Bis-sulfone-PEG8-NHS Ester - ≥95%

Cat. No.: B597104

GRADE & PURITY ≥95%

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Application

227,228,229

Panoramica

Bis-sulfone-PEG8-NHS Ester is a bis-alkylating labeling reagent that is selective for the cysteine sulfur atoms from a native disulfide. These reagents undergo bis-alkylation to conjugate both thiols derived from the two cysteine residues of a reduced native disulfide bond such as the interchain disulfide bonds of an antibody. The reaction results in covalent rebridging of the disulfide bond via a three carbon bridge leaving the protein structurally intact. The hydrophilic PEG spacer increases solubility in aqueous media.

Specifications

Specifiche e purezza

≥95%

Condizioni di conservazione di stoccaggio

Store at -20°C

Spedito in

Ice chest + Ice pads

Questo prodotto richiede spedizione a catena fredda. I servizi di terra e altri servizi economici non sono disponibili.

Purezza

≥95%

Application Protocols

Example procedures are provided as general guidance. Optimize for your system and consult the CoA/Spec Sheet for any item-specific parameters.

• Protocol A: NHS installation on an amine-bearing protein (literature/general)

  1. Dissolve protein in 50 mM phosphate buffer, pH 7.8–8.2, 150 mM NaCl.
  2. Prepare a 20–50 mM linker stock in anhydrous DMSO immediately before use.
  3. Add linker to protein to achieve 2–5 molar eq per accessible amine; keep final DMSO ≤10%.
  4. Incubate 30–60 min at room temperature with gentle mixing.
  5. Quench unreacted NHS by adding Tris (final 20 mM) or proceed directly to purification by desalting/SEC.

• Protocol B: Disulfide rebridging on a reduced protein/peptide (literature/general)

  1. Reduce target disulfide with 1–5 mM TCEP in pH 7.0–7.5 buffer for 15–30 min.
  2. Remove excess TCEP (spin column/desalting).
  3. Add bis-sulfone-PEG8–NHS linker-modified intermediate or the linker directly if strategy permits; maintain pH 7.2–8.0.
  4. Incubate 1–2 h at 20–25°C. Monitor by LC–MS or non-reducing SDS-PAGE.
  5. Purify conjugate by SEC or dialysis.

• Protocol C: Surface coupling to aminated beads/films (literature/general)

  1. Equilibrate surface in 0.1 M bicarbonate buffer, pH 8.3.
  2. Add linker from dry DMSO stock to 1–10 mM final; incubate 30–60 min.
  3. Rinse thoroughly; cap remaining amines with ethanolamine; proceed to thiol-capture step.

Note: Calculate molar amounts using the lot-specific molecular weight from the CoA.

Biological Roles

This is a synthetic bioconjugation reagent and does not have inherent physiological roles.

• Not a metabolite, cofactor, or signaling molecule. Its function is purely chemical: to form covalent bonds between biomolecules/materials bearing suitable reactive groups (general).
• In biochemical workflows (literature/general):
  • NHS ester end acylates primary amines on proteins/peptides, nucleic acids modified with amines, or amine-functional surfaces.
  • Bis-sulfone end engages cysteine thiols—often those generated by selective reduction of native disulfide bonds—to create robust thioether bridges that can restore structural integrity (rebridging) while adding functionality.
• PEG8 spacer (general):
  • Increases hydrophilicity and can reduce nonspecific interactions and aggregation during conjugation steps. Also provides distance to mitigate steric hindrance between coupled partners.

Note: All uses are for research and laboratory applications only; no medical or clinical claims are made.

Buffer Applications

Buffer choice is critical for controlling NHS ester reactivity and bis-sulfone thiol additions.

• NHS–amine coupling (literature/general):
  • pH: 7.5–8.5. Rate increases with pH but hydrolysis also accelerates above ~8.5; optimal window is typically 7.8–8.3.
  • Recommended buffers: Sodium phosphate, bicarbonate/carbonate, HEPES. Avoid primary amine buffers (Tris, glycine) and high concentrations of competing nucleophiles.
  • Additives: 0.1–0.5 M NaCl can improve solubility/ionic strength; keep organic co-solvent (DMSO/DMF) at the minimal level compatible with substrate stability.
• Thiol coupling/rebridging (literature/general):
  • pH: 7.0–8.5 in phosphate or HEPES. Maintain degassed buffers to limit thiol oxidation.
  • Reducing agents: Use TCEP to open disulfides; remove or quench excess prior to addition of the bis-sulfone reagent.
• Practical notes:
  • Prepare linker stocks in anhydrous solvent immediately before use; add to buffered solution last.
  • Control reaction time and temperature (e.g., 30 min to several hours at room temperature) to balance conversion and hydrolysis.

These are general guidelines; optimize for each substrate’s stability and reactivity.

Green Alternatives

Greener practice centers on solvent choice, aqueous compatibility, and minimizing protecting-group manipulations.

• Solvent considerations (general):
  • Prefer water-rich media with minimal DMSO/DMF when feasible; use acetonitrile over DMF where solubility permits due to lower toxicity and easier removal.
  • Employ micro-scale, high-concentration stocks to reduce total aprotic polar solvent use.
• Alternative chemistries (literature/general):
  • Sulfo-NHS variants of PEG linkers: Improved water solubility enables higher aqueous content, reducing reliance on organic co-solvents; tradeoff is potentially faster hydrolysis.
  • Thiol-selective handles such as pyridazinediones or dibromopyridazinediones can enable disulfide rebridging under aqueous conditions with good selectivity; availability and synthesis complexity may differ.
  • Enzymatic or bio-orthogonal approaches (e.g., transglutaminase, sortase A, SPAAC/DBCO–azide) can eliminate reducing agents but require engineered handles.
• Process improvements:
  • Use bicarbonate or phosphate buffers (pH 7.8–8.3) at ambient temperature to limit energy input and maintain NHS activity.
  • Implement in-line desalting or spin filtration to minimize organic solvent waste in workup.

Comparison snapshot (general):

• This product: High efficiency, broad compatibility; needs dry aprotic stock solutions and careful timing to avoid NHS hydrolysis.
• Sulfo-NHS analogs: More aqueous-friendly; faster hydrolysis demands just-in-time preparation.
• Maleimide linkers: Efficient thiol coupling; less stable to thiol exchange vs rebridged bis-sulfone systems.

Pharmaceutical Uses

No therapeutic or clinical use is claimed. In a research and development context, Bis-sulfone-PEG8-NHS Ester can be employed as a process reagent for preparing and characterizing conjugates.

• R&D formulation/manufacturing context (general):
  • Preparation of protein–polymer conjugates and prototype antibody/protein constructs to study conjugation patterns, stability, and analytics.
  • Surface activation of amine-bearing medical-material analogues for in vitro evaluation of ligand immobilization strategies.
  • Payload installation in model systems for analytical method development (e.g., LC–MS mapping of conjugation sites).
• Pharmacopeia status: Not specified for this item; refer to CoA/Spec Sheet. PEGylated heterobifunctional linkers of this class are typically research grade unless otherwise stated.
• Excipient role: None stated. Any use is for laboratory research only.

All applications must remain within non-clinical research; suitability for GMP or clinical manufacturing is not implied unless supported by specific documentation.

Physical Properties

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
• Melting point/Range: Not specified for this item; refer to CoA/Spec Sheet.
• Boiling point: Not applicable; decomposes before boiling (general for PEGylated activated esters; literature/general).
• Density: Not specified for this item; refer to CoA/Spec Sheet.
• Refractive index: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility (general/literature)
  • Organic: Typically soluble in anhydrous DMSO, DMF, acetonitrile; limited solubility in dichloromethane/THF may occur depending on exact substituents.
  • Aqueous: PEG8 improves dispersion in buffered aqueous media when pre-dissolved in DMSO/DMF (5–20% v/v co-solvent). NHS ester hydrolyzes in water—use promptly after dissolution (literature/general).
• pKa/logP: Not defined for the entire conjugate; effective behavior is amphiphilic due to PEG chain and polar end-groups (literature/general).
• Stability (general)
  • NHS ester: Moisture- and base-sensitive; hydrolyzes to carboxylate in water (faster at pH > 8.5). Store dry, cold.
  • Bis-sulfone: Forms a vinyl sulfone under mild base; reacts with thiols. More stable than maleimides toward retro-Michael exchange once rebridged (literature/general).

Note: Item-specific numeric specifications (e.g., water content, UV cutoff, metals) are not specified for this item; refer to CoA/Spec Sheet.

Quality and Grades

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
• What to look for in this class of reagents (general guidance):
  • Identity confirmation by 1H/13C NMR and HRMS (where applicable) and purity by HPLC/UPLC are common. For PEGylated linkers, single-species distribution is preferred over broad oligomer mixtures.
  • Low residual solvents and controlled water content improve shelf-life of the NHS ester.
  • Peroxide testing is generally more relevant to ethers; not typically critical here unless stored in peroxide-forming solvents.
• Stabilizers: None specified for this item; refer to CoA/Spec Sheet. NHS esters are usually packaged under inert gas with desiccant to minimize hydrolysis (general practice).
• Application implications:
  • Bioconjugation workflows (protein, peptide, nanoparticle, and surface modification) benefit from high chemical purity and narrow PEG dispersity to ensure reproducible spacing and loading.
  • If performing analytical separations post-conjugation, low UV background and minimal non-volatile impurities aid characterization.
• Documentation: For lot-specific assay values, residual solvents, and moisture, consult the CoA/Spec Sheet.

Reaction and Applications

This linker is designed for chemoselective bioconjugation, combining amine-coupling via NHS ester with cysteine-specific disulfide rebridging via a bis-sulfone.

• Application families (literature/general):
  • Protein modification: Attach the linker to lysine residues (NHS), then rebridge reduced disulfides on the same or another protein/peptide through the bis-sulfone, forming stable thioether linkages.
  • Disulfide rebridging: Following partial reduction of a native disulfide (e.g., with TCEP), the bis-sulfone evolves to a vinyl sulfone that reacts with two thiols sequentially, restoring connectivity with improved stability relative to maleimide adducts.
  • Surface and polymer functionalization: NHS attachment to amine-functional materials (e.g., PAMAM dendrimers, aminated silica, polymer films) leaving a cysteine-reactive handle for downstream capture of thiol-containing ligands.
  • Nanoparticle/biomaterials coupling: Introduce site-specific thiol-reactive points on amine-coated nanoparticles or hydrogels.
• Practical tips:
  • Sequence control: Perform NHS coupling first under mildly basic, anhydrous or mixed aqueous-organic conditions; purify; then conduct thiol addition under pH 7–8.5.
  • Reducing agents: Use TCEP for disulfide opening; remove excess before bis-sulfone addition to avoid side consumption.
  • Stoichiometry: 1.1–5.0 equivalents relative to accessible amines/thiols is commonly employed; optimize to control loading (literature/general).
  • Monitoring: Track NHS consumption by TNBS assay for free amines; follow thiol conversion by Ellman’s reagent or LC-MS of the construct.
• Advantages (general): Enhanced hydrolytic stability of the rebridged thioether and defined spacing from PEG8 can improve conjugate solubility and reduce aggregation.

Reaction Conditions

Typical conditions are summarized as general literature guidance; adjust to the specific substrate and scale.

• NHS–amine coupling (literature/general):
  • Solvent: Anhydrous DMSO or DMF stock; add to phosphate or bicarbonate buffer (pH 7.8–8.3). Avoid primary amine buffers.
  • Temperature: 20–25°C.
  • Time: 15–120 min depending on substrate accessibility; monitor by depletion of free amines (e.g., TNBS).
  • Stoichiometry: 1.2–5.0 eq linker per accessible amine. Excess accelerates reaction but can overlabel.
• Disulfide rebridging with bis-sulfone (literature/general):
  • Pre-treatment: Reduce target disulfide with TCEP (e.g., 0.5–5 mM) at pH 7–7.5, 15–60 min; remove excess reducing agent (desalting/spin column).
  • Reaction: Add linker (or a pre-installed linker-bearing intermediate) under pH 7.0–8.5 in phosphate/HEPES; optional slight base (e.g., 10–50 mM) facilitates formation of the vinyl sulfone.
  • Temperature: 20–25°C; sensitive proteins may prefer 4–10°C with longer times.
  • Time: 0.5–4 h, often complete within 1–2 h for accessible sites.
• Workup and purification (general):
  • Proteins: Desalt or size-exclusion chromatography to remove excess linker and small-molecule byproducts.
  • Small molecules/polymers: Precipitation into ether or chromatography (reverse-phase for PEGylated constructs) as appropriate.

Expected conversions and yields are system-dependent; verify by LC–MS, SDS-PAGE, or SEC as applicable.

Safety and Handling

• GHS classification, signal word, pictograms, and H-statements: Not specified for this item; refer to SDS.
• Anticipated hazards (general):
  • NHS esters are acylating agents and can irritate skin/eyes and respiratory tract; avoid contact and inhalation.
  • Bis-sulfone electrophiles can alkylate thiols; handle with care and avoid exposure to reducing agents that generate free thiols unintentionally.
• PPE recommendations (general best practice):
  • Use lab coat, nitrile gloves (change frequently), and safety glasses; handle powders/solutions in a fume hood.
• Handling notes:
  • Keep container tightly closed under dry inert atmosphere. Open only after equilibration to room temperature to minimize condensation.
  • Prepare solutions with anhydrous solvents (DMSO, DMF, acetonitrile). Filter-sterilize through 0.22 µm if sterility is required for sensitive applications.
• Incompatibilities (general):
  • Avoid strong bases/acids, nucleophiles (amines, thiols) unless intended; avoid prolonged aqueous exposure at elevated pH due to NHS hydrolysis.
  • Reducing agents (DTT, TCEP) liberate thiols; combine only under controlled conjugation workflows.
• First aid overview (general):
  • Skin/eye contact: Rinse with water for 15 minutes; remove contaminated clothing; seek medical attention.
  • Inhalation: Move to fresh air; seek medical attention if symptoms persist.
  • Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.

Always consult the product’s SDS for authoritative safety, disposal, and spill response information.

Solvent Selection

Choosing solvents for Bis-sulfone-PEG8-NHS Ester balances NHS stability, solubility, and compatibility with the target biomolecule or surface.

• Primary dissolution (general):
  • Anhydrous DMSO or DMF: Excellent solubility and NHS stability; prepare concentrated stocks (e.g., 10–50 mM) and use immediately.
  • Acetonitrile: Often suitable; may require gentle warming for complete dissolution.
• Aqueous use:
  • Introduce into buffered aqueous media by rapid dilution from a dry organic stock. Maintain 5–20% v/v DMSO/DMF co-solvent if target tolerates it.
  • For NHS-amine coupling, use pH 7.5–8.5 buffers (e.g., phosphate, bicarbonate, HEPES). Avoid primary amine buffers (e.g., Tris) during the coupling step as they compete.
• Polarity/miscibility (general):
  • PEG8 imparts amphiphilicity; the linker can distribute well between hydrophilic environments and organic co-solvents, aiding homogeneous reaction.
• When to choose alternatives:
  • If fully aqueous conditions are required, consider sulfo-NHS analogs of related linkers (higher water solubility; literature/general) or increase ionic strength to aid dispersion.
• Quick comparison (general):
  • DMSO: Maximal solubility; best for stock preparation.
  • DMF: Similar to DMSO; lower viscosity.
  • ACN: Cleaner volatility; moderate solubility.
  • Water/buffer: Use only transiently and at controlled pH to limit NHS hydrolysis.

Storage and Reconstitution

• Storage conditions (item-specific): Store at −20°C.
• Shipping (item-specific): Shipped in ice chest with ice pads.
• General stability/handling:
  • Keep tightly sealed under dry, inert atmosphere. Protect from moisture and light.
  • Allow vial to warm to room temperature before opening to prevent condensation.
• Reconstitution (general):
  • Dissolve in anhydrous DMSO or DMF to prepare concentrated stocks (e.g., 10–50 mM) immediately before use.
  • For aqueous applications, dilute the organic stock into pre-chilled, pH-controlled buffer (7.5–8.5 for NHS steps; 7.0–8.5 for thiol steps). Use promptly to minimize NHS hydrolysis.
• Aliquoting:
  • Prepare single-use aliquots; avoid repeated freeze–thaw. Store aliquots at −20°C in moisture-barrier containers with desiccant.
• Shelf-life:
  • Not specified for this item; refer to CoA/Spec Sheet. As a class, NHS esters exhibit improved longevity when kept dry and cold.
• Disposal:
  • Dispose of unused solutions and waste according to institutional and local regulations for reactive organic chemicals.

Research Use Note: For research use only.

Structure and Identity

A heterobifunctional PEGylated crosslinker combining a cysteine-reactive bis-sulfone motif with an amine-reactive NHS ester, separated by an octaethylene glycol (PEG8) spacer for enhanced aqueous compatibility.

• Item-specific identifiers

  • CAS: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
  • SMILES: Not specified for this item; refer to CoA/Spec Sheet.
  • InChIKey: Not specified for this item; refer to CoA/Spec Sheet.
  • Synonyms (general/literature): bis-sulfone–PEG8–NHS; NHS–PEG8–bis-sulfone; disulfide-rebridging PEG linker (literature/general).

• Structural features (general description)

  • One terminus: N-hydroxysuccinimide (NHS) active ester of a carboxylic acid for rapid acylation of primary amines (e.g., lysines, surface amines).
  • Spacer: PEG8 chain (–(CH2CH2O)8–) imparting hydrophilicity, flexibility, and ~3.5–4 nm reach (literature/general).
  • Other terminus: Bis-sulfone electrophile that can generate a vinyl sulfone under mildly basic conditions and undergo sequential Michael additions with two thiols, enabling disulfide rebridging to a stable three-carbon thioether bridge (literature/general).
  • Overall topology: Linear heterobifunctional linker with one amine-targeting end and one cysteine/disulfide-targeting end.

• 2D structure in words (general)

  • Succinimide leaving group attached to a carbonyl, followed by PEG8 chain, terminating in an aryl- or alkyl-substituted bis-sulfone unit bearing two electron-withdrawing sulfonyl groups that activate an adjacent methylene toward elimination/Michael addition (literature/general).

Synthetic Utility

Functional groups and reactivity provide versatile entry points for assembling complex bioconjugates and materials.

• Key functional groups (general):
  • NHS ester: Activated carboxyl for rapid amide formation with primary amines under mild conditions.
  • Bis-sulfone: Latent vinyl sulfone electrophile enabling sequential thiol additions and disulfide rebridging.
  • PEG8 spacer: Enhances solubility and reduces steric congestion.
• Applications in synthesis (literature/general):
  • Divergent conjugation: Install the linker on an amine-bearing scaffold (small molecule, peptide, polymer), then capture cysteine-containing partners site-selectively.
  • Macrocyclization/bridging: Rebridge intra- or inter-chain disulfides to form stable macrocycles or restore protein folds post-functionalization.
  • Orthogonal assembly: Combine with azide/alkyne-bearing partners introduced elsewhere to build multi-functional architectures.
• Retrosynthetic viewpoint (general):
  • Disconnect at the amide bond to reveal a carboxylic acid-PEG8-bis-sulfone precursor; NHS is introduced last for stability.
  • Bis-sulfone fragments often arise from di-sulfonylated benzyl or alkyl scaffolds bearing a leaving group adjacent to the sulfone units.
• Advantages vs alternatives:
  • Greater final linkage stability than maleimides (resistance to exchange), while maintaining efficient thiol reactivity.
  • Spacer length control through defined PEG repeat count improves reproducibility across constructs.

Target Specificity

This product is a chemical crosslinker, not a biological targeting reagent. It does not possess intrinsic target specificity beyond chemical selectivity:

• NHS ester: Reacts preferentially with primary amines (e.g., lysine side chains, N-termini) under mildly basic conditions over secondary amines/alcohols (literature/general).
• Bis-sulfone: Selective for thiols generated from cysteines/disulfide reduction, proceeding via a vinyl sulfone intermediate (literature/general).

No antigen/epitope specificity, clone, isotype, or species reactivity applies to this item.