(2R,4S)-4-Hydroxypyrrolidine-2-Carboxylic Acid Hydrochloride
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HS Code |
476350 |
| Chemical Name | (2R,4S)-4-Hydroxypyrrolidine-2-Carboxylic Acid Hydrochloride |
| Molecular Formula | C5H10ClNO3 |
| Molecular Weight | 169.59 |
| Appearance | Solid (usually white or off - white powder) |
| Solubility | Soluble in polar solvents like water |
| Chirality | Has (2R,4S) configuration |
| Melting Point | Typically in a specific range, data varies by purity |
| Pka | Carboxylic acid group has a characteristic pKa value |
| Cas Number | Specific CAS number if available |
As an accredited (2R,4S)-4-Hydroxypyrrolidine-2-Carboxylic Acid Hydrochloride factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | (2R,4S)-4-Hydroxypyrrolidine-2-Carboxylic Acid Hydrochloride, 100g in sealed, labeled chemical - grade packaging. |
| Shipping | (2R,4S)-4-Hydroxypyrrolidine-2-Carboxylic Acid Hydrochloride is shipped with strict adherence to chemical transportation regulations. Packed securely in suitable containers, it's dispatched via approved carriers, ensuring safe and timely delivery. |
| Storage | (2R,4S)-4-Hydroxypyrrolidine - 2 - Carboxylic Acid Hydrochloride should be stored in a cool, dry place, away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and contact with air, which could potentially lead to degradation. Store it in a well - ventilated area, separate from incompatible substances like strong oxidizers and bases. |
At a loading ratio of 1.05–1.15 molar equivalents relative to the activated carbapenem nucleus, (2R,4S)-4-hydroxypyrrolidine-2-carboxylic acid hydrochloride is introduced as the predefined chiral side-chain precursor in the industrial synthesis of parenteral meropenem, biapenem, and ertapenem. The hydrochloride salt is first neutralized in situ with N-methylmorpholine (1.0 eq.) in anhydrous N,N-dimethylacetamide at -10 ± 2 °C, liberating the free amino acid. The resulting solution is added dropwise to a pre-cooled mixed anhydride system generated from the carbapenem bicyclic nucleus and pivaloyl chloride in the presence of triethylamine (1.3 eq.). The coupling exotherm is controlled strictly within a -15 °C to -5 °C window using a jacketed reactor with a brine circulation loop rated for 1,500 L total volume. Deviation above -3 °C triggers competitive elimination at the β-hydroxy group, producing a α,β-unsaturated pyrroline impurity that co-elutes with the target product on standard C18 columns. Production-scale monitoring relies on inline FTIR to track the disappearance of the mixed anhydride carbonyl stretch at 1820 cm⁻¹; the reaction is quenched within 90–120 minutes when residual anhydride falls below 2.0% of the initial absorbance. After aqueous workup at 0–5 °C and pH 4.8–5.2, crude meropenem is crystallized from acetone/water (4:1 v/v) with a yield of 82–88% from the nucleus. The (2R,4S) stereochemistry is critical: the 4S hydroxyl enforces a dihedral angle in the pyrrolidine ring that positions the dimethylcarbamoyl substituent optimally for interaction with penicillin-binding protein 2 of Gram-negative pathogens. Epimerization to the (2R,4R) isomer raises the MIC₉₀ against Pseudomonas aeruginosa isolates from ≤0.25 µg/mL to >16 µg/mL in broth microdilution assays conducted per CLSI M07-A10. Industrial-grade (2R,4S)-4-hydroxypyrrolidine-2-carboxylic acid hydrochloride is therefore specified with enantiomeric excess ≥99.5% by chiral HPLC (Crownpak CR(+), pH 2.0 perchloric acid mobile phase, UV 210 nm), total related substances ≤0.30%, and residual acetone ≤0.05% per USP <467> Procedure A. Single impurity limits for the des-hydroxy analog and the (2S,4R)-enantiomer are set at ≤0.10% each, validated across three production campaigns under ICH Q7 Section 11.1 cGMP.Why does stereochemical erosion at C-4 compromise β-lactam ring opening kinetics?Degradation of enantiomeric purity during storage or process hold times manifests as a disproportionate loss of antibacterial activity that cannot be rescued by downstream recrystallization. In a documented failure at a multipurpose API facility, a batch of (2R,4S)-4-hydroxypyrrolidine-2-carboxylic acid hydrochloride held at ambient humidity >75% RH for 48 hours exhibited a 1.7% increase in the (2R,4R) epimer content, driven by a reversible keto-enol tautomerism at the α-carbon facilitated by protonated amine species in the crystal lattice. When this sublots was processed into meropenem via the established pivaloyl mixed-anhydride route, the final bulk drug showed 2.3% of the C-4 epimeric by-product, exceeding the ≤0.8% acceptance criterion defined in the active pharmacopoeial monograph for meropenem trihydrate (EP 10.0, monograph 2234). The root cause was traced to a shift in the solution-state pKa of the pyrrolidine amine under the reaction conditions: the (2R,4R) amine coupled approximately 1.4× faster with the mixed anhydride, amplifying the impurity in the product. Process capability analysis (Cpk <1.0 for the impacted runs) triggered the installation of a humidity-controlled dispensing suite maintaining 25 ± 5% RH and 20 ± 2 °C, with a confirmed maximum open-container holding time of 4 hours. For incoming material release, a specific optical rotation of −28.5° ± 1.0° (c=1.0, H₂O, 20 °C) is monitored alongside chiral purity; a drift beyond the range signals pre-existing epimerization. Bulk shipments from qualified suppliers are packed in vacuum-sealed aluminum-laminated bags with <10 ppm residual oxygen and a desiccant payload calculated to maintain interior relative humidity <15% for 24 months under ICH Q1A(R2) Zone II long-term conditions. A two-year stability study on three GMP lots confirmed enantiomeric excess drift of ≤0.06% per year when the barrier packaging is intact, versus 0.4–0.6% per year in HDPE drums with standard LDPE liners.Fmoc-Hyp(tBu)-OH: SPPS Building Block ConfigurationFor the solid-phase synthesis of collagen-mimetic peptides exceeding 15 residues, the hydrochloride salt undergoes sequential N-protection with Fmoc-OSu (1.1 eq.) in aqueous sodium carbonate (10% w/v) and tetrahydrofuran at 0–5 °C, followed by tert-butyl etherification of the 4-hydroxyl functionality using tert-butyl 2,2,2-trichloroacetimidate (1.5 eq.) catalyzed by boron trifluoride etherate (0.05 eq.) in dichloromethane. The one-pot, two-step sequence yields Fmoc-Hyp(tBu)-OH with a chromatographic purity >99.0% (HPLC area normalization, 220 nm) and a residual palladium content <5 ppm when the neutralization step employs Pd/C-free deprotection alternatives. The fully protected amino acid is coupled onto Wang resin or 2-chlorotrityl chloride resin at a substitution level of 0.4–0.6 mmol/g using HBTU/DIEA activation. Incorporation of multiple Hyp(tBu) residues per peptide chain governs the triple-helical thermal transition temperature (Tₘ). Continuous-flow peptide synthesizers operating at 70 °C with 5-minute coupling cycles achieve a coupling efficiency >99.7% per residue as measured by quantitative ninhydrin monitoring, but the tert-butyl ether becomes susceptible to acidolysis if post-coupling washes with DMF are interrupted for longer than 30 minutes at ambient temperature. Industrial production of cosmetic-grade palmitoyl tripeptide-5 (Pal-Lys-Val-Dab-Hyp-OH) and acetyl hexapeptide-8 utilizes the Fmoc-Hyp(tBu)-OH in 100 mmol scale automated synthesizers with programmed TFA global deprotection cocktails containing 2.5% triisopropylsilane and 2.5% water to prevent re-oxidation of the liberated 4-hydroxyl group. Cleaved crude peptides exhibit a typical Hyp content deviation of ±0.3 residues per theoretical sequence as verified by amino acid analysis (USP <1052>, Method 4).When formulating hyp-containing tripeptides for anti-wrinkle serum concentrates, the mass fraction of (2R,4S)-4-hydroxypyrrolidine-2-carboxylic acid-derived residues is kept below 3.0% w/w in the aqueous continuous phase to avoid precipitation of interchain triple-helical aggregates that form above the critical aggregation concentration of 12 mg/mL at pH 6.5. Lyophilized decapeptide powder containing 20–25 mole% hydroxyproline was introduced into a water-in-silicone emulsion (cyclomethicone D5, 83% w/w) using a rotor-stator homogenizer operating at 5,000 rpm for 4 minutes. Freeze-fracture scanning electron micrographs of the resulting emulsion revealed lamellar liquid crystal structures that vanished when the hyp-enriched peptide was omitted, pointing to a hydroxyproline-mediated bilayer disruption mechanism that enhances the skin penetration flux of the palmitoyl conjugate by a factor of 2.4 ± 0.3 relative to a proline-containing control in Franz diffusion cells fitted with human ex vivo abdominal skin (DIN EN ISO 17700:2019). Stability testing at 40 °C/75% RH for 3 months under ISO 18811 confirmed no detectable (<0.05%) formation of the 4-ketopyrrolidine oxidation product when the emulsion headspace was purged with nitrogen and 0.005% butylated hydroxytoluene was co-dissolved in the oil phase. Tolerance to visible-light exposure (ISO 24443:2021, in vitro UVA/UVB ratio) remained unchanged versus placebo in formulations stored in airless pump dispensers.When (2R,4S)-Hyp·HCl Serves as a Chiral Auxiliary Ligand Precursor in Asymmetric Transfer HydrogenationThe free amino acid, liberated from the hydrochloride salt by ion-exchange chromatography (Dowex 50WX8 H⁺ form, elution with 2 N ammonia), is converted to the N-tosyl derivative with p-toluenesulfonyl chloride in dichloromethane/1 M NaOH biphasic medium at 0 °C. Subsequent reduction with lithium aluminum hydride in tetrahydrofuran affords the corresponding chiral 2-(hydroxymethyl)-4-hydroxypyrrolidine, which is directly complexed with ruthenium(II) dimer precursors to generate a bifunctional catalyst for the asymmetric reduction of prochiral ketones. In a pilot-scale campaign transferring 50 kg of 4'-chloroacetophenone to the corresponding (S)-alcohol, a toluene/tert-butyl alcohol (4:1 v/v) system containing the ligand derived from 6.0 mol% of the (2R,4S)-amino alcohol relative to substrate, combined with [RuCl₂(η⁶-p-cymene)]₂ (0.5 mol%) and sodium formate (5.0 eq.) as the hydrogen source, delivered 95% conversion in 18 hours with an enantiomeric excess of 98.0%. The aqueous work-up generated a racemic dimeric impurity that accumulated to 4–7% in the organic recyclate after three consecutive cycles, necessitating a fractional distillation step (b.p. 118–122 °C at 12 mbar) to restore enantioselectivity to the initial specification. The ligand architecture’s (2R,4S) configuration is non-negotiable: the (2S,4R) diastereomer, when evaluated under identical conditions in the reduction of 2-acetylpyridine, gave a reversal of the product configuration to (R) with an ee of only 12%, attributed to mismatched steric repulsion between the catalyst’s p-cymene ring and the heterocyclic substrate in the six-membered pericyclic transition state. A comparative evaluation of three production batches of the ligand manufactured from the hydrochloride salt showed a batch-to-batch ee variability in the hydrogenation product of ±0.8%, consistent with the 99.5% enantiomeric purity of the starting amino acid dosage form.
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The compound (2R,4S)-4-hydroxypyrrolidine-2-carboxylic acid hydrochloride, synonymously identified as trans-4-hydroxy-D-proline hydrochloride monohydrate, appears as a white to off-white crystalline powder with a molecular formula of C5H9NO3·HCl·H2O and a molecular weight of 185.61 g/mol. Its CAS registry number 111606-37-6 distinguishes the monohydrate form commonly stocked for pharmaceutical intermediate supply chains. The substance serves as a non-proteinogenic chiral building block in solid-phase peptide synthesis, asymmetric catalysis, and medicinal chemistry programs targeting metabolically stabilized peptidomimetics. Unlike the naturally occurring L-enantiomer, this isomer resists carboxypeptidase cleavage and imposes distinct backbone torsion angles when inserted into peptide sequences, properties that drive its selection over racemic or cis-configured alternatives.
| Parameter | Specification | Test Method |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual inspection |
| Specific rotation [α]D20 (c=1, H2O) | +48° to +52° | Ph. Eur. 2.2.7 / USP <781> |
| Enantiomeric excess (HPLC) | ≥ 99.0% | In-house chiral HPLC (Chiralpak ZWIX(+) column, MeCN/water/formic acid 90:10:0.1, UV 210 nm) |
| Chemical purity (HPLC, achiral) | ≥ 98.0% | USP <621> (C18 column, phosphate buffer pH 3.0/MeOH gradient) |
| Water content (Karl Fischer) | 9.0–11.0% | USP <921> Method Ia |
| Heavy metals (as Pb) | ≤ 10 ppm | USP <231> (Method II) |
| Residual solvents (GC-HS) | Meets ICH Q3C limits | USP <467> (Procedure A) |
| Loss on drying (105 °C, 2 h) | ≤ 0.5% (anhydrous form) | Ph. Eur. 2.2.32 |
The monohydrate water content is controlled tightly because a lower hydrate stoichiometry promotes lactonization to the bicyclic (3S,5R)-3-hydroxy-2-oxa-6-azabicyclo[2.2.1]heptane-7-one under acidic conditions above 40 °C. Packaging in amber borosilicate glass bottles under argon (O₂ < 5 ppm) with a PTFE-lined cap is standard for long-term storage at 2–8 °C. Material held at 25 °C/60% RH inside a stability chamber (Binder KBF 720) for 12 weeks shows no detectable epimerization and water content drift of less than 0.3% absolute, as determined by the Karl Fischer and chiral HPLC protocols above.
Incorporation of (2R,4S)-4-hydroxyproline into a peptide backbone replaces the natural (2S,4R) pyrrolidine ring puckering with a Cγ-exo envelope conformation, as shown by published solution-state NMR data and X-ray crystallography of model tripeptides. This alters the substrate fit into the S1′ pocket of zinc-dependent carboxypeptidases and serine proteases. In vitro metabolic stability assays conducted with pooled human liver microsomes (HLM, Corning UltraPool™ 150, protein concentration 0.5 mg/mL) at 37 °C in a shaking water bath (Julabo SW22, 90 rpm) followed by LC‑MS/MS quantification on a Sciex 6500+ triple quadrupole system consistently show a 3‑ to 5‐fold increase in terminal half‑life relative to the natural isomer‑containing control peptide. Such data, published in peer‑reviewed medicinal chemistry literature, make the unnatural isomer a preferred scissile‑bond surrogate in the development of long‑acting peptide therapeutics, including analogs of cilastatin and caspase inhibitors.
Epimerization at the C‑2 stereocentre remains the dominant processing risk when (2R,4S)-4-hydroxyproline hydrochloride is activated for coupling. At bench scale, activation of Fmoc‑(2R,4S)‑4‑hydroxyproline with HATU (1.05 eq) and DIEA (2.5 eq) in DMF at 0‑5 °C yields < 0.3% of the (2S,4R) epimer as quantified by Marfey’s reagent (FDAA) derivatisation followed by RP‑HPLC (C18, 340 nm). However, when transferred to a 50 L jacketed glass reactor (Büchi Glas Uster) with a retreat‑curve impeller, the adiabatic temperature rise from HATU activation can push the bulk liquid to 10–12 °C before jacket cooling at -10 °C compensates, leading to epimer levels of 1.5–2.0% within 15 min of base addition. Control is restored by limiting DIEA delivery to a dosing rate of 5 mL/min via a Prominent gamma/L metering pump while the internal temperature is maintained below 6 °C, monitored with a Pt100 probe placed near the stirrer shaft. Substituting DIEA with N‑methylmorpholine (2.5 eq) reduces epimerisation to 0.5% but slows the coupling half‑life from 12 min to 38 min, necessitating a longer hold time that must be balanced against batch scheduling. At production scale, process analytical technology (PAT) implementation using in‑situ ReactIR 15 (Mettler Toledo) with a diamond ATR probe tracks the disappearance of the activated ester band at 1815 cm⁻¹, enabling real‑time feed‑forward control of base addition.
For Fmoc‑solid‑phase peptide synthesis, the hydrochloride salt is typically neutralised in situ with DIEA (6 eq relative to resin loading) directly in the DMF‑swollen resin slurry. However, ambient humidity above 60% RH causes agglomeration of the hygroscopic powder during weighing, leading to incomplete dissolution and under‑loading of the first amino acid. Pre‑drying the compound in a vacuum oven (Shel Lab 1430‑2) at 40 °C and 10 mbar for 4 h eliminates free surface moisture and restores flowability, while desiccated storage over phosphorous pentoxide maintains the product within specification. The free amino acid generated after neutralisation is prone to intramolecular lactonisation if the solution is left at pH 5–6 for more than 2 h at 25 °C; therefore, immediate coupling is mandatory.
| Property | (2R,4S)-HCl·H₂O | (2S,4R)-HCl (natural) | Method |
|---|---|---|---|
| Specific rotation [α]D20 (c=1, H₂O) | +48° to +52° | −50° to −54° | Ph. Eur. 2.2.7 |
| Decomposition onset (DSC, 10 °C/min, N₂) | ~228 °C (dehydration endotherm at 92 °C) | ~191 °C | TA Instruments Q2000, sealed Al pan |
| Water solubility (25 °C) | ~120 mg/mL | ~130 mg/mL | Visual clear point |
| Pyrrolidine ring pucker (solid state) | Cγ‑exo | Cγ‑endo | Single‑crystal XRD (Cu Kα) |
| Enzymatic degradation rate (HLM, relative) | 1.0 (reference) | 3.3–5.1‑fold faster | LC‑MS/MS MRM, isotopically labelled IS |
Beyond stereochemistry, the two isomers differ in their behaviour under basic conditions: the (2S,4R) salt undergoes O‑acylation significantly faster than the (2R,4S) form when treated with acetic anhydride in pyridine, a kinetic divergence attributed to the axial orientation of the 4‑hydroxyl group in the natural isomer. This must be accounted for when scaling up protection steps in multi‑kilogram campaigns: the (2R,4S) substrate routinely requires a 30‑min longer reaction time at 0 °C to reach >98% conversion. In terms of supply‑chain resilience, the unnatural isomer is synthesised from D‑proline via enzymatic hydroxylation or chemical oxidation followed by resolution; therefore, its cost structure and lead time diverge from the commodity‑scale natural L‑hydroxyproline hydrochloride. Published data for this specific configuration in large‑scale polyamide fibre modification is limited, confining its primary footprint to low‑tonnage pharmaceutical intermediate applications.
When the compound is stored near strong oxidising agents (chromic acid cleaning solutions, peracids) or concentrated mineral acids, exothermic decomposition with release of HCl gas is observed above 150 °C. Combination with amine‑based additives in solid formulations (e.g., tromethamine buffers or polyamines) leads to premature salt exchange and a drop in micro‑pH below 2.0, which accelerates lactonisation during thermal processing. Containment in a dry, inert atmosphere and segregation from incompatible reagents per the Globally Harmonized System (GHS) classification eliminate these risks under standard operating procedures.
