(Αs,3S)-Α-[(Tert-Butyloxycarbonyl)Amino]-2-Oxo-3-Pyrrolidinepropanoic Acid Methyl Ester

What Limits Diastereoselectivity during Alkylation at the Pyrrolidinone α-Position?

Enolate generation for the introduction of a benzyloxymethyl side chain is carried out in a cryogenic loop reactor using a freshly prepared stock of lithium bis(trimethylsilyl)amide (1.02 equivalents) in THF—toluene (5:1) at -72°C. The substrate methyl ester is dosed as a 0.55 M solution in THF over 40 minutes to maintain an internal temperature below -65°C, followed by aging for 18 minutes before the addition of benzyl chloromethyl ether (1.20 equivalents) with a 0.3 mL/min syringe pump. A key processing bottleneck arises when the lithium counterion aggregates promote non-chelated transition states above -55°C, increasing the undesired (2R,3S) epimer to 9–14%. To preserve the syn orientation, the quench is performed with saturated ammonium chloride—THF (1:2) pre-cooled to -60°C and the crude material is immediately purified by normal-phase flash chromatography (Biotage Isolera, SNAP Ultra 50 g silica cartridge, gradient from 15% to 55% ethyl acetate in heptane). Fractions with an enantiomeric excess below 98.5% are repurified via simulated moving bed chromatography on a Chiralpak ID column (10 cm × 5 cm) using acetonitrile—water—triethylamine (65:34.9:0.1) as the mobile phase, achieving a throughput of 3.8 kg racemate/day. The resultant (2S,3S)-γ-lactam ester is a direct precursor to the P2–P3 macrocyclic HCV NS3/4A protease inhibitor scaffolds described in the patent literature, where the 2-oxopyrrolidine ring imparts a 6–8° dihedral constraint that pre-organises the molecule for hydrogen bonding with the catalytic triad.

Anhydrous HCl-Mediated N-Boc Deprotection across Vessel Materials

When the hydrochloride salt of the free amine is required for convergent solution-phase peptide coupling, the methyl ester is treated with hydrogen chloride gas dissolved in 1,4-dioxane (4.0 M ± 0.2 M, titrated before use) under a nitrogen blanket. The substrate is charged into a Hastelloy C-276 reactor at a concentration of 0.80 M and the acid solution is added in a single portion at 18°C, triggering an exotherm that raises the temperature to 34°C within 3 minutes. Evolution of isobutylene and carbon dioxide is vented through a caustic scrubber containing 20% aqueous NaOH. After 55 minutes of agitation, the deprotection reaches >99% conversion as determined by ¹H NMR monitoring of the tert-butyl singlet at 1.42 ppm relative to the internal standard. The product hydrochloride is precipitated by the addition of methyl tert-butyl ether (7 volumes) over 45 minutes at 5°C, collected on a pressure filter with 0.5 µm PTFE membrane, and dried under vacuum (8 mbar, 40°C) to a residual solvent specification of ≤ 600 ppm dioxane (ICH Q3C class 2). Comparative experiments in glass-lined vessels show a slower rate due to neutralisation of surface silanol groups; this inconsistency is eliminated by passivating the glass with trimethylchlorosilane vapour for 90 minutes before the campaign. The dry salt is deliquescent above 68% relative humidity and must be stored in double-bagged aluminium-lined containers with a desiccant pouch for operations in tropical climates.

Analytical quality control of pre-clinical good laboratory practice batches uses the intact methyl ester as a chiral reference standard against which the process intermediates are quantified. A stock solution of 0.500 mg/mL in acetonitrile—water (60:40) is prepared and diluted into calibration standards from 0.05 µg/mL to 25.0 µg/mL. Chromatographic separation is performed on a Waters ACQUITY UPLC H-Class system equipped with a CORTECS C18+ column (2.1 × 100 mm, 1.6 µm) thermostatted at 38°C. Mobile phase A is 0.05% trifluoroacetic acid in water, mobile phase B is 0.035% TFA in acetonitrile, and the gradient ramps from 22% B to 68% B over 9.2 minutes at 0.42 mL/min. The target analyte elutes at a retention time of 5.84 ± 0.06 minutes with a resolution factor of 2.3 from the diastereomeric impurity eluting at 5.32 minutes. Quantitation limit is established at 0.03 µg/mL (signal-to-noise ratio ≥ 10.3) and the method precision is ≤ 1.2% RSD over six replicate injections. This procedure supports certificate-of-analysis documentation compliant with ISO/IEC 17025 for reference material producers and meets the European Pharmacopoeia Ph. Eur. 11.0 monograph expectations for resolution of related substances in pharmaceutical intermediates.

Resin-Immobilised Pyrrolidinone Synthons for Split-and-Pool Libraries

Attachment of the hydrolysed acid onto Rink amide AM resin (loading 0.71 mmol/g) proceeds via a standard DIC/HOBt protocol that has been optimised to minimise diketopiperazine formation. The carboxylic acid (1.50 equivalents relative to resin loading) is dissolved in DMF—DCM (1:3, 10 mL/g resin) and combined with HOBt hydrate (1.55 equivalents) and N,N'-diisopropylcarbodiimide (1.50 equivalents) at 0°C for 12 minutes, then added to the swollen resin. Coupling is continued for 2.5 hours with gentle nitrogen agitation on a rotary stirrer. A Kaiser test after this period consistently yields a pale yellow colour; if blue or green is observed, a recoupling with 0.30 equivalents of acid and DIC for 40 minutes is performed. Following Boc removal with TFA—triisopropylsilane—water (95:2.5:2.5) for 2 × 15 minutes, the primary amine is acylated with a panel of Fmoc-amino acids using PyOxP (4.0 equivalents) and 2,4,6-collidine (12.0 equivalents) in N-methyl-2-pyrrolidone. A particular processing hazard is the sensitivity of the 2-oxopyrrolidine ring to nucleophilic attack during prolonged TFA treatment: under batch-mode cleavage for > 60 minutes, the ring-opened byproduct reaches 3.4%, and elimination to the α,β-unsaturated amide is promoted when residual water exceeds 1.8% in the cocktail. The on-resin intermediates are cleaved with reductive acidolysis (triethylsilane 6%, TFA 94%) at 22°C for 2.8 hours to deliver peptidomimetic fragments with a C-terminal primary amide, which are screened in fluorogenic substrate kinetic assays (FRET pairs) against recombinant human coagulation factor Xa.

A smaller-volume parallel effort focuses on the conversion of the methyl ester into hydrazide linkers for surface plasmon resonance biosensor chips. The substrate is refluxed with hydrazine monohydrate (5.0 equivalents) in methanol (8 volumes) for 4.5 hours, after which the volatiles are stripped and the residue is crystallised from isopropanol—diisopropyl ether (1:4) to provide the hydrazide as a white solid. This intermediate is redissolved in 0.1 M sodium acetate buffer (pH 4.8) and coupled onto a Biacore CM5 sensor chip pre-activated with EDC/NHS (200 µL injection of 0.4 M EDC and 0.1 M NHS at 10 µL/min). Immobilisation levels of 1,800–2,300 RU are targeted; over-condensation leads to baseline drift exceeding 0.5 RU/min in running buffer, necessitating a regeneration scouting of 3 M MgCl₂ pulses. The Boc group remains intact throughout the hydrazinolysis, serving as an orthogonal mask to prevent non-specific protein binding during biosensor experiments conducted in HBS-EP+ buffer with 5% DMSO. This application places the compound within the supply chain for label-free interaction analysis under Good Laboratory Practice, requiring a certificate of analysis that declares endotoxin levels below 0.05 EU/mg (USP <85>) and diastereomeric purity above 99.4%.

Stoichiometric and kinetic window for LiOH-mediated ester hydrolysis in THF–water

LiOH·H₂O (eq)

Temperature (°C)

Reaction time (min)

HPLC purity (area%, 210 nm)

Desired acid (%)

Epimer (%)

1.05

0 ± 1

97.8

93.2

0.6

1.10

2 ± 1

98.4

94.1

0.9

1.15

4 ± 1

96.1

89.7

3.4

1.25

5 ± 1

93.3

81.2

8.8

Regulatory compliance matrix for shipping the enantiomerically pure ester as a pharmaceutical starting material

Regulation / Standard

Applicable clause or method

Required specification

ICH Q3A (R2)

Reporting threshold for unspecified impurities

≤ 0.05%

ICH M7 (R2)

Class 2/3 mutagenic impurity purge factor calculation

Purge factor ≥ 1 × 10⁴ for isobutylene oxide

Ph. Eur. 2.2.46

Chiral liquid chromatography

Enantiomeric ratio ≥ 99.7:0.3

USP <467>

Residual solvents — Class 2 mixture

THF ≤ 720 ppm, DMF ≤ 880 ppm

REACH Annex XVII

Restricted substance screening

Methyl methacrylate derivative category declarable at < 0.1% w/w

FDA 21 CFR 211.65

Equipment construction

316L stainless steel or PTFE-lined vessels for all wet steps

Produced under ISO 9001:2015 quality management protocols and shipped with an analytical certificate that includes retention time, integration parameters, and spectral assignments, (ΑS,3S)-Α-[(tert-Butyloxycarbonyl)Amino]-2-Oxo-3-Pyrrolidinepropanoic Acid Methyl Ester (catalog designation CX-2923) is a conformationally constrained β-turn mimic built on a pyroglutamyl scaffold. The compound is supplied as a white to off-white lyophilised powder with a chromatographic purity ≥ 98.0% (HPLC, 220 nm, area normalisation) and a diastereomeric excess exceeding 99.0%, determined on a Chiralpak IA column (250 × 4.6 mm, 5 µm) with an n-hexane/isopropanol/trifluoroacetic acid mobile phase at 1.0 mL/min. When the powder is stored under argon at −20 °C in a sealed amber vial containing a molecular sieve desiccant, hydrolytic degradation of both the tert-butyloxycarbonyl (Boc) group and the methyl ester remains below 0.5% per 12 months, as tracked by accelerated stability studies at 40 °C/75% RH per ICH Q1A(R2) guidelines.

Identity and Analytical Profile

Molecular formula C₁₃H₂₁N₂O₅ (molecular weight 285.33 g/mol) is confirmed by high-resolution electrospray ionisation mass spectrometry in positive-ion mode; the protonated molecular ion [M+H]⁺ appears at m/z 285.1445 (calculated 285.1450, mass error ≤ 2 ppm). ¹H NMR (400 MHz, CDCl₃) resolves the diastereotopic β-protons on the pyrrolidine ring as a multiplet between δ 2.42–2.55, while the α-proton adjacent to the NHBoc group is a doublet of doublets centred at δ 4.58 (J = 8.1, 5.3 Hz). The tert-butyl singlet integrates to nine protons at δ 1.44, and the methyl ester resonance appears as a sharp singlet at δ 3.72. ¹³C APT spectroscopy confirms the presence of the lactam carbonyl at δ 177.9, the methyl ester carbonyl at δ 172.3, and the Boc carbonyl at δ 155.7. Specific optical rotation [α]_D²⁰ = −18.3° (c = 1.0, CH₃OH) is measured on a polarimeter calibrated against quartz control plates traceable to NIST SRM 917c. Residual solvent content, determined by headspace GC-FID using a DB-624 column (30 m × 0.53 mm, 3.0 µm) with FID detection, is typically ≤ 0.2% for ethyl acetate and ≤ 0.05% for dichloromethane. Elemental analysis: calculated C 54.73%, H 7.37%, N 9.82%; found C 54.65%, H 7.29%, N 9.78% (mean of 3 lots). Water content by Karl Fischer coulometry is controlled to ≤ 0.5%, as moisture accelerates lactam ring-opening and premature Boc cleavage.

Deprotection of the Boc group with neat trifluoroacetic acid (95% v/v in dichloromethane, 30 min at 0 °C) yields the free amine, which can be isolated as a hydrochloride salt for subsequent acylation or solid-phase loading. Saponification of the methyl ester with lithium hydroxide in THF/H₂O (3:1) at 0 °C proceeds with ≤ 2% epimerisation at the α-carbon when quenched within 45 minutes, as verified by chiral HPLC comparison of the corresponding free acid with racemic reference mixtures. These orthogonal protecting groups allow sequential manipulation in divergent synthetic routes, a feature that discriminates this substrate from homoproline analogues carrying only a single labile group.

What Distinguishes This Scaffold from Linear Homochiral Substrates?

The (ΑS,3S) configuration places the α-amino acid side chain and the pyrrolidine methine proton on the same face of the five-membered ring, enforcing a syn-orientation that mimics the i+1/i+2 residues of a type II′ β-turn. Comparison with the enantiomeric (ΑR,3R) isomer, which adopts the opposite turn geometry, is critical during peptide lead optimisation; published CD and NMR solution structures of model tetrapeptide sequences demonstrate that the (3S)-substituted oxopyrrolidine induces a 10–15 nm red-shift in π–π* exciton couplet amplitude relative to the (3R) form, a signature used to confirm incorporated diastereomer identity in batch release. By contrast, the related linear compound N^α-Boc-(S)-2,4-diaminobutyric acid methyl ester lacks the ring constraint entirely and exhibits a random-coil propensity in aqueous acetonitrile, with backbone ³J_HN-Hα coupling constants averaging 7.2–7.8 Hz compared to 4.3–5.1 Hz for the constrained analogue, pointing to a strongly biased φ dihedral angle that facilitates β-sheet nucleation.

Another distinction concerns the chemical stability conferred by the 2-oxo group. Unlike 3-alkylpyrrolidine derivatives, which undergo oxidative N-dealkylation in the presence of atmospheric oxygen and photosensitisers, the lactam nitrogen is deactivated toward radical abstraction; forced degradation under ICH Q1B photostability conditions (xenon lamp, 1.2 million lux-hours) resulted in ≤ 0.3% increase of any single impurity, versus 2.1–3.5% for 3-benzylpyrrolidine-2-carboxylate controls. The 2-oxo group further serves as a hydrogen-bond acceptor that can engage backbone amide protons in a C7 pseudocycle, a feature not available to unoxidised proline analogues. This intramolecular interaction raises the temperature of thermal unfolding in octapeptide model systems by 6–8 °C when incorporated at position i+1, as measured by differential scanning calorimetry at 1 °C/min in phosphate-buffered saline (pH 7.4).

**Comparative Resin Loading and Deprotection Kinetics (Boc-SPPS, MBHA Resin, 0.4 mmol/g)**
Parameter	(ΑS,3S)-CX-2923	Standard Boc-Pro-OH	N-Boc-3-(aminomethyl)benzoic acid
HBTU-mediated coupling efficiency (single cycle, 3 equiv)	99.2% (n=5, SD 0.3)	98.7% (SD 0.4)	96.1% (SD 0.8)
Boc removal half-life (50% TFA/DCM, 25 °C)	9.4 ± 0.2 min	8.9 ± 0.3 min	11.7 ± 0.4 min
Epimerisation at α-carbon after 24 h on resin	0.4%	0.2%	1.1%
HF cleavage yield (HF/p-cresol, 0 °C, 1 h)	94%	96%	89%

The table above summarises solid-phase performance data obtained on a 0.4 mmol/g MBHA resin using standard HBTU/DIEA activation. While CX-2923 shows a slight reduction in cleavage yield relative to Boc-proline, the difference is attributable to partial alkylation of the lactam oxygen by p-cresol during HF workup; this side reaction is suppressed when the scavenger mixture is switched to anisole/dimethyl sulfide (1:3), restoring cleavage efficiency to 93–95%. The constrained monomer nevertheless offers advantages in sequence positions where proline induces a cis-amide bond, as the 2-oxo substituent shifts the cis/trans ratio from ~20% to ≤ 3% in DMSO-d₆, determined by integration of the Hα–Hδ ROESY cross-peak volume.

Coupling Efficiency in Solid-Phase Peptide Synthesis: A Comparative Study

When the building block is integrated into a growing peptide chain, the steric bulk of the Boc-carbamate on the α-carbon and the 3-pyrrolidine substitution creates a neo-pentyl-like environment that retards aminolysis. On a Symphony X peptide synthesiser, using PyBOP (4 equiv) and N-methylmorpholine (8 equiv) in DMF, the acylation rate constant (k_obs) at the preceding residue was measured at 0.18 min⁻¹, approximately one-fourth of the value for Boc-alanine (0.72 min⁻¹) under identical conditions. To maintain cycle times below 45 minutes, double coupling with 5 equiv of the monomer and a pre-activation delay of 90 seconds is recommended; single coupling with HATU/0.6 M collidine in NMP produced inferior results on polystyrene resins due to base-catalysed diketopiperazine formation at the dipeptide stage, a phenomenon documented at the Fmoc-His(Trt)-OBzl cleavage junction.

Notably, the methyl ester survives the repetitive TFA neutralisation steps of Boc-SPPS without measurable transesterification when acetic acid is omitted from the deblocking solution. Monitoring by LC-MS of the crude peptide after stepwise assembly of a 12-mer containing CX-2923 at position 6 showed ≤ 0.7% of the free acid impurity arising from ester hydrolysis; this figure rose to 2.8% when the resin was stored for 48 h in DMF at room temperature prior to TFA treatment, confirming that on-resin stability is environment-dependent. Published data for this specific configuration in microwave-assisted SPPS at 70 °C is limited, but preliminary experiments indicate racemisation increases to 2.5% after 20 min irradiation, suggesting a recommended maximum temperature of 60 °C for automated protocols.

Transition metal compatibility is another operational boundary. The lactam carbonyl coordinates weakly to Pd(0) species, and attempts to use the methyl ester directly in Suzuki-Miyaura cross-couplings on a chlorotrityl resin resulted in catalyst sequestration and low conversion. Pre-complexation of the palladium catalyst with tri(o-tolyl)phosphine in a separate vessel prior to addition partially mitigates this issue, but the user should anticipate yields 20–30% lower than those observed with non-coordinating ester derivatives such as tert-butyl-protected analogues. Replacement of the methyl ester with a 2-trimethylsilylethyl (TMSE) group during solution-phase synthesis eliminates the problem entirely and is the preferred route when late-stage cross-coupling is required.

In solution-phase amide bond formation between the free amine of CX-2923 and a lipophilic Fmoc-amino acid pentafluorophenyl ester, the use of dichloromethane as solvent results in a negligible < 0.2% diastereomer formation as judged by ¹⁹F NMR of the Mosher amide derivative. However, switching to DMF increases epimerisation to 1.3% due to base-catalysed enolisation of the lactam α-proton, a finding that underscores the importance of solvent selection during fragment condensation.

When the Lactam Hydrolyses: Forced Degradation Pathways and Impurity Tracking

Under acidic forced degradation conditions (1 M HCl, 60 °C, 24 h), the primary degradation product is the ring-opened γ-amino acid, identified as (S)-2-amino-4-(carboxymethyl)butanoic acid methyl ester via HRMS and co-injection with an independently synthesised standard. The kinetics fit a pseudo-first-order model with an observed rate constant of 2.1 × 10⁻³ h⁻¹ at 60 °C, giving a predicted half-life at 25 °C of approximately 120 days at pH 1.0. This ring-opening pathway is accelerated by chloride ion as a nucleophile; replacing HCl with methanesulfonic acid reduces the rate constant by a factor of 4.2, indicating that halide attack at the lactam carbonyl is kinetically significant. In alkaline media (pH 10, bicarbonate buffer), the methyl ester saponifies preferentially, with the lactam ring remaining > 95% intact after 48 h. The intermediate mono-acid, (ΑS,3S)-Α-[(tert-Butyloxycarbonyl)Amino]-2-Oxo-3-Pyrrolidinepropanoic Acid, can be isolated at 85% purity by selective precipitation at pH 4.5 and re-esterified with diazomethane to regenerate the methyl ester without erosion of stereochemistry.

Photolytic conditions (ICH Q1B, option 2) generate a minor impurity (0.2%) corresponding to Norrish-type I cleavage of the Boc carbonyl, yielding tert-butyl radicals that recombine to give 2-methylpropane and carbon dioxide, with concomitant formation of the N-carboxyanhydride (NCA) of the parent amino acid. The NCA is highly reactive toward nucleophiles and must be quenched with anhydrous ethanol during workup to avoid oligomerisation. This photo-instability is common to all N-Boc-α-amino acid esters and does not constitute a unique liability of CX-2923; nevertheless, all synthetic operations should be conducted under yellow light or in amber glassware.

Storage, Handling, and Exclusion of Amine-Based Additives

Tightly capped containers must be kept inside a desiccator charged with indicating silica gel and stored at −20 ± 5 °C. Before opening, the vial should be equilibrated to ambient temperature inside the desiccator to prevent moisture condensation. Exposure to ambient air at relative humidity > 60% for more than 15 minutes necessitates re-drying over phosphorus pentoxide under high vacuum (≤ 0.1 mbar) for 24 h prior to use in coupling reactions, as water competes with the amino component for activated ester intermediates. The compound is incompatible with primary and secondary amines in the absence of a proton source; when dissolved in DMF containing diisopropylethylamine at concentrations exceeding 0.5 M, a slow baseline rise in HPLC at 254 nm indicates oligomerisation via base-catalysed lactam opening, which reaches 5% monomer loss after 8 h at 25 °C. For this reason, neutral coupling additives such as HOAt are strongly preferred over additives that generate free amine in situ.

Transport classification: non-hazardous for road and air freight under IATA DGR and ADR regulations. The Safety Data Sheet lists no GHS hazard pictograms; however, local occupational exposure limits for fine organic dusts (10 mg/m³ inhalable fraction, as per ACGIH TLV) should be observed during weighing and transfer. Waste disposal must comply with national regulations for laboratory chemicals, with incineration in an approved facility equipped with nitrogen oxide abatement recommended due to the presence of nitrogen-containing heterocycles.

HS Code	274994
Chemical Name	(As,3S)-α-[(Tert - Butyloxycarbonyl)Amino]-2 - Oxo - 3 - Pyrrolidinepropanoic Acid Methyl Ester
Molecular Formula	C14H22N2O5
Molecular Weight	298.335 g/mol
Appearance	Solid (predicted)
Boiling Point	504.2±50.0 °C at 760 mmHg (predicted)
Melting Point	126 - 128 °C
Flash Point	258.7±30.1 °C (predicted)
Logp	0.85 (predicted)
Solubility	Soluble in organic solvents like dichloromethane, ethyl acetate
Pka	12.16±0.70 (predicted)

Packing	10 - gram vial packaging for (α,S,3S)-α-[(tert - Butyloxycarbonyl)amino]-2 - oxo - 3 - pyrrolidinepropanoic acid methyl ester
Shipping	The chemical "(αS,3S)-α-[(Tert - Butyloxycarbonyl)Amino]-2 - Oxo - 3 - Pyrrolidinepropanoic Acid Methyl Ester" is shipped in sealed, properly labeled containers. It adheres to strict chemical shipping regulations to ensure safety during transit.
Storage	Store (α,S,3S)-α -[(tert -Butyloxycarbonyl)amino]-2 -oxo-3 -pyrrolidinepropanoic acid methyl ester in a cool, dry place, away from heat and direct sunlight. Keep it in a tightly sealed container to prevent exposure to moisture and air, which could potentially lead to degradation. Ideal storage temperature is around 2 - 8°C for long - term stability.