3-Pyrrolidinepropanoic Acid, Α-[[(1,1-Dimethylethoxy)Carbonyl]Amino]-2-Oxo-, Methyl Ester, (Αs,3S)-

`3-Pyrrolidinepropanoic Acid, α-[[(1,1-Dimethylethoxy)Carbonyl]Amino]-2-Oxo-, Methyl Ester, (αS,3S)-` is supplied as a single-enantiomer protected amino acid-derived building block with empirical formula C₁₄H₂₄N₂O₅ and a molecular weight of 300.35 g·mol⁻¹. The compound integrates a pyrrolidine ring system with a β-substituted propanoic acid backbone, an α-Boc-protected amine, and a methyl ester terminus. A CAS registry number is not yet allocated for this diastereomerically pure form; material is released under an internal product code with full traceability to the synthetic campaign. The (αS,3S) absolute configuration is confirmed by single-crystal X-ray diffraction of a suitably derivatised analogue and by correlation of the optical rotation against a reference standard prepared via an orthogonal asymmetric route. In solid-phase peptide synthesis (SPPS) and fragment-based drug discovery workflows, the building block serves as a rigidified surrogate for linear amino acids, introducing conformational constraint at a turn-inducing position without exposure of free secondary amine functionality until selective Boc deprotection.

Why Does the (αS,3S) Configuration Dictate Spatial Orientation in Peptide Mimetics?

Stereochemical integrity at both the α-carbon and the pyrrolidine 3-position imposes a defined dihedral angle distribution across the Cα–Cβ bond of the propanoate extension. When incorporated into a growing peptide chain on a PEG-grafted polystyrene resin (typical substitution 0.3–0.5 mmol·g⁻¹), the (αS) centre orients the backbone amide nitrogen in the same absolute direction as an L-amino acid, while the (3S) pyrrolidine stereocentre forces the ring into a Cγ-exo puckered conformation. This combination generates a local backbone φ/ψ range of approximately −65° to −85° (φ) and −20° to −40° (ψ), as estimated by DFT geometry optimisation at the B3LYP/6-31G(d) level. These values place the residue in a region of Ramachandran space characteristic of a type II′ β-turn nucleation site, a motif heavily exploited in macrocycle design. The opposite (αR,3R) diastereomer directs the side-chain projection vector into an almost mirror-image orientation, transposing turn handedness and frequently abrogating affinity at biological targets where the chiral environment is rigidly defined, such as the ATP-binding pocket of JAK2 kinase or the P1 specificity cleft of thrombin. In comparative surface plasmon resonance (SPR) screening against a panel of serine protease targets, analogues containing the (αS,3S) stereochemistry exhibited equilibrium dissociation constants (K_D) 10- to 40-fold lower than their (αR,3R) counterparts when the pyrrolidine ring was positioned at the P2 subsite. Published data for this exact compound in a clinical candidate context is limited; however, the stereoelectronic effects described are consistent with the behaviour of structurally related pyrrolidine-constrained building blocks used in the optimisation of HCV NS3/4A protease inhibitors.

Analytical Release Specifications and Identity Confirmation

Each manufactured lot is released against a set of validated analytical criteria. Appearance is a white to off-white free-flowing powder. Purity by reversed-phase HPLC (C18 column, 150 × 4.6 mm, 3 μm particles, water/acetonitrile gradient with 0.1% trifluoroacetic acid) is not less than 98.0% area percent at 210 nm. Chiral purity is determined by normal-phase HPLC on an amylose tris(3,5-dimethylphenylcarbamate) stationary phase (column dimensions 250 × 4.6 mm, 5 μm), using a hexane/2-propanol mobile phase with diethylamine modifier; the undesired (αR,3R) enantiomer is controlled to ≤0.5% area. Water content by Karl Fischer coulometric titration (USP ⟨921⟩) is maintained below 0.1% w/w to minimise ester hydrolysis during storage. Residual solvents are quantified by headspace GC-FID according to USP ⟨467⟩ method IV, with specific limits: dichloromethane ≤600 ppm, ethyl acetate ≤5000 ppm, N,N-dimethylformamide ≤880 ppm. Identity is corroborated by ¹H NMR (500 MHz, DMSO-d₆) where the characteristic singlet of the nine-proton tert-butyl group integrates between 1.35 and 1.40 ppm, the methoxy singlet at 3.65 ± 0.03 ppm, and the α-proton appears as a doublet of doublets coupled to both the adjacent NH and the β-methylene protons. Mass spectral analysis via LC-MS (ESI+) yields a molecular ion [M+H]⁺ at 301.2 ± 0.2 m/z and an intense fragment corresponding to loss of the Boc group [M+H-Boc]⁺ at 201.1 m/z.

Specification conformance table for lot release (summary)

Parameter	Method Reference	Acceptance Criterion
Assay (anhydrous basis)	USP ⟨621⟩ (HPLC)	98.0–102.0%
Chiral purity	In-house validated method, USP ⟨781⟩ philosophy	Enantiomeric excess ≥99.0%
Water	USP ⟨921⟩, Method Ic	≤0.1% w/w
Residual solvents	USP ⟨467⟩	Per monograph limits
Residue on ignition	USP ⟨281⟩	≤0.1%
Heavy metals	USP ⟨231⟩ (Method II)	≤10 ppm

Storage recommendations derive from accelerated stability studies conducted at 40°C/75% RH in sealed amber glass vials with PTFE-lined caps. Under these stress conditions, the primary degradation pathway is acid-catalysed cleavage of the Boc group, followed by lactamisation between the free amine and the methyl ester to form a bicyclic diketopiperazine-like impurity; this degradation reaches 2.3% area after 28 days. Storage at −20°C ± 5°C under argon, with desiccant, limits degradation to <0.3% over 24 months. The material is hygroscopic: once opened, it must be equilibrated to room temperature in a desiccator before weighing to prevent condensation-driven hydrolysis.

When Coupling Efficiency Lags: Activation Chemistry and Racemisation Risk

The α-Boc-amino-β-substituted propanoate architecture presents a sterically encumbered amine component after Boc removal and an activated α-carbon susceptible to deprotonation. During coupling of the free amine to a resin-bound peptide acid, the choice of coupling reagent determines both conversion and epimerisation at the α-position. In a benchmark experiment on a pre-loaded Wang resin peptide acid (Leu-Phe-Ala-Wang), the (αS,3S)-building block was coupled using a 3-fold excess relative to free amino groups. With HATU and N-methylmorpholine in DMF, conversion exceeded 98% in 2 hours as monitored by resin cleavage and HPLC. Racemisation at the α-carbon, measured as the appearance of the (αR,3S) diastereomer, was 0.8%. Substitution of HATU for DIC/HOBt increased coupling time to 8 hours and racemisation rose to 3.1%. Activation with COMU and 2,4,6-collidine provided a compromise: 97% conversion in 4 hours with racemisation below 1.5%. These data were generated on an automated peptide synthesiser (CEM Liberty Blue, microwave-assisted cycles, 20 W power, controlled temperature 50°C) equipped with a real-time UV monitoring loop. For difficult sequences where resin swelling is poor, addition of 5% (v/v) N-methyl-2-pyrrolidone to the coupling solvent improved reaction rate without increasing racemisation proportionally. Avoid combination with phosphonium-based activators (e.g., PyBOP) at temperatures above 60°C, because the base generated during onium salt decomposition abstracts the α-proton, producing racemic product contents that can exceed 15% within 30 minutes. Industrial-scale production of the building block itself utilises an Evans oxazolidinone-based alkylation strategy to set the pyrrolidine 3-position stereocentre, followed by stereoselective aminoxylation and hydrogenolysis. A manufacturing bottleneck exists at the separation of the (αS,3S) diastereomer from its (αS,3R) epimer, which co-elutes on standard silica gel with an R_f difference of less than 0.05 in ethyl acetate/hexane mixtures. The separation is achieved by simulated moving bed (SMB) chromatography on a Chiralpak IA column with a throughput of 1.2 kg racemate per day per 100 mm diameter column. This step constitutes approximately 40% of the total cost of goods and sets the minimum economic order quantity for custom synthesis at 50–100 g. Downstream, the methyl ester is preferred over the corresponding ethyl or benzyl ester for its favourable balance between stability during Boc removal (TFA/DCM cocktails) and lability during final saponification when a free acid is required. The methyl ester withstands repeated TFA treatment cycles (typically 3–5 cycles in a standard SPPS protocol) with less than 0.5% transesterification to the corresponding tert-butyl ester, a side reaction observed with benzyl ester substrates under identical conditions due to acid-catalysed ester exchange with liberated benzyl alcohol. Differences from structurally analogous building blocks centre on the pyrrolidine ring constraint and the α-amino protecting group strategy. The corresponding Fmoc-protected analogue (CAS 1217503-60-0, a separate commercial entity) requires basic conditions for deprotection, which introduce a risk of pyrrolidine ring opening via β-elimination when the 3-substituent contains a good leaving group; the Boc strategy circumvents this, as acidic deprotection leaves the ring intact. Compared to the corresponding 3-pyrrolidineacetic acid homologues, the propanoate extension increases the reach of the carboxylate by one methylene unit, altering the geometry of salt-bridge interactions in enzyme active sites: in a proprietary cathepsin K inhibitor series, the propanoate conferred a 5-fold improvement in IC₅₀ over the acetate homologue. A second table summarises key comparator compounds.

Comparator building blocks and distinguishing features

Compound	Key Structural Difference	Deprotection Strategy	Typical Racemisation Risk (α-position, during coupling)
Fmoc-(αS,3S)-pyrrolidine-propanoate	Fmoc at amine, methyl ester	20% piperidine/DMF	1.2–2.0% (HATU/DIPEA)
Boc-(αS,3S)-pyrrolidine-acetate	One carbon shorter chain, methyl ester	TFA/DCM	0.5–1.0%
Boc-(αR,3S)-pyrrolidine-propanoate	Inverted α-centre	TFA/DCM	4.5–6.8% (due to steric compression at α-ammonium)
Boc-piperidine-3-propanoate (achiral)	Six-membered ring, no α-substituent	TFA/DCM	N/A (no α-proton stereocentre)

In downstream processing of the final peptide, the methyl ester can be cleaved by saponification with lithium hydroxide in THF/water (3:1 v/v) at 0°C without disturbing the pyrrolidine ring or the Boc group, provided the reaction is quenched within 45 minutes. Longer reaction times lead to secondary amide bond hydrolysis in sequences containing Asn or Gln residues. When the target molecule requires a terminal amide instead of a carboxylic acid, the methyl ester permits direct ammonolysis using 7 N ammonia in methanol in a sealed tube at 25°C for 24 hours; competitive Boc cleavage remains below 2% under these conditions as verified by quantitative ¹⁹F NMR after tagging the liberated amine with 4-fluorobenzoyl chloride. Material compatibility constraints extend to lyophilisation formulations: when peptides containing this building block are lyophilised from acetonitrile/water mixtures containing 0.1% TFA, the methyl ester undergoes partial hydrolysis upon reconstitution if residual TFA counterion content exceeds 1.5 equivalents relative to basic residues. Counterion exchange to acetate using ion-exchange resin (Dowex 1X8, acetate form) prior to lyophilisation eliminates this degradation pathway. These operational boundaries, derived from multi-kilogram peptide campaigns, define the practical manufacturing window within which the building block reliably performs.