(2S,4R)-1-Tert-Butyl2-Ethyl4-Hydroxypyrrolidine-1,2-Dicarboxylate

The chiral building block (2S,4R)-1-tert-butyl 2-ethyl 4-hydroxypyrrolidine-1,2-dicarboxylate—commonly catalogued as Boc-Hyp-OEt, CAS 90940-46-8, molecular formula C₁₃H₂₃NO₅, molecular weight 273.33 g mol⁻¹—is an orthogonally protected trans-4-hydroxy-L-proline derivative in which the secondary amine is masked by an acid-labile tert-butoxycarbonyl (Boc) group and the carboxylic acid is esterified as the ethyl ester. The product is supplied as a white to off-white crystalline powder with a melting onset of 68.5–71.0 °C (differential scanning calorimetry, 10 K min⁻¹, aluminum pan, nitrogen purge) and is freely soluble in dichloromethane, ethyl acetate, and tetrahydrofuran, but only sparingly soluble in water. Lot-release specifications are anchored to orthogonal analytical methods: purity by reversed-phase HPLC (C18 column, 4.6 × 250 mm, 5 µm, gradient of acetonitrile/water with 0.1% trifluoroacetic acid, UV detection at 210 nm) consistently exceeds 98.5 area%; enantiomeric excess, determined on a chiral stationary phase (Chiralpak AD-H, 250 × 4.6 mm, n-hexane/ethanol 90:10, 0.5 mL min⁻¹, 25 °C), is ≥99.0%; water content by Karl Fischer titration (USP <921>) is maintained below 0.5%; and residue on ignition (USP <281>) remains under 0.1%. Quantitative ¹H NMR (400 MHz, CDCl₃) is employed as a supplementary identity and purity check: the tert-butyl singlet appears at δ 1.40–1.44 (9H), the ethyl ester quartet at δ 4.24 (2H), and the α-proton doublet of doublets at δ 4.47; any lot exhibiting a deviation in the integration of N-Boc rotamer signals exceeding 2% relative to the reference is rejected, as this can indicate incomplete carbamate protection or premature diketopiperazine formation. Storage under an inert headspace is mandatory: the compound is filled into amber borosilicate glass containers, flushed with argon or nitrogen, sealed, and held at 2–8 °C. Long-term stability data at −20 °C over 24 months show no detectable racemization and negligible accumulation of the cyclic 2,5-diketopiperazine impurity; brief excursions to ambient temperature during weighing (≤4 h, relative humidity ≤50%) do not degrade enantiopurity, but prolonged exposure to moisture or alkaline surfaces must be avoided. Manufacturing follows ICH Q7 guidelines for active pharmaceutical ingredient starting materials, and the accompanying certificate of analysis reports residual solvents by headspace GC (USP <467>) with acceptance limits aligned to ICH Q3C. In production-scale campaigns—batches of up to 50 kg have been executed in a jacketed glass-lined reactor—maintaining a steady nitrogen blanket during dissolution has been documented to suppress oxidative discoloration, and filtration through a 0.2 µm inline capsule filter prior to crystallization consistently reduces particulate contamination to meet pharmacopeial limits for injectable-grade intermediates. In synthetic applications, the orthogonality of the Boc and ethyl ester protecting groups defines the compound’s utility. Selective removal of the Boc group proceeds quantitatively with 20% trifluoroacetic acid in dichloromethane at 0 °C to room temperature (typical reaction time 1–3 h), liberating the pyrrolidine nitrogen as a TFA salt while preserving the ethyl ester; subsequent neutralization with a tertiary amine resin or Amberlite IRA-67 yields the free amine for downstream coupling or acylation. Alternatively, mild alkaline saponification—LiOH·H₂O (1.2 equiv) in THF/water (3:1) at 0–5 °C over 4–6 h—cleaves the ethyl ester to the corresponding carboxylic acid without disturbing the Boc group, furnishing Boc-trans-4-hydroxy-L-proline, a direct precursor for solid-phase peptide synthesis of collagen-mimetic peptides and prolyl hydroxylase substrates. This divergent reactivity enables the compound to serve as a chiral pool starting material for structurally diverse targets. Under typical Mitsunobu conditions (PPh₃, DIAD, 4-nitrobenzoic acid, THF, 0 °C to rt), the trans-4-hydroxy group is inverted to the cis-4-acyloxy configuration with retention of the (2S) stereocenter, providing a route to cis-4-amino- or cis-4-azidoproline building blocks used in β-turn peptidomimetics. Direct fluorination with DAST in dichloromethane at −78 °C gives (2S,4R)-1-tert-butyl 2-ethyl 4-fluoropyrrolidine-1,2-dicarboxylate in isolated yields of 80–85% after silica gel chromatography (hexane/ethyl acetate 4:1), and silylation with TBDMSCl in the presence of imidazole yields the TBS ether, which withstands strongly basic alkylation conditions. Oxidative conversion of the 4-hydroxy group to the ketone—using pyridinium chlorochromate on Celite or Swern conditions—delivers the corresponding 4-oxoproline diester, a versatile intermediate for Horner–Wadsworth–Emmons olefinations en route to conformationally constrained pyrrolidine-based organocatalysts. The compound has been employed in medicinal chemistry programmes to construct macrocyclic HCV NS3/4A protease inhibitors and as an advanced intermediate for the synthesis of trans-4-alkylproline analogues that modulate the stability of collagen triple helices. When compared with structurally related protected hydroxyproline esters, several operational advantages and stereochemical boundaries become apparent. The N-Fmoc analogue, (2S,4R)-1-(9-fluorenylmethoxycarbonyl)-2-ethyl 4-hydroxypyrrolidine-2-carboxylate, requires strongly basic conditions (typically 20% piperidine in DMF) for deprotection, which can promote epimerization at the α-carbon of adjacent amino acid residues; the Boc group’s acidic deprotection avoids this risk, making the Boc-ethyl diester the preferred intermediate in solution-phase fragment condensations where retention of stereochemical integrity is paramount. The N-Cbz variant (CAS 13504-85-3) relies on hydrogenolysis (H₂, Pd/C, atmospheric pressure) for unmasking, a step that is incompatible with substrates containing reduction-sensitive functionalities such as alkenes, nitro groups, or benzyl ethers; the Boc compound bypasses this limitation and can be deprotected in the presence of benzylic groups. The ethyl ester itself confers a crystallinity advantage: compared with the methyl ester (CAS 62251-92-5), the ethyl homologue crystallizes reliably from ethyl acetate/hexane mixtures, enabling effective purification of intermediates without column chromatography, a factor that reduces process mass intensity in large-scale preparations. The ethyl ester also undergoes transesterification and aminolysis at rates that are sufficiently slower than those of the methyl ester to provide a wider process window during selective hydrolysis—under LiOH/THF/water conditions, the half-life of the ethyl ester is approximately 2.5 times that of the methyl ester at 0 °C, permitting controlled saponification even in the presence of other base-labile functionality. In contrast, the tert-butyl ester analogue (1,2-di-tert-butyl 4-hydroxypyrrolidine-1,2-dicarboxylate) resists nucleophilic attack under alkaline conditions but can be cleaved with formic acid or TFA; however, its diminished electrophilicity makes direct amidation sluggish and requires activation agents such as HATU or EDCI, whereas the ethyl ester can be directly treated with ammonia or primary amines in methanol with catalytic DMT-MM to give the corresponding amides in high yield. Stereochemistry is of course the decisive differentiator: the (2R,4S) enantiomer, sourced from D-proline, yields peptides with radically altered bioactivity and is rarely incorporated into natural proteinogenic motifs, while the cis-(2S,4S)-4-hydroxyproline isomer forces a pyrrolidine ring pucker that places the 4-substituent on the same face as the carboxylate, generating turn geometries distinct from the natural trans-configured building block. Batch-to-batch consistency across multiple manufacturers is further verified by monitoring the intensity of the νC=O stretching bands in the IR spectrum (Boc carbamate carbonyl at ~1695 cm⁻¹, ester carbonyl at ~1740 cm⁻¹) and by ensuring that the dominant mass spectral ion in ESI+ is the sodium adduct [M+Na]⁺ at m/z 296.1 with an isotopic distribution matching the theoretical pattern. Lower-grade or poorly stored lots often exhibit an additional ion at m/z 252.1 corresponding to the N-Boc-dehydropyrrolidine impurity generated by acid-catalyzed elimination of water from the hydroxyproline ring; such impurities are capped at ≤0.3 area% in current material specifications. Handling precautions are governed by GHS classification: the compound is categorised as a skin irritant (H315) and eye irritant (H319), assigned to GHS Category 2. Work must be conducted in a certified fume hood with powder-transfer operations performed under a ventilated balance enclosure. Nitrile gloves (minimum breakthrough time 240 min per EN 374) and safety goggles conforming to ANSI Z87.1 are mandatory. In the event of a spill, the material is rendered unreactive by adsorption onto a 3:1 mixture of vermiculite and sodium bicarbonate, swept into a chemical waste container, and disposed of via licensed high-temperature incineration complying with EU Directive 2008/98/EC. The product as supplied is intended solely for research and development purposes and is not to be administered to humans or animals; any downstream Good Manufacturing Practice validation of the compound in a drug substance context remains the responsibility of the end-user. Operational boundaries for bulk storage dictate that after removal of a required quantity, the original container must be resealed under an argon purge within 30 min to prevent moisture ingress beyond the 0.5% threshold; should the water content drift above 1.0%, the material can be reconstituted by dissolving in anhydrous dichloromethane, drying over molecular sieves (4 Å), and crystallizing from ethyl acetate/hexane, though enantiomeric excess must be re-confirmed post-treatment. This rigorous specification and application profile differentiates (2S,4R)-1-tert-butyl 2-ethyl 4-hydroxypyrrolidine-1,2-dicarboxylate from generic hydroxyl-proline esters and positions it as a precisely characterised enantiopure synthon for asymmetric synthesis and peptide engineering.