| HS Code | 927269 |
| Chemical Name | D-Alanine |
| Iupac Name | (2R)-2-aminopropanoic acid |
| Molecular Formula | C3H7NO2 |
| Molecular Weight | 89.09 g/mol |
| Cas Number | 338-69-2 |
| Pubchem Cid | 71080 |
| Melting Point | 291-293 °C (decomposes) |
| Density | 1.424 g/cm³ |
| Solubility | Soluble in water; slightly soluble in ethanol |
| Optical Rotation | [α]D -1.8° (c=2, H2O) |
| Pka | 2.35 (carboxyl), 9.87 (amino) |
| Isoelectric Point | 6.01 |
| Appearance | White crystalline powder |
As an accredited D-alanine factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | D-alanine, 25 g, packaged in a sealed polypropylene bottle with tamper-evident cap and clearly labeled purity information. |
| Container Loading (20′ FCL) | D-alanine is loaded into a 20′ FCL in 25 kg fiber drums, palletized, shrink-wrapped, and secured for safe transport. |
| Shipping | D-alanine, a non-essential amino acid, ships as a stable crystalline powder. Packaging uses sealed, moisture-resistant containers, often with desiccant. Store cool and dry, away from strong oxidizers. Not classified as dangerous goods for transport, but avoid dust inhalation and follow standard chemical hygiene. Ensure compliance with local regulations for laboratory reagents. |
| Storage | Store D-alanine in a tightly sealed, clearly labeled container in a cool, dry, well-ventilated area away from direct sunlight, heat, and moisture. Keep the container closed when not in use. Avoid contact with strong oxidizing agents, acids, and bases. Room temperature storage is generally suitable unless the product label specifies otherwise. Ensure proper handling to maintain purity and stability. |
| Shelf Life | D-alanine has a shelf life of approximately three to five years when stored in a cool, dry, sealed container. |
Alitame synthesis consumes D-alanine as the C-terminal amino acid fragment in a mixed anhydride coupling with protected L-aspartic acid. The charge ratio is fixed between 1.00 and 1.05 mol per mole of protected aspartate; wider excess produces crystalline agglomerates that carry unreacted D-alanine into the final sweetener matrix. Process control is governed by FSSC 22000, ISO 22000:2018, and the destination market’s food additive schedule, because regulatory authorization for alitame is jurisdiction-specific and EU-bound finished formulations require verification against Regulation (EC) No 1333/2008. The reaction is run in jacketed stainless steel at -15 to -10°C using isobutyl chloroformate for mixed anhydride formation in tetrahydrofuran; the D-alanine charge is pre-neutralized to pH 8.5–9.5 to maintain nucleophilicity without racemizing the aspartate residue. Residual free D-alanine is reduced by chilled water washing at 5°C and recrystallization from ethanol/water to below 0.5 wt% in the dried wet cake. Terminal product type is alitame high-intensity sweetener, used in carbonated beverages, tabletop sweeteners, and dry mix applications only where national regulations permit.
D-alanine for this route is normally supplied with a purity above 99.0% and a loss on drying below 0.20%; higher moisture would quench the mixed anhydride and shift the reaction toward free acid, generating L-aspartic acid-related impurities that are difficult to purge by crystallization. Production-scale campaigns of alitame typically monitor the consumption of D-alanine by OPA pre-column derivatization and RP-HPLC at 338 nm, allowing real-time adjustment of free monomer below the rejection limit. The downstream crystallization step uses controlled cooling at 0.2°C/min to maintain polymorph consistency, because rapid nucleation produces fines that entrain residual solvent and depress yield.
In pharmaceutical contract manufacturing, D-alanine (CAS 338-69-2) is charged as a chiral building block, not as a late-stage formulation ingredient. The typical feed ratio for amide bond-forming reactions lies between 1.0 and 1.2 molar equivalents relative to the activated carboxylic acid partner; the excess compensates for partial hydrolysis and adsorption losses during activated carbon treatment. Release methods for this downstream route follow ICH Q7 principles for active pharmaceutical ingredient intermediates, with chiral purity measured by HPLC against USP <621> and optical rotation controlled under USP <781> or Ph. Eur. 2.2.7. Production in multi-purpose glass-lined reactors begins with N-protection under pH-stat control at 8.0–9.0 and 0–5°C, followed by activation with a carbodiimide system such as EDCI in anhydrous 2-methyltetrahydrofuran. Residual water is held below 0.10% by Karl Fischer titration because higher moisture quenches the activation step and reduces coupling efficiency. Terminal product types include N-Boc-D-alanine, Fmoc-D-alanine, D-Ala-containing peptide fragments, and peptidomimetic scaffolds consumed in antimicrobial drug discovery and chiral peptide synthesis.
Process development records from pilot lines show that cooling rates during crystallization must remain below 0.3°C/min to avoid fines and that vacuum drying at 45°C for 4 h is required when the material has been stored above 60% relative humidity. The analytical release of D-alanine for this sector typically includes assay by non-aqueous titration at 98.5–101.0% and enantiomeric excess above 99.0%. These boundaries are not theoretical; they reflect the need to control downstream diastereomer ratios in coupling reactions where the chiral center is adjacent to an activated carbonyl. Batch-to-batch variance in residual chloride or sodium ion content is monitored because these species interfere with palladium-catalyzed deprotection stages used later in certain peptide sequences.
A racemic carboxylic acid stream that fails indirect resolution can be treated with D-alanine as a low-molecular-weight chiral base in the resolution train. The charge ratio moves across 0.5–1.5 molar equivalents relative to the racemic substrate; below 0.5 equivalents the recovery of the target enantiomer falls below process thresholds, while above 1.5 equivalents the excess precipitant contaminates the mother liquor and reduces filtration throughput. Compliance documentation for this sector aligns to ISO 9001:2015, REACH Regulation (EC) No 1907/2006 registration for tonnage above 1 t/a, and ICH Q11 where the resolved intermediate enters a drug substance route. The downstream procedure is run in a jacketed crystallizer with controlled cooling rates of 0.1–0.3°C/min and seeded at the cloud point; the resulting diastereomeric salts are isolated by pressure filtration, washed with chilled acetone, and dissociated with dilute HCl to release the target enantiomer. Terminal product types include single-enantiomer carboxylic acid building blocks and chiral auxiliaries for asymmetric induction. Published data for individual racemate matrices is limited; pilot-scale resolution trials are therefore required before fixing the D-alanine charge ratio for a specific commercial campaign.
The drying stage after dissociation is operated at 40–50°C under vacuum to avoid sublimation losses. Residual solvent limits for the resolved acid are typically set at ≤500 ppm for toluene and ≤100 ppm for acetone when the material is destined for pharmaceutical coupling. Equipment from fine chemical contract manufacturing lines is usually stainless steel rather than glass-lined, because the final dissociation step uses dilute hydrochloric acid and the primary corrosion risk shifts from reagent compatibility to chloride-induced pitting during repeated batch cycles.
D-Alanine enters cosmetic formulation not as a bulk filler but as a pH-sensitive skin-conditioning amino acid within aqueous-phase amino acid blends. The charge level in leave-on emulsions is typically held between 0.1 and 1.0 wt% of the final formulation; above this window the acid-base buffering required to keep pH stable can disturb the rheology of carbomer and acrylate copolymer systems. Compliance obligations under Regulation (EC) No 1223/2009 require a finished product safety assessment under Annex I, while manufacturing hygiene follows ISO 22716:2007; D-alanine is not listed as a prohibited or restricted substance under Annex II or Annex III, but the cosmetic safety assessor must still address the D-isomer-specific sensitization profile if source microbiota or fermentation residual data is not supplied. The production process dissolves D-alanine in deionized water at 40–50°C before combining with the oil phase at 75–80°C; the emulsion is then homogenized at 2,500–3,500 rpm and pH-adjusted to 5.0–5.8 with lactic acid or citrate buffer. Terminal product types include O/W skin-care emulsions, hydrogel moisturizers, and body lotions. Published data for D-isomer-specific performance differences in these matrices is limited, so formulators typically conduct paired comparative stability testing against L-alanine under accelerated storage at 45°C for 30 days.
Incompatibility boundaries matter more than addition level in this sector. D-alanine should not be pre-blended with strong oxidizing agents such as benzoyl peroxide or hypochlorite-based preservatives because oxidative deamination produces pyruvic acid and shifts the aqueous phase pH below 4.0. If the formulation contains reducing sugars or ascorbic acid, the temperature should remain below 60°C to limit Maillard-type browning; visual browning can be quantified by absorbance increase at 420 nm. These operational boundaries are part of the raw material handling specification supplied by D-alanine exporters to cosmetic manufacturers, not merely quality-control observations.
Enzymatic assay manufacturers consume D-alanine as a defined substrate in D-amino acid oxidase and alanine racemase activity measurements. The working concentration in reaction buffer commonly spans 0.1–10 mM, with the upper limit set by buffer capacity and the lower limit determined by the detection threshold of the coupled colorimetric or fluorometric readout. For research-use-only reagent packs, ISO 9001:2015 governs lot consistency; if the same substrate is incorporated into an in vitro diagnostic kit, ISO 13485:2016 and, where applicable, Regulation (EU) 2017/746 apply. The downstream production process for a D-alanine substrate solution includes dissolution in phosphate buffer at pH 8.0–8.3, sterile filtration through 0.22 µm PES membrane, and lyophilization or frozen aliquoting to prevent bacterial overgrowth. Terminal product types include D-amino acid oxidase assay kits, alanine racemase screening panels for antimycobacterial drug discovery, and chiral amino acid standard packs for HPLC calibration.
The substrate quality boundary is not merely chemical purity but also the absence of D/L interconversion during storage. Supplier release typically requires chiral purity above 99.0% with storage at 2–8°C for aqueous stocks and ≤25°C for dry powder. In racemase kinetic assays, any pre-existing L-alanine contamination above 0.5% creates measurable background at time zero and distorts initial velocity calculations; therefore OPA derivatization with RP-HPLC is used to confirm L-alanine content before batch acceptance. The material is also screened for acetate and chloride residuals because these ions alter ionic strength in enzyme kinetics and can shift apparent Km values by more than 10%.
| Downstream Sector | Reference Standard / Code | Control Parameter | Typical Acceptance Window |
|---|---|---|---|
| Peptidomimetic API building block | ICH Q7; USP <621>; USP <781>; Ph. Eur. 2.2.7 | Chiral purity / specific optical rotation | ≥99.0% ee; optical rotation within certified lot range |
| Alitame intermediate | ISO 22000:2018; FSSC 22000; EC No 1333/2008 verification | Residual free D-alanine in dried wet cake | ≤0.5 wt% |
| Chiral resolution | ISO 9001:2015; REACH (EC) No 1907/2006 | Diastereomeric salt purity after crystallization | ≥98.0% de |
| Cosmetic emulsion | EC No 1223/2009 Annex I; ISO 22716:2007 | Finished formulation pH after 30 days at 45°C | 5.0–5.8 |
| Enzymatic assay substrate | ISO 13485:2016 where IVD; ISO 9001:2015 | D-alanine chiral purity / L-alanine contamination | ≥99.0%; L-alanine ≤0.5% |
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Across specialty amino acid supply chains, D-alanine (CAS 338-69-2, (R)-2-aminopropanoic acid) is received as a white crystalline solid with a molecular weight of 89.09 g/mol and a nominal water solubility of approximately 165 g/L at 25 °C. Representative product models DA-98 and DA-99 designate assay and chiral-purity thresholds rather than official compendial grade definitions. The material is packaged in 25 kg fibre drums with double polyethylene liners and nitrogen overlay for pharmaceutical intermediate transfer, and in 1 kg high-density polyethylene bottles for laboratory-scale synthesis. The stereogenic centre at the α-carbon is in the (R) configuration, which separates D-alanine from the proteinogenic L-alanine; the α-amino position separates D-alanine from β-alanine. Particle-size control across batches is typically specified as D90 ≤ 74 µm for direct compression blends or 20–40 mesh for solid-phase peptide synthesis. Table 1 summarises representative release parameters for the two product models, based on supplier technical-data-sheet methods rather than a single pharmacopeial monograph.
| Parameter | DA-98 | DA-99 | Method |
|---|---|---|---|
| Appearance | White crystalline powder | White crystalline powder | Visual |
| Assay (dried basis) | 98.0–101.0% | 99.0–101.5% | Perchloric acid titration |
| Specific rotation [α]20D (c=10, 5 M HCl) | -14.0° to -15.0° | -14.0° to -15.0° | USP <781> |
| Loss on drying | ≤0.30% | ≤0.20% | USP <731> |
| Residue on ignition | ≤0.10% | ≤0.10% | USP <281> |
| L-Alanine isomer | ≤1.0% | ≤0.5% | Chiral HPLC, crown ether column |
| Heavy metals as Pb | ≤10 ppm | ≤10 ppm | USP <231> |
| Iron (Fe) | ≤20 ppm | ≤20 ppm | Atomic absorption |
| Bacterial endotoxins | Not specified | ≤0.5 EU/mg | USP <85> |
Chiral identity controls more than optical rotation. D-alanine has pKa1 2.35 and pKa2 9.69; β-alanine has pKa1 3.55 and pKa2 10.24 because its carboxyl and amine groups are separated by two methylene units. The corresponding isoelectric point for D-alanine is 6.02, while β-alanine has a pI of 6.90. These differences alter buffering profiles in fermentation feeds and require pH adjustment with hydrochloric acid or sodium hydroxide to maintain the free dissolved species. In chiral resolution, D-alanine exhibits [α]20D -14.5° (c=10, 5 M HCl), whereas L-alanine shows +14.5° and DL-alanine shows net 0°. β-Alanine is achiral and cannot be used as a stereochemical probe in enzymatic racemization studies. Table 2 provides a compressed comparison of the four common amino acid forms encountered in industrial specification.
| Compound | CAS | Molecular weight | Specific rotation | Decomposition range | Primary industrial distinction |
|---|---|---|---|---|---|
| D-Alanine | 338-69-2 | 89.09 g/mol | -14.5° (c=10, 5 M HCl) | 297 °C (dec) | Peptidoglycan synthon, chiral auxiliary |
| L-Alanine | 56-41-7 | 89.09 g/mol | +14.5° (c=10, 5 M HCl) | 314 °C (dec) | Parenteral nutrition, mammalian cell culture |
| DL-Alanine | 302-72-7 | 89.09 g/mol | 0° | 272 °C (dec) | Racemic process feed |
| β-Alanine | 107-95-9 | 89.09 g/mol | Optically inactive | 200 °C (dec) | Carnosine and pantothenic acid precursor |
For compendial identity testing, infrared absorption spectrophotometry per USP <197> and specific rotation per USP <781> are applied. When the product model DA-99 is designated for injectable synthesis routes, endotoxin release follows USP <85>; DA-98 is not released for parenteral-grade operations. L-Alanine is the proteinogenic isomer and is preferred in mammalian cell culture media, while DL-alanine is normally a racemic cost-reduction feed for applications where optical purity is not functionally significant. The D-isomer selected for antibiotic synthon work should not be assumed interchangeable with these grades without chiral purity verification by HPLC or polarimetry.
In solid-phase peptide synthesis lines, Fmoc-D-Ala-OH is charged at 4 equivalents relative to resin substitution to maintain coupling efficiency above 99% as monitored by Kaiser and chloranil tests. Coupling is executed in DMF at 0.1 M amino acid concentration with HBTU or HATU activation and DIPEA at 25±2 °C for 45–60 min. The D-enantiomer is selected for sequences requiring proteolytic resistance because human serum proteases exhibit stereospecific cleavage of L-peptide bonds; published data for specific clinical D-peptide candidates is limited. Process failures arise when free moisture exceeds 0.5%. In such cases, vacuum drying at 80±2 °C under <10 kPa for 3 h before activation is required. D-Alanine should not be combined with nitrous acid or strong oxidising agents because oxidative decarboxylation generates acetaldehyde and pyruvic acid derivatives that contaminate downstream crystalline intermediates.
Fermenter feed preparation in β-lactam antibiotic intermediate production uses sterile-filtered D-alanine solution at 50–100 g/L adjusted to pH 6.8–7.2. Batch-to-batch variance in free ammonia above 0.02% has been observed to depress alanine racemase turnover during cell-free enzyme assays in 1,000 L bioreactor campaigns; however, published data for this specific configuration is limited. A preferred feed strategy dissolves D-alanine in water-for-injection, adjusts to pH 2.8–3.2 with hydrochloric acid for bioburden hold, then neutralises with sodium hydroxide immediately before metering. This approach limits amine-carbamate formation and maintains the free D-amino acid concentration in solution.
D-Alanine is not incorporated into ribosomal protein in human metabolism but is a required precursor of the peptidoglycan stem peptide in Gram-positive and Gram-negative bacteria. Alanine racemase converts L-alanine to D-alanine, and D-alanyl-D-alanine ligase forms the dipeptide through ATP-dependent condensation. Vancomycin binds the terminal D-Ala-D-Ala with low-micromolar affinity; vancomycin-resistant enterococci that produce D-Ala-D-Lac reduce binding affinity by approximately 1,000-fold, a documented resistance mechanism. In microbiological media development, D-alanine supplementation at 0.1–0.5 mM is used to bypass alr knockout auxotrophy, but the exact supplementation level depends on bacterial strain, inoculum density, and defined growth medium composition.
Minimum inhibitory concentration assays per CLSI M07-A10 use cation-adjusted Mueller-Hinton broth. Exogenous D-alanine can antagonise D-cycloserine inhibition because D-cycloserine is a structural analogue of D-alanine and competes for alanine racemase and D-Ala-D-Ala ligase. Published data on the magnitude of this antagonism is limited to model organisms and should be generated for the specific production strain before defining media supplements. The D-enantiomer is also used as a starting material for D-cycloserine synthesis and for the preparation of D-alanyl-D-lactate analogues in antibiotic research.
For food, cosmetic, and nutraceutical applications, D-alanine does not enter the standard proteinogenic amino acid declaration path because it is not incorporated into human protein. The nitrogen contribution cannot be declared as protein nitrogen under 21 CFR 101.9. β-Alanine is the relevant positional isomer for carnosine synthesis in skeletal muscle and pantothenic acid production; D-alanine is not a direct substitute in these pathways. Where D-alanine is used as an analytical reference, isotopic purity or enantiomeric purity is certified by chiral HPLC/MS/MS and stable-isotope ratio analysis. Unpurified material containing L-alanine above 0.1% can bias stereospecific enzyme kinetic measurements using D-amino acid oxidase or alanine racemase.
Analytical release for non-compendial D-alanine typically uses amino acid analysis per AOAC 994.12 for free amino acid content and chiral HPLC with a crown ether stationary phase at 25 °C using aqueous perchloric acid mobile phase with detection at 200 nm. The absence of a dedicated D-alanine monograph in major pharmacopeias means that each batch must be qualified against the intended synthetic or fermentation route using a supplier technical data sheet. Regulatory compliance statements should be limited to REACH registration status and residual solvent levels per ICH Q3C, not extrapolated to food or injectable use without additional data. D-Alanine is also not a direct substitute for L-alanine in chemically defined mammalian cell culture media because the amino acid transport systems of CHO cells exhibit stereoselectivity, and excess D-alanine can be oxidised by D-amino acid oxidase to generate hydrogen peroxide.
D-Alanine is hygroscopic only at elevated relative humidity; production-scale storage at 25±2 °C and ≤60% RH maintains free moisture below 0.3% in closed containers. At RH ≥75%, surface moisture can rise above 0.5% within 24 h; the result is incomplete Fmoc activation and lower coupling yield in solid-phase synthesis. The corrective condition is vacuum drying at 80±2 °C and <10 kPa for 3 h, followed by Karl Fischer verification to ≤0.1%. D-Alanine is incompatible with nitrous acid, strong oxidisers, and carbodiimide crosslinkers in the absence of a protected amino function. Exposure to these agents generates acetaldehyde and carbon dioxide by oxidative decarboxylation and can exotherm in closed reactors. For downstream peptide coupling, moisture-sensitive reagent performance degrades if drums are repeatedly opened under ambient humidity; nitrogen-blanketed drum handling with −20 kPa differential pressure is used in pharmaceutical intermediates warehouses.
