iso-VQA-ACC TFA , CAS No.I1443153

CAS: I1443153 Cat. No.: I1443153 Formula: C₂₉H₃₅N₇O₉.xC₂HF₃O₂ Peso molecolare: 625.63 (free base)

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Panoramica

iso-VQA-ACC TFA is a constitutive proteasome substrate.

Specifications

Condizioni di conservazione di stoccaggio

Store at -20°C

Spedito in

Ice chest + Ice pads

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Nomi e identificatori

• Peso molecolare: 625.63 (free base)

Application Protocols

Item-specific, validated application protocols are not provided. The following is a general, literature-based workflow for fluorogenic protease assays using peptidyl-ACC substrates.

Basic microplate assay (general guidance):

• Reagents:
  • Substrate stock in DMSO (e.g., 10–20 mM).
  • Assay buffer (e.g., 50 mM HEPES, 100 mM NaCl, 1 mM DTT if needed, pH 7.5).
  • Protease of interest, quantified and pre-diluted on ice.
• Procedure (96- or 384-well black plate):
  1. Equilibrate buffer and enzyme to assay temperature (e.g., 25–30°C).
  2. Add buffer and enzyme to wells; initiate reaction by adding substrate to desired final concentration with ≤1% DMSO.
  3. Mix gently; measure fluorescence kinetically (Ex/Em appropriate for ACC) every 30–60 s for 10–30 min.
  4. Include blanks (no enzyme) and positive controls (known substrate/enzyme).
• Data analysis: Convert RFU/s to rate using a calibration curve for free ACC if available; calculate KM, kcat, or inhibitor IC50 with appropriate models.

Tips:

• Verify linearity of signal over time and with enzyme concentration.
• Minimize edge effects by using plate sealers and consistent incubation.
• Protect reagents from light and minimize freeze–thaw cycles by aliquoting stocks.

Biological Roles

Item-specific biological roles are not provided and, as a synthetic research reagent, this compound is intended for laboratory use only.

General/literature context for peptidyl-ACC substrates:

• Functional role: Serve as reporter substrates that release a fluorescent ACC moiety upon enzymatic cleavage by proteases, enabling real-time monitoring of catalytic activity in cell-free systems.
• Pathway interrogation: By varying the peptide sequence (e.g., VQA or its isomers), researchers probe substrate preferences of proteases involved in protein turnover, signaling, and antigen processing in vitro.
• Selectivity determinants: Residues at P3–P1 positions strongly influence recognition by serine, cysteine, or metalloproteases; ACC fluorophore generally does not dictate specificity but provides a sensitive readout.
• Biochemical advantages: High signal-to-background, straightforward kinetic analysis, compatibility with microplate readers, and low substrate consumption.

Important limitations:

• These substrates do not have inherent physiological roles; they are tools to study enzymes under controlled laboratory conditions.
• Cellular uptake and compartmentalization are typically poor for charged peptide salts; applications are primarily in purified enzyme or lysate assays unless delivery strategies are employed (not covered here).

Buffer Applications

Item-specific buffer recommendations are not provided. The following are general, literature-based practices for enzymatic assays with peptidyl-ACC substrates.

• Common buffers:
  • HEPES (10–50 mM), pH 7.0–8.0: Good buffering near neutrality; low metal binding.
  • Tris (25–50 mM), pH 7.5–8.5: Frequently used for serine/cysteine proteases; beware of temperature-dependent pKa.
  • Phosphate (10–50 mM), pH 6.5–7.5: Minimal autofluorescence; can coordinate metals.
• Ionic strength: 100–200 mM NaCl often stabilizes proteins and reduces nonspecific binding.
• Additives: 0.01–0.05% Tween-20 to reduce surface adsorption; 1 mM DTT or TCEP for cysteine proteases; avoid EDTA if metalloproteases are being studied unless intentional chelation is required.
• Solvent content: Keep DMSO ≤1–2% v/v to limit enzyme inhibition while maintaining substrate solubility.
• Light control: Use black-walled plates and protect solutions from ambient light to reduce photobleaching.

Practical notes:

• Adjust buffer pH at the assay temperature (e.g., 25–37°C) to maintain accuracy.
• Validate fluorescence background of each buffer in the absence of enzyme/substrate.
• If high background is observed, consider desalting to reduce residual TFA or switching to a buffer with lower intrinsic fluorescence.

Green Alternatives

While this product is itself a specialty research substrate, greener practices can be applied to its use.

Considerations and alternatives (general):

• Solvent choice:
  • Prefer water-based buffers with minimal DMSO (≤1–2%) for assays.
  • If organic solvent is required, DMSO is generally preferred over DMF or NMP for lower volatility and improved safety profile; 2-methyltetrahydrofuran (2-MeTHF) or CPME are not typically used for aqueous enzyme assays.
• Waste minimization:
  • Miniaturize assay volumes (e.g., 384- to 1536-well plates) to reduce solvent and plastic use.
  • Use multi-use, resealable amber vials to minimize container waste and light exposure.
• Energy and storage:
  • Store at –20°C as specified; avoid unnecessary ultra-low freezers unless stability data require it.
• Safer deprotection/counterion exchange (for synthesis or post-processing, literature):
  • If counterion exchange is desired, ion-exchange to acetate or chloride in aqueous ethanol can reduce TFA load in waste streams.

Trade-offs:

• Lower DMSO percentages improve eco-profile but may limit solubility and require lower stock concentrations.
• Replacing TFA counterion may improve assay performance for certain enzymes but adds processing steps and solvent use. Evaluate based on data-driven need.

Pharmaceutical Uses

No item-specific pharmacopeial status or excipient role is provided for this product. It is supplied for research use only.

General context (non-clinical):

• Role in drug discovery: Fluorogenic peptide–ACC substrates are widely used in screening and mechanistic enzymology to support lead identification/optimization of protease modulators.
• Manufacturing relevance: Not typically used as an excipient or process aid; rather, it serves as an analytical reagent for enzyme activity assays in development labs.
• Regulatory considerations: If data generated with this substrate are used to support regulated studies, document lot identity, purity, and analytical methods (HPLC, MS) and maintain chain-of-custody per QA procedures.
• Analytical method development: Substrate turnover can be used to qualify assay system suitability (Z′ factor, S/B) for HTS campaigns.

No therapeutic or clinical use is implied. For any application beyond research, full qualification and validation are required under appropriate quality systems.

Physical Properties

Item-specific physical constants are not provided and should be confirmed from the CoA/Spec Sheet for experimental design.

• 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: Not specified for this item; refer to CoA/Spec Sheet.
• Boiling point: Not applicable to peptides/ionic salts; decomposition is more typical (literature).
• Density: Not specified for this item; refer to CoA/Spec Sheet.
• Refractive index: Not applicable for solids; Not specified for this item; refer to CoA/Spec Sheet.
• Solubility (item-specific): Not specified for this item; refer to CoA/Spec Sheet.

General/literature guidance for peptidyl-ACC TFA salts (non-item-specific):

• State: usually solid; often off-white to light yellow due to coumarin chromophore.
• Solubility: typically soluble in polar aprotic solvents (DMSO, DMF); variable aqueous solubility that improves with co-solvent or buffering; insoluble in nonpolar solvents.
• Photophysics: ACC-type fluorophores typically exhibit excitation/emission in the near-UV/blue region; exact maxima depend on substitution and environment and must be verified for the specific compound.
• Hygroscopicity: TFA salts of peptides can be hygroscopic and may absorb moisture/CO2; handle under dry conditions.
• Stability: commonly stable at –20°C protected from light and moisture; solutions in DMSO are generally stable short-term at –20°C (literature).

Quality and Grades

• Grade/Purity (item-specific): Not specified for this item; refer to CoA/Spec Sheet.
• Research use note: For research use only.

Interpretation and context (general):

• Peptidyl fluorogenic substrates are often supplied at high synthetic purity to ensure low background fluorescence and consistent enzymatic kinetics. When a grade/purity is not specified, consult the CoA for HPLC purity, residual solvent profile, and counterion content (e.g., %TFA).
• TFA salt form: The trifluoroacetate counterion is a common result of final global deprotection/cleavage in solid-phase peptide synthesis. Residual TFA can influence mass balance, pH of aqueous solutions, and in some cases enzyme activity; desalting or counterion exchange (e.g., to acetate) may be considered if assay performance is affected.
• QC expectations (literature best practices): identity confirmation by MS and, where feasible, 1H NMR; purity by analytical RP-HPLC with UV/fluorescence detection; water content by Karl Fischer; counterion quantitation by IC or 19F NMR. UV/fluorescence spectra are often provided for fluorogenic substrates.
• Lot-to-lot consistency: For kinetic assays, verify kcat/KM comparability by running a reference enzyme and control substrate; minor differences in counterion or solvation can influence initial rates.

Reaction and Applications

Manufacturer text not provided for this SKU. Based on literature, peptidyl-ACC substrates are widely used as fluorogenic probes for protease activity measurements in biochemical assays.

General applications (non-item-specific):

• Enzyme kinetics: Measure initial rates of ACC fluorophore release upon proteolytic cleavage. Suitable for determining KM, kcat, IC50, and Ki in inhibitor screens.
• Enzyme profiling: Tripeptidyl sequences (e.g., VQA or isomeric variants) provide selectivity toward specific protease families depending on P3–P1 recognition; sequence selection tunes specificity and catalytic efficiency.
• High-throughput screening (HTS): Robust fluorescence readout enables 384/1536-well formats.
• Mechanistic studies: Substrate turnover can be used to probe active-site preferences and pH dependence of catalysis.

Practical tips (literature):

• Stock prep: Dissolve in anhydrous DMSO; quantify by weight and verify concentration spectrophotometrically if the extinction coefficient for the released ACC is known.
• Assay setup: Use matched buffer, ionic strength, and co-factors for the target protease; minimize DMSO to ≤1–2%.
• Controls: Include no-enzyme, heat-inactivated enzyme, and known substrate controls to benchmark signal-to-noise.
• Data quality: Monitor inner-filter effects at high substrate concentrations; keep fluorescence within the linear range of the detector.
• Interference: Some test compounds fluoresce or quench in the same spectral window; run counter-screens to identify artifacts.

Reaction Conditions

No item-specific reaction or processing conditions are provided. The following summarizes literature practices for preparing and handling peptidyl-ACC substrates.

• Stock solution preparation: Dissolve in anhydrous DMSO to 10–50 mM; vortex and, if needed, brief sonication. Filter (0.22 µm PTFE) to remove particulates. Protect from light.
• Enzymatic assay conditions (general):
  • Buffer: HEPES (25–50 mM) or Tris (25–50 mM) at pH 7.0–8.0; 100–150 mM NaCl.
  • Temperature: 25–37°C depending on enzyme; equilibrate plates and reagents.
  • Enzyme concentration: tuned to provide linear initial rates over the measurement window; determine empirically.
  • Substrate range: typically 0.1–5× KM for kinetic characterization; pre-determine KM for your enzyme/substrate pair.
  • Detection: Excitation/emission wavelengths appropriate for ACC; verify instrument settings to avoid saturation.
• Synthesis/processing (if modifying): Coupling in DMF/DCM with HATU/DIPEA at 0–25°C; deprotection with TFA-based cocktails followed by RP-HPLC purification (literature guidance).

Yields and exact parameters are enzyme- and sequence-dependent and must be optimized empirically. Always validate with controls and follow the SDS and CoA for item-specific constraints.

Safety and Handling

Authoritative safety information resides in the SDS. The following consolidates item data with general best practices.

Item-specific hazard data:

• GHS classification: Not specified for this item; refer to SDS.
• Signal word: Not specified for this item; refer to SDS.
• H-statements/Pictograms: Not specified for this item; refer to SDS.

General handling guidance for peptide TFA salts and fluorogenic substrates (literature/best practice):

• Likely hazards: may cause skin/eye/respiratory irritation; avoid dust formation and inhalation; coumarin derivatives can be light-sensitive.
• PPE: lab coat, nitrile gloves, safety glasses; use a chemical fume hood during weighing and solution prep.
• Incompatibilities: strong oxidizers; strong bases/acids may hydrolyze peptide bonds; moisture can degrade TFA salts or alter mass by hydration.
• Special risks: light sensitivity—store and handle under low light or amber containers; potential for DMSO solutions to enhance dermal absorption—avoid skin contact.
• First aid (overview; consult SDS): eye/skin contact—rinse with water for 15 minutes; inhalation—move to fresh air; ingestion—rinse mouth, seek medical attention.
• Spill response: avoid dust, collect with damp disposable towel or HEPA vacuum; dispose per institutional/chemical waste protocols.
• Fire safety: organic solid; use CO2, dry chemical, or foam; combustion may release irritating fumes including nitrogen oxides and HF from TFA-containing residues (general consideration).

Solvent Selection

Item-specific solubility data are not provided; consult the CoA/Spec Sheet. The following provides general guidance for peptidyl-ACC TFA substrates (literature):

• Primary solvents:
  • DMSO: excellent solvating power for peptide–fluorophore conjugates; common for making concentrated stocks (e.g., 10–50 mM).
  • DMF: alternative for stock preparation; similar solvency to DMSO.
• Aqueous use: Working solutions are typically prepared by diluting a DMSO stock into buffered aqueous media (e.g., HEPES, Tris, PBS) to a final DMSO content ≤1–2% to minimize enzyme perturbation.
• Miscibility/compatibility: DMSO is fully miscible with water; avoid high organic percentages for sensitive enzymes. Nonpolar solvents (hexanes, toluene) are generally unsuitable.
• pH considerations: Peptide bond stability is optimal near neutral pH; avoid prolonged exposure to pH >9 or <5 unless required by the enzyme and time is minimized.
• Light sensitivity: Prepare and store solutions in amber vials or wrap with foil.

Comparison (general):

• DMSO vs DMF: DMSO has lower volatility and is more commonly tolerated in biochemical assays; DMF can be more disruptive to some enzymes.
• Aqueous-only: Some highly polar substrates may dissolve in water alone, but peptidyl-ACC salts frequently require an organic co-solvent for reliable stock preparation.

Storage and Reconstitution

Item-specific storage instructions provided:

• Store at –20°C.
• Shipped in an ice chest with ice pads.

Additional best practices (general literature guidance for peptidyl-ACC TFA salts):

• Protection: Store desiccated under inert atmosphere if possible; keep in amber vials to protect from light.
• Reconstitution:
  • Prepare concentrated stocks in anhydrous DMSO (e.g., 10–50 mM).
  • Vortex to dissolve; brief sonication if needed. Avoid heating.
  • Optional: Determine concentration by UV/Vis if an extinction coefficient for the free ACC or conjugate is available; otherwise rely on gravimetric preparation with calibrated balances.
• Aliquoting: Dispense single-use aliquots to avoid repeated freeze–thaw; store aliquots at –20°C to –80°C as compatible with your workflow.
• Handling: Allow vial to warm to room temperature in a desiccator before opening to prevent condensation. Reseal promptly.
• Solution stability: Short-term stability at –20°C is generally good for weeks to months; verify by test assay or HPLC as needed. Discard if precipitation, discoloration, or abnormal background fluorescence is observed.

For any item-specific details (e.g., hygroscopicity, exact solubility, light sensitivity), refer to the CoA/Spec Sheet and SDS.

Structure and Identity

Item-specific identifiers for iso-VQA-ACC TFA are not provided in the product data. Where exact identity attributes are required for regulatory or analytical purposes, consult the CoA/Spec Sheet.

• Molecular formula (item-specific): Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight (item-specific): Not specified for this item; refer to CoA/Spec Sheet.
• SMILES (item-specific): Not specified for this item; refer to CoA/Spec Sheet.
• InChIKey (item-specific): Not specified for this item; refer to CoA/Spec Sheet.
• CAS: I1443153 (catalog identifier provided; not a registered CAS number).

General/literature context (non-item-specific):

• The name “iso-VQA-ACC TFA” suggests an isomeric variant of a peptidyl-ACC fluorogenic substrate, where “VQA” typically denotes a tripeptide (e.g., Val–Gln–Ala or an isomer thereof) and “ACC” commonly refers to 7-amino-4-carbamoylmethylcoumarin, a coumarin fluorophore released upon peptide bond cleavage.
• “TFA” indicates the trifluoroacetate salt form, frequently encountered after final deprotection in peptide synthesis.
• Structural features (typical for peptidyl-ACC TFA, literature): peptide backbone with amide bonds; terminal linkage to a coumarin fluorophore (ACC); potential N-terminal protonation balanced by trifluoroacetate counterion; multiple H-bond donors/acceptors; overall polar character with an embedded aromatic fluorophore.
• 2D description (literature): an N-acylated amino acid chain (V–Q–A isomer) terminating in an amide linkage to the amino-coumarin core; trifluoroacetate present as a counterion; no defined stereochemistry provided here, but peptide stereocenters are commonly L-configured unless otherwise noted (not specified for this item).

Synthetic Utility

Although primarily a biochemical tool, a peptidyl-ACC TFA salt provides useful synthetic handles when modifying or assembling related probes.

General synthetic considerations (literature):

• Functional groups: Multiple amide bonds (peptide), aromatic coumarin core (ACC), and a cationic site balanced by TFA in the salt form.
• Coupling strategies: Standard peptide coupling reagents (HATU, HBTU, PyBOP) for assembling the peptide sequence or for attaching ACC via amide bond formation to the C-terminus.
• Protecting groups: Fmoc/tBu orthogonal protection on solid phase; final global deprotection/cleavage commonly yields the TFA salt.
• Derivatization:
  • Counterion exchange (TFA→AcO−/Cl−) to modify solubility or assay performance.
  • PEGylation or addition of cell-penetrating motifs to alter uptake (for research tool development).
  • Installation of quenchers or FRET pairs for ratiometric readouts.
• Immobilization: Terminal functional groups enable attachment to resins or surfaces for protease profiling arrays.

Limitations:

• The fluorophore and peptide are sensitive to strong acids/bases and prolonged UV exposure; maintain mild conditions.
• Scale-up of peptide–fluorophore conjugates requires rigorous purification (prep RP-HPLC) to remove fluorescent impurities that can confound assays.

Target Specificity

Item-specific target, antigen, or enzyme selectivity is not provided for this product.

General/literature context:

• Peptide sequence dictates protease recognition. Variants of VQA (or isomeric permutations) can bias activity toward particular protease families, but specificity must be established experimentally with purified enzymes and select panels.
• Without validated data for this SKU, no claims are made regarding selectivity or potency. Users should determine target preferences under their assay conditions.

For detailed specificity data, refer to the product’s CoA/Spec Sheet or associated technical notes if available.