| HS Code | 473768 |
| Chemical Name | D-Asparagine |
| Cas Number | 2058-58-4 |
| Molecular Formula | C4H8N2O3 |
| Molecular Weight | 132.12 g/mol |
| Iupac Name | (2R)-2-amino-3-carbamoylpropanoic acid |
| Appearance | White crystalline powder |
| Melting Point | 234°C (decomposes) |
| Specific Rotation | [α]20/D = -34.5° (c=10, 5 M HCl) |
| Solubility | Soluble in water (approx. 20 g/L); practically insoluble in ethanol and ether |
| Pka | pKa1 ≈ 2.0 (carboxyl), pKa2 ≈ 8.8 (amino) |
| Storage Conditions | Store at 2-8°C in a dry, sealed container away from light |
| Typical Purity | ≥ 98% |
As an accredited D-Asparagine factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | D-Asparagine, 25 g, analytical grade, sealed in an amber glass bottle under nitrogen with desiccant and labeled safety information. |
| Container Loading (20′ FCL) | D-Asparagine loaded as 20′ FCL in sealed, palletized drums/bags, secured for safe transport, with proper labeling and ventilation. |
| Shipping | D-Asparagine ships as a non-hazardous chemical in sealed, labeled containers to prevent moisture absorption and contamination. Use clean, dry packaging, avoid extreme heat, and protect from light. Transport at ambient temperature in ventilated, covered vehicles. Keep away from incompatible substances and ensure proper documentation for traceability. |
| Storage | Store D-Asparagine in a tightly sealed container in a cool, dry, well-ventilated area away from direct sunlight, heat, and moisture. Keep it separate from strong oxidizing agents and incompatible materials. Ensure the container is clearly labeled and protected from physical damage. Use appropriate personal protective equipment when handling. |
| Shelf Life | D-Asparagine has a typical shelf life of 2–3 years when stored sealed in a cool, dry, dark place. |
D-Asparagine enters solid-phase peptide synthesis as Fmoc-D-Asn(Trt)-OH, not as the unprotected free amino acid. The trityl side-chain protection is retained because unprotected asparagine undergoes carbodiimide-mediated dehydration to β-cyanoalanine during activation, a side reaction that becomes analytically significant when D-Asn is coupled as the N-terminal residue or adjacent to sterically hindered amino acids. For pilot-scale and production-scale automated peptide synthesizers with working volumes from 2 L to 50 L, the resin substitution is typically controlled between 0.30 mmol/g and 0.50 mmol/g, with Fmoc-D-Asn(Trt)-OH charged at 2.0–4.0 equivalents relative to the resin-bound free amine. Coupling uses HBTU/HOBt/DIPEA at a 1:1:2 molar ratio in anhydrous dimethylformamide. The Karl Fischer water content of the solvent is maintained below 500 ppm, and the reaction temperature is held at 20–25 °C for 30–60 min. A second coupling cycle is applied when D-Asn occupies the N-terminus or sits adjacent to Val, Ile, or pseudoproline residues. Temperature excursions above 30 °C accelerate β-cyanoalanine formation and generate deletion sequences. Production failure data from CDMO batch records indicate that incomplete pre-drying of Fmoc-D-Asn(Trt)-OH when relative humidity exceeds 60% results in active ester hydrolysis and the appearance of des-Asn impurities detectable by LC-MS as a mass shift corresponding to the missing residue. The derivative is therefore dried at 25–30 °C under vacuum for 12–16 h before use. GMP compliance is anchored to ICH Q7, 21 CFR 210/211, ICH Q6B, and the FDA guidance for synthetic peptide drug substances. Deprotection of the Fmoc group uses 20% piperidine in DMF in two cycles of 5 min each, with UV monitoring at 301 nm for released dibenzofulvene. Cleavage from Rink amide resin proceeds in TFA/triisopropylsilane/water at 95:2.5:2.5 v/v for 2–4 h at 20–25 °C, followed by precipitation in cold methyl tert-butyl ether at −20 °C. Preparative reversed-phase HPLC on C18 columns with 0.1% TFA or acetic acid in acetonitrile/water gradients and UV detection at 214–220 nm yields peptide APIs, peptide-PEG conjugates, and peptidomimetic intermediates intended for protease-resistant drug development. The operational cleavage boundary is sequence-dependent: extended acid exposure beyond 4 h can promote acidolytic peptide backbone fragmentation when Asp residues are present elsewhere in the sequence, so pilot-scale time-course studies are required before scaling to production vessels.
D-Asparagine monohydrate is used as a chiral reference standard in the release testing of L-asparagine monohydrate, L-asparagine-containing APIs, and synthetic peptide intermediates where enantiomeric purity is a critical quality attribute. The system suitability solution is prepared at 0.1 mg/mL in 0.1 M hydrochloric acid, with serial dilutions from 0.005 mg/mL to 0.500 mg/mL for linearity assessment. Direct chiral HPLC is performed on crown ether-based chiral stationary phases with a perchloric acid mobile phase adjusted to pH 1.5, with UV detection at 210 nm. Alternatively, derivatization with Marfey’s reagent in 1 M sodium bicarbonate at pH 9.0 and 37 °C for 60 min is followed by reversed-phase C18 HPLC with UV detection at 340 nm. Method validation follows ICH Q2(R1) parameters for specificity, linearity, accuracy, precision, LOD, and LOQ, with system suitability limits consistent with USP <621> and Ph. Eur. 2.2.29. Optical rotation of the reference standard is verified by Ph. Eur. 2.2.7. Because D-asparagine monohydrate is hygroscopic, the material is weighed under dry nitrogen or after drying at 105 °C for 3 h according to USP <731>; water uptake shifts stock solution concentration and can change retention time reproducibility in chiral methods. The validated method outputs include certificates of analysis, chiral impurity method validation protocols, and reference standard qualification reports used by analytical quality control laboratories and contract research organizations. In the chromatographic procedure, the enantiomeric separation is preferentially run at column temperatures below 30 °C to limit mobile phase off-gassing and baseline drift in perchloric acid systems. Storage of prepared reference solutions is limited to 48 h at 2–8 °C because of microbial and hydrolytic instability concerns.
| Parameter | Typical acceptance criterion | Referenced procedure |
|---|---|---|
| Specificity | Resolution ≥ 2.0 between L- and D-asparagine | ICH Q2(R1) |
| Linearity range | 0.005–0.500 mg/mL | ICH Q2(R1) |
| LOD | ≤ 0.02% of L-asparagine | USP <621> |
| LOQ | ≤ 0.05% of L-asparagine | USP <621> |
| Recovery | 95–105% | ICH Q2(R1) |
| Solution stability | 48 h at 2–8 °C | Ph. Eur. 2.2.29 |
Chemical deamidation of D-asparagine monohydrate converts the terminal amide group into a carboxylic acid, yielding D-aspartic acid and its N-protected derivatives used as chiral pool intermediates. The reaction is run in 6 M hydrochloric acid at 10–20 mL/g starting material under reflux at 105–110 °C for 12–16 h. After acid removal by vacuum distillation at 60–70 °C and 20–30 mbar, the residue is redissolved in water and passed through a strong-acid cation-exchange column in H⁺ form. Elution with 0.5 M ammonia separates D-aspartic acid from residual neutral or unreacted material. Crystallization from water/ethanol gives D-aspartic acid, and the optical rotation is monitored by Ph. Eur. 2.2.7. The process is operationally straightforward, but production-scale yield data specific to D-asparagine deamidation is limited, so pilot batches should verify molar balance, residual chloride levels, and enantiomeric purity before scale-up. REACH registration obligations apply when the resulting D-aspartic acid derivative is placed on the EU market in quantities above 1 tonne per year. Downstream product types include D-aspartic acid, N-acetyl-D-aspartic acid, and D-aspartic acid dimethyl ester hydrochloride as chiral building blocks for pharmaceutical research, impurity marker synthesis, and specialized peptide intermediate preparation.
D-Asparagine-containing peptide actives are synthesized for topical formulations where D-residue substitution reduces proteolytic degradation in skin surface microenvironments. The synthetic route follows solid-phase assembly, preparative C18 RP-HPLC purification, lyophilization, and incorporation into leave-on cosmetic emulsions at final peptide concentrations commonly cited in the range of 5–50 ppm, though published data specific to D-asparagine-containing actives remains limited. Finished product types include peptide serums, dermal repair emulsions, and peptide mimetic skincare concentrates. Formulation temperature is held between 40–60 °C during the cooling phase, and the final pH is maintained between 4.5 and 6.5 because asparagine residues undergo accelerated deamidation at alkaline pH above 7.5. Stability is assessed under accelerated conditions at 40 °C and 75% RH following ICH Q1A(R2) principles, adapted for cosmetic product lifecycle expectations. The peptide active is specified with purity ≥ 95% by RP-HPLC and residual TFA ≤ 0.1%. Safety assessment and product notification follow EC 1223/2009, while manufacturing hygiene controls align with ISO 22716. The major operational boundary is cost-driven: D-asparagine is a specialty chiral amino acid, so its incorporation is reserved for short peptide sequences of 5–15 residues where one D-residue provides a measurable stability advantage. Protracted exposure of the finished emulsion to temperatures above 60 °C during homogenization is avoided because it promotes peptide aggregation and surface migration within the oil phase.
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D-Asparagine (CAS 2058-58-4; C4H8N2O3; 132.12 g/mol) is the non-proteinogenic D-enantiomer of the neutral amino acid asparagine. The monohydrate form has a molecular weight of 150.13 g/mol; its CAS registry entry should be verified against the supplier batch because hydrated forms can appear under multiple registry numbers. Commercial material is a white to almost white crystalline powder with aqueous solubility comparable to L-asparagine monohydrate, approximately 20 g/L at 20 °C. Polarimetry provides a primary identity test: the D-enantiomer shows a negative specific rotation under conditions where L-asparagine shows a positive value, typically reported as [α]20D −28.6° (c=5, 6 N HCl) for the monohydrate. The measured rotation is method-dependent, and the hydration state must be controlled before comparison.
Unlike L-asparagine (CAS 70-47-3), D-asparagine is not a substrate for therapeutic L-asparaginase preparations. That distinction restricts direct use in fermentation and mammalian cell culture media. Standard CHO and HEK293 growth media require L-asparagine for protein synthesis; substitution with D-asparagine removes the available L-enantiomer and cannot support equivalent growth unless the cell line expresses a racemase. Published data for this specific configuration in production-scale fed-batch platforms is limited. D-Asparagine is therefore positioned primarily as a chiral building block, resolution standard, or precursor to D-aspartic acid rather than as a nutrient supplement.
In Fmoc solid-phase peptide synthesis, the free amino acid is converted to Fmoc-D-Asn(Trt)-OH before coupling. The trityl side-chain protection is used to reduce deamidation and aspartimide formation during repeated piperidine exposure. Residual deamidation remains a process limit: prolonged treatment with 20% piperidine in N,N-dimethylformamide can slowly convert the side-chain amide to D-aspartate or imide species. This side reaction is not unique to D-asparagine, but it affects yield and impurity profiles in longer peptide sequences. On preparative scale, a coupling time of 2–4 h is common, and the washing cycle after deprotection should not exceed 30 min when the C-terminal residue is asparagine or aspartic acid to limit aspartimide opening. Automated peptide synthesizers with continuous-flow UV monitoring can detect premature Fmoc cleavage but do not distinguish enantiomeric contamination; chiral HPLC is required for that purpose.
Boc-D-asparagine is selected for solution-phase fragment coupling. Acidolytic Boc removal with 50% trifluoroacetic acid in dichloromethane is typically performed below 25 °C; prolonged exposure can hydrolyze the side-chain amide. In peptide synthesizers using uronium reagents, Fmoc-D-Asn(Trt)-OH lot-to-lot water content is more critical than the free amino acid because residual water above 0.1% reduces activation efficiency. The Fmoc derivative should be dissolved in DMF or N-methyl-2-pyrrolidone at concentrations up to 0.4 M; higher concentrations can cause gel formation with some activator combinations.
Because no current USP, Ph. Eur., or JP monograph specifically describes D-asparagine, manufacturers typically release the material against in-house criteria derived from the L-asparagine monograph. Peptide synthesis grades are commonly controlled to ≥99.0% assay and ≥99.0% enantiomeric excess; research grades may accept ≥98.0% assay and ≥98.0% ee. The difference is material in chiral resolution: a 1% L-asparagine impurity can propagate into a peptide as a diastereomer that co-elutes on standard C18 analysis, leaving only chiral HPLC or capillary electrophoresis for detection. The table below summarizes representative release criteria and the methods used to confirm them.
| Parameter | Method or standard designation | Typical criterion |
|---|---|---|
| Appearance | Visual inspection | White or almost white crystalline powder |
| Assay | HPLC area normalization | ≥98.0% to ≥99.0% |
| Specific rotation | Polarimetry, 6 N HCl, c=5 | −28.6° ± 1.0° (monohydrate) |
| Loss on drying | USP <731> / Ph. Eur. 2.2.32 | ≤0.5% anhydrous; ≤12.0% monohydrate |
| Residue on ignition | USP <281> / Ph. Eur. 2.4.14 | ≤0.1% |
| Enantiomeric purity | Chiral ligand-exchange HPLC or Chirobiotic T | L-asparagine ≤0.5% research grade; ≤0.1% peptide grade |
| Residual solvents | USP <467> / ICH Q3C | Report or ≤0.5% for selected Class 2 solvents |
Monohydrate and anhydrous grades differ in water content and molecular weight. Formulation calculations for peptide synthesis must use the anhydrous molecular weight 132.12 g/mol; use of the monohydrate weight 150.13 g/mol without correction introduces a 12% mass error. Available derivative forms include Fmoc-D-Asn(Trt)-OH and Boc-D-asparagine; these are released by peptide synthesis criteria rather than amino acid monograph criteria. Certificates of analysis for protected derivatives should also report residual piperidine, trifluoroacetic acid, and water content because the absence of a monograph makes a certificate claiming only “per amino acid test” insufficient for chiral peptide production.
D-Asparagine is not thermally robust under strongly alkaline conditions. In solution-phase peptide synthesis, saponification of methyl or ethyl esters in the presence of D-asparagine side chains is performed below 25 °C with lithium hydroxide at 1.2–1.5 equivalents; stronger hydroxide excess or heating above 40 °C accelerates deamidation and gives D-aspartic acid contamination. In enzymatic deamidation screens, L-asparaginase reactors operated at 37 °C and pH 7.4 show no conversion of D-asparagine, which is used as a negative control to confirm stereospecificity. For acid-catalyzed deamidation to D-aspartic acid, refluxing 6 N HCl for 8–12 h is representative; longer reflux times can cause partial racemization at the chiral center, reducing enantiomeric excess. The operational boundary is therefore controlled by chiral HPLC after hydrolysis rather than by gravimetric yield alone.
For asymmetric synthesis, D-asparagine and DL-asparagine are not interchangeable. DL-asparagine has a specific rotation of 0° and requires resolution or asymmetric transformation before chiral use. D-Asparagine is selected when a defined enantiomer is needed in a coupling sequence or when a chiral stationary phase standard is required. The following table compares enantiomer-specific properties that influence selection. Compendial monographs are absent for the D-enantiomer.
| Parameter | D-Asparagine | L-Asparagine | DL-Asparagine |
|---|---|---|---|
| Stereochemical configuration | D (R) | L (S) | Racemic mixture |
| CAS | 2058-58-4 (anhydrous) | 70-47-3 (anhydrous); 5794-13-8 (monohydrate) | Racemic reference grade; CAS varies by source |
| Specific rotation | [α]20D −28.6° (c=5, 6 N HCl) | [α]20D +28.6° (c=5, 6 N HCl) | 0° |
| L-asparaginase substrate | No | Yes | Partial, L-fraction only |
| Proteinogenic in standard CHO/HEK293 media | No | Yes | No net L-enantiomer benefit |
| Primary synthetic role | Chiral peptide fragment, D-aspartic acid precursor | Cell culture nutrient, parenteral amino acid component | Crystallographic reference, resolution studies |
The D-enantiomer is also preferred over DL-asparagine when enzymatic deamidation is used to generate D-aspartic acid. L-Asparaginase will not deamidate D-asparagine, so the D-enantiomer route avoids L-aspartic acid contamination. Acid-catalyzed deamidation of D-asparagine in 6 N HCl at reflux gives D-aspartic acid hydrochloride; this is a common preparative step in neurochemical intermediate synthesis. Because D-aspartic acid is a chiral pool intermediate for beta-lactam and peptide chemistry, the enantiomeric purity of the starting D-asparagine directly limits the achievable diastereomeric excess downstream.
Chiral purity measurement is frequently performed by ligand-exchange HPLC with copper(II) sulfate and a chiral amino acid ligand, or by reversed-phase HPLC after derivatization with Marfey’s reagent. The L-asparagine limit of ≤0.1% for peptide-grade material is achievable with refractive index detection, but UV detection at 200 nm can be affected by mobile-phase absorbance. Capillary electrophoresis with cyclodextrin-modified buffers offers orthogonal separation when HPLC co-elution is suspected. Process-scale quality control laboratories should qualify the chiral method against a spiked racemate at 0.1%, 0.5%, and 1.0% levels; published data for this specific configuration is limited, so site-specific validation is necessary.
Storage and handling of D-asparagine follow amino acid hygiene rules. The anhydrous material is stored in tightly closed containers at 15–25 °C and ≤60% relative humidity; the monohydrate is stable at ambient humidity but cycles through drying equipment can remove water and alter the certified water content. For peptide derivative Fmoc-D-Asn(Trt)-OH, long-term storage is recommended at −20 °C under inert gas to prevent slow detritylation. In aqueous solution, D-asparagine undergoes deamidation to D-aspartic acid under strong acid or base; the reaction rate is pH- and temperature-dependent, but published rate constants for the D-enantiomer across all relevant buffer systems are limited. Stability studies should therefore be performed with the actual process buffer. Filtration of aqueous D-asparagine through charged membranes can remove ionic impurities but may also alter pH and should be validated with conductivity and osmolality measurements if the solution is used in a bioreactor or chromatographic feed.
