Boc-2-Abz-OH: Properties, Handling & Lab Use Guidelines

Boc-2-Abz-OH is a Boc-protected 2-aminobenzoic acid building block widely used in solid-phase peptide synthesis and as a fluorescent FRET donor/quencher component in protease substrate design.

CAS / SKU

13734-34-4 / B139179

Chemical Name

N-(tert-Butoxycarbonyl)anthranilic acid (Boc-2-aminobenzoic acid)

Molecular Formula

C₁₂H₁₅NO₄

Molecular Weight

237.25 g/mol

Appearance

White to off-white crystalline powder

Recommended Storage

Store at 2–8 °C, protected from moisture and light

Primary Application

Fluorescent label in FRET-based protease substrates; peptide backbone modification

Chemical Identity and Key Properties

Boc-2-Abz-OH, formally N-(tert-butoxycarbonyl)anthranilic acid, is the Boc-protected form of 2-aminobenzoic acid (anthranilic acid). Its molecular formula is C₁₂H₁₅NO₄ with a molecular weight of 237.25 g/mol (CAS 13734-34-4). The compound presents as a white to off-white crystalline solid at room temperature.

The Boc carbamate group protects the aromatic amine during coupling reactions, preventing undesired side reactions while leaving the carboxylic acid available for activation. Upon standard Boc deprotection conditions (e.g., trifluoroacetic acid in DCM, or 4 M HCl in dioxane), the free 2-aminobenzoic acid (2-Abz) moiety is regenerated within the assembled peptide chain.

2-Abz is well-characterized as an intrinsic fluorophore (excitation ~320 nm, emission ~420 nm) frequently paired with quencher residues such as 3-nitrotyrosine (Tyr(3-NO₂)) or diaminopropionic acid-2,4-dinitrophenyl (Dap(Dnp)) in internally quenched FRET peptide substrates.

Storage, Handling and Safety Guidelines

Storage: Store Boc-2-Abz-OH at 2–8 °C in a tightly sealed container, protected from light and moisture. Prolonged exposure to humidity can promote hydrolysis of the Boc group. For long-term archival (>12 months), storage at −20 °C under inert atmosphere (argon or nitrogen) is advisable.

Handling: Use standard laboratory personal protective equipment: nitrile gloves, lab coat, and safety goggles. Work in a well-ventilated area or fume hood when weighing powders to minimize dust inhalation. Avoid contact with strong bases or strong oxidizing agents.

Stability: The compound is stable under recommended storage conditions. Solutions in DMSO or DMF should be prepared fresh or aliquoted and stored at −20 °C to prevent degradation. Repeated freeze–thaw cycles should be minimized.

Disposal: Dispose of waste material in accordance with local institutional regulations for organic chemical waste. Do not pour solutions down the drain without appropriate treatment.

Solubility and Solution Preparation

Boc-2-Abz-OH is sparingly soluble in water but dissolves well in common organic solvents used in peptide chemistry, including:

• DMF (N,N-dimethylformamide): Recommended solvent for coupling reactions in solid-phase peptide synthesis (SPPS); typical stock concentrations of 0.1–0.5 M are achievable.
• DMSO: Suitable for preparing stock solutions for biological assays; warm gently (37 °C) if dissolution is slow.
• DCM (dichloromethane): Suitable for solution-phase coupling reactions.
• Ethyl acetate / methanol mixtures: Useful for recrystallization or TLC analysis.

When preparing aqueous buffers for fluorescence assays, dissolve first in a minimum volume of DMSO, then dilute into the aqueous buffer. Keep the final DMSO concentration below 1% (v/v) to avoid interference with enzymatic assays.

Applications in Peptide Synthesis and FRET Assays

Solid-Phase Peptide Synthesis (SPPS): Boc-2-Abz-OH is compatible with both Boc-SPPS and Fmoc-SPPS strategies when used as a capping or N-terminal labeling reagent. In Boc-SPPS, it is incorporated using standard coupling reagents (e.g., DIC/HOBt, HATU, or HBTU/DIPEA). In Fmoc-SPPS workflows, it can be introduced as a C-terminal or internal non-natural residue after temporary Fmoc protection of the resin-bound amine.

Internally Quenched FRET Protease Substrates: The 2-Abz (anthranilic acid) fluorophore is one of the most widely used donors in internally quenched fluorescent (IQF) peptide substrates for protease activity assays. Upon protease cleavage, the donor and quencher separate, producing a measurable increase in fluorescence. Common pairings include 2-Abz / Tyr(3-NO₂) and 2-Abz / Lys(Dnp). These substrates are used to characterize endopeptidases including metalloproteinases (MMPs), serine proteases, and cysteine proteases.

Fluorescent Peptide Probes: Beyond protease substrates, 2-Abz-labeled peptides serve as fluorescent probes for receptor binding studies and as internal calibration standards in peptide HPLC analysis.

Quality, Purity and Analytical Verification

Research-grade Boc-2-Abz-OH (SKU B139179) from Aladdin Scientific is characterized by the following analytical methods:

• HPLC purity: Typically ≥98% by reverse-phase HPLC. Consult the Certificate of Analysis (CoA) for lot-specific data.
• Identity confirmation: ¹H NMR and/or LC-MS consistent with the target structure (MW 237.25 g/mol; [M+H]⁺ = 238.1).
• Appearance check: White to off-white crystalline powder; discoloration may indicate degradation.

Always request the CoA for each lot prior to use in critical applications. For GMP or regulated research environments, additional testing (e.g., Karl Fischer water content, residual solvent by GC) may be required — contact Aladdin Scientific technical support for custom specifications.

Perform a TLC or analytical HPLC spot-check upon receipt if the compound has been in transit under non-controlled temperature conditions.

Frequently asked questions

What deprotection conditions are recommended to remove the Boc group from Boc-2-Abz-OH after SPPS?

Standard Boc removal employs 20–50% trifluoroacetic acid (TFA) in dichloromethane (DCM) for 20–30 minutes at room temperature, or 4 M HCl in 1,4-dioxane. These conditions cleanly liberate the free 2-aminobenzoic acid residue within the peptide without affecting amide bonds. After deprotection, excess TFA is removed by evaporation or precipitation with cold diethyl ether.

Which quencher residues are most commonly paired with 2-Abz in FRET protease substrates?

The most commonly used quenchers paired with the 2-Abz fluorophore are 3-nitrotyrosine (Tyr(3-NO₂)) and dinitrophenyl-modified lysine (Lys(Dnp)). Both act as effective energy acceptors/quenchers for the 2-Abz emission (~420 nm). The choice depends on the peptide sequence requirements and the specific protease being studied.

Can Boc-2-Abz-OH be used in Fmoc-based solid-phase peptide synthesis?

Yes, Boc-2-Abz-OH can be incorporated into Fmoc-SPPS workflows, typically as an N-terminal capping residue or at internal positions after selective deprotection. Because the Boc group is acid-labile while Fmoc is base-labile, selective orthogonal removal is possible; however, care must be taken to avoid premature Boc loss during any acidic washes. For routine Fmoc-SPPS, Fmoc-2-Abz-OH may offer simpler handling and is worth considering as an alternative.

How should I prepare a stock solution of Boc-2-Abz-OH for coupling reactions?

Weigh the required amount of Boc-2-Abz-OH under dry conditions and dissolve in anhydrous DMF to achieve the desired concentration (typically 0.1–0.5 M for SPPS coupling). For biological assay stock solutions, dissolve in DMSO and then dilute into the appropriate aqueous buffer immediately before use, keeping the final DMSO content below 1% (v/v). Prepare fresh solutions where possible to ensure maximum coupling efficiency and fluorescence performance.

What is the fluorescence excitation and emission profile of the 2-Abz group?

The 2-aminobenzoic acid (2-Abz) moiety exhibits fluorescence with an excitation maximum near 320 nm and an emission maximum near 420 nm in aqueous solution. These values can shift slightly depending on solvent polarity and pH. This UV-range excitation is compatible with standard fluorescence plate readers and spectrofluorometers equipped with appropriate filters or monochromators.

References

1. Internally quenched fluorescent peptide substrates for protease activity assays — general review of 2-Abz/quencher pairs. (No specific citation from source material; textbook-level fact.)
2. Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 4th ed. Wiley-Interscience. (Boc protection/deprotection chemistry.)
3. Aladdin Scientific Certificate of Analysis for Boc-2-Abz-OH, SKU B139179. (Lot-specific purity and identity data.)

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.