This compound belongs to the class of organic compounds known as dipeptides. These are organic compounds containing a sequence of exactly two alpha-amino acids joined by a peptide bond.
External Descriptors
Not available
1. Djoumbou Feunang Y, Eisner R, Knox C, Chepelev L, Hastings J, Owen G, Fahy E, Steinbeck C, Subramanian S, Bolton E, Greiner R, and Wishart DS. ClassyFire: Automated Chemical Classification With A Comprehensive, Computable Taxonomy. Journal of Cheminformatics, 2016, 8:61.
Zertifikate (CoA, COO, BSE/TSE und Analyse-Diagramm)
C of A & Other Certificates(BSE/TSE, COO):
Analytical Chart:
Chemische und physikalische Eigenschaften
Molekulargewicht
397.300 g/mol
XLogP3
1.100
Hydrogen Bond Donor Count
2
Hydrogen Bond Acceptor Count
8
Rotatable Bond Count
3
Exact Mass
397.125 Da
Monoisotopic Mass
397.125 Da
Topological Polar Surface Area
102.000 Ų
Heavy Atom Count
28
Formal Charge
0
Complexity
694.000
Isotope Atom Count
0
Defined Atom Stereocenter Count
2
Undefined Atom Stereocenter Count
0
Defined Bond Stereocenter Count
0
Undefined Bond Stereocenter Count
0
The total count of all stereochemical bonds
0
Covalently-Bonded Unit Count
1
Lösungsrechner
Molarity Calculator
Determine the necessary mass, volume, or concentration for preparing a solution.
Dilution Calculator
Determine the dilution needed to prepare a stock solution.
Reconstitution Calculator
Bewertungen
Kundenbewertungen
Application Protocols
Example protocol: Continuous fluorogenic protease assay (general, literature-based)
Reagents:
H-Ala-Pro-AFC stock: 10–20 mM in anhydrous DMSO (protect from light).
Assay buffer: 50 mM HEPES, 100 mM NaCl, pH 7.5; 0.01% Tween-20; optional 1 mM EDTA (unless metalloprotease).
Enzyme: purified target protease, kept on ice.
AFC standard: prepare 0–5 µM free AFC in assay buffer with matched DMSO.
Plate setup (96-well):
Add 190 µL buffer ± enzyme to each well.
Initiate reaction by adding 10 µL substrate stock to give 5–50 µM final substrate and ≤1% DMSO.
Mix gently; read immediately.
Instrument settings:
Excitation 400–410 nm; emission 490–505 nm; read every 30–60 s for 10–30 min at 25–37°C.
Data analysis:
Subtract no-enzyme blank.
Convert RFU to µM product using AFC standard curve.
Fit initial rates to Michaelis–Menten or determine IC50 values for inhibitors.
Notes:
Verify linearity window and avoid substrate depletion (>10%).
If adsorption is observed, include 0.01–0.1% BSA.
This is a general protocol; optimize for your specific enzyme. Not a validated, item-specific method.
Biological Roles
This product is a synthetic fluorogenic substrate and does not have a native biological role. It serves as a probe to report on proteolytic activity in biochemical assays.
Background (general, literature):
Proline-containing dipeptides (e.g., Ala–Pro) are informative motifs for characterizing proteases that recognize N-terminal Xaa–Pro sequences. Such enzymes can play roles in peptide processing pathways in various organisms; however, H-Ala-Pro-AFC itself is only a tool compound to measure activity in vitro.
The AFC fluorophore is a coumarin derivative engineered for strong fluorescence when released as a free amine after proteolysis, providing sensitive readouts with minimal interference from typical protein autofluorescence.
Use context:
Employed in enzyme mechanism studies, inhibitor screening, and activity profiling in cell-free extracts or purified enzyme systems.
Note: No clinical or therapeutic claims are implied. For research use only.
Buffer Applications
This product is not a buffering reagent. However, buffer composition is critical for its use as a fluorogenic protease substrate.
General buffer guidance (literature):
Common assay buffers: 25–100 mM HEPES, Tris, or phosphate, pH 7.0–8.0, depending on enzyme optimum. Include 100–150 mM NaCl if required for stability.
Additives: 0.01–0.1% BSA or 0.01% Tween-20 can reduce nonspecific adsorption; include DTT or TCEP if the enzyme requires a reducing environment (avoid excess reductant that may affect fluorescence baselines).
Metal ions: chelators (e.g., 1 mM EDTA) are often included unless the target enzyme is metallodependent.
DMSO carryover: keep ≤1–2% v/v final to preserve enzyme activity while maintaining substrate solubility.
Always optimize pH/ionic strength for the specific protease under study. These are literature-based suggestions, not product specifications.
Green Alternatives
Sustainability considerations focus on solvent selection and assay design rather than the molecule itself.
Use water-based buffers with minimal organic cosolvent (e.g., ≤1% v/v DMSO), validating enzyme tolerance.
Prefer DMSO over DMF for stock solutions due to lower toxicity and better environmental profile.
Minimize plastic consumables by switching to reusable glass cuvettes/plates where feasible and validated for low background fluorescence.
Employ miniaturized assays (384- or 1536-well formats) to reduce reagent use and waste.
Comparative overview (informational):
Solvent choice for stocks
DMSO: greener of the two common options; fully miscible with water; high boiling point; biodegradable profile reported in literature.
DMF: effective but more toxic; stricter waste handling recommended.
Fluorophore alternatives
AFC vs AMC (7-amino-4-methylcoumarin): AMC is slightly less hydrophobic and may dissolve easier in aqueous media; spectral maxima are similar. Selection is typically driven by enzyme specificity data rather than “greenness.”
Waste and EHS:
Collect DMSO/DMF-containing aqueous waste as hazardous. Avoid chlorinated solvents for plate cleaning; use aqueous detergents or ethanol where compatible. Implement light shielding to extend reagent life and reduce discard rates.
Pharmaceutical Uses
This product is a research reagent used for in vitro enzymology and screening. It is not an active pharmaceutical ingredient or excipient.
Relevant roles in drug discovery workflows (general):
High-throughput screening (HTS) of protease inhibitors using fluorogenic readouts.
Mechanistic and kinetic characterization (KM, kcat, inhibition constants) for lead optimization.
Selectivity profiling of candidate inhibitors across panels of proline-preferring proteases.
Regulatory note:
No pharmacopeial monograph is referenced for this substrate. It is supplied for research use only. Any application in manufacturing or diagnostic settings would require independent validation and regulatory review.
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, density, refractive index: Not specified for this item; refer to CoA/Spec Sheet.
Solubility: Not specified for this item; refer to CoA/Spec Sheet.
General/literature guidance for peptide–AFC substrates (informational, not item specifications):
Physical form: typically pale solid; coumarin fluorophores are light-sensitive.
Solubility: commonly soluble in polar aprotic organics (DMSO, DMF) and sparingly soluble in aqueous buffers unless aided by organic cosolvent; final assay solutions often contain 0.1–2% v/v DMSO (literature).
Fluorescence (AFC): excitation ~400–410 nm; emission ~490–505 nm; Stokes shift is large, enabling sensitive detection above protein autofluorescence (literature).
pKa/logP: vary with peptide sequence; AFC itself is relatively hydrophobic due to CF3-substitution (literature).
Important: Use the SDS and CoA for definitive, lot-specific property data. The above general values are provided for planning only and are not product specifications.
Quality and Grades
Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
Guidance on quality considerations for fluorogenic peptide substrates (general):
Purity impacts assay background and enzyme kinetics. For sensitive fluorescence assays, peptides are commonly provided at >95% purity (HPLC) in the literature, minimizing non-fluorescent impurities and partially deprotected species.
Identity confirmation: LC–MS and analytical HPLC are the standard release tests. Many users verify the expected mass of the peptide–AFC conjugate prior to kinetic work.
Residual solvents and counterions: Peptide synthesis and workup can introduce TFA or other counterions; these can influence pH and enzyme activity at low buffer capacity. If critical, exchange or neutralize before use.
Stabilizers: none are typically required for dry peptide–AFC conjugates; avoid moisture. If a stabilizer is present for this item, it will be indicated on the CoA/Spec Sheet.
Fluorescence QC: Some suppliers report a fluorescence calibration factor for free AFC to aid in converting RFU to concentration. For conjugates, establish a standard curve by fully digesting a known amount to free AFC under non-limiting enzyme conditions.
Consult the lot-specific CoA/Spec Sheet from Aladdin Scientific for definitive grade, purity, and analytical data.
Reaction and Applications
Primary use (literature/general):
H-Ala-Pro-AFC is a fluorogenic peptide substrate designed for protease activity assays. Upon enzymatic cleavage of the peptide bond adjacent to the AFC-linked C-terminus, free AFC is released, producing a robust fluorescence increase (Ex ~400–410 nm, Em ~490–505 nm).
Enzyme classes commonly assessed with Xaa–Pro–AFC substrates (literature examples):
Dipeptidyl peptidases with preference for Ala–Pro at the N-terminus (e.g., DPP family variants in biochemical studies).
Prolyl oligopeptidase/prolyl endopeptidase systems that recognize proline-containing motifs.
General protease quality control: verifying proteolytic contamination or activity in enzyme preparations.
Assay formats:
Continuous kinetic assays in microplates (96/384-well) monitoring RFU over time.
Endpoint assays after defined incubation, quantified against an AFC standard curve.
Practical notes:
Prepare concentrated DMSO stocks (e.g., 10–20 mM) to minimize organic content in assays.
Maintain consistent ionic strength and pH (often pH 7.0–8.0, enzyme-dependent). Include BSA (0.01–0.1%) to reduce adsorption losses if needed.
Verify linearity with respect to time and enzyme concentration; avoid inner-filter effects by keeping absorbance at excitation wavelength <0.05 AU.
Calibrate using free AFC to convert fluorescence units to nmol product formed for accurate kcat/KM determinations.
Reaction Conditions
Kinetic assay conditions (general literature guidance; optimize per enzyme):
Temperature: 25–37°C commonly used; maintain constant temperature for kinetic reproducibility.
pH: Often 7.0–8.0 (HEPES or Tris buffers, 25–100 mM). Adjust to the enzyme’s reported optimum.
Substrate concentration: Start near or below the expected KM (e.g., 5–100 µM). Confirm no inner-filter effects by measuring absorbance at ~400 nm (<0.05 AU preferred).
Enzyme concentration: Choose to achieve linear product formation over the initial 5–10 minutes with ≤10% substrate consumption.
Cosolvent: ≤1–2% v/v DMSO typical to keep substrate in solution without major impact on most enzymes.
Detection: Excitation 400–410 nm; emission 490–505 nm; integrate 0.1–0.5 s per read. Include wells with free AFC standards to convert RFU to concentration.
Controls: No-enzyme blank, heat-inactivated enzyme control, and inhibitor control for assay validation.
Expected outcomes (illustrative):
Initial rates proportional to enzyme concentration over a defined range; Michaelis–Menten behavior with appropriate fit quality (R2 > 0.98). Inhibitor IC50 values can be derived via standard dose–response analysis.
These are literature-based norms for AFC-releasing substrates and not product-specific performance claims.
Safety and Handling
Product-specific hazard data:
GHS classification, signal word, pictograms, H-statements: Not specified for this item; refer to SDS.
General laboratory safety for peptide–coumarin fluorogenic substrates (literature/best practice):
Potential hazards: organic solids may cause skin/eye irritation and respiratory tract irritation if dust is formed. Coumarin fluorophores are often light-sensitive; avoid prolonged exposure to intense light.
PPE: lab coat, safety glasses, and suitable gloves (e.g., nitrile). Handle powders in a fume hood or with local exhaust to minimize inhalation of particulates.
Storage incompatibilities: segregate from strong oxidizers and strong acids/bases. Avoid moisture ingress for hygroscopic peptides. Protect from light.
First aid (overview):
Skin/eye contact: rinse with water for at least 15 minutes; 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 advice.
Spill/cleanup: avoid dust formation; collect mechanically or with damp disposable towels; dispose of in accordance with institutional and regional regulations.
Fire safety: treat as combustible organic solid; use CO2, dry chemical, or foam. Thermal decomposition may release irritating fumes.
Always consult the official SDS for authoritative safety and regulatory information. For research use only.
Solvent Selection
This product is a hydrophobic fluorogenic peptide substrate; solvent choice primarily affects stock preparation and assay compatibility.
Recommended stock solvents (literature):
DMSO: excellent solvating power for peptide–AFC conjugates; low volatility; miscible with water. Common for 10–50 mM stocks.
DMF: alternative to DMSO; good solubility; compatible with many enzyme assays at ≤2% v/v final.
Aqueous buffers: Direct dissolution can be limited; use small percentages of DMSO/DMF (typically 0.1–2% v/v final) to maintain solubility without inhibiting most enzymes.
Avoid: high percentages of organic co-solvent, strong acids/bases that may hydrolyze amide bonds, and alcohol-rich media if the enzyme is sensitive.
Polarity and miscibility (general, literature):
The AFC tag increases hydrophobicity vs. AMC analogs; peptide length (dipeptide) partially offsets this. Expect improved solubility in polar aprotic organics and moderate aqueous solubility with cosolvent.
Comparison notes (general):
DMSO vs DMF: DMSO is less volatile, more benign odor, and widely accepted for high-throughput screening; DMF may better dissolve some sequences but is more hygroscopic and toxic.
If aqueous-only is essential, consider raising temperature slightly (e.g., 25–30°C) and sonication during dissolution, while keeping enzyme stability in mind.
Protect from light to preserve fluorophore integrity. Store in a tightly closed container with desiccant to minimize moisture uptake. Although room temperature is indicated, many users keep working aliquots at 2–8°C or -20°C for long-term stability; avoid freeze–thaw cycling of solutions.
Reconstitution:
Prepare concentrated stocks (e.g., 10–20 mM) in anhydrous DMSO or DMF. Vortex and, if needed, sonicate briefly to aid dissolution. Filter through a low-protein-binding syringe filter if particulate persists.
Working solutions: Dilute into assay buffer immediately before use, keeping final organic cosolvent ≤1–2% v/v.
Stability of solutions: Use freshly prepared working solutions. For extended experiments, verify baseline stability by monitoring fluorescence of substrate-only wells.
Research Use Only: This product is intended for laboratory research. Refer to the CoA and SDS for definitive, lot-specific instructions.
Structure and Identity
Product name: H-Ala-Pro-AFC (L-alanyl-L-prolyl–AFC; AFC = 7-amino-4-trifluoromethylcoumarin, literature)
SKU: H931129
CAS: 125791-92-6
PubChem CID: 25108520
InChIKey: 101621 (provided)
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.
H-Ala-Pro-AFC is a dipeptidyl fluorogenic substrate composed of an N-terminal free amine (H-), followed by L-alanine and L-proline linked via a standard peptide bond, with the C-terminus coupled through an amide linkage to the amino group of the fluorophore AFC (7-amino-4-trifluoromethylcoumarin).
Key functional groups: secondary amide bonds (peptide), secondary amide linking peptide to the AFC moiety, pyrone ring of coumarin with a para-trifluoromethyl substituent (literature), tertiary amide adjacent to proline’s pyrrolidine ring.
Stereochemistry: typically L-Ala and L-Pro (literature norm for protease substrates). No additional stereocenters on the coumarin chromophore.
2D structure in words: H2N–CH(CH3)–CO–(NH)–(CH–(CH2)3–N in ring as L-Pro)–CO–NH–AFC (coumarin core bearing CF3 at the 4-position; the amide attaches at the 7-amino position) (literature).
Synthetic Utility
While primarily intended as a biochemical substrate, H-Ala-Pro-AFC also illustrates useful synthetic features (general):
Functional groups: two peptide amide bonds and a terminal amide linking to the AFC fluorophore provide handles for analog development (e.g., varying N-terminal residues, proline substitutions, or C-terminal modifications) to tune enzyme selectivity.
SAR exploration: swapping Ala for other small residues (Gly, Ser) or introducing N-terminal acyl caps (e.g., Ac-) can modulate recognition by different proteases (literature). The AFC tag can be replaced with other reporters (AMC, ACC, 7-amino-coumarin derivatives) while maintaining synthetic convergence.
Solid-phase peptide synthesis (SPPS) route (literature): assemble H-Ala–Pro on resin, then couple to AFC (as the amine component) after peptide activation at the C-terminus, followed by cleavage/deprotection and purification by RP-HPLC.
Protecting group strategy: orthogonal protection to preserve the AFC amino group until final coupling (or pre-activate peptide C-terminus for solution-phase coupling with AFC). Care is needed to avoid acyl migration or hydrolysis.
These synthetic notes are general literature guidance and not a manufacturing description for this specific catalog item.
Target Specificity
Item-specific target, antigen, epitope, clone, or isotype information: Not applicable; this product is a small-molecule/peptide substrate, not an antibody or biological targeting reagent.
General note (literature):
Xaa–Pro–AFC substrates are commonly used to probe proteases that recognize proline-containing motifs (e.g., certain dipeptidyl peptidases or prolyl oligopeptidases). Actual specificity must be empirically determined for the enzyme of interest.
Wir verwenden Cookies, um die ordnungsgemäße Funktion der Website sicherzustellen und, soweit zulässig, Ihr Nutzererlebnis zu verbessern. Sie können Ihre Einstellungen jederzeit unter „Einstellungen“ verwalten. Weitere Informationen finden Sie in unserer Cookie-Richtlinie.
Shall we send you a message when we have discounts available?
Remind me later
Thank you! Please check your email inbox to confirm.
Products are supplied to verified businesses, institutions, and qualified professionals for research and development use only. Not for use in humans, animals, diagnosis, or therapy.
