Dbco peg4 maleimide

Superoxide dismutase (SOD-1), obtained from bovine erythrocytes, (Sigma-Aldrich) was modified by mixing 2 mg/mL SOD-1 with a 60-fold excess of azido-PEG₄-NHS ester (Click Chemistry Tools) in PBS containing 0.1 M sodium bicarbonate, pH 8.3, and incubating for 1 h at room temperature. Unreacted NHS-ester was removed by repeated spins through a 10 kDa cutoff filter. Conjugation with nanobodies was carried out using strain-promoted alkyne–azide cycloaddition (SPAAC) (“click chemistry”) (Figure S8). As described previously,³⁴ a peptide containing a free C-terminal thiol (GGGK[5-FAM]GGSC) was modified with dibenzylocyclooctyne (DBCO) through maleimide chemistry, by reaction with DBCO-PEG₄-maleimide (Click Chemistry Tools, Scottsdale, AZ). This peptide was then site-specifically reacted with nanobodies using SrtA-mediated transpeptidation, as described above. Conjugation was carried out at varying ratios of nanobody:SOD-1, ranging from 1:1 to 4:1, in PBS, pH 7.4, and the degree of conjugation was assessed using SEC HPLC. For further evaluation, unreacted nanobody and SOD were removed by centrifugation through appropriate molecular weight cutoff filters.

DEX, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and sodium ascorbate (NaAsc) were purchased from Sigma-Aldrich (St. Louis, MO). Copper(II) sulfate pentahydrate (CuSO₄ • 5H₂O) was purchased from Acros Organics (Geel, Belgium). 2,5-dioxopyrrolidin-1-yl 2-azidoacetate, DBCO-PEG₄-Maleimide, DBCO-NH₂, and MMAE-DBCO were purchased from Click Chemistry Tools, LLC (Scottsdale, AZ). Doxorubicin hydrochloride was purchased from LC Laboratories (Woburn, MA). Mertansine (DM1) was purchased from Carbosynth Limited (Berkshire, UK). All other chemicals and reagents were analytical grade and were used as received without further purification.
The peptides PLP139–151-Alk and PLP139–151-N₃ have been synthesized in our laboratory via solid phase peptide synthesis on a Wang resin, but larger quantities of each peptide were obtained from Biomatik Corporation (Wilmington, DE). In each case, the linker 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid was purchased from PurePEG, LLC (San Diego, CA) and 4-pentynoic acid was purchased from Sigma-Aldrich (St. Louis, MO).