ZUSCHRIFTEN 2762  WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001 0044-8249/01/11314-2762 $ 17.50+.50/0 Angew. Chem. 2001, 113, Nr. 14 Spin-Labeled Dendrimers in EPR Imaging with Low Molecular Weight Nitroxides AlexanderT. Yordanov, Ken-ichi Yamada, Murali C. Krishna, James B. Mitchell, Eric Woller, Mary Cloninger, and Martin W. Brechbiel* Electron paramagnetic resonance (EPR) imaging is an emerging technology that shows great promise in medical research applications for measuring free radical distribution, metabolism, and extent of oxygenation in tumors, organs and tissues. [1] However, the stable nitroxyl radicals (nitroxides) which these applications utilize, such as 4-hydroxy-2,2,6,6- tetramethylpiperidine-1-oxyl (TEMPOL), are prone to bio- reduction to the analogous diamagnetic, EPR-silent hydroxyl- amines (i.e., TEMPOL-H; Scheme 1). The nitroxide and the hydroxylamine species rapidly establish an equilibrium that strongly favors the hydroxyla- mine in vivo. [2] Thus, the short half-life of nitroxides in vivo (t 1/2  3 min) has limited further development and wide applica- tion of EPR imaging in the bio- medical field. Recently it was reported that intravenous injection of polyni- troxyl-albumin (PNA) caused reoxidation of free hydroxyl- amine (shown to remain in the body for a relatively long period of time) back to the paramagnetic nitroxide. [3] This reagent has enabled high-resolution EPR imaging of rat heart and may also permit various therapeutic applications of nitroxides. Alternatively, polyamidoamine (PAMAM) Star- burst TM dendrimers are spherical macromolecules which can be produced in successive generations, each with a specified and defined size and molecular weight as well as a high number of terminal primary amino groups. [4] Because of these characteristics and the low immunogenicity, PAMAMs are finding utility in a variety of applications, many of which are biomedical. [5] Due to their macromolecular nature, dendrim- ers are impermeable and when injected intravenously distrib- ute primarily in the vascular space. This is in contrast to TEMPOL and TEMPOL-H, which are membrane permeable and move readily between the extra- and intracellular spaces, the latter being the major sites of nitroxide reduction. [6] Recently dendrimers labeled with 2,2,6,6-tetramethylpiper- idine-1-oxyl (TEMPO) were used to evaluate the heteroge- nicity of their carbohydrate-substituted surfaces. [7a] Previous EPR studies of lower generation dendrimers with 2,2,5,5- tetramethylpyrrolidin-1-oxyl (PROXYL) conjugates have shown that the large spin ± spin interaction encountered by dendrimer-labeled nitroxides causes extensive broadening of the EPR spectra, effectively making the latter indistinguish- able from the base line. [7b] In contrast, the low molecular weight nitroxides exhibit sharp EPR spectra, and the inten- sities of the bands can be easily measured. Here we report the preparation and characterization of two novel spin-labeled dendrimers, a polynitroxyl G-6 PAMAM TM dendrimer with 198 (G-6-TEMPO-198) or with 80 (G-6-TEMPO-80) free TEMPO radicals on the surface of the macromolecule. We show that both dendrimers reoxidize TEMPOL-H back to its EPR-active form, TEMPOL, and therefore are potential TEMPO free radical, life-supporting agents for EPR imaging. The commercially available G-6 PAMAM TM dendrimers are synthetic oblate spheroidal macromolecules composed of an ethylenediamine initiator core and repeating polyamido- amino units resulting in 256 amines on the surface. To attach TEMPO free radicals to the parent G-6 dendrimer, 4-amino- 2,2,6,6-tetramethylpiperidine-1-oxyl (1, 4-amino-TEMPO) was first converted into 2,2,6,6-tetramethylpiperidine-1-oxyl- 4-succinamic acid (2), which subsequently reacted to form the N-hydroxysuccinimidyl ester 3 (Scheme 2). Compounds 2 and 3 were characterized by their exact fast atom bombardment (FAB) mass spectra. No satisfactory elemental analyses could Lecommandoux, M. F. Archard, F. Hardouin, Liq. Cryst. 1998, 25, 85; d)A. Sunder, M.-F. Quincy, R. Mühlhaupt, H. Frey, Angew. Chem. 1999, 111, 3107; Angew. Chem. Int. Ed. 1999, 38, 2928; e) J.-L. Serrano, J. Barbera, personal communication. [6] M. Veith, R. Elsäûer, R.-P. Krüger, Organometallics 1999, 18, 656. [7] R. Elsäûer, G. H. Mehl, J. W. Goodby, D. J. Photinos, Chem. Commun. 2000, 851. [8] B. D. Karstedt, General Electrics, US-Patent 3.814.730, 1974 ;[Chem. Abstr. 1974, 80, 16134j]. [9] Compound 5 (150 mg, 6.1  10 5 mol) and compound A (1.3 g, 1.64  10 3 mol) were dissolved in dry toluene (10 mL). Fifteen drops of a solution of Karstedts catalyst in xylenes (3.5 %; Fluorochem) were added and the reaction mixture was stirred overnight at 45 8C. Volatiles were evaporated and the residue was purified by column chromatog- raphy on alumina (Brockmann II). The polarity of the eluent was varied from hexane/dichloromethane ratios of 3/1 to 1/3. Yield of isolated product: 60 mg (8 %). 1 H NMR (399.7 MHz, CD 2 Cl 2 , 25 8C): d  0.01 (s, 144 H; -OSi(CH 3 ) 2 -), 0.16 ± 0.17 (5 s (not resolved), 180 H; NSi(CH 3 ) 2 -), 0.35 ± 0.60 (m, 108H; SiCH 2 -), 0.88 (2t (not resolved), 72 H; -CH 3 ), 1.2 ± 1.6 (m, -CH 2 -, 360 H), 1.75 ± 1.84 (m, 72 H; CH 2 CH 2 O), 4.00 (3t (not resolved), -CH 2 O), 6.51 (m, 24H; CH, (carbonyl)), 6.95, 7.20, 7.50, 7.56 (4d, 4  24 H; CH, biphenyl unit), 7.98 (d, 12 H; CH, (carbonyl)); 13 C NMR (100.4 MHz, CD 2 Cl 2 , 25 8C): d  0.18, 0.53 (-Si(CH 3 ) 2 O- Si(CH 3 ) 2 -), 3.43, 3.50 (-N{Si(CH 3 ) 2 -} 2 ), 11.1, 12.7 (NSi(Me) 2 CH 2 CH 2 - Si(Me) 2 O-), 14.0 (NSi(Me) 2 CH 2 CH 2 Si(Me) 2 N-), 14.3 (CH 3 ), 18.7 (CH 2 Si(Me) 2 O), 23.1 ± 32.3 (-CH 2 -), 68.5, 68.7, 69.3 (-CH 2 O), 100.5, 105.5, 111.6, 115.1, 122.6, 127.7, 128.3, 133.0, 134.5, 138.5, 150.6, 159.2, 161.9, 164.8 (C aromatic), 164.3 (-C(O)O); GPC (THF, toluene standard) (hM w i/hM n i)  1.024. [*] Dr. M. W. Brechbiel, Dr. A.T. Yordanov Radioimmune & Inorganic Chemistry Section Bldg. 10, Room B3B69 Radiation Oncology Branch NCI, National Institutes of Health Bethesda, MD 20892 (USA) Fax: ( 1) 301-402-1923 E-mail : martinwb@box-m.nih.gov Dr. K.-i. Yamada, Dr. M. C. Krishna, Dr. J. B. Mitchell Radiation Biology Branch NCI, National Institutes of Health Bethesda, MD 20892 (USA) E. Woller, Dr. M. Cloninger Department of Chemistry and Biochemistry Montana State University Bozeman, MT 59717 (USA) Scheme 1. The equilibrium established between TEM- POL and TEMPOL-H. The latter is favored in vivo.