© 2012 Nature America, Inc. All rights reserved. PROTOCOL 1946 | VOL.7 NO.11 | 2012 | NATURE PROTOCOLS INTRODUCTION PET, a noninvasive imaging modality that has been used in clini- cal diagnostics for several decades, is able to visualize a variety of human afflictions such as cancer and cardiac and neurological dis- eases at a functional cell level and thus has increasing relevance in medical imaging. One of the most important radionuclides used in PET imaging is 18 F, which possesses very favorable physical and chemical characteristics. However, the introduction of 18 F into larger bioactive molecules such as peptides and proteins is gener- ally complex when using standard carbon- 18 F chemistry. An alternative is the SiFA methodology, which is characterized by the application of building blocks that comprise a central silicon (Si) atom surrounded by two tertiary butyl (tBu) groups, a phenyl (Ph) moiety and a nonradioactive fluorine (F) atom 1,2 . The Ph moiety is amenable toward chemical modifications for further con- jugation of the SiFA moiety to biomolecules 3 . The two necessary tBu groups impose kinetic stability on the Si-F bond—the centerpiece of the SiFA group. Shielded against hydrolysis under physiological conditions resulting in physiologically stable products 1,4,5 , the Si-F bond easily exchanges its 19 F atom with a radioactive 18 F isotope. Notably, the isotopic exchange (IE) reaction proceeds at very low substrate concentrations 6 . An experimentally determined low acti- vation energy and a large pre-exponential factor for the IE 7 confirm the results from density functional theory calculations in gas phase and solution 8 . This special feature of SiFA radiochemistry produces labeled compounds of high specific activities (SAs) and allows for a simple purification independent of HPLC. As the reaction condi- tions for the IE are very mild (room temperature and lower), no side reactions usually take place. Therefore, only unreacted 18 F − has to be separated from the final product. The SiFA protocols are perfectly suited to label peptides 4 in a single step (this protocol) and proteins with two different SiFA-labeling synthons 3,9 while requiring minimal technical skills. This was shown for several relevant peptides used in PET imaging of malignant diseases 4,8 . Technical developments of the SiFA method for peptide labeling Normally, IE reactions do not deliver labeled compounds of high SA. However, fluorosilanes irrespective of their chemical deriva- tization rapidly undergo IE with 18 F − in dipolar aprotic solvents such as acetonitrile, N,N-dimethyl formamide (DMF) and DMSO, although best results can normally be achieved using acetonitrile 5 . To impose kinetic stability to the SiFA unit under physiological conditions, two tBu groups have to be attached to the Si atom 1,10–13 . This structural prerequisite for hydrolytic stability increases the overall lipophilicity of the SiFA-conjugated peptides, leading to an unfavorable in vivo distribution of high liver uptake and reduced bioavailability 4,12 . To cope with high lipophilicity, sugar moieties and a PEG spacer are introduced, which alleviate this negative char- acteristic and improve the radioactivity accumulation in the target tissue (e.g., tumor). The most recent advancement in SiFA peptide labeling includes the additional introduction of hydrophilic aspar- tic acids counteracting the lipophilicity of a SiFA-derivatized pep- tide by introducing charged functional groups into the molecule. By using these three lipophilicity-reducing auxiliaries in variation or combination, the logD value of the peptide can be custom tai- lored. Lipophilicity is significantly reduced when aspartic acid is coupled to the peptide by standard solid-phase peptide synthesis before attaching the SiFA group. The labeling efficiency of these peptides with nucleophilic 18 F − remains unchanged. The general coupling of SiFA to peptides is achieved using a Ph group that is derivatized in accordance with the kind of click chemistry intended for bioconjugation. The most recent SiFA- labeling protocols for peptide labeling are easy to handle, even for One-step 18 F-labeling of peptides for positron emission tomography imaging using the SiFA methodology Carmen Wängler 1,2 , Sabrina Niedermoser 2,3 , Joshua Chin 4 , Katy Orchovski 4 , Esther Schirrmacher 4 , Klaus Jurkschat 5 , Liuba Iovkova-Berends 5 , Alexey P Kostikov 4 , Ralf Schirrmacher 4 & Björn Wängler 3 1 Biomedical Chemistry, Medical Research Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. 2 Department of Nuclear Medicine, Hospital of the Ludwig-Maximilians-University, Munich, Germany. 3 Molecular Imaging and Radiochemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. 4 McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada. 5 Lehrstuhl für Anorganische Chemie II, TU University of Dortmund, Dortmund, Germany. Correspondence should be addressed to C.W. (carmen.waengler@medma.uni-heidelberg.de) or B.W. (bjoern.waengler@medma.uni-heidelberg.de). Published online 4 October 2012; doi:10.1038/nprot.2012.109 Here we present a procedure to label peptides with the positron-emitting radioisotope fluorine-18 ( 18 F) using the silicon-fluoride acceptor (SiFA) labeling methodology. Positron emission tomography (PET) has gained high importance in noninvasive imaging of various diseases over the past decades, and thus new specific imaging probes for PET imaging, especially those labeled with 18 F, because of the advantageous properties of this nuclide, are highly sought after. N-terminally SiFA–modified peptides can be labeled with 18 F - in one step at room temperature (20–25 °C) or below without forming side products, thereby producing satisfactory radiochemical yields of 46 ± 1.5% (n = 10). The degree of chemoselectivity of the 18 F-introduction, which is based on simple isotopic exchange, allows for a facile cartridge-based purification fully devoid of HPLC implementation, thereby yielding peptides with specific activities between 44.4 and 62.9 GBq mmol - 1 (1,200–1,700 Ci mmol - 1 ) within 25 min.