D-Aspartic Acid Dimethyl Ester Hydrochloride is the hydrochloride salt of the dimethyl ester of (R)-2-aminosuccinic acid, catalogued under CAS 63167-85-1. The molecular formula C₆H₁₂ClNO₄ corresponds to a formula weight of 197.61 g mol−1. The product functions as a protected D-aspartic acid building block in which both the α- and β-carboxylic acid positions are esterified as methyl esters and the primary amine is isolated as the hydrochloride. The salt form is supplied as a white to off-white crystalline powder and is typically designated by the abbreviated notation D-Asp(OMe)-OMe·HCl rather than by a single proprietary model number. Because commercial vendors catalogue the substance under the same CAS registry number, model differentiation rests on enantiomeric purity, residual solvent profile, and lot-specific release data. The hydrochloride form is preferred over the neutral amino ester for handling stability because it suppresses atmospheric carbon dioxide uptake and avoids the strongly basic free amine.

The protonated amine imposes a stoichiometric boundary: 1.0–1.1 equivalents of a hindered tertiary amine such as N,N-diisopropylethylamine must be introduced to liberate the free amine immediately before acylation. Incomplete neutralisation leaves the ammonium salt unreactive toward carbonyl activation species, while delayed neutralisation exposes the base-labile methyl esters to saponification. This dual constraint is the central process issue for the compound and distinguishes it from neutral N-protected aspartate derivatives such as Boc-D-Asp(OMe)-OMe. In Boc-D-Asp(OMe)-OMe, the amine is blocked and requires acidic deprotection with trifluoroacetic acid or HCl/dioxane before coupling. In this hydrochloride, the amine is already deprotected but protonated, so the product is not directly compatible with carbodiimide activation unless neutralised. One-pot protocols therefore add the base before the coupling reagent or pre-neutralise the hydrochloride in a separate vessel at 0–5 °C. Pre-neutralised solutions cannot be stored beyond the racemization boundary because free aminomalonate-type esters undergo slow α-proton exchange in polar aprotic media.

What Release Specifications and Certificate-of-Analysis Limits Are Typically Applied?

Because no dedicated USP, Ph. Eur., or JP monograph exists for this exact hydrochloride salt, release testing is driven by in-house methods and the general raw material expectations of ICH Q7. The following acceptance criteria are recurrent in commercial certificates of analysis for laboratory and pilot-scale lots. Chiral identity is generally confirmed by chiral HPLC on a polysaccharide-based column, with L-aspartic acid dimethyl ester hydrochloride peak limited to ≤1.0% area. Chemical purity by reversed-phase HPLC is used to quantify total impurities; method precision is normally validated according to ICH Q2(R1), with reporting threshold at 0.05% and identification threshold at 0.10%.

Melting point is frequently omitted from release data because the hydrochloride can undergo thermal deesterification or decomposition before a sharp melt is observed. When reported, the value varies with heating rate and residual solvent. Identity in a regulated supply chain is better established by orthogonal methods such as ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry. The specific rotation of the D-enantiomer is negative in methanol, but inter-laboratory variation and residual solvent effects make the certificate of analysis the authoritative source for the lot-specific interval. Published data for this specific configuration is limited with respect to reproducibility of optical rotation across different polarimeter geometries.

Residual methanol is a common release variable because the product is generally prepared by acid-catalysed esterification of D-aspartic acid in methanol. Incomplete drying leaves methanol that can compete with the amino group during subsequent activation. Peptide-grade lots commonly apply a residual solvent limit of ≤0.10% w/w for methanol, although this is not a universal monograph requirement. Residual solvent control follows ICH Q3C when the material is used in pharmaceutical intermediate manufacture.

Hydrochloride Salt Behaviour in Peptide Coupling Systems

In a typical solution-phase peptide coupling, the hydrochloride is suspended in dichloromethane or N,N-dimethylformamide at 0–5 °C, and 1.05–1.10 equivalents of N,N-diisopropylethylamine are added at a rate that keeps the internal temperature below 5 °C. The carboxyl coupling partner is pre-activated separately with 1.0–1.1 equivalents of HBTU, HATU, PyBOP, or EDC/HOBt in the same solvent. Once activation reaches the O-acylisouronium or active ester stage, the neutralised amino ester is transferred by cannula into the activation vessel. Reaction progression at 20–25 °C is monitored by TLC or LC-MS; conversion above 90% is typically observed within 2–4 h for sterically unhindered acyl donors. The free amine should be generated no more than 15–30 min before coupling because delayed neutralisation results in methyl ester hydrolysis and lower isolated yield.

Isolated yields reported for this class of protected D-aspartate in solution-phase model couplings generally fall in the range of 85–95% when neutralisation is carried out in situ or immediately before addition. The coupled product is a neutral amide-ester that partitions into ethyl acetate or dichloromethane, while tertiary ammonium chloride by-products and water-soluble urea residues are removed by aqueous workup. Unlike unprotected D-aspartic acid, the dimethyl ester hydrochloride dissolves readily in polar aprotic media after neutralisation and does not form the highly hydrogen-bonded zwitterionic network that complicates filtration of the free amino acid. This solubility behaviour is the main reason the derivative is selected for homogeneous acylation.

The methyl esters remain sensitive to aqueous base. Extractive workup with cold saturated sodium bicarbonate should be limited to 5 min at 0–5 °C to avoid saponification. Strong aqueous sodium hydroxide should not be used during isolation because cleavage of both esters converts the product back to the water-soluble diacid and removes the protecting-group advantage. For pilot-scale batches, jacketed glass reactors of 50 L nominal volume with retreat-curve impellers are configured at 150–250 rpm; high-shear mixing is unnecessary because the neutralised derivative is molecularly dissolved. Nitrogen inerting is maintained throughout activation and coupling. The solid hydrochloride is charged through a nitrogen-purged side port, and the line is rinsed with dry solvent to prevent crust formation at the vessel wall. Published data for this specific equipment configuration is limited, but these settings are derived from standard handling practice for moisture-sensitive amino ester salts.

When Pre-Drying and Humidity Control Determine Suitability for Anhydrous Coupling

The methyl ester linkages are hydrolytically labile, and the hydrochloride salt is hygroscopic. Water uptake above 0.5% w/w can reduce coupling efficiency in carbodiimide-mediated reactions by hydrolysing the O-acylisourea intermediate before aminolysis occurs. At ambient relative humidity above 60%, the powder should be pre-dried in a vacuum oven at 40 °C under 10–20 mbar for 4–6 h before weighing. Drying above 45 °C is not recommended because thermal degradation of the hydrochloride can generate methanol and aspartic acid-derived by-products. After drying, the material is transferred to a desiccator over phosphorus pentoxide or activated molecular sieves.

Storage in the original amber glass container under nitrogen at 2–8 °C is the standard condition. The container should be warmed to ambient temperature before opening to prevent condensation. Multigram users often aliquot the powder into septum-capped vials under dry nitrogen so that a single campaign does not repeatedly expose the bulk supply to room air. Under these conditions, supplier certificates typically assign a retest interval of 12 months, but this interval is not an intrinsic stability guarantee and must be verified by loss-on-drying and HPLC assay before use in registered processes.

In comparison with L-aspartic acid dimethyl ester hydrochloride, the D-configuration is the sole structural discriminator but the controlling factor for biological recognition and conformational stability. The two isomers have the same molecular formula, the same salt stoichiometry, and essentially identical reversed-phase HPLC retention; conventional C18 HPLC cannot distinguish them without a chiral selector or chiral derivatisation. A chemical purity certificate of ≥98.0% area therefore does not by itself establish the desired enantiomeric identity. Suppliers use chiral HPLC or optical rotation to verify that the L-isomer content remains below the specified limit. In peptide synthesis, the D-residue is introduced when an unnatural backbone is required to resist protease cleavage or to stabilise a reverse turn; the corresponding L-isomer would not satisfy that structural requirement.

Compared with unprotected D-aspartic acid, the dimethyl ester hydrochloride removes both carboxylic acid protons and thereby eliminates the need for carboxyl-group protection during activation of the α-position. This is both an advantage and a limitation: the α- and β-esters are chemically indistinguishable, so the parent compound cannot be selectively deprotected at one position. If a free β-carboxyl group is required for on-resin cyclisation, branching, or subsequent conjugation, a regioisomerically protected form such as D-aspartic acid α-methyl ester or the β-tert-butyl ester should be used instead. The hydrochloride salt also differs from N-protected D-aspartate diesters by its lower molecular weight and its requirement for a base during coupling; the absence of an N-acyl group reduces steric hindrance but makes the amine nucleophilic only after neutralisation. In comparison with the racemic hydrochloride, the enantiopure D-form avoids the formation of enantiomeric peptide diastereomers that co-elute or co-crystallise and complicate preparative HPLC and crystallisation. Published data for this specific configuration is limited with respect to preparative chiral resolution of the dimethyl ester; the enantiopure material is generally prepared from D-aspartic acid by esterification under acid catalysis, preserving the α-carbon stereochemistry.

Chiral Purity Under Base-Mediated Activation: A Process Boundary

Racemization control in this substrate is dominated by the α-proton acidity in polar aprotic solvents. Once the hydrochloride is neutralised, the free amine is generated in the presence of a tertiary amine; prolonged standing under these conditions can abstract the α-proton and erode enantiomeric purity. The rate is temperature-dependent, and the operational boundary is defined by a neutralisation temperature of 0–5 °C and a maximum hold time of 15–30 min before the acylating species is added. Base choice matters: N-methylmorpholine is less sterically hindered than N,N-diisopropylethylamine and can still promote slow racemization if the hold time is extended. In carbodiimide-mediated couplings with EDC or DCC, auxiliary nucleophiles such as HOBt or HOAt are used to suppress loss of configuration during activation and to reduce the steady-state concentration of oxazolone precursors after the amine is acylated.

At the neutralisation step, the exotherm is significant when the amine is added rapidly to a slurry of the hydrochloride in DMF. In a 20 L jacketed reactor, rapid addition can raise the internal temperature by 8–12 °C unless the jacket is set to -5–0 °C and the addition is distributed over 30–45 min. The slurry should be stirred sufficiently to suspend the solid, but high-shear dispersion is not required. The hydrochloride is charged under nitrogen, and the base is added below the liquid surface through a dip tube to avoid local concentration gradients. After neutralisation, the solution is usually clear; residual solids after 30 min indicate incomplete salt dissociation, insufficiently dried solvent, or material with lower than specified chloride content.

Residual methanol from the esterification step is another process variable. Methanol above 0.1% w/w can compete with the amino group for activated carboxyl species and may generate methyl ester by-products that reduce yield and complicate impurity profiles. Vacuum-drying at 40 °C reduces residual methanol but cannot remove water of hydration if the salt has been exposed to humid air. The certificate of analysis should therefore list residual solvents according to ICH Q3C; common residual solvents are methanol, dichloromethane, or ethyl acetate depending on the vendor route. For pharmaceutical intermediate use, residual solvent limits are aligned with the intended downstream step and the final active pharmaceutical ingredient monograph.

The hydrochloride salt is incompatible with strong aqueous alkali, hydrazine, lithium aluminium hydride, and other strong nucleophiles that attack the ester carbonyls. Contact with aqueous solutions above pH 8 should be minimised because saponification of the methyl esters begins at a measurable rate. The product should also be kept away from amine-based scavenger additives because the free amine generated from the product itself can react prematurely with activated esters before the desired coupling partner is introduced. These process boundaries do not preclude use in standard peptide synthesis; they define the temperature, solvent dryness, and base-contact limits required to preserve structural integrity.

For regulatory compliance, the substance is treated as a chemical intermediate. When used in the manufacture of active pharmaceutical ingredients, the vendor should supply a certificate of analysis and batch documentation under ICH Q7. REACH status and specific transport classification must be confirmed with the supplier because the hydrochloride salt may not be separately registered from the parent amino acid derivative. The compound is not a finished drug product and has no assigned FDA Orange Book designation. Occupational exposure limits for the specific salt are generally not established; handling follows site chemical hygiene planning, local exhaust ventilation, and nitrile glove selection based on co-handled solvents. Safety data sheets for the product usually classify it as a skin, eye, and respiratory irritant due to the acidic chloride salt, though hazard statements must be read from the supplier SDS for the specific lot and jurisdiction.