs.
1
with H NMR, 13C NMR, 31P NMR, FT-IR spectroscopy and
(high resolution) mass spectrometry.
Supramolecular Synthesis of the Complexes. All complexes
were prepared in a similar fashion, in which the concentration
of the guest was kept constant. For both guest 2 and 3, 10 mg
were weighed and an amount of dendrimer was added to obtain
the desired guest/host ratio. The compounds were dissolved in
0.5 mL of CDCl3 and shaken for 5 min. Before we investigated
the complexation behavior of guest 2 and 3 with 13C NMR and
31P NMR, we have analyzed the complexation to dendrimer 1e
with several 1H NMR techniques to ascertain that binding
occurred. This was done for both guests on a sample with a
guest/host ratio of 32. This is denoted as 1e+232 and 1e+332,
and this notation is used for all guests and dendrimers throughout
this paper. The observed changes in 1H NMR upon addition of
When a guest molecule binds to fifth generation dendrimer
1e, its diffusion constant should change as the dendrimer has a
much higher molecular weight and consequentially diffuses
slower. Actually, if binding of a guest to 1e is strong enough,
both guest and host should diffuse with an equal rate as they
1
belong to the same supramolecular aggregate. Therefore, H-
DOSY-NMR has been performed on several complexes. When
1
samples 1e+932 and 1e+1032 are analyzed with H-DOSY-
NMR, different apparent diffusion constants were found for
guest and host (Figure 1). The apparent diffusion constants of
hydantoin 9 and phosphonic ester 10 are higher than that of
dendrimer 1e, as they are smaller molecules that diffuse faster.
These differences again support the idea that 9 and 10 do not
interact with the dendrimer. However, this is not the case for
guest molecules 2 and 3. When samples 1e+232 and 1e+332
(59) Geneˆt, J. P.; Uziel, J.; Port, M.; Touzin, A. M.; Roland, S.; Thorimbert,
S.; Tanier, S. Tetrahedron Lett. 1992, 33, 77-80.
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31P/13C-NMR Analysis of Dendritic Architectures
A R T I C L E S
Scheme 2. Synthesis of Guest 2 and 3a
a Reagents and Conditions: (a) p-toluenesulfonic acid, toluene; (b) H2NNH2, EtOH; (c) ethylchloroformate, triethylamine, THF; (d) NaN3, H2O; (e)
toluene, reflux; (f) CH2Cl2, triethylamine; (g) t-BuOH, H2O, H2(g), Pd/C; (h) 1 M NaOH(aq); (i) CH2Cl2; (j) TFA, CH2Cl2.
are analyzed with 1H-DOSY-NMR, the apparent diffusion
constants of 2 and 3 are similar to host 1e. This confirms that
guest and host are bound to each other.
in CDCl3 shows a signal at 172.5 ppm, which corresponds to
the carboxylic acid carbon (Figure 2).
For the free guest the line width of the peak at half-height is
approximately 3 Hz. For the sample with a guest/host ratio of
8, the peak shifts downfield to 175.6 ppm and broadens slightly.
The downfield shift is also observed when an excess of
triethylamine is added to guest 2, but not when hydantoin 9 is
added to 1e (S10). Clearly the shift is due to deprotonation of
the acid. As in chloroform a tight ion pair is formed between
an acid and a base, the downfield shift is a direct measure for
complexation of the guest to the dendrimer. When we look at
the results for increasing guest/host ratio, two trends are
observed. First of all, the carbonyl signal shifts upfield. The
change in chemical shift is small when 1e+28 is compared to
1e+216 but is clearly present when the guest/host ratio (from
now on referred to as G/H) is further increased to 32, 48, 64,
96, and 128. Furthermore, the signal broadens when G/H
increases until the value of 64 is reached. A further increase of
G/H results in a sharper signal again. We propose that the results
can be interpreted in the following manner. Only one peak is
observed, and this indicates that the signal is an average signal
for both free and bound guest. This shows that exchange
To exclude the possible formation of dendrimer-dendrimer
aggregates next to dendrimer-guest complexes, dynamic light
scattering experiments have been performed in nondeuterated
chloroform. Measurements performed on dendritic host 1e alone
in chloroform and complexes with guest 2 and 3 in different
guest/host ratios show one predominant process of small
particles. The hydrodynamic radius (RH) found for the sample
with only dendrimer 1e is 2.2 nm. This value is in good
agreement with the dimensions of a single dendrimer.3 For the
different host/guest complexes RH increases upon increasing
guest/host ratio, with RH ) 2.8 nm for 1e+234 and RH ) 3.0
nm for 1e+332 (S11). The results also show that no dendrimer
aggregation is taking place.
Complexation of Guest 2 to Dendrimer 1e Observed by
13C NMR. Having obtained evidence that guest 2 binds to
dendrimer 1e in chloroform, we can analyze the complexation
behavior with 13C NMR. This has been performed by investigat-
ing how the 13C chemical shift of the guest depends on the
composition of the sample. The 13C NMR spectrum of pure 2
9
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A R T I C L E S
Broeren et al.
Figure 1. 1H-1H-NOESY spectra (a-d) and 1H-DOSY spectra (e-h) recorded for (a) 1e+932, (b) 1e+232, (c) 1e+1032, (d) 1e+332, (e) 1e+932, (f) 1e+232,
(g) 1e+1032, and (h) 1e+332 in CDCl3 at 25°C.
between free and bound guest is fast on the spectral time scale.60
For increasing G/H the signal shifts back in the direction of the
free guest, indicating that the amount of unbound guest
increases.
broadening can also occur due to coalescence.61 In this case
the relative amount of free and bound guest should influence
the peak broadness, and this is exactly what happens when G/H
is increased. When we start at 1e+28, bound guest is the
predominant species. When the relative amount of 2 is increased
more unbound guest starts to become present, resulting in a
broader signal. At 1e+264 the line width is highest, which
suggests that the amount of free and bound guest is ap-
proximately equal in this case. A further increase in G/H results
in an excess of unbound guest and thus in a sharper signal. As
spectral line shape perturbations are dependent on the magnetic
field, complex 1e+264 was analyzed at different field strengths
(Figure 3). This resulted in a sharper signal at lower field
strength and is an indication that coalescence is indeed taking
place. This means that although T2 relaxation and coalescence
Two effects govern the broadening of the signal. Complex-
ation to a large macromolecule results in a decrease in mobility
of the headgroup of the guest. This causes an increase in T2
relaxation and consequentially in broadening of the signal.
However, this is not very plausible as the peak becomes sharper
again after 64 equiv of guest. When the exchange rate between
free and bound guest comes close to the spectral time scale,
(60) The spectral time scale represents the inverse width of the NMR spectrum,
measured in frequency units. The chemical shift difference between free
and bound guest corresponds to 390 Hz (3.1 ppm), which results in a
spectral time scale of 2.6 ms. Levitt, M. H.; Spin Dynamics, John Wiley
& Sons: 2002; p 485.
(61) Sandstro¨m, J. Dynamic NMR Spectroscopy; Academic Press, 1982.
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31P/13C-NMR Analysis of Dendritic Architectures
A R T I C L E S
Figure 2. 13C NMR spectra of different guest/host ratios of 2 and 1e. The concentration of guest is kept constant at 2.46 × 10-2 M. The line widths of the
peaks are indicated in the inset. The intensities are not normalized.
Figure 3. Dependency of the signal of 1e+264 on the magnetic field. The
signal becomes more narrow when the field strength of the spectrometer
decreases.
both play a role in line broadening, coalescence is the major
contributor of the two.
Figure 4. 31P NMR spectra of different G/H ratios of 3 and 1e. The
concentration of guest is kept constant at 2.36 × 10-2 M. The deconvolution
results are depicted in the table.
When guest 2 is added to lower generation dendrimers, the
same trend in chemical shift dependency is observed when G/H
is increased, and complexation can be followed in exactly the
same way. Apparently, the method is applicable to all dendrimer
generations, but this is beyond the scope of this paper. The
broadening of the signal upon increasing G/H has not been
observed for lower generation dendrimers, indicating that the
exchange kinetics are different for these dendrimers.
Complexation of Guest 3 to 1e Observed by 31P NMR. In
a manner similar to guest 2, we have investigated the complex-
ation of guest 3 to dendrimer 1e using 31P NMR (Figure 4).
Free guest 3 gives a sharp peak at 22.5 ppm in CDCl3. This
peak completely disappears when 16 (not depicted) or 32 guests
are added to 1e and is a broad signal at 16 ppm. When G/H is
increased to 56, no big changes occur. At G/H ) 64, two distinct
signals start to become present: a narrow peak at 21 ppm and
the very broad signal at 17 ppm. A further increase of G/H
shows that the peak at 21 ppm becomes more intense and shifts
a little downfield.
The broad peak remains present. These results show com-
pletely different trends than the observations for complexation
of guest 2 to 1e in 13C NMR. The chemical shift of the
phosphonic acid group shifts upfield instead of downfield due
to deprotonation. It is known that the chemical shift of
phosphonic acid derivatives strongly depends on the pH. Both
upfield and downfield shifts have been reported for different
compounds. In our case, deprotonation of the phosphonic acid
and the formation of a tight ion pair apparently results in an
upfield shift, and this has also been observed when an excess
of triethylamine was added to guest 3 (S10). We observe two
separate signals when G/H is increased, indicating that we now
have slow exchange on the spectral time scale.62 The signal of
the free guest is located around 22 ppm and relatively sharp,
(62) The chemical shift difference of 1315 Hz corresponds to a spectral time
scale of 0.76 ms.
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A R T I C L E S
Broeren et al.
Figure 5. (a) Chemical shift values of guest 2 as a function of G/H (points). The solid line represents the fit. (b) The fraction of bound guest 3 to host 1e
as a function of the amount of G/H. The solid line represents the fit.
and the signal of the bound guest is located around 16 ppm
and very broad. It is possible to deconvolute the two signals
and to directly determine the ratio of free and bound guest. This
has been done for G/H ) 64, 70, 96, and 128 (Figure 4, table)
and shows that the amount of bound guest remains fairly
constant around 60 equiv. The amount of free guest increases
from 5 to 67 guests. The numbers suggest that approximately
60 guests can bind to 1e. This amount comes very close to the
62 tertiary amines that are present inside the dendrimer.
The signal of the bound guest is very broad. Again, both T2
relaxation and coalescence can cause this broadening. However,
as we have slow exchange on the spectal time scale coalescence
is less likely to be the major contributor. This was confirmed
by the observation that measurements at other magnetic field
strengths did not show a change in the spectrum. To test whether
T2 relaxation could be the reason for the severe broadening,
we examined the phosphorus NMR spectra of guest 3 added to
the lower generation dendrimers 1c, 1a, and pincer molecule
1f (S10). In all cases the stoichiometry of one guest per two
endgroups was maintained. The spectrum of 1f+31 shows a
downfield shift of the phosphorus signal, but no big change in
line width, in contrast to 1a+32, 1c+38, and 1e+332. Clearly
the line width increases upon increasing size of the dendrimer
host. A decrease in mobility of the headgroup of the guest due
to complexation to such a large molecule could explain the
results.
For lower generation hosts a similar chemical shift depen-
dency is observed for different values of G/H, meaning that
the signal of the guest shifts upfield upon deprotonation.
However, the exchange kinetics also depend on the dendrimer
generation and are faster for lower generation dendrimers as
the two different signals for free and bound guest are not always
observed. For example, different amounts of guest added to
pincer 1f always results in one signal (S11). So, exchange is
faster for the pincer. In this case, the broadening is most likely
due to coalescence again.
Quantitative Analysis of Binding of Guest 2 and 3 to 1e.
The obtained chemical shift values can be used to analyze the
binding of guest 2 to dendrimer 1e in a quantitative manner.
The observed chemical shift (δ) can be seen as a mixture of
free and bound (deprotonated) guest since we are in a fast
exchange situation, and can be represented as
where pb ) fraction of bound guest, δb ) chemical shift of the
bound guest, pf ) fraction of free guest, and δf ) chemical
shift of the free guest.
The chemical shift of the free guest is known (δf ) 172.5
ppm), but that of the bound guest is not. However, most likely
it is very close to the 175.6 ppm obtained for 1e+28. We assume
there is no cooperativity: all binding sites in the host are
identical and do not influence each other. When a 1:1 binding
of guest and binding site is assumed, an equation can be derived
that gives the fraction of bound guest as a function of G/H (S12).
Fitting this equation to the measured chemical shift values with
a nonlinear least-squares fit (Figure 5a) gives us values for the
number of binding sites (n), the association constant for
carboxylic acid guest 2 (KC), and the chemical shift of the bound
guest (δb). We find
n ) 41 ( 2
δb ) 175.61 ppm
KC ) 400 ( 95 M-1
The obtained association constant is in the same order of
magnitude as the association constant found from fluorescence
measurements on model host compounds.30
For guest 3 we followed the same reasoning as that for guest
2, but now we have the fraction of bound guest (pb) directly
available from the deconvolution of the spectra. We can directly
fit pb to the equivalents of guest added (Figure 5b). We then
find
n ) 61 ( 1
KP ) (4 ( 3) × 104 M-1
The fit shows a higher association constant for guest 3 than
for guest 2. This is in agreement with earlier results that show
a stronger binding for the phosphonic acid guest. The results
are different from the traditional model as more than 32 guests
can apparently bind to the dendrimer. The discrimination
between guests bound at the periphery or the interior of the
dendrimer cannot be made based on 31P NMR.
Exchange Experiments. As we are now able to follow
complexation of guest 2 and 3 with two orthogonal NMR
techniques, we investigated what happens when both guests are
simultaneously added to dendrimer 1e. The amount of carboxylic
δ ) pbδb + pfδf
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31P/13C-NMR Analysis of Dendritic Architectures
A R T I C L E S
Figure 6. Influence of the addition of 3 to 1e+232 observed in 13C NMR (left) and 31P NMR (right). The concentration of guest 2 was kept constant at 2.46
× 10-2 M.
acid guest 2 was kept constant at G/H ) 32. To this sample
was added 16, 32, and 64 equiv of phosphonic acid guest 3,
and the results were monitored by both 13C NMR and 31P NMR
(Figure 6).
supramolecular aggregate in solution but a distribution in the
number of bound guests. When the amount of 2 is further
increased, more guests will be bound on average, but the amount
of free guest increases significantly too. For guest 3 the same
statistical rules apply, also resulting in a polydisperse supramo-
lecular aggregate. However, below G/H ) 60 virtually all guests
are bound to the dendrimer due to the higher association
constant. An almost monodisperse supramolecular aggregate can
be obtained for guest 3, but only at high guest/host ratios when
a lot of free guest is present (S14). Obviously, the number of
bound guests is based on a certain concentration and alters with
changes in the concentration.
To visualize the polydispersity and the ratio of bound and
free guest more clearly, two distributions have been depicted
in Figure 7. Although both graphs are based on simple statistical
calculations, we can use them to expatiate the often too
simplified view of a multicomponent aggregate, in which it is
presented as a monodisperse entity with complete binding.
The binding stoichiometries of guest 2 and 3 obtained with
NMR are in agreement with dynamic light scattering experi-
ments. The absolute scattering intensity Rvv/c, normalized to
the concentration of dendrimer at large probing lengths (q ≈
0), has been determined from several guest-host complexes.
It is proportional to the molar mass of the probed aggregate in
solution. This quantity is plotted against the number of guests
that are bound to the dendrimer based on the NMR results. A
monotonic, almost linear increase is observed for both guest-
host systems (Figure 8). This confirms the obtained relations
between the number of added guest and the number of bound
and free guest found for guests 2 and 3 to dendrimer 1e by
NMR. According to the fitted NMR data, 55 equiv of guest 2
added to dendrimer 1e should result in 32 equiv bound. For
guest 3, addition of 32 equiv to dendrimer 1e should also give
32 equiv bound. These points also overlap in the graph.
The differences between guest 2 and 3 are also reflected in
the exchange experiment. If we assume that the binding
equilibria are independent for 2 and 3, the amount of free and
When 32 equiv of 2 are added to 1e, the characteristic
downfield shift to 175.1 ppm is observed in 13C NMR. As 3 is
not present, the 31P NMR spectrum does not show anything.
When 16 equiv of 3 are added to this sample, resulting in 1e+-
(232+316), an upfield shift in 13C NMR is observed to 174.7
ppm, indicating that 2 dissociates partially from the dendrimer.
When 1e+(232+316) is analyzed with 31P NMR, a broad signal
at 16 ppm is observed, indicating that all of phosphonic acid
guest 3 is bound to 1e. An increase of the amount of 3 to 1e+-
(232+332) results in an upfield shift to 174.3 ppm in 13C NMR,
so 2 dissociates further from 1e due to 3. In 31P NMR we still
observe complete complexation of phosphonic acid guest 3 to
the dendrimer. When the amount of guest 3 is increased to 1e+-
(232+364) the signal of guest 2 in 13C NMR shifts to 173.1 ppm.
This is much further upfield than observed for 1e+296 or
1e+2128, which give values of 173.7 and 173.4 ppm, respectively
(Figure 2). Also the line width of the peak, 10 Hz, is much
lower than that for 1e+296 or 1e+2128 and indicates the amount
of bound 2 is lower. In 31P NMR, we now start to observe two
signals, so some free 3 is present.
Discussion
The results regarding the binding strength and binding
stoichiometry of guest 2 and 3 to urea-adamantyl dendrimer 1e
have some major implications for our molecular picture. With
the availability of association constant and number of binding
sites for both guests, we can calculate the amount of free and
bound guest for every guest/host ratio (S13, S14) at a certain
concentration. The relatively low association constant of guest
2 has the consequence that when 32 equiv of guest 2 are added
to 1e, only 26 guests are bound on aVerage and also free 2 is
present in solution. The term on average is deliberately used to
indicate that, as expected, we do not have a monodisperse
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A R T I C L E S
Broeren et al.
Figure 7. Calculated distributions of the number of bound guests 2 (left) or 3 (right) to dendrimer 1e in the case of noncooperative binding. In both cases
the guest/host ratio corresponds to 64. The concentration of 2 and 3 is 2.46 × 10-2 M and 2.36 × 10-2 M respectively.
Figure 8. Absolute scattering intensity Rvv/c plotted against the number
of bound guests 2 and 3 according to the fitted NMR data. The numbers in
the graph represent the guest/host ratio of the sample. With the number of
binding sites and the binding constant for guest 2 and 3 from NMR, the
Figure 9. Amounts of free and bound guests 2 and 3 as obtained from the
number of bound guests have been calculated and put on the x-axis. The
exchange experiment depicted in Figure 6.
line is to guide the eye.
bound carboxylic acid guest 2 can be determined from the
chemical shift, and the amount of free and bound phosphonic
acid guest 3 can be determined from deconvolution of the
spectra (Figure 9). The obtained numbers of bound and free
guest 2 and 3 show that initially guest 2 and 3 coexist on the
dendrimer. Although the association constant of guest 3 is
higher, it does not compete severely with carboxylic acid guest
2 as guest 3 has 61 possible sites to bind to. However, when 64
equiv of 3 are added to 1e+232, 3 competes with all the binding
sites of guest 2, and this results in almost complete dissociation
of 2 from 1e. The amounts of free and bound 2 and 3 that are
found in the mixing experiment are in accordance with the
expected values based on the obtained association constants.
From these experiments we can conclude that it is possible to
make mixed aggregates and that phosphonic acid guest 3 can
expel carboxylic acid guest 2 from the dendrimer.
1
the dendrimer, as previous H-1H-NOESY experiments have
shown that carboxylic acid based guest molecules are mainly
located at the periphery of the dendrimer. However, we must
conclude that as more than 32 equiv of guest 2 can bind to the
dendrimer, some guests should be bound to the inner tertiary
amines of the dendrimer. This does not exclude the formation
of hydrogen bonds with the dendrimer, as its structure is highly
flexible and certainly not always completely stretched, but
implies a more complex mode of binding than the initial “pincer
model” that has been presented (Introduction).
It is important to stress that the association constant 400 M-1
obtained for guest 2 is not general for every carboxylic acid
guest. For a cyanobiphenyl-containing guest21 the association
constant was estimated to be 105 M-1 in chloroform. The
discrepancy in association constant is caused by a difference in
solubility. The cyanobiphenyl guest has a low solubility in
chloroform but can be solubilized by complexation to the
dendrimer. Therefore the equilibrium (eq 1) is shifted in the
direction of the complex, resulting in a higher apparent
association constant.31 Clearly the apparent association constant
can be influenced over several orders of magnitude by the
Knowing all of these details, we can say that the number of
guests that bind to the dendrimer is mainly governed by the
acid-base interaction. When the acid strength is increased, the
amount of guests that bind to the dendrimer and the binding
strength increases similarly. The hydrogen bonds most likely
help to direct and strengthen the binding to the periphery of
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31P/13C-NMR Analysis of Dendritic Architectures
A R T I C L E S
solubility of guest and complex, as has been nicely demonstrated
by Gillies and Fre´chet.29
For guest 3 we have observed that it can bind to virtually all
tertiary amines, indicating that the acid-base interaction
dominates the binding to the host. The urea groups of guest 3
might still help to direct binding to the peripheral tertiary amines
of the dendrimer, but this effect is probably weaker than for
guest 2. Actually, based on our experiments we cannot
discriminate between binding to the peripheral tertiary amines
and binding to the amines further inside the dendrimer.
Especially for guest 3, Figure 10 gives a more realistic
representation for the binding to dendrimer 1e. Currently we
are making use of molecular simulations and crystal structures
to get detailed information on the way the guest molecules bind
to the dendrimer in three dimensions.
Conclusions
A new methodology has been introduced to get insights in
the binding of carboxylic and phosphonic acid type guest
molecules to dendrimers by making use of 13C NMR and 31P
NMR. The method is generally applicable for any type of guest
and host in chloroform and gives information about binding
strength, binding stoichiometry, and binding dynamics. In this
way it is possible to analyze multicomponent aggregates of
different guest molecules simultaneously bound to the dendrimer
in great detail. With simple statistical calculations, we have used
the results to expatiate the often too simplified view of a
multivalent aggregate, in which it is presented as a monodisperse
entity with complete binding.
Figure 10. Representation for the binding of guests to urea-adamantyl
dendrimers that shows that guest molecules (especially with phosphonic
acid headgroups) can also bind to the inner tertiary amines of the dendrimer.
Only a part of the dendrimer structure is depicted. The hydrogen bonds
between guest and host have been omitted for clarity.
Acknowledgment. The authors would like to thank Mrs.
Antje Larsen for technical assistance of the DLS measurements
and acknowledge financial support of the EU (HPRN-CT-2000-
2003) and grants from the department of Biomedical Engineer-
ing of the Eindhoven University of Technology and the Council
for Chemical Science of The Netherlands Organization for
Science Research (CW-NWO).
of triethylamine. Dynamic light scattering experiments of
complexes of 1e with 2 and 3 with different guest/host ratios.
Details on the equations derived for the quantitative analysis
of the binding of 2 and 3 to 1e. Calculations of the fractions of
bound and free guest for several G/H values and the corre-
sponding distributions. This material is available free of charge
via the Internet at http://pubs.acs.org.
Supporting Information Available: Experimental details for
1
the synthesis of compounds 2, 3, 4, 8, 9, and 10. H NMR
spectra for the samples 1e+232, 1e+332, 1e+932, and 1e+1032,
13C NMR spectra of samples 1e + 932 and 2 in CDCl3 with an
excess of triethylamine, and 31P NMR spectra of samples 1e +
1032, 1f+31, 1f+32, 1f+34, 1a+32, 1c+38, and 3 with an excess
JA052074M
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