Cysteine‐Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations

Abstract

We describe anew technique in protein synthesis that extends the existing repertoire of methods for protein modification:Achemoselective reaction that induces reactivity for as ubsequent bioconjugation. An azide-modified building blockr eacts first with an ethynylphosphonite through aS taudinger-phosphonite reaction (SPhR) to give an ethynylphosphonamidate.T he resulting electron-deficient triple bond subsequently undergoes ac ysteine-selective reaction with proteins or antibodies.W ed emonstrate that ethynylphosphonamidates display excellent cysteine-selective reactivity combined with superior stability of the thiol adducts,w hen compared to classical maleimide linkages.T his turns our technique into av ersatile and powerfult ool for the facile construction of stable functional protein conjugates.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.Figure 4. a) Synthetic scheme for phosphonamidate attachmentofthe Cy5f luorophore to trastuzumab(anti-Her2 antibody) to generate an AFC. b) Immunostaining of fixed cells either over-expressing the cell-surface receptor Her2 (BT474) or exhibiting low Her2 expression levels (MDAMB468). The merged images show the signal from the DNA stain DAPI in blue and the Cy5signal in red. Scale bar:10mm. c) Synthetic scheme for the attachmento fphosphonamidite-modified cCPPs 8 and 9 to aeGFP mutant with asingle addressable cysteine. d) Fluorescence imaging of HeLa cells after incubation with eGFP alone and eGFP-cTat at 50 mm.I mages show the GFP channel in green and the Hoechst 33342 nuclear stain in blue. Scale bar:2 0mm. Forfurther information see the SupportingInformation. Angewandte Chemie Communications

N‐Hydroxysuccinimide‐Modified Ethynylphosphonamidates Enable the Synthesis of Configurationally Defined Protein Conjugates

“…Next, l ‐glutathione was added to the triple bond and the corresponding products 4 were subjected to HPLC and 31 P NMR spectroscopic analysis (Figure A). Similar to our previous observations, we found that more than 90 % of the Z isomer formed upon thiol addition (Figure B and Figure S1 in the Supporting Information). In addition, the Z isomers that originated from the reaction of the enantiomerically pure phosphonamidates (+)‐ and (−)‐ 2 displayed a single resonance in the 31 P NMR spectrum at δ =17.58 and 17.51 ppm, respectively.…”

“…We started our investigations by preparative chiral HPLC separation of the two enantiomers of compound 2 that originated from the previously described Staudinger–phosphonite reaction of diethyl ethynylphosphonite and 4‐azidobenzoic acid NHS ester . After successful separation with a Chiralpak IA column, eluting with 30 % isopropanol in hexane, both enantiomers (+)‐ and (−)‐ 2 , as well as the racemic mixture, were reacted with the 5‐[(2‐aminoethyl)amino]naphthalene‐1‐sulfonic acid (EDANS) amine under basic conditions and the functional fluorescent phosphonamidates 3 were isolated in very good yields.…”

“…Here, we applied these building blocks for the synthesis of ADCs and antibody–protein conjugates and compared those to state‐of‐the‐art 1 . In contrast to the maleimide residue within 1 , the phosphonamidates are stable to hydrolysis under NHS conjugation conditions . We demonstrate that ethynylphosphonamidates exhibit a higher cysteine selectivity and thereby reduce undesired homo‐crosslinking side products at physiological pH (7.4).…”

“… Principle of the linkage of a lysine residue and a thiol with succinimidyl 4‐( N ‐maleimidomethyl)cyclohexane‐1‐carboxylate ( 1 ; left) and the NHS‐functionalized phosphonamidate 2 (right). In contrast to maleimides, phosphonamidates are not prone to hydrolysis and thiol exchange, and can deliver configurationally defined conjugates if starting from enantiomerically pure phosphonamidates.…”

“…Recently, we introduced unsaturated phosphonamidates for the selective and irreversible modification of cysteine residues in proteins and antibodies . We described the synthesis of NHS‐modified ethynylphosphonamidate building block 2 and demonstrated that hydrophilic diethylene glycol substitution at the phosphonamidate ester residue could drastically increase the water solubility . Here, we applied these building blocks for the synthesis of ADCs and antibody–protein conjugates and compared those to state‐of‐the‐art 1 .…”