Bis-MAL-Lysine-dPEG®₄-TFP ester

Cat. No.: B487870

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Specifications

Rechtliche Informationen

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

Storage

Store at -20°C

Verschickt in

Ice chest + Ice pads

Dieses Produkt erfordert Kühlkettenversand. Grundversand und andere Economy-Optionen sind nicht verfügbar.

Application Protocols

Not specified for this item; refer to CoA/Spec Sheet and SDS. No tested application protocols are provided in the product data.

Guidance: See the Reaction Conditions and Buffer Applications sections for general, literature-based workflows that can be adapted and optimized for your specific system.

Biological Roles

This product is a synthetic crosslinking reagent and does not have inherent biological roles. However, design elements influence bioconjugation outcomes (literature/general):

• PEGylation (dPEG4): Increases hydrophilicity, reduces nonspecific binding and aggregation, and provides spatial separation between conjugated partners, which can preserve biomolecular function.
• Maleimide–thiol chemistry: Enables selective modification of cysteine residues under mild, near-physiological conditions, commonly used in protein engineering and labeling.
• TFP ester–amine chemistry: Allows efficient coupling to lysine side chains or N-termini with improved hydrolytic stability and lower background than NHS esters.

Use context:

• Construction of multivalent ligands, protein–protein fusions via chemical bridges, labeling of peptides/oligonucleotides bearing thiols, and attachment of payloads to nanoparticles or surfaces through amine/thiol orthogonality.

No medical or clinical claims are made. This product is for laboratory research use only.

Buffer Applications

This reagent is not a buffer. However, buffer choice critically determines conjugation selectivity and efficiency (literature/general guidance):

• For TFP ester (amine coupling):

  • Use carbonate/bicarbonate (50–100 mM, pH 7.8–8.5) or borate buffers; HEPES at pH 8.0 can work but reacts more slowly.
  • Avoid primary amine buffers (Tris, glycine, ammonium salts) and high concentrations of competing nucleophiles.
  • Include minimal organic co-solvent (≤10–20% DMSO/DMF) only as needed for solubility.

• For maleimide–thiol coupling:

  • Use phosphate or HEPES, pH 6.5–7.2, with 0.5–5 mM EDTA to chelate metals that catalyze thiol oxidation.
  • Maintain reducing environment carefully: Prefer TCEP at low concentration for disulfide reduction, then remove/exhaust before coupling to avoid maleimide consumption.
  • Degas buffers and work under inert atmosphere if possible; keep reaction times short (15–60 min at RT).

• Quenching and cleanup:

  • After TFP coupling, residual activated ester can be quenched with ethanolamine; after maleimide coupling, excess thiol can be quenched with cysteine or DTT (post-conjugation, off-target risk considered).
  • Desalt via spin columns or dialysis promptly to remove TFP phenol and unreacted reagents.

Green Alternatives

Greener practice considerations focus on solvent choice, process intensification, and waste minimization (literature/general):

• Solvent selection:

  • Prefer aqueous buffers with minimal co-solvent where feasible; predissolve in dry DMSO, then dilute to ≤10% DMSO v/v.
  • Consider bio-based dipolar aprotics (e.g., Cyrene) or propylene carbonate for certain organic steps; validate stability of TFP ester and maleimide in these media.
  • Replace DMF where possible due to regulatory and environmental concerns.

• Process strategies:

  • Micro- to meso-scale continuous mixing (microfluidics) can reduce reagent excess and byproduct exposure times, lowering hydrolysis and waste.
  • On-column or in situ desalting (spin columns) minimizes buffer volumes and product losses.

• Comparative overview (general):

  Greener option | Benefit | Trade-off --- | --- | --- Aqueous buffer + ≤10% DMSO | Lowest environmental burden | Requires rapid handling to limit hydrolysis Cyrene | Bio-derived, good solvency | Higher viscosity; compatibility testing needed 2-MeTHF (for non-aqueous pre-steps) | Renewable, lower toxicity vs THF | Limited solubility for PEGylated linkers Propylene carbonate | Low volatility, greener profile | Removal can be challenging

• Waste reduction:

  • Use stoichiometric control and immediate quenching/cleanup after conjugation.
  • Collect fluorinated phenol waste from TFP cleavage separately for appropriate disposal.

Pharmaceutical Uses

No pharmacopeial status or excipient designation is provided for this item. Not specified for this item; refer to CoA/Spec Sheet.

General, non-clinical context (research/manufacturing development):

• Employed in discovery-stage bioconjugation workflows to prototype antibody–drug conjugates (ADCs), multivalent biologics, and PEGylated constructs under laboratory conditions.
• The TFP ester offers cleaner background than NHS in some formulations, potentially easing downstream purification during process development (literature/general).
• The dPEG4 spacer can improve aqueous processability of conjugates and reduce aggregation during formulation screening.

Important: This product is for research use only and is not intended for use in humans or for clinical or diagnostic applications.

Physical Properties

Item-specific numeric specifications (BP, MP, density, UV cutoff, water content, metals): Not specified for this item; refer to CoA/Spec Sheet.

Literature/general expectations for this class of PEGylated activated esters:

• Physical state: Typically an amorphous to microcrystalline solid; PEGylated linkers often appear as white to off-white solids (literature/general).
• Solubility profile (literature/general):
  • High solubility in polar aprotic organics such as anhydrous DMSO and DMF; moderate in acetonitrile.
  • Aqueous buffers: workable after predissolution in DMSO/DMF; overall solubility aided by the dPEG4 segment.
• Partitioning: PEG segments lower logP and increase hydrophilicity relative to purely hydrophobic crosslinkers (literature/general).
• Hydrolytic considerations (literature/general):
  • TFP ester hydrolyzes in water more slowly than NHS esters but still requires controlled basic pH (typically 7.5–8.5) and minimal exposure time to aqueous buffers.
  • Maleimide ring can hydrolyze to maleamic acid above pH ~8.0 and on prolonged aqueous exposure, diminishing thiol reactivity.
• Optical properties: No item-specific UV-vis or fluorescence characteristics provided.

Practical notes (general): Use freshly prepared, anhydrous stock solutions (e.g., 10–50 mM in dry DMSO/DMF), and minimize aqueous residence time to preserve both the TFP ester and maleimide functionalities.

Quality and Grades

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

Context for this product class (general):

• Common grades for bioconjugation linkers include “research grade,” “>95% purity,” or HPLC-purified material. When provided, these denote suitability for demanding labeling workflows and reduced byproducts.
• Stabilizers: None specified for this item. Some activated esters are supplied without stabilizers and rely on low-moisture handling and cold storage.
• Documentation: For exact assay method (HPLC/UPLC), residual solvents, and identity confirmation (NMR/MS), consult the CoA accompanying the lot.

What grade implies in practice (general guidance):

• High assay reduces heteroconjugate populations and minimizes unreactive hydrolyzed species (maleamic acid or phenol byproducts).
• Low water content and tight control of residual acids/bases improve shelf-life of both TFP ester and maleimides.

Recommendation:

• Verify lot-specific purity and identity before critical experiments (e.g., ADC prototype assembly, multivalent protein crosslinking). If UV requirements for analytical detection are critical, confirm UV background of the linker on your LC system.

Reaction and Applications

This linker is designed for orthogonal bioconjugation, enabling construction of complex architectures by pairing two thiol-selective maleimides with one amine-selective TFP ester (literature/general).

Key application families:

• Protein–protein or protein–polymer crosslinking: Site-specific cysteine coupling via maleimides at pH ~6.8–7.2; install the linker on an amine-bearing target via the TFP ester at pH ~7.8–8.5.
• Three-arm assembly: The lysine-based node allows sequential functionalization—e.g., first attach to a protein’s lysine (TFP), then bridge two thiol-bearing ligands/peptides via the two maleimides to form a trifunctional conjugate.
• Surface modification: Introduce maleimide handles onto amine-rich surfaces (proteins, polymers, nanoparticles), then capture thiol-bearing payloads.
• ADC and multivalent ligand research: Build well-defined multivalent constructs with controlled spacing afforded by the dPEG4.

Practical workflow (general guidance):

1. Amine-coupling step (TFP): Use 5–20 molar equivalents vs accessible amines or 1.1–2.0 eq vs targeted amine when site-selectivity is engineered; pH 7.8–8.5, 30–90 min at RT.
2. Desalt to remove TFP phenol and excess reagent.
3. Thiol-coupling steps (maleimide): pH 6.5–7.2 in degassed buffer with 1–5 mM EDTA; 1.1–1.5 eq maleimide per thiol; 15–60 min at RT.

Notes:

• Avoid thiol-containing reducers (DTT, βME) during maleimide steps. Use TCEP sparingly and remove before coupling, as it can add to maleimides at higher pH.
• Work under nitrogen/argon to limit thiol oxidation; confirm conjugation by LC–MS or SDS–PAGE as appropriate.

Reaction Conditions

General, literature-based guidance; adjust to your system and verify by small-scale scouting. No item-specific specs are provided.

• Stock solutions: Prepare 10–50 mM in anhydrous DMSO or DMF; store on ice during use. Use within the working day.

• Amine coupling (TFP ester):

  • Buffer: 50–100 mM carbonate/bicarbonate or borate, pH 7.8–8.5; avoid primary amine buffers.
  • Stoichiometry: 1.1–5 eq relative to targeted amines; lower excess for site-specific constructs.
  • Temperature/time: RT, 30–90 min (monitor by LC–MS/SEC/UV).
  • Notes: TFP phenol byproduct absorbs in UV; consider desalting post-reaction.

• Thiol coupling (maleimide):

  • Buffer: 50 mM phosphate or HEPES, pH 6.5–7.2, 0.5–5 mM EDTA; degas to limit oxidation.
  • Thiol generation: Reduce disulfides with minimal TCEP at pH ~7, then remove/exhaust prior to adding maleimide linker.
  • Stoichiometry: 1.1–1.5 eq maleimide per thiol site.
  • Temperature/time: RT, 15–60 min; rapid mixing benefits uniformity.

• Order of operations: Perform TFP–amine coupling first, purify/desalt, then execute one or two maleimide couplings.

• Quenching/cleanup: Ethanolamine (50 mM) to quench residual TFP ester; cysteine (5–10 mM) for unreacted maleimide if needed; follow with desalting/dialysis.

• Expected outcomes: With dry handling and correct pH, conversions are commonly high; verify by intact mass and, for proteins, peptide mapping if necessary.

Safety and Handling

Item-specific GHS classification, pictograms, and H-statements: Not specified for this item; refer to SDS.

General safety considerations for PEGylated bis-maleimide/TFP-ester crosslinkers (literature/general):

• Hazards: Activated esters and maleimides are electrophiles; may cause skin/eye irritation and can be sensitizers. Avoid inhalation of dust and contact with skin, eyes, and clothing.
• PPE: Lab coat, safety glasses or face shield, and appropriate chemically resistant gloves (e.g., nitrile). Handle in a fume hood when weighing and preparing solutions.
• Incompatibilities:
  • Moisture: Hydrolyzes TFP ester and deactivates maleimides; keep tightly closed with desiccant.
  • Nucleophiles: Primary amines (e.g., Tris buffer), thiols (e.g., DTT, β-mercaptoethanol), and strong bases will consume reactive groups.
  • Strong oxidizers/reducers: May degrade the linker or react with thiol-bearing targets.
• First-aid overview (general):
  • Skin/eye contact: Rinse with water for several minutes; remove contaminated clothing; seek medical attention if irritation persists.
  • Inhalation: Move to fresh air; obtain medical advice if symptoms occur.
  • Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.
• Spill response: Avoid dust; collect with inert absorbent; dispose per institutional and local regulations.

Always consult the product’s SDS for authoritative hazard classification, exposure limits, and disposal guidance. This product is for research use only.

Solvent Selection

General solvent behavior for Bis-MAL-Lysine-dPEG4-TFP ester (literature/general):

• Polarity class: Polar, due to the dPEG4 spacer; best handled in polar aprotic media.
• Preferred anhydrous solvents: DMSO and DMF (excellent), acetonitrile (good to moderate). These minimize premature hydrolysis and support high stock concentrations.
• Aqueous use: Prepare concentrated stocks in dry DMSO/DMF (e.g., 10–50 mM), then dilute into:
  • Thiol coupling: phosphate or HEPES, pH 6.5–7.2, with 0.5–5 mM EDTA to chelate metals that catalyze thiol oxidation.
  • Amine coupling (TFP ester): carbonate/bicarbonate or borate buffers, pH 7.8–8.5. Avoid Tris, glycine, or other free amine buffers.
• Co-solvent ceilings: Keep DMSO/DMF content ≤10–20% v/v in biological systems to maintain biomolecule integrity (general guidance).

Comparison (general):

• DMSO vs DMF: DMSO typically offers broader biocompatibility at low percentages; DMF can give higher solubility for some PEGylated linkers but is less favored in cell-facing workflows.
• ACN: Useful for HPLC and some conjugations but may have limited solubility relative to DMSO/DMF.

Practical tips:

• Dry solvents over molecular sieves and use septum-sealed vials. Pre-chill aqueous buffers and work quickly to reduce hydrolysis. Filter stocks (0.2 µm PTFE) if particulates are observed.

Storage and Reconstitution

• Storage conditions (item-specific): Store at -20°C.
• Shipping (item-specific): Shipped in ice chest with ice pads.
• Long-term handling (general best practice): Keep tightly capped in moisture-barrier packaging with fresh desiccant. Minimize freeze–thaw; aliquot upon first opening into dry, inert-atmosphere vials.
• Light/air: Protect from light and humidity; purge vials with nitrogen/argon after use.

Reconstitution (general guidance):

• Solvents: Reconstitute in anhydrous DMSO or DMF to prepare 10–50 mM stocks. Filter if needed through 0.2 µm PTFE.
• Aqueous dilution: Add stock to buffer immediately before use to achieve the desired final concentration. Limit organic co-solvent to ≤10–20% v/v for biomolecule compatibility.
• Working pH: For TFP ester reactions, pH 7.8–8.5; for maleimide–thiol, pH 6.5–7.2.
• Stability after reconstitution: Use promptly; avoid extended residence in aqueous media to limit hydrolysis of TFP ester and maleimide groups.

Disposal: Collect aqueous and organic wastes containing tetrafluorophenol and maleimide byproducts separately and dispose according to institutional and local regulations.

Research Use: For research use only; not for human or clinical use.

Structure and Identity

Brief description: Bis-MAL-Lysine-dPEG4-TFP ester is a multifunctional PEGylated crosslinker that combines two thiol-reactive maleimides with a highly active 2,3,5,6-tetrafluorophenyl (TFP) ester on a lysine-based scaffold separated by a discrete PEG4 spacer.

• 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.

• Structural features (general description)

  • Core scaffold: L-lysine serving as a trifunctional hub (α-amine/α-carboxyl and ε-amine derived linkages).
  • PEG spacer: Discrete polyethylene glycol segment with four oxyethylene units (–O–CH2–CH2– repeated 4×), enhancing hydrophilicity and flexibility.
  • Electrophiles:
    • Two maleimide rings (Bis-MAL) for rapid, chemoselective Michael addition to thiols (Cys) at near-neutral pH.
    • One TFP ester (2,3,5,6-tetrafluorophenyl ester), an amine-reactive activated ester with superior hydrolytic stability vs NHS.
  • Functional-group map: one amine-reactive site (TFP ester) orthogonal to two thiol-reactive maleimides, enabling convergent 3-point bioconjugation.
  • Stereochemistry: Lysine center is typically L; no additional stereocenters expected in PEG or maleimide/TFP motifs (literature/general).

• 2D description in words: A central lysine-derived node connects via a short dPEG4 chain to terminal maleimides on two arms, while a third arm terminates in a TFP-activated ester, offering orthogonal reactivity toward primary amines and sulfhydryls.

Synthetic Utility

Functional group complementarity makes Bis-MAL-Lysine-dPEG4-TFP ester a versatile node for modular assembly (literature/general):

• Orthogonal electrophiles:
  • TFP ester (amine-selective): Rapid acylation of primary amines to stable amide bonds under mild basic conditions; superior hydrolytic stability vs NHS enables longer working windows.
  • Maleimides (thiol-selective): Michael addition to cysteines proceeds quickly at pH ~6.5–7.2, forming thioether linkages.
• Retrosynthetic value: A single reagent installs two maleimide handles and an amide linkage from one operation, simplifying routes to tri-functional constructs and reducing protecting-group gymnastics.
• Scaffolding: The lysine core provides a defined three-arm topology, while dPEG4 imparts flexibility and water compatibility that can reduce steric hindrance in late-stage couplings.

Typical transformations:

• Install maleimide-bearing handles onto amine-rich biomolecules, then conjugate two thiolated ligands/labels.
• Bridge two cysteine-bearing fragments while anchoring the assembly to an amine-functional support or protein.
• Create heterobifunctional surfaces/nanomaterials with controlled spacing and reactivity.

Considerations:

• Sequence matters: Perform TFP–amine coupling first (basic pH), then maleimide–thiol additions (near-neutral pH) to minimize maleimide hydrolysis and off-target reactions.
• Avoid competing nucleophiles (free thiols/primary amines) in solvents and buffers during each step.

Target Specificity

Not applicable. This product is a chemical crosslinker and does not possess biological target specificity such as an antibody epitope or enzyme substrate profile. No item-specific target data are provided.