| Size | Price | Stock | Qty |
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| 50mg |
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| 100mg |
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| Targets |
The primary molecular target of 4-Ethynyl-L-phenylalanine is tryptophan hydroxylase (TPH), the enzyme responsible for the conversion of L-tryptophan to 5-hydroxytryptophan (5-HTP), which is the first and rate-limiting step in serotonin (5-HT) biosynthesis. TPH is a member of the aromatic amino acid hydroxylase family and requires tetrahydrobiopterin (BH4) and iron as cofactors. There are two isoforms of TPH in mammals: TPH1, which is primarily expressed in peripheral tissues such as the gastrointestinal tract and pineal gland, and TPH2, which is predominantly expressed in the central nervous system and is the major isoform responsible for serotonin production in the brain. 4-Ethynyl-L-phenylalanine acts as a competitive inhibitor with regard to the substrate tryptophan, meaning it binds to the active site of the enzyme and competes with tryptophan for binding. It does not appear to inhibit other aromatic amino acid hydroxylases, such as phenylalanine hydroxylase or tyrosine hydroxylase, at concentrations that effectively inhibit TPH. The compound is reversible, allowing for the recovery of enzyme activity upon removal of the inhibitor. This reversible and competitive inhibition profile makes it a valuable tool for studying the acute effects of TPH inhibition.
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| ln Vitro |
p-Ethynylphenylalanine pairs reconstitute multiple 5-HT kits (5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6). , and 5-HT7)[1].
In cell-free enzymatic assays, 4-Ethynyl-L-phenylalanine inhibits tryptophan hydroxylase (TPH) activity with a Ki of 32.6 μM. The compound acts as a competitive inhibitor with respect to the substrate tryptophan, meaning that increasing concentrations of tryptophan can overcome the inhibition. The assay typically involves incubating recombinant TPH with varying concentrations of the compound and its substrate, tryptophan, in the presence of cofactors such as 6-methyltetrahydropterin (a BH4 analog) and Fe²⁺. The reaction is initiated by the addition of the enzyme, and the production of 5-hydroxytryptophan (5-HTP) is measured by high-performance liquid chromatography (HPLC) with fluorescence or electrochemical detection. The Ki, which is the inhibition constant, is determined from Lineweaver-Burk or Dixon plots by analyzing the enzyme kinetics at different substrate and inhibitor concentrations. The compound's selectivity for TPH over other aromatic amino acid hydroxylases is an important aspect of its utility as a research tool. In cellular assays, the compound effectively reduces serotonin production in cells expressing TPH, such as serotonergic neurons or cell lines. |
| ln Vivo |
In midbrain non-tissue, p-Ethynylphenalanine (30 mg/kg; i.p.) lowers 5-HT and 5-HIAA levels [1]. Apoptotic enzymes controlled by aromatic compounds are not inhibited by p-Ethynylphenalanine [1].
In vivo, 4-Ethynyl-L-phenylalanine has been shown to decrease serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) levels in the rat midbrain. In a study using male Sprague-Dawley rats, administration of the compound at a dose of 30 mg/kg via intraperitoneal (i.p.) injection resulted in a significant reduction of 5-HT and 5-HIAA levels in the midbrain. The compound did not affect tissue levels outside the midbrain, suggesting some regional specificity or that the compound may not cross the blood-brain barrier uniformly. Importantly, p-Ethynylphenylalanine did not inhibit apoptotic enzymes controlled by aromatic compounds, indicating that its effects are relatively specific to TPH inhibition. The in vivo activity of this compound is consistent with its mechanism of action as a competitive TPH inhibitor. The reduction in 5-HT and 5-HIAA levels is dose-dependent and reversible, as the effects diminish upon discontinuation of the compound. This in vivo activity makes 4-Ethynyl-L-phenylalanine a valuable tool for studying the role of serotonin in various physiological processes and disease models. |
| Enzyme Assay |
In a cell-free enzymatic assay, the inhibition of tryptophan hydroxylase (TPH) by 4-Ethynyl-L-phenylalanine is typically evaluated using a recombinant enzyme preparation. The assay is performed in a 96-well plate format. The reaction mixture contains 50 mM HEPES buffer (pH 7.4), 1 mM DTT, 0.1 mM Fe(NH₄)₂(SO₄)₂, 0.1 mM 6-methyltetrahydropterin (BH4 analog), catalase, and varying concentrations of L-tryptophan and the test compound. The reaction is initiated by the addition of the TPH enzyme. The mixture is incubated at 37°C for 30 minutes. The reaction is terminated by the addition of perchloric acid. The amount of 5-hydroxytryptophan (5-HTP) produced is quantified by HPLC coupled with fluorescence detection (excitation at 280 nm, emission at 340 nm). The initial velocity of the reaction is determined for each substrate and inhibitor concentration. To determine the Ki, the data are fitted to a competitive inhibition model using nonlinear regression analysis. The selectivity of the compound for TPH over other aromatic amino acid hydroxylases, such as phenylalanine hydroxylase and tyrosine hydroxylase, is assessed using similar assays with the respective enzymes and substrates.
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| Cell Assay |
For in vitro cellular assays, cells expressing tryptophan hydroxylase (TPH), such as serotonergic neurons (e.g., RN46A cells) or cell lines engineered to express TPH, are used. Cells are seeded in multi-well plates and allowed to reach confluence. The cells are then treated with 4-Ethynyl-L-phenylalanine at various concentrations (e.g., 1-100 μM) for a defined period (e.g., 24-48 hours). The culture medium is collected, and the cells are lysed. The levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the medium and cell lysates are measured using enzyme-linked immunosorbent assay (ELISA) or HPLC with electrochemical detection. Cell viability is assessed using an MTT or resazurin assay to ensure that the observed effects on serotonin levels are not due to cytotoxicity. The percentage of inhibition of serotonin production is calculated relative to untreated control cells. The IC₅₀, representing the concentration that reduces serotonin levels by 50%, is determined from the dose-response curve. These assays provide a measure of the compound's cellular activity and its ability to inhibit serotonin biosynthesis in a cellular context.
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| Animal Protocol |
Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rats (200 g) [1]
Doses: 30 mg/kg Route of Administration: intraperitoneal (ip) injection Experimental Results: The levels of 5-HT and 5-HIAA in the midbrain of rats were diminished. For in vivo animal studies, the effect of 4-Ethynyl-L-phenylalanine on serotonin levels is typically assessed in rodents, such as male Sprague-Dawley rats. The compound is administered at a dose of 30 mg/kg via intraperitoneal (i.p.) injection. A control group receives the vehicle (e.g., saline) alone. At various time points after administration (e.g., 1, 2, 4, 8 hours), the animals are euthanized, and the brain regions of interest (e.g., midbrain, hippocampus, prefrontal cortex) are dissected. The tissue samples are homogenized, and the levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) are measured using HPLC with electrochemical detection. The concentrations of 5-HT and 5-HIAA in the treated animals are compared to those in the control group to determine the degree of TPH inhibition. The time course of the effect is also evaluated to determine the duration of action. To assess reversibility, animals may be allowed to recover for several days after the last dose, and serotonin levels are measured again. The compound's effect on other neurotransmitters, such as dopamine and norepinephrine, may also be measured to assess its selectivity. |
| ADME/Pharmacokinetics |
As a small amino acid derivative with a molecular weight of 189.21 and a logP of 1.29, 4-Ethynyl-L-phenylalanine is expected to be readily absorbed and distributed in the body. Its relatively high water solubility (due to the amino acid structure) suggests that it may have good bioavailability. The compound likely crosses the blood-brain barrier to some extent, although this may be limited, as evidenced by its effect on midbrain serotonin levels but not tissue levels. Specific pharmacokinetic parameters, such as half-life, volume of distribution, and clearance, have not been extensively reported in the available literature. The compound's metabolism is likely to involve pathways common to amino acids, such as deamination or transamination. The alkyne group may also undergo metabolism. The compound's rapid clearance from the body is suggested by the reversibility of its effects. Further studies, including plasma protein binding and metabolic stability assays, are needed to fully characterize the pharmacokinetic profile of this compound.
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| Toxicity/Toxicokinetics |
Toxicological data for 4-Ethynyl-L-phenylalanine is limited, as it is a research chemical. Standard safety precautions should be observed when handling this compound. No specific toxicity studies, such as acute or chronic toxicity in animal models, have been detailed in the public domain. However, based on its structure as a derivative of the natural amino acid phenylalanine, it is likely to have low inherent toxicity. The compound's selectivity for TPH over other enzymes suggests a reduced risk of off-target effects. Nonetheless, as with all research chemicals, it should be handled with care, using appropriate personal protective equipment and following institutional safety guidelines. The compound is not approved for clinical use and should only be used in preclinical research settings. The potential for the compound to affect serotonin levels in vivo could lead to physiological effects, such as changes in mood, sleep, or appetite, which are important considerations for in vivo studies.
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| References | |
| Additional Infomation |
4-Ethynyl-L-phenylalanine is a research tool for studying serotonin biosynthesis. Its mechanism of action involves competitive inhibition of TPH. It is a reagent for click chemistry and is not approved for clinical use.
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| Molecular Formula |
C₁₁H₁₁NO₂
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| Molecular Weight |
189.21
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| Exact Mass |
189.078
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| CAS # |
278605-15-5
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| Related CAS # |
p-Ethynylphenylalanine hydrochloride;188640-63-3
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| PubChem CID |
25216972
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
343.2±37.0 °C at 760 mmHg
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| Flash Point |
161.4±26.5 °C
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| Vapour Pressure |
0.0±0.8 mmHg at 25°C
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| Index of Refraction |
1.595
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| LogP |
1.29
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
14
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| Complexity |
245
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C#CC1=CC=C(C=C1)C[C@@H](C(=O)O)N
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| InChi Key |
PPDNGMUGVMESGE-JTQLQIEISA-N
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| InChi Code |
InChI=1S/C11H11NO2/c1-2-8-3-5-9(6-4-8)7-10(12)11(13)14/h1,3-6,10H,7,12H2,(H,13,14)/t10-/m0/s1
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| Chemical Name |
(2S)-2-amino-3-(4-ethynylphenyl)propanoic acid
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| Synonyms |
4EthynylLphenylalanine; 4 Ethynyl L phenylalanine
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
1M HCl : 240 mg/mL (~1268.43 mM)
1M NaOH : 240 mg/mL (~1268.43 mM) DMSO : ~2.22 mg/mL (~11.73 mM) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 5.2851 mL | 26.4257 mL | 52.8513 mL | |
| 5 mM | 1.0570 mL | 5.2851 mL | 10.5703 mL | |
| 10 mM | 0.5285 mL | 2.6426 mL | 5.2851 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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