Elastase Substrate, Fluorogenic (MeOSuc-AAPV-AFC) - ≥99% , CAS No.E276322

CAS: E276322 Cat. No.: E276322 Summenformel: C₃₁H₃₈F₃N₅O₉ Molekulargewicht: 681.67

QUALITÄT & REINHEIT ≥99%

Synonyms

N-(Methoxysuccinyl)-Ala-Ala-Pro-Val-7-amino-4-trifluormethylcoumarin

Storage

Store at -20°C,Desiccated

Shipped In

Ice chest + Ice pads

Übersicht

Store at -20°C. Store under desiccating conditions. The product can be stored for up to 12 months.

Spezifikationen

Synonyme

N-(Methoxysuccinyl)-Ala-Ala-Pro-Val-7-amino-4-trifluormethylcoumarin

Spezifikationen & Reinheit

≥99%

Quelle

Synthetic

Storage

Store at -20°C,Desiccated

Verschickt in

Ice chest + Ice pads

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

Reinheit

≥99%

Namen und Kennungen

Molekulargewicht

681.67

Chemische und physikalische Eigenschaften

• Löslichkeit: Soluble in DMSO

Application Protocols

No vendor-validated protocols are provided for this SKU; the following is a general microplate assay workflow (literature/general guidance):

• Stock preparation:
  • Dissolve MeOSuc-AAPV-AFC in anhydrous DMSO to 5–10 mM. Prepare single-use aliquots; protect from light.
• Assay buffer (example):
  • 50 mM Tris–HCl, pH 8.0; 150 mM NaCl; 0.1 mg/mL BSA. Avoid detergents unless validated.
• Plate setup (96- or 384-well black, flat-bottom):
  • Pre-equilibrate buffer and enzyme to assay temperature (e.g., 25 or 37°C).
  • Add enzyme to wells; initiate reaction by adding substrate to reach 1–20 μM final with ≤1% DMSO.
  • Include blanks (no enzyme), negatives (with known elastase inhibitor), and a free AFC standard curve (e.g., 0.05–5 μM) for calibration.
• Read parameters:
  • Kinetic read at Ex 400–410 nm / Em 490–505 nm every 30 s for 10–30 min; maintain linear range (≤10% substrate consumption).
• Data analysis:
  • Convert RFU to μM AFC using the standard curve. Fit initial rates to Michaelis–Menten; determine IC50 or Ki as appropriate.
• Notes:
  • Verify inner-filter effects at high substrate concentrations; adjust gain/slit widths accordingly.
  • Keep plate edge effects low by using incubated plate shakers and consistent volumes (e.g., 50–200 μL per well depending on format).

Biological Roles

This substrate is an analytical tool that mimics preferred cleavage motifs of elastase-like serine proteases.

• Target enzymes (biology overview):
  • Neutrophil elastase (ELANE) and pancreatic elastase cleave after small hydrophobic residues; the MeOSuc–AAPV motif reflects this specificity (literature).
• Mechanism of signal generation:
  • Proteolysis at the peptide–AFC junction liberates free AFC, which fluoresces strongly at ~500 nm; the intact conjugate is weakly fluorescent.
• Experimental relevance:
  • Enables quantification of protease activity in biochemical assays, cell lysates, or conditioned media (with appropriate controls and calibration).
  • Supports studies on protease regulation, inhibitor SAR, and substrate specificity.
• Notes:
  • Substrate usage reports should correct for possible off-target cleavage by other serine proteases; include selective inhibitors or counter-substrates.
  • AFC fluorescence increases with pH across neutral–basic range; maintain consistent pH to compare rates.

No physiological or clinical claims are implied; this material is provided strictly for research use.

Buffer Applications

This is not a buffer component per se; however, appropriate buffer selection is critical for enzymatic assays with this substrate.

• Common assay buffers (literature/general):
  • Tris–HCl: 50 mM, pH 7.5–8.5; often with 150 mM NaCl and 0.01–0.1% Tween-20 only if validated (detergents can affect fluorescence and enzyme activity).
  • HEPES–NaOH: 50 mM, pH 7.4–8.0; good buffering near neutral pH with lower temperature sensitivity.
  • Additives: 0.1 mg/mL BSA to minimize adsorption; Ca2+ or Na+ as required by specific protease systems (verify with enzyme supplier).
• Practical tips:
  • Filter (0.22 μm) and degass buffers to reduce light scatter.
  • Maintain ionic strength and pH constant across test and control wells.
  • Avoid high phosphate at pH >8 if observing reduced signal (possible inner-filter/complexation effects; validate conditions).

If a validated buffer recipe is required for compliance or comparability, consult method-specific literature or your internal SOPs.

Green Alternatives

While the substrate itself is fixed, greener choices can be made for solvents and workflows.

• Solvent considerations:
  • Prefer DMSO over DMF where possible (lower toxicity profile and better biodegradability profile among polar aprotic solvents; literature consensus), and keep final organic content minimal.
  • Explore aqueous-first methods: dissolve minimal DMSO stock and immediately dilute into buffer to reduce organic solvent usage.
• Process optimizations:
  • Miniaturize assays (e.g., 384/1536-well) to reduce per-experiment solvent and plastic waste.
  • Implement wash-free kinetic readouts to avoid quenching/extraction steps.
• Waste handling:
  • Segregate DMSO/DMF waste; use designated non-halogenated solvent streams where allowed.
• Trade-offs:
  • Lowering organic cosolvent improves sustainability but may limit solubility at high substrate loads; balance concentration with enzyme sensitivity and instrument dynamic range.

Comparison (general):

• DMSO vs DMF:
  • Solubility: both high; DMSO typically sufficient.
  • Enzyme tolerance: DMSO often superior at equal % v/v.
  • EHS profile: DMSO generally preferred; DMF is a reproductive hazard (consult SDS).

Pharmaceutical Uses

No excipient or therapeutic use is claimed. For research use only.

• Relevance to pharmaceutical R&D (general):
  • Lead discovery and optimization: HTS and kinetic assays for elastase inhibitors using MeOSuc-AAPV-AFC as a readout.
  • Selectivity panels: Profiling compound activity across serine proteases to assess off-target liability.
  • Assay development/validation: Determination of kinetic parameters and mechanism-of-action for protease modulators.
• Formulation context:
  • Not used as a formulation excipient; applied solely as an analytical substrate in preclinical research workflows.

Physical Properties

• 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 / boiling point: Not specified for this item; refer to CoA/Spec Sheet.
• Density: Not applicable for a solid peptide-like substrate; Not specified for this item.
• Solubility (literature/general guidance):
  • Highly soluble in anhydrous DMSO or DMF for stock solutions (e.g., 1–10 mM typical for microplate assays).
  • Aqueous buffers: working dilutions prepared by adding a small aliquot of DMSO stock into assay buffer (common final DMSO ≤1–2% v/v to preserve enzyme activity; literature guidance).
• Spectral properties (AFC, literature values):
  • Excitation max ~400–410 nm; emission max ~490–505 nm upon release of free AFC; extinction and quantum yield depend on buffer pH (signal increases at neutral–basic pH).
• pKa/logP/refractive index: Not typically specified for peptide–coumarin substrates; Not specified for this item.

Note: Values above labeled as “literature/general guidance” are provided to aid method development; consult the item’s CoA/SDS for any definitive specifications for this specific SKU.

Quality and Grades

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
• Typical expectations for fluorogenic peptide substrates (general guidance):
  • High chemical purity and single major HPLC peak are desirable to minimize background signal and non-specific hydrolysis.
  • Low residual free AFC content to ensure high signal-to-background upon enzymatic cleavage.
  • Identity confirmation by LC–MS and analytical HPLC; optional NMR on protected intermediates.
• Stabilizer/antioxidant: Not specified for this item; refer to CoA/Spec Sheet. Absence or presence of stabilizers can influence baseline fluorescence and storage stability.
• UV/fluorescence background: For enzyme kinetics or HTS, low baseline fluorescence and well-defined emission at ~500 nm (AFC) are important; lot-specific spectra may be provided on request.
• Documentation: For regulated workflows or cross-lab comparability, request the Certificate of Analysis (CoA) for exact purity, identity data, and assay acceptance criteria applicable to SKU E276322.

Reaction and Applications

This material is a fluorogenic reporter for enzymatic reactions, not a general synthetic reagent.

• Primary application (literature/general):
  • Real-time monitoring of elastase-like serine protease activity. Cleavage at the Val–AFC amide liberates free AFC, increasing fluorescence (Ex ~400–410 nm/Em ~490–505 nm).
• Use cases:
  • Enzyme kinetics (Km, kcat, Ki) for neutrophil elastase (human), pancreatic elastase, and engineered variants with elastase-like specificity.
  • High-throughput screening (HTS) for inhibitors/activators of elastase.
  • Selectivity profiling of related serine proteases (trypsin/chymotrypsin controls) to validate sequence specificity.
• Practical considerations:
  • Maintain substrate concentrations below Km for inhibitor IC50 determinations; use initial-rate conditions (≤10% substrate consumption).
  • Control final DMSO ≤1–2% v/v; confirm enzyme tolerance.
  • Include no-enzyme and no-substrate wells to quantify background/bleed-through.
  • AFC fluorescence is pH-dependent; standardize buffer pH and ionic strength.
• Data quality:
  • Calibrate instrument with free AFC standard curve to convert RFU to molar product and to assess inner-filter effects.
  • Monitor linearity over time; avoid substrate depletion and product inhibition.

Manufacturer Applications: Not specified in the catalog entry beyond “For research use only.” The above expands on typical literature use of MeOSuc-AAPV-AFC.

Reaction Conditions

Enzymatic assay conditions (literature/general guidance for elastase using MeOSuc-AAPV-AFC):

• Temperature: 25–37°C; maintain constant temperature for kinetic comparability.
• pH: 7.5–8.5 (Tris or HEPES buffers commonly used). Activity of elastases often increases toward mildly basic pH.
• Ionic strength: 100–200 mM NaCl typical; optimize per enzyme source.
• Substrate concentration: 1–50 μM common for kinetic assays; keep ≤10% conversion during initial-rate measurement.
• Enzyme concentration: Adjust to achieve linear product formation for ≥5–10 min. Start with low-nanomolar active enzyme and titrate.
• Cosolvent: Final DMSO ≤1–2% v/v preferred; verify enzyme tolerance.
• Readout: Fluorescence Ex 400–410 nm / Em 490–505 nm; read every 15–60 s for kinetics.
• Controls: No-enzyme, no-substrate, and free AFC calibration curve (e.g., 0.05–5 μM) to convert RFU to μM product.
• Expected outcomes: Under optimized conditions, linear rates and robust Z′-factors (>0.5) are routinely achievable in HTS (literature benchmark, not a specification for this item).

These parameters are guidance only; optimize for your specific enzyme source and instrument.

Safety and Handling

• GHS classification, signal word, pictograms, and H-statements: Not specified for this item; refer to the SDS for authoritative safety information.
• General precautions (good laboratory practice):
  • Handle under low moisture and reduced light to preserve activity and prevent degradation of the fluorophore.
  • Avoid inhalation of dust and contact with skin/eyes. Use PPE: lab coat, safety glasses, and appropriate gloves.
  • Work with stock solutions in a fume hood when using organic solvents (DMSO/DMF).
• Incompatibilities (general):
  • Strong acids/bases and oxidizing agents may degrade the peptide or fluorophore.
  • Avoid prolonged exposure to aqueous buffers at elevated temperature or high pH in the absence of enzyme (hydrolysis/bleaching risk).
• First aid (overview – defer to SDS):
  • Skin/eye contact: Rinse with water for several minutes; remove contaminated clothing; seek medical attention if irritation persists.
  • Ingestion/inhalation: Seek medical attention; move to fresh air if inhaled.
• Spill & waste: Collect solids or absorb liquids with inert material; dispose of according to institutional and local regulations.
• Peroxide formation: Not applicable to this peptide substrate; however, check any ether-containing cosolvents separately per their SDS.

Always consult and follow the SDS for the specific lot prior to use.

Solvent Selection

• Primary stock solvents (literature/general):
  • DMSO (anhydrous): Preferred for 1–10 mM stocks owing to excellent solubility and enzyme compatibility at ≤1–2% v/v final.
  • DMF (anhydrous): Alternative to DMSO; similar handling, sometimes slightly lower enzyme tolerance.
• Aqueous buffers for assays:
  • Tris, HEPES, or phosphate buffers at pH ~7.5–8.5 commonly used for serine elastases; include 0.1 mg/mL BSA to reduce surface adsorption where needed.
• Miscibility/compatibility (general):
  • DMSO and DMF are fully miscible with water; minimize final organic fraction to maintain enzyme activity and reduce background.
• When to choose which:
  • Select DMSO for routine HTS and kinetic assays; robust, low volatility, better enzyme tolerance.
  • Choose DMF if compound-solubility dictates or when DMSO interference is suspected.
• Practical tips:
  • Prepare small single-use aliquots to avoid water uptake and freeze–thaw.
  • Filter aqueous buffers (0.22 μm) and degas if high precision fluorescence is required to reduce scatter.
  • Avoid detergents with aromatic fluorophores unless validated (can alter quantum yield).

Storage and Reconstitution

• Storage (item-specific): Store at -20°C, desiccated. Protect from light. Shipped in an ice chest with ice pads.
• Stability notes (general guidance):
  • Keep container tightly closed under dry, inert atmosphere if possible to prevent moisture uptake and hydrolysis.
  • Avoid repeated freeze–thaw cycles; AFC conjugates can degrade with moisture and light.
• Reconstitution (general guidance for assay use):
  • Dissolve in anhydrous DMSO to prepare concentrated stock (e.g., 5–10 mM). Vortex gently until fully dissolved.
  • Dispense single-use aliquots (e.g., 10–50 μL) in amber vials or light-protective tubes; store at -20°C or below.
  • For working solutions, dilute DMSO stock into pre-warmed assay buffer to the desired final concentration, keeping final DMSO ≤1–2% v/v.
• Handling tips:
  • Thaw aliquots at room temperature, brief spin, use immediately. Do not refreeze partially used aliquots.
  • Monitor baseline fluorescence of fresh buffer + DMSO to ensure low background before adding enzyme.

For definitive, lot-specific stability and reconstitution instructions, consult the CoA and SDS for SKU E276322.

Structure and Identity

Fluorogenic peptide substrate designed for serine elastases; consists of an N-terminal MeOSuc cap on the tetrapeptide AAPV linked via the C-terminus to the fluorophore AFC (7-amino-4-trifluoromethylcoumarin). Upon proteolytic cleavage after the Val residue, free AFC is released, yielding a fluorescence increase.

• Product name: Elastase Substrate, Fluorogenic (MeOSuc-AAPV-AFC)
• Common abbreviation: MeOSuc-AAPV-AFC
• Structural features (general description):
  • MeOSuc: 2-methoxy-succinyl N-cap that enhances selectivity toward elastase-like serine proteases.
  • Peptide: Ala–Ala–Pro–Val (AAPV), a canonical elastase recognition sequence.
  • Fluorophore: AFC (7-amino-4-trifluoromethylcoumarin), released upon cleavage to generate strong green fluorescence.
  • Architecture: MeOSuc–Ala–Ala–Pro–Val–AFC (peptide–fluorophore amide linkage at the C-terminus).
• Stereochemistry: peptide residues are typically L-amino acids (literature, unless otherwise noted on CoA).
• 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.

2D structure in words: MeOSuc carbonyl attached to the N-terminus of a linear L-Ala–L-Ala–L-Pro–L-Val tetrapeptide; the Val C-terminus forms an amide with the amino group of a 7-amino-4-trifluoromethyl-substituted coumarin ring.

Synthetic Utility

This product is primarily an assay substrate rather than a building block; however, its chemical architecture informs custom probe design (literature/general guidance):

• Functional groups:
  • Amide bonds connecting MeOSuc cap, AAPV peptide, and AFC fluorophore.
  • Coumarin core (AFC) bearing a trifluoromethyl group, providing high quantum yield upon amide cleavage.
• Utility considerations:
  • Sequence and capping group can be varied synthetically to tune protease selectivity (e.g., substitutions at P4–P1 positions, alternate N-caps).
  • The AFC leaving group allows direct fluorescence readout without coupled enzymes.
• For custom synthesis:
  • Standard Fmoc solid-phase peptide synthesis (SPPS) of AAPV, on-resin N-capping with MeOSuc derivative, followed by C-terminal coupling to AFC (e.g., via activated ester or carbodiimide coupling) and global deprotection.
  • Purification by preparative RP-HPLC with gradient water/acetonitrile + 0.1% TFA or alternatives (e.g., formic acid for MS-compatibility).

Note: These are general synthetic insights and not procedures for this specific catalog item.

Target Specificity

This section is adapted for a substrate (not an antibody). No clone/epitope data apply.

• Enzyme target (literature): Elastase-like serine proteases with preference for small hydrophobic residues at P1–P4, exemplified by the AAPV motif.
• Cleavage site: Typically at the Val–AFC amide bond (P1 = Val). Cleavage liberates free AFC.
• Selectivity notes:
  • The MeOSuc N-cap enhances selectivity for elastase over chymotrypsin- or trypsin-like proteases, but cross-reactivity can occur; confirm with inhibitor controls (e.g., elastase-selective inhibitors vs serine protease pan-inhibitors; literature guidance).
  • Substrate selectivity can vary with enzyme source (human neutrophil vs porcine pancreatic elastase). Validate under your assay conditions.

All specificity statements above are literature-based generalizations and are not lot-specific certifications.