1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride edc hcl

CAL-B (Novozyme 435), which is a lipase derived from Candida Antarctica type B and immobilized on acrylic resin, was purchased from Sigma Aldrich, St. Louis, MO, USA. Prior to its use, it was first vacuum-dried over Phosphorous pentoxide for 24 h. Sorbitol (98%) and divinyl adipate (96%) were purchased from Sigma Aldrich (Steinheim, Germany) and TCI GmbH (Eschborn, Germany), respectively. Phosphorous pentoxide (≥99%), 4-(dimethylamino)pyridine (DMAP), anhydrous N,N-dimethylformamide (DMF, 99.8%), anhydrous tetrahydrofuran (THF, 99.9%), acetonitrile (anhydrous, 99.8%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), dialysis membranes with 1000 g·mol⁻¹ molecular weight cut off (MWCO), and 10,000 g·mol⁻¹ MWCO (Spectra/Por^®, made from regenerated cellulose) were purchased from Carl Roth, Karlsruhe, Germany. α,ω-bis-hydroxy poly(ethylene glycol)_n (where n = 9, 23, and 45) and α- methoxy,ω-hydroxy poly(ethylene glycol)_n (where n = 12) (mPEG 550) were purchased from Alfa Aesar, Kandel, Germany. DY-781 Amine (molecular weight: 781 g·mol⁻¹) and DY-784 NHS-ester (molecular weight: 1188 g·mol⁻¹) were purchased from Dyomics GmbH (Jena, Germany), while BSA-TMR was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

Novozyme 435, Lipase derived from Candida Antarctica type B (CAL-B) and immobilized on acrylic resin, was purchased from Sigma Aldrich, St. Louis, MO, USA. It was vacuum dried over Phosphorous pentoxide for 24 h prior to use. Sorbitol (98%) and divinyl adipate were purchased from Sigma Aldrich (Steinheim, Germany) and TCI GmbH (Eschborn, Germany), respectively. Phosphorous pentoxide (≥99%), 4-(dimethylamino)pyridine (DMAP), anhydrous N,N-dimethylformamide (DMF, 99.8%), anhydrous tetrahydrofuran (THF, 99.9%), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), dialysis membranes with 1000 g·mol⁻¹ molar mass cut off (MWCO) and 10,000 g·mol⁻¹ MWCO (Spectra/Por^®, made from regenerated cellulose) were purchased from Carl Roth, Karlsruhe, Germany. Deuterated chloroform (CDCl₃) and deuterated dimethyl sulfoxide (DMSO-d₆) were purchased from Armar (Europa) GmbH (Leipzig, Germany). α,ω-bis-hydroxy poly(ethylene glycol)_n (OH-PEG_n-OH, with n = 9, 23, and 45) and α-methoxy,ω-hydroxy poly(ethylene glycol)₁₂ (mPEG₁₂-OH) were purchased from Alfa Aesar (Kandel, Germany).

Inorganic salts, sucrose, Tween 80, human serum albumin, and gamma globulin (IgGHum) (AppliChem GmbH, Darmstadt, Germany and Sigma–Aldrich, St. Louis, MO, USA) were used in this work. A commercial set of bovine blood serum samples was used (Biolot, Saint Petersburg, Russia).
All experiments were performed in ultrapure water prepared with Milli-Q equipment (Merck KGaA, Darmstadt, Germany). PBS was prepared from tablets from EcoService (Saint Petersburg, Russia). HEPES buffer, pH 7.5, from AppliChem (Darmstadt, Germany), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCl) from Roth (Karlsruhe, Germany), and sodium salt of N-hydroxysulfosuccinimide (s-NHS) from Chem-Impex Int’l (Wood Dale, IL, USA) were used.
Xeomin 50 Units per vial (Merz therapeutics, Frankfurt am Main, Germany) contains BoNT serotype A in human serum albumin with sucrose. The powder was dissolved in 125 µL Ringer’s solution (Grotex, Saint Petersburg, Russia) providing a solution with 400 Units/mL BoNT, 8 mg/mL human serum albumin, and 38 mg/mL sucrose. The solution was exploited immediately after the preparation. According to Frevert [21 (link)], 400 Units/mL of Xeomin corresponds to 1.76 ng/mL or 11.7 pM of BoNT serotype A.