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Bis-PEG1-NHS ester, chemically known as N-(2-aminoethyl)-2-[2-(2-aminoethoxy)ethoxy]acetamide di(N-hydroxysuccinimide ester), is a versatile homobifunctional crosslinker widely utilized in bioconjugation and chemical biology. Its molecular formula is C14H27N3O8, and it carries the CAS registry number 106596-45-4. This reagent features two reactive N-hydroxysuccinimide (NHS) ester groups positioned at opposite ends of a short, hydrophilic polyethylene glycol (PEG) spacer consisting of a single ethylene glycol unit. The NHS esters are highly efficient at reacting with primary amines found on lysine residues of proteins, antibodies, or other amine-containing biomolecules under physiological pH conditions to form stable amide bonds.
The primary application of Bis-PEG1-NHS ester lies in the covalent linking of two distinct molecules or the intramolecular cyclization of a single protein. Due to the presence of the PEG chain, this crosslinker introduces a degree of flexibility and hydrophilicity into the conjugate, which helps maintain the solubility and biological activity of the resulting complex. It is particularly valuable for labeling therapeutic proteins, creating antibody-drug conjugates, or immobilizing enzymes onto solid supports where minimal steric hindrance is required. The short one-unit PEG spacer ensures that the distance between the two linked moieties remains minimal compared to longer PEG variants, making it ideal for applications requiring precise spatial control without excessive bulk.
In laboratory settings, the reaction is typically performed in aqueous buffers such as phosphate-buffered saline (PBS) or borate buffer at a pH range of 7.2 to 9.0. Unreacted NHS esters must be quenched promptly using excess amine-containing agents like Tris-HCl or glycine to prevent non-specific background reactions. While highly effective, researchers must consider the stability of the NHS ester in aqueous environments, as it can undergo hydrolysis over time if not used immediately after dissolution in anhydrous organic solvents like DMSO or DMF. Overall, Bis-PEG1-NHS ester serves as a fundamental tool for generating stable, defined bioconjugates with enhanced biocompatibility and controlled intermolecular spacing.