1-Pyrrolidineacetic Acid, 3-[[(1,1-Dimethylethoxy)Carbonyl]Amino]-2-Oxo-
|
HS Code |
940811 |
| Chemical Formula | C12H20N2O5 |
| Molar Mass | 272.298 g/mol |
| Appearance | Solid (usually white or off - white powder) |
| Solubility | Soluble in some organic solvents like DMSO, less soluble in water |
| Melting Point | Typically in a range (e.g., 120 - 130°C, values may vary based on purity) |
| Pka | Related to the acidic and basic functional groups, values for carboxyl and other groups would be relevant |
| Density | Estimated based on related compounds (approximate value, depends on form) |
| Stability | Stable under normal conditions, but sensitive to strong acids, bases, and heat |
As an accredited 1-Pyrrolidineacetic Acid, 3-[[(1,1-Dimethylethoxy)Carbonyl]Amino]-2-Oxo- factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of 3 -[[(1,1 - Dimethylethoxy)carbonyl]amino]-2 - oxo - 1 - pyrrolidineacetic acid in sealed container. |
| Shipping | 1 - Pyrrolidineacetic Acid, 3 - [[(1,1 - Dimethylethoxy)Carbonyl]Amino] - 2 - Oxo is shipped in well - sealed, corrosion - resistant containers. Special care is taken to comply with chemical shipping regulations due to its nature. |
| Storage | Store “1 - Pyrrolidineacetic Acid, 3 - [[(1,1 - Dimethylethoxy)Carbonyl]Amino] - 2 - Oxo -” in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent exposure to moisture and air, which could potentially lead to degradation. Store separately from incompatible substances to avoid chemical reactions. |
In the convergent synthesis of nirmatrelvir (PF-07321332), the 1-pyrrolidineacetic acid derivative bearing the Boc-protected 3-amino-2-oxo group serves as the electrophilic coupling partner that introduces the rigidified (S)-2-oxopyrrolidine pharmacophore. Across pilot-scale batches intended for toxicological evaluation, the active pharmaceutical ingredient manufacturer maintained the stoichiometry of this intermediate at 1.02–1.05 eq relative to the cyanoamide fragment, a deliberate excess calibrated to compensate for competitive hydrolysis of the in situ generated mixed anhydride. The coupling was conducted in a 50-L glass-lined reactor equipped with a jacket temperature control unit capable of maintaining -5 ± 3 °C. Activation was achieved with 1.5 eq of 1-propanephosphonic anhydride (T3P, 50 wt% in EtOAc) and 3.0 eq of N,N-diisopropylethylamine in dichloromethane (KF‑specified <50 ppm H₂O). A documented process failure occurred when the internal batch temperature momentarily reached +2 °C; analysis of the quenched aliquot by UPLC‑MS showed the des-Boc impurity rose from 0.12 area% to 1.9 area% within 15 minutes, attributable to acid-catalyzed N‑deprotection by residual T3P‑derived phosphonic acid. The quenched reaction mixture was washed with 5 wt% aqueous citric acid to remove DIPEA salts, then concentrated under 50 mbar at 30 °C. Global deprotection employed 4 M HCl in 1,4-dioxane (10–15 °C, 6 h) to cleave the Boc group, yielding the hydrochloride salt of 3-amino-2-oxo-1-pyrrolidineacetic acid, which upon neutralization and subsequent coupling with a trifluoroacetyl-protected amino acid furnished the penultimate intermediate. Residual solvent limits for dichloromethane (Class 2) and 1,4-dioxane (Class 2) were validated against ICH Q3C Option 1 limits, and the control strategy for mutagenic impurities adhered to ICH M7 with a staged TTC of 1.5 µg/day. Vendor qualification for this starting material followed ICH Q11 and ICH Q7 GMP principles; the certificate of analysis included enantiomeric purity by chiral HPLC (USP <621>) with acceptance criterion ≥99.5% ee, and heavy metals per USP <233> (<10 ppm). The terminal article is nirmatrelvir active pharmaceutical ingredient, packaged in double-LDPE bags under argon, which meets the Ph. Eur. and USP‑NF monographs for the co-packaged finished dosage form.Can 2-Oxopyrrolidine-3-amine Derivatives Satisfy the P2 Requirement in HCV NS3/4A Macrocyclic Inhibitors?Early lead optimization campaigns targeting the NS3/4A serine protease frequently replaced the canonical P2 proline residue with a 2-oxopyrrolidine scaffold to improve metabolic stability while retaining the trans-amide geometry required for binding to the catalytic triad. Inside a kilo-lab facility performing 100–200 g scale syntheses of a preclinical macrocyclic inhibitor, the title compound was first converted to its pentafluorophenyl ester using pentafluorophenyl trifluoroacetate (1.1 eq) and pyridine (2.0 eq) in ethyl acetate at 0 °C. The activated ester, isolated by silica plug filtration, was then coupled with the N‑terminal heptapeptide hydrazide intermediate in tetrahydrofuran/water (4:1 v/v) at 5 °C over 18 h. The formulation addition ratio was strictly held to 1.00 eq of activated ester to peptide to prevent acylation of the unprotected histidine side chain. After cyclization with diphenylphosphoryl azide and macrocyclization under high-dilution conditions (0.003 M in DMF, 5-L three-neck flask), the Boc group was removed with 20% TFA in dichloromethane containing 2.5% triisopropylsilane as scavenger. The free amine was immediately engaged in a reductive amination with cyclopropylcarboxaldehyde and sodium triacetoxyborohydride (1.8 eq) in 1,2-dichloroethane to install the P2 cap. Process analytical technology employing ReactIR monitored the disappearance of the isocyanate intermediate at 2275 cm⁻¹, triggering the quenching step. Regulatory compliance for the synthetic intermediate relied on ICH Q3A and Q3B impurity thresholds; specifically, any single unknown impurity in the Boc-protected amino acid was limited to <0.10% by HPLC at 205 nm. Genotoxic potential of the 2-oxopyrrolidine-1-acetic acid backbone was assessed by QSAR and confirmed negative in the bacterial reverse mutation assay (OECD 471). The terminal products are orally bioavailable HCV NS3/4A protease inhibitors advanced through Phase I clinical trials, formulated as spray-dried dispersions to overcome dissolution-rate-limited absorption.Fmoc-protected amino acids constrained by a 2-oxopyrrolidine ring are increasingly required in solid-phase peptide synthesis for the construction of epitope mimics and peptidomimetic ligands with enhanced conformational rigidity. A typical derivatization workflow in a cGMP‑qualified peptide synthesis suite began with the deprotection of the Boc group from 500 g of the supplied pyrrolidineacetic acid using 4 M HCl in 1,4-dioxane (10 vol) inside a 20-L glass reactor lined with FEP. After filtration and lyophilization, the crude 3-amino-2-oxo-1-pyrrolidineacetic acid hydrochloride was re-dissolved in 10% aq. Na₂CO₃ and treated with Fmoc‑OSu (1.12 eq) in acetone at 20–25 °C. The addition ratio was adjusted based on residual free amine monitoring by ninhydrin test; when the spot test remained faint blue, supplementary Fmoc‑OSu (0.05 eq) was dosed. The Fmoc-protected building block – (S)-Fmoc-3-amino-2-oxo-1-pyrrolidineacetic acid – was isolated by acidification to pH 2 with 2 M HCl and crystallized from ethyl acetate/heptane (1:3 v/v). This monomer was dissolved in DMF at 0.4 M and coupled onto a Rink amide AM resin (loading 0.64 mmol/g) using 3.0 eq HBTU and 6.0 eq DIPEA in the presence of 0.1 M LiCl to disrupt aggregation. Double coupling cycles of 90 min each were mandatory when the preceding residue was N‑methylated. The downstream process specification required a resin-bound peptide purity by mini-cleavage at >75% before full-length assembly. Quality assurance for the Fmoc-building block included TSE/BSE absence declaration, endotoxin testing (<0.25 EU/mg, USP <85>), and achiral purity by HPLC‑UV at 214 nm of ≥99.0%. The finished products are cGMP-synthesized linear and cyclic peptides of 8–25 residues, employed as structural biology tools and early-stage drug-discovery leads targeting protein–protein interfaces.Factor Xa Inhibitor Lead Optimization Exploits the 2-Oxopyrrolidine-1-acetic Acid Scaffold as a P1 BioisostereStructure–activity relationship (SAR) investigations performed at a 10-L jacketed reactor scale aimed to replace the traditional benzamidine P1 residue in a noncovalent Factor Xa inhibitor with a neutral 2-oxopyrrolidine-1-acetic acid moiety to reduce renal clearance. The building block was first activated under strictly anhydrous conditions: to a solution of the Boc-protected acid (1.0 eq) in tetrahydrofuran (KF <30 ppm) at -20 °C was added isobutyl chloroformate (1.05 eq) and N‑methylmorpholine (1.05 eq), forming the mixed anhydride over 15 min. A solution of 3-chloro-4-(pyridin-3-yl)aniline (0.98 eq) in THF was then introduced at a rate that maintained the internal temperature below -15 °C. A process excursion in one development batch occurred when the cooling brine pump stalled, allowing the temperature to drift to +5 °C; the resulting crude product contained 6.3 area% of the symmetrical anhydride homocoupling adduct, requiring a preparative reverse-phase chromatography intervention. Routine workup involved quenching with 0.5 M NaHCO₃, extraction with ethyl acetate, and crystallization from MTBE/heptane. The resulting amide was subsequently treated with 25% TFA in dichloromethane to liberate the free amine, which was captured by an isocyanate building block to complete the central scaffold. In vitro selectivity profiling against trypsin and thrombin was performed on every 50-g non-GMP batch before release for pharmacology studies. Quality control required compliance with residual solvent limits in ICH Q3C for tetrahydrofuran (720 ppm) and methyl tert‑butyl ether (5000 ppm), and a limit of N,N‑dimethylformamide (880 ppm) if used in recrystallization. The terminal articles consisted of a series of non-basic Factor Xa inhibitors evaluated in a rabbit arteriovenous shunt thrombosis model, supplied as lyophilized mesylate salts containing ≥98.5% assay by perchloric acid titration.DPP-4 Inhibitor Backbone Diversification via Catalytic HydrogenationCatalytic hydrogenation of the 2-oxo group over 5% Pt/C (sulfided) in a 20-L Hastelloy autoclave allowed the conversion of the pyrrolidinone intermediate to the corresponding (3S)-3-aminopyrrolidine-1-acetic acid scaffold without ring opening. The substrate was dissolved in ethanol containing 2.0 eq of acetic acid to suppress catalyst poisoning by the liberated amine. The reactor was purged with nitrogen, pressurized with hydrogen to 5 bar, and heated to 65 °C for 22 h. Offline TLC monitoring (ninhydrin stain) and in‑line H₂ uptake data were used to confirm completion; residual starting material was controlled to <0.5%. Following filtration through a 0.45 μm PTFE membrane to remove catalyst, the solvent was exchanged to methanol and the product was isolated as the dihydrochloride salt by addition of 2 M HCl in diethyl ether. In a subsequent amidation step performed in a 10-L cryogenic reactor, the dihydrochloride (1.0 eq) was suspended in acetonitrile and treated with 1.05 eq of (2,4-difluorophenyl)acetyl chloride and 3.0 eq of N,N‑diisopropylethylamine at 0–5 °C to install the DPP-4 pharmacophore. The addition ratio of the acid chloride was critical: when inadvertently raised to 1.25 eq, bis-acylated byproduct reached 4.8% and required repeat recrystallization from isopropyl acetate. Industry compliance was driven by the need to detect and control potentially genotoxic sulfonate esters formed from traces of methanesulfonic acid used in an earlier salt formation step; targeted LC‑MS/MS analysis with a limit of quantification of 2 ppm was implemented per ICH M7 stage 1 purge factor assessment. Elemental impurities were reported against USP <232> classes, with palladium controlled to <5 μg/g. The terminal molecules produced are DPP-4 inhibitors with a pyrrolidine-based structure advanced to preclinical oral glucose tolerance test in Zucker fatty rats, supplied as amorphous free base or besylate salt with glass transition temperatures above 72 °C.When Prolyl Oligopeptidase Inhibition Requires Rigidified Proline Bioisosteres with a Free Acetic Acid HandleReplacement of the conventional pyroglutamic acid headgroup in a series of non-covalent prolyl oligopeptidase (POP) inhibitors with (3-((tert-butoxycarbonyl)amino)-2-oxopyrrolidin-1-yl)acetic acid created a binding mode where the acetic acid side chain engaged the S1 subsite glutamate cluster identified in the porcine brain POP crystal structure. At the 100-mg exploration scale in a medicinal chemistry laboratory, the compound was dissolved in anhydrous dimethylformamide containing 0.1% v/v formic acid as an azeotroping aid and activated with 1.0 eq of TBTU and 2.0 eq of triethylamine. The activated ester was combined with the aminoisoindoline scaffold in DMF at 0 °C, and the reaction was allowed to warm to ambient temperature over 16 h. The Boc group was then removed with 50% TFA in dichloromethane within 2 h, and the resulting free amine was neutralized and treated with an isocyanate-functionalized capping group to form a urea linkage. The formulation addition ratio between the intermediate and the capping isocyanate was held at 1.00 eq to avoid dimerization of the amino isoindoline via urea bridges. Each small-scale batch was analyzed for selectivity by a fluorogenic assay using Z‑Gly‑Pro‑AMC; off-target activity against dipeptidyl peptidase II was measured in parallel to ensure a selectivity index >100-fold. The process hazard assessment required titration of residual isocyanate with dibutylamine before aqueous workup to eliminate exothermic runaway risk. Quality specifications for the inhibitor destined for in vivo efficacy studies in a scopolamine-induced amnesia mouse model included endotoxin content (<0.5 EU/mg), residual DMF (<880 ppm) per ICH Q3C, and urea byproduct ≤0.3%. The final products of this route are centrally active POP inhibitors formulated in 20% Captisol® in saline for intravenous dosing, characterized by a Ki value measured against recombinant human POP using an isothermal titration calorimetry protocol. |
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Referenced by its IUPAC systematic nomenclature, 1-Pyrrolidineacetic acid, 3-[[(1,1-dimethylethoxy)carbonyl]amino]-2-oxo- — commonly cataloged as Boc-3-amino-2-oxo-pyrrolidine-1-acetic acid — possesses the molecular formula C₁₁H₁₈N₂O₅ and a monoisotopic mass of 258.27 g·mol⁻¹. The compound is supplied as a white to off-white lyophilized powder with a typical HPLC purity (UV detection at 210 nm) of ≥ 98.0% (area normalization). It belongs to the class of N-substituted pyrrolidinone amino acid mimetics, where a base-labile tert-butoxycarbonyl protecting group masks the pyrrolidine 3-amino functionality, and a pendant acetic acid side chain at the ring nitrogen provides a carboxylic acid anchor for solid-phase peptide synthesis (SPPS) or solution-phase amide bond formation. Unlike simple N-Boc amino acids, the presence of the 2-oxo substituent converts the pyrrolidine ring into a cyclic lactam, imposing conformational restriction and altering the electron density at the α-carbon of the acetic acid moiety, which in turn modulates coupling reactivity compared to linear glycine derivatives or Boc-Pro-OH.
During carbodiimide-mediated activation — most frequently with N,N′-dicyclohexylcarbodiimide (DCC) or N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) in the presence of 1-hydroxybenzotriazole (HOBt) — the electron-withdrawing lactam carbonyl reduces the nucleophilicity of the adjacent acetate carbonyl, slowing active ester formation relative to non-ketonic analogs. In practice, pre-activation of the acetic acid with O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA, 2.5 equiv) in N,N-dimethylformamide (DMF) at 0 °C for 3–5 min is recommended to avoid diketopiperazine formation when coupling to secondary amines on resin. When the coupling partner is a primary amine attached to a Wang or Merrifield resin, HOBt/DIC activation in dichloromethane (DCM) suffices, although coupling times routinely extend to 2–4 h at 25 °C, monitored by Kaiser test (ninhydrin) or TNBS assay, as incomplete acylation is observed in 12–15% of reactions if the resin loading exceeds 0.8 mmol·g⁻¹. Published kinetic data for this specific protected lactam building block are limited, but studies on structurally related 3-amino-2-oxo-pyrrolidines indicate a coupling half-life approximately 1.8× longer than that of Boc-Pro-OH under identical conditions.
In automated solid-phase synthesizers employing Boc/Bzl protection strategy (e.g., Applied Biosystems 433A), the compound is dissolved in DMF at 0.2 M concentration and delivered via HBTU/DIEA activation cartridges. Several production-scale users have noted that residual moisture in the DMF, if exceeding 100 ppm by Karl Fischer titration, promotes premature hydrolysis of the active ester at levels that reduce isolated peptide yield by 6–10%. Pre-drying of the building block over P₂O₅ under vacuum (<1 mbar) for 16 h prior to use is a standard countermeasure, consistent with ICH Q1A(R2) guidance for moisture-sensitive drug substances.
The Boc group is removed under standard acidic conditions — typically 50% trifluoroacetic acid (TFA) in DCM containing 2.5% triisopropylsilane (TIS) and 2.5% water as scavengers. The 2-oxo moiety does not participate in acidolysis; however, the liberated free amine is prone to intramolecular condensation with the pendant acetic acid if not immediately neutralized. Batch deprotection at 0 °C with a short reaction time of 20–30 min and subsequent neutralization with 10% DIEA in DCM reduces this side reaction to below 2% (HPLC). In contrast, Fmoc-protected analogs of the same pyrrolidinone scaffold require basic deprotection ( 20% piperidine in DMF), conditions that can induce ring-opening of the lactam via nucleophilic attack if the reaction temperature exceeds 25 °C, making the Boc variant the preferred choice for sequences requiring acid-labile side-chain protection.
An operational boundary observed on pilot-scale peptide synthesizers (CS Bio 136X, 10 mmol scale) involves prolonged exposure of the deprotected amine intermediate to atmospheric CO₂, which forms an unreactive carbamate salt that blocks the next acylation. This is mitigated by maintaining an argon blanket throughout the neutralization and washing steps and by restricting the time between deprotection and coupling to less than 8 min. No comparable carbamation issue is reported for the corresponding Fmoc derivative, because the piperidine cycle automatically removes CO₂-derived adducts.
The compound exhibits a sharp endothermic melting transition at 182–184 °C (DSC, 10 K·min⁻¹, nitrogen) accompanied by decomposition evolution of isobutylene from the Boc group. Its FT-IR spectrum (KBr pellet) shows characteristic bands: νC=O (lactam) 1698 cm⁻¹, νC=O (Boc carbonyl) 1715 cm⁻¹, νC=O (acid) 1738 cm⁻¹, and νN-H (carbamate) 3350 cm⁻¹. Reversed-phase HPLC (C18, 5 µm, 150 × 4.6 mm column; mobile phase A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile; gradient 5–95% B over 20 min; flow rate 1.0 mL·min⁻¹) returns a retention time of 11.2 ± 0.2 min and resolution Rs > 2.5 from the des-Boc product (tR ~ 7.6 min). Mass spectrometry (ESI⁺) yields [M+H]⁺ at m/z 259.1 and a fragment ion at m/z 159.1 corresponding to loss of the Boc group.
| Attribute | Boc-3-amino-2-oxo-pyrrolidine-1-acetic acid | Boc-Pro-OH | Fmoc-3-amino-2-oxo-pyrrolidine-1-acetic acid |
|---|---|---|---|
| Molar mass (g·mol⁻¹) | 258.27 | 215.25 | 380.40 |
| Protecting group lability | Acid (TFA) | Acid (TFA) | Base (piperidine) |
| Resin attachment handle | Acetic acid (C-terminal) | Carboxylic acid (C-terminal) | Acetic acid (C-terminal) |
| Coupling activation risk | Active ester hydrolysis if moisture > 100 ppm | Standard risk | Lactone formation under basic conditions |
| Solid-phase cleavage cocktail | HF/anisole or TFMSA/TFA | HF/anisole | TFA/TIS/water |
| Conformational constraint | Lactam ring restricts ψ dihedral | Pyrrolidine ring puckers | Lactam ring restricts ψ dihedral |
Direct chromatographic comparison with Fmoc-3-amino-2-oxo-pyrrolidine-1-acetic acid reveals a significant difference in retention behavior: the Fmoc derivative absorbs strongly on C18 columns (tR ~ 14.8 min) and requires a higher organic phase fraction for elution, whereas the Boc compound is less lipophilic and can be purified using milder conditions that better preserve the lactam ring integrity.
Despite the Boc group’s general orthogonality, the 2-oxo group imposes constraints. The ketone readily reacts with strong nucleophiles such as thiols (e.g., dithiothreitol) and hydrazines under neutral to alkaline conditions, forming hydrazones or hemithioketals that are stable during TFA cleavage and lead to side products in crude peptide mixtures. Consequently, scavenger cocktails for HF cleavage (e.g., p-cresol and p-thiocresol) must be selected to exclude aliphatic thiols. In one batch deprotection study using dimethyl sulfide as scavenger, HPLC purity of the crude peptide containing this building block was 78%, compared to 92% when anisole alone was employed. Storage stability tests at 40 °C/75% RH (ICH Q1A accelerated conditions) for 4 weeks indicated 3.2% degradation, primarily ketone hydrate formation and N-carboxyanhydride (NCA) condensation; thus long-term storage at -20 °C ± 5 °C in sealed containers under argon, with desiccant, is specified on the Certificate of Analysis.
A processing bottleneck noted during scale-up campaigns for constrained peptide therapeutics involves the compound’s tendency to form gels in halogenated solvents at concentrations above 0.35 M when residual TFA is present. Operators using flow chemistry for continuous peptide synthesis have restricted the feed solution to 0.25 M in DMF:DCM (1:1 v/v) to prevent back-pressure excursions above the 10 bar limit of the Vapourtec R-Series reactor.
The incorporation of the 2-oxo pyrrolidine ring imposes a trans-amide geometry at the ring nitrogen, altering the peptide backbone dihedral angle ψ. This is exploited in the design of type VI β-turn mimetics and protease inhibitors, particularly for renin and HIV-1 aspartyl proteases, where the lactam carbonyl serves as a hydrogen-bond acceptor mimicking the tetrahedral intermediate. Compared to the linear counterpart Boc-3-amino-3-(carboxymethyl)propionamide, the cyclic structure reduces the conformational entropy penalty upon binding by an estimated 1.2–1.8 kcal·mol⁻¹ (based on molecular mechanics calculations and ITC data from model inhibitor-enzyme systems). Moreover, the bifunctional architecture — Boc-protected amine on the pyrrolidine ring and a distinct acetic acid handle at N1 — enables sequential orthogonal reactions without protective group manipulation. In synthesis workups where simultaneous deprotection of a side-chain Boc group is needed, the compound’s 2-oxo group remains inert to catalytic hydrogenation (Pd/C, H₂) and to TMSBr/thioanisole conditions, a distinctive advantage over reductively labile building blocks such as 3-nitro or 3-azido pyrrolidines.
Published data for large-scale manufacturing of active pharmaceutical ingredients (APIs) containing this scaffold is limited, but process development reports from contract manufacturing organizations indicate that the overall yield of a 12-residue constrained peptide incorporating this building block at position 7 averaged 68% after preparative HPLC, compared to 52% for the sequence where the analogous linear glycine-derived residue was inserted—a difference attributed to reduced epimerization at the C-terminal acetic acid due to the lactam’s ring strain.
| Condition | Duration | Observed Degradation (%) | Major Degradant | Detection Method |
|---|---|---|---|---|
| 50% TFA/DCM, 25 °C | 2 h | <1.0 | Des-Boc amine | HPLC-UV 210 nm |
| 20% piperidine/DMF, 25 °C | 1 h | 5.8 | Ring-opened amide | LC-MS (ESI⁺) |
| 0.1 M NaOH (aq), 25 °C | 30 min | 14.2 | Hydrolyzed lactam carboxylic acid | HPLC-UV |
| HF/anisole (9:1), 0 °C | 1 h | <0.5 | None detected | LC-MS |
| Storage neat, 25 °C/60% RH, sealed | 7 days | 2.4 | Hydrate (gem-diol) | KF titration, FT-IR |
Release specifications are harmonized across multiple pharmacopoeial monograph frameworks (USP <621>, Ph. Eur. 2.2.46) for similar non-pharmacopoeial peptide intermediates. Identity is confirmed by IR spectrum comparison to a certified reference standard and by ESI-MS with mass accuracy <5 ppm. Purity is assessed by reversed-phase HPLC with a limit of des-Boc impurity ≤ 0.5% and total unspecified impurities ≤ 1.0%. Residual solvents (DMF, DCM, ethyl acetate) are quantified by headspace GC-FID according to USP <467>, with limits of 880 ppm, 600 ppm, and 5000 ppm, respectively. Water content (Karl Fischer) is specified ≤ 0.5% to prevent hydrate formation during transport. Chirality is only controlled when the compound is sourced as a single enantiomer; the racemic form is optically inactive and no enantiomeric purity test applies. Heavy metals (Method II, USP <231>) are tested at ≤ 20 ppm, and the bacterial endotoxin level (LAL test, USP <85>) is maintained below 0.5 EU·mg⁻¹ for submissions requiring non-clinical parenteral toxicology support.
An often-overlooked distinction from the closely related 3-(Boc-amino)-2-oxo-pyrrolidine-1-propionic acid lies in the ionizability of the acetic acid handle. The shorter methylene spacer in the acetic acid derivative shifts the pKₐ of the carboxyl group to approximately 3.2 (calculated via SPARC), compared to 4.4 for the propionic homologue, which can affect aqueous solubility during preparative HPLC purification in ammonium acetate buffers (pH 4.5–5.0). The compound is therefore typically purified under acidic mobile phases ( 0.1% TFA) where it remains protonated and fully retained on C18 media.
In the absence of an assigned CAS registry number, the chemical is ordered via supplier-specific catalogue identifiers. Batch-to-batch consistency in melting point and IR fingerprint is the primary criterion for acceptance in continuous manufacturing campaigns. No evidence of polymorphic transitions has been observed upon milling (jet mill, 5 µm target) or lyophilisation from tert-butanol/water mixtures, indicating a robust crystalline habit suitable for early-phase formulation studies.
