F-18 Labeled RGD Probes Based on Bioorthogonal Strain-Promoted Click Reaction for PET Imaging.

A series of fluorine-substituted monomeric and dimeric cRGD peptide derivatives, such as cRGD-ADIBOT-F (ADIBOT = azadibenzocyclooctatriazole), di-cRGD-ADIBOT-F, cRGD-PEG5-ADIBOT-F, and di-cRGD-PEG5-ADIBOT-F, were prepared by strain-promoted alkyne azide cycloaddition (SPAAC) reaction of the corresponding aza-dibenzocyclooctyne (ADIBO) substituted peptides with a fluorinated azide 3. Among these cRGD derivatives, di-cRGD-PEG5-ADIBOT-F had the highest binding affinity in a competitive binding assay compared to other derivatives and even the original cRGDyk. On the basis of the in vitro study results, di-cRGD-PEG5-ADIBOT-(18)F was prepared from a SPAAC reaction with (18)F-labeled azide and subsequent chemo-orthogonal scavenger-assisted separation without high performance liquid chromatography (HPLC) purification in 92% decay-corrected radiochemical yield (dcRCY) with high specific activity for further in vivo positron emission tomography (PET) imaging study. In vivo PET imaging study and biodistribution data showed that this radiotracer allowed successful visualization of tumors with good tumor-to-background contrast and significantly higher tumor uptake compared to other major organs.

Development of a simple method for protein conjugation by copper-free click reaction and its application to antibody-free Western blot analysis.

There are currently many methods available for labeling proteins in order to study their structure and function. However, the utility of these methods is hampered by low efficiency, slow reaction rates, nonbiocompatible reaction conditions, large-sized labeling groups, and the requirement of specific side chains such as cysteine or lysine. In this study, a simple and efficient method for protein labeling was developed, in which an azide-containing amino acid was introduced into a protein and conjugated to a labeling reagent by strain-promoted azide-alkyne cycloaddition (SPAAC). This method allowed us to label proteins by simply mixing a protein and a labeling reagent in physiological conditions with a labeling yield of approximately 80% in 120 min. In addition, the specificity of SPAAC made it possible to analyze the expression level of a protein quantitatively by simple mixing and SDS-PAGE analysis with no need for antibodies or multistep incubations. Because the genetic incorporation of the azide-containing amino acid can be generally applied to any protein and the SPAAC reaction is highly specific, this method should prove useful for labeling and analyzing proteins.

F-18 labeling protocol of peptides based on chemically orthogonal strain-promoted cycloaddition under physiologically friendly reaction conditions.

We introduce the high-throughput synthesis of various (18)F-labeled peptide tracers by a straightforward (18)F-labeling protocol based on a chemo-orthogonal strain-promoted alkyne azide cycloaddition (SPAAC) using aza-dibenzocyclootyne-substituted peptides as precursors with (18)F-azide synthon to develop peptide based positron emission tomography (PET) molecular imaging probes. The SPAAC reaction and subsequent chemo-orthogonal purification reaction with azide resin proceeded quickly and selectively under physiologically friendly reaction conditions (i.e., toxic chemical reagents-free, aqueous medium, room temperature, and pH ≈7), and provided four (18)F-labeled tumor targetable bioactive peptides such as cyclic Arg-Gly-Asp (cRGD) peptide, bombesin (BBN), c-Met binding peptide (cMBP), and apoptosis targeting peptide (ApoPep) in high radiochemical yields as direct injectable solutions without any HPLC purification and/or formulation processes. In vitro binding assay and in vivo PET molecular imaging study using the (18)F-labeled cRGD peptide also demonstrated a successful application of our (18)F-labeling protocol.

Synthesis of N4'-[¹8F]fluoroalkylated ciprofloxacin as a potential bacterial infection imaging agent for PET study.

Syntheses and evaluation of fluoroalkylated ciprofloxacin analogues are described. Among these analogues, N4'-3-fluoropropylciprofloxacin (16) showed the most efficient antibacterial activity against E. coli strains (DH5α and TOP10) and a high binding affinity for DNA gyrase of bacteria. To develop bacteria-specific infection imaging agents for positron emission tomography (PET), no-carrier-added N4-3-[¹8F]fluoropropylciprofloxacin ([¹8F]16) was prepared in two steps from N4-3-methanesufonyloxypropylciprofloxacin, resulting in a 40% radiochemical yield (decay corrected for 100 min) via the tert-alcohol media radiofluorination protocol with high radiochemical purity (> 99%) as well as high specific activity (149 ± 75 GBq/µmol). The agent was stable (> 90%), as shown by an in vitro human serum stability assay. A bacterial uptake and blocking study of [¹8F]16 using authentic compound 16 in TOP10 cells demonstrated its high specific bacterial uptake. The results suggest that this radiotracer holds promise as a useful bacterial infection radiopharmaceutical for PET imaging.