MAL-dPEG ®₂₄-amido-dPEG ®₂₄-TFP ester

Cat. No.: M487908

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Specifications

法的情報

Products Protected under U.S. Patents # 7,888,536 B2dPEG is a registered trademark of Quanta BioDesign

保管条件

Store at -20°C

入荷

Ice chest + Ice pads

この製品は冷冷この冷このこの製品の運送が必要です。地上およびその他の経済サービスは利用できません。

Application Protocols

The following are generalized, literature-based protocols for research use with heterobifunctional TFP/maleimide dPEG linkers. Optimize for your specific system.

A. Installing the linker onto an amine-bearing protein (TFP step), then coupling to a thiolated ligand (maleimide step):

1. Dissolve linker at 10–50 mM in anhydrous DMSO.
2. Exchange protein into 50 mM phosphate, pH 8.0 (amine-free), 150 mM NaCl if needed. Optional: include 0.5 mM EDTA.
3. Add linker to protein to achieve 2–5 molar eq per target amine; mix gently at 4–25°C for 30–120 min.
4. Remove excess linker by desalting (spin column, SEC). Verify incorporation by intact LC-MS if available.
5. Reduce partner ligand to generate free thiols (e.g., TCEP, brief), then remove reducing agent by desalting.
6. Maleimide coupling: combine maleimide-bearing protein with thiolated ligand in phosphate/citrate buffer, pH 6.8–7.0, at 4–25°C for 15–60 min.
7. Quench residual maleimide with excess cysteine if appropriate. Purify final conjugate.

B. Surface/nanoparticle functionalization:

1. Prepare amine-functionalized surface/beads.
2. React with linker (TFP step) in amine-free buffer with ≤10% DMSO, pH 8.3, RT, 1–2 h; rinse thoroughly.
3. Introduce thiol-bearing biomolecule in pH 6.8–7.0 buffer; incubate 30–60 min; wash to remove unbound species.

Notes:

• Avoid Tris/glycine buffers during TFP coupling.
• Avoid thiol reductants during maleimide coupling. Work quickly and keep solutions oxygen-reduced to maintain thiols.

Biological Roles

Item-specific biological data are not provided for this reagent. As a synthetic heterobifunctional crosslinker, it does not possess an intrinsic biological role. The following are general, research-focused considerations for PEG-based linkers (literature/general knowledge; no clinical claims):

• PEG as a biophysical modulator: Attaching long, hydrophilic PEG chains reduces protein surface charge density, steric shields hydrophobic patches, and minimizes nonspecific adsorption. This often improves solubility and decreases aggregation in vitro.
• Spacing and flexibility: Two dPEG24 segments confer an extended, flexible tether that can spatially separate functional components (e.g., enzyme from surface, fluorophore from quencher), mitigating steric hindrance and enabling more accurate biophysical measurements.
• Reduced immunogenic adsorption and fouling (in research systems): PEG brushes reduce protein fouling on surfaces and nanoparticles, improving signal-to-noise in biosensing and analytical assays.
• Site-selective conjugation: Maleimide chemistry targets free thiols (cysteine residues or thiolated probes), whereas TFP esters acylate primary amines (lysines, N-termini). Orthogonality allows sequential, controlled modifications in complex biomolecules.
• Enzymology and structural biology: Defined-length spacers assist in single-molecule studies, FRET distance calibration, and crosslinking-mass spectrometry where precise linker length influences distance constraints.

Note: All uses are for research and laboratory purposes only. No medical, clinical, or in vivo performance claims are made or implied.

Buffer Applications

This product is not a buffering reagent. However, buffer selection is critical to its successful use in bioconjugation.

General/literature guidance for buffer systems (not product specifications):

• For TFP–amine coupling:
  • Use amine-free buffers such as 50–100 mM phosphate (pH 7.5–8.2) or 50–100 mM bicarbonate/carbonate (pH 8.3–8.5). Avoid Tris, glycine, imidazole, or ammonium salts which compete with the coupling.
  • Include 50–150 mM NaCl as needed to maintain protein stability; add minimal organic cosolvent (≤10% DMSO/DMF) to solubilize the linker.
• For maleimide–thiol coupling:
  • Employ mildly acidic to neutral buffers, typically phosphate or citrate at pH 6.5–7.2, to limit maleimide hydrolysis and preserve thiol reactivity.
  • Avoid thiol-containing additives (DTT, β-mercaptoethanol). If reduction is required, use TCEP briefly, then remove via desalting before adding the linker.
• Degassing and oxygen control: For thiol chemistry, degas buffers and include EDTA (0.5–1 mM) to chelate metals that catalyze thiol oxidation.

Preparation hints:

• Filter (0.22 µm) and pre-chill buffers when working with sensitive proteins.
• Validate pH at working temperature; amine coupling is pH sensitive, and small deviations can alter kinetics substantially.

Green Alternatives

Sustainability considerations for PEGylation linkers focus on solvent choice, atom economy of coupling chemistry, and minimizing reagent excess/hydrolysis waste.

Greener practice options (literature/general guidance):

• Solvent substitution:
  • Prefer aqueous buffers with minimal cosolvent when feasible; keep anhydrous DMSO/DMF stock volume ≤10% v/v in the reaction.
  • Evaluate GVL or propylene carbonate for small-molecule amination steps (if biomolecule absent), acknowledging limited biocompatibility compared to DMSO.
• Chemistry choice:
  • TFP esters offer improved hydrolytic stability vs NHS, reducing waste from premature hydrolysis and lowering required excess.
  • Employ on-demand micro-aliquoting (pre-aliquoted dry vials) to reduce repeated freeze–thaw and disposal of degraded material.
• Process intensification:
  • Use continuous desalting (spin columns) to curtail large buffer volumes.
  • Consider shorter dPEG lengths when application tolerates it, cutting embodied mass without sacrificing function.

Comparison (general attributes; not product specifications):

• TFP vs NHS ester
  • Hydrolytic stability: TFP > NHS (at neutral to mildly basic pH)
  • Leaving group toxicity: both generate low-tox aryl/OSu byproducts; handle as organic waste.
  • Coupling efficiency in amine-free buffer: TFP often attains high conversions with less excess reagent.

Trade-offs:

• Higher PEG length improves solubility and reduces nonspecific binding but increases synthetic footprint. Balance spacer length with performance needs.

Pharmaceutical Uses

No pharmacopeial grade or clinical application details are provided for this item. The statements below refer to general research and process-development contexts only (no therapeutic claims):

• Conjugation process development: Such heterobifunctional PEG linkers are commonly evaluated during early-stage research to optimize conjugation strategies for proteins, peptides, and particles by exploring spacer length, hydrophilicity, and orthogonal reactivity.
• Formulation studies (in vitro): The long PEG spacer can reduce aggregation and improve solubility in analytical formulations used to characterize conjugates (e.g., SEC, DLS stability screens).
• Analytical reference materials: Defined-length dPEG reagents aid in establishing LC/MS and HPLC methods for monitoring conjugation extent, residual linker, and byproducts.
• Device and surface research: In fabrication of diagnostic prototypes or research-grade biosensors, these linkers are used to derivatize amine-functionalized surfaces and subsequently attach thiol-bearing capture ligands.

Compliance note:

• Any use in GMP settings requires qualification of supplier, traceability, and specification control (identity, purity, residual solvents, bioburden/endotoxin if relevant). For SKU-specific certification, Grade/Purity is Not specified for this item; refer to CoA/Spec Sheet.
• For clinical, diagnostic, or therapeutic applications, additional validation and regulatory review are mandatory; this product is provided strictly for research use only as stated in the Product Data.

Physical Properties

Item-specific physicochemical specifications (from Product Data):

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.

General/literature expectations for this class of heterobifunctional dPEG reagents (informational only; not product specifications):

• Physical state: typically off-white to white solid or waxy solid for long dPEG linkers; hygroscopic; readily absorbs moisture.
• Solubility: high in polar aprotic solvents (anhydrous DMSO, DMF, NMP) and mixed aqueous buffers after initial dissolution; miscible behavior improved by PEG segments.
• Water solubility: generally good for long dPEG linkers; stock solutions commonly prepared in anhydrous DMSO/DMF then diluted into aqueous buffer.
• Partitioning: PEGylated structures exhibit very low logP and high hydrophilicity versus hydrocarbon linkers.
• Stability: TFP ester is more hydrolytically robust than NHS esters but still undergoes hydrolysis in water, especially at elevated pH and temperature; maleimide slowly hydrolyzes to maleamic acid above neutral pH and can participate in thiol exchange under certain conditions.
• Volatility: non-volatile; decomposition over melting rather than true boiling (typical for high-MW PEGylated reagents).

Do not treat the above as specifications for SKU M487908. For exact values (water content, purity, residual solvents, melting range, UV cutoff, etc.) consult the item’s CoA/Spec Sheet.

Quality and Grades

Item-specific quality attributes (from Product Data):

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

General notes on quality for discrete PEG (dPEG®) crosslinkers (literature/general guidance):

• Discrete PEG (dPEG®) design provides a single, defined oligomer length, minimizing polydispersity and enabling reproducible conjugation stoichiometry and biophysical properties compared to conventional polymeric PEGs.
• Typical QC elements for high-end PEGylation linkers include:
  • Identity and homogeneity by NMR and high-resolution MS (e.g., ESI, MALDI).
  • Purity by HPLC/UPLC with low levels of related PEG oligomers/functional group hydrolysis products.
  • Functional group integrity: active ester content (e.g., TFP) and maleimide content by titration or LC methods.
  • Residual solvent and inorganic content within internal limits.
• UV characteristics: TFP-aryl and maleimide exhibit modest UV absorbance; HPLC methods often monitor at 214–260 nm (literature), but actual UV cutoff/ε for this item is Not specified for this item; refer to CoA/Spec Sheet.
• Stabilization: Some suppliers package reactive esters under inert atmosphere with desiccant. If present, stabilizers would be listed on the CoA; for this item, any stabilizer use is Not specified for this item; refer to CoA/Spec Sheet.

Recommendation: Use freshly opened material or validated aliquots. Verify functional activity (e.g., amine/quenching test, thiol reactivity test) prior to critical conjugations when required by QA.

Reaction and Applications

This heterobifunctional linker enables site-selective, orthogonal bioconjugations that bridge thiols and amines across an extended, hydrophilic spacer.

Key application families (literature/general knowledge):

• Thiol-to-amine crosslinking: Couple cysteine residues (proteins/peptides) to primary amines (lysine-bearing proteins, amine-functionalized polymers, nanoparticles, or surfaces). Sequence: amine coupling via TFP to introduce linker, then thiol coupling via maleimide (or vice versa using protecting groups).
• Surface and nanoparticle modification: Immobilize biomolecules onto amine-functionalized beads, quantum dots, silica, or polymer films; PEG spacer minimizes nonspecific adsorption and enhances probe accessibility.
• Antibody/oligonucleotide conjugation (research use only): Introduce thiol-reactive maleimide onto an amine-bearing oligo or, conversely, append an amine-reactive TFP onto a thiolated oligo; the long dPEG reduces aggregation and improves solubility.
• Multistep assemblies: Central amide and dual PEG24 segments furnish length for spanning crowded interfaces, enabling FRET pair spacing, biosensor construction, or enzyme–substrate tethering.

Practical tips:

• Keep stocks anhydrous; prepare immediately before use. Titrate equivalents to limit over-modification (typical 1.1–5.0 eq depending on substrate accessibility; literature guidance).
• For proteins: perform TFP–amine coupling at pH ~8.3 in amine-free buffer on ice/RT; remove excess reagent; then reduce cysteines (if necessary) and perform maleimide coupling at pH ~6.8–7.0.
• Avoid reducing agents containing thiols (DTT, βME). If using TCEP to generate free thiols, remove by desalting prior to maleimide coupling to minimize side reactions.
• Monitor by SDS-PAGE, LC-MS (intact mass), or UV/fluorescence if a co-label is present.

Reaction Conditions

The following conditions are general literature guidance for maleimide/TFP-ester linkers and should be optimized per system. They are not specifications for this SKU.

TFP–amine coupling (forming an amide):

• Buffer: 50–100 mM phosphate or bicarbonate, amine-free, pH 7.5–8.5.
• Solvent: add freshly prepared anhydrous DMSO/DMF stock to ≤10% v/v final.
• Temperature/time: 0–25°C, 0.5–2 h typical for small molecules; 0.5–4 h for proteins (monitor to avoid over-modification).
• Stoichiometry: 1.1–5.0 eq of linker vs available amines depending on accessibility; for proteins, start low and titrate.
• Quench: remove unreacted linker by desalting; residual active ester can be quenched with ethanolamine if desired (post-purification).

Maleimide–thiol coupling (forming a thioether):

• Buffer: 50 mM phosphate or citrate, pH 6.5–7.2; include 0.5–1 mM EDTA to limit metal-catalyzed oxidation.
• Temperature/time: 4–25°C, 15–120 min; reaction is typically rapid.
• Stoichiometry: 1.1–2.0 eq vs thiol. Excess can increase off-target reactions if other nucleophiles are present.
• Reducing agents: avoid DTT/βME. If using TCEP to generate thiols, remove by desalting prior to coupling.

Analytics and controls:

• Monitor by LC-MS (intact mass), capillary electrophoresis, or SDS-PAGE.
• Verify maleimide integrity by test coupling with cysteine or small-molecule thiols.

Notes:

• Oxygen exclusion and degassing improve thiol stability. Light protection is advisable to reduce side reactions in sensitive systems.

Safety and Handling

Product-specific hazard information (from Product Data):

• Signal Word: Not specified for this item; refer to SDS.
• H-Statements: Not specified for this item; refer to SDS.
• GHS Classification/Pictograms: Not specified for this item; refer to SDS.

General safety considerations for PEGylation crosslinkers bearing maleimide and TFP ester (literature/general guidance):

• Potential hazards: irritant to skin/eyes/respiratory tract; can react with biological nucleophiles (amines/thiols). Avoid inhalation of dust and contact with skin or eyes.
• PPE: lab coat, safety glasses, and suitable chemical-resistant gloves (e.g., nitrile). Handle powders in a fume hood to minimize exposure and moisture uptake.
• Storage incompatibilities: avoid moisture, strong bases (accelerate ester hydrolysis and maleimide ring opening), strong nucleophiles, and free thiols/amine-containing buffers in storage.
• Reaction cautions: buffers containing primary amines (e.g., Tris, glycine) will quench the TFP ester; free thiols (e.g., DTT, 2-mercaptoethanol) will consume the maleimide.
• First aid (overview; defer to SDS):
  • Skin/eye contact: rinse with plenty of water for ≥15 min; remove contaminated clothing; seek medical attention if irritation persists.
  • Inhalation: move to fresh air; seek medical attention if symptoms occur.
  • Ingestion: rinse mouth; do not induce vomiting; seek medical attention.
• Fire: not highly flammable; treat as organic combustible. Use CO2, dry chemical, or foam.
• Waste: segregate solutions containing residual active ester/maleimide; quench intentionally with aqueous ethanolamine or cysteine, then dispose per institutional chemical waste procedures.

Always consult the SDS for authoritative hazard, toxicological, and first-aid information.

Solvent Selection

This reagent is a highly hydrophilic, heterobifunctional dPEG linker that dissolves readily in polar aprotic solvents and can be transitioned into aqueous media for bioconjugation.

General/literature guidance:

• Primary stock solvents (anhydrous):
  • DMSO or DMF: excellent solubility; minimize water to preserve TFP/maleimide functionality.
  • NMP or acetonitrile can be used for certain workflows; verify compatibility with biomolecules.
• Aqueous compatibility: After forming a concentrated stock (e.g., 10–50 mM) in anhydrous DMSO/DMF, dilute into buffered aqueous solutions immediately before use.
• Buffer selection by reaction:
  • TFP–amine coupling: pH 7.5–8.5 in amine-free buffers (e.g., phosphate, HEPES, bicarbonate); avoid Tris, glycine, or other nucleophilic buffers that quench the ester.
  • Maleimide–thiol coupling: pH 6.5–7.2 (to limit maleimide hydrolysis and thiol oxidation) in phosphate or citrate; avoid free thiol reductants (DTT, βME). Use TCEP cautiously and remove prior to conjugation if possible.
• Miscibility profile (literature): miscible with water upon dilution; excellent in DMSO/DMF; poor in nonpolar solvents (hexanes, toluene).

Selection tips vs alternatives:

• Choose this long dPEG24–amido–dPEG24 spacer when maximal hydrophilicity, flexibility, and long reach are desired. For shorter reach or reduced hydrodynamic radius, consider shorter dPEGn analogues. For purely aqueous workflows, NHS-ester analogs hydrolyze faster; TFP provides improved hydrolytic stability at comparable pH.

Storage and Reconstitution

Item-specific conditions (from Product Data):

• Storage Conditions: Store at -20°C.
• Shipped In: Ice chest + Ice pads.

General best practices for this class of reagents (literature/general guidance):

• Packaging/handling: Keep tightly sealed under dry inert atmosphere if possible. Include desiccant. Allow vial to equilibrate to room temperature in a desiccator before opening to prevent condensation.
• Reconstitution:
  • Prepare concentrated stocks (e.g., 10–50 mM) in anhydrous DMSO or DMF immediately before use.
  • Filter if necessary using PTFE syringe filters pre-rinsed with dry solvent. Avoid aqueous contact until ready to react.
• Aliquoting:
  • Dispense single-use aliquots into dry, inert vials to minimize freeze–thaw and moisture exposure.
  • Store aliquots at −20°C (or lower) protected from light. Do not store diluted aqueous solutions—TFP and maleimide hydrolyze.
• Stability notes:
  • Maleimide slowly hydrolyzes to maleamic acid, especially at pH >7.5 and elevated temperature.
  • TFP ester hydrolyzes in water; maintain neutral-to-slightly basic pH only during active coupling and limit exposure time.
• Thawing/use:
  • Warm frozen aliquots to room temperature before opening to avoid moisture ingress. Use promptly after opening; discard any remaining solution that has absorbed moisture or shows diminished activity.

For definitive shelf-life and retest dates, as well as any stabilizers or specification limits, consult the item’s CoA/Spec Sheet.

Structure and Identity

Item-specific identifiers (from Product Data):

• CAS: Not specified for this item; refer to CoA/Spec Sheet.
• InChIKey: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES: 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.

General structural description (literature/general knowledge):

• MAL-dPEG®24-amido-dPEG®24-TFP ester is a heterobifunctional, discrete-length polyethylene glycol (dPEG) crosslinker bearing two distinct reactive termini:
  • A maleimide (MAL) group for selective conjugation to thiols via Michael addition.
  • A 2,3,5,6-tetrafluorophenyl (TFP) ester for amine coupling via acyl substitution to form amides.
• The central scaffold consists of two dPEG24 segments (each with 24 ethylene glycol repeat units) joined by a central amide linkage (–CONH–), yielding a long, highly hydrophilic, flexible spacer with low nonspecific binding.
• Functional group inventory: activated aryl ester (TFP-ester), maleimide ring (electron-deficient alkene), multiple ether oxygens (PEG), and an internal amide.
• 2D structure (described): TFP-phenyl–O–C(=O)–(PEG)24–C(=O)–NH–(PEG)24–maleimide. One terminus is an activated carbonate-like aryl ester; the opposite terminus is a substituted maleimide ring; between them lie two PEG chains bridged by an amide.
• Stereochemistry: none expected (achiral linker; PEG segments are achiral, maleimide/TFP units planar).

Notes:

• Discrete PEG (dPEG®) specifies a single, defined oligomer length rather than a broad polymer distribution (literature).

Synthetic Utility

From a synthetic chemistry perspective, MAL-dPEG®24-amido-dPEG®24-TFP ester is an orthogonally reactive spacer enabling modular assembly of complex conjugates through two high-yielding bond-forming reactions (literature/general knowledge):

• Amide bond formation via TFP ester: The TFP-activated ester undergoes nucleophilic acyl substitution with primary amines (alkyl or aryl) to form robust amide linkages. TFP esters are known for greater hydrolytic stability and reactivity balance compared to NHS esters, permitting cleaner couplings at lower reagent excess.
• Thiol–maleimide Michael addition: Selective addition of thiolates to the electron-deficient double bond of maleimide creates stable thioether linkages. Reaction is rapid at near-neutral pH and proceeds efficiently with minimal catalyst.
• Spacer engineering: Two dPEG24 segments separated by an amide provide long reach and high hydrophilicity, decoupling conjugated entities and improving aqueous handling. The internal amide increases chemical stability and introduces a defined hinge point.
• Orthogonality in sequences: One can first install the linker onto an amine-bearing substrate (via TFP), purify, then couple to a thiol-bearing partner; or reverse the order if amine reactivity must be preserved, using thiol-protecting strategies.
• Surface chemistry: Readily attaches to amine-terminated SAMs, polymers, or nanoparticles, followed by thiol capture of biomolecules—useful in analytical and materials chemistry.

This linker is highly valuable for retrosynthetic planning of bioconjugates where aqueous compatibility and minimized nonspecific interactions are required.

Target Specificity

Not applicable. This product is a chemical crosslinker, not a biological targeting reagent. It has no inherent target, antigen, epitope, clone, isotype, or species specificity. Reactivity is governed by functional group chemistry:

• The TFP ester reacts broadly with accessible primary amines.
• The maleimide reacts selectively with free thiols under near-neutral conditions. For selective bioconjugation, control is achieved through reaction conditions, protection/deprotection strategies, and substrate engineering rather than biological target recognition.