Synthesis of Biotin3xNHS-X-Linker

Biotinyl N-Tris((2-(2.5-dioxopyrrolidin-1-yl propionate triethyleneglycolamido) ethoxy) methyl) methylamide, herein referred to as Biotin_3xNHSX-Linker, was synthesized as described in detail in the Supplementary Information (SI). Briefly, spacer synthesis started with 2- (2- (2-chloroethoxy) ethoxy) ethanol, which was converted into the ester (1) in a MICHAEL reaction with t-butyl acrylate and sodium in tetrahydrofuran (THF) (Figure 1). The chloride (1) was then mixed with sodium azide (NaN₃) and the azide obtained was reduced to the amine-terminated spacer with triphenylphosphine. In the convergent procedure, Tris (hydroxymethyl) aminomethane (THAM) was converted with t-butyl acrylate and sodium hydroxide (NaOH) in THF to the triple-functionalized amine (3), which was then protected with benzyl chloroformate (4). This protective group shows stable behavior in the case of tri-fluoro acetic acid (TFA) initiated acidic hydrolysis. This was followed by selective TFA deprotection of the ester-protected hydroxyl group with subsequent peptide coupling of the molecule (4) and the spacer (2). In the next step, the N-Cbz protective group was removed under reductive conditions using hydrogen and palladium on activated carbon (Pd/C). The amine (5) obtained was further processed under peptide coupling conditions with D(+)biotin, diisopropylethylamine (DIPEA), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) to yield the biotin-functionalized product (7). The activated crosslinker (Biotin_3xNHSX-linker) was obtained after deprotection of the ester units and direct functionalization of the carboxylic acids with N-hydroxysuccinimide (NHS) and N, N′-dicyclohexylcarbodiimide (DCC) in THF.