PAMAM Dendrimers Conjugated with an Uncharged Gadolinium(III) Chelate with a Fast Water Exchange: The Inﬂuence of Chelate Charge on Rotational Dynamics Miloslav Pola´sˇek, † Petr Hermann,* ,† Joop A. Peters, ‡ Carlos F. G. C. Geraldes, § and Ivan Lukesˇ † Department of Inorganic Chemistry, Faculty of Science, Universita Karlova (Charles University), Hlavova 2030, 128 40 Prague 2, Czech Republic, Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands, and Department of Biochemistry, Faculty of Science and Technology, Center of Neurosciences and Cell Biology, University of Coimbra, 3001-401 Coimbra, Portugal. Received July 20, 2009; Revised Manuscript Received October 9, 2009 A bifunctional ligand, DO3A-py NO-C (DO3A-py NO-C ) 10-[(4-carboxy-1-oxidopyridin-2-yl)methyl]-1,4,7,10- tetraazacyclododecane-1,4,7-triacetic acid), was attached to different generations (G0, G1, G2, and G4) of ethylenediamine-cored PAMAM dendrimers (PAMAM ) polyamidoamine). The gadolinium(III) complex of this ligand possesses one molecule of water in its ﬁrst coordination sphere and has a unique combination of a short water residence lifetime (τ M ) 34 ns), a neutral overall charge, and a possibility for rigid attachment to molecules bearing primary amino groups. These favorable properties predestine the ligand for constructions of highly efﬁcient nanosized contrast agents for magnetic resonance imaging (MRI). The coupling reaction between the carboxylic group on the pyridine-N-oxide moiety of the protected ligand molecule and primary amines in the dendrimers was achieved by an active ester method under nonaqueous conditions using the coupling agent TBTU. This reaction afforded conjugates with high loadings (80-100% of the theoretically available primary amines) and of high purity. The gadolinium(III) complexes of the conjugates were studied by variable-temperature 17 O NMR and 1 H NMRD measurements. The water residence lifetime (τ M ) 55 ns) found in the largest conjugate G4- [Gd(H 2 O)(do3a-py NO-C )] 57 , though somewhat longer compared to the “monomeric” complex, is still short enough not to limit the relaxivity. Surprisingly, compared with analogous conjugates with negatively charged chelates, the prepared uncharged compounds displayed much faster global rotational correlation times (τ g ) and lower relaxivities. This phenomenon can be explained on the basis of Coulomb interactions. The motion of the charged chelates is restrained due to interactions with their counterions and the chelates themselves, while the uncharged chelates are not affected. Comparison of the PAMAM-based conjugates bearing uncharged and (1-)-charged chelates based on relaxometric data, 1 H DOSY spectra, and SAXS measurements reveals that τ g reﬂects the rotational motion of large segments (dendrons) of the conjugates rather than that of the whole macromolecule. INTRODUCTION Magnetic resonance imaging (MRI) is a powerful and rapidly developing diagnostic technique, which is indispensable for imaging of soft tissues. The basic MRI image reﬂects the concentration of 1 H nuclei in the 3D space; however, employing T 1 and T 2 relaxation times of protons helps to signiﬁcantly improve the quality of the images. Though the use of contrast agents (CAs) is not essential to this technique, it is advantageous to use paramagnetic CAs to accelerate the relaxation of the 1 H nuclei. Among the various available MRI contrast agents, the gadolinium(III) chelates with polydentate ligands, such as the macrocyclic DOTA and open-chain DTPA (Chart 1) and their various derivatives, are probably the most popular ones. These paramagnetic chelates act mainly as T 1 -contrast agents. Since the discovery of these ﬁrst-generation CAs, there has been a continuous effort to improve the properties of the chelates in order to obtain CAs with higher relaxivity (i.e., the efﬁciency expressed as relaxation rate enhancement per milimolar con- centration of metal ion) and higher selectivity (1-5). The latest development moves toward targeted agents and the ﬁeld of molecular imaging (6-10), which allows in vivo investigation of cellular molecular events involved in normal and pathological processes. However, the applicability of MRI contrast agents in molecular imaging encounters a tremendous obstacle in an unfavorable combination of low efﬁciency of the CAs and low concentration of the target receptors (6, 7). There are, in principle, two basic strategies to overcome this limitation. (i) A high payload of paramagnetic metal ions can be delivered at the target by conjugating a large number of CA molecules with a macromolecule or a particle that is linked to a targeting vector. (ii) The efﬁciency of the individual CA molecules can be enhanced by rational design of the Gd(III) chelates. Preferably, both approaches should be combined in order to attain the maximum relaxivity. Important physico- chemical parameters in this respect are the molecular tumbling (characterized by the rotational correlation time τ R ) and the mean residential time of the water molecule(s) in the coordination sphere of the Gd(III) ion (related to the water exchange by τ M ) 1/k ex ). Both parameters should be optimized in order to attain the high relaxivity. Too fast or two slow water exchange can both limit the relaxivity; therefore, the values of τ M should ideally be within the range 10-30 ns. The optimum of τ R somewhat varies with the magnetic ﬁeld and can be tuned by an attachment of the Gd(III) chelate to another molecule. * E-mail: petrh@natur.cuni.cz. Tel: (+420) 22195-1263. Fax: (+420) 22195-1253. † Universita Karlova. ‡ Delft University of Technology. § University of Coimbra. Bioconjugate Chem. 2009, 20, 2142–2153 2142 10.1021/bc900288q CCC: $40.75  2009 American Chemical Society Published on Web 11/02/2009