SPDP-PEG6-NHS ester - ≥95% , CAS No.1818294-32-4

CAS: 1818294-32-4 Cat. No.: S597540 Formula: C₂₇H₄₁N₃O₁₁S₂ Molecular Weight: 647.8

GRADE & PURITY ≥95%

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Overview

SPDP-PEG6-NHS ester is a PEG derivative with SPDP and NHS ester moieties. The SPDP is an amine and thiol reactive crosslinker. It is also membrane permeable, allowing it to participate in intracellular crosslinking reactions. The NHS ester is amine reactive and forms stable amide bonds. The hydrophilic PEG linker increases the solubility of the compound in aqueous media.

Specifications

Specifications & Purity

≥95%

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

This product requires cold chain shipping. Ground and other economy services are not available.

Purity

≥95%

Names and Identifiers

Molecular Weight

647.8

Chemical and Physical Properties

• Solubility: Solubility in DCM

Application Protocols

The following is a representative, literature-based protocol for amine-to-thiol bioconjugation using SPDP-PEG6-NHS ester. Adjust to your system.

• Stock preparation:
  • Equilibrate vial to room temperature in a desiccator. Dissolve in anhydrous DMSO at 50 mM. Prepare single-use aliquots under dry nitrogen.
• Step 1: Amine labeling
  • Prepare target protein in 50 mM phosphate, 100 mM NaCl, pH 7.4 at 1–10 mg/mL.
  • Add linker stock to achieve 2–10 molar equivalents per protein amine site (start with 5–10 eq per lysine if DOF is unknown); keep DMSO ≤10% v/v.
  • Incubate 30–60 min at 20–25°C with gentle mixing.
  • Remove excess linker by desalting (e.g., size-exclusion spin column) into pH 7.0 buffer.
• Optional: Quantify incorporated SPDP by reducing a test aliquot and measuring the released pyridine-2-thione at 343 nm (literature method).
• Step 2: Thiol conjugation
  • Prepare thiol partner in pH 6.8–7.4 buffer. Ensure thiols are free (reduce and desalt if necessary).
  • Mix with the SPDP-activated protein at 1–2 eq thiol per SPDP; incubate 30–60 min at room temperature.
  • Monitor A343 nm to follow progress; remove byproducts by desalting/dialysis.
• QC and storage
  • Analyze by SDS-PAGE, SEC, and/or LC–MS to assess conjugation state.
  • Store conjugate at 2–8°C (short term) or frozen for long term, per biomolecule stability.

Note: Parameters are starting points; verify compatibility with sensitive biomolecules.

Biological Roles

This product is a synthetic chemical tool and does not have an intrinsic biological role. It is used to construct defined, cleavable covalent linkages between biomolecules.

General/literature context:

• Bioconjugation utility: Targets primary amines on proteins (lysine residues, N-termini) and free thiols (cysteines), enabling formation of amide-linked disulfide bridges that can be selectively reduced to release the cargo in reductive environments.
• Impact of PEG6 spacer: The hydrophilic PEG architecture can decrease nonspecific binding and improve solubility of conjugates, often preserving biomolecular activity relative to more hydrophobic linkers.
• Reductive sensitivity: Disulfide-linked constructs can be engineered for triggered release in reducing milieus (e.g., intracellular, cytosolic environments), but such uses must be validated in context and are for research only.

Note: No therapeutic, diagnostic, or clinical performance is claimed. All applications are limited to research and laboratory investigations.

Buffer Applications

Although not a buffer itself, this reagent’s performance is highly dependent on buffer choice during conjugation.

General/literature guidance for coupling:

• NHS–amine step:
  • Recommended buffers: 50–100 mM phosphate or HEPES, pH 7.2–8.0. Avoid primary amines (e.g., Tris, glycine) that compete with coupling.
  • Ionic strength: 50–150 mM NaCl is typically compatible; excessive salt can reduce solubility of some targets.
  • Additives: Minimal (<10% v/v) anhydrous DMSO/DMF to keep linker in solution.
• Thiol–disulfide exchange step:
  • Buffers: Phosphate, HEPES, or citrate/phosphate at pH 6.5–7.5. Avoid reducing agents (DTT, TCEP, βME) unless intentionally used for cleavage.
  • Monitoring: Track pyridine-2-thione release at 343 nm to follow reaction progress (literature).
• Quenching/Blocking:
  • Residual NHS esters can be quenched with ethanolamine (10–50 mM, pH 8.0) after the amine-coupling step.

Recipe hint (example, literature): 50 mM sodium phosphate, 100 mM NaCl, pH 7.4; prepare fresh, degas with argon, and pre-chill to slow NHS hydrolysis during setup.

Green Alternatives

When selecting a crosslinker/solvent system, consider greener choices that maintain performance while reducing hazard and environmental burden.

• Solvent considerations (literature):

  • Prefer anhydrous DMSO over DMF/NMP where feasible due to regulatory scrutiny of DMF/NMP. Use minimal organic cosolvent (≤10% v/v) in water to lower VOC load.
  • Employ buffered aqueous media (phosphate, HEPES) post-dissolution to minimize organic waste.

• Crosslinker alternatives (functional tradeoffs):

  • Non-cleavable PEG–maleimide NHS linkers (e.g., SM(PEG)n) avoid pyridine-2-thione byproduct and provide rapid, selective thiol addition (Michael addition). Less hazardous byproducts; however, linkages are non-reducible.
  • Water-soluble cleavable analogs (e.g., sulfo-SPDP-PEG variants) can reduce organic solvent use during protein labeling.
  • Zero-length EDC/NHS couplings avoid additional linker mass but do not enable thiol targeting or cleavability.

Comparison (general):

• SPDP-PEG6-NHS: Cleavable disulfide; broad thiol compatibility; releases pyridine-2-thione (monitorable at 343 nm); requires control of reducing environment.
• Maleimide–PEG–NHS: Non-cleavable thioether; faster thiol coupling at pH 6.5–7.5; sensitive to hydrolysis to maleamic acid; typically fewer odorous byproducts.
• Sulfo variants: Improved water solubility; can reduce DMSO content but may change membrane permeability and reaction distribution.

Choose based on downstream need for cleavability, solvent minimization, and biocompatibility constraints.

Pharmaceutical Uses

For research use only (per Product Data). This product is not an API or excipient for clinical use.

Research and development context (general/literature):

• Conjugate discovery: Useful for preparing proof-of-concept antibody–protein, peptide–polymer, and nanoparticle–ligand conjugates featuring cleavable disulfide linkages for release studies.
• Process development: Evaluating capture-and-release workflows on chromatographic resins or beads via reducible linkers.
• Analytical tools: The 2-pyridinethione leaving group provides a built-in spectrophotometric handle for reaction monitoring and load estimation during process screening.

If translation to regulated manufacturing is contemplated, a different grade and full regulatory documentation (e.g., GMP manufacture, residual solvent profiles, elemental impurities) would be required. No claims of safety or efficacy in humans or animals are made.

Physical Properties

Item-specific numerical specifications were not provided for this listing; consult the CoA/SDS for definitive values.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• Melting point: Not specified for this item; refer to CoA/Spec Sheet.
• Boiling point: Not applicable (decomposes before boiling; general for crosslinkers).
• Density/refractive index/UV cutoff: Not specified for this item; refer to CoA/Spec Sheet.

General/literature characteristics for PEGylated NHS–pyridyldithiol crosslinkers:

• Physical form: Typically a white to off-white solid; amorphous (literature, varies by lot and counterions).
• Solubility (literature): High in anhydrous polar aprotic solvents (DMSO, DMF, NMP); miscible to workable concentrations in aqueous buffers after pre-dissolution in DMSO. Limited stability in water due to NHS hydrolysis.
• Stability (literature trends): NHS ester half-life decreases sharply with increasing pH and in aqueous media; improved stability when stored dry, under inert gas, and cold. Pyridyldithiol is stable to mild bases and acids but susceptible to reduction.
• Spacer length (geometric, literature): PEG6 provides an extended contour length of roughly 24–30 Å, depending on solvation and conformation.

Note: Treat all numerical performance attributes (e.g., solubility limits, half-lives) as method- and system-dependent; verify experimentally under your conditions.

Quality & Grades

• Grade/purity for this specific item: Not specified for this item; refer to CoA/Spec Sheet.

Interpretation and guidance (general for crosslinkers):

• Analytical expectations: For bioconjugation, vendors typically control purity by HPLC/LC–MS and report assay (%), identity (MS), and residual solvents. Low water content is important to limit NHS hydrolysis; verify via Karl Fischer if your application is moisture-sensitive.
• UV trace/adsorbance: Not specified for this item; refer to CoA/Spec Sheet. Note that pyridine-2-thione release at ~343 nm (literature) provides a convenient quality check for thiol-reactivity in use.
• Stabilizers: None are typically added to NHS esters; if any are present, they will appear on the CoA. Absence/presence of stabilizer may impact long-term stability.
• Batch-to-batch control: For quantitative protein conjugations, confirm functionality via:
  • NHS content by amine-reactivity test (e.g., glycine quench + HPLC).
  • Thiol-reactivity by exchange with a low-MW thiol and monitoring A343 nm.
• Regulatory: For research use only (per Product Data). Not intended for human or veterinary use. If your workflow requires GMP, request documentation and additional testing from the manufacturer.

Reaction & Applications

SPDP-PEG6-NHS ester is a heterobifunctional, cleavable crosslinker that links primary amines to free thiols via a disulfide bridge, with a hydrophilic PEG6 spacer improving solubility and reducing nonspecific interactions.

• Core reaction chemistry (literature):
  • NHS ester + R–NH2 → stable amide (pH ~7.2–8.5).
  • 2-pyridyldithiol + R′–SH → R′–S–S–(linker) with release of pyridine-2-thione (A343 nm).
• Typical applications:
  • Protein and peptide bioconjugation: Lysine (or N-terminus) modification followed by conjugation to cysteine-containing partners; enables cleavable linkage for downstream release.
  • Surface and material functionalization: Introduce thiol- or disulfide-switchable handles onto amine-presenting polymers, nanoparticles, and hydrogels.
  • Affinity and pull-down workflows: Cleavable capture of thiol-bearing targets with reductive elution.
• Advantages of PEG6 spacer:
  • Enhanced aqueous compatibility; decreased aggregation.
  • Increased effective reach (~24–30 Å, literature) improving accessibility to buried residues.
• Practical guidance:
  • Stage 1: Couple to amine-bearing biomolecule at pH 7.4–8.0 for 30–120 min at 0–25°C; remove excess by desalting.
  • Stage 2: React with thiol partner at pH 6.5–7.5; monitor release of pyridine-2-thione at 343 nm to track completion.
  • Use slight molar excesses (e.g., 2–10 eq to lysine content) depending on desired substitution; optimize empirically to preserve activity.
  • Avoid reducing agents until after the thiol-exchange step; quench unreacted NHS with ethanolamine if needed.

Reaction Conditions

The following are general literature guidelines; optimize for your specific system.

• NHS–amine coupling:
  • Buffer: 50–100 mM phosphate or HEPES, pH 7.2–8.0; avoid primary amine buffers.
  • Solvent: Pre-dissolve linker in anhydrous DMSO or DMF (10–100 mM); add to buffer to ≤10% v/v organic.
  • Temperature/time: 0–25°C for 15–120 min, depending on reactivity and stability of the target.
  • Stoichiometry: 2–10 eq per accessible amine site for proteins; 1.1–2.0 eq for small molecules/surfaces.
  • Quench: Ethanolamine or hydroxylamine to cap residual NHS esters after coupling.
• Thiol–disulfide exchange:
  • Buffer: pH 6.5–7.5, 20–25°C.
  • Stoichiometry: 1.0–2.0 eq thiol per SPDP unit.
  • Monitoring: Measure A343 nm of released pyridine-2-thione (ε343 ≈ 8,080–8,500 M−1 cm−1; literature ranges) to estimate conversion.
  • Time: 15–90 min typical; ensure absence of competing reductants.
• Reduction/cleavage (if desired): DTT or TCEP under mild conditions (e.g., pH 7–8, 1–10 mM) cleaves the disulfide linkage; verify compatibility with cargo.

Notes:

• Keep solutions cold and anhydrous until dosing to extend NHS half-life.
• Desalt or dialyze between steps to remove excess linker/byproducts and prevent cross-reactivity.
• For surfaces/polymers, longer times and higher organic content may be required to ensure wetting.

Safety & Handling

Item-specific GHS details were not provided. Always review the product SDS before use.

• GHS classification, pictograms, H/P statements: Not specified for this item; refer to SDS.
• Anticipated hazards (general for NHS–pyridyldithiol crosslinkers):
  • NHS esters are acylating agents; can cause skin/eye irritation and sensitization upon contact.
  • 2-pyridyldithiol component and byproduct (pyridine-2-thione) are harmful if inhaled/ingested; irritant to eyes/skin.
  • Dust may be combustible; avoid dust formation and ignition sources.
• PPE: Use lab coat, nitrile gloves (change regularly), safety glasses or face shield. Handle powders in a fume hood.
• Handling practices:
  • Work anhydrously to minimize NHS hydrolysis; keep containers tightly closed with desiccant.
  • Avoid amine-containing buffers (e.g., Tris) during activation. Prevent contact with reducing agents that cleave disulfides (e.g., DTT, TCEP) until intended.
• Incompatibilities (general): Strong bases/acids (accelerate decomposition), nucleophilic amines (uncontrolled acylation), reducing agents (disulfide cleavage), strong oxidizers.
• First aid overview (general):
  • Skin/eye contact: Rinse with water for ≥15 minutes; remove contaminated clothing; seek medical advice.
  • Inhalation: Move to fresh air; seek medical attention if symptoms persist.
  • Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.
• Spill response: Avoid dust, collect with inert absorbent, dispose per institutional and local regulations.
• Waste: Treat as hazardous organic waste; segregate solutions containing DMSO/DMF and pyridine-2-thione byproduct.

Solvent Selection

SPDP-PEG6-NHS ester is best handled in anhydrous, polar aprotic solvents that dissolve PEG chains and preserve the NHS functionality.

• Preferred solvents (general/literature):
  • Anhydrous DMSO, DMF, or NMP for stock solutions (10–100 mM). These provide excellent solubility for PEGylated linkers and minimize premature hydrolysis when dry.
  • Aqueous buffers (after dilution): Phosphate or HEPES (pH 7.2–7.8) for NHS–amine coupling; avoid primary amine buffers (e.g., Tris) and high bicarbonate when selectivity is critical.
• Miscibility profile (general): DMSO/DMF stocks are miscible with water and most biological buffers, enabling direct dosing into reaction media.
• Choosing among options:
  • Use DMSO when biocompatibility of the cosolvent is important (e.g., protein conjugation ≤10% v/v).
  • Use DMF/NMP for polymer/surface modification where higher organic content is acceptable.
  • For very hydrophobic targets, incorporate a small percentage of acetonitrile or isopropanol alongside DMSO to aid wetting (verify compatibility).
• Practical tips:
  • Prepare small, single-use aliquots in a dry glovebox or under dry nitrogen.
  • Chill and dry buffers (argon-sparged) to slow NHS hydrolysis during setup.
  • Final pH window for amine acylation: ~7.2–8.5 (literature). Higher pH increases rate but reduces NHS half-life.

Storage & Reconstitution

• Storage (item-specific): Store at -20°C (per Product Data). Shipped cold (ice chest + ice pads).
• Container handling: Keep tightly capped with desiccant in the original amber vial. Minimize headspace and moisture ingress; consider storing under dry nitrogen/argon.
• Light/moisture sensitivity: NHS esters hydrolyze upon exposure to moisture; 2-pyridyldithiol is sensitive to reduction. Protect from ambient humidity and light.
• Reconstitution (general guidance):
  • Dissolve immediately before use in anhydrous DMSO or DMF to prepare 10–100 mM stocks. Mix gently; avoid prolonged warming.
  • Filter small particulates through a PTFE syringe filter if needed under dry conditions.
  • For aqueous applications, add the organic stock to buffered solutions to a final organic content typically ≤10% v/v.
• Aliquoting and stability (general):
  • Prepare single-use aliquots to avoid repeated freeze–thaw and moisture exposure.
  • Store aliquots at -20°C in tightly sealed microvials; bring to room temperature in a desiccator before opening to prevent condensation.
• Disposal: Discard unused solutions and byproducts (including pyridine-2-thione–containing fractions) as hazardous organic waste per institutional guidelines.

Note: Exact shelf life, assay, and residual solvent limits are not specified for this item; refer to CoA/Spec Sheet and SDS for definitive stability and safety information.

Structure & Identity

SPDP-PEG6-NHS ester is a heterobifunctional, cleavable, amine-to-thiol crosslinker comprising an NHS-activated carboxylate on one end and a 2-pyridyldithiol (SPDP) thiol-reactive group on the other, separated by a hexaethylene glycol (PEG6) spacer for increased hydrophilicity and extended reach.

• Item-specific identifiers
  • 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.
• Structural features (general/literature description)
  • Functional groups: N-hydroxysuccinimide (NHS) activated ester; hexaethylene glycol spacer; 3-(2-pyridyldithio)propionyl (SPDP) motif.
  • Reactivity: NHS ester acylates primary amines (e.g., lysines, N-termini); 2-pyridyldithiol undergoes disulfide exchange with free thiols to form a disulfide linkage while releasing pyridine-2-thione (chromophore ~343 nm, literature).
  • Architecture: Linear amphiphilic linker; PEG6 confers water compatibility and reduces nonspecific adsorption compared with purely aliphatic linkers.
  • Cleavability: The final amine–thiol connection is a disulfide that can be reductively cleaved (e.g., DTT, TCEP; literature).

Synthetic Utility

From a synthetic perspective, SPDP-PEG6-NHS ester is a modular tool to install a cleavable disulfide handle via amide formation on amine-bearing substrates.

• Functional group leverage:
  • NHS ester enables rapid acylation of primary amines on small molecules, peptides, polymers, and surfaces to append the PEG6–SPDP moiety.
  • The distal 2-pyridyldithiol allows subsequent exchange with thiols, forging a disulfide linkage under mild, aqueous conditions.
• Strategic uses:
  • Temporarily connect two entities with a reductively cleavable tether (capture, delivery, release studies).
  • Introduce protected thiols: After installing the SPDP arm, reduction (e.g., DTT/TCEP) yields a free thiol on the modified substrate for further chemistry (thiol–ene, Michael addition; literature).
  • Polymer/material modification: Generate disulfide-crosslinked hydrogels or attach ligands to nanoparticle shells with built-in cleavability.
• Advantages of PEG6: Enhances solubility and decreases steric hindrance versus short aliphatic SPDP variants, often improving conversion on crowded biomolecular surfaces.
• Notes and caveats:
  • Control substitution degree to preserve function (optimize equivalents and reaction time).
  • Protect sensitive thiols until the intended exchange step; avoid reductants and high pH which can prematurely scramble disulfides.

Target Specificity

Not applicable. This product is a chemical crosslinker and does not have antigen/epitope specificity, clone, isotype, or species reactivity. For targeting considerations, see Reaction & Applications and Synthetic Utility for functional selectivity (amines and thiols).