Methyltetrazine-PEG12-acid - ≥98% , CAS No.2183440-39-1

CAS: 2183440-39-1 Cat. No.: M596566 Formula: C₃₆H₆₀N₄O₁₅ Peso molecolare: 788.9

GRADE & PURITY ≥98%

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Application

227,228,229

Panoramica

Methyltetrazine-PEG12-Acid is an amine-reactive reagent and is one of the most stable tetrazines commercially available containing a terminal carboxylic acid. The terminal carboxylic acid can be reacted with primary amine groups in the presence of activators (e.g. EDC, or DCC) to form a stable amide bond. The hydrophilic PEG spacer increases water-solubility and methyl group enhances the stability of Methyltetrazine-PEG4-Acid.

Specifications

Specifiche e purezza

≥98%

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

≥98%

Nomi e identificatori

Peso molecolare

788.9

Application Protocols

The following are general literature protocols intended as starting points; optimize for your system.

• A. Installing Methyltetrazine-PEG12 onto a lysine-bearing protein (EDC/sulfo-NHS):

  1. Dissolve protein at 1–5 mg/mL in 50 mM MES, pH 6.0 (no primary amines in buffer).
  2. Prepare reagent at 5–20 mM in DMSO. Separately dissolve EDC·HCl (100 mM) and sulfo‑NHS (100 mM) in water.
  3. Add EDC (5–10 equiv vs. reagent) and sulfo‑NHS (5–10 equiv) to the protein solution, then add the methyltetrazine reagent (5–20 equiv per intended labeling site) with gentle mixing; keep DMSO ≤10% v/v.
  4. React 1–2 h at RT protected from light.
  5. Quench residual active esters with 10 mM hydroxylamine (optional), then purify by desalting/SEC. Determine degree of labeling by UV–vis or MS.

• B. IEDDA labeling of a methyltetrazine-modified biomolecule with a TCO probe:

  1. Prepare biomolecule (e.g., 1–10 µM) in PBS, pH 7.4.
  2. Add TCO probe at 1.2–2.0 equiv.
  3. Incubate at RT for 5–30 min (longer for BCN/norbornene). Protect from light.
  4. Remove excess probe by desalting or dialysis; analyze by SDS-PAGE, fluorescence, or MS.

• C. Small-molecule coupling (amide formation):

  1. Dissolve amine substrate and Methyltetrazine-PEG12-acid (1.0–1.5 equiv) in DMF or 1:1 DMF/DCM.
  2. Add HATU (1.1–1.5 equiv) and DIPEA (2–4 equiv); stir at RT for 1–3 h.
  3. Work up and purify by preparative HPLC.

Note: These are general guidelines; consult the SDS and CoA and perform controls.

Biological Roles

This reagent is a chemical tool; it does not have intrinsic biological function. Its value lies in enabling bioorthogonal labeling within complex biological systems (literature/general):

• Bioorthogonal handle: Methyltetrazine reacts selectively with TCO/BCN-modified biomolecules without interacting with native cellular functionalities, allowing spatiotemporally controlled tagging.
• Probe assembly: Facilitates two-step imaging or pull-down workflows—first install methyltetrazine on a biomolecule, then add a TCO-bearing fluorophore, affinity tag, or payload.
• Minimal perturbation: The PEG12 spacer enhances aqueous compatibility and often reduces perturbation to protein structure compared with shorter linkers, improving accessibility of the reactive site.
• Kinetic advantage: Fast IEDDA kinetics enable labeling at low micromolar or even nanomolar concentrations under physiological conditions, reducing reagent exposure.

No medical or clinical claims are made; this product is provided strictly for research use only, as stated in the Product Data.

Buffer Applications

While not a classical buffer, Methyltetrazine-PEG12-acid is routinely used in buffered aqueous media for bioconjugation. Buffer choice is critical for efficient coupling and subsequent IEDDA labeling (literature/general):

• For carbodiimide coupling of the terminal acid to amines:

  • Use MES buffer, 25–100 mM, pH 5.0–6.0, to favor O-acylisourea/NHS ester formation and minimize amine protonation balance.
  • Alternatively, phosphate buffer at pH 6.0–7.2 can be used when substrates are base-sensitive; avoid Tris, glycine, or other primary amine buffers, which compete with the coupling target.

• For IEDDA reaction after installation:

  • Perform tetrazine–TCO ligations in PBS or HEPES at pH 6.5–7.8. Reaction proceeds efficiently at room temperature; inclusion of 0.01–0.1% nonionic surfactant (e.g., Tween-20) can reduce nonspecific adsorption for surface work.

• Practical notes:

  • Prepare reagent stocks in DMSO and add to buffer to ≤10% v/v final to maintain solubility without denaturing proteins.
  • Control ionic strength (50–150 mM) to balance protein stability and reaction rate.
  • Remove excess carbodiimide/NHS prior to IEDDA to prevent unwanted side chemistry.

Green Alternatives

The core chemistry (IEDDA) is inherently green—copper-free, room-temperature, and water-compatible. Environmental impact mainly arises from solvent choice and carbodiimide activation for installing the methyltetrazine.

• Greener choices (literature/general):

  • Prefer aqueous buffers (MES, phosphate, HEPES) with minimal co-solvent during EDC/NHS coupling; avoid DMF when DMSO or water suffice.
  • Use water-soluble carbodiimides (EDC) instead of DCC to simplify workup and reduce hazardous waste.
  • Employ micro-scale reactions and high-concentration stocks to reduce solvent volumes.

• Comparison (general guidance): | Aspect | Less green option | Greener alternative | Trade-offs | |---|---|---|---| | Coupling solvent | DMF | Aqueous MES ± small % DMSO | May need pH control, slower dissolution | | Activator | DCC/NHS (organic) | EDC/NHS (aqueous) | Urea byproduct still formed; pH critical | | Copper-catalyzed click | CuAAC | IEDDA (tetrazine–TCO) | Requires strained partner chemistry |

• Waste minimization tips:

  • Quench and segregate carbodiimide wastes; neutralize before disposal per regulations.
  • Choose volatile or recyclable co-solvents when possible; recover acetonitrile by distillation in process settings.
  • Design two-step labeling to avoid excess of expensive strained partners.

Pharmaceutical Uses

No therapeutic use is implied. In pharmaceutical research and development contexts (literature/general):

• Tool for discovery: Employed in the assembly of antibody–drug conjugates (ADCs) and targeted delivery systems during lead optimization and analytical characterization, using IEDDA as a bioorthogonal ligation step.
• Surface/particle modification: Used to decorate liposomes, nanoparticles, or solid supports with methyltetrazine groups for subsequent attachment of TCO-bearing ligands, enabling modular screening of targeting moieties.
• Analytical tagging: Facilitates site-selective labeling for mass spectrometry standards, DAR determination, or fluorescence-based assays after installing tetrazine handles on biomolecules.
• Process considerations: The PEG12 spacer often improves aqueous handling and reduces aggregation in protein formulations compared with shorter linkers.

Note: Any use in drug manufacturing must establish internal specifications (identity, purity, residual reagents) and follow GMP/GxP frameworks. This product is provided for research use only.

Physical Properties

• Item-specific values (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.
  • Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.

• Literature/general expectations for PEG12-tetrazine acids (guidance; not product specifications):

  • Physical state: typically a pink to magenta solid or viscous syrup due to tetrazine chromophore and PEG chain; color intensity can vary with substitution (literature).
  • Solubility: high in polar organics (DMSO, DMF, acetonitrile) and miscible/soluble in aqueous buffers when neutralized or when formulated from a DMSO stock (literature). Aqueous solubility improves at pH > pKa of the terminal carboxyl (estimated pKa ~4–5 for aliphatic carboxylates; literature, not product-specific).
  • LogP: strongly negative/apparent hydrophilic due to PEG; exact value depends on chain length (literature trend).
  • UV–vis: tetrazines absorb in the visible (often 520–540 nm band with ε 10^3–10^4 M−1 cm−1, literature range), useful for monitoring.

• Practical notes:

  • Hygroscopicity: PEGylated reagents may absorb moisture; handle quickly and cap tightly (general guidance).
  • Do not treat literature values above as lot specifications; consult the CoA for exact analytical data when needed.

Quality and Grades

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

• Interpreting typical grades for this class (general guidance):

  • Research grade reagents for bioorthogonal chemistry are commonly supplied at high chemical purity with LC/MS and HPLC area % reported on the CoA. When stated, “>95%” or higher indicates minimal low-MW impurities that could compromise conjugations.
  • Residual solvents, water content, and homolog distribution (for PEG12) are often characterized; exact acceptance criteria are lot-specific.
  • Color can vary from pink to deep magenta due to the tetrazine chromophore; minor color variations do not necessarily indicate impurities if chromatographic purity is within spec.

• Stabilizer/antioxidant: Not specified for this item; refer to CoA/Spec Sheet.

• What to verify before critical work:

  • Review the CoA for identity (HRMS/ESI-MS), purity (HPLC), and if applicable, assay for active tetrazine (e.g., reaction with TCO test).
  • Confirm water content if moisture-sensitive downstream steps are planned; dry under vacuum if appropriate.
  • For quantitative bioconjugation, determine exact concentration by UV–vis using literature ε for the tetrazine chromophore or by weight after desiccation.

Reaction and Applications

Primary role: methyltetrazine enables rapid, catalyst-free bioorthogonal ligation via inverse electron-demand Diels–Alder (IEDDA) with strained dienophiles such as trans‑cyclooctene (TCO), bicyclononyne (BCN), and norbornene (literature). The PEG12‑acid terminus allows installation onto amine-bearing targets by amide coupling.

• Application families (literature/general):

  • Bioconjugation: install the methyltetrazine on proteins, antibodies, peptides, polymers, nanoparticles via EDC/NHS activation of the acid and reaction with lysine ε‑amines or surface amines.
  • Two-step labeling: first conjugate methyltetrazine to the biomolecule; then add a TCO/BCN‑functional probe (fluorophore, payload) for rapid, selective ligation under physiological conditions.
  • Surface modification: functionalize hydrogels, microbeads, or planar substrates bearing amines to create tetrazine-presenting surfaces for patterning with TCO‑decorated ligands.
  • Polymer chemistry: introduce reactive handles into PEGylated scaffolds for post-polymerization modification.

• Mechanistic notes (literature):

  • IEDDA rate constants with TCO can reach 10^4–10^6 M−1 s−1 depending on substitution and solvent; BCN/norbornene are slower but still practical.
  • The cycloaddition is followed by N2 extrusion to give a dihydropyridazine/pyridazine product, typically with high yield and orthogonality to thiol–maleimide and azide–alkyne chemistries.

• Practical tips:

  • Protect from light; avoid strong reductants which quench tetrazines.
  • For coupling the acid, use freshly prepared NHS esters (in situ with EDC/NHS) to minimize hydrolysis; remove excess reagents before the IEDDA step to avoid off‑target reactions.

Reaction Conditions

General literature guidance; adjust to your system and verify by small-scale trials.

• Amide coupling using EDC/NHS (install methyltetrazine onto amines):

  • Solvent/buffer: 50 mM MES, pH 5.5–6.0; optional 0–20% DMF or DMSO.
  • Reagents: 1.0 equiv Methyltetrazine-PEG12-acid; 1.2–2.0 equiv EDC·HCl; 1.5–3.0 equiv NHS (or sulfo‑NHS for aqueous systems).
  • Temperature/time: 20–25°C, 15–60 min activation; then add amine substrate (1–10 mM) and react 1–4 h (proteins may need lower concentrations and longer times).
  • Notes: Remove excess carbodiimide before IEDDA; monitor by UV–vis (tetrazine band ~520–540 nm, literature) or MS.

• IEDDA ligation with TCO/BCN (after installation):

  • Solvent: PBS or HEPES, pH 6.8–7.8; ≤10% DMSO acceptable.
  • Concentrations: 0.5–50 µM partners; use 1.2–2.0 equiv of the faster-reacting partner for complete conversion.
  • Temperature/time: RT; reactions with TCO often complete within seconds to minutes at µM levels (k 10^4–10^6 M−1 s−1, literature). BCN/norbornene require longer.
  • Compatibility: No metal catalyst required; tolerant of salts and many additives; avoid strong reductants and excess free thiols if undesired side reactions are a concern.

• Workup/purification:

  • Small molecules: preparative HPLC or SPE.
  • Proteins: desalting columns, dialysis, or SEC; verify degree of labeling by UV–vis or MS.

Safety and Handling

• Item-specific hazard data (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: Not specified for this item; refer to SDS.
  • Pictograms: Not specified for this item; refer to SDS.

• General safety guidance for tetrazine–PEG acids (literature/practice; defer to SDS for authority):

  • Tetrazines are reactive dienes; avoid contact with strong nucleophiles, strong reducing agents (which can form dihydrotetrazines), and strong bases for prolonged periods.
  • Protect from light to minimize photodegradation and color changes; store cold and dry.
  • Avoid oxidants and metal powders that could promote side reactions; although methyltetrazines are generally stable under standard lab conditions, handle with care.
  • PPE: laboratory coat, safety glasses, and appropriate chemically resistant gloves (e.g., nitrile). Work in a fume hood to avoid inhalation of dust/aerosols.
  • First aid (overview; follow institutional protocol):
    • Skin/eye contact: rinse with water for at least 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.
  • Spill/cleanup: absorb with inert material, avoid generating dust, collect for disposal according to local regulations.

• Storage incompatibilities (general): strong bases, strong reducing agents, and amine-containing buffers if long-term storage in solution (risk of unintended coupling when activated).

Solvent Selection

This reagent combines a hydrophilic PEG12 chain with a terminal acid and an aromatic tetrazine. Selection of solvent depends on task (stock preparation, activation, conjugation).

• Polarity/miscibility (general/literature):

  • Highly soluble in DMSO and DMF; typically soluble in acetonitrile and mixtures with water.
  • Readily formulated into aqueous buffers from DMSO stocks; solubility in pure water increases at pH > pKa (carboxylate form).

• Recommended use cases:

  • Stock solutions: 5–50 mM in anhydrous DMSO; aliquot and store at −20°C protected from light.
  • EDC/NHS activation: MES (pH 5.0–6.0) or phosphate buffers lacking primary amines; co-solvent 0–20% DMF or DMSO if needed.
  • Direct IEDDA labeling (after coupling methyltetrazine to a biomolecule): aqueous buffers (PBS, HEPES) at neutral pH.

• Comparison (general guidance):

  • DMSO: maximal solubility; benign to most biomolecules at ≤10% v/v final.
  • DMF: strong solvent for coupling setups; consider toxicity and removal.
  • Aqueous (MES/PBS): preferred for bioconjugation steps once dissolved; avoid Tris or other primary amine buffers during carbodiimide chemistry.

• Practical tips:

  • Filter stocks (0.22 µm PTFE) if particulate forms.
  • Limit water exposure before coupling to reduce premature hydrolysis of activated esters (if generated in situ).

Storage and Reconstitution

• Item-specific storage and shipping (from Product Data):

  • Storage Conditions: Store at −20°C.
  • Shipped In: Ice chest + Ice pads.
  • Research Use Note: For research use only.

• General handling and reconstitution (literature/practice):

  • Protect from light and moisture. Keep container tightly closed; consider desiccant in the secondary container.
  • Allow vial to warm to room temperature in a desiccated environment before opening to minimize moisture uptake/condensation.
  • Prepare concentrated stocks (e.g., 5–50 mM) in anhydrous DMSO. Vortex until fully dissolved; if needed, sonicate briefly.
  • Aliquot stocks to single-use vials to avoid repeated freeze–thaw; store aliquots at −20°C protected from light. Typical stability of tetrazine stocks is improved under inert gas (N2/Ar); verify activity periodically by test reaction with TCO (literature practice).
  • For aqueous working solutions, dilute DMSO stocks into buffer immediately before use. Avoid prolonged storage in basic solutions or with reducing agents.

• Shelf-life and specifications:

  • No item-specific shelf-life, stabilizers, or assay values are provided here. For exact stability and analytical acceptance criteria, consult the product’s CoA/Spec Sheet and SDS.

Structure and Identity

Brief description: Methyltetrazine-PEG12-acid is a bioorthogonal conjugation handle combining a reactive methyltetrazine ring with a 12-unit polyethylene glycol (PEG12) spacer terminating in a carboxylic acid.

• Item-specific identifiers (from Product Data):

  • CAS: 2183440-39-1
  • Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular Weight: 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.

• Structural features (general/literature description):

  • Contains a 1,2,4,5-tetrazine ring substituted with a methyl group ("methyltetrazine"), which functions as an electron-poor diene for inverse electron-demand Diels–Alder (IEDDA) reactions.
  • A flexible, hydrophilic PEG12 chain (approximately 12 ethylene oxide repeats) provides a ~5–6 nm spacer (literature) improving aqueous solubility and reducing steric hindrance in bioconjugations.
  • Terminal carboxylic acid enables amide-bond formation to primary amines after activation (e.g., EDC/NHS), providing a route to introduce the methyltetrazine tag onto biomolecules, polymers, or surfaces.
  • No stereogenic centers expected in the PEG chain; the tetrazine ring is planar and aromatic. The material may exist as a distribution of PEG homologs centered on 12 EO units (typical for PEGylated reagents; literature note).

• 2D structure in words: Methyltetrazine ring — flexible poly(ethylene glycol) chain — terminal –CH2–CO2H (carboxylic acid).

Synthetic Utility

Functional groups and reactivity (literature/general):

• Carboxylic acid terminus: amenable to standard amide/ester coupling (EDC/NHS, HATU, mixed anhydrides). Enables grafting onto amines on biomolecules, polymers, and surfaces.
• Methyltetrazine ring: highly electron-deficient diene for IEDDA ligations with strained alkenes/alkynes (TCO, BCN, norbornene). Post-ligation, forms pyridazine derivatives after N2 extrusion.
• PEG12 spacer: increases hydrophilicity and provides a conformationally flexible linker to mitigate steric hindrance and preserve biomolecule function.

Transformations enabled:

• NHS ester formation in situ or via isolated active esters for controlled bioconjugations.
• Sequential orthogonal modifications: e.g., install tetrazine via amide bond, then click to TCO-bearing fluorophores; compatible with azide–alkyne chemistries on different residues.
• Solid-support chemistry: immobilize on amine-functional resins to create tetrazine-bearing scavengers or capture media for TCO-tagged entities.

Retrosynthetic perspective:

• The molecule can be viewed as three modules: (i) methyltetrazine cap, (ii) PEG12 linker, (iii) terminal acid. Modularity allows adaptation to other handles (e.g., swap acid for azide/alkyne) while preserving the bioorthogonal diene functionality.

Target Specificity

Not applicable. This product is a small-molecule chemical reagent, not a biological targeting agent or antibody. It exhibits chemical selectivity for strained alkene/alkyne partners (e.g., TCO, BCN) via IEDDA, but no biological epitope or species specificity applies.