A “Click Chemistry” Approach to the Efficient Synthesis of Multiple Imaging Probes Derived from a Single Precursor Thomas L. Mindt,* ,†,§ Cristina Mu ¨ller, | Florian Stuker, ⊥ Jean-Fre ´de ´ric Salazar, ‡,§ Alexander Hohn, | Thomas Mueggler, ⊥ Markus Rudin, ⊥,# and Roger Schibli* ,§,| Department of Chemistry and Applied Biosciences, ETH Zurich, Center for Radiopharmaceutical Science ETH-PSI-USZ, Paul Scherrer Institute, Villigen, Institute for Biomedical Engineering and Institute of Pharmacology and Toxicology, University and ETH Zurich, Switzerland. Received June 24, 2009; Revised Manuscript Received September 3, 2009 Different imaging modalities can provide complementary information on biological processes at the cellular or molecular level in Vitro and in ViVo. However, specific molecular probes suitable for a comparison of different imaging modalities are often not readily accessible because their preparation is usually accomplished by individually developed and optimized syntheses. Herein, we present a general, modular synthetic approach that provides access to multiple probes derived from a single precursor by application of the same, efficient functionalization strategy, the Cu(I)-catalyzed cycloaddition of terminal alkynes and azides (click chemistry). To demonstrate the viability and efficiency of this approach, folic acid (FA) was selected as a targeting vector because the preparation of FA-based imaging probes used for SPECT, PET, MRI, and NIRF by reported synthetic strategies is usually difficult to achieve and often results in low overall yields. We prepared a versatile γ-azido-FA precursor as well as a set of alkyne functionalized probes and precursors including ligand systems suitable for the chelation of various (radio)metals, an NIR dye and 18 F- and 19 F-derivatives, which enabled the parallel development of new FA-imaging probes. The Cu(I)-mediated coupling of the alkynes with the γ-azido-FA precursor was accomplished in high yields and with minimal use of protective groups. The various probes were fully characterized spectroscopically as well as in Vitro and in ViVo. In Vitro, all new FA-derivatives exhibited high affinity toward the folic acid receptor (FR) and/or were specifically internalized into FR-overexpressing KB cells. In ViVo experiments with nude mice showed that all probes (except the MRI probes which have not been tested yet) accumulated specifically in FR-positive organs and human KB-cell xenografts. However, in ViVo imaging revealed significant differences between the various FA-derivatives with respect to unspecific, off-target localization. In general, the comparison of different probes proved the superiority of the more hydrophilic, radiometal-based imaging agents, a result which will guide future efforts for the development of FA-based imaging probes and therapeutic agents. In addition, the strategy presented herein should be readily applicable to other molecules of interest for imaging and therapeutic purposes and thus represents a valuable alternative to other synthetic approaches. INTRODUCTION Molecular imaging can be defined as the noninvasive visualization of biological processes at the cellular or molecular level in ViVo (1). To this end, different imaging modalities can be applied (e.g., single photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging (MRI), and near-infrared fluorescence imag- ing (NIRF)), each of which has certain advantages as well as limitations (2, 3). Regardless the modality employed, targeted imaging approaches require the use of specific molecular probes. These are usually composed of a suitable imaging entity which is covalently linked to a targeting (bio)molecule (vector). The multifunctional character of both components can complicate the preparation of such conjugates, in particular, because of potential side reactions or the lack of specificity of the coupling step. As a consequence, multistep syntheses including the use of protective groups are often employed that can lower significantly the overall efficiency of the preparation procedure. In addition, the syntheses of related imaging probes (e.g., for a comparison of different imaging modalities) are usually devel- oped and optimized individually, which reflects the lack of general synthetic transformations for the connection of structur- ally diverse precursors and building blocks. Therefore, new, efficient, and universally applicable synthetic strategies are needed. The discovery of the Cu(I)-catalyzed version of Huisgen’s dipolar [2 + 3] cycloaddition of terminal alkynes and azides by the groups of Sharpless (4) and Meldal (5) has had a great impact on the scientific community as demonstrated by countless reports describing its application in different areas of research (6). In general, the formation of 1,4-disubstituted 1,2,3-triazoles by the cycloaddition proceeds efficiently and selectively under benign (aqueous) reaction conditions and in the presence of various other functionalities (7). Termed a “click reaction” (8), this transformation has also found application in the develop- ment of targeted or nontargeted imaging agents (9, 10). The majority of reported applications of the click cycloaddition * Thomas L. Mindt, Department of Medical Radiology, Division of Radiological Chemistry, University Hospital Basel, Telephone: +41- 61-556-53-80, Fax: +41-61-265-55-59, E-mail mindtt@uhbs.ch; Roger Schibli, Department of Chemistry and Applied Biosciences, ETH Zurich, Telephone: +41-56-310-28-37, Fax: +41-56-310-2849, E-mail roger.schibli@pharma.ethz.ch. † Current address: Department of Medical Radiology, Division of Radiological Chemistry, University Hospital Basel, Basel (Switzerland). ‡ Current address: GlaxoSmithKline R&D, Clinical Imaging Centre, Hammersmith Hospital, London (UK). § Department of Chemistry and Applied Biosciences, ETH Zurich. | Paul Scherrer Institute. ⊥ Institute for Biomedical Engineering, University and ETH Zurich. # Institute of Pharmacology and Toxicology, University and ETH Zurich. Bioconjugate Chem. 2009, 20, 1940–1949 1940 10.1021/bc900276b CCC: $40.75  2009 American Chemical Society Published on Web 10/05/2009