Synthesis of cholera toxin B subunit glycoconjugates using site-specific orthogonal oxime and sortase ligation reactions

The chemoenzymatic synthesis of a series of dual N- and C-terminal–functionalized cholera toxin B subunit (CTB) glycoconjugates is described. Mucin 1 peptides bearing different levels of Tn antigen glycosylation [MUC1(Tn)] were prepared via solid-phase peptide synthesis. Using sortase-mediated ligation, the MUC1(Tn) epitopes were conjugated to the C-terminus of CTB in a well-defined manner allowing for high-density display of the MUC1(Tn) epitopes. This work explores the challenges of using sortase-mediated ligation in combination with glycopeptides and the practical considerations to obtain high levels of conjugation. Furthermore, we describe methods to combine two orthogonal labeling methodologies, oxime- and sortase-mediated ligation, to expand the biochemical toolkit and produce dual N- and C-terminal–labeled conjugates.


Imidazole-sulfonyl-azide hydrogen sulfate salt (S1)
Imidazole-1-sulfonyl azide is highly explosive in its neutral form. Reaction should only be performed behind Perspex blast shield and no attempt to concentrate solutions of imidazole-1sulfonyl azide should be made. Sodium azide and imidazole were dried overnight (12-14 hrs) in a vacuum desiccator over phosphorous pentoxide prior to use. Extra dry EtOAc over molecular sieves was purchased from Acros Organics; a fresh bottle was used for each reaction.
Sodium azide (5.00 g, 77 mmol, 1.0 eq) was placed in a dry 500 mL three neck RBF with a dry stirrer bead. Extra dry EtOAc (77 mL) was added to the flask and the resulting suspension cooled to 0 °C. Sulfuryl chloride (6.2 mL, 77 mmol, 1.0 eq) was added dropwise over 5 minutes and the mixture allowed to warm to room temperature and stirred for 24 hrs. The suspension was recooled to 0 °C and imidazole (10.00 g, 146 mmol, 1.9 eq) was added continuously over 5 minutes. The suspension was stirred at RT for 3 hrs. The mixture was basified by the addition of sat. aq. NaHCO3 solution (150 mL). Once bubbling had ceased, the mixture was separated, the organic portion washed with water (150 mL), dried over MgSO4 and filtered. Filtrate was recooled to 0 °C and placed under N2 atmosphere. Conc. H2SO4 (4.1 mL, 77 mmol, 1.0 eq) was added dropwise over the course of 5 minutes and gradually warmed to room temperature with vigorous stirring. Over the course of 30 minutes, a colourless precipitate formed and was collected by vacuum filtration. Precipitate was washed with a small amount of ice-cold EtOAc and the crystals were dried under high vacuum to yield imidazole-1-sulfonyl azide hydrogen sulfate salt, S1 (12.71 g, 46.8 mmol, 61%).
NMR data is in agreement with reported data (Potter et al., 2016).
NMR data is in agreement with reported data for the α-anomer (Koeller et al., 2000).
NMR data is in agreement with reported data (Koeller et al., 2000).
NMR data is in agreement with reported data (Koeller et al., 2000).
NMR data is in agreement with reported data (Koeller et al., 2000).

Solid Phase Peptide Synthesis (SPPS)
General Reagents & Equipment for SPPS All amino acids, resins and coupling reagents were purchased from Novabiochem or Fluorochem and used without further purification. Fritted polypropylene tubes (2.5 mL) were purchased from Supelco (Merck) and were used for all solid phase reactions. Agitation of solid phase reaction mixture was achieved by rotation on a Stuart blood rotator at room temperature.

General procedure for Solid Phase Glycopeptide Synthesis
Low loading Rink Amide MBHA resin (loading: 0.33-0.35 mmol/g, 100 µmol) was shaken in DMF (5 mL) for 2 hours, filtered and washed with DMF. Fmoc deprotectionresin bound peptide intermediates were shaken in 20% piperidine in DMF (3 × 5 mL × 3 min) and washed with DMF, CH2Cl2 then DMF. Couplingsthe resin was treated with a 5-fold excess of the Fmoc-amino acid (except for the Fmoc-Ser/Thr(GalNAc)-OH which was employed in a 2-fold excess) in 2 mL DMF containing HCTU (5 equiv.) and DIPEA (10 equiv). The mixture was shaken and coupling times were 40 mins (except for the Fmoc-Ser/Thr(GalNAc)-OH and the subsequent coupling each of which were 12 hours in duration). The excess reagents removed by filtration and the resin washed with DMF, CH2Cl2 then DMF. O-Acetyl Deprotection -Following coupling of the final amino acid, resin was washed with methanol, shaken in 70% hydrazine hydrate in methanol (3 × 5 mL × 5 min) and washed with methanol. Glycopeptide Cleavage -Following Fmoc and O-Acetyl deprotection, the resin was washed with CH2Cl2 and then methanol, before drying under vacuum. The resin was suspended in TFA:H2O:TIS (95:2.5:2.5, 5 mL) and shaken for a maximum of 2 hours. The mixture was filtered and washed with TFA. The solution was concentrated under a stream of N2 to <1 mL. Glycopeptide obtained by ether precipitation, isolated by centrifugation and pellet further washed with ether. The white precipitate was dissolved in H2O and lyophilized to give white foams.

General Biochemical Methods and Equipment
Sterilisation of media and equipment was carried out in either a Prestige Medical bench top autoclave or an LTE Touchclave-R autoclave at 121 ºC for 15 minutes. Thermo Electron Corporation SAFE 2010 Class II laminar flow cabinet or bench top bunsen burner were using to maintain a sterile environment. Bacterial cultures were incubated in a Kuhner ShakerX ISF1-X or Stuart Orbital incubator. LB-agar plates and enzymatic reactions were incubated in a Binder BD23 incubator.
Centrifugation was performed using either a Beckman Coulter TM Avanti TM JXN-30 centrifuge, Heraeus mutifuge 3S-R centrifuge or Heraeus pico centrifuge. Gel filtration chromatography was achieved using a GE Pharmacia ÄKTA FPLC system or BioRad NGC FPLC system. Spectrophotometric readings were recorded using a Thermoscientific Nanodrop 2000. SDS-PAGE was carried out using Bio-Rad mini protean 3 apparatus, and a Bio-Rad imager Gel Doc TM XR was used to visualise polyacrylamide gels. Proteins were concentrated either using 10k or 30k MWCO Amicon® Ultra-15 centrifugal filter device or Amicon® Stirred Ultrafiltration Cell (10k MWCO membrane). High resolution mass spectrometry of protein samples was performed using a Bruker Daltonics MicroTOF mass spectrometer. Protein samples were loaded at a concentration of 20-40 µM (made up with H2O) into the instrument before being automatically diluted into 0.1% TFA/50% MeCN (v/v) in H2O prior to analysis. Analytical grade reagents were supplied by commercial suppliers.

Media & Buffers
Buffers and media were made in-house unless otherwise stated using analytical grade reagents supplied by commercial suppliers. All common buffers and media were prepared with 18.2 MΩ water to the required volume. The pH of the solutions was adjusted using 5 M NaOH or 5 M HCl. Gel filtration buffers were filtered through a 0.22 µm membrane under reduced pressure. Media was sterilised by autoclave at 121 °C for 20 minutes.

Expression of CTB-LPETGA
A glycerol stock of C41 cells containing the mutant plasmid pSAB2.2-LPETGA was used to inoculate 2 x 5 ml of LB media containing 100 µg/ml ampicillin. These cultures were incubated at 37 °C overnight, shaking at 200 rpm and then used to inoculate 4 x 1 L of LB media containing 100 µg/ml of ampicillin. The culture was incubated at 37 °C with shaking (200 rpm) and overexpression by the addition of IPTG (0.5 mM) when OD600 = 0.6-0.8 was reached. The cells were incubated overnight at a 25 °C before being collected by centrifugation (10,000 × g, 20 minutes, 4 °C). The E. coli cell pellet was discarded and media retained. Solid ammonium sulfate was added and dissolved to a final concentration of 57% (w/v) to precipitate the protein of interest. The saturated solution was stirred overnight at 4 °C. The solution was centrifuged (17,600 × g, 1 hour, 4 °C), and the supernatant discarded. The protein pellet was resuspended in sodium phosphate buffered saline (10 mL per 1 L LB media) and centrifuged (17,600 × g, 10 minutes, 4 °C) to remove insoluble material and to allow the suspension to be filtered through a 0.8 µm Sartorius Minisart filter.
The protein was purified by Ni-NTA affinity chromatography washing with sodium phosphate buffered saline (no imidazole). CTB has native surface histidine residue meaning the pentamer is able to bind to Ni-NTA resin without the presence of a His-tag. The protein was then eluted from the column by washing with sodium phosphate buffered saline containing 200 mM imidazole, before further purification by size exclusion chromatography on Superdex S75 in sodium phosphate buffered saline.
A glycerol stock of BL21(DE3) cells containing the mutant plasmid pET30b-Srt7M (Wuethrich et al., 2014) or pET28a-CBD-Srt7M was used to inoculate 5 ml of LB media containing 50 µg/ml kanamycin. These cultures were incubated at 37 °C overnight, shaking at 200 rpm and then 2 mL used to inoculate 1 L of autoinduction-LB medium (Studier, 2005) containing 100 µg/ml of kanamycin. The culture was incubated at 37 °C with shaking (200 rpm) for 5 hours then the incubator temperature was reduced to 25 °C. The cells were incubated overnight before being collected by centrifugation (13,000 × g, 20 minutes, 4 °C). Cells were either used immediately or stored at -80 °C for future lysis and purification.
E. coli cells were resuspended in Tris buffered saline at 4 °C and treated with DNase (10 µg/mL) and protease inhibitors. The cell mixture was lysed mechanically using a Constant Systems cell disruptor (20 kpsi). The cell debris was pelleted by centrifugation (30,000 × g, 45 mins, 4 °C) and the cell lysate was decanted off. Purification of the clarified lysate performed by Ni-NTA affinity chromatography washing with Tris buffered saline containing 50 mM imidazole. The protein was then eluted from the column by washing with Tris buffered saline containing 250 mM imidazole, before further purification by size exclusion chromatography on Superdex S75 in Tris buffered saline. Sortase 7M & CBD-Sortase 7M was stored at -80 °C containing 10% (v/v) glycerol.