Design, Synthesis, Conjugation, and Reactivity of Novel trans,trans-1,5-Cyclooctadiene-Derived Bioorthogonal Linkers.

The tetrazine/trans-cyclooctene (TCO) inverse electron-demand Diels-Alder (IEDDA) reaction is the fastest bioorthogonal "click" ligation process reported to date. In this context, TCO reagents have found widespread applications; however, their availability and structural diversity is still somewhat limited due to challenges connected with their synthesis and structural modification. To address this issue, we developed a novel strategy for the conjugation of TCO derivatives to a biomolecule, which allows for the creation of greater structural diversity from a single precursor molecule, i.e., trans,trans-1,5-cyclooctadiene [(E,E)-COD] 1, whose preparation requires standard laboratory equipment and readily available reagents. This two-step strategy relies on the use of new bifunctional TCO linkers (5a-11a) for IEDDA reactions, which can be synthesized via 1,3-dipolar cycloaddition of (E,E)-COD 1 with different azido spacers (5-11) carrying an electrophilic function (NHS-ester, N-succinimidyl carbonate, p-nitrophenyl-carbonate, maleimide) in the ω-position. Following bioconjugation of these electrophilic linkers to the nucleophilic residue (cysteine or lysine) of a protein (step 1), the resulting TCO-decorated constructs can be subjected to a IEDDA reaction with tetrazines functionalized with fluorescent or near-infrared (NIR) tags (step 2). We successfully used this strategy to label bovine serum albumin with the TCO linker 8a and subsequently reacted it in a cell lysate with the fluorescein-isothiocyanate (FITC)-derived tetrazine 12. The same strategy was then used to label the bacterial wall of Gram-positive Staphylococcus aureus, showing the potential of these linkers for live-cell imaging. Finally, we determined the impact of structural differences of the linkers upon the stability of the bioorthogonal constructs. The compounds for stability studies were prepared by conjugation of TCO linkers 6a, 8a, and 10a to mAbs, such as Rituximab and Obinutuzumab, and subsequent labeling with a reactive Cy3-functionalized tetrazine.

Comparison of analytical methods for antibody conjugates with application in nuclear imaging - Report from the trenches.

INTRODUCTION: Monoclonal antibodies (mAbs) are widely used in nuclear imaging. Radiolabelling with positron emitting radionuclides, typically radiometals, requires the incorporation of a bifunctional chelator for the formation of the radiometal-mAb complex. Additionally, mAbs can be conjugated with small molecules capable to undergo bioorthogonal click reactions in vivo, enabling pre-targeting strategies. The determination of the number of functionalities attached to the mAb is critically important to ensure a good labelling yield or to guarantee pre-targeting efficacy. In this work, we compare three different analytical methods for the assessment of average functionalisation and heterogeneity of the conjugated mAbs. METHODS: Two selected mAbs (Trastuzumab and Bevacizumab) were randomly conjugated through lysine residues with 3-10 equivalents p-isothiocyanatobenzyl-desferrioxamine (p-NCS-Bz-DFO) or 20-200 equivalents trans-cyclooctene-N-hydroxysuccinimide ester (TCO-NHS). The DFO- or TCO-to-mAb ratio were determined using three different methods: direct titration (radiometric for DFO-conjugated mAbs, photometric for TCO-conjugated mAbs), MALDI/TOF MS mass analysis (Matrix-Assisted Laser Desorption-Ionization/Time of Flight Mass Spectrometry), and UPLC/ESI-TOF MS mass analysis (Ultra High Performance Liquid Chromatography/Electrospray Ionization-Time of Flight Mass Spectrometry). RESULTS: Radiometric and photometric titrations provided information on the average number of DFO and TCO functionalities per mAb respectively. MALDI/TOF MS provided equivalent results to those obtained by titration, although investigation of the heterogeneity of the resulting mixture was challenging and inaccurate. UPLC/ESI-TOF MS resulted in good peak resolution in the case of DFO-conjugated mAbs, where an accurate discrimination of the contribution of mono-, di- and tri-substituted mAbs could be achieved by mathematical fitting of the spectra. However, UPLC/ESI-TOF MS was unable to discriminate between different conjugates when the smaller TCO moiety was attached to the mAbs. CONCLUSIONS: The three techniques offered comparable results in terms of determining the average number of conjugates per mAb. Additionally, UPLC/ESI-TOF MS was able to shed a light on the heterogeneity of the resulting functionalised mAbs, especially in the case of DFO-conjugated mAbs. Finally, while using a single analytical method might not be a reliable way to determine the average functionalisation and assess the heterogeneity of the sample, a combination of these methods could substantially improve the characterization of mAb conjugates.

Synthesis and Characterization of a Click-Assembled 18-Atom Macrocycle That Displays Selective AXL Kinase Inhibitory Activity.

A novel macrocyclic construct consisting of a pyrazolopyrimidine scaffold concatenated to a benzene ring through two triazoles has been developed to investigate uncharted chemical space with bioactive potential. The 18-atom macrocycle was assembled via a double copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction between 1,3-bis(azidomethyl)benzene and a bis-propargylated pyrazolo[3,4-d]pyrimidine core. The resulting macrocycle was functionalized further into a multicyclic analog that displays selective inhibitory activity against the receptor tyrosine kinase AXL.