Diethynyl Phosphinates for Cysteine-Selective Protein Labeling and Disulfide Rebridging

Abstract

Diethynyl phosphinates were developed as bisfunctional electrophiles for the site-selective modification of peptides, proteins and antibodies. One of their electron-deficient triple bonds reacts selectively with a thiol and positions an electrophilic moiety for a subsequent intra- or intermolecular reaction with another thiol. The obtained conjugates were found to be stable in human plasma and in the presence of small thiols. We further demonstrate that this method is suitable for the generation of functional protein conjugates for intracellular delivery. Finally, this reagent class was used to generate functional homogeneously rebridged antibodies that remain specific for their target. Their modular synthesis, thiol selectivity and conjugate stability make diethynyl phosphinates ideal candidates for protein conjugation for biological and pharmaceutical applications.

Keywords: antibody rebridging; bioconjugation; cysteine-selective modification; phosphorous-based electrophiles; protein double-modification.

Figure 1

a) Electrophilic phosphonamidates and phosphonothiolates, generated from azides or electrophilic disulfides and vinyl or ethynyl phosphonites, react selectively with cysteine residues on proteins.[¹⁴, ²⁵, ²⁶] b) Development of substituted diethynyl phosphinates as reagents for selective thiol–thiol bioconjugation and rebridging of native disulfides, for example, in therapeutic antibodies.

Scheme 1

Synthesis of diethynyl phosphinates. a) Generation of phosphinate 1 and the formation of its thiol adducts. b) Synthetic route towards functional diethynyl phosphinates 2–5. c) Sequential thiol addition to diethynyl phosphinates allows to generate the quenched FRET pairs F1 and F2.

Figure 2

a) FRET‐quenching assay to investigate the stability of the thiol conjugates. b),c) Observed EDANS fluorescence for constructs F1 and F2 in PBS, PBS supplemented with glutathione, human serum and in 0.1 m aq. NaOH over 72 h. d) General scheme for the site‐selective protein modification using diethynyl phosphinates and deconvoluted intact protein MS spectra of successfully labeled proteins. e) Conjugation of a cell‐penetrating R₁₀‐peptide to mCherry‐5 allows delivery of mCherry into living cells with nucleolar localization and co‐localization of mCherry with NBD (scale bar 20 μm).

Figure 3

Reaction of Trastuzumab with diethynyl phosphinate 1 and subsequent analysis. a) General procedure for antibody rebridging using compound 1. b) Analysis of Trastuzumab before and after the reaction via SDS‐PAGE. c) Deconvoluted intact protein MS of the rebridged half antibody (2× modified with 1) after deglycosylation by PNGaseF.

Figure 4

Functional modification of Trastuzumab and its biological evaluation. a) Two‐step modification of the antibody with phosphinate 2, followed by on antibody CuAAC forming the fluorescein conjugate (half and full antibody). b) Analysis of the conjugate via SDS‐PAGE using coomassie staining (B) and in‐gel fluorescence (C). c) UV/Vis spectrum of the fluorescein‐conjugated antibody. d) Cell‐membrane labeling of Her2‐positive cells without any observed staining of Her2‐negative cells (scale bar 20 μm).