General Conjugation Protocols of PEG linkers——PEG ONH2
Polyethylene glycol (PEG) linker is a chemical sensing the carrier of polyethylene glycol (PEG), due to its solubility in water and non-immunogenicity. In the field of scientific research, it is widely used in chemical coupling, drug delivery, nanoparticles functionalized modifications and chemical biology. Its powerful function has attracted intense research interest. In the following, we will show the general conjugate reactions of nine common polyethylene glycol linkers one by one.
PEG ONH2
Introduction
Aminooxy PEG Linkers can be used in bioconjugation through the reaction of the aldehyde group with aminooxy to form aldoxime. It is much more stable than hydrazone and imine. In most of cases, it can be used directly without reduction while hydrazine and imines bond normally need be reduced to form stable C-N bond.
Alkoxyamines react with carbonyls most efficiently in amine-free, neutral conditions (pH 6.5-7.5). Carbonyls may exist at the reducing end of polysaccharides. To create additional carbonyls, oxidize sugar groups using either a specific oxidase, such as galactose oxidase, or 1-10 mM sodium meta-periodate or use Ald-PEG-NHS ester to introduce an aldehyde group. Oxidation with periodate is most efficient in acidic conditions (e.g., 0.1 M sodium acetate, pH=5.5), although neutral buffers such as phosphate- buffered saline can be used. If oxidation is performed in acidic conditions, buffer exchange by dialysis or gel filtration into neutral buffer might be necessary to obtain the optimal alkoxyamine reaction. Sometimes, aniline can be used to accelerate the coupling rate of hydrazide and alkoxyamine moieties with reactive aldehydes/ ketones (carbonyls).
- Figure 1.Oxime bond formation is a specific, bioorthogonal reaction thatoxime can be greatly accelerated by the use of suitable catalysts.
- a. Scheme for the formation of aldoximes, when an aminooxy compound reacts with an aldehyde.
- b. stability comparison of imine, hydrazone, and oxime.
- c. Common catalysts used to accelerate oxime bond formation.
- d. Recently
discovered low-toxicity catalysts for oxime bond formation.
Example protocol for labeling glycoproteins with an alkoxyamine-biotin reagent
Note: The optimal alkoxyamine-biotin concentration and reaction conditions depend on the specific protein and downstream application. For best results, empirically optimize the molar ratio of reagent and glycoprotein.
A. Materials required
1) Alkoxyamine-biotin Solution: 50 mM alkoxyamine-biotin reagent in DMSO. Prepare a volume sufficient to achieve the desired final concentration in step B.3.
Note: Alkoxyamine biotin reagents are hygroscopic solids that are difficult to weigh and dispense. To facilitate handling, make a 250 mM stock solution by dissolving the entire contents of the vial (50 mg) in a dry (anhydrous, molecular sievetreated) organic solvent such as DMSO. Store the stock solution at -20 °C for up to 1 month; warm the vial to room temperature before opening to prevent moisture condensation.
2) Oxidation Buffer: 0.1 M sodium acetate, pH 5.5.
3) Sodium meta-periodate at 20 mM in Oxidation Buffer: Prepare solution immediately before use in an amber vial or other light-protecting vessel.
4) Coupling Buffer: 0.1 M sodium phosphate, 0.15 M sodium chloride; pH 7.2 (PBS) or other neutral or slightly alkaline, non-amine buffer.
5) Glycoprotein at 2 mg/mL in Oxidation Buffer.
6) Dialysis cassette or desalting column.
B. Procedure
1) Add 1 mL of cold sodium meta-periodate solution to 1 mL of cold glycoprotein solution and mix well. Incubate mixture for 30 minutes on ice or at 4 °C protected from light.
Note: To oxidize only sialic acid groups, add 50 μL of sodium meta-periodate instead of 1 mL, which results in 1 mM periodate final concentration rather than 10 mM.
2) Remove excess sodium meta-periodate and exchange the buffer by dialysis against coupling buffer or with a desalting column equilibrated with coupling buffer.
3) Add 1 part of Alkoxyamine-biotin Solution to 9 parts oxidized protein post buffer-exchange (results in 5 mM alkoxyamine biotin). Mix the reaction for 2 hours at room temperature.
4) Separate the biotinylated protein from non-reacted material by dialysis or desalting. Store the biotinylated protein using the same conditions as for the non-biotinylated sample.