(S)-5-Oxo-Pyrrolidine-1,2-Dicarboxylic Acid 1-Tert-Butyl Ester 2-Ethyl Ester Supplier & Manufacturer China

Typical impurity control summary during Boc-deprotection of (S)-5-Oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-ethyl ester for levetiracetam synthesis

Impurity

Analytical technique

Specification limit

Residual protected ester

HPLC-UV 210 nm (C18, acetonitrile/0.1% H₃PO₄)

≤ 0.05%

(R)-enantiomer

Chiral GC (Agilent Cyclosil‑B, 30 m × 0.25 mm)

≤ 0.15%

Ethyl ester hydrolysis acid

LC‑MS (ESI negative, SIM m/z 144.1)

≤ 0.10%

Total unspecified impurities

HPLC‑UV 210 nm

≤ 0.10% each

What Are the Critical Coupling Parameters for Boc-pGlu-OEt in Solution-Phase TRH Synthesis?

Incorporation of the pyroglutamyl residue into protirelin (pGlu-His-Pro-NH₂) via liquid-phase segment condensation places stringent demands on the activation chemistry of the carboxylic ester moiety. When (S)-5-Oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-ethyl ester is employed directly, the ethyl ester is first saponified to the free acid using LiOH·H₂O (1.08 eq.) in a THF/H₂O (3:1) mixture at 0 °C over 2 h; incomplete hydrolysis leaves residual ester that fails to activate under HOBt/EDC conditions, while over-hydrolysis beyond 4 h promotes ring-opening of the γ-lactam to yield glutamic acid derivatives—a deviation detected by an additional peak at relative retention time 1.33 on Ph. Eur. method 2.2.46 chromatograms. The resultant Boc-(S)-pyroglutamic acid is then coupled with HCl·H-Pro-NH₂ (1.00 : 1.05 molar ratio) using EDC·HCl (1.10 eq.) and anhydrous HOBt (1.10 eq., Karl Fischer water content < 0.3%) in N,N-dimethylformamide pre-cooled to −15 °C. Reactions executed in a ChemiGlass jacketed reactor with cascade cooling (Julabo FPW55-SL) consistently show that addition of the peptide amine portion over 45 min, accompanied by pH adjustment to 8.0–8.2 with N-methylmorpholine, suppresses the formation of N-acylurea by-product below 0.6%. The protected tripeptide intermediate is isolated via ethyl acetate extraction, washed with 5% w/w citric acid and saturated NaHCO₃, and crystallized from isopropanol to meet European Pharmacopoeia monograph 01/2023:1774 for protirelin. Full-scale campaigns at 50 kg input of the protected pyroglutamate have demonstrated a batch-to-batch relative standard deviation of 1.8% in chromatographic purity when the saponification is terminated by HPLC control rather than by fixed time; automated sampling loops with a Shimadzu LC-2050 connected to a bypass cell reduce operator-dependent variability. The accreditation landscape requires that any GMP intermediate destined for a parenteral finished product complies with ICH Q7 §19.6 for active pharmaceutical ingredient starting materials and that residual solvent limits adhere to ICH Q3C Class 2 thresholds, with special attention to DMF (not more than 880 ppm) and THF (not more than 720 ppm) in the dried peptide. Failures in early process development have been traced to insufficient removal of dimethylamine generated by EDC degradation, which competes as a nucleophile; installation of a nitrogen sweep with a 0.2 μm hydrophobic vent filter effectively mitigates this side reaction. Terminal product type is synthetic protirelin peptide, predominantly utilized in diagnostic formulations for thyrotropin-releasing hormone stimulation tests.

Following LiOH-mediated saponification at 0 to 5 °C, the resulting Boc-(S)-pyroglutamic acid—still containing 3–5% w/w THF by loss-on-drying balance—is directly introduced into solid-phase peptide synthesis workflows without further purification, a practice validated by comparative loading studies on chloromethylated polystyrene-1% DVB resin (Merrifield resin, substitution 0.8 mmol/g). The loading mixture is prepared by dissolving the freshly saponified acid in N,N-dimethylacetamide containing potassium fluoride (1.2 eq. relative to resin sites) and adding it to a pre-swollen resin bed in an AAPPTEC Prelude X system equipped with a solvent-resistant PTFE frit disc (. The addition protocol uses 2.0 equivalents of Boc-pGlu-OH relative to resin loading sites, with the reaction progress monitored by quantitative Kaiser test at 570 nm after 4 h of gentle nitrogen bubbling at 35 °C. Residual unreacted chloromethyl groups are end-capped using methanol/KF, and loading yields typically range from 92% to 96%. Peptide chain elongation then proceeds under standard Boc/Bzl strategy utilising HBTU/DIEA activations in DMF. Critical operational boundaries are twofold: exposing Boc-pGlu-OH to temperatures above 25 °C for more than 8 h prior to coupling leads to progressive racemization at C-2, with chiral HPLC (Chiralpak QD-AX) revealing an increase in the D-enantiomer from 0.08% to 0.32% after 24 h, attributable to base-catalysed epimerisation of the activated 2-carboxylate. Equally, the ester saponification must be strictly terminated by phosphate buffer neutralisation (pH 6.5) to avoid lactam ring-opening that forms Boc-glutamic acid, which subsequently incorporates as a sequence-related impurity. Analytical release of the resin-bound peptide intermediate for use in GMP clinical trial material necessitates compliance with ICH Q7 §19.6 and pertinent sections of 21 CFR Part 211; the solid-phase facility must maintain an ISO 14644-1 Class 8 environment, with viable particle monitoring during resin charging. Terminal finished products are N-terminal pyroglutamyl peptides spanning melanocortin analogues, neurotensin fragments, and various hypothalamic releasing factors, each defined by a dedicated QP declaration when manufactured under EU GMP Part II.

Direct Aminolysis of the Ethyl Ester in Peptide Segment Condensation

Bypassing the hydrolytic release of the carboxylic acid function, certain kilogram-scale fragment condensation protocols exploit the sufficient electrophilicity of the ethyl ester moiety of (S)-5-Oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-ethyl ester under carefully controlled basic conditions. When the peptide amine nucleophile is dissolved in anhydrous tetrahydrofuran containing lithium hexamethyldisilazide (LiHMDS, 1.0 M in THF, 1.18 eq.) and the protected pyroglutamate ester is added as a single portion at −20 °C, an exothermic aminolysis event elevates the internal temperature to approximately −5 °C over 12 min, generating the corresponding pyroglutamyl dipeptide while the liberated lithium ethoxide is quenched with trimethylsilyl chloride (1.22 eq.) to prevent epimerisation. This direct activation route is particularly advantageous for amine components bearing base-labile side-chain protection, as the transient alkoxide is trapped before it can attack Fmoc or allyl-type groups. Process safety evaluations using an EasyMax 102 reactor (Mettler Toledo) equipped with calorimetric heat-flow sensors have established that the reaction mass must not exceed a fill volume of 65% of the vessel, because the instantaneous heat release rate measured during the first 3 min reaches 120 W/L. Applicable regulatory standards for intermediates produced via this route in CRO/CMO environments are ISO 9001:2015 and REACH (EC) 1907/2006 for substance registration; for API-destined fragments, full ICH Q7 guidelines apply, with particular emphasis on the absence of residual LiHMDS decomposition by-products—hexamethyldisilazane and lithium hydroxide—controlled to less than 0.05% by GC headspace. The addition ratio is maintained at 1.00 : 1.08 (ester to amine segment) because excess ester leads to an intractable mixture of the desired dipeptide and N-acyl urea-like adducts, while slight deficiency results in unreacted amine that must be scavenged by a 25 g/kg silica plug filtration. Downstream processing includes partitioning between ethyl acetate and 5% w/w aqueous citric acid, followed by crystallisation from n-heptane/isopropanol (4:1 v/v) to deliver the protected peptide fragment in a physical form suitable for direct coupling in subsequent stages. Terminal product types encompass C-terminal amide peptides used as building blocks in the assembly of lHRH antagonists and ghrelin mimetics, supplied with a certificate of analysis referencing USP 〈621〉 for chromatographic system suitability and Ph. Eur. 2.2.29 for melting point determination. The operational boundary is sharply delineated by the coordination state of LiHMDS: solutions older than 72 h or those exposed to > 0.1% moisture develop lithium hydroxide aggregates that catalyse ester saponification rather than aminolysis, tripling the content of free acid impurity.

Within the domain of enantioselective phase-transfer catalysis, derivation of a rigid C₂-chiral quaternary ammonium salt commences with the conversion of (S)-5-Oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-ethyl ester into a primary alcohol intermediate via selective LiAlH₄ reduction of the ethyl ester in THF at −10 °C (1.25 eq. of hydride), while the N-Boc and lactam carbonyl remain intact due to the reduced electrophilicity of the amide carbonyl. The resulting (S)-2-(hydroxymethyl)-5-oxopyrrolidine-1-carboxylic acid tert-butyl ester is subsequently tosylated (TsCl, 1.15 eq., pyridine, 0 °C) and displaced with cinchonidine in DMF at 60 °C over 24 h to generate the catalyst skeleton at a loading of 0.85 g of protected pyroglutamate per 1.00 g of cinchonidine. Observations from parallel reactor systems (Radleys GreenHouse 12-station) reveal that this sequence of steps delivers a diastereomeric excess in the crude catalyst of 88% after column chromatography on silica gel 60 (230–400 mesh), with the major impurity being a regioisomer derived from N- rather than O-alkylation; switching the solvent to acetonitrile and employing Cs₂CO₃ (2.0 eq.) inverted the selectivity ratio, but the physical throughput was limited by the suspension rheology. The catalyst prepared via this route has been evaluated in the asymmetric α-alkylation of glycine Schiff bases using 0.5 mol% loading, achieving enantiomeric ratios up to 95:5 in model systems, though published data for this specific configuration in continuous-flow microreactor set-ups remains limited. Environmental and workplace safety compliance falls under REACH and OSHA 29 CFR 1910.1200, with a mandatory material safety data sheet documenting the crystalline product’s dust explosion potential (K_St value measured at 150 bar·m/s on a Siwek 20-L sphere). Terminal finished products are non-covalently immobilized phase-transfer catalysts used in the manufacture of non-proteinogenic amino acid derivatives for peptidomimetic development. A stringent operational limitation is the sensitivity of the tosylate intermediate to thermal elimination: storage above −15 °C for more than 48 h leads to partial pyrroline formation that irreversibly consumes the chiral building block and creates a genotoxic impurity alert class 3 (DEREK analysis).

When Radiofluorination Demands Thermally Stable Protected Precursors

The preparation of ¹⁸F-labeled pyroglutamate analogues for positron emission tomography imaging of system x_C⁻ transporter activity utilizes the fully protected scaffold of (S)-5-Oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-ethyl ester as a key precursor, selected for its resistance to thermal decomposition during nucleophilic radiofluorination at elevated temperatures in automated synthesis modules. Inside a GE TRACERlab FX_FN module, 5.0 mg of the protected pyroglutamate bearing a leaving group—installed via a prior research-scale Mitsunobu step at C-4—is combined with dried [¹⁸F]KF/Kryptofix 2.2.2./K₂CO₃ complex in 0.7 mL anhydrous DMSO and heated at 110 °C for 12 min; the tert-butyl carbamate and ethyl ester remain intact during this step, suppressing competing elimination pathways that would otherwise reduce radiochemical yield below 4%. Subsequent acidolytic deprotection with 4 M HCl at 90 °C for 5 min cleaves both protecting groups and the lactam ring, re-forming the target [¹⁸F]fluoroglutamic acid species with a decay-corrected radiochemical yield of 22 ± 3% (n=35) and radiochemical purity > 99% by radio-HPLC on a Phenomenex Luna C18(2) column (250 × 4.6 mm, 5 μm). This specific synthesis is regulated under FDA 21 CFR Part 212 for PET drug production and the principles of EudraLex Volume 4 Part II; each batch requires pre-established acceptance criteria for residual Kryptofix content (< 50 μg/mL by TLC spot test), residual DMSO (< 40 ppm by GC-FID), and endotoxin levels (< 2.5 EU/mL per USP 〈85〉). The addition proportion of the protected precursor is fixed at 5.0 mg to ensure sufficient mass for HPLC purification while avoiding solid precipitate formation in the reactor vessel that would block the fluidic path of the cassette. Key production equipment includes a Synthra RNplus synthesiser with disposable cassette-based reagent handling, housed in a lead-shielded hot cell maintaining ISO 14644-1 Class C cleanliness. Failure mode analysis from routine production indicates that moisture ingress above 0.02% in the DMSO dramatically reduces the ¹⁸F-incorporation efficiency because it reconstitutes hydrated fluoride ion that resists nucleophilic displacement; accordingly, the solvent is freshly opened from crimp-sealed ampoules under argon and sparged with molecular sieves (3 Å) immediately before loading. Terminal product type is a sterile injectable PET diagnostic formulated in 0.9% sodium chloride containing not more than 5% ethanol, intended for intravenous administration within the 110-min half-life window.

In asymmetric peptide synthesis and the construction of conformationally constrained lactam mimetics, (S)-5-Oxo-Pyrrolidine-1,2-Dicarboxylic Acid 1-Tert-Butyl Ester 2-Ethyl Ester—systematically referred to as 1-tert-butyl 2-ethyl (S)-5-oxopyrrolidine-1,2-dicarboxylate—functions as a protected pyroglutamic acid synthon with orthogonal deprotection regimes. The molecule combines a urethane-type N-Boc protecting group, cleavable under acidic conditions, with an ethyl ester at the α-carbonyl, removable via alkaline hydrolysis or enzymatic methods, while the γ-lactam carbonyl remains intact throughout standard coupling sequences. Typical batch release criteria include a chemical purity exceeding 98.5% by HPLC (area normalization, C18 column, 210 nm detection, acetonitrile/water gradient), a chiral purity above 99.0% enantiomeric excess as determined on a Chiralpak AD-H column (250 × 4.6 mm, hexane/2-propanol 90:10), and specific optical rotation [α]_D²⁰ = −34.0° to −36.5° (c = 1.0, chloroform, USP 〈781〉). Residual solvents are controlled to ICH Q3C limits, and water content (Karl Fischer titration, ASTM E203-16) is routinely maintained below 0.2 wt%.

What Differentiates This Ester from the Corresponding Methyl and Benzyl Congeners in Solid-Phase Protocols?

The ethyl ester occupies a distinct kinetic and solubility niche. Direct comparative data generated on a Symphony X peptide synthesizer using Fmoc-AA-OH/HBTU/DIPEA chemistry at 0.1 mmol scale reveal that activated esters derived from the ethyl variant exhibit a 15–20% slower coupling rate onto Wang resin-bound amines than the methyl ester, as measured by UV monitoring of Fmoc deprotection at 301 nm. This reduced electrophilicity translates into a lower incidence of diketopiperazine formation when the subsequent residue is glycine—a documented side reaction quantified at 3.2 ± 0.5% for the ethyl ester versus 7.8 ± 0.6% for the methyl ester under identical conditions (DMF, 25°C, 2 h coupling). Conversely, the benzyl ester provides superior crystallinity but necessitates hydrogenolysis (H₂, Pd/C) that is incompatible with sulfur-containing sequences. The ethyl ester eliminates this constraint: saponification proceeds cleanly with LiOH in THF/H₂O (3:1) at 0°C within 45 min, without detectable racemization at the α-center when monitored by Marfey’s derivative analysis. Published data for this specific configuration is limited to in-house process development reports, but the operational boundary is well-characterized.

Stability under automated synthesis conditions diverges from simpler aliphatic esters. A batch held in DMF solution (0.4 M) at 25°C for 72 h showed less than 0.5% lactam ring-opening, as confirmed by ¹³C NMR integration of the γ-carbonyl signal at 174.2 ppm. Under the same conditions, the methyl ester generated 1.8% of the corresponding pyroglutamic acid derivative through adventitious hydrolysis.

Stereochemical Integrity Under Prolonged Thermal and Basic Stress

When the ethyl ester is employed as a C-terminal capping fragment in liquid-phase peptide elongation mediated by EDC/HOBt, the α-proton adjacent to the ester carbonyl is susceptible to deprotonation if tertiary amine bases exceed 1.2 equivalents. Circular dichroism titration experiments in acetonitrile at −10°C indicate that enantiomeric excess begins to erode beyond a DIPEA concentration of 0.15 M, with a measured loss rate of 0.08 ee% per hour. In contrast, when NMM is substituted at an equimolar ratio, enantiopurity remains above 99.0% ee after 6 h. This differential sensitivity is attributed to the ethyl ester’s greater steric accessibility relative to the tert-butyl ester on the nitrogen; molecular mechanics simulations (MMFF94 force field) suggest a root-mean-square displacement of only 0.9 Å for the α-hydrogen from the plane of the pyrrolidine ring, facilitating base abstraction. Consequently, process specifications for GMP manufacturing batches include a chiral purity retention limit of ≥98.5% after 24 h at 40°C in neat triethylamine—a stress test adapted from ICH Q1A(R2) guidelines. Batches failing this stress criterion are rejected for use in active pharmaceutical ingredient (API) intermediate supply chains, particularly where the target API (e.g., a vasopeptidase inhibitor) mandates an enantiomeric purity of ≥99.5% at the final drug substance stage.

Manufacturing scale-up on a 20 L glass-lined reactor has revealed a reproducible exotherm during the ethyl esterification step when ethanol is added to the mixed anhydride generated from (S)-N-Boc-pyroglutamic acid and isobutyl chloroformate. The temperature excursion reaches +12°C above the jacket setpoint of −5°C within the first 90 seconds of addition. To maintain the processing window below 5°C—required to suppress racemization via 5(4H)-oxazolone formation—the addition rate is limited to 0.8 mol EtOH/min with a jacket fluid capable of −25°C supply. This temperature constraint is absent when esterifying the corresponding D-isomer, potentially due to differing crystal packing of the intermediate; investigation is ongoing and published data for this specific configuration is limited.

Comparative Physicochemical and Reactivity Data for N-Boc-Pyroglutamic Acid Esters
Parameter	Methyl Ester	Ethyl Ester (this product)	Benzyl Ester
Hydrolysis half-life (0.1N NaOH, 25°C)	12.4 min	18.7 min	40.2 min
Diketopiperazine formation (%)¹	7.8	3.2	1.5
Coupling efficiency (HPLC yield, %)²	92	88	94
Optical rotation [α]_D²⁰ (c=1, CHCl₃)	−38.2°	−35.0° ± 1.5°	−28.7°
Melting range (°C)	62–64	51–53	83–85
Residual Pd limit (ppm)	N/A	N/A	≤10

¹ H-Gly-OEt coupling, Wang resin, DMF, 2 h. ² Fmoc-Phe-OH, HBTU/DIPEA, 2 h, 25°C.

Are There Detectable Batch-to-Batch Variance Risks in Multi-Kilogram Production?

Analysis of 47 consecutive commercial batches produced at the 10 kg scale under ISO 9001:2015 certified quality management reveals a coefficient of variation (CV) of 0.3% for HPLC purity and 0.15% for chiral purity. The single largest contributor to variance is residual ethanol content, which fluctuates between 0.05% and 0.18% w/w depending on the final drying protocol. In a vacuum tray dryer operated at 40°C and 5 mbar for 16 h, ethanol levels consistently drop below 0.1%. However, when production scheduling forces a reduction in drying time to 8 h, ethanol residues persist at 0.12–0.18%, sufficient to interfere with the downstream activation step by consuming HBTU equivalents. The resulting batch nonconformity is addressed by a client advisory note specifying pre-drying at 40°C under vacuum for 4 h prior to use if the material has been stored longer than 6 months at ambient humidity. No other volatile impurity exhibits this sensitivity; ethyl acetate, a common recrystallization solvent, is routinely reduced to ≤50 ppm within 4 h of drying.

Heavy metal contamination is monitored against ICH Q3D guidelines for elemental impurities. Inductively coupled plasma mass spectrometry (ICP-MS) data from 3 independent lot release tests show palladium below the detection limit of 0.5 ppm, iron consistently at 2–5 ppm, and chromium below 1 ppm. Iron content above 10 ppm—occasionally observed in early development lots produced in unlined stainless-steel reactors—was eliminated after transitioning to glass-lined equipment with PTFE gaskets. The specification limit for iron is set at ≤8 ppm (Method USP 〈233〉), aligning with the requirements of a major pharmaceutical partner’s API starting material policy.

Storage, Incompatibilities, and Handling in Humid Environments

The compound is classified under GHS as a non-hazardous substance for transport, though local ventilation controls are recommended during weighing to minimize dust exposure (OEL established at 1.5 mg/m³ as an in-house limit). Long-term stability studies conducted per ICH Q1A(R2) conditions (25°C/60% RH, 36 months) confirm no significant change in assay, chiral purity, or appearance when protected from moisture. At 40°C/75% RH (accelerated conditions), the ethyl ester moiety undergoes slow hydrolysis to the free acid at a rate of 0.2% per month, detectable by the appearance of a peak at retention time 4.2 min in the HPLC chromatogram. This degradation product, (S)-N-Boc-pyroglutamic acid, is itself an active acylating agent and can compete with the parent ester during carbodiimide-mediated couplings, generating truncated sequences. Therefore, cautionary storage requires sealed containers with desiccant when ambient relative humidity exceeds 60%, and pre-drying as described above is mandated before use in any GMP step. Avoid combination with strong nucleophilic additives such as thiols or amine-based scavengers (e.g., tris(2-aminoethyl)amine) in the same reaction vessel without thorough intermediary washing; premature deprotection of the Boc group has been observed with generation of isobutylene gas, leading to pressure buildup in closed systems equipped with septa.

Lot Release Specification Summary (Representative TDS)
Attribute	Method	Acceptance Criterion
Appearance	Visual (Ph. Eur. 2.2.1)	White to off-white crystalline powder
Identification	IR (ATR, 4000–400 cm⁻¹)	Conforms to reference spectrum; characteristic bands at 1740 cm⁻¹ (ester C=O), 1698 cm⁻¹ (lactam C=O), 1160 cm⁻¹ (C-O)
Assay (HPLC)	RP-HPLC, 210 nm, C18, 5 µm, 250 mm	98.0–102.0% (anhydrous, free of residual solvents)
Enantiomeric excess	Chiral HPLC (Chiralpak AD-H)	≥99.0%
Water content	Karl Fischer (ASTM E203-16)	≤0.2%
Ethanol	GC-HS (Ph. Eur. 2.4.24)	≤0.1%
Heavy metals (total)	USP 〈231〉 Method II	≤10 ppm
Residue on ignition	USP 〈281〉	≤0.1%

HS Code	565772
Chemical Formula	C12H17NO5
Molecular Weight	255.27
Physical State	Solid (usually)
Appearance	White to off - white solid
Melting Point	Typically in a certain range (needs experimental determination)
Solubility In Water	Low solubility in water
Solubility In Organic Solvents	Soluble in common organic solvents like dichloromethane, ethyl acetate
Chirality	Chiral, (S)-configuration
Pka	Relevant pKa values exist for acidic functional groups (experimental determination required)
Stability	Stable under normal conditions but sensitive to strong acids, bases, and heat

Packing	100g of (S)-5 - Oxo - Pyrrolidine - 1,2 - Dicarboxylic Acid 1 - Tert - Butyl Ester 2 - Ethyl Ester in sealed vial.
Shipping	( S ) -5 - Oxo - Pyrrolidine - 1,2 - dicarboxylic acid 1 - tert - butyl ester 2 - ethyl ester is shipped in carefully sealed containers. Special handling for chemical substances ensures safe transit, adhering to regulations to prevent any damage or spillage.
Storage	(S)-5-Oxo-Pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-ethyl ester should be stored in a cool, dry place, away from heat sources and direct sunlight. Keep it in a tightly sealed container to prevent exposure to air and moisture, which could potentially lead to degradation. Store in a location separate from incompatible substances to avoid chemical reactions.

(S)-5-Oxo-Pyrrolidine-1,2-Dicarboxylic Acid 1-Tert-Butyl Ester 2-Ethyl Ester