Human Endogenous Metabolite
Ac-Phe-OH is classified as an N-acetylated amino acid and phenylalanine derivative. It does not have a specific biological receptor target. Its primary application is as a building block in peptide synthesis for introducing N-acetylated phenylalanine residues. N-Acetylation can modulate peptide properties and protect the N-terminus. It has anti-inflammatory properties and prevents protein denaturation and aggregation.

In vitro, Ac-Phe-OH has been shown to have anti-inflammatory effects, preventing protein denaturation and aggregation, and possesses efficient moisturizing and free radical scavenging functions. It has been tried for the treatment of various inflammatory conditions such as allergic rhinitis, allergic dermatitis, and chronic obstructive pulmonary disease. Amino acids and amino acid derivatives have been commercially used as ergogenic supplements.

In vivo, Ac-Phe-OH has been studied for its anti-inflammatory properties and potential in treating various inflammatory diseases. N-Acetyl-L-phenylalanine has been investigated for therapeutic applications in inflammatory conditions. As an N-acetylated amino acid, it may have different metabolic and pharmacokinetic properties compared to L-phenylalanine, potentially offering advantages in drug development.

Transacylase assay: The reaction mixture for transacylase assay contained tris-HCl buffer pH 8.3 (100 μmol), N-acetyl-L-amino acid (10 μmol for L-isomer or 20 μmol for DL-isomer), DL-[1-14C]phenylalanine (8×10^4 c.p.m.) or L-[U-14C]phenylalanine (4×10^4 c.p.m.), and transacylase (400-500 μg protein) in 1 ml final volume. Incubation was at 37°C for 15 min. The reaction was terminated by boiling for 3 min, centrifuged, and the supernatant applied to a Dowex 50 (H+ form) column (1 cm × 4 cm), eluted with water, and the effluent radioactivity counted. One unit of enzyme activity is defined as the amount catalyzing formation of 1 μmol of N-acetyl-L-[14C]phenylalanine under these conditions. [1]
Acetyl-CoA-L-phenylalanine α-N-acetyltransferase assay: The reaction mixture contained (in 1 ml): tris-HCl buffer pH 8.3 (100 μmol), L-phenylalanine (10 μmol) containing L-[U-14C]phenylalanine (4×10^4 c.p.m.) or DL-[1-14C]phenylalanine (8×10^4 c.p.m.), acetyl-CoA (100 μg), and purified acetyltransferase (10-100 μg). Incubation at 37°C for 15 min, terminated by boiling for 3 min, centrifuged, supernatant applied to Dowex 50 (H+ form) column (1 cm × 4 cm), eluted with water to 25 ml, and radioactivity counted. One unit of activity is the amount catalyzing formation of 1 μmol of N-Acetyl-L-Phenylalanine per min. [1]
Reversibility assay: Reaction mixture (1 ml) contained TME buffer (50 μmol), L-phenylalanine (20 μmol), N-acetyl-DL-[1-14C]phenylalanine (6×10^4 c.p.m.), MgCl2 (10 μmol), CoA (100 μg), and α-N-acetyltransferase (100 μg). Incubation at 37°C for 15 min, stopped by heat, centrifuged, supernatant applied to Dowex 50 (H+ form) column (1 cm × 5 cm), washed with water (25 ml), then amino acids eluted with 2 M NH3 (25 ml). Eluates concentrated and radioactivity counted. No reversibility was detected. [1]
In vitro enzyme/receptor binding protocols for Ac-Phe-OH involve studying its interactions with enzymes involved in amino acid metabolism and inflammatory pathways. The compound can be used in receptor binding studies to investigate the effects of N-acetylation on ligand-receptor interactions. For enzyme activity studies, Ac-Phe-OH can be used as a substrate or inhibitor to probe enzyme specificity.

Cell culture protocols for Ac-Phe-OH involve dissolving the compound in sterile water or cell culture medium. Cells are treated with concentrations ranging from 0.1-100 µM for 24-72 hours. Effects on inflammatory markers, cell viability, and signaling pathways are assessed. The compound is soluble in water and ethanol. Standard assays such as ELISA for cytokine measurement and Western blotting for protein expression can be employed.

In vivo animal study protocols for Ac-Phe-OH involve administration via oral gavage or injection in saline. Dosing typically ranges from 10-100 mg/kg depending on the study. Studies may assess effects on inflammation, allergic responses, and oxidative stress. Blood and tissue samples are collected for analysis of compound distribution, inflammatory markers, and histopathological evaluation.

Pharmacokinetic properties of Ac-Phe-OH are characteristic of N-acetylated amino acids. The compound has a molecular weight of 207.23 g/mol, formula C11H13NO3, and melting point of 163-169°C. It is soluble in water and ethanol. Storage is recommended at room temperature in a cool, dark place below 15°C. It is absorbed and distributed to tissues, with elimination via renal excretion.

Toxicological data for Ac-Phe-OH indicates it has low acute toxicity. The compound has been studied for therapeutic applications in inflammatory conditions. Standard safety precautions for handling amino acid derivatives should be observed. No significant toxicity has been reported in research use.

[1]. Enzymic synthesis of N-acetyl-L-phenylalanine in Escherichia coli K12. Biochem J. 1971 Oct;124(5):905-13.

N-acetyl-L-phenylalanine is an N-acetyl derivative of L-phenylalanine and a metabolite. It belongs to the N-acyl-L-phenylalanine, N-acetyl-L-amino acid, and N-acetylphenylalanine families. It is the conjugate acid of N-acetyl-L-phenylalanine and an enantiomer of N-acetyl-D-phenylalanine. N-acetyl-L-phenylalanine has been reported in Daphnia pulex, Bombyx mori, and other organisms with relevant data. N-acetyl-D-phenylalanine is a metabolite found or produced in Saccharomyces cerevisiae. See also: aflanin (note moved here).

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N-Acetyl-L-Phenylalanine was identified as the principal acylamino acid in Escherichia coli K12 both free and in proteins. [1]
The compound is synthesized from acetyl-CoA and L-phenylalanine by α-N-acetyltransferase, and also participates in transacylation reactions with transacylase. [1]
The purified α-N-acetyltransferase had pH optimum 8, Km for L-phenylalanine 3.3 mM and for acetyl-CoA 0.13 mM. [1]
The enzyme is a thiol enzyme inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and iodoacetamide (0.08 mM gives 80-90% inhibition). [1]
Ac-Phe-OH (CAS#: 2018-61-3) is also known as N-Acetyl-L-phenylalanine, Acetylphenylalanine, and (2S)-2-acetamido-3-phenylpropanoic acid. Its molecular formula is C11H13NO3. It has anti-inflammatory properties and has been investigated for treating allergic and inflammatory diseases. It has no approved therapeutic indications.

C11H13NO3

207.2258

207.089

2018-61-3

74839

White to off-white powder

1.2±0.1 g/cm3

453.9±38.0 °C at 760 mmHg

171-173 °C(lit.)

228.3±26.8 °C

0.0±1.2 mmHg at 25°C

1.548

0.78

2

3

4

15

234

1

O([H])C([C@]([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C(C([H])([H])[H])=O)=O

CBQJSKKFNMDLON-JTQLQIEISA-N

InChI=1S/C11H13NO3/c1-8(13)12-10(11(14)15)7-9-5-3-2-4-6-9/h2-6,10H,7H2,1H3,(H,12,13)(H,14,15)/t10-/m0/s1

(2S)-2-acetamido-3-phenylpropanoic acid

N-Acetylphenylalanine; N-Acetyl-L-phenylalanine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~482.56 mM)
H2O : ~7.69 mg/mL (~37.11 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.06 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (12.06 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (12.06 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 7.14 mg/mL (34.45 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)