| HS Code | 710532 |
| Chemical Name | D-Phenylalanine Ethyl Ester Hydrochloride |
| Cas Number | 17333-58-9 |
| Molecular Formula | C11H16ClNO2 |
| Molecular Weight | 229.70 g/mol |
| Appearance | White crystalline powder |
| Purity | ≥98% |
| Melting Point | 156-158 °C |
| Specific Optical Rotation | -8.0° (c=1, ethanol) |
| Solubility | Soluble in water, methanol, ethanol, and DMSO |
| Storage Conditions | Store at 2-8 °C, sealed, protected from moisture |
| Synonyms | D-Phe-OEt·HCl; H-D-Phe-OEt·HCl |
As an accredited D-phenylalanine Ethyl Ester Hydrochloride factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | Packaged as 25 g in a sealed amber glass bottle with nitrogen purge for stability. |
| Container Loading (20′ FCL) | 20' FCL container loading of D-phenylalanine Ethyl Ester Hydrochloride: palletized drums, secure bracing, moisture-proof lining, and proper labeling. |
| Shipping | Ship D-phenylalanine Ethyl Ester Hydrochloride in airtight, moisture-resistant containers away from heat and light. Ensure proper labeling, safe packing to prevent leakage, and compliant transport documentation. Although not typically classified as hazardous, avoid ingestion, inhalation, and skin contact. Follow local, national, and international shipping regulations for fine chemicals. |
| Storage | Store D-phenylalanine Ethyl Ester Hydrochloride in a tightly sealed container in a cool, dry, well-ventilated area. Protect from moisture, light, and heat. Keep away from incompatible materials and strong oxidizing agents. Ensure the container is clearly labeled and stored under inert gas if possible to maintain stability and prevent decomposition. |
| Shelf Life | Stable for at least 2 years when stored dry, airtight, and protected from light at -20°C. |
Solution-phase carbodiimide-mediated coupling of H-D-Phe-OEt·HCl to N-α-Fmoc-protected amino acids is carried out in anhydrous dichloromethane or dichloromethane/dimethylformamide 4:1 v/v at 0–5°C. The hydrochloride salt is suspended and treated with 2.0–2.5 equivalents of N-methylmorpholine or diisopropylethylamine relative to the salt; apparent pH is maintained above 7.5 to release the free amino ester before activation. Fmoc-L-leucine, Fmoc-L-proline, and Fmoc-D-phenylalanine are typical carboxyl components. Activation with N,N'-diisopropylcarbodiimide in the presence of ethyl 2-cyano-2-(hydroxyimino)acetate is performed at 1.05–1.2 molar equivalents relative to the N-protected amino acid. The reaction is tracked by thin-layer chromatography on silica gel 60 F254 using ethyl acetate/n-hexane 1:1 v/v; free amino ester is visualised with ninhydrin at Rf 0.40. The resulting dipeptide ethyl ester is used in solution-phase synthesis of bradykinin receptor ligands and peptide-based enzyme inhibitors. Residual water in the solvent above 800 ppm promotes saponification to D-phenylalanine; solvent is dried over 3 Å molecular sieves before use. Incoming lot release of the hydrochloride salt includes loss on drying per USP <731>; if moisture exceeds 0.5%, drying in a vacuum oven at 40°C and 10 mbar for 12 h is specified. Bulk storage at relative humidity above 60% requires double-lined LDPE bags and nitrogen blanketing to prevent ester hydrolysis and caking. The table below consolidates in-process controls for this coupling.
| Control parameter | Specification | Test method |
|---|---|---|
| Reaction temperature | 0–5°C | Jacketed reactor data logger, ISO 9001 calibration |
| Apparent pH after base addition | 7.5–8.5 | pH electrode, calibrated at 4.01 and 7.00 |
| Dichloromethane moisture | ≤ 800 ppm | Karl Fischer titration, USP <921> |
| Free amino ester Rf | 0.40 in ethyl acetate/n-hexane 1:1 v/v | TLC silica gel 60 F254, UV 254 nm |
| Dipeptide ethyl ester purity | ≥ 95.0% area | HPLC-UV, EP 2.2.46 |
| Residual H-D-Phe-OEt | ≤ 2.0% area | HPLC-UV, EP 2.2.46 |
| Loss on drying of input salt | ≤ 0.5% | USP <731> |
The ethyl ester is hydrolysed to D-phenylalanine under low-temperature alkaline conditions in tetrahydrofuran/water 3:1 v/v with lithium hydroxide monohydrate at 1.0–1.05 molar equivalents relative to ester. The suspension is held at 0–2°C for 2–4 h. Higher temperatures or sodium hydroxide/methanol systems at 20–25°C raise the L-enantiomer content above 0.5% area in the resulting acid, as detected by chiral HPLC. On 500 L glass-lined reactors, batch-to-batch variation in optical purity was traced to localised hot spots near the bottom outlet when one-third of the alkali was charged too rapidly. Installation of a retreat-curve impeller at 90–110 rpm and an internal cooling coil reduced the temperature differential to ±2°C. The cooling jacket is operated with a 40% ethylene glycol/water mixture supplied at −10°C. Conversion is monitored by TLC and stopped at complete disappearance of the ester spot in ethyl acetate/methanol 9:1 v/v. The free acid is isolated by acidification to pH 3.0 with 6 M hydrochloric acid and filtration; the crude wet cake is washed with deionised water until chloride content in the filtrate is ≤ 100 ppm. The dried D-phenylalanine is then N-protected with Fmoc-OSu in acetonitrile/water 2:1 v/v using sodium carbonate at 1.2 equivalents. The Fmoc-D-Phe-OH product is crystallised from ethyl acetate/n-heptane 1:5 v/v; residual tetrahydrofuran is controlled at ≤ 720 ppm, methanol ≤ 3000 ppm, and dichloromethane ≤ 600 ppm according to ICH Q3C. Published data for this specific configuration is limited; process validation studies should use a bracketing approach over 0–5°C and 1.00–1.10 equivalents of lithium hydroxide monohydrate.
D-Phe-OEt·HCl is converted to N-acyl-D-phenylalanine ethyl esters by Schotten-Baumann reaction with aliphatic or cycloalkyl carbonyl chlorides. In a typical batch, the salt is dissolved in tetrahydrofuran/water 2:1 v/v at 0–5°C; sodium hydrogen carbonate is charged at 2.5–3.0 molar equivalents relative to the hydrochloride to neutralise both the salt and evolving hydrogen chloride. The acyl chloride, for example trans-4-isopropylcyclohexanecarbonyl chloride, is added dropwise over 90–120 min while maintaining the aqueous phase at pH 8.0–9.5. If pH is allowed to fall below 7.0, the free amino ester converts back to the hydrochloride and the N-acylation rate collapses. Above pH 10.0, the ethyl ester undergoes competitive hydrolysis. pH-stat control with 1 M sodium hydroxide is therefore used on production vessels larger than 2000 L. The acyl chloride line is fitted with a mass flow meter and a dip tube positioned below the aqueous surface. The resulting N-acyl ester is extracted into ethyl acetate, washed with 0.5 M hydrochloric acid and 5% w/w sodium chloride, and concentrated under reduced pressure at ≤ 45°C. This intermediate class is used for glinide-type insulin secretagogue APIs and for chiral building blocks in endopeptidase inhibitors. Impurity control is performed by HPLC-UV using a C18 column of 150 mm length and 4.6 mm internal diameter; mobile phase is acetonitrile/0.1% phosphoric acid 60:40 v/v, flow rate 1.0 mL/min, detection at 220 nm. Residual starting material is controlled to ≤ 1.0% area; the corresponding free carboxylic acid is controlled to ≤ 0.5%. Process water content is verified by Karl Fischer titration, USP <921>, before solvent swap to toluene for crystallisation.
Quantitative determination of enantiomeric excess in bulk D-phenylalanine derivatives is performed by direct chiral stationary-phase HPLC without pre-column derivatisation. H-D-Phe-OEt·HCl is dissolved in mobile phase at 0.1 mg/mL and used as the D-enantiomer reference marker; the L-enantiomer reference is prepared from the corresponding L-phenylalanine ethyl ester hydrochloride. The method uses a 250 mm × 4.6 mm chiral column with amylose tris(3,5-dimethylphenylcarbamate) covalently bonded to silica, 5 µm particles. The mobile phase is n-hexane/ethanol/diethylamine 80:20:0.1 v/v/v at 1.0 mL/min and 25°C. Detection at 254 nm yields system suitability resolution ≥ 2.0 between D and L peaks; the minor L-enantiomer is quantifiable at 0.05% area. Injection precision for a 0.1% L-enantiomer standard is assessed using six replicate injections with relative standard deviation ≤ 2.0%. Column temperature is controlled with a Peltier compartment to ±0.5°C to avoid retention time drift. Sample preparation uses amber vials because the ethyl ester hydrochloride is light-sensitive in solution; samples are analysed within 8 h of preparation. This procedure is applied for lot release of chiral intermediates intended for peptide active pharmaceutical ingredients and complies with system suitability requirements described in USP <621>.
D-Phenylalaninamide is produced by pressure aminolysis of H-D-Phe-OEt·HCl in 7 M methanolic ammonia at −10 to −5°C for 24–48 h. The ester is charged to a stainless-steel reactor with a design pressure of 20 bar at 50°C. Ammonia is charged at 10–12 molar equivalents relative to ester. The reaction mixture is held under mechanical agitation at 120–150 rpm; the amide is formed without intermediate isolation of the free base. Ammonia dissolution is exothermic, so the headspace is cooled to −20°C before ammonia is admitted through a dip tube below the liquid level. After venting, the methanolic solution is concentrated at ≤ 40°C under reduced pressure. The residue is dissolved in dichloromethane and washed with water to remove ammonium chloride. The organic layer is dried over sodium sulfate, filtered, and crystallised by adding diisopropyl ether at 0–5°C. Ester-to-amide conversion is monitored by FTIR disappearance of the ester carbonyl near 1735 cm⁻¹ and appearance of the amide carbonyl near 1650 cm⁻¹. Residual methanol in the final product is controlled at ≤ 3000 ppm according to ICH Q3C. The amide is used in opioid peptide research and for preparing peptidomimetic inhibitors with C-terminal amide bonds. Published data for this specific configuration is limited; reaction calorimetry should be performed to determine the adiabatic temperature rise and to size the pressure relief system.
In the in vitro D-amino acid transaminase assay, H-D-Phe-OEt·HCl is dissolved in 50 mM potassium phosphate buffer pH 7.4 to a final substrate concentration of 2.0 mM. The assay is incubated at 37°C for 30 min with purified D-amino acid transaminase or whole-cell lysate. Formation of D-phenylalanine or phenylpyruvate is quantified after o-phthaldialdehyde derivatisation by HPLC with fluorescence detection at excitation 340 nm and emission 450 nm. The ethyl ester resists hydrolysis by carboxypeptidase A, while the corresponding free acid and amide serve as control substrates. Kinetic parameters are obtained from six substrate concentrations in the range 0.05 mM to 5.0 mM using Lineweaver-Burk analysis. Control experiments with L-phenylalanine ethyl ester hydrochloride are conducted to confirm stereoselectivity and to detect contaminating L-specific aminotransferase activity. The procedure is applicable to biocatalytic process development and to enzyme kinetic quality control. Because the hydrochloride salt shifts the assay pH, stock solutions are prepared in 50 mM phosphate buffer and adjusted to pH 7.4 with 1 M sodium hydroxide before addition. Data for this specific hydrochloride salt is limited in public literature; enzyme batch activity must be normalised against an internal standard such as D-norleucine.
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D-Phenylalanine ethyl ester hydrochloride is distributed under the catalogue designation H-D-Phe-OEt·HCl and is identified by CAS 6301-81-9. The IUPAC name is ethyl (2R)-2-amino-3-phenylpropanoate hydrochloride, and the molecular formula C₁₁H₁₅NO₂·HCl corresponds to a molecular mass of 229.70 g/mol. The free amino ester has a molecular mass of 193.25 g/mol; the difference is 36.46 g/mol of hydrochloride. The theoretical chloride content is 15.43% by mass and the theoretical HCl content is 15.87% by mass. These values are required to calculate the base charge for neutralization in coupling steps. The model field in supplier catalogues is not an industry-wide standard; the product is normally keyed as H-D-Phe-OEt·HCl, with supplier-specific codes designating package size, purity grade, and shipment condition. The compound is a chiral amino acid ester salt, provided as a white to off-white crystalline powder. The carboxyl group is protected as the ethyl ester, the amino group is present as the hydrochloride salt, and the D-configuration is the principal stereochemical identifier. The product is used as a C-terminal protected D-phenylalanine building block in solution-phase peptide synthesis, as a precursor for N-protected D-phenylalanine derivatives, and as a starting point for reduction to D-phenylalaninol after neutralization. Its selection over the L-enantiomer, D-phenylalanine free acid, methyl ester, benzyl ester, or alternative salt forms is determined by stereochemical need, C-terminal protection strategy, residual solvent risk, and handling constraints.
No individual USP or Ph. Eur. monograph exists for D-phenylalanine ethyl ester hydrochloride; therefore specification limits are supplier-defined and must be confirmed against the certificate of analysis. The most critical quality attribute is enantiomeric purity. Infrared and proton NMR spectra do not distinguish the D and L enantiomers, so chiral HPLC is required. Typical research-grade acceptance criteria are ≥98.0% chemical purity by HPLC area percent and ≤0.5% of the L-enantiomer by chiral HPLC, but these values are not universal. Identification is confirmed by Fourier transform infrared spectroscopy in accordance with Ph. Eur. 2.2.24. HPLC methods align with USP 621 and typically use a reversed-phase C18 column with UV detection at 210 nm or 254 nm. For chiral separation, polysaccharide or macrocyclic glycopeptide chiral stationary phases are used under normal-phase or polar-ionic conditions; the mobile phase and retention time are method-specific and are reported in the certificate of analysis. Water content is determined by Karl Fischer titration according to USP 921 Method Ic. Residual solvents are determined by headspace gas chromatography according to USP 467. If the product is used in a drug-substance route, the supplier documentation should include an ICH Q3D elemental impurity risk statement and ICH Q3C residual solvent classification. Analytical laboratories supporting this specification are typically accredited to ISO/IEC 17025:2017; the manufacturer’s quality system is commonly certified to ISO 9001:2015. Where applicable to finished pharmaceuticals, manufacturing may also fall under 21 CFR 210/211 or ICH Q7 for active pharmaceutical ingredients.
| Parameter | Representative acceptance limit | Method |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual inspection |
| Identification | Matches reference spectrum | FTIR, Ph. Eur. 2.2.24 |
| Chemical purity | ≥98.0% area | HPLC, USP 621 |
| Enantiomeric purity | ≤0.5% L-isomer | Chiral HPLC |
| Water content | ≤0.5% | Karl Fischer, USP 921 Method Ic |
| Residual solvents | Complies with USP 467 limits | Headspace gas chromatography |
| Specific rotation | Reported on COA in defined solvent, concentration, temperature, and wavelength | Ph. Eur. 2.2.7 |
| Chloride content | Batch-specific; salt stoichiometry confirmation | Potentiometric titration or ion chromatography |
| Elemental impurities | ICH Q3D risk statement required for drug use | ICP-MS or ICP-OES |
Chiral HPLC area percent alone is not a complete assay of the salt; the counterion should be confirmed separately if salt stoichiometry is critical. Chloride content can be determined by potentiometric titration or ion chromatography. The sum of HPLC area percent, water, residual solvents, chloride, and non-chromophoric impurities is used to establish mass balance in the batch. The optical rotation sign should not be used as the sole proof of D-configuration because the observed rotation depends on solvent, concentration, and wavelength. When enantiomeric excess above 99.0% is required, the chiral method should be validated for limit of quantitation and detector linearity under ICH Q2(R1). Published stability and method robustness data for this specific configuration are limited; each manufacturing site should establish batch release criteria suitable for the intended transformation.
Moisture, pH, and temperature are the main degradation boundaries. The ethyl ester group can hydrolyze to D-phenylalanine and ethanol. Hydrolysis is slow in cold neutral or acidic media but accelerates under alkaline conditions. Above pH 8, the ester is increasingly subject to saponification, and the α-carbon becomes more susceptible to base-catalyzed racemization. The product should be stored in tightly closed, desiccated containers under inert gas, preferably at 2–8 °C. Prolonged exposure to relative humidity above 60% can produce caking and raise the water content; if the Karl Fischer value exceeds 0.5%, drying under reduced pressure at 25–30 °C may be considered, but published drying data for this specific salt are limited. The hydrochloride salt is not exposed to ambient air for extended periods on production lines; after weighing, the container should be resealed and returned to cold storage. The product is incompatible with strong bases, aqueous alkali above pH 8, and direct addition of strong hydride reagents before neutralization.
In solution-phase peptide synthesis, D-Phe-OEt·HCl is converted to the free amino ester immediately before coupling. The release of the free base requires a tertiary amine such as N-methylmorpholine or N,N-diisopropylethylamine at 1.05–1.20 molar equivalents relative to the hydrochloride. The neutralization should be performed in anhydrous dichloromethane or dimethylformamide at 0–4 °C under nitrogen. The free amine is then treated with an N-protected amino acid and a coupling reagent. Representative published conditions for analogous D-amino acid ester hydrochlorides use EDC hydrochloride/HOBt or HATU with 1.1–1.2 equivalents of coupling reagent in DMF at 0–4 °C, followed by slow warming to 20–25 °C. Published data for this specific configuration are limited, and coupling protocols should be verified on the intended scale. The ethyl ester remains intact during short exposure typical of Fmoc removal with piperidine in DMF and during Boc removal with trifluoroacetic acid; this acid-stability is a major reason to select the ethyl ester over a tert-butyl ester. The ethyl ester is not removable by catalytic hydrogenolysis, so it is not a replacement for a benzyl ester when a neutral C-terminal deprotection is required. Final hydrolysis to the free acid is usually carried out with lithium hydroxide in tetrahydrofuran-water at 0 °C, with the reaction stopped as soon as conversion is complete; prolonged base contact can reduce enantiomeric purity. Acylation with acid chlorides or anhydrides can be performed under Schotten-Baumann conditions at pH 8–9 using sodium carbonate in dichloromethane-water. The pH window is narrow because the free amine must be present but the ethyl ester must not be hydrolyzed. The resulting N-acyl D-phenylalanine ethyl ester remains C-terminal protected and can be further hydrolyzed or aminolyzed. In pilot-scale reactors, jacket temperature control and controlled dosing of base are used to maintain the 0–5 °C window. Because the salt releases chloride upon neutralization, glass-lined or Hastelloy equipment is preferred for long campaigns; carbon steel is generally not recommended for aqueous acidic stages.
Reduction of the ester is another application. After neutralization and extraction into an aprotic solvent, the free amino ester can be reduced to D-phenylalaninol with lithium aluminium hydride in tetrahydrofuran or with sodium borohydride–calcium chloride in ethanol/tetrahydrofuran. Direct addition of hydride reagents to the hydrochloride salt is not appropriate because the acidic salt protonates hydride and releases hydrogen. The reduction product should be isolated under nitrogen and protected from atmospheric carbon dioxide. The reaction is exothermic; controlled addition at −5 °C to 5 °C is typical. Published protocols vary in stoichiometry and work-up.
The D-enantiomer and L-enantiomer have identical molecular mass and salt stoichiometry but differ in configuration and chiral recognition. L-Phenylalanine ethyl ester hydrochloride, CAS 3182-93-2, is the enantiomeric counterpart; it cannot be substituted for the D-form in a peptide that requires the D configuration. In non-biological synthetic steps the two enantiomers may appear similar, but in drug substance synthesis the configuration affects final drug identity and must be controlled. D-Phenylalanine ethyl ester hydrochloride differs from D-phenylalanine free acid, CAS 673-06-3, in that the free acid has a free carboxyl group and requires carboxyl activation for peptide bond formation; the ethyl ester salt supplies the carboxyl group already protected and the amino group available after neutralization. Compared with the methyl ester analogue, the ethyl ester releases ethanol during hydrolysis. Ethanol is an ICH Q3C Class 3 solvent with a permitted daily exposure of 50 mg/day, whereas methanol is an ICH Q3C Class 2 solvent with a permitted daily exposure of 30 mg/day; this difference can influence final API residual solvent risk. In homogeneous alkaline hydrolysis, methyl esters are generally more easily saponified than ethyl esters because of reduced steric hindrance, but published kinetic data for D-phenylalanine derivatives are not compiled in public monographs. The hydrochloride salt provides a defined crystalline solid with a theoretical HCl mass fraction of 15.87%, which simplifies neutralization calculations compared with a free base of uncertain hydration state.
| Product | CAS | Molecular mass | Key functional difference |
|---|---|---|---|
| D-Phenylalanine ethyl ester hydrochloride | 6301-81-9 | 229.70 g/mol | C-terminal ethyl ester protection; D configuration; hydrochloride salt |
| L-Phenylalanine ethyl ester hydrochloride | 3182-93-2 | 229.70 g/mol | Enantiomeric opposite; otherwise similar salt and ester form |
| D-Phenylalanine | 673-06-3 | 165.19 g/mol | Free acid; no ester protecting group; no hydrochloride counterion |
In a production campaign, the material should be charged under dry nitrogen into a reactor previously rinsed with dry solvent and vacuum-stripped. If the charging room exceeds 60% relative humidity, the product may cake and absorb moisture; weighed portions should be retested if exposure exceeds the site-defined hold time. The neutralization and coupling steps should be maintained at 0–5 °C as a conservative process window. At temperatures above 10 °C and in the presence of water, ester hydrolysis and racemization risk increase once the free base is generated. The hydrochloride salt releases chloride upon neutralization, and chloride-containing aqueous streams should be handled in glass-lined or Hastelloy reactors; unlined carbon steel is not recommended. Filtration and drying from ethyl acetate or methyl tert-butyl ether under nitrogen reduces hygroscopic uptake, but prolonged hot drying may increase hydrolysis. The product should not be milled in humid air; if particle size control is required, it should be conducted under dry nitrogen. These limits are operational boundaries, not compendial requirements. Published pilot-scale data for this exact salt are limited, so site-specific validation and stability data are required before implementing large-scale use. The material can be converted to N-protected intermediates such as N-Boc-D-phenylalanine ethyl ester by treatment with di-tert-butyl dicarbonate and a mild base in anhydrous solvent; in aqueous base the ethyl ester may hydrolyze, so anhydrous or mixed-solvent systems with controlled pH are used. Subsequent Boc deprotection with acid regenerates the hydrochloride or acetate salt and leaves the ethyl ester intact under typical conditions.
