Application of the Ugi four-component reaction to the synthesis of ditopic bifunctional chelating agents

Article abstract of DOI:10.1039/b907932g

The Ugi four-component reaction (Ugi 4CR) was exploited for the first time to obtain in a single synthetic step bifunctional ditopic chelators by using DOTA monoamide (DOTAMA) derivatives as amino and acid components. A number of ditopic systems in which

Full text of DOI:10.1039/b907932g

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Application of the Ugi four-component reaction to the synthesis of ditopic
bifunctional chelating agents†
Lorenzo Tei,a Giuseppe Gugliotta,a Stefano Avedano,a Giovanni B. Giovenzanab and Mauro Botta*a
Received 21st April 2009, Accepted 24th July 2009
First published as an Advance Article on the web 19th August 2009
DOI: 10.1039/b907932g
The Ugi four-component reaction (Ugi 4CR) was exploited for the first time to obtain in a single
synthetic step bifunctional ditopic chelators by using DOTA monoamide (DOTAMA) derivatives as
amino and acid components. A number of ditopic systems in which the two DOTAMA units are
connected by a central a-acylaminoamide group were synthesized by reacting different aldehydes,
isocyanides and two DOTAMA chelates containing amino and acid functionalities. Variation of the
components allows the insertion of another functional group into the a-acylaminoamide skeleton for
further conjugation to biomolecules. The optimal reaction conditions were found by using methanol as
solvent and ultrasound irradiation at a power of 60 W (20 kHz) for 3 h. The Gd(III) complexes of the
dimeric ligands L1 and L2 (bearing a cyclohexyl ring and an octadecyl chain on the central
a-acylaminoamide moiety, respectively) were fully characterized in aqueous media by relaxometric
techniques with varying temperature and magnetic field strength. The relaxivity of Gd2L1 and Gd2L2
(in the aggregated form), at 20 MHz and 310 K, are 5.6 and 20.0 mM-1 s-1, respectively. The enhanced
value found for Gd2L2 indicates that this lipophilic complex forms micelles at concentrations
<0.1 mM. Finally, the binding of Gd2L2 to human serum albumin (HSA) was investigated by proton
relaxometry, and the affinity constant of the complex and the relaxivity of the macromolecular adduct
(rb1p = 38.1 mM-1 s-1; 20 MHz and 310 K) derived.
only great molecular complexity and diversity per step but also
the possibility of introducing matched functionalities suitable for
Introduction
Multicomponent reactions (MCRs)1 have experienced fast-
growing interest in the last decade, mainly because they allow the
assembly of complicated structures from simple building blocks
in a single operation. One of the best-known MCRs is the Ugi
four-component reaction (Ugi 4CR)2 whose great potential in
synthetic organic chemistry has been widely explored.3 In fact,
it is possible to synthesize a large number of different systems
by the reaction of a number of carbonyl compounds, amines,
isocyanides and carboxylic acids together to form a-acylamino
amides. These libraries have been investigated either with enzymes
or living organisms to find new bioactive leads.4 The exploitation
of the Ugi 4CR provides the rapid generation of molecular
complexity and diversity from simple and readily accessible
starting materials. The one-pot preparation of variably substituted
polyamides and/or of functionalized macrocyclic ligands allows
one to avoid the time- and chemical-consuming steps needed to
discriminate the chemically equivalent nitrogen atoms of di-, tri-
or poly-amines.5 Indeed, being capable of combining three or
more reactants together in an ordered process, MCRs offer not
further transformations.3 In fact, the combination of an MCR
with an efficient post-transformation, for example the conjugation
to macromolecules or biomolecules, could represent a powerful
tool for the synthesis of molecular probes useful for biomedical
applications.
In a parallel context, the synthesis of functionalized polyazacy-
cloalkanes continues to see growing interest, since they represent a
class of chelating agents able to form stable complexes with a large
variety of metal ions, ranging from transition metals to lanthanides
and other heavy metals. The uses of these chelates as contrast-
enhancing agents (CAs) for magnetic resonance imaging (MRI)6
and as radiopharmaceuticals for the diagnosis and therapy of
tumours7 represent the most relevant biomedical applications. An
important challenge in the development of novel chelates is the im-
provement of their capability to target certain organs and tissues, in
order to allow precise delivery to specific cellular targets.8 The gen-
eral strategy is the synthesis of a chelating unit linked to a suitable
function through which it can be covalently bound to the specific
biological carrier, leading to the so-called bifunctional chelating
agents (BFCAs).9 Macrocyclic BFCAs are often based on DOTA
or DOTA monoamide (DOTA = 1,4,7,10-tetraazacyclododecane-
N,N¢,N¢¢,N¢¢¢-tetraacetic acid) derivatives bearing a large variety
of functional groups available for conjugation, such as carboxyls,
amines, aldehydes or ketones, isothiocyanates, maleimides, alkynes
and vinylsulfone.10,11
aDipartimento di Scienze dell’Ambiente e della Vita, Universita` degli Studi
del Piemonte Orientale “Amedeo Avogadro”, Viale T. Michel 11, I-15100,
Alessandria, Italy. E-mail: mauro.botta@mfn.unipmn.it
bDiSCAFF & DFB Center, Universita` degli Studi del Piemonte Orientale
“Amedeo Avogadro”, Via Bovio 6, I-28100, Novara, Italy
1
† Electronic supplementary information (ESI) available: H relaxometric
In the search for the optimal Gd(III) chelate for MRI appli-
cations, one strategy is based on the increase of the number of
Gd(III) units per CA through the formation of multimers, of
titration of Gd2L2 with HSA (Fig. S1); VT 1H NMR profile of Gd2L2
conjugated to HSA (Fig. S2); 1/T1 NMRD profiles of Gd2L2 conjugated
to HSA (Fig. S3). See DOI: 10.1039/b907932g
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covalent or non-covalent conjugates between the paramagnetic
chelate and slowly moving substrates, and the self-assembly of
amphiphilic Gd(III) chelates to form micelles.12
4CR by using three bifunctional chelating agents11,16 based on a
DOTA-monoamide platform containing a primary amino group
(DOTAMA(OtBu)3En (1) and DOTAMA(OtBu)3C6NH2 (2)) or
a carboxylic acid (DOTAMA(OtBu)3C6OH (3)) functional group,
respectively (Scheme 2). The reacting functionality is separated
from the DOTA monoamide moiety by a two-carbon spacer group
in 1 and a six-carbon spacer in 2 and 3. In detail, by using one of
the BFCAs with the free NH2 as the amino component and 3 as
the acid component and varying the aldehyde and the isocyanide
components, we were able to synthesize a series of ditopic systems
in which the two chelating units are connected by a central
a-acylaminoamide group (Scheme 2).
Initially, we sought the best conditions to obtain the product
with yields as high as possible. For this reason, we pursued the Ugi
4CR reaction either by using conventional heating or alternative
activation tools, i.e.: microwaves (MW) and ultrasound. MW and
ultrasound have in fact been shown to improve many organic
reactions in terms of reaction times, product yields and purity
substantially by reducing or even eliminating side reactions.
In this context, the synthesis of multimeric BFCAs, which can
combine the search for more efficient CA and the targeting of
these CA to specific receptors, has never been pursued. Thus,
although several ditopic cyclen-based ligands and their Ln(III)
complexes have been reported in the literature,13 none of them
offers the possibility to anchor the dimeric system to vectors or
biomolecules through orthogonally protected functional groups.
We have therefore approached this problem through the use of
the Ugi 4CR, which has permitted the synthesis of a series of
dimeric ligands with a hydrophobic chain or different functional
groups. The synthesis was achieved by the use of simple monomeric
BFCAs containing a primary amino group in one case and a
carboxylic acid in another and by changing the aldehyde and/or
the isocyanide employed for the Ugi 4CR.
Results and discussion
In the commonly accepted mechanism of the Ugi 4CR, the
first step is the condensation of the primary amine (Scheme 1)
with the carbonyl compound generating an intermediate imine,14
protonated by the carboxylic acid moiety to give an iminium
ion. The latter undergoes a nucleophilic attack by the isocyanide
leading to the formation of a nitrilium ion intermediate, sub-
sequently intercepted by the corresponding carboxylate anion.
The resulting iminoanhydride typically proceeds through the
irreversible transacylation known as a Mumm rearrangement15
to give the final Ugi 4CR product.
Synthetic procedures
The first attempt was made by treating 1 with equimolar amounts
of paraformaldehyde, 3, and cyclohexylisocyanide in refluxing
methanol. Indeed, we observed the formation of the Ugi 4CR
product by ESI-MS, although only after 6 h with by-products
and residual starting materials. The dimer 4 was isolated after
column chromatography in quite low yield (18%). Either using
other solvents (acetonitrile, THF) or increasing the reaction time
did not improve significantly the yield. Switching the aldehyde
component to decanal increased the yield slightly (28%), but
this was not yet satisfactory. Replacing the amino component
1 with the ligand DOTAMA(OtBu)3C6NH2 (2) gave the product
in 20% yield. We surmised that the amino and acid functional
groups on the DOTA monoamide derivatives 1 and 3 were not
easily available for the multicomponent reaction. In fact, the NH2
group can interact with carboxylate or amide oxygen atoms, and
the carboxylic acid with nitrogen atoms of the macrocycle, thus
wrapping the pendant arms and closing the molecule to the action
of external reactants (molecular “pangolinization”). Therefore,
we were prompted to explore alternative heating techniques such
as MW and ultrasound in order to provide enough energy to
dissociate these intramolecular H-bonds. Several examples of the
Scheme 1 Classic Ugi four-component reaction.
Although the Ugi 4CR has been one of the most used
MCRs during the last decades, to the best of our knowl-
edge this is the first report in which the Ugi 4CR is ap-
plied to poly(aminocarboxylate) ligands for the preparation of
multimeric systems. Recently, S. Faulkner and coworkers re-
ported the use of the Ugi reaction for preparing chromophore-
appended DOTA-monoamide ligands.5 We exploited the Ugi
Scheme 2 General synthesis of ditopic bifunctional chelating agents through Ugi 4CR (see Table 1 for different R1 and R2 groups employed).
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Table 1 Compounds obtained varying the aldehyde and/or isocyanide components
Amine
Acid
Aldehyde R1–CHO
(CH2O)n
Isocyanide R2–NC
Product
Yield (%)
48
1
3
4
5
6
1
1
3
3
CH3(CH2)8CHO
45
42
Cbz-NH(CH2)5CHO
1
1
3
3
(CH2O)n
(CH2O)n
C18H37NC
7
8
52
38
use of MW in MCR have been reported to improve reaction rates
and yields.17 Due to the marked polarity of the functionalized
polyaminocarboxylic substrates, we envisaged that the use of
MW as source of power would be useful to the progress of the
Ugi 4CR unfolding of the pendant arms. The reactions were
carried out in a CEM Discover microwave oven using 10 mL
Pyrex reaction vessels sealed with pressure caps. Thus, 1 was
reacted with equimolar amounts of paraformaldehyde, 3, and
cyclohexylisocyanide for 1 h at 100 W and 2 bar in methanol.
With conventional heating, the product was collected only in
low yield (24%) after evaporation of the solvent and purification
by column chromatography. No significant improvement in the
yield was observed using decanal instead of paraformaldehyde.
However, using MW, we observed the formation of a large amount
of by-products, whereas the starting materials were still present in
the reaction mixture when the reaction was carried out under
conventional heating. This behavior may be ascribed to a stronger
activation of reagents and intermediates by MW, leading to a
reactivity enhancement and a resulting selectivity loss. Isocyanides
and some of the reaction intermediates are indeed known to
undergo side-reactions such as oligo- and polymerization.1 Finally,
we exploited ultrasound,18 a tunable high-energy power source
which might break up the intramolecular interactions and help
the formation of the desired Ugi 4CR product. The reaction
components (1, paraformaldehyde, 3, and cyclohexylisocyanide)
dissolved in methanol were therefore mixed in equimolar amounts
in a cup horn and sonicated at a power of 60 W (20 kHz)
for 3 h. Under these conditions, the product was obtained in
moderately good yields (48%) after column chromatography
and was characterized by ESI-MS, and 1H and 13C NMR
spectroscopy.
Synthesis of ditopic BFCAs
Once we had found the best conditions for the Ugi 4CR, we
checked the generality of this reaction for DOTA monoamide
amino (1) and acid (3) derivatives, by varying the aldehyde and
isocyanide components. Different components were studied in
order to demonstrate the possibility of inserting a functional group
into the a-acylaminoamide skeleton for further conjugation to
biomolecules. In addition to paraformaldehyde (Table 1, line 1),
alkanals (Table 1, line 2) and aminoaldehydes (Table 1, line 3) were
successfully employed as the carbonyl compound.
Modification of the isocyanide was similarly performed by using
octadecylisocyanide (Table 1, line 4) or methyl isocyanoacetate
(Table 1, line 5) instead of cyclohexylisocyanide (Table 1, lines
1–3). The presence of protected amino (6) or carboxylate (8)
groups in the dimeric system makes these compounds the first
examples of dimeric BFCA. In fact, orthogonal removal of the
Cbz protecting group from 6 by catalytic hydrogenation or alkaline
hydrolysis of the methyl ester in 8 would yield products able to
react respectively with carboxylic acid or amino functionalities
of biomolecules. These systems, containing two chelates for the
coordination of metal ions of medical interest and a suitable
biological carrier, are of great interest for possible use in molecular
imaging and therapy applications.
Moreover, with the idea to build a tetrameric system with four
DOTA monoamide units in a one-pot reaction, we performed the
Ugi 4CR with a diisocyanide and two molar equivalents of 1,
formaldehyde and 3. Unfortunately, this attempt, which was per-
formed using two different diisocyanides (1,4-diisocyanobenzene
and 1,4-diisocyanohexane), did not succeed, since the reaction
stopped at the dimeric systems, possibly due to steric hindrance of
the DOTA units.
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Finally, two of the synthesized compounds (4 and 7) were
successfully deprotected by hydrolysis of the t-butyl esters with
TFA, and the dimeric ligands L1 and L2 were obtained as
analytically pure white solids by precipitation with diethyl ether
(Scheme 3).
intramolecular interactions cannot occur when all the donor
atoms ofthe DOTA–monoamide cage are involvedincoordination
of the metal ion.
Relaxometric characterization of the Gd(III) complexes
NMR relaxometry is a powerful tool for obtaining detailed struc-
tural and dynamic information on a paramagnetic system through
the measurement and analysis of the nuclear magnetic longitudinal
and transverse relaxation rates (R1 and R2) of the solvent nuclei
as a function of the applied field strength, temperature and pH.19
The water proton relaxation enhancement ability of a Gd(III)
complex is expressed by the parameter relaxivity, rip (i = 1,2),
which represents the increase of the water proton relaxation rates
observed in a 1 mM solution of the paramagnetic system at a given
temperature and magnetic field strength (typically 298/310 K and
20 MHz).
As representative examples, we have considered the Gd(III)
complexes of two ditopic chelators, L1 and L2, differing in the
chemical nature and size of the central moiety. Whereas for the
ligand L1 the central pendant moiety is a cyclohexyl group, for L2
it is a long C18 alkyl chain that provides the corresponding complex
with the possibility to form aggregates (micelles) and to interact
with the hydrophobic pockets of HSA.12 All the ligand subunits
of these systems are octadentate, and thus the corresponding
Gd(III) complexes are expected to be coordinated by a single water
molecule (q = 1). The longitudinal relaxivity values (20 MHz,
310 K and pH = 7.2; per Gd) of the two ditopic complexes are 5.6
(Gd2L1) and 20.0 mM-1 s-1 (Gd2L2).
Scheme 3 Synthesis of ditopic ligands L1 and L2.
Heteroditopic Y–Gd complex.
An additional advantage of this synthetic approach is the easy
access to heteroditopic complexes. In fact, carrying out the Ugi
4CR directly on metal–DOTAMA complexes we were able to
obtain heteroditopic complexes (Scheme 4). Thus, we synthesized
the Y(III) complex of DOTAMAEn (9) and the Gd(III) complex
of DOTAMAC6OH (10),16 which were then reacted together with
paraformaldehyde and cyclohexylisocyanide.
The NMRD (Nuclear Magnetic Relaxation Dispersion) profile
of Gd2L1, measured at 298 K and neutral pH and reported in
Fig. 1a, is characterized by the typical shape and amplitude of
mono-aqua, low molecular weight complexes, featuring a single
dispersion around 4–6 MHz. The relaxivity of Gd2L1 (per Gd) is
higher than that of a typical monomeric GdDOTAMA complex
(i.e. 3.9 mM-1 s-1 at 310 K)20 to indicate that the difference is due
to the different rotational dynamics of the two complexes. The
best fit parameters, listed in Table 2, have been obtained using the
standard equations for the inner sphere (IS) and outer sphere (OS)
relaxation contributions and by fixing the values of the following
parameters: the hydration number (q = 1), the Gd–Hw distance
˚
(r = 3.0 A), the distance of closest approach of the bulk water
Table 2 Selected relaxation parameters for Gd2L1 and Gd2L2 derived
from the analysis of 1H relaxivity as a function of temperature (20 MHz)
and magnetic field strength (NMRD)a
Scheme 4 Synthesis of a heteroditopic Y–Gd complex by Ugi four-
Parameter
Gd2L1
Gd2L2
component reaction.
310
r
(mM-1 s-1)
5.6
20.0
1p
298
k
ex
(106 s-1)
1.1 0.2
39.3 1.8
2.2 0.1
22
156
19
1.4 0.2
38.2 2.1
0.60 0.03
This reaction was performed both with ultrasound and with
conventional heating but with different outcomes in respect of
what was observed previously on the protected 1 and 3 derivatives.
Thus, while using ultrasound, the Ugi 4CR product 11 was
obtained only in low amounts due to the known problem of metal–
ligand bond cleavage caused by ultrasound,18 whereas by refluxing
in methanol we observed the formation of 11 in good yields
(47%). This is good evidence of our hypothesis of intramolecular
interactions as the origin of the low yields in the conventional
heating Ugi 4CR protocol using 1 and 3 derivatives. In fact,
DHM (kJ mol-1)
D2 (1019 s-2)
298
tV (ps)
tRg (ns)
1
4
1
19
2
298
3710 203
21.2 1.8
400 28
DHRg (kJ mol-1)
298
tRl (ns)
—
—
—
DHRl (kJ mol-1)
17
1
S2
0.36 0.02
a For the parameters q, r, a, DHV and D, values of 1, 3.0 A, 4.0 A,
298
˚
˚
2.0 kJ mol-1 and 2.24 ¥ 10-5 cm2 s-1 were used, respectively.
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to its aggregated form. The NMRD profiles (298 and 310 K)
were recorded in the frequency range 1–70 MHz and are shown
in Fig. 2. The profiles are characterized by different amplitudes
but present a common general shape with a broad hump centered
at ~25–30 MHz, typical of slowly tumbling systems. The analysis
of the NMRD profile takes into account the relatively fast local
rotation of the complex about the main axis of the aliphatic chain
superimposed on the global motion of the micellar aggregate
(Lipari–Szabo approach).23 This model allows one to separate the
contribution of the overall global rotation of the paramagnetic
micelle (tRG = 3710 ps) from the contribution of a faster local
motion (tRL = 400 ps) due to the rotation of the coordination
polyhedron about the pendant arm. The large difference between
the two correlation times indicates that the system is highly flexible
and its relaxivity strongly limited by the relatively short value
of tRL. The correlation of the two types of motions is described
by the parameter S2, whose value is between zero (completely
independent motions) and one (totally correlated motions). Data
relative to micellar assemblies formed by ditopic complexes are
reported here for the first time and can only be compared to those
for corresponding aggregated monomeric complexes.22 Larger
values for the parameters tRG and tRL are calculated for Gd2L2
to indicate both the presence of larger aggregates and a more
restricted local rotational motion. This is supported by the larger
value of S2 and can be interpreted as the consequence of larger
steric interactions involving the bulkier dimeric units. It is worth
noting that the relaxivity values at 310 K are higher than those
at 298 K over the entire range of frequencies. This indicates some
limiting effects arising from the non-optimal value of the rate of
water exchange.
Fig. 1 a) 1/T1 1H NMRD profile for Gd2L1 at 298 K and pH = 7.2. The
solid line represents the best-fit to the experimental points (see Table 2).
b) Proton relaxivity as a function of temperature for Gd2L1 at 20 MHz.
The solid line represents the best-fit to the experimental data (Table 2),
whereas the dotted and dashed lines represent the calculated outer- and
inner-sphere contributions to the relaxivity.
˚
molecules (a = 4.0 A) to the metal ion, and the relative diffusion
coefficient (D = 2.4 ¥ 10-5 cm2 s-1).6 For Gd2L1, due to its rather low
solubility, the information on the water exchange rate (kex = 1/tM)
was extracted from the analysis of the temperature dependence
of the 1H relaxivity at 20 MHz (as detailed in ref. 20). As shown
in Fig. 1b, the r1p increases by lowering the temperature from
335 to ca. 290 K, then it flattens and begins to decrease below
~280 K. This behavior is consistent with an intermediate rate of
exchange of the bound water molecule. The overall behavior is the
results of the different temperature-dependence of the IS and OS
contributions to r1p: the OS relaxivity decreases with increasing
temperature, whereas the behavior of the IS term depends on the
relative values of the longitudinal relaxation time of the bound
water proton, T1M, and its mean residence lifetime, tM.21 When
tM < T1M, r1p increases with decreasing temperature (>300 K in
Fig. 3), whereas when tM > T1M (<285 K in Fig. 3) the relaxivity
has the opposite behavior, and the contribution of the IS term
diminishes with decreasing temperature.
The relaxivity of Gd2L2 is significantly higher than that of
Gd2L1. This is the result of the presence of an octadecyl aliphatic
chain that has the tendency to aggregate and form micellar systems
of large dimension and thus slow tumbling rate (large tR value).
The observed proton relaxation rate of Gd2L2 (20 MHz; 298 K)
shows a linear dependence from the concentration in the range
0.10–1.0 mM, and so an accurate determination of the critical
micellar concentration (cmc) was not possible. However, cmc
values lower than 0.10 mM are in agreement with typical values
of previously investigated systems conjugated to long lipophilic
chains.12,22 Then, all the experimental data relative to Gd2L2 refer
Fig. 2 1/T1 1H NMRD profiles of Gd2L2 at 298 (᭹) and 310 K (᭺). The
solid lines are calculated with the parameters of Table 2.
The temperature dependence of the proton relaxivity was also
measured in order to obtain an independent evaluation of the
residence lifetime of the coordinated water molecule, as the value
of this parameter is generally not determined with high accuracy
from the NMRD profiles. The profile is characterized by a broad
peak centered at about 310–315 K (Fig. 3): this implies that around
physiological temperatures the relaxivity reaches the maximum
value and it is not limited by the relatively slow exchange of the
bound water molecule. A good fit of the data was obtained with
a 298tM value of 700 ns (Table 2), to confirm that this parameter is
not significantly affected by the aggregation process. Finally, it is
worth noting that the satisfactory fit of the calculated curve to the
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Conclusions
The Ugi four-component reaction has been exploited to obtain,
in a single pot, bifunctional ditopic chelators by using DOTA
monoamide derivatives as amino and acid components. This
synthetic approach offers several advantages: i) quick reac-
tion times with reasonable yields; ii) the possibility of modulating
the chemical properties of the chelator (ligating groups, charge,
denticity) and the pendant central functional moiety; and iii)
the improved possibility of rapidly generating a large number
of derivatives to meet different chemical needs. In addition, this
synthetic approach may involve the use of Ln(III) complexes
as reactants and this allows the easy preparation of a variety
of heterodimetallic derivatives. The wide variety of magnetic,
optical and nuclear properties of the lanthanide cations could then
be exploited to prepare homo- and heterodimetallic systems for
both diagnostic and therapeutic applications. Two representative
Gd(III) complexes, Gd2L1 and Gd2L2, were investigated in detail
by relaxometric techniques in aqueous solution. Gd2L1 shows the
typical properties of ditopic complexes: a higher relaxivity (per
Gd) with regard to the monomer as a consequence of the slower
rotation. Gd2L2 forms micelles at concentrations <0.1 mM, whose
relaxivity is ca. 25% higher (per Gd) than that of corresponding
monomeric complexes in the aggregated form.12,21 This enhanced
relaxivity appears to be the consequence of the combined effect
of i) a larger molecular dimensions of the micelles and ii) more
hindered local rotation of the dimer about the alkyl chain. Then,
micellar aggregates formed by multimeric complexes have the
favorable property of being characterized by a larger number
of paramagnetic units (higher molecular relaxivity) and by a
lower anisotropic rotational dynamics (higher relaxivity per Gd).
Finally, it is worth noting that this approach has general validity,
and it could be easily applied to the preparation of a large variety
of bifunctional ditopic metal chelates characterized by different
size, charge, coordination polyhedra and hydration number.
Fig. 3 Variable-temperature 1H NMR relaxation data for Gd2L2 at
20 MHz normalized to 1 mM concentration of gadolinium. The solid
line represents the best results of the fitting to the experimental data (see
Table 2), whereas the dotted and dashed lines represent the calculated
outer- and inner-sphere contributions to the overall relaxivity.
experimental data provides further support to the results of the
NMRD study.
Binding to HSA. The presence of a long-chain alkyl group
offers to Gd2L2 the possibility to form supramolecular adducts
with HSA characterized by enhanced relaxivity.12 The non-
covalent hydrophobic interaction was investigated by titrating
a dilute solution of the complex (0.07 mM) with the protein
up to a 1.1 mM concentration, and by following the resulting
increase of the observed longitudinal proton relaxation rate, R1,
at 20 MHz and 298 K (Fig. S1†). A pronounced enhancement of
R1 is observed at low concentration of the protein, indicating a
rather strong affinity of the complex for the hydrophobic sites of
HSA. The binding isotherm was analyzed in terms of the PRE
equations to yield a value of 2.6 ¥ 105 M-1 for the association
constant, KA, and a value of 34.1 mM-1 s-1 for the relaxivity of
the protein-conjugate, r1bp.12 The KA value provides just a simple
indication of the binding strength since different binding sites
of different affinity are likely to be present on the protein.24
However, the value is quite similar to that found for the interaction
Experimental
Materials and instrumentation
All chemicals were purchased from Sigma-Aldrich Co. and were
used without purification unless otherwise stated. NMR spectra
were recorded on a on a JEOL Eclipse Plus 400 (operating
at 9.4 Tesla). ESI mass spectra were recorded on a Waters
Micromass ZQ. Infrared (IR) spectra were recorded in the range
4000–400 cm-1 at 4 cm-1 resolution using a Bruker Equinox 55
spectrometer. MW-promoted reactions were carried out in a CEM
Discover oven. Sonochemical apparatus used in the present work
was developed by Prof. G. Cravotto’s group (Department of Drug
Science and Technology, University of Torino) in collaboration
with Danacamerini s.a.s. (Torino).25 DOTAMA(t-BuO)3C6OH,
DOTAMA(t-BuO)3En and DOTAMA(OtBu)3C6NH2 were
prepared following a reported procedure.11
24
with high-affinity fatty acid sites on HSA of GdDOTA-C11 and
GdAAZTA-C17 derivatives.12
The value of rb1p is in the upper range of values of the HSA-
conjugates of complexes with q = 1, where the relaxivity is limited
by both the presence of a fast internal motion and a relatively
long residence lifetime of the bound water molecule. In agreement
with the results obtained on the micellar system, the rb1p of the
HSA-conjugated complex appears to be limited mostly by the
high flexibility of the alkyl chain. In fact, the rb1p increases with
temperature up to ca. 310 K and then it decreases (Fig. S2†) again.
The maximum of relaxivity is then at physiological temperatures,
where it has a value of 38.1 mM-1 s-1. The NMRD profiles do
not add further information. The relaxivity presents a broad peak
centered around 25 MHz, typical of this type of slowly tumbling
macromolecular systems, whose value is higher than those found
for several other Gd(III) chelates conjugated to HSA, suggesting
a more restricted internal rotation of the dimeric unit about the
alkyl chain.
General Ugi 4CR – Conventional heating protocol
A solution of DOTAMA(OtBu)3En (61.4 mg; 0.1 mmol), aldehyde
(0.1 mmol), DOTAMA(OtBu)3C6OH (68.6 mg; 0.1 mmol) and
isocyanide (0.1 mmol) in MeOH (3 mL) was refluxed for 6 h. Then,
the solvent was evaporated in vacuo and the residue was purified
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by column chromatography (silica gel, eluent: CH2Cl2–MeOH,
95:5, v/v) to give a light yellow oil.
1,15-Bis[4,7,10-tris(t-butoxycarbonylmethyl)-1,4,7,10-
tetraazacyclododec-1-yl]-6-{[N-cyclohexyl-1-(N-Cbz-5-
aminopentyl)]carboxamidomethyl}-2,7,14-trioxo-3,6,13-
triazapentadecane (6)
General Ugi 4CR – Microwave protocol
1H NMR (CDCl3, 400 MHz): d = 8.56 (d, 1H, J = 7.3 Hz),
8.05 (t, 2H, J = 5.8 Hz), 7.30 (m, 5H), 5.03 (s, 2H), 3.67–1.72
(m, 58H), 1.68 (m, 2H), 1.66–0.74 (m, 22H), 1.41 (s, 36H), 1.40
(s, 18H). 13C NMR (CDCl3, 100 MHz): d = 175.1, 172.6, 172.5,
172.1, 171.5, 156.6, 156.4, 128.6, 128.5, 128.1, 81.9, 81.8, 66.5, 66.4,
56.3, 55.8, 55.8, 53.5, 52.7-48.3 (bs), 40.9, 38.8, 38.6, 38.4, 37.6,
32.8, 32.6, 32.5, 29.7, 29.6, 29.4, 28.1, 28.0, 26.4, 26.0, 25.4, 25.2,
24.7, 22.7, 14.2. ESI-MS (m/z): found 1642.45 (M + H+, 8%),
(calc. 1641.19), found 1663.37 (M + Na+, 3%), (calc. 1663.10),
found 821.79 [(M + 2H+)/2, 100%], (calc. 821.06). IR spectrum
(KBr disk) 3240, 3062, 2973, 2925, 2861, 1731, 1674, 1544, 1455,
1367, 1230, 1165, 1108 cm-1. Elemental analysis calcd (%) for
C85H149N12O16: C, 62.21; H, 9.15; N, 11.09; found: C, 62.16; H,
9.21; N, 10.97.
A solution of DOTAMA(OtBu)3En (61.4 mg; 0.1 mmol), aldehyde
(0.1 mmol), DOTAMA(OtBu)3C6OH (68.6 mg; 0.1 mmol) and
isocyanide (0.1 mmol) in MeOH (4 mL) was irradiated with
microwaves at 100 W (Pmax = 2 bar) for 1 h. The solvent was
then evaporated in vacuo and the residue was purified by column
chromatography (silica gel, eluent: CH2Cl2–MeOH, 95:5, v/v) to
give a light yellow oil.
General Ugi 4CR – Power ultrasound protocol
A solution of DOTAMA(OtBu)3En (61.4 mg; 0.1 mmol), aldehyde
(0.1 mmol), DOTAMA(OtBu)3C6OH (68.6 mg; 0.1 mmol) and
isocyanide (0.1 mmol) in MeOH (4 mL) was sonicated in an
ultrasound reactor at 60 W (20.3 kHz) for 3 h. Then, the solvent
was evaporated in vacuo and the residue was purified by column
chromatography (silica gel, eluent: CH2Cl2–MeOH, 95:5, v/v) to
give a light yellow oil.
1,15-Bis[4,7,10-tris(t-butoxycarbonylmethyl)-1,4,7,10-
tetraazacyclododec-1-yl]-6-[(N-octadecyl)carboxamidomethyl]-
2,7,14-trioxo-3,6,13-triazapentadecane (7)
1H NMR (CDCl3, 400 MHz): d = 8.68 (t, 1H, J = 5.5 Hz), 8.06
(t, 1H, J = 5.8 Hz), 7.99 (t, 1H, J = 5.5 Hz), 3.67–2.00 (m, 60H),
1.80–1.45 (m, 8H) 1.41 (s, 36H), 1.39 (s, 18H), 1.19 (m, 30H),
0.82 (t, 3H, J = 7.0 Hz). 13C NMR (CDCl3, 100 MHz): d =
175.1, 172.6, 172.5, 171.4, 171.1, 170.4, 169.3, 82.0, 81.9, 81.8,
64.3, 56.3, 55.8, 55.7, 53.1-47.6 (bs), 39.8, 39.4, 38.7, 33.1, 33.0,
31.9, 29.8, 29.4, 28.7, 28.2, 28.1, 28.0, 27.2, 26.4, 25.4, 24.6, 22.7,
14.2. ESI-MS (m/z): found 1592.50 (M + H+, 5%), (calc. 1592.20),
found 1614.52 (M + Na+, 2%), (calc. 1614.18), found 797.30 [(M +
2H+)/2, 25%], (calc. 796.60), found 531.66 [(M + 3H+)/3, 100%],
(calc. 531.40). IR spectrum (KBr disk) 3444, 3271, 2970, 2931,
2848, 1725, 1660, 1367, 1231, 1163, 1110 cm-1. Elemental analysis
calcd (%) for C84H158N13O17: C, 63.36; H, 10.00; N, 10.56; found:
C, 63.56; H, 10.11; N, 10.64.
1,15-Bis[4,7,10-tris(t-butoxycarbonylmethyl)-1,4,7,10-
tetraazacyclododec-1-yl]-6-[(N-cyclohexyl)carboxamidomethyl]-
2,7,14-trioxo-3,6,13-triazapentadecane (4)
1H NMR (CDCl3, 400 MHz): d = 8.58 (d, 1H, J = 8.0 Hz), 8.09 (t,
2H, J = 5.5 Hz), 3.76–1.91 (m, 57H), 1.86 (m, 2H), 1.72 (m, 2H),
1.63–1.08 (m, 14H), 1.42 (s, 36H), 1.41 (s, 18H). 13C NMR (CDCl3,
100 MHz): d = 175.1, 172.6, 172.5, 171.4, 169.5, 82.0, 81.9, 56.8,
56.3, 55.8, 55.7, 53.5–48.0 (bs), 38.7, 37.6, 33.3, 32.9, 32.6, 29.7,
28.5, 28.1, 28.0, 26.3, 25.5, 25.4, 25.2, 24.8, 24.5, 22.7, 14.2. ESI-
MS (m/z): found 1422.56 (M + H+, 10%), (calc. 1422.00), found
1444.53 (M + Na+, 5%), (calc. 1443.98), found 711.85 [(M +
2H+)/2, 100%], (calc. 711.50). IR spectrum (KBr disk): 3245, 2978,
2930, 2880, 1748, 1668, 1367, 1231, 1165, 1108 cm-1. Elemental
analysis calcd (%) for C72H132N12O16: C, 60.82; H, 9.36; N, 11.82;
found: C, 60.76; H, 9.21; N, 11.65.
1,15-Bis[4,7,10-tris(t-butoxycarbonylmethyl)-1,4,7,10-tetraaza-
cyclododec-1-yl]-6-[(N-methoxycarbonylmethyl)carboxamido-
methyl]-2,7,14-trioxo-3,6,13-triazapentadecane (8)
1,15-Bis[4,7,10-tris(t-butoxycarbonylmethyl)-1,4,7,10-
tetraazacyclododec-1-yl]-6-[(N-cyclohexyl)(1-nonyl)carbox-
amidomethyl]-2,7,14-trioxo-3,6,13-triazapentadecane (5)
1H NMR (CDCl3, 400 MHz): d = 8.75 (d, 1H, J = 7.4 Hz), 8.06 (t,
2H, J = 5.5 Hz), 3.72 (s, 3H), 3.68–2.10 (m, 60H), 1.95–1.55 (m,
6H), 1.42 (s, 36H), 1.41 (s, 18H). 13C NMR (CDCl3, 100 MHz):
d = 175.1, 172.6, 172.5, 171.4, 170.2, 169.5, 81.9, 81.9, 56.8, 56.3,
55.8, 55.7, 53.5–48.0 (bs), 41.8, 38.7, 37.6, 33.3, 32.9, 29.7, 28.5,
28.1, 28.0, 26.3, 25.5, 25.4, 25.2, 24.5, 14.2. ESI-MS (m/z): found
1412.55 (M + H+, 8%), (calc. 1411.94), found 1434.52 (M + Na+,
10%), (calc. 1433.92), found 706.81 [(M + 2H+)/2, 100%], (calc.
706.47). IR spectrum (KBr disk) 3252, 2975, 2935, 2862, 1728,
1666, 1365, 1229, 1164, 1107 cm-1. Elemental analysis calcd (%)
for C69H126N12O18: C, 58.70; H, 9.00; N, 11.91; found: C, 58.86; H,
9.07; N, 12.04.
1H NMR (CDCl3, 400 MHz): d = 8.65 (d, 1H, J = 5.5 Hz),
8.16 (t, 1H, J = 5.5 Hz), 8.06 (t, 1H, J = 5.1 Hz), 3.86–1.89 (bs,
56H), 1.83 (m, 2H), 1.74–1.34 (m, 20H), 1.44 (s, 36H), 1.43 (s,
18H), 1.32–1.08 (m, 12H), 0.85 (t, 3H, J = 7.0 Hz). 13C NMR
(CDCl3, 100 MHz): d = 172.9, 172.7, 172.5, 172.2, 171.6, 81.9,
81.8, 56.4, 55.9, 55.8, 55.7, 53.5, 58.9-47.5 (bs), 34.0, 32.9, 32.6,
31.9, 29.8, 29.6, 29.4, 28.2, 28.1, 28.0, 26.6, 26.5, 26.3, 26.2, 25.6,
25.4, 25.3, 25.2, 25.1, 24.9, 24.7, 24.6, 22.7, 14.2. ESI-MS (m/z):
found 1548.46 (M + H+, 6%), (calc. 1548.14), found 1570.49 (M
+ Na+, 3%), (calc. 1570.12), found 775.27 [(M + 2H+)/2, 100%],
(calc. 774.57). IR spectrum (KBr disk) 3439, 3258, 2974, 2925,
2852, 1723, 1665, 1367, 1230, 1165, 1109 cm-1. Elemental analysis
calcd (%) for C81H150N12O16: C, 62.84; H, 9.77; N, 10.86; found: C,
62.91; H, 9.88; N, 10.94.
Synthesis of ligands L1 and L2
The compound obtained by the Ugi four-component reaction was
dissolved in a mixture of CH2Cl2 and trifluoroacetic acid (1:1 v:v,
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2 mL) and stirred at room temperature overnight. The solution was
then evaporated in vacuo and the product was precipitated with
excess diethyl ether, isolated by centrifugation, washed thoroughly
with diethyl ether and dried in vacuo, to give the pure desired
product as amorphous white solid.
concentrations of gadolinium were determined by measurement of
bulk magnetic susceptibility shifts of a tBuOH signal and/or ICP-
1
MS. The H T1 relaxation times were acquired by the standard
inversion recovery method with typical 90◦ pulse width of 3.5 ms,
16 experiments of 4 scans. The reproducibility of the T1 data
was 5%. The temperature was controlled with a Stelar VTC-
91 airflow heater equipped with a calibrated copper–constantan
thermocouple (uncertainty of 0.1 ◦C). The data were measured in
the range of magnetic fields from 0.00024 to 1.6 T (corresponding
to 0.01–70 MHz proton Larmor frequencies).
1,15-Bis[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
1-yl]-6-[(N-cyclohexyl) carboxamidomethyl]-2,7,14-
trioxo-3,6,13-triazapentadecane (L1)
1H NMR (CD3OD, 400 MHz): d = 4.11–3.20 (m, 54H), 2,48 (m,
1H), 2.28 (m, 2H), 1.84 (m, 2H), 1.75 (m, 2H), 1.68–1.17 (m,
14H). 13C NMR (CD3OD, 100 MHz): d = 175.7, 175.2, 169.9,
169.0, 65.6, 54.8–52.6 (bs), 51.1–49.0 (bs), 48.3, 48.1, 39.0, 37.5,
37.1, 32.6, 33.3, 28.7, 27.2, 26.6, 26.1, 25.2, 24.8, 24.6, 24.4, 14.1.
ESI-MS (m/z): found 1086.01 (M + H+, 8%), (calc. 1085.62),
found 1108,05 (M + Na+, 10%), (calc. 1107.60), found 543.49 [(M
+ 2H+)/2, 100%], (calc. 543.31). IR spectrum (KBr disk) 3361,
3199, 2917, 2853, 1649, 1463, 1383, 1092 cm-1. Elemental analysis
calcd (%) for C48H84N12O16·4CF3COOH: C, 43.64; H, 5.75; N,
10.90; found: C, 43.35; H, 5.51; N, 10.75.
Acknowledgements
We thank the support of the Regione Piemonte (Ricerca Sanitaria
Finalizzata 2007 and Converging Technologies 2007-NanoIGT
project). This work was carried out under the auspices of CIR-
CMSB and COST Action D38. We also would like to thank Prof.
G. Cravotto (University of Torino) for the use of his sonochemical
apparatus.
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J = 7.0 Hz), 2.30 (m, 1H, J = 7.0 Hz), 1.62–1.39 (m, 8H), 1.28
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Synthesis of the heteroditopic Y–Gd complex
A
solution of Y–DOTAMAEn (10 mg; 0.017 mmol),
paraformaldehyde (0.5 mg, 0,017 mmol), Gd-DOTAMAC6OH
(11.5 mg; 0,017 mmol) and cyclohexylisocyanide (2.1 mg,
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©