Chloromethyl Acryl Reagents for Simple and Site-Selective Cysteine and Disulfide Modification

The generation of protein biotherapeutics with improved features compared to the synthetic drugs has received emerging interest. The conjugation of various synthetic functionalities to proteins provides access to new classes of protein conjugates, where the advantages from both the synthetic world and Nature can be combined in a synergistic fashion. Here, we reported that 2-chloromethyl acryl scaffold can serve as a simple yet versatile platform for synthesizing acrylamide or acrylate derivatives by coupling with different end-group functionalities (amino group or hydroxyl group) via a one-pot reaction. The chemical properties of the amide or ester linkage influence their inherent reactivity as bioconjugation reagents, which in turn allows synthetic customization of their features to achieve selective protein modification at cysteine or disulfide sites on demand. 2-Chloromethyl acrylamide reagents with amide linkage favors selective modification at cysteine site with high efficiency and the resultant bioconjugates exhibit superior stability compared to commonly employed maleimide-thiol conjugates. In contrast, 2-chloromethyl acrylate reagents bearing ester linkage can undergo two successive Michael reaction, allowing the selective modification of disulfides with high labelling efficiency and conjugate stability. These reagents could outperform widely applied maleimide reagents in terms of stability of the resultant bioconjugates without compromising on the ease of reagent preparation, reactivity and reaction speed. <br>

Chloromethyl Acryl Reagents for Simple and Site-Selective Cysteine and Disulfide Modification

The generation of protein biotherapeutics with improved features compared to the synthetic drugs has received emerging interest. The conjugation of various synthetic functionalities to proteins provides access to new classes of protein conjugates, where the advantages from both the synthetic world and Nature can be combined in a synergistic fashion. Here, we reported that 2-chloromethyl acryl scaffold can serve as a simple yet versatile platform for synthesizing acrylamide or acrylate derivatives by coupling with different end-group functionalities (amino group or hydroxyl group) via a one-pot reaction. The chemical properties of the amide or ester linkage influence their inherent reactivity as bioconjugation reagents, which in turn allows synthetic customization of their features to achieve selective protein modification at cysteine or disulfide sites on demand. 2-Chloromethyl acrylamide reagents with amide linkage favors selective modification at cysteine site with high efficiency and the resultant bioconjugates exhibit superior stability compared to commonly employed maleimide-thiol conjugates. In contrast, 2-chloromethyl acrylate reagents bearing ester linkage can undergo two successive Michael reaction, allowing the selective modification of disulfides with high labelling efficiency and conjugate stability. These reagents could outperform widely applied maleimide reagents in terms of stability of the resultant bioconjugates without compromising on the ease of reagent preparation, reactivity and reaction speed. <br>

Engineering of extracellular vesicles for display of protein biotherapeutics

Extracellular vesicles (EVs) have recently emerged as a highly promising cell-free bio-therapeutics. While a range of engineering strategies have been developed to functionalize the EV surface, current approaches fail to address the limitations associated with endogenous surface display, pertaining to the heterogeneous display of commonly used EV-loading moieties among different EV subpopulations. Here we present a novel engineering platform to display multiple protein therapeutics simultaneously on the EV surface. As proof-of-concept, we screened multiple endogenous display strategies for decorating the EV surface with cytokine binding domains derived from tumor necrosis factor receptor 1 (TNFR1) and interleukin 6 signal transducer (IL6ST), which can act as decoys for the pro-inflammatory cytokines TNFα and IL6, respectively. Combining synthetic biology and systematic screening of loading moieties, resulted in a three-component system which increased the display and decoy activity of TNFR1 and IL6ST, respectively. Further, this system allowed for combinatorial functionalization of two different receptors on the same EV surface. These cytokine decoy EVs significantly ameliorated disease phenotypes in three different inflammatory mouse models for systemic inflammation, neuroinflammation, and intestinal inflammation. Importantly, significantly improved in vitro and in vivo efficacy of these engineered EVs was observed when compared directly to clinically approved biologics targeting the IL6 and TNFα pathways.