Site-Specific Glycoconjugation of Protein via Bioorthogonal Tetrazine Cycloaddition with a Genetically Encoded trans-Cyclooctene or Bicyclononyne

Graphical Abstract


General procedures
Azido glycan 1 were prepared according to the procedure reported by Shoda et al. [2] Briefly, a mixture of reducing glycan 4, Et 3 N, 2-chloro-1.3-dimethylimidazolium chloride (DMC), NaN 3 , in 1,4-dioxane/ H 2 O=1/1 was stirred for 1 h at -10 ºC. The solvent was evaporated and the residue was purified directly by column chromatography. In some cases, it was found to be more practical to transiently acetylate the product for purification purposes. For convenience, the reactions were also performed in D 2 O which facilitates reaction monitoring by NMR. In a 2.0ml Eppendorf tube, glycan 4, NaN 3 , 2-chloro-1.3-dimethylimidazolium chloride (DMC), and Et 3 N or 2,6-lutidine were added to D 2 O. The mixture was shaken vigorously until the mixture became homogeneous. The mixture was then transferred to an NMR tube and the progress was monitored by H 1 -NMR with intermittent shaking. Upon completion, the reaction was transferred back to the Eppendorf and lyophilized.
To the residue was added Ac 2 O (300 µl) and pyridine (1 ml) and the reaction mixture was stirred overnight at room temperature. The reaction was concentrated in vaccuo and the product was isolated by reverse phase chromatography (12g of C18 column, 50 ml of H 2 O, then 100 ml of acetonitrile). The acetonitrile fraction was concentrated in vacuo then lyophilized. To the residue was added sodium methoxide (6 mg, 0.1 mmol) and dry methanol. The reaction mixture was stirred overnight under nitrogen atmosphere. The reaction mixture was neutralized with acidic resin (DOWEX ® 50WX2), filtered and lyophilized.
After filtration, the mixture was concentrated in vacuo and the residue was purified by reverse phase column (12g of C18 column, flowed 200 ml of H 2 O ). Lyophilizing solvent gave desired azide-suger.
The mixture was then tra 1 M NaOH (100 µl) and stirred 3 h.

L-Fucopyranosyl azide (1d)
L-Fucose (48 mg, 0.3 mmol), DMC (153 mg, 0.9 mmol), NaN 3 (291 mg, 4.5 mmol), triethylamine (375 µl, 2.7 mmol) in deutrium oxide (1.2 ml) were shaken for 1 h at room temperature. The solvent was evaporated in vacuo and to the residues were treated with Ac 2 O (900 µl) and pyridine (3 ml). After stirring overnight at room temperature, the solvent was removed in vacuo and the residues were purified by silica gel column chromatography  , triethylamine (208 µl, 1.5 mmol) in deutrium oxide (500 µl) were shaken for 1 h and lyophilized. To the residue was added Ac 2 O (300 µl) and pyridine (1 ml) and the reaction mixture was stirred overnight at room temperature. The reaction was concentrated in vaccuo and the product was isolated by reverse phase chromatography (12g of C18 column, 50 ml of H 2 O, then 100 ml of acetonitrile). The acetonitrile fraction was concentrated in vacuo then lyophilized. To the residues was added sodium methoxide (6 mg, 0.1 mmol) and dry methanol. The reaction mixture was stirred overnight under nitrogen atmosphere. The reaction mixture was neutralized with acidic resin (DOWEX ® 50WX2), filtered and lyophilized to afford β-D-cellobiosyl azide 1h (30 mg, 82%) as colorless solid. Spectral characteristics of this compound matched the reported data.  To the residue was added Ac 2 O (50 µl) and pyridine (150 µl) and the reaction mixture was stirred overnight at room temperature. The reaction was concentrated in vaccuo and the product was isolated by reverse phase chromatography (1g of C18 column, 5 ml of H 2 O, then 10 ml of acetonitrile).

Fucα1-2Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc-1N 3 (1j)
The acetonitrile fraction was concentrated in vacuo then lyophilized. To the residues was added sodium methoxide (2 mg) and dry methanol (100 µl).  pyridine (450 µl) and the reaction mixture was stirred overnight at room temperature. The reaction was concentrated in vaccuo and the product was isolated by reverse phase chromatography (2g of C18 column, 10 ml of H 2 O, then 20 ml of acetonitrile). The acetonitrile fraction was concentrated in vacuo then lyophilized. To the residues was added LiOH (2 mg) and methanol:water (1:1, 100 µl) and stirred overnight. The reaction mixture was neutralized with acidic resin (DOWEX ® 50WX2), filtered, washed with water and lyophilized to afford

Synthesis of trans-cyclooctene derivative S7 for kinetic study (Z)-cyclooct-4-en-1-ylmethanol (S7)
Compound S6 [5] (1.1 g, 8.15 mmol) in CH 2 Cl 2 (33 ml) was treated dropwise with diisobutylalminium hydride (1.0 M in toluene, 12.2 ml, 12.2 mmol) at -78 ºC and stirred for 1 h under nitrogen atmosphere. The reaction mixture was warmed up to 0 ºC and 2 M HCl (20 ml) was added dropwise to the reaction mixture. The mixture was stirred for another 30 min at room temperature then the organic compounds were extracted with ethyl acetate, washed with brine, dried over Na 2 SO 4 , filtrated and concentrated in vacuo. The residue were taken up

(E)-(cyclooct-4-en-1-ylmethoxy)triethylsilane (S9)
According to the previously reported procedure, [7] to chlorodiphenylphosphine (723 µl, 4.0 mmol) in dry THF (5 ml) was slowly added lithium wire (62 mg, 8.8 mmol) washed in dry THF (5 ml). The mixture was stirred vigorously at room temperature overnight to give dark red lithium diphenylphosphide solution. This solution was added to S8 (471 mg, 1.74 mmol) dissolved in a few drops of THF and the reaction mixture was stirred for 2 d at room

S9
H H OTES temperature (the color changed to pale yellow). The reaction mixture was cooled to 0 ºC and AcOH (251 µl, 4.4 mmol) followed by 35% H 2 O 2 (466 µl, 4.8 mmol) were slowly added to the stirring for 1 h at room temperature. The organic compounds were extracted with CH 2 Cl 2 , washed with brine, dried over Na 2 SO 4 , filtrated and concentrated in vacuo. To the residue dissolved in DMF (10 ml) were added to NaH (<60%, 209 mg, 5.22 mmol, washed with dry hexane before using) and stirred for 1 h at room temperature. The reaction was quenched with saturated NH 4 Cl and the organic compounds were extracted with ethyl acetate, washed with brine, dried over Na 2 SO 4 , filtrated and concentrated in vacuo. Purification by silica gel column chromatography (pentane : ethyl acetate = 10 : 1) gave S9 (113 mg, 26%, mixture of atropisomers) as colorless oil.

Kinetic study of tetrazine-TCO cycloaddition
Kinetic studies of tetrazine-trans-cyclooctene (TCO) cycloaddition were performed by monitoring the change in CY3 fluorescence resulting from the cycloaddition [8] (Figure SI1).

Protein conjugation with purified sfGFP-TCOK
Purified sfGFP-BCNK were prepared according to published protocols. [8] To the solution of sfGFP-BCNK in Tris buffer pH 7.4 (13.5, 1, 0.1 µM, respectively) was added tetrazine-glycan at 10 equivalents (entry 1) or 5 equivalents (entry 2-4) and the solution was incubated for 10 min at room temperature with gentle agitation on an orbital shaker. The reaction was quenched by addition of 100 eq. of TCO-OH S10 and the product was analyzed by MALDI-TOF. The same procedure was performed for azide-glycan (entry 5) for 10 min and 3 hours and the reaction was quenched by addition of 100 eq. of tetrazine 2 instead of TCO-OH S10.

Protein modification of E. coli expressing sfGFP-TCOK
E. coli pellet (50 µl) expressing sfGFP-TCOK or sfGFP-BocK were obtained as previously described from 3 mL of culture. [8] The pellets were washed 3x with PBS to remove excess TCOK or BOCK. An aliquot of each E. coli pellet (15 µl) were treated with 100 µl of a 25 µM solution of 3-GluNAc-Cy3 in PBS buffer and incubated for 8h at room temperature with gentle agitation on an orbital shaker. The E. coli were re-pelleted by centrifugation and washed twice with PBS (centrifuge and discard supernatant). Then, 30 µl of LDS buffer was added to the pellet and the mixture was incubated for 10 min at room temperature. The