Chemoselective bioconjugation reactions of tyrosine residues for application in PET radiochemistry

• Author / Creator

  Leier, Samantha A

• Achieving chemoselectivity while maintaining bioorthogonality are among some of the major challenges in bioconjugation chemistry. Bioconjugation chemistry has important applications in PET radiochemistry. While modern bioconjugation techniques rely predominantly on lysine and cysteine residues as targets for bioconjugation, other bioconjugation techniques that selectively target other amino acids would contribute greatly to the development of new PET radiotracers. Although only modestly prevalent and often buried within the protein structure, tyrosine represents an attractive target for bioconjugation. Tyrosine residues can be selectively targeted by both luminol derivatives and aryl diazonium salts. Luminol derivatives react via an ene-like reaction where as diazonium compounds react via electrophilic aromatic substitution to produce a stable conjugate. Recently, luminol derivatives have been reported for the introduction of fluorescent probes into proteins and diazonium salts have been reported for the introduction of radiometals into tyrosinamide-containing polymers. To the best of our knowledge, neither technique has been applied to PET radiotracer synthesis. Therefore, the aim of this project is the chemoselective introduction of radionuclides onto tyrosine residues under mild conditions. Specifically, we describe the preparation of 18F-labeled luminol derivatives as well as 64Cu- and 68Ga-labeled diazonium salts as novel building blocks for subsequent coupling with tyrosine residues under mild conditions. Luminol derivative N-(4-[18F]fluorobenzyl)-2-methyl-1,4-dioxo-1,2,3,4-tetrahydro-phthalazine-6-carboxamide (29) was synthesized in 48% radiochemical yield. The use of H2O2 in the presence of hemin as an oxidizing agent resulted in improved oxidation when compared with 1,3-dibromo-5,5-dimethylhydantoin, N-bromosuccinimide, 1,3,4,6-tetrachloro-3α,6α-diphenyl-glucoluril coated iodination tubes and N-chloro-benzenesulfonamide coated iodination beads. Tyrosine-click chemistry of compound 29 with acetyl-L-tyrosine methyl amide afforded the expected conjugate in 27% radiochemical yield. As well, 68Ga- and 64Cu-labeled NOTA-diazonium salts were synthesized from 20 µg of macrocyclic chelator 2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH2-Bn-NOTA). 68Ga- and 64Cu-labeled NOTA complexes were prepared in high radiochemical yields >80% and in situ conversion of 68Ga- and 64Cu-labeled p-NH2-Bn-NOTA complexes into corresponding diazonium salts was carried out with NaNO2 under acidic conditions. Azo coupling with L-tyrosine, neurotensin NT(8-13), human serum albumin (HSA) and tobacco mosaic virus (TMV) afforded the expected conjugates in radiochemical yields upwards of 80%, 45%, 20% and 9%, respectively. Coupling reactions proceeded under mild, aqueous conditions within 60 min. The described luminol-based tyrosine-click chemistry technique and azo coupling technique provide innovative bioconjugation strategies for the chemoselective introduction of various radionuclides onto tyrosine residues under mild and aqueous reaction conditions. The applied mild reaction conditions make these novel bioconjugation strategies also compatible with delicate tyrosine-based biomolecules such as proteins and may be expanded to antibodies.

• Subjects / Keywords

  • Bioconjugation
  • PET radiochemistry
  • Tyrosine-click
  • Azo coupling

• Graduation date

  Spring 2018

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3707X382

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.