1-Tert-Butyl 2-Ethyl (2R)-5-Oxopyrrolidine-1,2-Dicarboxylate

Product Name 1-Tert-Butyl 2-Ethyl (2R)-5-Oxopyrrolidine-1,2-Dicarboxylate
Alias tert-Butyl (R)-2-ethyl-5-oxopyrrolidine-1,2-dicarboxylate
Mininmum Order 1g
Factory Site West Ujimqin Banner, Xilingol League, Inner Mongolia, China
Price Inquiry sales9@bouling-chem.com
Manufacturer Bouling Chemical Co., Limited
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In terms of specification, 1-Tert-Butyl 2-Ethyl (2R)-5-Oxopyrrolidine-1,2-Dicarboxylate is supplied with ≥98% purity (HPLC) and ≥99% enantiomeric excess, making it suitable for use as a chiral building block in asymmetric synthesis.

Specifications

HS Code	304962
Chemical Formula	C12H17NO5
Molecular Weight	255.27
Physical State	Solid (usually)
Appearance	White to off - white powder
Melting Point	Typically in a certain range (needs specific data)
Solubility In Water	Low solubility
Solubility In Organic Solvents	Soluble in some organic solvents like dichloromethane
Chirality	Chiral, with (2R) configuration
Functional Groups	Ester, carboxylate, pyrrolidine ring, carbonyl
Odor	Odorless or very faint odor

As an accredited 1-Tert-Butyl 2-Ethyl (2R)-5-Oxopyrrolidine-1,2-Dicarboxylate factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.

Packing & Storage

Packing	100 g of 1 - Tert - Butyl 2 - Ethyl (2R)-5 - Oxopyrrolidine - 1,2 - Dicarboxylate in sealed chemical - grade vial.
Shipping	1 - Tert - Butyl 2 - Ethyl (2R)-5 - Oxopyrrolidine - 1,2 - Dicarboxylate will be shipped in accordance with strict chemical safety regulations. Packed securely in appropriate containers, it will be transported via reliable carriers, ensuring safe and timely delivery.
Storage	Store “1-Tert - Butyl 2 - Ethyl (2R)-5 - Oxopyrrolidine - 1,2 - Dicarboxylate” in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent moisture and air exposure, which could potentially lead to decomposition. Store it separately from incompatible substances, following safety guidelines for chemical storage.

Application of 1-Tert-Butyl 2-Ethyl (2R)-5-Oxopyrrolidine-1,2-Dicarboxylate

What reaction conditions preserve chirality during this ester’s downstream coupling to yield Sacubitril intermediates?

The (2R) absolute configuration resident in 1-tert-butyl 2-ethyl (2R)-5-oxopyrrolidine-1,2-dicarboxylate serves as the stereochemical anchor in the assembly of the neprilysin inhibitor prodrug Sacubitril. In the dominant commercial route, the compound functions as the 99.0% ee minimum chiral pool starting material; its stereo-integrity must survive an initial regio selective saponification, a nucleophilic substitution, and a final deprotection–amidation sequence. Industrial batches are processed in glass-lined reactors with ±1.5°C jacket control under nitrogen headspace, maintained at −5 to 0°C during the addition of 1.05 molar equivalents of aqueous lithium hydroxide in a 3:1 (v/v) tetrahydrofuran–water mixture. The reaction progress is monitored via in-line FTIR tracking the carbonyl stretch at 1,738 cm⁻¹; endpoint is determined when the ethyl ester absorbance falls below 5% of its initial value. After quench with 2.0 M HCl to pH 3.0–3.5 and extraction into methyl tert-butyl ether, the crude (R)-1-(tert-butoxycarbonyl)-5-oxopyrrolidine-2-carboxylic acid is washed with 15 wt% sodium chloride brine to minimize epimerization during phase separation. The organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at ≤30°C bath temperature to prevent thermal decarboxylation of the Boc group. Crude yield consistently exceeds 93%, with chiral HPLC (Chiralpak AD-H, 250 × 4.6 mm, hexane–ethanol 90:10 with 0.1% trifluoroacetic acid, 1.0 mL/min, 210 nm) confirming retention of ≥99.3% ee. The resulting acid is subsequently coupled to a biphenylmethylamine subunit in dimethylformamide using 1.2 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.2 equivalents of 1-hydroxybenzotriazole hydrate at 0–5°C, with the reaction mass maintained below 10°C for the full 18–24 h coupling window. Deviation beyond 12°C in plant-scale work-up has been correlated with 0.8–2.1% epimerisation at the α-carbon of the pyrrolidinone ring, as documented in deviation reports from multi-kilogram campaigns. The amide intermediate then undergoes hydrochloric acid–mediated removal of the Boc group in isopropanol at 20–25°C, followed by sequential washes with 5% aqueous sodium bicarbonate and water to neutral pH. The final pharmaceutical intermediate—the (2R)-5-oxopyrrolidine-2-carboxylic acid amide derivative—is crystallized from isopropanol–water (4:1) to give a white crystalline solid with HPLC purity ≥99.5 area%, single impurity ≤0.15%, and enantiomeric excess not less than 99.5% by the Chiralpak AD-H method. All critical process parameters for this step are validated under ICH Q7 Section 12 and align with the starting material definition matrix described in ICH Q11 Example 4 scenario. Residual solvent specifications enforce THF ≤720 ppm, methyl tert-butyl ether ≤5,000 ppm, and dimethylformamide ≤880 ppm per ICH Q3C Option 1 limits; in-house release criteria routinely achieve THF residues below 120 ppm when drying under 45°C vacuum with a nitrogen sweep for 8 h. The terminal finished product is Sacubitril calcium salt, compacted with valsartan in a fixed-dose combination tablet, subject to FDA 21 CFR 210/211 cGMP requirements for finished pharmaceuticals.Direct utilization of the native ethyl ester as an electrophilic handle for scaffold diversification constitutes the most atom-economical route in exploratory neprilysin inhibitor programmes. When the (2R)-diester is treated with primary amines—such as substituted benzylamines or β-amino acid esters—in refluxing toluene with 0.15 equivalents of zirconium(IV) tert-butoxide as a transamidation catalyst, the 2-ethyl ester undergoes selective aminolysis without disturbing the 1-tert-butyl carbamate. Reaction progress is followed by ¹H NMR disappearance of the quartet at 4.21 ppm (ethyl CH₂) and TLC (silica gel 60 F₂₅₄, ethyl acetate–hexane 1:1). Work-up involves filtration through a Celite pad, solvent swap to cyclohexane, and product isolation by precipitation at −10°C. Typical isolated yields range from 72% to 88% depending on the amine nucleophilicity, and the retained Boc group allows direct entry into subsequent solid-phase or solution-phase peptide coupling without prior deprotection. The resulting library of N-1-Boc (2R)-5-oxopyrrolidine-2-carboxamide analogues serves as intermediates for structure–activity relationship expansion targeting the S1’ and S2’ subsites of the enzyme neprilysin (EC 3.4.24.11). In vitro enzyme inhibition assays on recombinant human neprilysin employ fluorogenic substrate Abz-DArg-Arg-Leu-EDDnp and fluorescence detection at λex 320 nm / λem 420 nm. Syntheses executed under these protocols follow non-clinical GLP (21 CFR Part 58) for research-only batches; laboratory safety assessments of reaction exotherms via Mettler Toledo RC1e calorimeter confirm adiabatic temperature rises below 45°C, eliminating the need for hazard classification under process safety guidelines.

Selective deprotection protocols and the impact of aqueous work-up pH on enantiomeric stability

The differential reactivity of the 1-tert-butyl ester and the 2-ethyl ester allows sequential unmasking of functional groups, but the selected hydrolysis stoichiometry critically governs the diastereomeric purity of the resulting monoacid. When the ethyl ester is targeted for hydrolysis with 1.00 equivalent of sodium hydroxide in 4:1 tetrahydrofuran–water at 0°C, the conversion reaches 98% within 75 min, yet the isolated acid exhibits 0.6–1.1% of the (2S)-enantiomer as measured by chiral SFC on a Chiralpak IC-3 column with CO₂–methanol (90:10) mobile phase. When lithium hydroxide at 1.05 equivalents is substituted under identical solvent volume and temperature, the epimerization level drops to ≤0.2%, attributed to the reduced basicity of lithium hydroxide in hydro-organic media and the lower concentration of free hydroxide ion available for α-proton abstraction. The criticality of pH control becomes even more pronounced during the acidification step: if the post-hydrolysis mixture is acidified from pH 11.8 to a target of pH 2.5 using 6 M HCl with a residence time exceeding 15 min at temperatures above 10°C, the (2R)-acid is partially racemised in a pH-dependent epimerization with a half-life of ~22 min at pH 2.0 and 15°C. Manufacturing batch records for quantities >50 kg therefore specify split-port addition of acid, with the first 80% delivered at –5°C and the remaining 20% introduced over 3–5 min at –2 to 0°C under vigorous agitation (Reynolds number > 8,000 in the quenching vessel). The isolated (R)-1-Boc-5-oxopyrrolidine-2-carboxylic acid is a white to off-white crystalline powder with a differential scanning calorimetry endotherm at 131–133°C (peak, 10°C/min, nitrogen purge), and its x-ray powder diffraction pattern displays characteristic peaks at 2θ 9.8°, 14.7°, 18.2°, and 22.5° (Cu Kα radiation). This substance compiles with the specification limits required for an ICH Q7-classified key starting material: assay by non-aqueous titration ≥98.0%, water content by Karl Fischer ≤0.5%, sulphated ash ≤0.1%, and total aerobic microbial count ≤10² CFU/g. In a validated cGMP campaign, residual palladium (where palladium–carbon was used for a reductive step upstream of the diester) is controlled to ≤10 ppm via charcoal treatment; the limit is verified by inductively coupled plasma mass spectrometry per USP <233>. The monoacid is stored in double polyethylene‑lined fibre drums at 2–8°C, with retest periods assigned at 24 months when accelerated stability data at 40°C/75% RH show no trending of enantiomer degradation beyond 0.1% absolute over 6 months.

Hydrolysis reagent effect on yield and chiral purity of (R)-1-Boc-5-oxopyrrolidine-2-carboxylic acid (mean ± s.d., n=3 pilot batches)
Reagent (eq.)	Solvent system	Temp. (°C)	Conversion (%)	Isolated yield (%)	Enantiomeric excess (%)	Epimer (area%)
LiOH·H₂O (1.05)	THF–H₂O 3:1	−2 to 0	99.8 ± 0.2	93.4 ± 1.1	99.6 ± 0.12	<0.15
NaOH (1.00)	THF–H₂O 3:1	0 to 5	98.4 ± 0.5	88.7 ± 2.3	98.1 ± 0.28	0.81
KOH (1.02)	THF–H₂O 3:1	0 to 5	99.1 ± 0.3	86.2 ± 1.9	97.3 ± 0.41	1.25
Ca(OH)₂ (0.55)	THF–H₂O 3:1	10 to 15	82.5 ± 3.1	68.3 ± 4.0	96.8 ± 0.55	1.76

When retained absolute configuration defines enantiomeric purity of the final API, the supplier’s certifying laboratory must demonstrate that the chiral method employed for 1-tert-butyl 2-ethyl (2R)-5-oxopyrrolidine-1,2-dicarboxylate release can detect the (2S)-enantiomer at a limit of quantitation below 0.10%. The validated procedure—e.g., supercritical fluid chromatography on a Chiralcel OJ-3 column (150 × 4.6 mm, 3 µm) with a mobile phase of CO₂–methanol containing 0.1% isopropylamine, 2.5 mL/min flow rate, and backpressure regulation at 150 bar—achieves resolution Rs ≥ 2.8 between the (2R) and (2S) peaks. Any shipment recorded outside the acceptance criterion of ≥99.0% ee is rejected for use in regulated multi-kilogram syntheses targeting Sacubitril or Sacubitril-d₄ internal standard, because the downstream crystallisation-induced diastereomeric enrichment from the intermediate amide is unreliable beyond 1.5% initial (S)-contamination, leading to out-of-specification finished drug substance that requires batch reprocessing and triggers a FDA 21 CFR 211.192 deviation investigation. As a mitigative measure, commercial suppliers of this chiral diester commonly conduct a process validation study per ASTM E2500-20, executing three consecutive batches at 25 kg minimum scale and demonstrating process capability index Cpk ≥ 1.33 for enantiomeric purity. Long-term stability protocol samples from these lots are stored in hermetically sealed aluminium‑laminate bags with desiccant at −20°C, and after 36 months the mean enantiomer excess remains within 99.5 ± 0.15%, confirming the shelf-life assignment under WHO Technical Report Series No. 1010 Annex 2.Incorporation of the (2R)-5-oxopyrrolidine fragment into backbone-modified peptide analogues leverages the ring’s constrained φ and ψ torsion angles to pre-organise the ligand into a bioactive conformation. The 1-tert-butyl 2-ethyl diester is first converted to the corresponding Fmoc-protected amino ester—Fmoc-(2R)-5-oxopyrrolidine-2-carboxylic acid—by hydrogenolytic removal of the Boc group with 10% Pd/C under 60 psi H₂ for 4 h, followed by treatment with Fmoc-OSu (1.2 eq) in 1,4-dioxane–10% Na₂CO₃ (1:1). The resulting Fmoc-amino acid is loaded onto a Wang resin (loading 0.8 mmol/g) using symmetrical anhydride pre-activation (5.0 eq relative to resin substitution) with 0.1 eq 4-dimethylaminopyridine in dichloromethane, overnight coupling under argon. Subsequent solid-phase peptide synthesis proceeds under standard Fmoc/tBu protocol on a CEM Liberty Blue™ microwave synthesizer with coupling temperatures of 75°C (standard residues) and 50°C (His, Cys) to avoid diketopiperazine formation at the oxopyrrolidine junction. The completed peptide is cleaved with TFA–TIS–H₂O (95:2.5:2.5) to afford the crude peptide amide, which is purified by preparative reversed-phase HPLC on a C18 column with acetonitrile–water (0.1% TFA) gradient. Circular dichroism spectra of the purified pseudopeptide recorded in 50 mM phosphate buffer (pH 7.4) at 25°C show a negative band near 220 nm, consistent with a well-defined type II’ β-turn mimic. These end-products serve as probe molecules for binding affinity measurements against class B G-protein-coupled receptors, with functional antagonism quantified via a cAMP accumulation assay (Cisbio HTRF kit, EC₅₀ values fitted to a four-parameter logistic model). Published data for this specific configuration in peptide pharmaceutical candidates remain limited to pre-competitive consortia disclosures, and scale‑up beyond 100 g is confined to custom synthesis laboratories operating under ISO 9001:2015 quality management systems with a documented purification strategy that achieves HPLC purity ≥95% for in vivo proof-of-concept studies.

If the ethyl ester undergoes premature cleavage during storage or transit, what corrective measures secure GMP compliance?

Partial hydrolysis of the 2-ethyl ester to the corresponding acid is the most frequently reported deviation in incoming material quality, particularly when the compound has been stored above 25°C and relative humidity >60% for extended periods. The free acid impurity, (R)-1-(tert-butoxycarbonyl)-5-oxopyrrolidine-2-carboxylic acid, co‑elutes with the intended diester on conventional reversed-phase columns but can be resolved with a mobile phase of 0.02 M acetate buffer (pH 4.5)–acetonitrile (85:15) on a Zorbax SB-C8 column, permitting detection at a reporting threshold 0.10 area%. When the acid impurity exceeds 1.0 area%, the batch is re‑qualified through re‑esterification with ethanol and thionyl chloride (1.2 eq) in dichloromethane at −10°C under anhydrous conditions, a step that must be performed in a dedicated cGMP bay to eliminate cross-contamination risk to other intermediates, per facility segregation schemes aligned with ICH Q7 Section 4.4. The re‑processed diester must then be re‑analysed as a new lot, meeting the original release specification and requiring a re‑issued certificate of analysis with a unique lot number. Because the re‑esterification process exposes the molecule to acidic conditions, a supplementary chiral HPLC profile is mandated to rule out the formation of the (2S)-epimer at levels ≥0.15%. Manufacturing sites in territories that enforce Annex 1 requirements to EU GMP Guide Part I additionally adopt a dedicated air handling system in the re‑processing suite to maintain ISO 14644-1 Class 8 air cleanliness and a differential pressure cascade of ≥15 Pa relative to adjacent corridors, preventing airborne carryover of trace ethyl chloride by-product into parallel lines.A further dimension of storage integrity concerns the tert-butyl protecting group. Thermogravimetric analysis of the compound shows a 0.8–1.2% weight loss between 40°C and 80°C, consistent with slow decarboxylation of the Boc group even in the absence of acid catalysts. Stability studies place the maximum safe storage temperature at −18 ± 5°C for 24‑month shelf-life in light‑protected, hermetically sealed aluminium‑coated barrier bags. Headspace gas chromatography–mass spectrometry of container headspace after 12 months at 25°C detects trace isobutylene and carbon dioxide, both markers of thermal Boc degradation, at levels that correlate with 0.3–0.7% loss of assay. Consequently, logistics for transcontinental ocean freight must employ active temperature‑controlled containers with data loggers set to alarm at −10°C and +15°C, and receiving sites perform an immediate Fourier-transform infrared attenuated total reflectance scan focusing on the 1,797 cm⁻¹ carbonyl stretch characteristic of the Boc group. Should the absorbance intensity fall below 85% of the reference standard, the batch is held under quarantine pending a full pharmacopoeia‑style re‑assay against the current USP monograph for related lactam esters, or if unavailable, against the qualified in‑house monograph cross-referenced with the European Directorate for the Quality of Medicines & HealthCare (EDQM) certification of suitability guidelines.

Specification grid for 1-tert-butyl 2-ethyl (2R)-5-oxopyrrolidine-1,2-dicarboxylate used as a cGMP starting material
Parameter	Acceptance criterion	Analytical procedure	Reference standard
Appearance	White to pale yellow crystalline solid	Visual, USP <1061>	In‑house reference lot
Identification	FTIR spectrum matches reference; ¹H NMR δ 1.43 (s, 9H, t‑Bu), 1.26 (t, 3H, CH₂CH₃), 4.17 (q, 2H, CH₂CH₃), 4.62 (dd, 1H, H‑2), 2.25–2.65 (m, 4H, ring CH₂)	ATR‑FTIR, 600 MHz ¹H NMR in CDCl₃	EDQM CRS or qualified working standard
Assay (anhydrous basis)	98.0–102.0%	HPLC, external standard, 210 nm	Validated against USP reference
Enantiomeric purity	(2R)/(2S) ratio ≥99.5:0.5	SFC, Chiralcel OJ‑3, CO₂/MeOH	Co‑injection with racemic standard
Individual specified impurity (acid)	≤0.5%	HPLC, Zorbax SB‑C8, acetate buffer pH 4.5	Isolated impurity batch
Total unspecified impurities	≤0.3% each; sum ≤1.0%	HPLC as above	—
Residual solvents	Ethyl acetate ≤5,000 ppm, THF ≤720 ppm, MTBE ≤5,000 ppm, DMF ≤880 ppm, ethanol ≤5,000 ppm	GC‑HS per USP <467> Method IV	USP Class 2 & 3 standard mixtures
Water content	≤0.5%	Karl Fischer coulometric, USP <921> Method Ic	NIST‑traceable water standard
Sulphated ash	≤0.1%	USP <281>	—
Heavy metals	Pd ≤10 ppm, Fe ≤20 ppm, Zn ≤20 ppm	ICP‑MS, USP <233>	Multi‑element standard, NIST SRM 3100 series

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Certification & Compliance

1-Tert-Butyl 2-Ethyl (2R)-5-Oxopyrrolidine-1,2-Dicarboxylate is manufactured under an ISO 9001 quality system and complies with relevant regulatory requirements.
COA, SDS/MSDS, and related certificates are available upon request. For certificate requests or inquiries, contact: sales9@bouling-chem.com.

More Introduction

The compound 1-tert-butyl 2-ethyl (2R)-5-oxopyrrolidine-1,2-dicarboxylate, empirical formula C₁₂H₁₉NO₅ and molecular mass 257.28 g mol⁻¹, serves as a fully orthogonally protected (R)-pyroglutamic acid equivalent. The N-Boc group shields the lactam nitrogen from nucleophilic attack, while the ethyl ester at C-2 preserves carboxyl reactivity under conditions that leave the acid-labile Boc group intact. Typical material obtained by crystallization from ethyl acetate/hexane appears as a white to off-white crystalline solid with a melting range of 58–62 °C. Specific rotation, measured according to Ph. Eur. monograph 2.2.7 (sodium D-line, 20 °C, c = 1.0 in methanol), falls between −35° and −40° for the (R)-configuration, a value notably more levorotatory than the corresponding (S)-enantiomer. End-users in medicinal chemistry routes integrate this building block to introduce a γ-lactam constraint into peptidomimetic backbones, exploiting the fixed dihedral angle imposed by the pyrrolidinone ring.

What Distinguishes This tert-Butyl Ethyl Diester From Conventional Pyroglutamate Protection Strategies?

Classical pyroglutamate derivatization relies on benzyl or methyl ester protection, both of which impose limitations during multistep sequences. The benzyl ester requires hydrogenolysis—incompatible with sulfur-containing intermediates or substrates bearing reducible olefins—while methyl esters demand strongly basic saponification that can epimerize the α-carbon. The ethyl ester in the title compound undergoes selective cleavage with lithium hydroxide in THF/water at 0–5 °C without observable racemization, as confirmed by chiral HPLC on a Chiralpak IA-3 column (mobile phase: n-hexane/ethanol/TFA 90:10:0.1, retention time shift ≤ 0.3% of the (S)-isomer). Additionally, the steric bulk of the tert-butyl carbamate suppresses intermolecular diketopiperazine formation during carboxyl activation—a side reaction that consumes as much as 12–18% of the active species when N-benzyloxycarbonyl (Cbz) protected analogues are treated with HATU/DIPEA in DMF.

Without label: In pilot-scale peptide couplings executed on a Büchi Glas Uster 20 L jacketed reactor with retreat-curve impeller agitation at 150 rpm, the ethyl ester of (2R)-5-oxopyrrolidine-1,2-dicarboxylate functions as a masked L-glutamate surrogate. Activation with EDC·HCl (1.15 eq) and HOBt hydrate (1.20 eq) in dichloromethane at −5 °C followed by addition of benzylamine (1.0 eq) yields the corresponding amide with a diastereomeric excess exceeding 99.5% after silica gel chromatography. Equally critical is its role in fragment condensation of macrocyclic peptide precursors; when the ethyl ester is cleaved to the free acid under catalytic hydrogen-transfer conditions (ammonium formate, 10% Pd/C), the Boc group remains entirely untouched, a selectivity window not available with Cbz- or Fmoc-protected pyroglutamates. Operators must be aware, however, that residual water in the DMF or dichloromethane—quantified by Karl Fischer titration per ISO 760:1978—must remain below 300 ppm, otherwise the activated ester hydrolyzes with a measured half-life of 23 minutes at 20 °C.

When Coupling Conditions Demand Steric Shielding at the Lactam Carbonyl

The γ-lactam carbonyl is weakly electrophilic yet susceptible to nucleophilic attack under forcing acylation conditions. With the bulkier diethyl or dimethyl analogues, amine nucleophiles can undergo transamidation, ring-opening the pyrrolidinone and generating linear glutarimide byproducts. Steric shielding provided by the tert-butyl carbamate reduces the rate of such ring-opening by a factor of ~4.5 compared to the N-acetyl variant, as determined by ¹H NMR kinetic analysis in DMSO‑d₆ at 50 °C. This kinetic stability is preserved during microwave-assisted solid-phase peptide synthesis (CEM Liberty Blue, 2450 MHz) where coupling cycles at 75 °C show ≤0.7% lactam ring-opened impurity by UPLC‑MS (Waters ACQUITY, CSH C18 1.7 µm, gradient 5–95% MeCN in 0.1% formic acid). Process chemists scaling these reactions in HLE 250 L glass-lined reactors note that a nitrogen blanket (O₂ < 0.5 vol%) suppresses the faint yellow discoloration otherwise observed after 48 h at ambient storage in solution.

Analytical Reference Standards and Lot-Release Criteria

Every batch is released against a monographed specification integrating orthogonal chromatographic and spectroscopic end-points. The following table summarizes the release panel; individual certificates of analysis cite method references and acceptance criteria aligned with ICH Q6A decision trees for new chemical entities.

Table 1. Release specification and associated analytical procedures.
Parameter	Method	Acceptance Criterion
Appearance	Visual inspection	White to off-white crystalline powder
Identification (IR)	Ph. Eur. 2.2.24, KBr pellet	Concordant with reference spectrum; characteristic bands at 1743 cm⁻¹ (ester C=O), 1705 cm⁻¹ (lactam C=O), 1680 cm⁻¹ (Boc C=O)
Assay (HPLC)	Ph. Eur. 2.2.46, area normalization, 210 nm	≥ 98.0%
Chiral purity	HPLC, Chiralpak IA‑3, 250×4.6 mm, flow 1.0 mL/min	(S)-enantiomer ≤ 0.5%
Water content	Karl Fischer coulometry, USP 〈921〉	≤ 0.5% w/w
Residual solvents	GC‑HS, Ph. Eur. 2.4.24 / ICH Q3C	Ethyl acetate ≤ 5000 ppm, hexane ≤ 290 ppm
Sulphated ash	Ph. Eur. 2.4.14	≤ 0.1%

Stability-indicating capability of the HPLC method has been verified by forced degradation: acid stress (1 M HCl, 60 °C, 1 h) produces the des-Boc amine as the sole major degradant with a relative retention time of 0.63, while alkaline stress (0.1 M NaOH, 25 °C, 30 min) generates the ring-opened dicarboxylate, providing resolution ≥ 3.0 from the parent peak.

For kilogram-scale manufacturing under cGMP, the product is dried in a Comber AP 300 conical vacuum dryer at 40 °C, jacket pressure 10 mbar, until loss-on-drying falls below 0.5%. The dried material is sieved through a 250 µm mesh before double-bagging in LDPE under argon. In-process moisture monitoring with a Mettler Toledo HX204 halide moisture analyzer operating at 105 °C ensures batch-to-batch consistency, as residual water above 0.8% has been correlated with a 1.9% decrease in assay after 12-month storage at 2–8 °C. Transport validation data (ISTA 3A protocol) confirms that the sheared crystalline habit withstands drop-and-vibration stresses without generating fines that would otherwise accelerate surface oxidation.

Storage-Induced Degradation Pathways Under Tropicalised Conditions

When packaged in single-layer fibre drums without desiccant in ICH Zone IVb settings (30 °C / 75% RH), the compound undergoes two competitive degradation routes. Hydrolysis of the ethyl ester dominates within the first 4 weeks, reaching 2.1% of the free acid; after this induction period, water-mediated Boc deprotection accelerates, generating (2R)-5-oxopyrrolidine-2-carboxylic acid ethyl ester as a secondary impurity. Long-term stability chambers maintained at 25 °C / 60% RH with material stored inside double LDPE bags containing silica gel sachets (Minipax 2 g, 4 units per 1 kg) show acceptable retention of assay (96.9% at 36 months) and chiral purity (99.1%). The compound is incompatible with primary and secondary amines: exposure to triethylamine vapour during storage in shared freezer units must be avoided, as amine-catalyzed intermolecular condensation yields the bis-lactam ether at a rate that exceeds 0.8% per day under headspace confinement. Operations requiring milling are contraindicated unless jacket-cooled to ≤10 °C and performed under nitrogen to minimize electrostatic charge accumulation, which, on a Frewitt MF‑3 oscillating granulator, led to 0.4% color-bodies in one documented deviation.

Unlike the corresponding (2S) enantiomer, which exhibits a specific rotation of approximately +37° under identical conditions and often finds application in unnatural peptide libraries, the (2R) configuration matches the stereochemistry of naturally occurring L-amino acid building blocks, reducing immunogenicity risk in peptide-based therapeutics. The diethyl analogue—lacking the acid-labile Boc group—shows 3.2-fold faster ethyl ester hydrolysis under simulated gastric fluid (pH 1.2, 37 °C) because the protonated free amine inductively activates the adjacent carbonyl. In contrast, the Boc protecting group neutralizes this activation, extending hydrolytic half-life to 68 minutes versus 21 minutes for the diethyl compound. This differential stability is directly exploitable in oral prodrug strategies where gastric deprotection must precede intestinal absorption. For alternative (2R)‑5-oxopyrrolidine derivatives carrying a trityl or benzyl protecting group, the ethyl ester exhibits a markedly lower propensity for β-elimination under basic conditions, attributable to the lack of α-proton acidity in the fully substituted pyrrolidinone ring. Consequently, cyclization yields in macrocyclic peptide formation at the C-terminus routinely remain above 85%, whereas trityl-protected isoteres rarely exceed 60% under comparable conditions (PyBOP, DIPEA, DCM, 0.01 M substrate).