Vivaspin turbo

Triethylamine (3.0 equiv)
was added to a 9:1 DMSO/water solution of hexasaccharide followed
by di-N-hydroxysuccinimidyl adipate (12 equiv.).
The reaction was stirred for 3 h, then the product was precipitated
at 0 °C by adding ethyl acetate (9 volumes). The solid was washed
10 times with ethyl acetate (5 volumes each) and lyophilized. The
activated sugar was conjugated to CRM197 or HSA in sodium phosphate
100 mM at a protein concentration of 20 mg/mL, using the saccharide/protein
molar ratio of 75:1. After incubating overnight, the glycoconjugate
was purified by dialysis against 10 mM sodium phosphate buffer pH
7.2 (30 washings) in 30 kDa Vivaspin Turbo (Sartorius) centrifugal
concentrators and reconstituted in the same buffer. Protein content
in the purified glycoconjugates was determined by micro-BCA (Thermo-scientific).
The saccharide content was estimated by CE-MS analysis.

The production of Cys and HCys generated upon α,γ‐ and α,β‐elimination of Cth, respectively was analyzed through reverse phase HPLC (Conter et al., 2020 (link)). A 500 μL reaction mixture containing 0.1–8 mM Cth and 30 μM enzyme variants in 50 mM HEPES pH 8.0 was incubated at 37°C for 60 min. Where indicated 0.05–0.5 mM PPG was incubated with the enzyme for 30 min at 25°C. The enzyme was removed from the reaction mixture by centrifugation using a Vivaspin Turbo centrifugal concentrator (10 kDa cutoff, Sartorius). Dansyl chloride (stock 1.5 mg/mL in acetonitrile) was then reacted with the amino acids in the filtered solution following the protocol in Mothersole and Wolthers (2019 (link)) and Tapuhi et al. (1981 (link)). Following a 30‐min incubation at 25°C, the reaction was quenched with 30 μL of pyridine 4% and 20 μL of the mixture was injected onto a C‐18 column (Agilent Poroshell 120 HPH RP‐C18, 4 μm, 4.6 × 250 mm). The dansylated products were eluted at a flow rate of 1 mL/min at 40°C with a mobile phase of 33.25/66.75 (vol/vol) methanol/water containing 0.008% (vol/vol) triethylamine and 0.6% (vol/vol) glacial acetic acid. The fluorescence detector was set at 335 and 522 nm for excitation and emission wavelengths, respectively.