Chemoselective Methionine Bioconjugation: Site-Selective Fluorine-18 Labeling of Proteins and Peptides.

Chemoselective methionine bioconjugation with alkyne-bearing oxaziridine and alkyne-bearing iodonium salts was investigated as a new platform for site-selective radiolabeling of proteins and peptides with fluorine-18. Alkyne-bearing sulfimide conjugates, resulting from oxaziridine modification, underwent copper-assisted alkyne-azide cycloaddition (CuAAC) with an 18F-labeled PEGylated azide to afford 18F-labeled triazoles in excellent radiochemical yields. Diazoester sulfonium salt bioconjugates, formed from alkyne-bearing 2-diazoiodonium salts, gave low yields of 18F-labeled triazoles and were shown to be unstable to CuAAC conditions. Photolytic removal of the diazo group, however, afforded the trialkylsulfonium salt which smoothly underwent CuAAC with the 18F-labeled PEGylated azide to afford high radiochemical yields of the desired 18F-labeled click product. Overall, the results establish the viability of chemoselective methionine bioconjugation as a method for preparing site-selective 18F-labeled PET radioligands.

Direct Carbon Isotope Exchange through Decarboxylative Carboxylation.

A two-step degradation-reconstruction approach to the carbon-14 radiolabeling of alkyl carboxylic acids is presented. Simple activation via redox-active ester formation was followed by nickel-mediated decarboxylative carboxylation to afford a range of complex compounds with ample isotopic incorporations for drug metabolism and pharmacokinetic studies. The practicality and operational simplicity of the protocol were demonstrated by its use in an industrial carbon-14 radiolabeling setting.

Adnectin-drug conjugates for Glypican-3-specific delivery of a cytotoxic payload to tumors.

Tumor-specific delivery of cytotoxic agents remains a challenge in cancer therapy. Antibody-drug conjugates (ADC) deliver their payloads to tumor cells that overexpress specific tumor-associated antigens-but the multi-day half-life of ADC leads to high exposure even of normal, antigen-free, tissues and thus contributes to dose-limiting toxicity. Here, we present Adnectin-drug conjugates, an alternative platform for tumor-specific delivery of cytotoxic payloads. Due to their small size (10 kDa), renal filtration eliminates Adnectins from the bloodstream within minutes to hours, ensuring low exposure to normal tissues. We used an engineered cysteine to conjugate an Adnectin that binds Glypican-3, a membrane protein overexpressed in hepatocellular carcinoma, to a cytotoxic derivative of tubulysin, with the drug-to-Adnectin ratio of 1. We demonstrate specific, nanomolar binding of this Adnectin-drug conjugate to human and murine Glypican-3; its high thermostability; its localization to target-expressing tumor cells in vitro and in vivo, its fast clearance from normal tissues and its efficacy against Glypican-3-positive mouse xenograft models.

Synthesis of 2-(14)C-iminothiolane and 2-(13)C,(15)N-iminothiolane (Traut's reagent).

2-Iminothiolane has found utility in the growing area of antibody-drug conjugates by serving as a lysine-thiolating agent and the junction between the antibody and the cytotoxic payload during random conjugation of a monoclonal antibody. 2-(14)C-Iminothiolane was prepared from commercially available [(14)C]KCN using a four-step sequence in an overall 10% radiochemical yield. Stable-labeled 2-(13)C,(15)N-iminothiolane was also prepared from [(13)C(15)N]KCN in a similar manner. The ˙ labeled Traut's reagent produced by this sequence showed comparable reactivity as the commercially available unlabeled reagent with a representative monoclonal antibody and could serve as highly informative analytical tools to investigate antibody-drug conjugate formation via the random conjugation process.

Synthesis of a stable isotopically labeled universal surrogate peptide for use as an internal standard in LC-MS/MS bioanalysis of human IgG and Fc-fusion protein drug candidates.

The synthesis of a 16-residue, stable isotopically labeled peptide is described for use as a LC-MS/MS (Liquid chromatography-mass spectrometry/mass spectrometry) internal standard in bioanalytical studies. This peptide serves as a single universal surrogate peptide capable of quantifying a wide variety of immunoglobulin G and Fc-fusion protein drug candidates in animal species used in pre-clinical drug development studies. An efficient synthesis approach for this peptide was developed using microwave-assisted solid phase peptide synthesis (SPPS) techniques, which included the use of a pseudoproline dipeptide derivative. The corresponding conventional room temperature SPPS was unsuccessful and gave only mixtures of truncated products. Stable-labeled leucine was incorporated as a single residue via manual coupling of commercially available Fmoc-[(13) C6 , (15) N]-l-leucine onto an 11-unit segment followed by automated microwave-assisted elaboration of the final four residues. Using this approach, the desired labeled peptide was prepared in high purity and in sufficient quantities for long-term supplies as a bioanalytical internal standard. The results strongly demonstrate the importance of utilizing both microwave-assisted peptide synthesis and pseudoproline dipeptide techniques to allow the preparation of labeled peptides with highly lipophilic and sterically hindered side-chains.