| HS Code | 877217 |
| Product Name | D-Alanine Ethyl Ester Hydrochloride |
| Cas Number | 17407-54-0 |
| Molecular Formula | C5H11NO2·HCl |
| Molecular Weight | 153.61 g/mol |
| Appearance | White to off-white crystalline powder |
| Melting Point | 84-86 °C |
| Optical Rotation | [α]D20 = -8.5° (c=2, H2O) |
| Solubility | Soluble in water, methanol, ethanol, and DMF |
| Storage Conditions | Store at 2-8 °C, under inert atmosphere, protected from light and moisture |
| Purity | ≥98% |
| Hygroscopicity | Hygroscopic |
| Physical State | Solid |
| Smiles | Cl.C[C@@H](N)C(=O)OCC |
As an accredited D-alanine 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 of white crystalline powder in a sealed amber glass bottle, protected from moisture and light. |
| Container Loading (20′ FCL) | Container Loading (20′ FCL): 20-foot full container load of D-Alanine Ethyl Ester Hydrochloride, packed in sealed drums, palletized, secured, dry and ventilated. |
| Shipping | D-Alanine Ethyl Ester Hydrochloride is typically shipped at ambient temperature in a tightly sealed, moisture-resistant container with desiccant. Avoid exposure to heat, humidity, and prolonged light. Ensure proper labeling and follow standard chemical handling protocols. It is generally not classified as hazardous for transport under normal conditions. |
| Storage | Store D-alanine ethyl ester hydrochloride in a tightly sealed container under an inert atmosphere (e.g., nitrogen or argon). Keep refrigerated at 2–8 °C, protected from moisture, light, and air. Ensure the storage area is cool, dry, and well-ventilated. Avoid contact with strong oxidizing agents. Always open and handle in a dry environment to prevent hydrolysis. |
| Shelf Life | Stable for up to two years when stored sealed, dry, at room temperature, protected from moisture and light. |
In solution-phase peptide synthesis, D-alanine ethyl ester hydrochloride is charged as the D-Ala-OEt building block after in situ freebase generation. The hydrochloride is suspended in anhydrous N,N-dimethylformamide or dichloromethane at 0–5 °C. N-methylmorpholine or N,N-diisopropylethylamine is added in 1.1–1.3 molar equivalents relative to the hydrochloride. Nα-protected amino acid is dissolved separately. Coupling reagent system may be EDC·HCl with HOBt monohydrate at 1.0–1.2 molar equivalents or HBTU at 1.1–1.5 molar equivalents. Activation is carried out at 0–5 °C for 15–30 min. The freebased ester solution is added dropwise over 30–60 min. The reaction mixture is stirred at 20–25 °C for 4–18 h. Completion is monitored by IP-HPLC or TLC. The terminal product class comprises D-Ala-containing peptide APIs and peptide hormone analogues. These sequences exploit D-configuration for resistance to aminopeptidase degradation. Compliance under drug master file frameworks follows ICH Q7 for active pharmaceutical ingredient intermediates. Residual solvent testing is performed according to USP <467>. Elemental impurities are controlled per ICH Q3D. The ethyl ester protecting group requires strict moisture control during storage and use. Hydrolysis to D-Ala accelerates above 5 wt% water in DMF at room temperature.
| Control point | Standard or method | Typical limit |
|---|---|---|
| Chiral purity | HPLC, ligand-exchange or chiral column | ≥99.0% ee |
| Residual solvents | USP <467> | Class 2 solvents per ICH Q3C |
| Elemental impurities | ICH Q3D | Route-specific PDE limits |
| Water content | Karl Fischer titration | ≤0.5 wt% |
| Assay | HPLC, external standard | ≥98.0% anhydrous basis |
Racemization risk during activation is the primary constraint in solution-phase peptide assembly. Activation of Nα-protected D-Ala with uranium reagents under excess N,N-diisopropylethylamine can promote oxazolone formation at the α-carbon. Published data for D-alanine ethyl ester hydrochloride under HATU/DIPEA activation at ambient temperature is limited. Analogous L-alanine ethyl ester systems have shown that oxazolone-mediated racemization increases when activation temperature is raised from 0 °C to 20 °C. Process development reports from pilot-scale campaigns indicate that racemization can be maintained below 0.3% when activation temperature does not exceed 5 °C and coupling is initiated within 30 min of freebase generation. A jacketed glass reactor with PTFE baffles and a recirculating chiller is used for tight thermal control. High-shear mixing is not required. The hydrochloride should be neutralized immediately before coupling. Prolonged standing of freebased D-Ala-OEt in polar aprotic solvents increases moisture uptake and epimerization risk. Solvent selection also alters reaction rate. Dichloromethane reduces oxazolone formation relative to DMF but may require longer coupling times of 12–24 h. The operational boundary is therefore a temperature window of 0–5 °C and a freebase hold time below 30 min.
For Fmoc solid-phase synthesis, D-alanine ethyl ester hydrochloride is first hydrolysed to free D-alanine and then protected as N-Fmoc-D-Ala. The hydrochloride is dissolved in tetrahydrofuran/water at 0–5 °C. Lithium hydroxide solution is added in 2.0–2.2 molar equivalents. The mixture is stirred for 1–2 h until TLC shows complete ester cleavage. Tetrahydrofuran is removed under reduced pressure at 30–35 °C. The aqueous phase is adjusted to pH 8.5–9.0 with sodium carbonate. Fmoc-OSu is added in 1.0–1.2 molar equivalents in dioxane. The reaction is maintained at 0–5 °C for 4–6 h and then at 20–25 °C for 6–12 h. The product is isolated by ethyl acetate extraction followed by recrystallization. The resulting N-Fmoc-D-Ala is used as the first amino acid loaded onto Wang resin or 2-chlorotrityl chloride resin. Typical loading density is 0.4–0.8 mmol/g. Peptide chain elongation then proceeds stepwise under standard Fmoc deprotection cycles with 20% piperidine in DMF. Terminal products include D-Ala-containing antimicrobial peptides and peptidomimetic hormone analogues. This conversion is covered by the same ICH Q7 requirements for GMP intermediates. Batch-to-batch variance in loading density is controlled by UV quantification of the dibenzofulvene adduct at 301 nm.
Bioanalytical method development for chiral carboxylic acid metabolites can use D-alanine ethyl ester hydrochloride as a pre-column derivatization reagent. The freebased D-Ala-OEt is reacted with carboxy-containing analytes via EDC/HOBt in acetonitrile at 20–25 °C for 60–120 min. The resulting diastereomeric amides are separated on a conventional C18 column. This strategy avoids expensive chiral stationary phases for routine metabolite screening. Derivatization must be performed with enantiomerically pure reagent. Any D-Ala ethyl ester hydrochloride with enantiomeric excess below 99.0% creates quantitation bias between diastereomer pairs. Method validation follows ICH M10 for bioanalytical method validation. Injection sequences include blank plasma and quality control samples at 3 concentration levels. The derivatization reagent is stored desiccated at 2–8 °C. Working solutions are prepared fresh daily because the freebased ester degrades in aqueous acetonitrile by ester hydrolysis. This application is concentrated in pharmacokinetic laboratories and drug metabolism stability studies.
Chiral selector synthesis for ligand-exchange chromatography uses D-alanine ethyl ester hydrochloride as the chiral amine precursor. The hydrochloride is freebased with N-methylmorpholine in DMF. HBTU activation with 1.0–1.1 equivalents is used to form the amide bond with 3-aminopropyl silica. Coupling proceeds at 20–25 °C for 12–18 h with gentle rotary agitation. Unreacted amino groups are end-capped with acetic anhydride to reduce non-specific retention. The resulting chiral stationary phase is slurry-packed into stainless steel columns of 150 mm × 4.6 mm internal diameter. Mobile phase is copper(II) acetate at 1.0–5.0 mmol/L in water/methanol. Flow rate is 1.0 mL/min. Detection is by UV at 254 nm. Under these conditions, underivatized D/L amino acid enantiomers are separated via ternary copper-ligand complexes. Resolution depends on column temperature and mobile phase pH. Optimal pH is between 4.5 and 5.5. Below pH 4.0, copper complexes dissociate and peak shape deteriorates. Above pH 6.0, ligand exchange slows and retention times increase. Column qualification follows ICH Q2(R1) for system precision, repeatability, and limit of detection. This stationary phase is used for enantiomeric excess testing of alanine and other native amino acids in fermentation broths and peptide hydrolysates.
D-Alanine ethyl ester hydrochloride is a starting material for D-Ala-D-Ala dipeptide ester synthesis used in peptidoglycan enzymology. The hydrochloride is freebased in tetrahydrofuran with N-methylmorpholine at 0–5 °C. Isobutyl chloroformate is added in 1.0–1.1 molar equivalents to form a mixed anhydride. After activation for 10–15 min, D-alanine methyl ester hydrochloride is added as the second component with additional N-methylmorpholine. The coupling is maintained at 0–5 °C for 2–4 h. The terminal product is D-Ala-D-Ala methyl ester, which serves as a substrate analogue for D-Ala carboxypeptidase and peptidoglycan transpeptidase assays. These enzymes are targets of β-lactam antibiotic research. The synthesis is performed under ISO 9001 laboratory control. Enzymatic assays require product purity by HPLC of ≥95.0%. Storage conditions are −20 °C under argon. Exposure to moisture leads to methyl ester hydrolysis and reduced enzyme inhibition readout. This application is highly specific to antibiotic resistance research and is not interchangeable with large-scale peptide manufacturing. Equipment requirements are limited to standard glassware with inert atmosphere capability. No high-pressure hydrogenation or high-temperature distillation is needed.
For screening D-amino acid aminotransferase variants, D-alanine ethyl ester hydrochloride is hydrolysed in situ to D-alanine and used as the donor substrate. Volumetric productivity is enzyme-specific. Published data for this ester hydrochloride configuration in evolved aminotransferase variants is limited.
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D-Alanine ethyl ester hydrochloride is the hydrochloride salt of the ethyl ester of D-alanine, with molecular formula C5H12ClNO2 and molecular weight 153.61 g/mol. The CAS registry number is 6331-09-5. Commercial material is typically supplied as a white to off-white crystalline powder with an assay specification of 98.0% to 102.0% by non-aqueous titration. The salt form is selected because it converts a volatile, alkaline free amino ester into a crystalline solid that can be handled with standard powder-transfer equipment and dispensed with improved accuracy. The compound is soluble in water, methanol, N,N-dimethylformamide, and dimethyl sulfoxide; solubility is limited in ethyl acetate and methyl tert-butyl ether. As an α-amino ester, the molecule contains a protected carboxyl group and a masked amino group; the ethyl ester can be removed by hydrolysis, and the protonated amine can be neutralized in situ before acylation.
Because the material is a hydrochloride salt, aqueous solutions are acidic, and neutralization with a tertiary amine liberates the free ester. The free base is more soluble in organic media but is also more mobile and must be consumed immediately. These differences are amplified at production scale, where the crystalline hydrochloride is preferred for metering, containment, and reduction of amine exposure.
The D-configuration is the primary structural distinction from the L-enantiomer. L-Alanine ethyl ester hydrochloride is used for L-amino acid sequences, whereas the D-compound introduces the opposite stereochemical sense. Enantiomeric purity is specified by chiral HPLC or chiral GC and is commonly controlled at ≥99.0% enantiomeric excess, with the L-isomer treated as a specified impurity. The α-carbon configuration influences biological recognition, crystallinity of downstream diastereomers, and retention time in chiral chromatographic systems.
The hydrochloride form differs from the free base in volatility, amine release, and storage behavior. The free base is a low-melting solid or liquid with measurable vapor pressure, whereas the hydrochloride is a crystalline salt that can be stored under desiccant at 2–8 °C. Compared with the methyl ester hydrochloride, the ethyl ester has one additional carbon, which increases molecular weight and reduces vapor pressure. The ethyl ester also hydrolyzes more slowly than the methyl ester under equivalent aqueous alkaline conditions; however, published data for the isolated product in complex peptide-coupling media are limited, and solvent-specific hold times should be verified during pilot studies.
The product is supplied in research-grade, development, kilo-lab, and cGMP grades depending on the intended use. Research-grade material may have assay and chiral purity specifications comparable to larger quantities but with less comprehensive documentation. cGMP material includes batch records, change control, and a certificate of analysis that reports the parameters in Table 1. Large-scale campaigns typically use the cGMP grade when the compound is a regulatory starting material or an advanced intermediate; process development orders are often supplied as development-grade to permit method qualification before scale-up.
Representative release parameters are shown in Table 1. These parameters are typical of development-quantity and cGMP-grade material and should be verified against the certificate of analysis for a specific lot.
| Parameter | Typical acceptance criterion | Analytical reference |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual inspection |
| Assay | 98.0–102.0% | Non-aqueous titration |
| Enantiomeric purity | ≥99.0% ee | Chiral HPLC |
| Water content | ≤0.5% | USP 921 Karl Fischer |
| Loss on drying | ≤0.5% | USP 731 |
| Residue on ignition | ≤0.1% | USP 281 |
| Elemental impurities | Conforms to ICH Q3D option 1 limits | USP 233 |
| Residual solvents | Conforms to ICH Q3C limits | USP 467 |
| Specific rotation | Reported as [α]D20 | USP 781 |
For a solution-phase coupling sequence, the hydrochloride is suspended in anhydrous N,N-dimethylformamide or dichloromethane and neutralized with N-methylmorpholine or diisopropylethylamine. The base charge is typically 1.0–1.2 mol per mol of hydrochloride. Neutralization is performed at 0–5 °C under nitrogen because the liberated free amine can absorb carbon dioxide or promote ester aminolysis. After neutralization, the solution is transferred directly to a coupling vessel containing a preactivated N-protected amino acid. HATU or HBTU is used with a second equivalent of tertiary base to generate the active ester; the free amino ester then attacks the activated carboxyl component to form the amide bond.
The pH and temperature boundaries are more important than the choice of coupling additive. Ethyl esters of primary amino acids hydrolyze rapidly at pH above 8.5, especially when water is present. In preparative campaigns, the free-amine intermediate should not be held for more than 30–60 min before coupling. If the coupling is delayed, D-alanine and ethanol are generated, reducing yield and introducing the free acid into downstream workups.
On jacketed reactors with a 50–100 L working volume, temperature control is achieved with a circulating bath set below 0 °C while internal process temperature is maintained at 2–5 °C. Addition of diisopropylethylamine is exothermic; a controlled feed over 20–30 min prevents local pH excursions and thermal loss of the free base. These boundaries are derived from the known behavior of primary amino acid ethyl esters; published data for this specific salt under all solvent ratios are limited, and new processes should verify stability at the intended pH and temperature.
In mixed-mode process development, D-alanine ethyl ester hydrochloride is sometimes converted to the free amine by partition between an organic solvent and a cold aqueous carbonate or bicarbonate solution. The extraction must be performed with short contact times because the ester partitions into the organic layer while the aqueous layer hydrolyzes the free ester. Ethyl acetate or dichloromethane is used, and the organic layer is dried over anhydrous sodium sulfate or molecular sieves before coupling. This operation is avoided when possible by in situ neutralization in the coupling solvent.
The ester group provides a masked carboxyl handle for condensation with acid chlorides, sulfonyl chlorides, isocyanates, and electrophilic heterocycles. The free amino group can be derivatized while the carboxyl remains protected, allowing selective formation of amides, sulfonamides, ureas, and carbamates. Subsequent hydrolysis of the ethyl ester liberates D-alanine derivatives or D-alanine itself after acidification. This sequence is used to introduce chirality into non-peptide scaffolds where the D-configuration is required for target binding or metabolic stability.
Compared with D-alanine itself, the ethyl ester hydrochloride is soluble in organic reaction media and avoids zwitterionic behavior that can limit reactivity. The hydrochloride is neutralized by the same bases used in peptide coupling; however, the neutralization can also generate the free amine for later N-functionalization. The ethyl ester is generally retained during anhydrous N-acylation, while the carboxyl can be unmasked later under mild saponification conditions. Ethanol released by hydrolysis is easier to remove by vacuum distillation than methanol released from the methyl ester in some pilot-plant operations.
A comparison with common alanine ester forms is provided in Table 2. Selection of the D-ethyl ester hydrochloride is driven by chirality, salt handling, and the required deprotection conditions.
| Product form | Molecular weight | CAS registry number | Key functionality | Typical selection driver |
|---|---|---|---|---|
| D-Alanine ethyl ester hydrochloride | 153.61 g/mol | 6331-09-5 | D chiral amine, ethyl ester, hydrochloride salt | D-peptide and chiral-intermediate synthesis |
| L-Alanine ethyl ester hydrochloride | 153.61 g/mol | 1115-59-9 | L chiral amine, ethyl ester, hydrochloride salt | L-peptide and natural-product analogue synthesis |
| D-Alanine methyl ester hydrochloride | 139.58 g/mol | 14316-06-4 | D chiral amine, methyl ester, hydrochloride salt | Lower ester steric bulk and more active acyl acceptor |
| Alanine ethyl ester free base | 117.15 g/mol | — | Free amine, ethyl ester, no hydrochloride counterion | Direct use where salt removal is undesirable |
The free base of alanine ethyl ester is a low-melting solid or liquid with amine odor and a tendency to absorb carbon dioxide. The hydrochloride salt is crystalline, non-volatile, and easier to isolate by filtration. At kilogram scale, the salt can be dispensed from screw feeders or weighed under laminar flow without the high vapor exposure associated with the free amine. This reduces operator exposure and improves batch-to-batch mass balance because material losses from evaporation and surface film formation are less pronounced. In manufacturing suites with open transfer, the crystalline powder is also easier to contain with local exhaust ventilation.
Metering accuracy is particularly relevant in peptide and chiral-intermediate campaigns where the amine component must be charged at stoichiometric control. A crystalline hydrochloride can be assayed and then weighed to the exact molar quantity; the free base may require cold storage, Schlenk handling, and an additional assumption about purity after partial evaporation. Salt-form use also removes the need for in situ salt formation from the free base and hydrogen chloride, which can introduce water and cause ester hydrolysis.
Structural confirmation is performed by FTIR and electrospray ionization mass spectrometry. The ester carbonyl stretching absorption appears near 1730 cm−1, and the protonated amine produces broad N–H absorption in the 2500–3000 cm−1 region. Positive-mode ESI shows the protonated free-base ion at 118.1 m/z. Chiral HPLC methods usually employ a polysaccharide-based column with an alcohol-hexane mobile phase containing a small amount of trifluoroacetic acid or diethylamine; retention times are column-specific and are not transferable across manufacturers. For release, a racemic alanine ethyl ester standard is used to confirm resolution between D and L peaks.
Analytical batch-to-batch variation is most often observed in water content and residual solvent profile rather than in assay or chiral purity. Process development samples should therefore be characterized by Karl Fischer titration and static headspace gas chromatography before use in water-sensitive coupling chemistry. In dimethylformamide, water above 1000 ppm can hydrolyze the activated acid or the free amino ester; molecular sieves are not always sufficient once the hydrochloride has been neutralized.
Storage and handling boundaries are governed by hygroscopicity and ester reactivity. The compound should be stored under inert gas at 2–8 °C with desiccant, and containers should be closed immediately after dispensing. At relative humidity above 60%, water uptake can cause caking and reduce assay. Prolonged contact with water or aqueous buffer at pH above 8.0 hydrolyzes the ester, releasing ethanol and D-alanine. Aqueous process solutions should be kept cold and held for the shortest practical interval.
The product is incompatible with strong oxidizing agents and should not be mixed with anhydrous acidic or basic dehydrating agents without controlled conditions. Primary and secondary amines can attack the ester carbonyl at elevated temperature; therefore, storage with amine-containing materials and formulation adjuvants is avoided. For waste handling, the compound should be segregated as a corrosive solid due to the hydrochloride counterion, and local regulations for chlorinated organic salts should be followed.
