Aminooxy-PEG3-methane: Properties, Uses & Lab Guide

Aminooxy-PEG3-methane is a short-chain polyethylene glycol reagent bearing a reactive aminooxy (–ONH₂) group and a terminal methane moiety, designed for selective oxime-bond formation with aldehyde- or ketone-containing biomolecules under mild aqueous conditions.

Product SKU

A596575

Chemical Class

Aminooxy-functionalized PEG (PEG3) linker

Reactive Group

Aminooxy (–O–NH₂) — reacts selectively with aldehydes and ketones to form stable oxime bonds

PEG Chain Length

PEG3 (three ethylene oxide repeat units)

Reaction Type

Bioorthogonal oxime ligation; proceeds efficiently at mildly acidic to neutral pH (4.5–7.0)

Key Application

Site-specific bioconjugation of peptides, proteins, glycans, and small molecules

Reference Catalog (Supplier)

BroadPharm BP-22985

Chemical Structure and Key Properties

Aminooxy-PEG3-methane belongs to the family of aminooxy-functionalized polyethylene glycol (PEG) linkers. Its backbone consists of three ethylene oxide (–CH₂CH₂O–) repeat units, providing a hydrophilic, flexible spacer between the reactive aminooxy head group (–O–NH₂) and the terminal methane (methyl) cap.

The aminooxy group is the chemically active component of this molecule. Unlike primary amines, the alpha-effect of the adjacent oxygen atom renders the nitrogen nucleophile substantially more reactive toward carbonyl compounds (aldehydes and ketones), enabling clean, selective condensation under mild conditions. The resulting oxime bond (–CH=N–O–) is hydrolytically stable over a broad pH range once formed, making it suitable for downstream biochemical assays.

The short PEG3 spacer contributes water solubility, reduces steric hindrance relative to bulkier PEG chains, and minimizes non-specific interactions with biological matrices. The methyl cap at the distal terminus renders that end chemically inert, ensuring reactivity is confined to the aminooxy group.

Bioconjugation Applications and Reaction Mechanism

Aminooxy-PEG3-methane is principally employed in bioorthogonal oxime ligation, a strategy that exploits the high chemoselectivity of aminooxy groups for aldehyde and ketone carbonyls. Because aldehydes and unmasked ketones are rare in native proteomes, this reaction can be conducted in complex biological mixtures with minimal off-target reactivity.

Typical applications include:

• Glycoprotein and glycan labeling: Periodate-oxidized carbohydrates present aldehyde handles that react efficiently with aminooxy reagents to form oxime-linked conjugates.
• Site-specific protein modification: Proteins engineered to carry an N-terminal glyoxylyl group or an unnatural keto-amino acid can be selectively modified at a single, defined site.
• Peptide conjugation: Synthetic peptides carrying aldehyde or levulinoyl tags are conjugated to payloads, surfaces, or carrier proteins via oxime ligation.
• Small-molecule and drug conjugation: The reagent serves as a PEGylating linker, attaching drug molecules bearing carbonyl groups to larger scaffolds while improving aqueous solubility.

The reaction proceeds by nucleophilic addition of the aminooxy nitrogen to the carbonyl carbon, forming a hemiorthoamide intermediate that rapidly dehydrates to yield the stable oxime. Mildly acidic conditions (pH 4.5–6.5) accelerate the reaction by protonating the carbonyl oxygen, increasing electrophilicity. Aniline or similar aromatic amine catalysts can be used to further accelerate the ligation at near-neutral pH.

Handling, Storage, and Practical Considerations

Storage: Store Aminooxy-PEG3-methane in a tightly sealed container under inert atmosphere (argon or nitrogen) at –20 °C to prevent oxidation of the aminooxy group and moisture uptake by the PEG chain. Allow the vial to equilibrate to room temperature before opening to avoid condensation. Aliquoting into single-use portions is recommended to minimize freeze-thaw cycles.

Solubility: The compound is readily soluble in water and common polar organic solvents such as dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). Prepare stock solutions in dry DMSO when maximum stability is required; aqueous stocks should be used promptly.

Reaction setup: For oxime ligation, combine the aminooxy reagent with the carbonyl-bearing substrate in an aqueous buffer at pH 4.5–6.5 (e.g., 100 mM sodium acetate or sodium phosphate). Use a molar excess of the aminooxy reagent (typically 5–20 equivalents relative to the carbonyl substrate) to drive the reaction to completion. Reaction times range from 1 hour to overnight depending on substrate concentration and temperature. Aniline (10–100 mM) may be included as a nucleophilic catalyst to accelerate ligation at pH values closer to neutral.

Quenching and purification: Excess reagent is readily removed by size-exclusion chromatography, dialysis, or ultrafiltration, owing to the size difference between the small linker and macromolecular substrates. For small-molecule applications, HPLC or solid-phase extraction is appropriate.

Safety: Handle in accordance with standard laboratory safety practices. Consult the Safety Data Sheet (SDS) for specific hazard information before use.

Advantages Over Alternative Bioconjugation Chemistries

Aminooxy-PEG3-methane offers several practical advantages compared with other bioconjugation strategies:

• High chemoselectivity: The aminooxy–carbonyl reaction is highly selective in the presence of amines, thiols, hydroxyls, and other common biological functional groups, unlike NHS ester or maleimide chemistries which can react with multiple nucleophiles.
• Stability of the oxime product: Oxime bonds are significantly more hydrolytically stable than hydrazone bonds formed by hydrazide reagents, making oxime conjugates better suited for in vivo or long-term in vitro applications.
• Mild, aqueous-compatible conditions: No harsh reagents, elevated temperatures, or organic co-solvents are required, preserving the integrity of sensitive biomolecules such as antibodies and enzymes.
• PEG3 spacer benefits: The short PEG linker improves water solubility of the conjugate, reduces immunogenicity, and adds conformational flexibility without introducing excessive steric bulk.
• Compatibility with metabolic labeling: Aminooxy reagents can capture ketone or aldehyde handles introduced into cell-surface glycans via unnatural sugar biosynthesis, enabling cell-surface glycan imaging and profiling.

Frequently asked questions

What is the optimal pH for oxime ligation using Aminooxy-PEG3-methane?

Oxime ligation with aminooxy reagents proceeds most efficiently at mildly acidic pH, typically in the range of 4.5–6.5. At this pH, protonation of the carbonyl oxygen increases its electrophilicity, accelerating the condensation reaction. At neutral or basic pH, the reaction rate decreases significantly, but nucleophilic catalysts such as aniline (10–100 mM) can compensate by forming a reactive imine intermediate that is more rapidly captured by the aminooxy group.

How should I store Aminooxy-PEG3-methane to maintain its reactivity?

Store the compound at –20 °C under an inert atmosphere (argon or nitrogen) in a tightly sealed container, protected from light and moisture. The aminooxy group is susceptible to oxidation upon prolonged air exposure. Aliquot the material into single-use portions upon first opening, and allow vials to warm to room temperature under inert gas before opening to prevent condensation-related degradation.

Can Aminooxy-PEG3-methane be used to label oxidized glycoproteins?

Yes. Periodate oxidation of cis-diol groups on glycoprotein carbohydrates generates aldehyde handles that are highly reactive toward aminooxy reagents. Aminooxy-PEG3-methane can be conjugated to periodate-oxidized glycoproteins in mildly acidic aqueous buffer to yield stable oxime-linked products. This strategy is widely used for site-specific attachment of labels, drugs, or other functional groups to the glycan portion of glycoproteins, preserving the protein backbone's integrity.

What is the difference between aminooxy and hydrazide reagents for carbonyl conjugation?

Both aminooxy and hydrazide groups react with aldehydes and ketones to form imine-type bonds—oxime and hydrazone, respectively—but oxime bonds are considerably more hydrolytically stable than hydrazone bonds across a broad pH range. This makes aminooxy reagents such as Aminooxy-PEG3-methane preferable when conjugate stability is critical, for example in in vivo applications or long-term storage. Hydrazone bonds can be stabilized by reduction with sodium cyanoborohydride, but this adds an extra processing step.

What molar ratio of Aminooxy-PEG3-methane to substrate is recommended?

A molar excess of 5- to 20-fold of Aminooxy-PEG3-methane relative to the carbonyl-bearing substrate is typically recommended to drive the reaction toward completion, given the reversible equilibrium nature of oxime formation. The optimal ratio depends on the specific substrate, reaction conditions, and acceptable levels of excess reagent. Excess linker can be removed after the reaction by size-exclusion chromatography, dialysis, or HPLC, depending on the application.

References

1. Dirksen A, Dawson PE. Rapid Oxime and Hydrazone Ligations with Aromatic Aldehydes for Biomolecular Labeling. Bioconjugate Chemistry. 2008;19(12):2543–2548. doi:10.1021/bc800310p
2. Zeng Y, Ramya TNC, Dirksen A, Dawson PE, Bhaskaran S. High-efficiency labeling of sialylated glycoproteins on living cells. Nature Methods. 2009;6(3):207–209. doi:10.1038/nmeth.1305
3. Kalia J, Raines RT. Hydrolytic Stability of Hydrazones and Oximes. Angewandte Chemie International Edition. 2008;47(39):7523–7526. doi:10.1002/anie.200802651
4. BroadPharm Product Listing: Aminooxy-PEG3-methane, Catalog No. BP-22985.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.