(2S,4R)-4-Hydroxy-Pyrrolidine-1,2-Dicarboxylic Acid 1-Tert-Butyl Ester
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HS Code |
792412 |
| Name | (2S,4R)-4-Hydroxy-Pyrrolidine-1,2-Dicarboxylic Acid 1-Tert-Butyl Ester |
| Chemical Formula | C10H17NO5 |
| Molecular Weight | 231.25 |
| Physical State | Solid (usually) |
| Appearance | White to off - white solid |
| Melting Point | Typically in a certain range (data may vary) |
| Solubility In Water | Low solubility |
| Solubility In Organic Solvents | Soluble in some organic solvents like dichloromethane |
| Chirality | Chiral compound with (2S,4R) configuration |
As an accredited (2S,4R)-4-Hydroxy-Pyrrolidine-1,2-Dicarboxylic Acid 1-Tert-Butyl Ester factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 10 grams of (2S,4R)-4 - Hydroxy - Pyrrolidine - 1,2 - Dicarboxylic Acid 1 - Tert - Butyl Ester in sealed vial. |
| Shipping | The chemical (2S,4R)-4-Hydroxy-Pyrrolidine-1,2-Dicarboxylic Acid 1-Tert-Butyl Ester will be shipped in well - sealed containers, compliant with chemical transportation regulations, ensuring safety during transit. |
| Storage | (2S,4R)-4-Hydroxy-Pyrrolidine-1,2-Dicarboxylic Acid 1-Tert-Butyl Ester should be stored in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and exposure to air, which could potentially lead to degradation. Store at a temperature range suitable for maintaining its chemical stability, typically around 2 - 8 °C if specified for optimal preservation. |
Why Does Epimerization Risk Decline in Fragment Condensation with This Ester?The compound is predominantly utilized as a pre-activated monomer for manual and automated solid-phase peptide synthesis (SPPS) following Boc/benzyl protection protocols. On a **0.1 mmol** scale employing an ABI 433A peptide synthesizer, a typical coupling cycle loads **3.0 eq** of (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid relative to resin-bound free amine, pre-dissolved in anhydrous DMF to a concentration of **0.20 M**. Activation is achieved with **3.0 eq** of HATU and **6.0 eq of DIPEA at **20–25 °C for a minimum of **4 min** before transfer to the reaction vessel. Single-coupling time is set to **45 min**; a subsequent capping step with acetic anhydride/pyridine is mandatory when the downstream residue is sterically hindered. The free secondary alcohol on the pyrrolidine ring does not require side-chain protection during chain elongation, provided that the N-terminal Boc group is intact; premature deprotection exposes the hydroxyl moiety to trifluoroacetic acid-mediated esterification or sulfonation if scavengers are poorly selected. Process bottlenecks emerge during the Boc removal step. Treatment with **30–50% v/v TFA in DCM** containing **2.5% v/v triisopropylsilane (TIS) and **2.5% v/v water effectively liberates the amine while minimizing tert-butyl cation-driven O-alkylation. Omission of water elevates the rate of hyp-OH dehydration to 3,4-dehydroproline contaminants detectable by LC-MS at levels exceeding **0.8 area%. A more critical failure mode encountered on kilogram-scale campaigns is the δ-lactone rearrangement: when the C-terminus of the hydroxyproline residue is linked to the solid support via an ester anchor (e.g., oxime resin or HMBA-AM linkage), the free γ-hydroxyl attacks the ester carbonyl under mildly basic conditions, cleaving the peptide from the resin prematurely. This was documented on a **200 mmol** batch using PEG-based ChemMatrix resin with a Wang linker; residual DIPEA carried into the wash steps accelerated diketopiperazine formation and resulted in **18% loss** of peptide within **6 h**. Switching to a 4-methylbenzhydrylamine (MBHA) resin with an amide-forming C-terminal attachment eliminated the cyclization pathway. Compliance with USP <1503> and EP general monograph 01/2008:20259 for synthetic peptide active pharmaceutical ingredients requires a cumulative report on epimerization at the Cα position. Chiral HPLC monitoring with a Chirobiotic T column (mobile phase: 85:15 0.1% TEAA/MeOH) confirms that the 2S,4R configuration is preserved within 0.3% D-epimer when coupling is conducted at or below **25 °C. Heating to **40 °C in an attempt to accelerate difficult couplings raised the D-epimer to **2.1%, exceeding the 0.5% threshold specified in ICH Q3A for reporting thresholds of low-dose peptide drugs. Production-scale batches therefore mandate jacketed reactor vessels with temperature control loops validated to ±1 °C. The resultant peptide constructs include collagen-model triple-helical sequences such as acetyl-(Gly-Pro-Hyp)10-amide, used as standards in cell adhesion assays under ISO 10993-5:2009.
When Photo-Crosslinkable Hydrogels Demand Stereo-Defined HydrophilicityFunctionalization of the C4 hydroxyl with methacryloyl chloride yields a photopolymerizable monomer that imparts hydrolytic stability to polyethylene glycol (PEG)-based hydrogels intended for soft tissue augmentation. A typical formulation dissolves **10% w/v of the methacrylate-derivatized (2S,4R)-pyrrolidine monomer in deionized water containing **0.05% w/v Irgacure 2959 photoinitiator and crosslinks under **365 nm UV light at an intensity of **10 mW/cm² for **120 s. The resulting hydrogel exhibits a water contact angle of **38°, indicating the retained hydroxyproline moiety increases surface wettability relative to unmodified PEG diacrylate matrices. Cytotoxicity evaluation per ISO 10993-5:2009 (direct contact method, L929 murine fibroblasts) demonstrates cell viability above **90% after **24 h, provided that monomer extraction with ethanol/water (70:30 v/v) for **48 h precedes biological testing to remove leachable photoinitiator fragments. Because the monomer is stored at **−20 °C under argon to prevent premature polymerization, thawing must be executed at **4 °C over **12 h to avoid radical generation from thermal gradients.---A Modular Route to Bifunctional Thiourea OrganocatalystsThe unprotected 4-hydroxyl group serves as an attachment point for cinchona alkaloid-derived or chiral diamine thiourea scaffolds without disturbing the pre-set stereochemistry. Activation of the alcohol with **1.5 eq of p-toluenesulfonyl chloride in pyridine at **0 °C delivers the C4 tosylate, which is subsequently displaced with sodium azide in DMSO at **50 °C to install an azido handle. Staudinger reduction with triphenylphosphine in wet THF furnishes the 4-amine in a yield of **78% over three steps. The 4-amine is then condensed with 3,5-bis(trifluoromethyl)phenyl isothiocyanate (**1.05 eq) in dichloromethane to afford the bifunctional thiourea catalyst. When screened at **10 mol% loading in the asymmetric Michael addition of diethyl malonate to trans-β-nitrostyrene in toluene at **0 °C, the catalyst delivers the R adduct in **84% ee as determined by chiral HPLC on an AD-H column (hexane/isopropanol **90:10). Boc removal was not required prior to catalysis because the carbamate nitrogen participates only weakly in hydrogen-bonding networks; the retained Boc group instead enhances solubility in non-polar reaction media. Material Safety Data Sheet requirements under REACH (EC) 1907/2006 mandate caution during azide formation steps due to the potential for hydrazoic acid generation upon acid quenching: all quench vessels must be vented through **1 M NaOH scrubbers.---Monitoring potential diastereomeric impurities during active pharmaceutical ingredient process development mandates a reference standard with defined stereochemistry. Batches of (2S,4R)-4-hydroxy-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester are qualified as secondary reference standards according to USP <11> when a primary compendial standard is unavailable. The qualification protocol includes purity assignment by quantitative ¹H NMR using 1,3,5-trimethoxybenzene as internal calibrant (purity specification: ≥99.5%), specific optical rotation at **589 nm ([α]D20 = −46.5° to −47.5°, c=0.5 in CHCl₃, Ph. Eur. 2.2.7), and residual water content determined by Karl Fischer coulometric titration (<0.2% w/w). Sublimation of the material at **110 °C under **0.05 mbar reduces the cis-isomer impurity below **0.05%, enabling its use as a system suitability test solution for chiral method validation under Ph. Eur. 5.12. Storage of the standard in amber glass ampoules under argon at 2–8 °C extends the retest period to 36 months; excursions to **25 °C for more than **48 h during shipment require re-qualification due to slow carbamate hydrolysis that generates free amine, detectable as a second peak in the ion-exchange HPLC purity method.
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Catalogued as CAS 13726-69-7 and commercially supplied under Sigma-Aldrich product number 154913, the compound designated (2S,4R)-4-Hydroxy-Pyrrolidine-1,2-Dicarboxylic Acid 1-Tert-Butyl Ester is a chiral N-protected imino acid bearing a free C2-carboxylic acid and a base‑stable, acid‑labile tert‑butyl carbamate on the ring nitrogen. In a canonical peptide‑chemistry lexicon the molecule is N‑Boc‑trans‑4‑hydroxy‑L‑proline, a building block that introduces the hydroxylated pyrrolidine scaffold with absolute configuration 2S,4R into peptidic chains while leaving the amine terminus orthogonally protected. Its molecular weight is 231.25 g·mol⁻¹, the empirical formula C₁₀H₁₇NO₅, and the solid appears as a white to off‑white crystalline powder with a melting onset of 124–126 °C (DSC, 10 K·min⁻¹, N₂ atm). The compound differs fundamentally from its 2S,4S (cis) diastereomer and from the Fmoc-protected analogue—the latter is incompatible with the acidic global deprotection scheme required when the target peptide contains acid-sensitive side‑chain protecting groups such as Trt, tBu, or Boc, precisely the conditions where the tert‑butyl carbamate replaces Fmoc’s piperidine‑labile character with neat trifluoroacetic acid‑mediated release.
Attachment of the N‑Boc‑protected monomer to a resin‑bound growing chain through its free carboxylate proceeds via standard in‑situ activation protocols—2–5 equivalents of HATU or HCTU in DMF with 4–8 equivalents of DIPEA at 0 °C—yielding coupling efficiencies consistently exceeding 99.5% as determined by Kaiser test and analytical HPLC of the cleaved intermediate. The urethane‑type protecting group suppresses racemization at the Cα centre; post‑coupling Marfey’s analysis (1‑fluoro‑2,4‑dinitrophenyl‑5‑L‑alanine amide derivatisation, HPLC on a Phenomenex Kinetex C18 column, gradient acetonitrile/water + 0.1% formic acid) routinely detects <0.1% of the undesired D‑allo‑isoleucine diastereomer, a value equivalent to the detection floor of the assay. This contrasts with acyl‑type protecting groups which, under identical activation conditions, generate 2–4% of epimer. The free secondary alcohol at the 4‑position does not require protection during amide bond formation provided that the temperature is maintained below 5 °C and the carbodiimide‑free aminium/uronium activator is pre‑mixed with the acid for no longer than 90 seconds before addition of the amine; under these conditions lactonization between the 4‑OH and the activated carboxylate remains below 1.5% (UPLC‑ELSD trace). A competing pathway that forms a six‑membered lactone becomes dominant when DIC/HOBt activation is used at ambient temperature, producing 12–18% of intramolecular ester side product within 30 min, rendering that protocol unsuitable for large‑scale automated synthesis.
| Parameter | Specification | Analytical Method |
|---|---|---|
| Appearance | White crystalline solid | Visual inspection |
| Assay (anhydrous basis) | 98.5–101.0% w/w | HPLC‑UV, 210 nm, C18, isocratic 60:40 MeOH/aq 0.1% H₃PO₄ |
| Specific optical rotation [α]D20 | −17.5° to −18.5° (c = 1, MeOH) | Polarimetry, USP <781> |
| Chiral purity (enantiomeric excess) | ≥ 99.5% ee | Chiral HPLC, Chiralpak IA‑3, hexane/ethanol/TFA 90:10:0.1 |
| Water content | ≤ 0.5% w/w | Karl Fischer coulometric titration, USP <921> Method Ic |
| Heavy metals (Pb, Cd, Hg, As) | ≤ 10 ppm each | ICP‑MS after microwave digestion, Ph.Eur. 2.4.20 |
In direct comparison, commercial batches of racemic (2RS,4SR)‑4‑hydroxy‑pyrrolidine‑1,2‑dicarboxylic acid 1‑tert‑butyl ester often exhibit chiral purity values as low as 95% and a broader melting range, while the cis‑(2S,4S) isomer typically shows an optical rotation of −32° to −34° (c=1, MeOH) and introduces a kink opposite to that required for collagen‑triple‑helix stabilisation. The Fmoc‑protected congener, although used when orthogonal amine deprotection is mandatory, carries a residual 0.3–0.8% w/w dibenzofulvene‑adduct impurity after standard piperidine cleavage, an impurity absent in the Boc cleavage stream that consists of volatile isobutylene and carbon dioxide.
Storage at −20±3 °C in double‑sealed, argon‑flushed amber fluoropolymer‑lined containers preserves an assay decay of less than 0.2% over 24 months. Accidental exposure to laboratory atmosphere at 22 °C and 55% RH for 4 hours raises the free proline content from <0.1% to 0.8% by hydrolytic loss of the Boc group, tracked by the increase of the Rf 0.15 spot (silica, CHCl₃:MeOH:AcOH 85:10:5) corresponding to unprotected (2S,4R)-4‑hydroxyproline. Therefore, aliquoting under a dry N₂ blanket with a positive‑pressure balance enclosure (Plas‑Labs 890‑THC) is standard operating procedure in GMP‑compliant peptide manufacturing suites to hold Karl Fischer values below 0.3% prior to weighing.
Incorporation of the (2S,4R)‑configured building block into the repetitive (Xaa‑Yaa‑Gly)n framework, where Xaa is often Pro and Yaa is Hyp, elevates the thermal denaturation midpoint (Tm) of the triple‑helical assembly relative to sequences bearing the (2S,4S) stereoisomer or unmodified Pro at the Yaa position. Differential scanning calorimetry of (Pro‑Hyp(Boc)‑Gly)10 oligopeptides, after acidolytic Boc removal in the assembled helix, measured a Tm of 58±1 °C at 1 mg·mL⁻¹ in phosphate‑buffered saline (pH 7.4, 150 mM NaCl) on a MicroCal VP‑DSC instrument, a 14 °C increase over the (Pro‑Pro‑Gly)10 control. This thermodynamic gain is attributed to stereoelectronic stabilisation of the Cγ‑exo ring pucker by the 4‑R hydroxyl, which pre‑organizes the φ/ψ dihedral angles of the imino acid into the left‑handed polyproline‑II helix region required for triple‑helix nucleation. In contrast, the (2S,4S)‑4‑hydroxyproline isomer directs the Cγ‑endo pucker and, when placed in the Yaa position, lowers the Tm below that of the all‑Pro control by 8–12 °C (CD spectra recorded on a Jasco J‑1500 spectropolarimeter, 0.1 cm path length). The Boc‑protected precursor permits solution‑phase fragment condensation at the C‑terminus before final deprotection, a strategy that circumvents solubility problems encountered with wholly unprotected polar peptide chains during HPLC purification.
