Design of a New Multifunctionalized PAMAM Dendrimer with Hydrazide-Terminated Spacer Arm Suitable for Metal-Carbonyl Multilabeling of Aldehyde-Containing Molecules Nathalie Fischer-Durand, ² Miche ` le Salmain, ² Bogna Rudolf, ‡ Lauriane Juge ´ , § Vincent Gue ´ rineau, | Olivier Lapre ´ vote, | Anne Vessie ` res,* ,² and Ge ´ rard Jaouen ² Ecole Nationale Supe ´ rieure de Chimie de Paris (ENSCP), Laboratoire de Chimie et Biochimie des Complexes Mole ´ culaires CNRS UMR 7576, 11 rue P. et M. Curie, 75231 Paris cedex 05, France, UniVersity of Lodz, Department of Organic Chemistry, 90-136 Lodz, Narutowicza 68, Poland, ENSCP, SerVice de RMN, 11 rue P. et M. Curie, 75231 Paris cedex 05, France, and Institut de Chimie des Substances Naturelles (ICSN), UPR-2301 CNRS, Laboratoire de Spectrome ´ trie de Masse, AV. de la Terrasse, 91198 Gif sur YVette Cedex, France ReceiVed July 20, 2007; ReVised Manuscript ReceiVed September 17, 2007 ABSTRACT: We describe herein the synthesis of generation 4 (G4) poly(amidoamine) (PAMAM) dendrimers bearing (1) a nitroaniline derivative as an NMR probe, (2) (η 5 -cyclopentadienyl)iron dicarbonyl succinimidato complexes as infrared probes for carbonyl metallo immunoassay, (3) acetamide groups for increased solubility, and (4) hydrazide functions at the extremity of five-carbon atom spacer arms for the labeling of aldehyde-containing molecules. The step-by-step building of these multifunctionalized dendrimers was monitored by 1 H NMR spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, and by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Substitution of terminal amine groups of PAMAM dendrimers with appropriate functional groups provides complex dendrimeric structures with a wide range of applications. The final goal of this study is the development of new reagents suitable for the metal-carbonyl multilabeling of immunological reagents such as antibodies. Introduction Poly(amidoamine) (PAMAM) dendrimers are new synthetic polymers with well-defined molecular structures and polydis- persities close to unity. 1-3 They are constructed from an amine core (ammonia, ethylenediamine, cystamine) through alternate reactions of methyl acrylate and ethylenediamine to produce generation 0 to generation 10 (G0-G10) PAMAM dendrimers, corresponding to each two-step reaction sequence, with amine surface functionalities that double at each generation. Their water solubility, combined with their lack of immunogenicity, has led to a wide range of biomedical applications by func- tionalization of their peripheral groups with various compounds, such as targeting, therapeutic, and diagnostic agents, alone or in combination. 4-11 Their use in the field of biosensors has also been investigated, for instance, as building blocks for the design of biosensing interfaces that favor ligand functionalization and biospecific interaction. 12-17 Dendrimers functionalized with fluorescent probes have also found application in the determi- nation of environmental contaminants. 18-20 In our laboratory, we have been studying for some time the potential use of dendrimers as carriers of transition-metal- carbonyl complexes, with the objective of developing new generations of universal immunodetection reagents. This re- search is aimed at the amplification, by multiple labeling, of the infrared detection signal in the carbonyl metallo immunoas- say (CMIA) we have developed since the early 1990s. 21-24 For this purpose, we have recently described the preparation of a bifunctional G4-PAMAM dendrimer bearing 45 (η 5 -cyclo- pentadienyl)iron dicarbonyl succinimidato (Fp) units and four biotin entities, 25 and the synthesis of G3- and G4-PAMAM dendrimers labeled with (η 5 -cyclopentadienyl)rhenium tri- carbonyl [CpRe(CO) 3 ] units with or without attachment of polyethylene glycol (PEG) moieties of various chain lengths. 26 We have also reported the conjugation of G4-PAMAM bearing 10-25 Fp units to the glycosylated part of secondary antibodies. 27 In this latter report, we showed that direct conjugation of an amine-terminated dendrimer to periodate- oxidized immunoglobulin G (IgG) through reductive amination afforded immunoconjugates with a metal-carbonyl dendrimer to IgG ratio that was related to the steric hindrance around the remaining amino groups on the surface of the dendrimer. For instance, with a dendrimer carrying 10 Fp complexes, we were able to conjugate 1.4 dendrimers per IgG, whereas the use of a dendrimer carrying 25 Fp complexes led to the attachment of only 0.5 dendrimer per IgG. These results prompted us to engineer a more advanced generation of metallocarbonyl PAMAM dendrimers bearing a spacer arm so as to move the dendrimer away from the IgG, thus decreasing steric hindrance between the two macromolecules. The same anchoring site for the antibody molecule will be used via oxidation of the glycosylated moieties. Here we describe the design, synthesis, and characterization of a hydrazide-based trifunctional G4-PAMAM dendrimer bearing Fp complexes (Scheme 1) intended to be used for the labeling of aldehyde-containing molecules, such as oxidized antibodies. Characterization of the dendrimer after the successive functionalization steps was achieved by combining several spectroscopic techniques, including 1 H NMR, UV-vis, FT-IR, and MALDI-TOF mass spectrometry. * Corresponding author. E-mail: a-vessieres@enscp.fr. ² Laboratoire de Chimie et Biochimie des Complexes Mole ´culaires, ENSCP. ‡ University of Lodz. § Service de RMN, ENSCP. | ICSN. 8568 Macromolecules 2007, 40, 8568-8575 10.1021/ma071621g CCC: $37.00 © 2007 American Chemical Society Published on Web 11/01/2007